copyright 2018 seán martin mythen

CHARACTERIZATION OF ENZYMATIC PATHWAYS INVOLVED IN CORTISOL AND BILE ACID METABOLISM BY THE GUT MICROBIOTA

BY

SEÁN MARTIN MYTHEN

THESIS

Submitted in partial fulfillment of the requirements for the degree of Master of Science in Animal Sciences

in the Graduate College of the University of Illinois at Urbana-Champaign, 2018

Urbana, Illinois

Advisor:

Assistant Professor Jason M. Ridlon

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ABSTRACT

The gut microbiota consists of a complex network of distinct bacterial taxa that

together affect the physiology of the human host. Of the many facets affected by gut

microbiota, their impact on the endocrine system is both significant and understudied.

The diversity observed in microbial isolates and operational taxonomic units from one

human gut to another is due largely in part by the strain specific proteins expressed by

the microbes that inhabit them. Some of these unique proteins have evolved to

biotransform host sterols which can be reabsorbed by the body and potentially affect

endocrine functions.

Bacterial hydroxysteroid dehydrogenases (HSDHs) have the potential to

significantly alter the physicochemical properties of bile acids with implications for

increased/decreased toxicity for gut bacteria and the host. We located a gene-cluster in

Eggerthella CAG:298 predicted to encode three HSDHs (CDD59473, CDD59474,

CDD59475), synthesized the genes for heterologous expression in E. coli and then

screened bile acid substrates against the purified recombinant enzymes, revealing

novel 3α-, 3β-, and 12α-HSDHs.

We also developed an enzyme-linked continuous spectrophotometric assay to

quantify steroid-17,20-desmolase activity from recombinant enzymes encoded by the

desA and desB genes from Clostridium scindens ATCC 35704. Steroid-17,20-

desmolase is responsible for the side chain cleavage that biotransforms cortisol into

11β-hydroxyandrostenedione (11β-OHAD).

The reaction performed by the steroid-17,20-desmolase appears to be regulated

via pyridine nucleotide-dependent HSDHs, which are capable of converting cortisol into

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a form unable to be utilized by steroid-17,20-desmolase. A recently identified 20β-

HSDH from Bifidobacterium adolescentis strain L2-32 has been biochemically

characterized and crystallized. This 20β-HSDH has the potential to directly affect

androgen formation by functioning as a metabolic rheostat controlling the steroid-17,20-

desmolase activity of Clostridium scindens ATCC 35704.

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ACKNOWLEDGMENTS

This research would not have been possible without the support of my adviser,

Dr. Jason Ridlon, who took me under his wing when I barely knew how to use a pipette.

I am also thankful for the guidance provided by Dr. Roderick Mackie and Dr. Isaac

Cann. I would like to thank Dr. Saravanan Devendran for his teachings over the past

few years. I would also like to thank each of the students and post-docs in the Ridlon,

Mackie, and Cann labs, with whom I have shared many great moments. I would also

like to thank my family and friends for their endless encouragement. Finally, I would like

to thank my girlfriend, Samantha, for her patience, love, and support.

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TABLE OF CONTENTS

LIST OF FIGURES ......................................................................................................... vii

LIST OF TABLES ............................................................................................................ix

CHAPTER 1: Literature Review ...................................................................................... 1

INTRODUCTION .................................................................................................... 1

STEROIDS ............................................................................................................. 1

STEROID HORMONES .......................................................................................... 7

BILE ACIDS .......................................................................................................... 12

THE GUT MICROBIOTA ...................................................................................... 17

Clostridium scindens ............................................................................................. 17

Eggerthella lenta ................................................................................................... 19

Butyricicoccus desmolans .................................................................................... 20

Bifidobacterium adolescentis ................................................................................ 21

OBJECTIVES ....................................................................................................... 23

CHAPTER 2: Targeted synthesis and characterization of a gene-cluster encoding NAD(P)H-dependent 3α-, 3β-, and 12α-hydroxysteroid dehydrogenases from Eggerthella CAG:298, a gut metagenomic sequence ................................................... 25

ABSTRACT .......................................................................................................... 25

INTRODUCTION .................................................................................................. 26

MATERIALS AND METHODS .............................................................................. 29

RESULTS ............................................................................................................. 33

DISCUSSION ....................................................................................................... 54

CHAPTER 3: The desA and desB genes from Clostridium scindens ATCC 35704 encode steroid-17,20-desmolase .................................................................................. 60

ABSTRACT .......................................................................................................... 60

INTRODUCTION .................................................................................................. 61


RESULTS ............................................................................................................. 69

DISCUSSION ....................................................................................................... 79

CHAPTER 4: Structural and biochemical characterization of 20β-hydroxysteroid dehydrogenase from Bifidobacterium adolescentis strain L2-32 ................................... 95

ABSTRACT .......................................................................................................... 95

INTRODUCTION .................................................................................................. 95

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RESULTS ........................................................................................................... 104

DISCUSSION ..................................................................................................... 114

SUMMARY OF RESEARCH ....................................................................................... 118

FUTURE RESEARCH ................................................................................................. 124

REFERENCES ............................................................................................................ 126

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LIST OF FIGURES

Figure 1.1 Steroid classifications .................................................................................... 3

Figure 1.2 Steroid numbering ......................................................................................... 4

Figure 1.3 Alpha and beta isomeric variation of the C-7 hydroxyl group ........................ 6

Figure 1.4 Phylogenetic analysis of identified 20β-hydroxysteroid dehydrogenases .... 22

Figure 2.1 SDS-PAGE of purified recombinant 3β-, 3α-, and 12α-hydroxysteroid dehydrogenases from a gene cluster in Eggerthella CAG:298 ...................................... 38

Figure 2.2 Representative TLC of bile acid reaction products from recombinant CDD59473 (A), CDD59474 (B), and CDD59475 (C) .................................................... 39

Figure 2.3 Mass spectrometry of enzymatic reaction products ..................................... 42

Figure 2.4 pH optima for purified recombinant CDD59473 (A), CDD59474 (B), and CDD59475 (C) in the oxidative and reductive directions. .............................................. 44

Figure 2.5 Kinetic analysis of recombinant CDD59473, CDD59474, and CDD59475 from metagenomic sequence of Eggerthella CAG:298 ................................................. 46

Figure 2.6 Schematic representation of bile acid metabolism by Eggerthella lenta. ..... 50

Figure 2.7 iTOl representation of FastTree maximum likelihood phylogeny from MUSCLE alignment of bacterial hydroxysteroid dehydrogenases and the placement of CDD59473, CDD59474, and CDD59475. ..................................................................... 51

Figure 2.8 CLUSTAL alignment of biochemically characterized gut bacterial hydroxysteroid dehydrogenases ................................................................................... 57

Figure 3.1 Steroid-17,20-desmolase (DesAB) shares conserved catalytic and co-factor binding residues with transketolases ............................................................................. 70

Figure 3.2 SDS-PAGE of affinity purified rDesA and rDesB and native molecular mass estimate of rDesAB. ...................................................................................................... 72

Figure 3.3 Liquid chromatography-mass spectrometry of rDesAB reaction products ... 73

Figure 3.4 SDS-PAGE of purified recombinant 17β-HSDH from Cochliobolus lunatus (rCL17HSDH) and schematic representation of enzyme-coupled continuous spectrophotometric steroid-17,20-desmolase activity assay. ........................................ 74

Figure 3.5 Biochemical characterization of rDesAB by enzyme-coupled continuous spectrophotometric assay. ............................................................................................ 76

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Figure 3.6 Determination of steroid-17,20-desmolase activity against substrates lacking a 17-hydroxy, and tetrahydro(deoxy)cortisol by LC/MS................................................. 80

Figure 3.7 Functional groups important for rDesAB specificity ..................................... 82

Figure 3.8 Mass spectra of rDesAB catalyzed reactions. ............................................. 83

Figure 3.9 Gene organization and domain architecture of steroid-17,20-desmolase ... 91

Figure 3.10 Schematic representation of side-chain cleavage reactions for steroid-17,20-desmolase from C. scindens ATCC 35704, steroid-transaldolase from Mycobacterium tuberculosis, and Cytochrome P450 monooxygenase (CYP11A1) in Eukaryotes. ................................................................................................................... 93

Figure 4.1 Reaction mechanism of 20β-HSDH ........................................................... 105

Figure 4.2 Whole cell assays of B. adolescentis strain L2-32, B. desmolans ATCC 43058, and C. scindens ATCC 35704 ......................................................................... 106

Figure 4.3 Purified 20β-hydroxysteroid dehydrogenase (20β-HSDH) from Bifidobacterium adolescentis strain L2-32 ................................................................... 108

Figure 4.4 pH optimum and Michaelis-Menten saturation curve of wild-type 20β-HSDH .................................................................................................................................... 109

Figure 4.5 Circular dichroism spectroscopy ................................................................ 112

Figure 4.6 20β-HSDH crystals .................................................................................... 115

Figure 4.7 Competing cortisol metabolizing reactions by B. adolescentis, E. lenta, B. desmolans, and C. scindens ....................................................................................... 123

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LIST OF TABLES

Table 1.1 Concentrations of major steroids found in humans ......................................... 9

Table 2.1 Oligonucleotides used for amplification and directional cloning .................... 30

Table 2.2 Sequences utilized in phylogenetic analysis ................................................. 34

Table 2.3 Kinetic parameters of recombinant CDD59473, CDD59474, and CDD59475 ...................................................................................................................................... 45

Table 2.4 Substrate-specificity of Eggerthella CAG:298 HSDHs .................................. 48

Table 3.1 Oligonucleotides and synthetic gene sequences used in this study .............. 64

Table 3.2 Substrate specificity for rDesAB from C. scindens ATCC 35704 .................. 78

Table 4.1 Kinetic parameters of cortisol reduction by wild-type and mutant 20β-HSDH .................................................................................................................................... 111

Table 4.2 Substrate specificity of 20β-HSDH .............................................................. 113

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CHAPTER 1: Literature Review

INTRODUCTION

It is now widely recognized that the community of microbes that inhabit the guts

of higher eukaryotes function as a “virtual organ”, with many metabolic, immunologic,

and endocrine influences (1). Many microbes in the gut are known to benefit the host

by fermenting indigestible food, synthesizing vitamins, and protecting against

pathogens. It is known that steroids circulating throughout the body alter metabolism,

salt and water balance, immune functions, digestion, and sexual characteristics. The

effect of steroid biotransformations performed by microbes within the gut is significantly

understudied. We have proposed the term “sterolbiome” to describe the repertoire of

human gut microbiome genes involved in the uptake and metabolism of host,

pharmaceutical, and diet derived steroids (2). Small changes in steroid structure can

have significant consequences on both host physiology and disease, as well as

microbial diversity, spore germination, and microbial growth (3). Therefore, there is a

pressing need to understand the nature of steroid biotransformation in the gut and the

biological significance it has on the host. Here, we characterize important gut microbe

enzymatic pathways involved in the biotransformation of steroids.

STEROIDS

Steroids are biologically important molecules utilized by eukaryotic organisms as

essential signaling molecules (4). They exert their effects on the host by binding to

intracellular nuclear receptors which can either induce or repress gene transcription (5).

Small changes in the structure of a steroid determines its overall biological effect and

functional class to which it belongs. The functional classes discussed here are

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glucocorticoids, mineralocorticoids, androgens, estrogens, progestogens, and bile acids

(Figure 1.1) (6).

Steroid Structure and Host Steroid Diversity- Each of these steroid groups is

a biological derivative of cholesterol, and because of that, every steroid shares a

common cyclopentanophenanthrene parent ring structure of three six-carbon

cyclohexane rings and one five-carbon cyclopentane ring, denoted as the A, B, C, and

D rings, respectively (Figure 1.2). Host enzymes create steroid diversity via

modifications to the eight-carbon aliphatic side-chain (carbon positions 20-27), and the

hydroxylation of carbons on the parent ring structure resulting in variations in steroid

length, branching, and oxidation. The steroids secreted into the human gut are uniquely

modified by anaerobic microbes that are capable of producing steroids that the host is

incapable of synthesizing. This explains the differences in gut steroid composition

observed in germ-free versus conventional animals (7).

Steroid Biotransformation by Gut Microbes- A multitude of microbial enzymes

exist that can perform various transformations to the ring system and the side chain of

steroid molecules (8). The main enzymatic reactions catalyzed by gut microbial

enzymes tend to be net reductive and hydrolytic, whereas host enzymatic reactions

tend to be oxidative and conjugative. It is possible to form more than 230 steroids If you

take into account the interplay between host and microbial enzymes (6).

Hydroxysteroid dehydrogenases (HSDHs) are a class of pyridine nucleotide-dependent

oxidoreductases that are involved in the reversible oxidation and epimerization of host

steroids and are the most common type of steroid metabolizing enzymes.

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Figure 1.2 Steroid numbering At its core, a steroid is composed of 17 carbons organized into 4 rings (A, B, C, and D). Rings A, B, and C are cyclohexanes. Ring D is a cyclopentane. Steroids are made unique by the addition or removal of functional groups, by reduction or oxidation of carbons on the 4 ring structure, and many other modifications.

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Hydroxysteroid dehydrogenases- HSDHs expressed by the host are a means

of rapidly regulating the intracellular concentration of ligands for various host nuclear

receptors by functioning as enzymatic “switches” that shuttle steroids between active

and inactive forms. The best example is the interconversion of cortisol (11β-hydroxyl) to

cortisone (11-keto) by the human 11β-HSD1 and 2 isoforms (9). Cortisol is capable of

binding to and activating both the glucocorticoid and the mineralocorticoid receptor, but

cortisone is not a ligand nor an activator of these receptors. In this way, reversible

oxidation and epimerization by HSDHs produce slight, readily reversible, structural

modifications that determine the biological activity of a population of steroid molecules

in host tissues and cells (Figure 1.3). By contrast, HSDHs expressed by microbes

function as redox sinks and have the potential to indirectly affect host physiology and

the surrounding microbial community.

Most HSDHs belong to the superfamily of proteins known as the short-chain

dehydrogenases/reductases (SDRs) (10). Proteins in the SDR superfamily are typically

composed of 250-350 residues and present highly similar tertiary structure folding

patterns consisting of a characteristic Rossmann-fold; which consists of a central β-

sheet (composed of 6-7 β-strands), flanked by α-helices (11). HSDHs are important

SDR type enzymes with a role in various processes such as host growth, energy

homeostasis, reproduction, and cell signaling (12). HSDHs are also widespread in the

gut microbiome, with the appearance of having been randomly scattered between the

various genomes of gut species. This random appearance is largely due to an

inadequate understanding of the function(s) of HSDHs for particular isolates and the

6

Figure 1.3 Alpha and beta isomeric variation of the C-7 hydroxyl group The hydroxyl (-OH) group bound to carbon 7 (red) of ring B can be in one of two isomeric orientations. Alpha (α) is represented by dashes, signifying the bond going into the plane of reference or away from the reader. Beta (β) is represented by a wedge, signifying the bond coming out of the plane of reference or toward the reader. In this example, distinct 7α- or 7β-HSDHs perform the biotransformations.

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effect of bile acid chemistry on gut bacterial fitness (13). Many of the genes encoding

HSDHs have yet to be identified. It is imperative that we characterize these enzymes if

we are to understand the full extent to which they modify steroids and affect the host

and surrounding microbial community.

STEROID HORMONES

Many steroids act as hormone signaling molecules that bind to receptors and

cause physiological changes to the host. These “steroid hormones” are produced in

numerous host tissues, including the adrenal cortex, gonads, and placenta, and are all

synthesized from cholesterol (5). Steroids can then be transported through the blood

via plasma transport proteins to target tissues that have receptors for that specific

steroid hormone (6). Steroids exert their regulatory effects by binding to cell surface

receptors or entering target cells by passive diffusion and associating with and

activating the nuclear receptors therein (5, 14, 15). Once bound to the receptor, the

steroid induces a conformational change in the receptor which then generates a specific

biological response based on the target tissue, receptor, and steroid in question (6).

Steroid hormones are categorized into two classes and five types according to

the receptors to which they bind. The two classes are corticosteroids and sex steroids.

The 5 types are: glucocorticoids, mineralocorticoids, androgens, progestogens, and

estrogens (Figure 1.1).

Corticosteroids- The corticosteroids consist of glucocorticoids and

mineralocorticoids. Corticosteroids are synthesized in the adrenal cortex in mammals

(16). Corticosteroids contain 21 carbons and are characterized by an oxo group at C-3

and 20, a Δ4,5 double bond, a two-carbon side chain on C-17, and a hydroxyl on C-21.

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Glucocorticoids- Glucocorticoids are categorized by the characteristics

described in corticosteroids, and by the presence or absence of hydroxyls at C-11 and

C-17. Production of glucocorticoids is a classic endocrine response to stress.

Glucocorticoids synthesized in the adrenal cortex stimulate gluconeogenesis to provide

energy for the “flight or fight” response (17). In humans and animals, it has been shown

that excess stress or excess exogenous glucocorticoids cause hyperglycemia,

hypertension, and increased anxiety-related behavior (18). Glucocorticoids act by

binding to glucocorticoid receptors, which subsequently act as transcription factors to

alter gene expression. Various stressors, including infection, malnutrition, anxiety, and

depression, trigger a rise in glucocorticoids (19, 20).

Cortisol- In tetrapods, the active glucocorticoid can be either cortisol or

corticosterone (16). But rodents, birds, lizard, and other animals only secrete

corticosterone (21). In species that secrete both steroids, cortisol is often regarded as

the main glucocorticoid, while corticosterone is just considered the second

glucocorticoid with similar properties to cortisol (22). But, corticosterone actually has

different properties than cortisol. Cortisol and its metabolites cannot enter

enterohepatic circulation, whereas corticosterone can; meaning that it can be recycled

throughout the body instead of being excreted. Overall though, it is still not known why

humans and many other mammals produce both glucocorticoids. Many more studies

have been done on the physiological effects of cortisol relating to obesity and stress,

(23, 24). The amount of cortisol synthesized daily in humans is listed in Table 1.1.

Mineralocorticoids- Mineralocorticoids are a class of corticosteroids, with a

hydroxyl group at carbon 11 and an aldehyde on carbon 18. The primary active

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Table 1.1 Concentrations of major steroids found in humans

Concentration Produced Per Day

(nM)

Steroid Hormone Male Female Major Route of Excretion

Cortisol 55.18 - 772.52 Urine Aldosterone 0.0277 - 0.582.5 Urine Testosterone 10.4 - 41.6 0.3 - 2.1 Urine Estradiol 0.0367 - 0.146 0.09912 - 1.6007 Urine Progesterone ≤ 3.18 ≤ 3.18 - 954 Urine

Bile Acid Concentration Measured in Human Feces (µM)

Major Route of Excretion

Cholic Acid 0 - 97 (25) (26) Feces Deoxycholic Acid 30 - 700 (25) (26) Feces Chenodeoxycholic Acid 0 - 94 (25) (26) Feces Lithocholic Acid 1 - 450 (25) (26) Feces Iso Deoxycholic Acid 0 - 390 (26) Feces Iso Lithocholic Acid 0 - 260 (26) Feces

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mineralocorticoid is aldosterone (16). The amount of aldosterone synthesized daily in

humans is listed in Table 1.1. Mineralocorticoids contribute to electrolyte homeostasis

(4). Uniquely, the mineralocorticoid receptor responds to both aldosterone and cortisol

(corticosterone in rodents), a glucocorticoid (27).

Sex Steroids- The sex steroids are comprised of androgens, estrogens, and

progestogens. Typically, estrogens and progestogens are thought of as the female sex

steroids, and androgens are thought of as the male sex steroids. Sex steroids are

essential for reproductive function but can act on other organ systems as well. Each

sex steroid is produced by both sexes, though the quantity differs by age and sex (28).

Sex steroids are not only produced by the gonads, but by other organs as well,

including the central nervous system (29, 30).

Androgens- The androgens are steroids that contain 19 carbons. Androgens

are characterized by the absence of a carbon side chain on C-17, and by the presence

of a ketone functional group at C-3 (6). Androgens are important steroid hormones that

control normal male development and reproductive function (31). Androgens are

produced in males in the testes and in females by the ovaries and placenta. The most

important androgens are 5α-dihydrotestosterone (DHT) and testosterone. The amount

of testosterone synthesized daily in humans is listed in Table 1.1. The biological actions

of DHT and testosterone are facilitated by androgen receptors, which upon activation,

regulate gene expression in target tissues. Although testosterone and DHT have affinity

for the androgen receptor, they interact differently. Testosterone has a two-fold lower

affinity than DHT, while the dissociation rate of testosterone from the receptor is five-

11

fold faster than DHT (32). Overall, androgens are important for normal development

and function of male reproductive tissues and for development of muscles and bone.

11β-hydroxyandrostenedione- An important androstane synthesized in the

adrenal glands that is often metabolized by steroidogenic enzymes is 11β-

hydroxyandrostenedione (11β-OHAD). The biological role of 11β-OHAD specifically is

still being elucidated, but it is known that 11β-OHAD is a precursor for many recently

recognized novel androgens (33). These androgens bind to and activate the human

androgen receptors similarly to testosterone and DHT (34). This is significant when

considering androgen dependent diseases such as prostate cancer and other diseases

associated with androgen excess such as polycystic ovary syndrome (35). Bacterial

enzymes have been identified that cleave the side chain of cortisol, converting cortisol

into 11β-OHAD in the gut. It is important to further understand 11β-OHAD and the

enzyme responsible for its synthesis from cortisol. The synthesis of 11β-OHAD in the

gut highlights an alternative pathway for the formation of potent androgens, the majority

of which, were classically thought to be synthesized in the adrenal glands.

Estrogens- The estrogens are steroids that contain 18 carbons. They are

produced in females in the ovaries and the placenta, and in the testes in males.

Estrogens are also produced in the adrenal cortex. Estrogens are characterized by a

loss of C-19, the presence of an aromatic A ring, the absence of a carbon side chain on

C-17, and the presence of a hydroxyl group at both C-3 and C-17 (6). One of the

principal regulators of circulating estrogens is the gut microbiome (36). The three major

estrogens produced are, estrone, estradiol, and estriol, the most common of which is

estradiol. The amount of estradiol synthesized daily in humans is listed in Table 1.1.

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Estradiol is required for breast development as well as body fat and skin changes seen

in females. Pregnancy is characterized in humans by a massive increase in the

production of estradiol (6). Overall, the ovaries are the principle source of estradiol,

which functions as a circulating hormone to act on near or distant target tissues (29).

Progestogens- Progestogens bind and activate the progesterone receptor.

Progesterone, the primary progestogen, is produced in the corpus luteum of the ovaries

and the placenta, as well as the adrenal cortex (37). The amount of progesterone

synthesized daily in humans is listed in Table 1.1. Prevotella intermedius has been

shown to take up progesterone, which is then metabolized to enhance the growth of the

microbe (38). The synthesis of every other steroid hormone starts with the production

of progesterone from cholesterol; progesterone leads to the production of

mineralocorticoids (aldosterone), glucocorticoids (cortisol and corticosterone), other

progestogens, estrogens (estradiol), and androgens (testosterone). Similarly to

estradiol, pregnancy is characterized in humans by a massive increase in the

production of progesterone (6). Progesterone has also been shown to be protective

and preventative of breast cancer (39).

BILE ACIDS

Historically, bile acids were viewed solely as detergents, but it is now widely

recognized that bile acids can regulate various metabolic processes, by acting as

nutrient signaling hormones (40). Bile acids, are synthesized from cholesterol in the

liver (41). From there they are stored in the gallbladder and released upon consumption

of a meal to aid in digestion and the absorption of lipids (6, 42).

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Bile acid synthesis and enterohepatic circulation- In the liver, bile acids are

conjugated with either taurine or glycine, which increases their hydrophilicity, improving

their detergent properties, and preventing them from penetrating cell membranes.

Individuals that consume a “western diet” have predominantly taurine conjugated bile

acids, while individuals who consume a vegetarian diet have predominantly glycine

conjugated bile acids (43). Bile salts are then secreted into bile and stored in the

gallbladder (41). Upon consumption of a meal, the gallbladder contracts and bile salts

enter the duodenum of the small intestine where they aid in the absorption of fat-soluble

vitamins and cholesterol (8, 40, 44). Upon reaching the distal ileum, more than 95% of

the bile salts are reabsorbed and returned to the liver via portal veins (8, 45). The bile

salts are transported across the sinusoidal membrane and re-secreted into the

gallbladder. This cyclical process is known as enterohepatic circulation (EHC) and

allows for the recycling of bile salts multiple times throughout the day (8, 46). The

process of EHC occurs between four and twelve times per day and helps to maintains a

bile acid pool of about 2 to 4 grams in humans (45).

EHC is about 95% efficient with most bile acids reabsorbed in the terminal ileum,

however, about 400-800 mg of bile salts enter the large intestine per day (26). In the

large intestine, bile salts come into contact with a large community of microbes capable

of metabolizing the bile salts (42). In the gut, the bile salts are deconjugated back into

bile acids by microbial enzymes called bile salt hydrolases (BSH) (47). The bile acids

are then further metabolized by a small group of gut bacteria within the genus

Clostridium via 7α-dehydroxylating enzymes, creating toxic secondary bile acids.

Additionally, microbial HSDHs can create toxic secondary bile acids. Secondary bile

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acids are then mostly excreted in the feces, but a certain proportion is also absorbed in

the colon and sent back to the liver via EHC. In the liver, bile acids are re-conjugated

and secreted into the biliary pool along with the primary bile acids (48).

Primary bile acids- The major primary bile acids synthesized in humans, are

cholic acid (CA) and chenodeoxycholic acid (CDCA). When produced, they are

conjugated with either taurine or glycine and then referred to as bile salts. Classically,

these bile salts are known to function as detergents that aid in the absorption of

nutrients (49). The ability of bile salts to aid in digestion is due to their amphipathic

structure, characterized by the orientation of the hydroxyl groups toward one side of the

steroid structure and the methyl groups toward the other when viewed edge-on. More

importantly, however, is the recent recognition that the various bile acids have their own

unique set of biological activities which are able to regulate gene expression by serving

as ligands for various nuclear receptors.

Secondary bile acids- Secondary bile acids are derivatives of primary bile acids

resulting from bacterial metabolism in the colon. The major secondary bile acids in

humans are deoxycholic acid (DCA), produced from CA; and lithocholic acid (LCA) and

ursodeoxycholic acid (UDCA), produced from CDCA. There are additional important

secondary bile acids called iso bile acids which are 3β-hydroxylated. In fact, after DCA

and LCA, isoDCA and isoLCA are the most abundant secondary bile acids. For

secondary bile acids to be produced; however, primary bile salts have to first be

deconjugated by an enzyme called bile salt hydrolase (BSH). The primary bile acids

can then be converted into more than twenty different secondary bile acid metabolites

by various gut microbes (45). The enzymes responsible for the multitude of secondary

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bile acids include those that function to dehydroxylate the 7 position, to epimerize the C-

5 hydrogen, and to oxidize or epimerize hydroxyl groups at position 3, 7, or 12 (50).

Some of these modifications make secondary bile acids more hydrophobic, thereby

increasing their toxicity. Hydrophobic secondary bile acids such as DCA and LCA can

lead to the development of apoptosis resistance and, therefore, colon cancer (42).

Other secondary bile acids appear to bind and activate various host receptors to an

even greater extent than primary bile acids (47). Thus, it is necessary to study these

secondary bile acids if we want to understand their full effect on the host. Overall, of the

main human bile acids, the circulating bile acid pool is comprised of about 35% CA and

CDCA each, about 25% of DCA, and less than 5% of LCA (51). The concentration of

bile acids in the large intestine of humans is listed in Table 1.1.

Iso Bile Acids- Following DCA and LCA, isoDCA and isoLCA are the most

abundant secondary bile acids in humans (26). It is hypothesized that the formation of

iso bile acids is a pathway evolved by gut bacteria to detoxify DCA or LCA (13). DCA

and LCA act as detergents, which can solubilize membrane lipids causing cells to lyse

and die (52). By flipping the C3-hydroxyl stereocenter to form iso bile acids, the

amphipathic nature of the bile acids is altered, decreasing the molecule’s detergent

properties, which in turn decreases toxicity to bacteria (26). It has been shown in

Ruminococcus gnavus that isoDCA results in less membrane damage than DCA,

meaning isoDCA is less toxic (26). The formation of an iso bile acid requires two

concerted enzymatic reactions performed by 3α- and 3β- HSDHs, respectively. In order

for DCA or LCA to be converted into isoDCA or isoLCA, the C3-hydroxyl group, which is

in an α orientation in DCA and LCA, has to first be oxidized by a 3α-HSDH. Then, a 3β-

16

HSDH can reduce the C3-ketone into β oriented hydroxyl. Iso bile acid pathways have

been found in Eggerthella lenta (previously Eubacterium lentum) and R. gnavus.

Bile acid signaling- Many of the genes induced by bile acids encode enzymes

involved in their own regulation, transport, conjugation, and metabolism (53). Bile acids

can function as important regulatory molecules in the small intestine by inducing genes

that are involved in resistance to bacterial growth (54). Bile acids have been found to

have affinity for the Farnesoid X Receptor (FXR) and G-protein coupled receptors such

as TGR5, and the sphingosine phosphate receptor (S1PR2). Further, the bile acid

structure determines its affinity for a certain receptor and therefore extent of agonism or

antagonism.

FXR is a member of the nuclear receptor superfamily and controls multiple

metabolic pathways. FXRs are mostly found in the liver and intestine. Upon activation

of the FXR by bile acids, FXR can regulate bile acid synthesis and conjugation in the

liver and transport, as well as aspects of lipid and glucose metabolism (40, 53, 55)

Activation of TGR5 has been shown to have beneficial effects on body weight as well as

glucose homeostasis (47, 56). Lithocholic acid (LCA) is the most potent natural agonist

of TGR5. Conjugated and unconjugated forms of DCA, CDCA, and CA have 53%,

12%, and 7% potency, respectively, compared to LCA (57). S1PR2 is only activated by

taurine or glycine conjugated bile acids. Activation of S1PR2 may help these

conjugated bile acids control hepatic glucose levels, reduce bile acid levels overall, and

increase lipid metabolism. These bile acid signaling pathways elicit hormone-like

regulating mechanisms, highlighting their importance in the sterolbiome.

17

THE GUT MICROBIOTA

The intestinal tract of human adults contains about 1000 g of bacteria, which

equates to about 1014 organisms from more than 400 distinct species (58). More than

99% of these organisms are obligate anaerobes (58). Adult colons are predominantly

colonized with members of two phyla; the Bacteroidetes, comprising of mainly gram-

negative bacteria, and the Firmicutes, comprising of mainly gram-positive clostridia (59).

The gut microbial community encodes about 150 times more genes than are present in

the human genome (60). Steroids in the gut can undergo drastic transformative

changes caused by the strain specific enzymes certain microbes express. If we are to

understand the impact bacteria and their metabolites have on the host, characterization

of bacteria is needed at the strain level. Both dietary and endogenous steroids come

into contact with the microbial population in the gut (8). The resulting metabolites affect

the overall microbial community and, through passive diffusion, can also circulate

throughout the body and alter the host endocrine system. One of the most important

goals in the field of gut microbiology is to gain a better understanding of microbial

interactions with the host in the hopes of modulating or reengineering the microbiome to

improve health states in the host.

Clostridium scindens

Early studies during the 1950’s revealed that rectal infusion of hydrocortisone

(cortisol), a C-21 steroid, resulted in a 100-fold increase in urinary excretion of C-19 17-

ketosteroids (61, 62). Rectal infusion of cortisol and concomitant oral antibiotic

treatment with neomycin, however, ablated the formation of 17-ketosteroids, suggesting

that gut bacteria were responsible for the biotransformation (62). Twenty years later,

18

similar findings were reported when cortisol was incubated with human and rat fecal

samples, resulting in a notable conversion of cortisol to various other C-19 metabolites

(63, 64). These studies led to the hypothesis that the side chain of cortisol was being

enzymatically removed by intestinal bacteria. It was postulated that intestinal bacteria

encode an enzyme known as steroid-17,20-desmolase, which is responsible for the

conversion of cortisol into the 17-ketosteroid metabolite known as 11β-OHAD. In 1984,

the first gut bacterium that expresses steroid-17,20-desmolase activity was isolated and

was named Clostridium scindens (type strain ATCC 35705), which means “to cut” (65,

66). C. scindens is a gram-positive, spore forming, motile, obligate anaerobe. C.

scindens is a known bile acid 7α-dehydroxylating species. In addition to steroid-17,20-

desmolase activity, C. scindens expressed cortisol-inducible 20α-hydroxysteroid

dehydrogenase (20α-HSDH) activity. The two enzymes were originally characterized

from whole cell extracts; substrate-specificity and cofactor requirements were reported

(67). Native 20α-HSDH was thereafter purified to apparent electrophoretic homogeneity

(although 2D-gel revealed a major contaminant of identical molecular weight) and

characterized from cell extracts of C. scindens ATCC 35704 (68). Up until recently, the

genes encoding these enzymes and the mechanism by which steroid-17,20-desmolase

proceeded was unclear. Using RNA sequencing, Ridlon et al (2013), identified a

cortisol-inducible operon (desABCD) hypothesized to encode the steroid-17,20-

desmolase and 20α-HSDH (69). Genes involved in the biosynthesis of thiamine

pyrophosphate (TPP) and the expression of pentose-phosphate pathway enzymes were

notably upregulated. The desA and desB genes are annotated as N-terminal and C-

terminal transketolases, respectively, and it is hypothesized that DesA and DesB form a

19

complex that has steroid-17,20-desmolase activity that functions similarly to the

transketolases found in the pentose-phosphate pathway. The desC gene was cloned,

overexpressed, purified, and shown to encode the cortisol 20α-HSDH (69). The desD

gene is hypothesized to encode a cortisol transporter.

Eggerthella lenta

Eggerthella lenta (formerly Eubacterium lentum) is a gram-positive, non-spore

forming, non-motile, obligate anaerobic bacterium whose growth is stimulated by

arginine (70, 71). E. lenta is a particularly important participant in the gut sterolbiome,

various strains of which have the potential to encode enzymes that metabolize cardiac

glycosides (72, 73), 21-dehydroxylate corticosteroids (74), hydrolyze conjugated bile

acids (75), and oxidize bile acid hydroxyl groups (76, 77). E. lenta strains have

previously been grouped according to their pattern of hydroxysteroid dehydrogenase

(HSDH) activities; some strains having no HSDH activity (Group V), with others

expressing 3α-HSDH, 3β-HSDH, 7α-HSDH, and 12α-HSDH (Group III) (77). E. lenta

and other members of the Coriobacteriaceae family have been correlated with changes

in host liver metabolome, specifically through reduction in serum triglycerides,

cholesterol excretion, and metabolism of xenobiotics (78).

Of the mentioned enzymatic activities expressed by E. lenta, 21-dehydroxylation

is of notable importance. 21-dehydroxylation of the glucocorticoid cortisol forms 11β-

hydroxyprogesterone. Metabolites of 21-dehydroxylation have been shown to be potent

inhibitors of host 11β-HSD2 (79, 80). 11β-HSD2 is responsible for the conversion of

cortisol to an inactive form known as cortisone. Inhibition of 11β-HSD2 allows cortisol to

20

accumulate and continuously occupy glucocorticoid and mineralocorticoid receptors,

potentially causing hypertension (81).

The E. lenta sterolbiome offers a potential link between associations with strains

of this species and changes in host liver metabolome and physiology. Alteration of

steroid structure, and absorption of E. lenta steroid metabolites (and derivatives from

additional microbial biotransformations) from the colon and transport to the liver via

portal circulation may alter nuclear receptor and G-protein coupled receptor activation,

as well as affect liver enzyme activities, leading to altered lipid and steroid metabolism.

Currently, the sterolbiome of E. lenta is poorly characterized. Genes encoding HSDHs,

particularly 7α-HSDH and 12α-HSDH, have yet to be identified. It may be expected that

diverse isoforms of currently identified 3α-HSDH and 3β-HSDH may exist, requiring

functional characterization of genes whose protein products are predicted to encode

these enzymes, namely those within the short chain dehydrogenases/reductases (SDR)

family (82). A major barrier to such mechanistic study is the need to identify and

characterize much of the E. lenta sterolbiome.

Butyricicoccus desmolans

B. desmolans is a gram-positive, non-spore forming, non-motile obligate

anaerobic and butyrate-producing bacterium recovered from a healthy human’s feces

(83). Previously, the bacterium was named Eubacterium desmolans (83). Steroid-

17,20-desmoalse activity had been reported in B. desmolans, but unlike C. scindens

ATCC 35704, which expresses cortisol 20α-HSDH activity, B. desmolans was

demonstrated to express cortisol 20β-HSDH activity (84). Soon after the reporting of

the desABCD operon from C. scindens ATCC 35704, the genome sequence for B.

21

desmolans ATCC 43058 became available and the desAB gene in B. desmolans ATCC

43058 was shown to encode steroid-17,20-desmolase (85). The desE gene, not

present in C. scindens ATCC 35704, but present in B. desmolans ATCC 43058 was

shown to encode cortisol 20β-HSDH (85). The 20β-HSDH from B. desmolans 43058

was overexpressed and purified from E. coli. The enzyme was determined to be a

homotetramer with cortisol as the preferred substrate. Phylogenetic analysis of 70

amino acid sequences with at least 30% identity to the gene encoding cortisol 20β-

HSDH from B. desmolans ATCC 43058 was performed. A phylogenetic tree was

constructed with cortisol 20β-HSDH from B. desmolans and closest neighboring

sequences, forming three major clusters (Figure 1.4). The cluster containing the most

distantly related proteins relative to cortisol 20β-HSDH from B. desmolans was

composed exclusively of polypeptides from Mycobacterium spp., followed by a cluster of

proteins from anaerobic gut bacteria that inhabit ruminants and monogastrics. The final

cluster, populated by B. desmolans ATCC 43058 and other steroid-17,20-desmolase

expressing strains, also harbored polypeptides from a number of species of

Bifidobacterium. The desA and desB genes, hypothesized to encode steroid-17,20-

desmolase were not located in the available genomes from Bifidobacterium represented

in the tree. B. scardovii DSM 13734 (ATCC BAA-773) was tested for cortisol

metabolism and after 48 hours of incubation in BHI medium, cortisol was converted to

20β -dihydrocortisol, but did not form 11β- OHAD (85).

Bifidobacterium adolescentis

In 1982, a strain of B. adolescentis was isolated from human feces that

22

Figure 1.4 Phylogenetic analysis of identified 20β-hydroxysteroid dehydrogenases 70 amino acid sequences with at least 30% identity to the gene encoding cortisol 20β-HSDH from B. desmolans ATCC 43058 are aligned in a phylogenetic tree constructed using FastTree v2.1.7. Steroid-17,20-desmolase expressing strains are denoted by a red “x” through a C-C bond. Predicted active-site residues are depicted adjacent to strain designations

23

displayed 20β-HSDH activity (86). Early studies have shown that Bifidobacterium spp.

are among the first bacteria to colonize the gastrointestinal tract of breast-fed infants

and are regularly used as probiotics in products such as milk, yogurt, and dietary

supplements (87). The recent phylogenetic analysis of the 20β-HSDH gene from B.

desmolans ATCC 43058 and B. scardovii DSM 13734 (ATCC BAA-773) resulted in the

identification of several Bifidobacterium strains that are predicted to express 20β-HSDH

activity (85). The fact that some bifidobacteria strains encode and express 20β-HSDH,

but not steroid-17,20-desmolase is interesting. It is hypothesized that 20β-HSDH

activity in bifidobacteria may reduce the conversion of cortisol to 11β-OHAD in guts

dominated by 20α-HSDH expressing bacteria such as C. scindens ATCC 35704.

We identified particular B. adolescentis strains that possess a gene predicted to

encode 20β-HSDH based on the phylogenetic tree with B. desmolans ATCC 43058.

Specifically, B. adolescentis strain L2-32 was selected for further study because this

strain is predicted to encode 20β-HSDH. B. adolescentis is a gram-positive, non-motile,

non-spore forming, obligate anaerobic bacteria. Because of the important pathways

20β-HSDHs mediate in cortisol metabolism and the properties conferred intrinsically by

Bifidobacteria, the 20β-HSDH expressed by B. adolescentis strain L2-32 has the

potential to be of great pharmaceutical interest.

OBJECTIVES

There are a plethora of steroids circulating throughout the human host which are

constantly being modified by microbes in the gut. The overall impact these steroids

have on the surrounding microbial community and the host is still understudied and

predicted to be of significant biomedical consequence. Currently, it is difficult to

24

bioinformatically determine the function of many predicted steroid metabolizing genes,

therefore, it is important that these genes are biochemically characterized so that their

functions can be tested. The desAB genes cluster with the desC (20α-HSDH) and desE

(20β-HSDH) genes in C. scindens and B. desmolans, respectively, and are strong

candidates for genes encoding steroid-17,20-desmolase. By characterizing the desAB

genes and other steroid metabolizing genes predicted to be HSDHs of various stereo

and regiospecificity, we can begin to understand and potentially control the steroid

profile by mediating receptor activation and downstream effects such as metabolism,

immune responses, and inflammation in the host. In an effort to elucidate the

importance of steroid metabolizing genes, we will:

1. Characterize a gene cluster predicted to encode multiple HSDHs in

Eggerthella:CAG 298, a gut metagenomic sequence.

2. Determine whether the desA and desB genes encode steroid-17,20-

desmolase. Develop an enzyme-coupled continuous spectrophotometric

assay to simplify enzyme characterization of DesAB.

3. Characterize 20β-HSDH from Bifidobacterium adolescentis strain L2-32 and

attempt to crystallize and determine the structure.

25

CHAPTER 2: Targeted synthesis and characterization of a gene-cluster encoding

NAD(P)H-dependent 3α-, 3β-, and 12α-hydroxysteroid dehydrogenases from

Eggerthella CAG:298, a gut metagenomic sequence

ABSTRACT

Gut metagenomic sequences provide a rich source of microbial genes, the majority of

which are annotated by homology or unknown. Genes and gene pathways that encode

enzymes catalyzing biotransformation of host bile acids are important to identify in gut

metagenomic sequences due to the importance of bile acids on gut microbiome

structure and host physiology. Hydroxysteroid dehydrogenases (HSDH) are pyridine

nucleotide-dependent enzymes with stereo-specificity and regio-specificity for bile acid

and steroid hydroxyl groups. HSDHs have been identified in several protein families

including medium chain and short chain dehydrogenase/reductase families as well as

aldo-keto reductase family. These protein families are large and contain diverse

functionalities making prediction of HSDH-encoding genes difficult, necessitating

biochemical characterization. We located a gene-cluster in Eggerthella CAG:298

predicted to encode three HSDHs (CDD59473, CDD59474, CDD59475) and

synthesized the genes for heterologous expression in E. coli. We then screened bile

acid substrates against the purified recombinant enzymes. CDD59475 is a novel 12α-

HSDH and we determined that CDD59474 (3α-HSDH) and CDD59473 (3β-HSDH)

constitute novel enzymes in an iso-bile acid pathway. Phylogenetic analysis of these

HSDHs with other gut bacterial HSDHs and closest homologues in the database

revealed predictable clustering of HSDHs by function and identify several likely HSDH

26

sequences from bacteria isolated or sequenced from diverse mammalian and avian gut

samples.

Bacterial HSDHs have the potential to significantly alter the physicochemical

properties of bile acids with implications for increased/decreased toxicity for gut bacteria

and the host. The generation of oxo-bile acids is known to inhibit host enzymes involved

in glucocorticoid metabolism and may alter signaling through nuclear receptors such as

farnesoid X receptor and G-protein coupled receptor TGR-5. Biochemistry or similar

approaches are required to fill in many gaps in our ability to link a particular enzymatic

function with a nucleic acid or amino acid sequence. In this regard, we have identified a

novel 12α-HSDH, and a novel set of genes encoding an iso-bile acid pathway (3α-

HSDH, 3β-HSDH) involved in epimerization and detoxification of harmful secondary bile

acids.

INTRODUCTION

Metagenomic sequencing of microbial DNA in environmental samples, including

human stool, has provided a massive gene catalogue to the microbiologist, only an

estimated 50% of which have been annotated; much of these homology-based, and a

miniscule number of these characterized biochemically (88). The Human Microbiome

Project (89) and Metagenomics of the Human Intestinal Tract (MetaHIT) have provided a

‘second human genome’ to decode (90). One set of functional pathways within this

‘second human genome’ is the gut sterolbiome (2) which participates in the endocrine

function of the host as well as, in the case of bile acids, regulation of microbiome structure

and functions. Gut bacterial metabolism of bile acids is known to affect signaling through

the farnesoid-X-receptor (91), G-coupled protein receptor TGR5 (57), muscarinic receptor

27

(92), and Vitamin-D-receptor (93). In addition, the formation of oxo-bile acids has been

shown to inhibit the enzyme 11β-hydroxysteroid dehydrogenase-1 (11β-HSD1), which

has been reported to alter gut microbiome community structure (94).

The oxidation of bile acid hydroxyl groups by gut bacteria also allows epimerization

by the concerted action of two regio-specific enzymes differing in stereo-specificity such

that the conversion of, for instance, 7α-hydroxylated chenodeoxycholic acid (CDCA) to

7β-hydroxylated ursodeoxycholic acid (UDCA) bile proceeds through a stable 7-

oxolithocholic acid intermediate (95). Epimerization of the 3α-hydroxy results in formation

of so called iso-bile acids (3β-hydroxyl) (96) which are more hydrophilic and less toxic

than the 3α-hydroxyl host-derived bile acid (26, 97), as is also the case with UDCA which

is used therapeutically in hepatobiliary diseases (98). The least studied pathway is the

epimerization of the 12α-hydroxyl 12-oxo 12β-hydroxyl of cholic acid derivatives (99,

100).

Eggerthella lenta (formerly Eubacterium lentum) is a gram-positive, non-spore

forming, non-motile, obligate anaerobic bacterium whose growth is stimulated by arginine

(70, 71). E. lenta is a particularly important participant in the gut sterolbiome, various

strains of which have the potential to encode enzymes that metabolize cardiac glycosides

(72, 73), 21-dehydroxylate corticosteroids (74), hydrolyze conjugated bile acids (75), and

oxidize bile acid hydroxyl groups (76, 77). E. lenta strains have previously been grouped

according to their pattern of hydroxysteroid dehydrogenase (HSDH) activities; some

strains having no HSDH activity (Group V), with others expressing 3α-HSDH, 3β-HSDH,

7α-HSDH, and 12α-HSDH (Group III) (77). E. lenta and other members of the

Coriobacteriaceae family have been correlated with changes in host liver metabolome,

28

specifically through reduction in serum triglycerides, cholesterol excretion, and

metabolism of xenobiotics (78).

The E. lenta sterolbiome offers a potential link between associations with strains

of this species and changes in host liver metabolome and physiology. Alteration of steroid

structure, and absorption of E. lenta steroid metabolites (and derivatives from additional

microbial biotransformations) from the colon and transport to the liver via the portal

circulation may alter nuclear receptor and G-protein coupled receptor activation, as well

as affect liver enzyme activities, leading to altered lipid and steroid metabolism. Currently,

the sterolbiome of E. lenta is poorly characterized. Genes encoding HSDHs, particularly

7α-HSDH and 12α-HSDH, have yet to be identified. It may be expected that diverse

isoforms of currently identified 3α-HSDH and 3β-HSDH may exist, requiring functional

characterization of genes whose protein products are predicted to encode these

enzymes, namely those within the family of short chain dehydrogenases/reductases

(SDR) (82). A major barrier to such mechanistic study is the need to identify and

characterize much of the E. lenta sterolbiome.

In the current study, we queried the non-redundant database with the amino acid

sequence from the only characterized 12α-HSDH to date, identified in Clostridium sp.

strain ATCC 29733/VPI C48-50 (99). We located an open reading frame (ORF) in a gut

metagenome of Eggerthella CAG:298, a sequence from Metagenomics of the Human

Intestinal Tract (MetaHIT), that is part of a gene cluster with additional NAD(P)H-

dependent dehydrogenases in the short-chain dehydrogenase/reductase (SDR) and

aldo-keto reductase protein families. Utilizing targeted gene synthesis, we identified and

cloned these three synthesized genes for heterologous expression in E. coli. We report

29

the characterization of a gene encoding a novel 12α-HSDH as well as two genes

encoding an iso-bile acid pathway (3α-HSDH, 3β-HSDH), distinct from those previously

reported (26).

MATERIALS AND METHODS

Bacterial strains and chemicals- Escherichia coli DH5α and E. coli BL21-

CodonPlus(DE3) RIPL competent cells. The pET-46 Ek/LIC vector kit was obtained

from Novagen (San Diego, CA). The QIAprep Spin Miniprep kit was obtained from

Qiagen (Valencia, CA). Isopropyl β-D-1-thiogalactopyranoside (IPTG) was purchased

from Gold Biotechnology (St. Louis, MO). Bile acid substrates were purchased from

Steraloids, Inc (Newport, RI, USA).

Targeted gene synthesis, cloning, expression and purification recombinant

proteins- Gene fragments (GenBlock®) encoding 3α-HSDH, 3β-HSDH and 12α-HSDH

from E. lenta CAG:298 (Bio Project PRJEB816) were synthesized by Integrated DNA

Technologies (IDT) (Coralville, IA, USA). Gene fragments were amplified with primers

synthesized by IDT (Table 2.1), using the Phusion high fidelity polymerase (Stratagene,

La Jolla, CA) and cloned into pET-46b vector (Novagen), as described by the

manufacturer’s protocol. Cultivation, plasmid isolation and sequence confirmation were

as previously described (85). HSDH enzymes were expressed as previously described

(85). The recombinant proteins were then purified using TALON® Metal Affinity Resin

(Clontech Laboratories, Mountain View, CA) as per manufacturer’s protocol. The

recombinant protein was eluted using an elution buffer composed of 20 mM Tris-HCl, 150

mM NaCl, 20% glycerol, 10mM 2-mercaptoethanol pH 7.9 and 250 mM imidazole. The

protein purity was assessed by sodium dodecyl sulfate-polyacrylamide gel

30

Table 2.1 Oligonucleotides used for amplification and directional cloning

31

electrophoresis (SDS-PAGE), and protein bands were visualized by staining with

Coomassie brilliant blue G-250. Subunit molecular mass was calculated using three

independent SDS-PAGE gels with Biorad Precision Plus ProteinTm Kaleidoscope

Prestained Protein Standards (Bio Rad Laboratories, Inc, Hercules, CA) using the image

processing software ImageJ (https://imagej.nih.gov/ij/index.html). Recombinant protein

concentrations were calculated based on their molecular mass and extinction coefficients

(Table 2.1). Briefly, deduced amino acid sequence was used as input for Expasy’s

ProtParam tool (https://web.expasy.org/protparam/) and subunit mass and extinction

coefficient (mM-1 cm-1) are utilized to determine enzyme concentration (mg ml-1) in a

Nanodrop 2000c with 10 mm pathlength cuvette at 280 nm.

Enzyme Assays- The buffers used to study the pH profiling of each enzyme

contained 150 mM NaCl, 20% glycerol and 10 mM 2-mercaptoethanol and varied as

follows: 50 mM sodium citrate, (pH 4.0–6.0), 50 mM sodium phosphate (pH 6.5–7.5), 50

mM Tris-Cl (pH 8–9), and 50 mM glycine-NaOH (pH 10–11). Linearity of enzyme activity

with respect to time and enzyme concentration was determined aerobically by monitoring

the oxidation/reduction of NAD(P)(H) at 340 nM (ε=6,220 M-1.cm-1) in the presence of bile

acid and steroid substrates. The standard reaction mixtures were 50 mM sodium

phosphate buffer, pH 6.5 (CDD59473 reductive direction) or 50 mM sodium phosphate

buffer, pH 7.0 (CDD59474 in reductive direction, CDD59475 in both oxidative and

reductive direction) or 50 mM Tris-HCl buffer pH 9.5 (CDD59473 oxidative direction) or

pH 10 (CDD59474 (oxidative direction) each containing 150 µM pyridine nucleotide

cofactor and initiated by addition of the enzyme (50 nM). Kinetic parameters were

estimated by fitting the data to the Michaelis-Menten equation by non-linear regression

https://imagej.nih.gov/ij/index.html

https://web.expasy.org/protparam/

32

method using the enzyme kinetics module in GraphPad Prism (GraphPad Software, La

Jolla, CA). Substrate-specificity studies were performed with 150 µM pyridine nucleotide,

and 50 µM bile acid substrate at the respective pH optimum for the enzyme and each

reaction direction.

Thin Layer Chromatography (TLC) and Mass Spectrometry (MS) of Bile Acid

Metabolites- The standard reaction mixtures with different pyridine nucleotide were setup

as described above and incubated overnight at room temperature. The reactions were

stopped by adding 100 μL of 1N HCl. The reaction mixtures were extracted by vortexing

with 2x volumes of ethyl acetate for 1-2 mins and then the organic phase was recovered.

The organic phase was evaporated under nitrogen gas. The residue was dissolved in 50

μL ethyl acetate and spotted along with standards on TLC plate (Silica Gel IB2-F Flexible

TLC Sheet 20 x 20 cm with 250 µm analytical layer, J.T. Baker, Avantor Performance

Materials, LLC. PA, USA). A mobile phase consisting of 70:20:2 v/v/v toluene:1,4-

dioxane:acetic acid was used. Bile acid metabolites separated on the TLC plate were

visualized by spraying 10% phosphomolybdic acid in ethanol and heated at 100⁰C for 15

min. The spots corresponding to substrate and product were isolated, extracted, and

underwent MS analysis. LC-MS analysis was run on a Shimadzu LCMS-IT-TOF System

(Shimadzu Corporation, Kyoto, Japan). The mass spectrometer was operated with an

electrospray ionization (ESI) source in negative ionization mode. The nebulizer gas

pressure was set at 150kPa with a source temperature of 200°C and the gas flow at 1.5

L/min. The detector voltage was 1.65kV. High-purity nitrogen gas was used as collision

cell gas. The mass spectrogram data was processed with the LC solution Workstation

software.

33

Phylogenetic reconstruction of bacterial SDR-family and aldo-keto

reductase family protein sequences- Protein sequences were aligned with MUSCLE

using the web service from the EMBL-EBI (http://www.ebi.ac.uk/Tools/msa/muscle/). The

alignment was manually explored to verify the absence of inaccuracies. Phylogeny

reconstruction was performed using both FastTree v2.1 (101, 102) and IQ-TREE, a time-

efficient tool to generate maximum likelihood phylogenies (103). The two methods were

used for comparative purposes. FastTree ran locally using default settings. IQ-TREE ran

locally using the following options: -st AA -m TEST -bb 1000 -alrt 1000 (-st, sequence

type; -m, automatic model selection; -bb, number of bootstrap replicates). The generated

trees were exported in newick format and uploaded to iTOL v3 for visualization (104). The

trees were compared using Phylo.io (105). Sequence information is found in Table 2.2.

RESULTS

Identification of a gene cluster predicted to encode multiple hydroxysteroid

dehydrogenases in a metagenomic sequence- 12α-HSDHs (EC 1.1.1.176) have been

reported in gut bacteria such as Clostridium perfringens (106), C. leptum (107), E. lenta

(76), Clostridium sp. strain ATCC 29733/VPI C48-50 (99). To date, the only reported 12α-

HSDH whose gene sequence is known is that of Clostridium sp. strain ATCC 29733/VPI

C48-50 (108). Previously, a phylogenetic analysis of the 12α-HSDH amino acid sequence

(ERJ00208.1) was performed, suggesting this function may be widespread in gut

microbiota (12); however, biochemical characterization has yet to confirm this. An

updated query of the non-redundant database identified numerous gut microbial phyla,

particularly Firmicutes and Actinobacteria. One particular sequence, CDD59475,

belonging to Eggerthella CAG:298, and not reported previously (12),

34

Table 2.2 Sequences utilized in phylogenetic analysis

Phylogenetic tree identifier

Protein Locus Tag Function Organism Ref

3A_CDD59473 CDD59473 3α-HSDH Eggerthella sp. CAG:298 TS



3A_Elen_0690 Elen_0690 3α-HSDH Eggerthella lenta DSM 2243 (26)

3B_Elen_1325 Elen_1325 3b-HSDH Eggerthella lenta DSM 2243 (26)

3B_Elen_0198 Elen_0198 3b-HSDH Eggerthella lenta DSM 2243 (26)

3A_Rumgna_02133 Rumgna_02133 3α-HSDH Ruminococcus gnavus ATCC 29149 (26)

3B_Rumgna_00694 3B_Rumgna_00694 3b-HSDH Ruminococcus gnavus ATCC 29149 (26)

12A_COLAER_RS06615 COLAER_RS06615

putative 12α-HSDH Collinsella aerofaciens ATCC 25986 (12)

12A_COPCOM_01375 COPCOM_01375 putative 12α-HSDH Coprococcus comes ATCC 27758 (12)

7A_Ecoli BAA01384 7α-HSDH Escherichia coli (109)

3A_BAIA2Csi BAIA2_CLOSV 3α-HSDH Clostridium scindens ATCC 35704 (110, 111)

3A_BAIAChyl ACF20977 3α-HSDH Clostridium hylemonae DSM 15053 (112)

3A_BAIAChir EEA86309 3α-HSDH Clostridium hiranonis DSM 13275 (113)

3A_RUMHYD_02185 RUMHYD_02185 putative 3α-HSDH Blautia hydrogenotrophica DSM 10507 (12)

12A_Csp29733 ERJ00208.1 12α-HSDH Clostridium sp. strain ATCC 29733 (99, 108)

3A_DORFOR_00173 DORFOR_00173 putative 3α-HSDH Dorea formicigenerans ATCC 27755 (12)

3A_RUMTOR_01195 RUMTOR_01195 putative 3α-HSDH Ruminococcus torques ATCC 27756 (12)

3A_ RUMOBE_03494 RUMOBE_03494 putative 3α-HSDH Ruminococcus obeum ATCC 29174 (12)

7A_NP_810824Bthet NP_810824 putative 7α-HSDH Bacteroides thetaiotaomicron VPI-5482 (12, 95, 114)

3A_EDT24590.1Cperf EDT24590.1 putative 3α-HSDH Clostridium perfringens B str. ATCC 3626 (106), (115)

7A_AAA53556.1Csord AAA53556.1 7α-HSDH Clostridium sordellii ATCC 9714 (116)

7A_CsciAAB61151.1 AAB61151.1 7α-HSDH Clostridium scindens VPI 12708 (117)

35

Table 2.2 Continued

Phylogenetic tree identifier Protein Locus Tag Function Organism Ref

7A_CLOHIR_RS06190 CLOHIR_RS06190 putative 7α-HSDH

Clostridium hiranonis DSM 13275 (12)

7A_ BACOVA_01473

BACOVA_01473 putative 7α-

HSDH Bacteroides ovatus ATCC 8483 (12)

7A_BF9343_RS16010 7A_BF9343_RS16010

putative 7α-HSDH Bacteroides fragilis NCTC 9343 (12)

7A_BACFIN_RS13120 BACFIN_RS13120 putative 7α-HSDH

Bacteroides finegoldii DSM 17565 (12)

7A_ANACAC_03661 7A_ANACAC_03661 putative 7α-HSDH

Anaerostipes caccae DSM 14662 (12)

7A_CD630_00650 CD630_00650 putative 7α-HSDH Clostridium difficile 630 (12)

7B_COLAER_RS09325 COLAER_RS09325 7b-HSDH Collinsella aerofaciens ATCC 25986 (118), (119)

7B_RUMGNA_RS11340 RUMGNA_RS11340 7b-HSDH Ruminococcus gnavus ATCC 29149 (120)

12A_CLOHYLEM_04236

12A_CLOHYLEM_04236 12α-HSDH Clostridium hylemonae DSM

15053 (12), unpublished

data

12A_CLOSCI_02455 CLOSCI_02455 putative 12α-HSDH

Clostridium scindens ATCC 35704

(12), unpublished data

12A_COLAER_01483 COLAER_01483 putative 12α-HSDH

Collinsella aerofaciens ATCC 25986 (12)

20B_Bdes WP_051643274.1 20b-HSDH Butyricicoccus demolans ATCC 43058 (85)

20B_Ccad1 WP_051196374.1 20b-HSDH Clostridium cadaveris AGR2141 (85)

20B_Ccad2” WP_027640050 20b-HSDH Clostridium cadaveris AGR2141 (85)

20B_BScar BAQ31198.1 20b-HSDH Bifidobacterium scardovii DSM 13734 (85)

20B_Plym WP_024111275.1 20b-HSDH Propionimicrobium sp. BV2F7 (85)

WP_025022266.1 WP_025022266.1 aldo/keto reductase Lactobacillus hayakitensis TS

KRM19149.1 KRM19149.1 oxidoreductase Lactobacillus hayakitensis DSM 18933 TS

KRM65245.1 KRM65245.1 oxidoreductase Lactobacillus agilis DSM 20509 TS

WP_050611824.1 WP_050611824.1 aldo/keto reductase Lactobacillus agilis TS

WP_019206370.1 WP_019206370.1 aldo/keto reductase Lactobacillus ingluviei TS

36

Table 2.2 Continued

TS=This Study

Phylogenetic tree identifier

Protein Locus Tag Function Organism Ref

KRL88436.1 KRL88436.1 organophosphate reductase Lactobacillus ingluviei DSM 15946 TS

ASN60792.1 ASN60792.1 aldo/keto reductase Lactobacillus curvatus TS

WP_085844664.1 WP_085844664.1 aldo/keto reductase Lactobacillus curvatus TS

WP_054778020.1 WP_054778020.1 aldo/keto reductase Lactobacillus saniviri TS

BAN77682.1 BAN77682.1 conserved hypothetical protein Adlercreutzia equolifaciens DSM 19450 TS

WP_041715350.1 WP_041715350.1 SDR family oxidoreductase Adlercreutzia equolifaciens TS

CDD77593.1 CDD77593.1 putative uncharacterized protein Cryptobacterium sp. CAG:338 TS

CDD68244.1 CDD68244.1 putative uncharacterized protein Eggerthella sp. CAG:368 TS

CDB33831.1 CDB33831.1 putative uncharacterized protein Eggerthella sp. CAG:209 TS

CCY06514.1 CCY06514.1 putative uncharacterized protein Eggerthella sp. CAG:1427 TS

CDD56334.1 CDD56334.1 putative uncharacterized protein

Bacteroides pectinophilus CAG:437 TS

WP_044925038.1 WP_044925038.1 short-chain dehydrogenase Anaerostipes hadrus TS

WP_008116638.1 WP_008116638.1 NAD(P)-dependent oxidoreductase Bacteroides pectinophilus TS


OKZ9660.1 OKZ9660.1 short-chain dehydrogenase Clostridiales bacterium Nov_37_41 TS



CCX88977.1 CCX88977.1 dehydrogenases with different specificities Clostridium sp. CAG:590 TS

WP_009265642.1 WP_009265642.1 short-chain dehydrogenase Lachnospiraceae bacterium 5_1_63FAA TS

37

shared 71% identity (E value = 1e-134) with ERJ00208.1. This predicted 266 amino acid

protein in the 3-ketoacyl-(acyl-carrier protein) reductase/SDR family was unique among

gene products that populated the BLAST query in that it is part of a three-gene cluster

containing two additional putative NAD(P)(H)-dependent oxidoreductases. Directly

upstream is a gene (BN592_00769) encoding a 250 amino acid protein (CDD59474.1)

annotated as a “7α-HSDH” by homology in the SDR-family, and upstream

(BN592_00768) a 280 amino acid protein (CDD59473.1) predicted to encode aldo/keto

reductase. Downstream of BN592_00768 is a gene (BN592_00767) predicted to encode

a gamma-glutamylcysteine synthetase, and upstream on the opposite strand of the gene-

cluster is an ORF (BN592_00771) encoding a hypothetical protein in the Phasin

superfamily involved in intracellular storage of polyhydroxyalkanoates (Figure 2.1).

Targeted gene synthesis, cloning, and overexpression of CDD59475 in E.

coli- Since Eggerthella CAG:298 is a metagenomic sequence and a microbial isolate is

not available, the genes were synthesized (GenBlock® IDT, Inc) and PCR amplified for

ligation-independent cloning (LIC) into pET46(+) for overexpression of recombinant N-

terminal His-tagged protein in E. coli (Figure 2.1). Purified recombinant CDD59475

separated as a single band on SDS-PAGE with a subunit molecular mass of 28.0 ± 0.6

kDa (theoretical molecular weight of 28.30 kDa) (Figure 2.1). We initially screened

CDD59475 dehydrogenase activity spectrophotometrically at 340 nm in the presence of

oxo-bile acids at a concentration of 50 µM, including 3-oxoDCA, 7-oxoDCA, and 12-

oxoDCA with 150 µM NADH or NADPH cofactor. We did not detect enzyme activity when

3-oxoDCA or 7-oxoDCA were substrates; however, CDD59475 clearly displayed

NAD(P)H-dependent activity in the presence of 12-oxoDCA (Figure 2.2C). To initially

38

Figure 2.1 SDS-PAGE of purified recombinant 3β-, 3α-, and 12α-hydroxysteroid dehydrogenases from a gene cluster in Eggerthella CAG:298

3β-HSDH

3α-HSDH

12α-HSDH

39

Figure 2.2 Representative TLC of bile acid reaction products from recombinant CDD59473 (A), CDD59474 (B), and CDD59475 (C) Standard reactions contained 50 μM bile acid substrates, 150 μM pyridine nucleotide, and 50 μM enzyme. Experiments also include a no enzyme control. Reactions were incubated at 37°C for 12 hours. Reaction products that co-migrated with authentic standards were scraped from the TLC plate, extracted with ethyl acetate and dried, and subjected to MS analysis after re-suspension in mobile phase. Experiments were repeated three times.

A.

B.

40

Figure 2.2 Continued

C.

41

confirm activity, we ran several substrates in the oxidative direction and separated the

reaction products by thin layer chromatography, the products of which were subjected to

mass spectrometry (Figure 2.3). In the presence of NADP+, CDD59475 was capable of

oxidizing cholic acid (CA; 3α,7α,12α-trihydroxy-5β-cholan-24-oic acid) and deoxycholic

acid (DCA; 3α,12α-dihydroxy-5β-cholan-24-oic acid), but not CDCA (3α,7α-dihydroxy-

5β-cholan-24-oic acid). Thin layer chromatography (TLC) of overnight reactions

confirmed the formation of a product, co-migrating with authentic DCA standard, from 12-

oxoDCA in the presence of NAD(P)H (Figure 2.2). The reaction product was scraped from

the TLC plate and mass spectrometry (MS) identified a major mass ion in negative mode

at 391.2831 m/z (DCA molecular weight is 392.57 amu) (Figure 2.3B).

Next, we optimized pH in both the oxidative and reductive directions. In the

oxidative direction, with DCA as a substrate and NADP+ as a co-factor, the optimum

enzyme activity was observed at pH 7.0. Enzyme activity declined sharply from pH 7.0 to

pH 8.0 (< 10% relative activity) and ~50% residual activity remained at pH 6.0 (Figure

2.4C). In the reductive direction, with 12-oxoDCA as a substrate and NADPH as a co-

factor, the optimum was also determined to be pH 7.0, with a similar sharp decline to <5%

residual activity at pH 8.0, but ~80% maximal activity between pH 6.5-6.0. We therefore

chose pH 7.0 to run steady-state kinetics in both directions.

Kinetic parameters are listed in Table 2.3; substrate-saturation curves and

Lineweaver-Burke plots are displayed in Figure 2.5. Recombinant CDD59475 had an

order of magnitude higher KM for DCA (139.71 ± 14.64 μM) relative to 12-oxoDCA (11.41

± 0.68 μM). The native 12α-HSDH characterized from Clostridium sp. strain ATCC

29733/VPI C48-50 had a comparable KM value for DCA in the presence of

42

Figure 2.3 Mass spectrometry of enzymatic reaction products A. DCA standard, scraped from TLC plate after resolution in solvent system 75:20:2 toluene:dioxane:glacial acetic acid (v/v/v). B. MS analysis of reaction product co-migrating with iso-DCA, catalyzed by rCDD59473 in the presence of NADPH and 3-oxoDCA (see Figure 2.2A) C. MS analysis of reaction product co-migrating with 3-oxoDCA, catalyzed by rCDD59474 in the presence of NADP+ and DCA (see Figure 2.2B). D. MS analysis of reaction product co-migrating with DCA, catalyzed by rCDD59475 in the presence of NADPH and 12-oxoDCA (see Figure 2.2C).

A.

B.

43


C.

D.

44

Figure 2.4 pH optima for purified recombinant CDD59473 (A), CDD59474 (B), and CDD59475 (C) in the oxidative and reductive directions. CDD59473 was tested with 50 μM 3-oxoDCA (reductive) and isoDCA (oxidative). CDD59474 was tested with 50 μM 3-oxoDCA (reductive) and DCA (oxidative). CDD59475 was tested with 50 μM 12-oxoDCA (reductive) and DCA (oxidative). See Materials and methods for buffer compositions. Experiments were repeated three times and represented as ± standard error of the mean (S.E.M.).

A.

B.

C.

45

Tabl

e 2.

3 Ki

netic

par

amet

ers

of re

com

bina

nt C

DD

5947

3, C

DD

5947

4, a

nd C

DD

5947

5

46

Figure 2.5 Kinetic analysis of recombinant CDD59473, CDD59474, and CDD59475 from metagenomic sequence of Eggerthella CAG:298 Michaelis-Menten and Lineweaver-Burk plot of initial velocity with varying concentrations of substrate in the oxidative (left panels) and reductive (right panels) directions. The oxidation/reduction of NAD(P)(H) was measured by continuous spectrophotometry at 340 nm for 5 min and the initial velocities were used to calculate kinetic constants (Table 2.3). Each data-point represents ± S.E.M. from four independent experiments.

47

NADP+ (12 μM). The catalytic efficiency (kcat/KM) for CDD59475 in the reductive direction

was nearly an order of magnitude greater than in the oxidative, but turnover number (kcat)

was on the same order in both directions.

In the oxidative direction, CDD59475 recognized substrates regardless of side-

chain conjugation to glycine or taurine or presence/absence of 7α-hydroxyl group, which

was reported for the native 12α-HSDH from Clostridium sp. strain ATCC 29733/VPI C48-

50. Interestingly, in the reductive direction, the enzyme only displayed 4.06% activity for

12-oxoCA relative to 12-oxoDCA, suggesting steric hindrance from the 7α-hydroxyl group

(Table 2.4).

Characterization of recombinant CDD59473 and CDD59474 purified from E.

coli- We then overexpressed and purified recombinant CDD59473 (34.2 ± 0.4 kDa;

theoretical 31.59 kDa) and CDD59474 (28.0 ± 0.6 kDa; theoretical 26.40 kDa) in E. coli

(Figure 2.1) and tested these enzymes against a panel of bile acids and their oxo-

derivatives. Separation of CDD59473 and CDD59474 reaction products by TLC revealed

formation of iso-DCA and DCA, respectively, when 3-oxoDCA was the substrate (Figures

2.2A and 2.2B). The CDD59473 reaction product co-migrated with authentic iso-DCA

(392.57 amu) with major mass ion of 391.2849 m/z in negative ion mode (Figure 2.3C),

consistent with iso-DCA formation. The reaction product from CDD59474 co-migrated

with DCA and product in the oxidative direction co-migrated with 3-oxoDCA (390.56 amu)

with major mass ion of 389.2688 m/z in negative ion mode (Figure 2.3D), consistent with

formation of 3-oxo-DCA. A pH optimum for CDD59474 in the oxidative direction between

9 and 10 was observed, and optimum in the reductive direction was pH 7.0. With

NADP(H) as co-substrate,

48

Table 2.4 Substrate-specificity of Eggerthella CAG:298 HSDHs

49

CDD59474 favored the reductive direction, but mass action should favor the oxidative

direction in the anaerobic environment of the gut (Table 2.3; Figure 2.3).

E. lenta DSM 2243T was shown to express two 3β-HSDHs (Elen_0198,

Elen_1325) in the SDR family, sharing 38.1% amino acid sequence identity, involved in

iso-DCA formation (26). By contrast, the aldo/keto reductase family member, CDD59473,

shares only 20% amino acid sequence identity with the 3β-HSDHs Elen_0198 and

Elen_1325. The pH optimum of CDD59473 in the oxidative direction was between pH 9-

10, as observed in CDD59474, with optimum at pH 6.5 in the reductive direction (Figure

2.4). The 3β-HSDH activity of CDD59473 displayed a KM ~2.5 fold higher, a Vmax an order

of magnitude lower, and kcat four orders of magnitude lower than CDD59474 (3α-HSDH)

when 3-oxoDCA was substrate. CDD59473 shares with Elen_0198 and Elen_1325 the

ability to form small quantities of DCA (Figure 2.2), and thus low epimerase activity.

Previous work (77) and unpublished data in our laboratory with cultures of E. lenta DSM

2243T demonstrate that iso-CDCA or iso-DCA are minor metabolites, and that the mono-

keto and diketo intermediates predominate.

The high KM and low turnover number and catalytic efficiency of CDD59473 in the

reductive direction (Table 2.2), which partially agrees with values previously reported for

E. lenta DSM 2243T 3β-HSDH, may further explain why iso-CDCA does not predominate

in culture (26). Taken together, this three-gene cluster accounts for three of the four

HSDH activities reported in E. lenta strains (26, 76, 77) (Figure 2.6).

Phylogeny of Eggerthella HSDH enzymes- Next, the phylogenetic relationship

of these novel Eggerthella HSDH enzymes to other E. lenta SDR family proteins, and

bacterial HSDH proteins was determined (Figure 2.7; Table 2.2). The 3β-HSDH,

50

Figure 2.6 Schematic representation of bile acid metabolism by Eggerthella lenta. Accession numbers of proteins we identified in Eggerthella CAG:298 and the reactions they catalyze.

51

Figure 2.7 iTOl representation of FastTree maximum likelihood phylogeny from MUSCLE alignment of bacterial hydroxysteroid dehydrogenases and the placement of CDD59473, CDD59474, and CDD59475. Phylogeny is the result of 100 bootstraps compared via Phylo.io. Clusters are shaded according to function (see Key). See Table 2.1 for additional sequence information.

52

CDD59473, had by far the longest branch length of any cluster in the tree, distinct from

other 3β-HSDHs characterized in E. lenta DSM 2243T and Ruminococcus gnavus ATCC

29149T (26). Proteins clustering with CDD59473 include predicted aldo/keto reductases

and SDR family members. A BLAST search of CDD59473 against the non-redundant

database revealed amino acid sequences (61-59% identity) that cluster with CDD59473

encoded by Lactobacillus sp. isolated from the gut of pigeon, such as L. agilis

(KRM65245.1; WP_050611824.1) and L. ingluviei (WP_019206370.1; KRL88436.1)

(121, 122), L. hayakitensis (WP_025022266.1; KRM19149.1) from healthy equine (123),

L. saniviri isolated form human feces (124), and L. curvatus isolated from kimchi

(WP_089557116.1; WP_085844664.1) (125) (Figure 2.7).

CDD59474 is located within a cluster of confirmed and predicted 3α-HSDHs and

shares a node and 65.46% amino acid sequence identity with a reported 3α-HSDH

(Rumgna_02133) (26). An enzyme EDT24590.1 from C. perfringens clusters closely with

Rumgna_02133, suggesting this may be a 3α-HSDH whose activity was previously

reported (106). Clustering closely with CDD59474 are uncharacterized, potential 3α-

HSDH candidates from species including the equol-utilizing bacterium Adlercreutzia

equolifaciens (BAN77692.1) which shares much of its protein-coding genes with E. lenta

strains (126), the “Eubacterium-like” arginine-utilizer Cryptobacterium (CDD77593.1)

involved in periodontal disease (127). Also represented in this cluster are the BaiA

enzymes from Clostridium scindens, C. hylemonae, and C. hiranonis involved in the multi-

enzyme bile acid 7α-dehydroxylation pathway responsible for conversion of CA to DCA

and CDCA to lithocholic acid (LCA). BaiA enzymes are NAD(H)-dependent SDR-family

enzymes with strict specificity for bile acid coenzyme A thioesters, which is unique among

53

bacterial HSDHs (110, 111). Our phylogeny also revealed distinct clustering of recently

reported 20β-HSDHs from gut and urinary tract isolates (85).

Predicted 12α-HSDHs (12) also formed a separate branch which includes the 12α-

HSDH, CDD59475, and the characterized 12α-HSDH from Clostridium sp. ATCC 29733

(99), (108) (Figure 2.7). We have recently confirmed 12α-HSDH activity in whole cells

and pure CLOHYLEM_04236 and CLOSCI_02455 from C. hylemonae DSM 15053T and

C. scindens ATCC 35704T, respectively (Doden H, Sallam SA, Devendran S, Ly L, Doden

G, Mythen SM, Daniel SL, Alves JMP, Ridlon JM, unpublished data). Collinsella

aerofaciens is reported to encode 7β-HSDH (COLAER_RS09325) (120) but is also

predicted to encode a 12α-HSDH (COLAER_RS06615) based on homology with

CDD59475. We also identified a putative 12α-HSDH in a strain of the butyrate-producing

species Anaerostipes hadrus, an organism reported to exacerbate colitis in DSS-treated

mice (128). Human gut metagenomic sequences from Clostridiales bacterium

Nov_37_41 (OKZ96620.1), Lachnospiraceae bacterium 5_1_63FAA

(WP_009265642.1), and Clostridium sp. CAG:590 (CCX88977.1) are also predicted to

encode 12α-HSDH. Interestingly, while the majority of sequences were represented by

Firmicutes and Actinobacteria, a human gut metagenomic sequence from a pectin-

degrading bacterium, Bacteroides pectinophilus CAG:437 (CDD56334.1) was also

represented.

Previously characterized 7α-HSDHs formed two distinct clusters in our

phylogenetic analysis: one containing sequences from Firmicutes (gram-positive), the

other Bacteroidetes and E. coli (gram-negative); both clusters sharing a common node.

54

While 7α-HSDH activity has been reported in numerous E. lenta strains, a gene encoding

7α-HSDH has yet to be reported in E. lenta (76, 77).

DISCUSSION

In the current study, we utilized targeted gene synthesis to clone and

heterologously express genes from a gene-cluster (BN592_00768-BN592_00770) in

Eggerthella CAG:298, hypothesized to encode multiple HSDHs (CDD59473, CDD59474,

CDD59475) (Figure 2.1; Table 2.2). Characterization of the purified recombinant enzymes

revealed that each enzyme catalyzed a distinct oxidation/reduction of bile acid hydroxyl

groups. CDD59473 catalyzed NADPH-dependent reduction of 3-oxoDCA yielding iso-

DCA (3β-hydroxyl) as the main reaction product, co-migrating on TLC and with mass

spectrum consistent with iso-DCA (Figures 2.2 and 2.3). CDD59473 is a member of the

aldo-keto reductase family, and is distantly related to previously characterized 3β-HSDHs

from E. lenta DSM 2243T (Elen_0198, Elen_1325) and R. gnavus (Rumgna_00694), all

of which are SDR-family enzymes (Figure 2.7). Interestingly, despite the divergence in

sequence, the epimerase activity of CDD59473 (Figure 2.2; Table 2.2) is consistent with

gut microbial 3β-HSDHs characterized previously (26).

The host generates α-hydroxyl bile acids at C3, C7, and in half of the bile acid pool

also at C12 (cholic acid). Each α-hydroxyl group and the carboxyl group (C24) is on the

same face of the ABCD ring system resulting in a polar, hydrophilic side (Figure 2.6).

Below the ABCD ring system is the hydrophobic face which interact with lipids and

cholesterol packaged into mixed micelles. The isomerization of the 3α-hydroxyl to 3β-

hydroxyl decreases the amphipathic nature of bile acids making them less effective

detergents, and also less toxic to bacteria as well as host cells (26), (128, 129). The high

55

KM value (Table 2.3) suggests that this enzyme functions at high concentrations of toxic

secondary bile acids. The low turnover number (kcat) and catalytic efficiency (kcat/KM) of

CDD59473 vs. SDR-family enzymes (Elen_0198, Elen_1325, Rumgna_00694) provide a

contrast that structural biology might illuminate in an attempt to optimize the reductase

direction of 3β-HSDH and also to establish the basis for the stereo-specificity of the

reaction that yields low 3α-HSDH (epimerase) activity. Currently, there are no structures

reported for gut bacterial 3β-HSDH. Our phylogenetic analysis identified multiple

Lactobacillus spp. isolated from diverse avian and mammalian GI tracts encoding putative

3β-HSDHs with sequence identities at or below 71% (Figure 2.7). A similar targeted gene-

synthesis approach would be useful in determining the function and catalytic potential of

these putative 3β-HSDHs. Isomerization at C3 of primary bile acids may be important in

precluding bile acid 7α-dehydroxylation, a microbial biochemical pathway responsible for

formation of DCA and LCA, bile acids that are causally associated with cancers of the

colon and liver (2).

In contrast to CDD59473, CDD59474 converted 3-oxoDCA to DCA and thus

BN592_00769 encodes 3α-HSDH (Figure 2.6). DCA is generated by the multi-step bile

acid 7α-dehydroxylation of CA by a few species of intestinal clostridia (2) and is found in

fecal water at ~50 µM (130) but can reach several hundred micromolar (25). With KM

values for CDD59474 at ~150 µM and specificity for DCA, these results indicate that at

high DCA concentrations, CDD59473 and CDD59474 work in concert to epimerize DCA

to iso-DCA, potentially as a means of detoxification in E. lenta. Interestingly, CDD59474

was clustered with 3α-HSDH from R. gnavus (Rumgna_02133) but more distantly related

to a 3α-HSDH characterized in E. lenta DSM 2243T (Elen_0690) (Figure 2.7) (26). E.

56

lenta has been reported to express 7α-HSDH (76, 77); however, genes encoding 7α-

HSDH in E. lenta have yet to be identified. CDD59474 was annotated as “7α-HSDH” due

to sequence homology, yet expression and characterization of this protein revealed

specificity for 3α-hydroxyl groups providing a cautionary tale and further reinforcing the

need to functionally characterize genes predicted to encode particular functions by

annotation.

To our knowledge, BN592_00770 is the only reported sequence so far

demonstrated to encode 12α-HSDH (CDD59475) in E. lenta (Figures 2.2 and 2.6; Tables

2.3 and 2.4), and sequence comparison with CDD59475 is expected to elucidate further

gut bacterial 12α-HSDHs (Figure 2.7). CDD59475 shares with 12α-HSDH from

Clostridium sp. strain ATCC 29733/VPI C48-50 and the 3α-HSDH CDD59474 a

conserved N-terminal pyridine nucleotide binding domain (GX3GXG) and active-site

catalytic triad (SYK) (Figure 2.8). Particular bile acid structures trigger germination of C.

difficile spores. Recent work on a germinant receptor (CspC) in C. difficile determined that

bile acid specificity was dependent on 12α-hydroxylation (131). Binding to this receptor

led to the release of Ca2+ dipicolinic acid from the inside of the spore and subsequent

influx of water, ultimately leading to growth into a vegetative cell (131). Gut bacterial 12α-

HSDH activity may therefore affect C. difficile germination.

Thus, our analysis demonstrates that E. lenta strains harbor distinct forms of 3α-

HSDH, 3β-HSDH and 12α-HSDH enzymes (Figures 2.1 & 2.6). Critical at the intersection

between next-generation sequencing data (metagenomics, metatranscriptomics) and

metabolomics is biochemistry, providing the ability to experimentally demonstrate that a

57

Figure 2.8 CLUSTAL alignment of biochemically characterized gut bacterial hydroxysteroid dehydrogenases Red asterisks (*) represent active-site residues conserved in SDR-family. “7BCsard” AET80684.1 7-beta-hydroxysteroid dehydrogenase Clostridium sardiniense; “7BRgnav” AGN52919.1 7-beta-hydroxysteroid dehydrogenase Ruminococcus gnavus; “7BCaero” CZQ36308.1 unnamed protein product Collinsella aerofaciens; “3AComtest” WP_003078312.1 3-alpha-hydroxysteroid dehydrogenase/carbonyl reductase Comamonas testosteroni; “12A29733” ERJ00208.1 oxidoreductase, short chain dehydrogenase/reductase family protein Clostridium sp. ATCC 29733; “3ACSci” AAC45414.1 3-alpha hydroxysteroid dehydrogenase Clostridium scindens; “7AEcoi” AKF67908.1 7-alpha-hydroxysteroid dehydrogenase, NAD-dependent Escherichia coli; “7ABfrag” AAD49430.2 7-alpha-hydroxysteroid dehydrogenase Bacteroides fragilis; “7ACADSO” 5EPO_A Chain A, The Three-dimensional Structure of Clostridium absonum 7alpha-hydroxysteroid dehydrogenase.

58


59

particular amino acid sequence catalyzes a particular biochemical reaction. A major task

in gut microbiology research today is linking nucleic and amino acid sequences in

databases to function, and determining how functions affect microbiome structure and

host physiology. A major question in the field of bile acid metabolism (132) that we are

currently working to address is why bacteria, such as E. lenta, encode numerous HSDHs

and oxidize bile acids in an anaerobic environment.

60

CHAPTER 3: The desA and desB genes from Clostridium scindens ATCC 35704

encode steroid-17,20-desmolase

ABSTRACT

Clostridium scindens is a gut microbe capable of removing the side-chain of cortisol,

forming 11β-hydroxyandrostenedione. A cortisol-inducible operon (desABCD) was

previously identified in C. scindens ATCC 35704 by RNA-Seq. The desC gene was shown

to encode a cortisol 20α-hydroxysteroid dehydrogenase (20α-HSDH). The desD encodes

a protein annotated as a member of the major facilitator family, predicted to function as a

cortisol transporter. The desA and desB genes are annotated as N-terminal and C-

terminal transketolases, respectively. We hypothesized that the DesAB forms a complex

and has steroid-17,20-desmolase activity. We cloned the desA and desB genes from C.

scindens ATCC 35704 in pETDuet for overexpression in E. coli. The purified recombinant

DesAB was determined to be a 142 ± 5.4 kDa heterotetramer. We developed an enzyme-

linked continuous spectrophotometric assay to quantify steroid-17,20-desmolase. This

was achieved by coupling DesAB-dependent formation of 11β-hydroxyandrostenedione

with the NADPH-dependent reduction of the steroid 17-keto group by a recombinant 17β-

HSDH from the filamentous fungus, Cochliobolus lunatus. The pH optimum for the

coupled assay was 7.0 and kinetic constants using cortisol as substrate were KM of 4.96

± 0.57 µM and kcat of 0.87 ± 0.076 min-1. Substrate-specificity studies revealed that

rDesAB recognized substrates regardless of 11β-hydroxylation, but had an absolute

requirement for 17,21-dihydroxy 20-ketosteroids.

61

INTRODUCTION

Clinical studies in the 1950s reported urinary excretion of 17-ketosteroids following

rectal infusion of cortisol for the treatment of ulcerative colitis. Concomitant neomycin

treatment precluded urinary 17-ketosteroid excretion, implicating gut bacteria in the

conversion of a C21 glucocorticoid to a C19 androstane/androgenic metabolite (61), (62).

Later, it was shown that rat and human fecal bacteria were capable of this cortisol side-

chain cleavage reaction, which became known as steroid-17,20-desmolase (63), (64).

Serial dilutions of human and rat fecal samples revealed that ~ 106 bacterial gram-1 of

wet-weight express steroid-17,20-desmolase activity (65). Subsequently, an organism

was isolated in pure culture that expressed steroid-17,20-desmolase and 20α-

hydroxysteroid dehydrogenase (20α-HSDH) activities, and was named Clostridium

scindens, whose species epithet means “to cut”, a reference to separation of the side-

chain from the steroid D-ring (66).

Cortisol-inducible steroid-17,20-desmolase was partially purified, and 20α-HSDH

was purified to apparent electrophoretic homogeneity (SDS-PAGE) by traditional

chromatographic separation (67), (68). The application of genome-wide transcriptomics

(RNA-Seq) identified a polycistronic cortisol-inducible operon (desABCD) which included

a gene encoding 20α-HSDH (desC) (69). The desA and desB genes are annotated as N-

terminal and C-terminal transketolases, respectively. Transketolase catalyzes the TPP-

dependent transfer of glycol aldehyde to an aldose acceptor. Comparison of the

transketolation reaction to that of steroid-17,20-desmolase suggests an analogous

reaction, and that desAB encodes a novel steroid transketolase (69). C. cadavaris and

B. desmolans were shown previously to generate 20β-dihydrocortisol as well as 11β-

62

hydroxyandrostenedione (84). When the genomes of C. cadavaris and B. desmolans

were sequenced and reported, the desAB genes were located via BLAST search, and

flanked a gene encoding a short-chain dehydrogenase (SDR) family protein. We

overexpressed the SDR gene from B. desmolans in E. coli and showed that the enzyme

is a pyridine nucleotide-dependent 20β-HSDH (85). The clustering of genes encoding

cortisol 20α-HSDH or 20β-HSDH with desAB in different gut microbial species suggests

that desAB encodes steroid-17,20-desmolase; however, this function has not yet been

demonstrated. Here, we report that the desAB genes encode steroid-17,20-desmolase.

In addition, we developed a novel, enzyme-coupled continuous spectrophotometric assay

to quantify steroid-17,20-desmolase activity.


Bacterial strains and chemicals- Escherichia coli DH5α (turbo) competent cells

from New England Biolab (NEB) (Ipswich, MA) was used for cloning, and E. coli BL21-

CodonPlus (DE3) RIPL was purchased from Stratagene (La Jolla, CA) and used for

protein overexpression. The pETDuet and pET-51b(+) vectors were obtained from

Novagen (San Diego, CA). Restriction enzymes were purchased from NEB, QIAprep Spin

Miniprep kit was obtained from Qiagen (Valencia, CA). Isopropyl β-D-1-

thiogalactopyranoside (IPTG) was purchased from Gold Biotechnology (St. Louis, MO).

Streptactin resin was purchased from IBA GmbH, (Goettingen, Germany). Steroids were

purchased from Steraloids (Newport, RI). Amicon Ultra-15 centrifugal filter units with 30-

kDa- and 50-kDa-molecular-mass cutoffs (MMCOs) were obtained from Millipore

(Billerica, MA). All other reagents were of the highest possible purity and were purchased

from Fisher Scientific (Pittsburgh, PA).

63

Cloning of desA and desB from C. scindens and 17β-hydroxysteroid

dehydrogenase (17β-HSDH) from C. lunatus- Genomic DNA (gDNA) was extracted

from C. scindens ATCC 35704 using Fast DNA isolation kit from Mo-Bio (Carlsbad, CA,

USA) according to the manufacturer's protocol. The nucleotide sequence of the coding

region of a cDNA encoding 17β-HSDH from C. lunatus was entered into the Codon

Optimization Tool from Integrated DNA Technologies (IDT, Coralville, IA, USA). The

resulting sequence, whose deduced amino acid sequence was identical to the wild type

17β-HSDH sequence was synthesized as double-strand DNA (dsDNA) by gBlocks(R)

gene fragments service from IDT. C. scindens ATCC 35704 gDNA and gBlocks 17β-

HSDH dsDNA were used as templates to amplify the desAB and 17β-HSDH genes using

oligonucleotide primers reported in Table 3.1 Amplification of desAB and 17β-HSDH

inserts was performed on an Applied Biosystems ProFlex PCR System (Foster City, CA,

USA) using Phusion High-Fidelity DNA Polymerase (Stratagene, La Jolla, CA). Inserts

were gel-purified from agarose and cloned into the pETDuet and pET-51b(+) vectors,

respectively, using appropriate restriction enzymes to digest insert and vector and ligated

with DNA ligase. Recombinant plasmids were transformed into chemically competent E.

coli DH5α cells via the heat shock method, plated, and grown for 16 hours at 37°C on

lysogeny broth (LB) agar plates supplemented with ampicillin (100 µg/mL). A single

colony from each transformation was inoculated into LB medium (5 ml) containing

ampicillin (100 µg/ml) and grown to saturation. The cells were subsequently centrifuged

(3,220 x g, 15 min, 4°C) and plasmids were extracted from the resulting cell pellet using

the QIAprep Spin Miniprep kit (Qiagen, Valencia, CA). The sequences of the inserts were

64

Table 3.1 Oligonucleotides and synthetic gene sequences used in this study

Restriction sites are in bold and italics

65

determined via Sanger sequencing (W. M. Keck Center for Comparative and Functional

Genomics at the University of Illinois at Urbana-Champaign).

Gene expression and purification of recombinant steroid-17,20-desmolase

and 17β-HSDH- For protein expression, plasmids extracted containing correct insert from

the E. coli DH5α cells were transformed into E. coli BL-21 CodonPlus (DE3) RIPL

chemically competent cells via the heat shock method (42°C for 30 s) and grown

overnight at 37°C on LB agar plates supplemented with ampicillin (100 µg/mL) and

chloramphenicol (50 µg/mL). After 16 hours, five isolated colonies were used to inoculate

10 mL of fresh LB medium supplemented with antibiotics and grown at 37°C for 6 hours

with vigorous aeration. The pre-cultures were then added to fresh LB medium (1 L)

supplemented with the same antibiotics at the same concentrations and grown with

vigorous aeration at 37°C. At OD600 of 0.3, IPTG was added to each culture at a final

concentration of 0.1 mM and the temperature was decreased to 16°C. Following 16 hours

of culturing, cells were pelleted by centrifugation (4,000 x g, 30 min, 4°C) and re-

suspended in 30 mL of binding buffer (20 mM Tris-HCl, 150 mM NaCl, 20% glycerol, and

10 mM 2-mercaptoethanol at pH 7.9). The cell suspension was subjected to four

passages through an EmulsiFlex C-3 cell homogenizer (Avestin, Ottawa, Canada), and

the cell lysate was clarified by centrifugation at 20,000 x g for 30 min at 4°C. The

recombinant steroid-17,20-desmolase was then purified using TALON® Metal Affinity

Resin (Clontech Laboratories, Mountain View, CA) as per the manufacturer’s protocol.

The recombinant protein was eluted using an elution buffer composed of 20 mM Tris-HCl,

150 mM NaCl, 10% glycerol, 10 mM 2-mercaptoethanol, and 250 mM imidazole at pH

7.9. The recombinant 17β-HSDH was then purified using Strep-Tactin® resin (IBA GmbH,

66

Goettingen, Germany) as per manufacturing protocol. The recombinant protein was

eluted using an elution buffer composed of 20 mM Tris-HCl, 150 mM NaCl, 10% glycerol,

10 mM 2-mercaptoethanol, and 2.5 mM desthiobiotin at pH 7.0. The purity of the proteins

were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-

PAGE), and protein bands were visualized by staining with Coomassie Brilliant Blue G-

250 Dye. The protein concentrations were calculated based on the computed molecular

mass and extinction coefficient. The observed subunit mass for each protein was

calculated by migration distance of purified protein to standard proteins using ImageJ

(https://imagej.nih.gov/ij/index.html).

Gel filtration chromatography- Gel filtration chromatography was carried out on

a Superose-6 10/300 analytical column (GE Healthcare, Piscataway, NJ) attached to an

ÄKTAxpress chromatography system (GE Healthcare, Piscataway, NJ) at 4°C. The

eluted recombinant steroid-17,20-desmolase from the metal affinity resin was

concentrated and loaded on to the column with a buffer composed of 50 mM Tris-Cl, 150

mM NaCl, 10% glycerol, and 10 mM 2-mercaptoethanol at pH 7.0. The native molecular

mass of the protein was calculated by peak retention volume of protein to retention

volume of standard proteins.

Liquid chromatography-mass spectrometry analysis- Steroid-17,20-

desmolase activity was determined by incubating 1 μM recombinant enzyme with 50 μM

cortisol in the presence of 1 µM MnCl2 and 10 µM TPP in 50 mM glycine-glycine buffer

(pH 7.0) at room temperature overnight. After incubation, the products were extracted by

vortexing the reaction with 2 volumes of ethyl acetate for 1-2 mins and then recovering

the organic phase. The organic phase was then evaporated under nitrogen gas. The

67

residue was dissolved in 50 μL methanol and analyzed using a Shimadzu UHPLC system

with DAD detector coupled with a Shimadzu LCMS-IT-TOF System (Shimadzu

Corporation, Kyoto, Japan). The LC operating conditions were as follows: LC column, C-

18 analytical column (Capcell Pak C18, Shiseido, Japan), 250 mm × 2 mm i.d., particle

size - 3 μm; mobile phase, H2O containing 0.1% formic acid (A), and acetonitrile

containing 0.1% formic acid (B); total flow rate of mobile phase, 0.2 mL/min; total run time

including equilibration, 41 min. The initial mobile phase composition was 70% mobile

phase A and 30% mobile phase B. The percentage of mobile phase B was changed

linearly over the next 5 min until 35%. Over the next 25 min, the percentage was increased

to 98% linearly. After that the percentage was maintained for 5 min, the mobile phase

composition was allowed to return to the initial conditions and allowed to equilibrate for 5

min. The injection volume was 10 μL. The DAD detector was used at a wavelength of 254

nm. Peak retention times and peak areas of samples were compared with standard

steroid molecules. The mass spectrometer (LCMS-IT-TOF) was operated with an

electrospray ionization (ESI) source in positive mode. The nebulizer gas pressure was

set at 150 kPa with the source temperature of 200 °C and the gas flow at 1.5 L min-1. The

detector voltage was 1.65 kV. High-purity nitrogen gas was used as collision cell gas. The

raw chromatogram and mass spectrogram data were processed with the LC solution

Workstation software (Shimadzu).

Continuous enzyme-coupled spectrophotometric assay- Steroid-17,20-

desmolase activity was measured aerobically at 25°C by monitoring the conversion of

NADPH to NADP+ 340 nM (ε=6,220 M-1.cm-1) reflecting DesAB-catalyzed conversion of

cortisol to 11β-hydroxyandrostenedione followed by NADPH-dependent 17β-HSDH

68

conversion of 11β-hydroxyandrostenedione to a 11β-testosterone. Control reactions were

run leaving out one component at a time. Linearity with respect to time and enzyme

concentration were determined. The standard reaction mixture contained 50 mM MOPS

buffer, pH 7, 50 μM of cortisol, 1 µM MnCl2, 10 µM TPP, 200 μM NADPH, 1 μM each

enzyme, and buffer to a final volume of 0.5 ml. The reaction was initiated by the addition

of the steroid-17,20-desmolase. The initial velocity of enzyme catalyzed reactions was

plotted against the substrate concentrations, and the kinetic parameters were estimated

by fitting the data to the Michaelis-Menten equation by non-linear regression method

using the enzyme kinetics module in GraphPad Prism (GraphPad Software, La Jolla, CA).

An alternative methodology was utilized for substrates lacking a 17-hydroxyl, or those

substrates whose 17-keto products had low specificity for recombinant C. lunatus 17β-

HSDH (rCL17HSDH), was to run the basic assay without rCL17HSDH and NADPH.

Reactions were terminated with 1N HCl and reaction products extracted and separated

by LC/MS as described above. A standard curve of peak area vs. concentration of the

reaction products was generated to quantify reaction rates.

Determination of optimal pH- The buffers used to study the pH profiling of the

coupled enzymatic assay contained 50 mM of buffering agent, 1 µM MnCl2, 10 µM TPP,

and 150 mM NaCl. Buffering agents used were as follows: Sodium Citrate (pH 6.0),

MOPS (pH 6.5 – 7.0), Glycine-Glycine (pH 6.5 – 8.5), Tris-Cl (pH 9), and Glycine-NaOH

(pH 9.5 – 10). To determine the optimal pH, purified recombinant steroid-17,20-

desmolase and 17β-HSDH at final concentrations of 1 μM each, were added to 50 μM

cortisol and 200 μM NADPH in 500 μL of buffer. The oxidation of NADPH was measured

69

continuously at 340 nM for 5 mins by spectrophotometry and initial velocities were

calculated.

RESULTS

Cloning, overexpression of desAB, and purification of recombinant DesAB-

The desA gene encodes a 296 amino acid protein (CLOSCI_00899) with predicted

thiamine pyrophosphate (TPP) binding site (H74, G154, N183, K253), metal-binding site

(D153, E155) and active-site (H37, H268) based on multiple sequence alignment with

characterized transketolases and organisms expressing steroid-17,20-desmolase

activity, or encoding a predicted desABE operon (Figure 3.1). The desB encodes a 327

amino acid protein (CLOSCI_00900) comprising the C-terminal portion of a typical full-

length transketolase, and includes conserved residues with transketolases as diverse as

Lactobacillus salivarius UCC118 (YP_536833) (133) and Homo sapiens (P29401.3) (134)

including TPP-binding (F95), and active-site residues (E70). Also included in this

alignment are DesA and DesB sequences from known steroid-17,20-desmolase

expressing strains such as gut bacteria B. desmolans (WP_031476415.1,

WP_031476417.1), C. cadaveris (WP_027640053.1, WP_027640052.1), urinary isolate

Propionimicrobium lymphophilum ACS-093-V-SCH5 (135, 136) (WP_016455355.1,

WP_016455356.1), and inferred by homology and desABE operon structure from urinary

tract isolate Arcanobacterium urinimassiliense (137) (WP_084789932.1,

WP_073996552.1).

Since key conserved transketolase catalytic and co-factor binding residues are

distributed across both the desA and desB genes, we chose the pETDuet expression

vector, designed for overexpression of two target genes in E. coli BL21(DE3)RIL as His-

70

Figure 3.1 Steroid-17,20-desmolase (DesAB) shares conserved catalytic and co-factor binding residues with transketolases Sequences in partial CLUSTAL-Omega alignment include Homo sapien transketolase (TKT_Hsap), transketolase from Lactobacillus salivarius (TKT_Lsa1), DesAB from C. scindens ATCC 35704 (desAB_Csci), DesAB from Butyricicoccus desmolans (desAB_Bdes), DesAB from Clostridium cadavaris (desAB_Ccad), DesAB from Propionimicrobium lymphophilum (desAB_Plym), and DesAB from Arcanobacterium urinimassiliense (desAB_Aaur). Conserved residues are highlighted based on functional role depicted by the color key.

71

tagged recombinant proteins. SDS-PAGE analysis of the elution fraction following affinity

chromatography revealed two bands at 32.7 kDa and 38.4 kDa, corresponding to the

predicted molecular mass of rDesA and rDesB, respectively (Figure 3.2A). Gel filtration

chromatography determined the native molecular mass of rDesAB to be 142 ± 5.4 kDa,

suggesting that the rDesAB is a heterotetramer (Figure 3.2B).

Development of a coupled, continuous spectrophotometric assay to

measure steroid-17,20-desmolase activity- Next, we incubated recombinant DesAB

overnight in the presence of cortisol, TPP and Mn2+ in glycine-glycine buffer, pH 7.0.

Extraction of the reaction buffer with ethyl acetate and separation by reverse-phase HPLC

revealed net conversion of cortisol (RT = 17.5 min) to a single product, with retention time

identical to 11β-hydroxyandrostenedione standard (RT =23.5 min) (Figure 3.3). The

control cortisol peak was subjected to electrospray ionization-time of flight-mass

spectrometry (ESI-TOF-MS) and resulted in a major mass ion in positive mode of 363.17

m/z, consistent with the molecular weight of cortisol (362.46 amu). The major mass ion

of the rDesAB product was 303.15 m/z, consistent with side-chain cleavage and formation

of 11β-hydroxyandrostenedione (302.414 amu). These results demonstrate that the

DesAB is the steroid-17,20-desmolase.

The steroid-17,20-desmolase assay previously utilized is discontinuous, time-

consuming, and expensive. We therefore sought another approach to quantify steroid-

17,20-desmolase activity using a continuous enzyme-coupled spectrophotometric assay.

The steroid-17,20-desmolase reaction yields a 17-ketosteroid product which, if coupled

to an NAD(P)H-dependent 17α- or 17β-HSDH, could be used to measure reaction

velocity by monitoring NAD(P)H oxidation at 340 nm (Figure 3.4A). Consultation of the

72

A. B.

Figure 3.2 SDS-PAGE of affinity purified rDesA and rDesB and native molecular mass estimate of rDesAB. A. SDS-PAGE of pETDuet co-expressed rDesA and rDesB (Lane DesAB), protein molecular weight marker (M). B. Native molecular size analysis of rDesAB by size-exclusion chromatography.

73

Figure 3.3 Liquid chromatography-mass spectrometry of rDesAB reaction products Standards include authentic cortisol and 11β-hydroxyandrostenedione. Reaction assay conditions include 1 μM recombinant enzyme with 50 μM cortisol in the presence of 1 µM MnCl2 and 10 µM TPP in 50 mM glycine-glycine buffer (pH 7.0) at room temperature overnight. Control reactions omitted rDesAB. Mass spectra corresponding to HPLC peaks depicted in inset.

74

Figure 3.4 SDS-PAGE of purified recombinant 17β-HSDH from Cochliobolus lunatus (rCL17HSDH) and schematic representation of enzyme-coupled continuous spectrophotometric steroid-17,20-desmolase activity assay. A. Schematic of conversion of cortisol to 11β-hydroxyandrostenedione, catalyzed by rDesAB, followed by NADPH-dependent conversion of 11β-hydroxyandrostenedione to 11β-hydroxytestosterone by rCL17HSDH. B. SDS-PAGE of rCL17HSDH. Protein molecular weight marker (M).

75

relevant literature revealed a well characterized NADP(H)-dependent 17β-HSDH from the

filamentous fungus Cochliobolus lunatus, whose 1.044 kB cDNA sequence is available

at NCBI (Accession number AAD12052.1) (17). We trimmed the 5’- and 3’- untranslated

regions (UTR) sequences and optimized the resulting 813 bp coding sequence for codon-

usage in E. coli with the Codon Optimizer tool from IDT and the gene insert was

synthesized using the GenBlock synthetic biology service from IDT. The insert was PCR

amplified using primers designed for bi-directional cloning into pET51b(+) vector, allowing

for expression of rCL17HSDH as a 32.2 kDa N-terminal streptavidin affinity-tagged

protein (Figure 3.4B).

rCL17HSDH showed NADPH-dependent activity against 11β-

hydroxyandrostenedione, but not cortisol in 50 mM glycine-glycine buffer, pH 7.0.

Oxidation of NADPH was observed at 340 nm only in the presence of rDesAB, cortisol,

and rCL17HSDH. Previous characterization of rCL17HSDH showed pH optimum

between 7.0 and 8.0 (18). We determined that DesAB was most active between pH 7.0

and pH 7.5 by observing conversion of cortisol to 11β-hydroxyandrostenedione between

pH 6.0 to 8.5 (data not shown). Therefore, the pH optima of both rDesAB complex and

rCL17HSDH overlap. After optimization of the coupled assay with respect to enzyme

concentration, we determined pH optimum for the coupled reaction was pH 7.0 in MOPS

buffer. There was a precipitous decline in activity from pH 6.5 to pH 6.0, nearly equivalent

to the loss of activity from pH 7.0 to 8.0. At pH 10.0, the coupled assay retained ~5% of

its maximum activity (Figure 3.5).

76

Figure 3.5 Biochemical characterization of rDesAB by enzyme-coupled continuous spectrophotometric assay. (Top) Effect of pH on steroid-17,20-desmolase activity (Bottom) Michaelis-Menten of rDesAB activity with increasing cortisol.

77

Determination of kinetic constants and substrate-specificity- We varied the

concentration of cortisol in the presence of 200 µM NADPH, 1 µM MnCl2, 10 µM TPP in

50 mM MOPs buffer pH 7.0. Michaelis–Menten saturation curve is shown in Figure 3.5.

The KM for the rDesAB is 4.96 ± 0.57 µM and Vmax of 12.76 ± 1.12 nmol.min-1.mg-1. The

turnover number (kcat) was determined to be 0.87 ± 0.076 min-1 and catalytic efficiency

(kcat/KM) was 0.17 ± 0.029 min-1.μM-1. Suspecting that an aldose-acceptor may be utilized

in the reaction, glyceraldehyde-3-phosphate and ribose-5-phosphate were tested at 50

µM, 100 µM, and 500 µM; however, we did not observe increased enzymatic activity.

We tested substrate-specificity using a combination of methods, depending on whether

steroids were 17-hydroxylated. The enzyme-coupled continuous spectrophotometric

assay was relegated to 17-hydroxylated substrates. Those reaction products from

substrates lacking 17-hydroxy were characterized by LC-MS. Furthermore, we tested

NADPH-dependent rCL17HSDH activity against the expected C-19 steroid-17,20-

desmolase products to determine whether we could make a direct comparison between

substrates whose products differed structurally. We determined that Ring-A reduced

products (50 µM concentration), such as 5β-androsterone and 11β-hydroxyandrosterone,

are not substrates for rCL17HSDH at, resulting in 8.06 ± 2.65% and 4.73 ± 0.68% activity,

respectively, relative to 11β-hydroxyandrostenedione (three technical replicates). These

substrates were therefore tested for activity using rDesAB with separation of steroids by

LC/MS.

We determined that 11β-hydroxylation is not necessary for steroid-17,20-

desmolase (Table 3.2). 20α-Hydroxy or 20β-hydroxy derivatives of cortisol were not

78

Table 3.2 Substrate specificity for rDesAB from C. scindens ATCC 35704

79

substrates. Those substrates lacking 17-hydroxyl group, and/or 21-hydroxyl group, such

as corticosterone and progesterone metabolites were not side-chain cleaved (Figures 3.6

- 3.7). Mass spectra are presented in Figures 3.8. Interestingly, tetrahydrocortisol and

tetrahydrodeoxycortisol were also not recognized by rDesAB, indicating 3α-hydroxy, 5β-

reduced metabolites are not substrates.

DISCUSSION

In the current study, we report that the desA and desB genes from C. scindens

ATCC 35704 encode steroid-17,20-desmolase. Recombinant DesAB displayed

substrate-specificity consistent with native steroid-17,20-desmolase: the enzyme

recognizes 20-keto-17, 21-dihydroxy steroids (67, 68). C. scindens ATCC 35704

expresses NAD(H)-dependent 20α-HSDH (69), while B. desmolans, C. cadavaris, and P.

lymphophilum express 20β-HSDH (85) in addition to steroid-17,20-desmolase. Oxidation-

reduction of the C20 oxygen is predicted to function as a regulatory “switch”,

interconverting cortisol between DesAB substrate and non-substrate forms. The desD

gene is co-expressed with desABC (69) and encodes a predicted 51.2 kDa transport

protein in the major facilitator superfamily. DesD is predicted to transport cortisol from the

gut lumen into the bacterial cytoplasm. RNA-Seq analysis also identified a cortisol-

inducible ABC-type multidrug transport system predicted to export 11β-

hydroxyandrostenedione (69).

The conversion of cortisol to 11β-hydroxyandrostenedione by C. scindens is an

important step in a multi-species gut microbial endocrine pathway that results in the

formation of 11β-hydroxyandrogens (69). Previous studies reveal a number of cortisol

80

Figu

re 3

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. HPL

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B. O

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81

B. Fi

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3.6

con

tinue

d

82

Figure 3.7 Functional groups important for rDesAB specificity

83

Mass spectra data for 11-epicorticosterone

Mass spectra data for 11β-hydroxyprogesterone

Figure 3.8 Mass spectra of rDesAB catalyzed reactions. MS spectra are from major peaks depicted in Figure 3.6A. Conditions for MS and LC-MS are described in Materials and Methods in the main text.

84

Mass spectra data for tetrahydrocortisol

Mass spectra data for tetrahydrodeoxycortisol


85

Mass spectra data for corticosterone

Mass spectra data for cortisol with rDesAB


86

Mass spectra data for cortisol

Mass spectra data for 11β-androstenedione


87

Mass spectra data for desoxycortisol with rDesAB

Mass spectra data for desoxycortisol


88

Mass spectra data for androstenedione


89

metabolites following incubation with mixed human or rat fecal bacteria included

3α,11β,17β-trihydroxy-5β-androstane, 3α,11β,17β-trihydroxy-5α-androstane,

demonstrating the ability of gut microbes to form 5α-, 17β-reduced metabolites (63),

(64). Some of the taxa responsible for these biotransformations have been identified. C.

paraputrificum is known to express 3α-HSDH and 5β-reductase (138), C. innocuum is

reported to encode 3βHSDH and 5β-reductase (139), Clostridium sp. Strain J-1 encodes

3α-HSDH and 5α-reductase (138), capable of reducing the 3-oxo-Δ4 bonds in ring-A. 17α-

HSDH has been reported in C. scindens VPI 12708 (140) and has been detected in E.

lenta DSM 2243 and C592 (141). The genes encoding these reductases have yet to be

identified, but are likely to affect steroid-17,20-desmolase activity in the gut. E. lenta also

expresses 21-dehydroxylase (74), and 21-dehydroxylated metabolites are detected

following incubation of cortisol in fecal suspensions (63, 64). Due to the absolute

requirement for the C21-hydroxy group, steroid-17,20-desmolase and cortisol 21-

dehydroxylation by Eggerthella lenta are competing reactions in the gut (74).

Extrapolations from radiometric studies in non-human primates (142) and data

from humans (143) suggest that 15% of the 15 mg of cortisol are detected in the gut.

Based on these studies, the concentration of cortisol metabolites in the ~0.4L human

colon (144) is ~15 µM. 11β-hydroxyandrostenedione is now viewed as a pro-androgen,

whose downstream 5α-, 17β-reduced metabolites, are now thought to play an important

role in activation of androgen receptor (33, 145). The field of microbial endocrinology has

emerged and recognizes the need to characterize androgen production by C. scindens

(38, 146–150). Many host immune cells express androgen-receptor (151, 152), and it is

possible that C. scindens affects immune function through generation of androgens.

90

Androgens directly affect bacterial growth and virulence (153, 154). Intriguingly, steroids

have been shown to influence C. difficile germination (3). 11β-hydroxy-androgens are

also inhibitors of the host enzyme 11β-HSD2 (80, 155), but in kidney and vascular smooth

muscle may be pro-hypertensive (155). Inhibition of 11β-HSD2 in colonocytes may be

protective against colorectal cancer (156). Prostate tissue contains 11β-

hydroxyandrogens despite inhibition of host enzymes involved in synthesizing androgens

(chemical castration) (33, 145). The role of 11β-OHAD formation by the gut microbiota in

androgen accumulation in the prostate is unknown; however, we discovered that bacteria

isolated from urine also express the bacterial steroid-17,20-desmolase pathway (85), and

the majority of cortisol excretion from the body is via the urine (142, 143). Of note, we

recently reported identification of the steroid-17,20-desmolase gene cluster in the

genomes of urinary bacterial isolates and confirmed steroid-17,20-desmolase activity in

an isolate of Propionimicrobium lymphophylum (85). Verification that desA and desB

genes encode steroid-17,20-desmolase is an important step towards the application of

nucleic acid-based approaches such as quantitative polymerase chain, and metagenomic

sequencing in determining whether correlations exist between levels/expression of

desAB and host phenotypes and disease states.

As gut metagenomic sequencing becomes less costly, and more widespread,

assigning function to particular nucleic acid and deduced amino acid sequences will be

important for hypothesis generation, and testing. The desA and desB genes are a clear

case in point. The desABCD operon is annotated as participating in carbohydrate

metabolism, due largely to the desA and desB gene product homology to N-terminal and

C-terminal TPP-dependent transketolases, respectively (Figure 3.9). Early culture-based

91

Figure 3.9 Gene organization and domain architecture of steroid-17,20-desmolase

92

studies established that steroid-17,20-desmolase activity is induced by cortisol (66–68).

Utilization of RNA-Seq to identify differentially expressed genes, coupled with biochemical

characterization of the recombinant gene products was necessary to determine the

nucleic acid sequences encoding this bacteria steroid biotransforming pathway (69).

A recent report describes side-chain cleavage of cholesterol by Mycobacterium

tuberculosis and revealed a thiolase-derived enzyme, a steroid-aldolase, involved in

microbial steroid-degradation (157) (Figure 3.10). It is therefore possible that additional

side-chain cleaving gut microbial members will be identified that are capable of aldolase

cleavage of host and dietary steroids in the gut. This is in contrast to eukaryotic cells

which typically utilize oxygen-dependent P450 monooxygenases (158).

The low turnover number is within the range of previously reported prokaryotic

enzymes involved in secondary metabolism (159), and may indicate that selection

pressure may be less stringent for optimization of enzyme rate for functions that may not

contribute greatly to organismal fitness. Studies are now ongoing to understand important

amino acid residues involved in substrate-binding to steroid-17,20-desmolase, and

catalysis, as well as the identity and fate of the side-chain. Development of a novel

enzyme-coupled continuous spectrophotometric assay to quantify steroid-17,20-

desmolase activity against cortisol is expected to hasten results from these efforts.

Current limitations of the assay with respect to androgen specificity of rCL17HSDH may

be addressed in the future either through protein engineering, or by identifying naturally

occurring 17α- or 17β-HSDHs. In addition, future studies will be required to determine the

identity of the side-chain and whether an aldose acceptor is utilized in vivo. A detailed

understanding of the steroid-17,20-desmolase will be important in uncovering how

93

Figure 3.10 Schematic representation of side-chain cleavage reactions for steroid-17,20-desmolase from C. scindens ATCC 35704, steroid-transaldolase from Mycobacterium tuberculosis, and Cytochrome P450 monooxygenase (CYP11A1) in Eukaryotes.

94

anaerobic bacteria co-opted pentose-phosphate pathway enzymes for steroid side-

chain cleavage.

95

CHAPTER 4: Structural and biochemical characterization of 20β-hydroxysteroid

dehydrogenase from Bifidobacterium adolescentis strain L2-32

ABSTRACT

A novel 20β-hydroxysteroid dehydrogenase (20β-HSDH) from Bifidobacterium

adolescentis strain L2-32 has been enzymatically characterized and crystallized. 20β-

HSDH catalyzes the reduction of the carboxyl group at position 20 of the steroid cortisol,

thereby producing 20β-dihydrocortisol. The enzyme was determined to be tetrameric in

structure, with a subunit molecular mass of 32 ± .12 kDa, and exhibits a pH optimum of

5.0 in the reductive direction and 5.5 in the oxidative direction. Cortisol was determined

to be the preferred substrate with a KM of 24.07 μM. Vmax, kcat, and kcat/KM values were

determined to be 21.08 µmol.min-1.mg-1, 668.3 min-1, and 27.765 μM-1.min-1, respectively.

Mutation of the putative catalytic serine confirmed its functional role in reaction catalysis.

The enzyme was shown to have coenzyme specificity for NADH rather than NADPH. The

apo and holo forms of the enzyme were crystallized and shown to diffract at 2.1 and 2.5

Å resolution, respectively.

INTRODUCTION

Hydroxysteroid dehydrogenases (HSDH) are pyridine nucleotide-dependent

enzymes classified into three superfamilies which include aldo-keto reductase (AKR),

long/medium chain dehydrogenase/reductase (MDR), and short-chain

dehydrogenase/reductase (SDR) ((11, 69, 160) ). The gut microbiome is exposed to a

mixture of corticosteroids and bile acids and much research has focused on bacteria

strains of diverse taxa which express stereo- and regio-specific HSDHs in the SDR and

AKR families capable of oxidation and epimerization of bile acid hydroxyl groups at C-3,

96

C-7, and C-12 (161, 162). Recent attention has been focused on the bacterial cortisol

steroid-17,20-desmolase pathway in Clostridium scindens, capable of converting cortisol

to 11β-hydroxyandrostenedione (11 β-OHAD), and the zinc-dependent MDR family 20α-

HSDH which is predicted to enzymatically regulate the pathway (67, 69). 11β-OHAD is a

pro-androgen whose formation in the gut is predicted to alter host physiology (33).

Bacterial steroid-17,20-desmolase has also been reported in Clostridium cadaveris and

Butyricicoccus desmolans (formerly Eubacterium desmolans) and is associated with

NADH-dependent cortisol 20β-HSDH activity (85). Previous studies also identified strains

of Bifidobacterium adolescentis isolated from human and rat fecal dilutions which

expressed cortisol 20β-HSDH activity alone (86). Recently, the gene encoding gut

bacterial NADH-dependent 20β-HSDH activity was reported in B. desmolans (85).

Phylogenetic analysis identified numerous Bifidobacterium spp., including strains of B.

adolescentis, encoding putative cortisol 20β-HSDH (85).

Gut bacterial 20β-HSDH is a member of the SDR family of proteins (11, 85). The

SDR family constitute one of the largest of protein superfamilies distributed across all

three domains of life, comprising of functionally diverse members (12, 162–164). SDR

family enzymes include several EC classifications including lyases, isomerases, and

pyridine nucleotide-dependent oxidoreductases constituting the majority of enzymes.

Currently in Uniprot there are 308,095 entries and members only share ~20-30% residue

identity in pairwise comparisons. Typical SDR family enzymes are 250-350 amino acids

in length, with secondary structure consisting of one-domain Rossmann folds containing

6-7 beta-strands flanked by three alpha helices. Conserved N-terminal Gly-X3-Gly-X-Gly

motif critical in NAD(P)(H) binding, as well as catalytic tetrad composed of Ser, Tyr, Lys,

97

and Asn residues (11, 165). SDR family enzymes vary in substrate recognition, including

sugars, dyes, prostaglandins, porphyrins, alcohols, and steroids (11). As a consequence,

substrate-binding residues are not highly conserved. Differentiating HSDHs from other

functional classes of SDR family enzymes is important for identifying steroid biochemical

pathways in gut metagenomes. At present, this task is impeded by a lack of primary

amino acid sequences for several HSDHs whose activities have been reported. If we are

to determine the residues important in differentiating HSDHs into functional categories

(i.e. 7α-HSDH from 7β-HSDH and both from 20β-HSDH), the genes encoding these

enzymes have to be located.

The first SDR family enzyme crystallized was 3α,20β-HSDH from the soil

bacterium Streptomyces hydrogenans (166). The 3α,20β-HSDH is able to reversibly

oxidize the 3α-hydroxyl and 20β-hydroxyl groups of androgens and progestogens. The

S. hydrogenans 3α,20β-HSDH shares 31% amino acid identity with the 20β-HSDH from

B. adolescentis, which does not appear to function as a dual 3α-HSDH. It is important to

obtain a mechanistic understanding of 20β-HSDH because this enzyme is capable of

regulating the major gut microbial side-chain cleavage of cortisol steroid-17,20-

desmolase resulting in pro-androgens, as well as cortisol 21-dehydroxylation resulting in

formation of progestogens (74). Herein, we report the characterization of 20β-HSDH from

Bifidobacterium adolescentis strain L2-32.


Bacterial strains, enzymes, reagents, and materials- Bifidobacterium

adolescentis, strain L2-32 was purchased through BEI Resources (Catalog No. HM-633).

Turbo Competent Escherichia coli DH5α (High Efficiency) cells were purchased from New

98

England Biolabs (NEB). BL21-CodonPlus (DE3)-RIPL Competent E. coli cells were

purchased from Agilent Technologies (Catalog No. 230280). Phusion High-Fidelity DNA

Polymerase, Restriction Endonucleases, and T4 DNA Ligase were purchased from NEB.

Isopropyl β-D-1-thiogalactopyranoside (IPTG) was purchased from Gold Biotechnology.

TALON His Tag Purification Resin was purchased from Takara Bio USA, Inc. Corning

Spin-X UF concentrators with a 10 kDa molecular weight cut-off (MWCO) were purchased

from MilliporeSigma. Steroids were purchased from Steraloids. Further materials were

purchased from Fisher Scientific and MilliporeSigma.

Whole cell steroid conversion assay- B. adolescentis, strain L2-32, B.

desmolans ATCC 43058, and C. scindens ATCC 35704 were cultivated in anaerobic

brain heart infusion (BHI) broth. Steroids were dissolved in methanol and added to sterile

anaerobic BHI medium at a concentration of 50 μM. The samples were then seeded

separately with 0.1 ml of each bacterial culture and incubated at 37°C for 48 hours. After

incubation, the products were extracted by vortexing the samples with 2 volumes of ethyl

acetate for 3 min followed by recovery of the organic phase. The organic phase was then

evaporated under nitrogen gas. The remaining residues were dissolved in 100 μL of

methanol and analyzed using a high-performance liquid chromatography (HPLC) system

(Shimadzu, Japan) equipped with C-18 analytical column (Capcell Pak c18, Shiseido,

Japan). The mobile phase contained acetonitrile/water with 0.01% formic acid and the

flow rate was maintained at 0.2 ml.min−1. A DAD detector was used at a wavelength of

254 nm. Peak retention times and peak areas of samples were compared with standard

steroid molecules.

99

Cloning and formation of the 20β-HSDH expression vector- Genomic DNA

was extracted and purified from B. adolescentis, strain L2-32 using the FastDNA Spin Kit

from MP Biomedicals. The gene encoding 20β-HSDH (accession number

WP_003810233.1) was amplified using Phusion High Fidelity DNA Polymerase and the

following primers: 5'-ATATATcatatgATGGCAGTTGAATCATCGCAGATTCCA-3' and 5'-

ATATATctcgagTTAGAAGACGCTGTAGCCGCCATCTAC-3'. The restriction

endonuclease sites for NdeI and XhoI are shown in lower-case respectively. The PCR

product was purified and double digested with NdeI and XhoI restriction endonucleases.

The digested PCR product was purified and cloned into the pET-28(+) (Novagen,

Madison, WI, USA) vector using T4 DNA Ligase to create the expression construct. The

pET-28(+) vector used had previously been re-engineered with an ampicillin resistance

gene. The resulting construct was transformed into Turbo Competent E. coli DH5α (High

Efficiency) cells by heat shock at 43°C for 35 sec and outgrown form 1 hour in Super

Optimal Broth with Catabolite Repression (SOC) (NEB) before growing on LB agar plates

supplemented with ampicillin (100 µg/ml) for 16 hours at 37°C. Single colonies from the

LB agar plates where inoculated into 5 mL of LB medium containing ampicillin (100 µg/ml)

and grown overnight at 37°C at 220 rpm. The cells were then centrifuged at 4000 x g for

10 minutes at 4°C and plasmids were extracted and purified from the pellets using the

QIAprep Spin Miniprep Kit. Both strands of the plasmid DNA were sequenced for

confirmation of the insert and absence of mutations using T7 promoter and T7 terminator

primers (W. M. Keck Center for Comparative and Functional Genomics at the University

of Illinois at Urbana-Champaign). Once overexpressed, the protein will include an N-

terminus 6xHis-tag and a thrombin cleavage site.

100

Site-directed mutagenesis- Plasmids containing the 20β-HSDH gene construct

were mutated using the QuikChange Lightning Site-Directed Mutagenesis Kit. Primers

containing the desired mutations were designed as follows: 5'-

CTGATCTTCACAGCCgcgCTGTCCGGACACAA-3' and 5'-

TTGTGTCCGGACAGcgcGGCTGTGAAGATCAG-3' for Serine 181 to Alanine (S181A);

and 5'-TCTTCACAGCCTCCCTGgcgGGACACAACGCGAACTA-3' and 5'-

TAGTTCGCGTTGTGTCCcgcCAGGGAGGCTGTGAAGA-3' for Serine 183 to Alanine

(S183A). Mutated bases are shown in lowercase. The resulting mutated constructs were

transformed into Turbo Competent E. coli DH5α (High Efficiency) cells by heat shock at

43°C for 35 sec and outgrown form 1 hour in Super Optimal Broth with Catabolite

Repression (SOC) (NEB) before growing on LB agar plates supplemented with ampicillin

(100 µg/ml) for 16 hours at 37°C. Single colonies from the LB agar plates where

inoculated into 5 mL of LB medium containing ampicillin (100 µg/ml) and grown overnight

at 37°C at 220 rpm. The cells were then centrifuged at 4000 x g for 10 minutes at 4°C

and plasmids were extracted and purified from the pellets using the QIAprep Spin

Miniprep Kit. Mutations were confirmed by sequencing of the mutated plasmid using T7

promoter and T7 terminator primers (W. M. Keck Center for Comparative and Functional

Genomics at the University of Illinois at Urbana-Champaign).

Overexpression and purification of wild-type and mutant 20β-HSDH

proteins- For protein expression, recombinant plasmids extracted from the E. coli DH5α

cells were transformed into BL21-CodonPlus (DE3)-RIPL Competent E. coli cells by heat

shock at 43°C for 35 sec and outgrown for 1 hour in Super Optimal Broth with Catabolite

Repression (SOC) (NEB) before growing on LB agar plates supplemented with ampicillin

101

(100 µg/ml) and chloramphenicol (50 µg/ml). After 16 hours of growth at 37°C, five

colonies were picked to inoculate 10 mL of LB medium containing ampicillin (100 µg/ml)

and chloramphenicol (50 µg/ml) and were grown for 6 hours at 37°C at 220 rpm. The

turbid pre-culture was then added to 1 L of LB medium containing the same antibiotics

and shaken at 37°C at 220 rpm. At an OD600 of 0.6, the culture was induced with 0.25

mM IPTG, after which, the culture was incubated at 16°C at 220 rpm for 16 h. Cells were

pelleted by centrifugation at 4,000 x g for 30 minutes at 4°C. The pellets were

resuspended in 25 mL of buffer containing 50 mM Tris-Cl and 300 mM NaCl at a pH of

7.9. The resuspended cells were homogenized using an EmulsiFlex C-3 homogenizer

by passing the samples through the homogenizer four times and the lysate was refined

by centrifugation at 20,000 x g for 30 minutes at 4°C. The soluble lysate was then purified

using metal chelate chromatography followed by removal of the 6xHis-tag by use of

thrombin. The lysate was further purified using anion exchange chromatography to

remove the excess thrombin. The resulting purified protein was analyzed using sodium

dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Gel filtration chromatography- Gel filtration chromatography was performed on

a Superdex 200 analytical column (GE Healthcare, Piscataway, NJ) attached to an

ÄKTAxpress chromatography system (GE Healthcare, Piscataway, NJ) at 4°C. The

purified protein was concentrated and loaded onto the analytical column equilibrated with

50 mM tris-Cl and 300 mM NaCl at a pH of 7.5 and was eluted at a flow rate of 1 ml/min.

The native molecular mass of the 20β-HSDH protein was determined to by comparing its

elution volume with that of standard proteins.

102

Optimal pH determination- The 20β-HSDH pH optimum was measured

spectrophotometrically by monitoring the reductive biotransformation of cortisol to 20β-

dihydrocortisol as well as the reverse oxidative biotransformation of 20β-dihydrocortisol

to cortisol. For the reductive direction, measurements were taken by analyzing the

decrease of NADH at 340 nm. For the oxidative direction, measurements were taken by

analyzing the increase of NADH at 340 nm. The buffers used in the pH profiling contained

50 mM of buffering agent and 150 mM NaCl. Buffering agents used were as follows:

Sodium acetate trihydrate (pH 3.5 – 5.5), Sodium phosphate monobasic monohydrate

(pH 6.5 – 7.5), Tris (pH 8.0 – 9.0), Glycine (pH 10 – 11). For the reductive reaction 50

nM of enzyme was added to 50 μM cortisol and 150 μM NADH in 200 μL of buffer. For

the oxidative reaction 50 nM of enzyme was added to 50 μM 20β-dihydrocortisol and 150

μM NAD+ in 200 μL of buffer. For each reaction the decrease or increase of NADH was

measured continuously for five minutes and initial velocities were calculated.

Enzyme activity analyses and determination of kinetic parameters- 20β-

HSDH wild-type and mutant activity was measured spectrophotometrically by monitoring

the reductive biotransformation of cortisol to 20β-dihydrocortisol as well as the reverse

oxidative biotransformation of 20β-dihydrocortisol to cortisol by measuring the levels of

NADH at 340 nm. The standard reaction mixture contained 50 mM sodium acetate

trihydrate buffer (pH 5.0), 150 μM of cofactor (NADH), 10 nM of enzyme, and a varied

substrate concentration between 5 μM and 90 μM. GraphPad Prism was used to plot

data, create Michaelis-Menten saturation curves, Lineweaver-Burk plots, and to calculate

kinetic parameters.

103

Substrate-specificity assay- A saturating concentration of 50 μM of substrate as

determined by the enzyme activity analyses, was used as the concentration for analyzing

various other steroid substrates and their activity. Like the enzyme activity analysis, 20β-

HSDH activity was measured spectrophotometrically by monitoring the biotransformation

of substrate by measuring the levels of NADH at 340 nm. The standard reaction mixtures

contained sodium acetate trihydrate buffer (pH 5.0 or pH 5.5), 150 μM of cofactor (NADH

or NAD+), 50 nM of enzyme, and 50 μM of substrate.

Circular dichroism spectroscopy- Circular dichroism (CD) spectroscopy was

performed using Jasco model J-815 circular dichroism spectropolarimeter (Jasco,

Japan). Purified recombinant 20β-HSDH enzyme samples were analyzed from 190 to

260 nm in 10 mM potassium phosphate buffer (pH 7.9) at a concentration of 0.2 mg/mL.

Crystallization- 20β-HSDH crystallization conditions were obtained using the

Protein Crystallography System from Art Robbins Instruments (PHOENIX). Purified

recombinant wild-type 20β-HSDH protein was concentrated between a range of 15-20

mg/ml. Utilizing the PHOENIX and dispensing in 96 - 3 well Intelli-Plates, sitting drops

were prepared comprising of 30 μL of well condition and 300 nL of protein mixed with 300

nL of well condition. Plates were placed in 4°C and crystals were grown in condition 69

of PEGRX HT containing 20% v/v 2-propanol, 0.1 M MES monohydrate pH 6.0, and 20%

w/v Polyethylene glycol monomethyl ether 2,000. The crystals continued to grow up to

day 7 and no further optimization was necessary. The crystals were overlaid with paraffin

oil to prevent evaporation and 20% ethylene glycol as a cryoprotectant. Crystals were

picked and immediately flash-cooled in liquid nitrogen. X-ray data was collected at the

Life Sciences Collaborative Access Team (LSCAT) of the Advanced Photon Source at

104

Argonne National Laboratory. For mutant S181A 20β-HSDH, purified recombinant

protein was incubated with 2.5 mM NADH for 2 hours. Crystals were grown in condition

86 of PEG/Ion containing 0.05 M Citric acid, 0.05 M BIS-TRIS propane / pH 5.0, and 16%

w/v Polyethylene glycol 3,350. The condition was then optimized in hanging-drop format

using 18-20% w/v Polyethylene glycol 3,350. S181A crystals were collected using the

same process outlined for the wild-type 20β-HSDH.

RESULTS

Bifidobacterium adolescentis expresses 20β-HSDH activity- Based on our

previous phylogenetic analysis (85), we predicted that B. adolescentis strain L2-32

expresses cortisol 20β-HSDH (Figure 4.1). In order to determine whether B. adolescentis

strain L2-32 expresses 20β-HSDH activity, we grew this strain in BHI in the presence of

50 μM cortisol, 20α-dihydrocortisol, or 20β-dihydrocortisol for 48 hours. After 48 hours of

growth, we observed total conversion of cortisol to 20β-dihydrocortisol by B. adolescentis

strain L2-32 (Figure 4.2). When 20β-dihydrocortisol was a substrate, we did not observe

formation of cortisol, suggesting that the enzyme reaction equilibrium greatly favors the

reductive direction under anaerobic conditions. 20α-dihydrocortisol is not a metabolite

for B. adolescentis strain L2-32 or B. desmolans ATCC 43058. As controls, B. desmolans

ATCC 43058 and C. scindens ATCC 35704 were shown to metabolize 20β-

dihydrocortisol and 20α-dihydrocortisol, respectively. 20β-dihydrocortisol is not a

metabolite for C. scindens ATCC 35704. Steroid-17,20-desmolase is present in C.

scindens ATCC 35704 and B. Desmolans 43058. Consistent with this, net conversion of

20α-dihydrocortisol to 11β-OHAD was observed in cultures of C. scindens ATCC 35704,

and net conversion of 20β-dihydrocortisol to 11β-OHAD was observed in cultures

105

Figure 4.1 Reaction mechanism of 20β-HSDH 20β-HSDH oxidizes the coenzyme NADH to transform cortisol into 20β-dihydrocortisol. Cortisol + NADH ⇌ 20β-dihydrocortisol + NAD+. This reaction reduces the double bonded oxygen at C-20 (highlighted in red) on cortisol and is fully reversible.

106

Figure 4.2 Whole cell assays of B. adolescentis strain L2-32, B. desmolans ATCC 43058, and C. scindens ATCC 35704 (A) B. adolescentis completely converts cortisol into 20β-dihydrocortisol. 20α-dihydrocortisol is not metabolized. In a highly reductive environment, B. adolescentis does not convert 20β-dihydrocortisol into cortisol. (B) B. desmolans completely converts cortisol into 11β-OHAD and 20β-dihydrocortisol into 11β-OHAD. 20α-dihydrocortisol is not metabolized. (C) C. scindens completely converts cortisol into 11β-OHAD and 20α-dihydrocortisol into 11β-OHAD. 20β-dihydrocortisol is not metabolized.

107

of B. desmolans ATCC 43058. We did not observe formation of 11β-OHAD by B.

adolescentis strain L2-32 which lacks desA and desB genes, recently reported to encode

steroid-17,20-desmolase activity (167).

Identification and biochemical characterization of recombinant 20β-HSDH- In

our previously reported phylogenetic analysis, we identified a putative 20β-HSDH gene

from B. adolescentis strain L2-32 annotated as an SDR family oxidoreductase

(WP_003810233.1) with amino acid identity of 59% with the characterized 20β-HSDH

from B. desmolans ATCC 43058 (WP_051643274.1) (85). To determine whether the

SDR family oxidoreductase from B. adolescentis encodes a 20β-HSDH, the gene

(WP_003810233.1) was cloned into the pET-28(+) expression vector for overexpression

in E. coli BL21-CodonPlus (DE3)-RIPL competent cells. The N-terminus 6xHis-tagged

recombinant protein was purified via TALON® metal affinity chromatography. The

purified protein was resolved as a single band using SDS-PAGE and three independent

gels show an average subunit molecular mass of 32 ± 0.12 kDa (Figure 4.3A), on par with

the theoretical calculated subunit molecular mass of 31.7 kDa.

The native molecular mass of recombinant 20β-HSDH protein was determined

using gel filtration chromatography and elute at 1.036 Ve/Vo which corresponds to a

molecular mass of 132 ± 3 kDa, eluting shortly after γ-globulin (158 kDa) (Figure

4.3B). The gel filtration data, incorporated with the subunit molecular mass determined

by SDS-PAGE, suggests that the 20β-HSDH forms a homotetrameric quaternary

structure. 20β-HSDH displayed a pH optimum in the reductive direction at a pH of 5.0

and in the oxidative direction at a pH of 5.5 (Figure 4.4A). The Michaelis-Menten

saturation curve for the primary reaction of cortisol reduction performed by 20β-HSDH is

108

Figure 4.3 Purified 20β-hydroxysteroid dehydrogenase (20β-HSDH) from Bifidobacterium adolescentis strain L2-32 (A) SDS-PAGE gel of crude and purified 20β-HSDH showing a subunit size of 32 ± 0.12 kDa. (B) Native molecular size analysis of purified 20β-HSDH via size-exclusion chromatography.

A. B.

109

Figure 4.4 pH optimum and Michaelis-Menten saturation curve of wild-type 20β-HSDH (A) pH optimization of 20β-HSDH in the reductive (blue) and oxidative (red) direction. (B) Saturation curve showing the reduction of cortisol by wild-type 20β-HSDH.

A. B.

110

shown in Figure 4.4B. The saturation curve was used to calculate the kinetic

constants for wild type and mutant 20β-HSDH proteins and are shown in Table 4.1. 20β-

HSDH shows a KM of 24.07 μM. Vmax, kcat, and kcat/KM values were deduced to be 21.08

µmol·min-1·mg-1, 668.3 min-1, and 27.765 μM-1·min-1, respectively. Mutating the predicted

catalytic serine 181 to alanine (S181A) completely abolished activity. Mutating a nearby

serine 183 to alanine (S183) decreased the catalytic efficiency by 44% when compared

to the wild type 20β-HSDH. The site-directed mutants did not affect the secondary

structures of the wild-type protein, as shown by circular dichroism (CD) spectroscopy

(Figure 4.5).

Next, we determined substrate specificity for the wild type 20β-HSDH. Cortisol

was determined to be the primary substrate utilized by 20β-HSDH (Table 4.2). The

enzyme requires NAD(H) as a cofactor, but did not recognize NADP(H). Substrates

identical to cortisol, but lacking an 11β-hydroxyl group showed ~34% loss of relative

activity whereas loss of 17-hydroxyl resulted in ~60% loss in relative activity. Reduction

of the 3-oxo-Δ4,5 to 3α-hydroxy-5β results in loss of ~46% activity suggesting the A/B ring

orientation (trans vs. planar) and C-3 oxygen are recognized by the enzyme. Consistent

with whole-cell experiments, the enzyme displayed negligible activity in the oxidative

direction with NAD+ as co-factor. Substrates with 20α-hydroxyl groups are not substrates.

These results are consistent with substrate-specificity analysis of the purified recombinant

20β-HSDH from B. desmolans ATCC 43058 (85).

Crystallization of 20β-HSDH from Bifidobacterium adolescentis strain L2-32-

Wild type 20β-HSDH and mutant S181A were crystallized using the PHOENIX, followed

111

Tabl

e 4.

1 Ki

netic

par

amet

ers

of c

ortis

ol re

duct

ion

by w

ild-ty

pe a

nd m

utan

t 20β

-HSD

H

Prot

ein

Vm

axK

Mk

cat

Rel

ative

kca

tk

cat/K

MR

elat

ive k

cat/K

M

Wild

-type

21.0

8 +

1.13

124

.07

+ 3.

797

668.

3 +

50.5

3510

027

.765

+ 3

.648

100

S181

A0

00

00

0

S183

A4.

691

+ 0.

097

9.48

5 +

2.59

814

8.7

+ 11

.244

2215

.677

+ 2

.06

56Al

l rea

ctio

ns w

ere

perfo

rmed

in 5

0 m

M s

odiu

m a

ceta

te tr

ihyd

rate

buf

fer (

pH 5

.0) a

nd w

ere

repl

icat

ed a

tleas

t thr

ee ti

mes

kca

t/Km

valu

es a

re in

µM

-1.m

in-1

Rel

ative

kca

t/Km

valu

es a

re s

peci

fied

as a

per

cent

age

of th

e w

iild-

type

Rel

ative

kca

t val

ues

are

spec

ified

as

perc

enta

ge o

f the

wild

-type

kca

t val

ues

are

in m

in-1

Km

valu

es a

re in

µM

Vm

ax v

alue

s ar

e in

µm

ol.m

in-1

.mg-1

112

Figure 4.5 Circular dichroism spectroscopy CD spectra of purified recombinant wild-type 20β-HSDH, and S181A and S183 mutants from Bifidobacterium adolescentis strain L2-32.

113

Tabl

e 4.

2 Su

bstra

te s

peci

ficity

of 2

0β-H

SDH

St

eroi

dTr

ivial

Nam

eC

oenz

yme

Rel

ative

Act

ivity

%

4-pr

egne

n-11

β, 1

7, 2

1-tri

ol-3

, 20-

dion

eC

OR

TISO

LNA

DH

100.

00 ±

0.7

7

4-pr

egne

n-17

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114

by larger scale hanging-drops (Figure 4.6). For the wild-type, crystals grew in condition

69 of PEGRX containing 20% v/v 2-propanol, 0.1 M MES monohydrate pH 6.0, and 20%

w/v Polyethylene glycol monomethyl ether 2,000. Wild-type 20β-HSDH crystals

appeared within 24-48 hours and were allowed to grow up to 7 days. Crystals were then

overlaid with paraffin oil to prevent evaporation and 20% ethylene glycol as a

cryoprotectant, picked, and immediately stored in liquid nitrogen.

Prior to crystallizing S181A, the pure recombinant S181A was incubated with 2.5

mM NADH for 2 hours. S181A crystals were initially grown in condition 86 of PEG/Ion

containing 0.05 M Citric acid, 0.05 M BIS-TRIS propane / pH 5.0, and 16% w/v

Polyethylene glycol 3,350. S181A was then optimized to grow in condition containing

0.05 M Citric acid, 0.05 M BIS-TRIS propane / pH 5.0, and 18-20% w/v Polyethylene

glycol 3,350. Thin plate crystals appeared within 48 hours and were allowed to grow up

to 14 days. Crystals were then overlaid with paraffin oil to prevent evaporation and 20%

ethylene glycol as a cryoprotectant, picked, and immediately stored in liquid nitrogen.

Wild-type 20β-HSDH and mutant S181A were diffracted at 2.1 and 2.5 Å resolution,

respectively.

DISCUSSION

B. adolescentis is among the first bacteria to colonize the gastrointestinal tract of

breast-fed infants; the species was first isolated from the stool of a healthy two-year-old

infant in 1996 (168, 169). B. adolescentis, is a gram-positive, non-motile, anaerobic

bacterium found among the normal human microbiota. The presence of B. adolescentis

has been associated with numerous health benefits, however, the mechanisms by which

this species and other bifidobacteria confer these benefits on the host are still yet to be

115

Figure 4.6 20β-HSDH crystals (A) Wild-type 20β-HSDH crystals. (B) S181A mutant 20β-HSDH crystals bound to NADH

A.

B.

116

fully understood. Bacterial 20β-hydroxysteroid dehydrogenase (20β-HSDH) is an NADH-

dependent oxidoreductase belonging to the short-chain dehydrogenases/reductases

(SDR) family isolated from B. adolescentis. 20β-HSDH catalyzes the reduction of the

carboxyl group at position 20 of the steroid cortisol, thereby producing 20β-

dihydrocortisol. 20β-HSDH plays an important role in steroid hormone and bile acid

metabolism in the gut.

There are currently 31 known Bifidobacterium spp. (69). Of those species, only a

select few strains exhibit this potential probiotic 20β-HSDH activity. Based on a non-

redundant protein search from a recently characterized 20β-HSDH (WP_051643274.1)

from B. desmolans ATCC 43058, a gene annotated to encode an SDR family

oxidoreductase (WP_003810233.1) with 59% homology to the 20β-HSDH gene from B.

desmolans ATCC 43058 was identified in B. adolescentis strain L2-32 (87). The 20β-

HSDH gene from B. adolescentis strain L2-32 was cloned, overexpressed, and purified

from E. coli BL21-CodonPlus (DE3)-RIPL competent cells. Using whole cell and

enzymatic assays, the microbe and purified protein were shown to have 20β-HSDH

activity identical to that of 20β-HSDH found in B. desmolans ATCC 43058.

We mutated the predicted catalytic serine 181 to alanine (S181A) and activity was

completely ablated. This allowed for crystallization studies that would bind NADH and

cortisol, but would not convert them to NAD+ and 20β-dihydrocortisol, respectively. We

also mutated serine 183 to alanine (S183A) which is one residues away from the catalytic

serine 181. This mutation decreased the turnover rate by 5x, but decreased the KM about

3x. Meaning that without serine 183, substrate is able to bind easier. It is predicted that

117

serine 183 is important in hydrogen binding. Overall, the catalytic efficiency of S183A is

only 64% as efficient as wild-type 20β-HSDH.

The purified recombinant 20β-HSDH and co-factor-bound mutant S181A from B.

adolescentis strain L2-32 were crystallized after screening 1,250 unique conditions. The

crystal structure of 20β-HSDH in apo and holo form will allow us to visualize secondary

structure changes when co-factor binds to the enzyme. This information brings us one

step closer to predicting the reaction mechanism by which 20β-HSDH proceeds and the

residues involved in the reaction. It will also show us the exact location NADH binds to

the enzyme. The next step is to crystalize the ternary complex of 20β-HSDH which

contains co-factor (NADH) and substrate (cortisol), elucidating the location cortisol binds

on the enzyme and what conformational changes are involved. HSDHs follow an ordered

bi-bi mechanism where the co-factor (NAD(H) or NADP(H)) binds to the enzyme first,

followed by the substrate. Once the reaction is completed, the product leaves first, and

the co-factor leaves last. It is predicted that 20β-HSDH follows the same ordered reaction

mechanism. Isothermal titration calorimetry will be performed to test this hypothesis.

There are many B. adolescentis strains, however, only a select few express 20β-

HSDH activity. When B. adolescentis strain L2-32 metabolizes cortisol utilizing 20β-

HSDH, it is predicted to compete with cortisol metabolism by steroid-17,20-desmolase

from C. scindens and 21-dehydroxylation from E. lenta. The reason is simple: there are

three alternative pathways for side chain metabolism of cortisol by gut microbes. If the

rate of 20β-dihydrocortisol formation is greater than the rate of steroid-17,20-desmolase

or 21-dehydroxylase, then these pathways are prevented because 20β-dihydrocortisol is

not a substrate for either of these enzymes and there is no way for them to utilize cortisol

118

while it is in that form. By understanding 20β-HSDH and how it interacts with other steroid

metabolizing pathways, we may be able to alter the composition of toxic steroid hormones

and bile acids.

SUMMARY OF RESEARCH

In this work, we utilized targeted gene synthesis to clone and heterologously

express genes from a gene-cluster (BN592_00768-BN592_00770) in Eggerthella

CAG:298, hypothesized to encode multiple HSDHs (CDD59473, CDD59474,

CDD59475). Characterization of the purified recombinant enzymes revealed that each

enzyme catalyzed a distinct oxidation/reduction of bile acid hydroxyl groups. CDD59473

catalyzed NADPH-dependent reduction of 3-oxoDCA yielding iso-DCA (3β-hydroxyl) as

the main reaction product, co-migrating on TLC and with mass spectrum consistent with

iso-DCA. The formation of 3β-hydroxyl decreases the amphipathic nature of bile acids

making them less effective detergents, and also less toxic to bacteria as well as host cells

(26), (128, 129). Currently, there are no structures reported for gut bacterial 3β-HSDH.

Our phylogenetic analysis identified multiple Lactobacillus spp. isolated from diverse

avian and mammalian GI tracts encoding putative 3β-HSDHs with sequence identities at

or below 71%. A similar targeted gene-synthesis approach would be useful in

determining the function and catalytic potential of these putative 3β-HSDHs.

Isomerization at C3 of primary bile acids may be important in precluding bile acid 7α-

dehydroxylation, a microbial biochemical pathway responsible for formation of DCA and

LCA, bile acids that are causally associated with cancers of the colon and liver (2).

In contrast to CDD59473, CDD59474 converted 3-oxoDCA to DCA and thus

BN592_00769 encodes 3α-HSDH. DCA is generated by the multi-step bile acid 7α-

119

dehydroxylation of CA by a few species of intestinal clostridia (2) and is found in fecal

water at ~50 µM (130) but can reach several hundred micromolar (25). CDD59473 and

CDD59474 work in concert to epimerize DCA to iso-DCA, potentially as a means of

detoxification in E. lenta. Interestingly, CDD59474 was clustered with 3α-HSDH from R.

gnavus (Rumgna_02133) but more distantly related to a 3α-HSDH characterized in E.

lenta DSM 2243T (Elen_0690) (26). E. lenta has been reported to express 7α-HSDH (76,

77), however, genes encoding 7α-HSDH in E. lenta have yet to be identified. CDD59474

was annotated as “7α-HSDH” due to sequence homology, yet expression and

characterization of this protein revealed specificity for 3α-hydroxyl groups providing a

cautionary tale and further reinforcing the need to functionally characterize genes

predicted to encode particular functions by annotation. To our knowledge, BN592_00770

is the only reported sequence so far demonstrated to encode 12α-HSDH (CDD59475) in

E. lenta, and sequence comparison with CDD59475 is expected to elucidate further gut

bacterial 12α-HSDHs.

Thus, our analysis demonstrates that E. lenta strains harbor distinct forms of 3α-

HSDH, 3β-HSDH and 12α-HSDH enzymes. Critical at the intersection between next-

generation sequencing data (metagenomics, metatranscriptomics) and metabolomics is

biochemistry, providing the ability to experimentally demonstrate that a particular amino

acid sequence catalyzes a particular biochemical reaction. A major task in gut

microbiology research today is linking nucleic and amino acid sequences in databases to

function, and determining how functions affect microbiome structure and host physiology.

A major question in the field of bile acid metabolism (132) that we are currently working

to address is why bacteria, such as E. lenta, encode numerous HSDHs and oxidize bile

120

acids in an anaerobic environment.

We also report that the desA and desB genes from C. scindens ATCC 35704

encode steroid-17,20-desmolase. Recombinant DesAB displayed substrate-specificity

consistent with native steroid-17,20-desmolase: the enzyme recognizes 20-keto-17,21-

dihydroxy steroids (67, 68). C. scindens ATCC 35704 expresses NAD(H)-dependent 20α-

HSDH (69), while B. desmolans, C. cadavaris, and P. lymphophilum express 20β-HSDH

(85) in addition to steroid-17,20-desmolase. Oxidation-reduction of the C-20 oxygen is

predicted to function as a regulatory “switch”, interconverting cortisol between DesAB

substrate and non-substrate forms.

The conversion of cortisol to 11β-hydroxyandrostenedione by C. scindens is an

important step in a multi-species gut microbial endocrine pathway that results in the

formation of 11β-hydroxyandrogens (69). E. lenta expresses 21-dehydroxylase (74), and

21-dehydroxylated metabolites are detected following incubation of cortisol in fecal

suspensions (63, 64). Due to the absolute requirement for the C21-hydroxyl group,

steroid-17,20-desmolase and cortisol 21-dehydroxylation by Eggerthella lenta are

competing reactions in the gut (74).

Extrapolations from radiometric studies in non-human primates (142) and data

from humans (143) suggest that 15% of the 15 mg of cortisol are detected in the gut.

Based on these studies, the concentration of cortisol metabolites in the ~0.4L human

colon (144) is ~15 µM. 11β-hydroxyandrostenedione is now viewed as a pro-androgen,

whose downstream 5α-, 17β-reduced metabolites, are now thought to play an important

role in activation of androgen receptor (33, 145). The field of microbial endocrinology has

emerged and recognizes the need to characterize androgen production by C. scindens

121

(38, 146–150). Many host immune cells express androgen-receptor (151, 152), and it is

possible that C. scindens affects immune function through generation of androgens.

Androgens directly affect bacterial growth and virulence (153, 154). Intriguingly, steroids

have been shown to influence C. difficile germination (3). 11β-hydroxy-androgens are

also inhibitors of the host enzyme 11β-HSD2 (80, 155), but in kidney and vascular smooth

muscle may be pro-hypertensive (155). Inhibition of 11β-HSD2 in colonocytes may be

protective against colorectal cancer (156). Prostate tissue contains 11β-

hydroxyandrogens despite inhibition of host enzymes involved in synthesizing androgens

(chemical castration) (33, 145). The role of 11β-OHAD formation by the gut microbiota in

androgen accumulation in the prostate is unknown; however, we discovered that bacteria

isolated from urine also express the bacterial steroid-17,20-desmolase pathway (85), and

the majority of cortisol excretion from the body is via the urine (142, 143). Of note, we

recently reported identification of the steroid-17,20-desmolase gene cluster in the

genomes of urinary bacterial isolates and confirmed steroid-17,20-desmolase activity in

an isolate of Propionimicrobium lymphophylum (85). Verification that desA and desB

genes encode steroid-17,20-desmolase is an important step towards the application of

nucleic acid-based approaches such as quantitative polymerase chain, and metagenomic

sequencing in determining whether correlations exist between levels/expression of

desAB and host phenotypes and disease states.

Finally, we characterized and crystallized purified recombinant 20β-HSDH from B.

adolescentis strain L2-32 in the apo and holo forms. This will allow for predicting of the

reaction mechanism by which 20β-HSDH proceeds and the residues involved in the

reaction. It will also show us the exact location NADH binds to the enzyme. The next

122

step is to crystalize the ternary complex of 20β-HSDH which contains co-factor (NADH)

and substrate (cortisol), elucidating the location cortisol binds on the enzyme and what

conformational changes are involved. As discussed, there are many B. adolescentis

strains, however, only a select few express 20β-HSDH activity. When B. adolescentis

strain L2-32 metabolizes cortisol utilizing 20β-HSDH, it is predicted to compete with

cortisol metabolism by steroid-17,20-desmolase from C. scindens and 21-

dehydroxylation from E. lenta. By understanding 20β-HSDH and how it interacts with

other steroid metabolizing pathways, we may be able to alter the composition of toxic

steroid hormones and bile acids (Figure 5.1).

123

Figure 4.7 Competing cortisol metabolizing reactions by B. adolescentis, E. lenta, B. desmolans, and C. scindens

124

FUTURE RESEARCH

This research has laid the groundwork for future studies aiming to understand the

enzymes and pathways involved in steroid metabolism by gut microbes. The majority of

steroid biotransforming enzymes that gut microbes employ have yet to be explored. The

characterization of the steroid-17,20-desmolase and various other important HSDHs is

only one small piece of the puzzle. Microbes within the gut form complex interactions,

resulting in a myriad of steroid metabolites in different proportions and concentrations

which affect the health of the host. A major task in gut microbiology research today is

linking nucleic and amino acid sequences in databases to functions, and determining how

functions affect microbiome structure and host physiology. By experimentally

demonstrating that a particular amino acid sequence catalyzes a particular biochemical

reaction we get one step closer to predicting the biochemical potential of the gut

microbiome.

Future short-term studies will aim to further characterize novel steroid metabolizing

enzymes. We will crystallize steroid-17,20-desmolase from C. scindens ATCC 35704

and work to further elucidate the structure and catalytic mechanism of steroid

transketolation. Currently there are no crystal structures for 3β-, and 12α- Hydroxysteroid

dehydrogenases. By identifying the 3α-, 3β-, and 12α-hydroxysteroid dehydrogenases

from Eggerthella CAG:298 we can begin to crystallize these enzymes to gain structural

insight into the stereo- and regio-specificity of HSDHs. The structure of 20β-HSDH from

B. adolescentis strain L2-32 will be determined using the diffraction data collected from

the apo and holo enzyme crystals obtained in these studies. The 20β-HSDH from B.

adolescentis strain L2-32 will also be crystallized in the ternary form to determine binding

125

locations of substrate to analyze important conformational changes. We will perform

isothermal titration calorimetry to determine the binding order and affinity of ligands for

the serine active site mutant S181A. Our existing collaboration with the Theoretical and

Computational Biophysics group at the Beckman institute on campus will facilitate the

development of catalytic models based on the crystal structure and molecular dynamics.

A major long-term goal in the field of microbial endocrinology is to be able to

engineer microbiotas to generate steroid end products associated with health to reduce

the concentration of metabolites deemed to harmful to the hosts. The application of germ-

free animals and defined microbial communities will be utilized to determine how this will

be accomplished.

126

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