steroids represent a wide distributed and very diverse group of

1

Molybdoenzyme that catalyzes the anaerobic hydroxylation of a tertiary

carbon atom in the side chain of cholesterol Juri Dermer and Georg Fuchs

From Lehrstuhl Mikrobiologie, Fakultät Biologie, Universität Freiburg,

Schänzlestr. 1, D-79104 Freiburg, Germany

Running title: Steroid C25 dehydrogenase

Address correspondence to: Georg Fuchs, Mailing address: Mikrobiologie, Fakultät Biologie,

Schänzlestr. 1, D-79104 Freiburg, Germany; Phone: 49-761-2032608; Fax: 49-761-2032626; E-mail:

[email protected]

Keyword: molybdenum hydroxylase; anaerobic steroid metabolism

Background: Cholesterol degradation is

challenging due to its complex structure and low

water solubility.

Results: C25 dehydrogenase is a novel

molybdenum/iron-sulfur/heme-containing

enzyme that hydroxylates the tertiary C25 of the

steroid side chain.

Conclusion: C25 dehydrogenase and related

enzymes identified in the genome of

Sterolibacterium denitrificans replace

oxygenases in anaerobic and even aerobic steroid

metabolism.

Significance: O2-independent hydroxylations by

molybdoenzymes probably represent a general

strategy to activate steroid substrates

anaerobically.

SUMMARY

Cholesterol is a ubiquitous hydrocarbon

compound that can serve as substrate for

microbial growth. This steroid and related

cyclic compounds are recalcitrant owing to

their low solubility in water, complex ring

structure, the presence of quaternary carbon

atoms, and the low number of functional

groups. Aerobic metabolism therefore makes

use of reactive molecular oxygen as co-

substrate of oxygenases to hydroxylate and

cleave the sterane ring system. Consequently,

anaerobic metabolism must substitute oxyge-

nase catalyzed steps by O2-independent

hydroxylases. Here we show that one of the

initial reactions of anaerobic cholesterol

metabolism in the betaproteobacterium

Sterolibacterium denitrificans is catalyzed by

an unprecedented enzyme that hydroxylates

the tertiary C25 atom of the side chain

without molecular oxygen forming a tertiary

alcohol. This steroid C25 dehydrogenase

belongs to the dimethylsulfoxide dehydro-

genase molybdoenzyme family, the closest

relative being ethylbenzene dehydrogenase. It

is a heterotrimer, which is probably located at

the periplasmic side of the membrane and

contains 1 molybdenum cofactor, 5 [Fe-S]-

clusters, and 1 heme b. The draft genome of

the organism contains several genes coding

for related enzymes that likely replace

oxygenases in steroid metabolism.

INTRODUCTION

Cholesterol is one of the most abundant

and ubiquitous steroids. It serves as an essential

constituent of eukaryotic membranes and acts as

a precursor molecule for the biosynthesis of

steroid hormones, oxysterols, and bile acids.

Steroids are also formed by plants (1) and some

prokaryotes (2), but their degradation is mostly

limited to microorganisms. Microbial degrada-

tion of the omnipresent steroids like cholesterol

and the analogous plant and fungal steroids

(stigmasterol, β-sitosterol, ergosterol) is an

important issue of the global carbon cycle.

Furthermore, microbial transformation of steroid

molecules is an essential part of the biotechno-

logical production of steroid drugs (3).

Degradation of cholesterol is challenging

because of its low solubility in water (95 µg l-1

,

0.25 µM) and particularly because of its complex

chemical structure harboring quaternary carbon

atoms and a low number of functional groups.

Owing to this recalcitrance, cholesterol and

related steroids are used as biological markers in

studying the biological origin and fate of organic

compounds in geological records (4). Never-

theless, the ability to grow on cholesterol as a

sole carbon source is widespread in the microbial

world. Nocardia, Mycobacterium, Pseudomonas,

Arthrobacter, and Rhodococcus species are well

known cholesterol degrading aerobes (5), some

of those bacteria being used in biotechnology

(3). Under aerobic conditions, mono- and dioxy-

http://www.jbc.org/cgi/doi/10.1074/jbc.M112.407304The latest version is at JBC Papers in Press. Published on September 1, 2012 as Manuscript M112.407304

Copyright 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

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genases are involved in cholesterol metabolism.

They catalyze crucial hydroxylation steps that

initiate degradation of the side chain and bring

about the cleavage of the sterane ring (5).

Obviously, the anoxic metabolism needs

to substitute all essential O2-dependent steps by

an oxygen-independent strategy to overcome the

chemical recalcitrance of the molecule. This

challenge prompted us to study the anoxic

metabolism of cholesterol in the β-proteo-

bacterium Sterolibacterium denitrificans (6), one

of two strains that are known to grow an-

aerobically (but also aerobically) on cholesterol

as sole carbon source (7). In a previous work, the

initial steps of this anoxic metabolism were

identified (8-10) (Fig. 1). Cholesterol is

converted to cholest-4-en-3-one by a

bifunctional cholesterol dehydroge-

nase/isomerase. Cholest-4-en-3-one is further

oxidized to cholesta-1,4-dien-3-one catalyzed by

cholest-4-en-3-one-∆1-dehydrogenase. Both

transformations show analogy to the aerobic

cholesterol metabolism. Recently, similar

reactions were identified in anaerobic degra-

dation of testosterone in Steroidobacter

denitrificans (11).

Intriguingly, the following degradation

of cholesterol involves an unprecedented

anaerobic hydroxylation of the tertiary C25 atom

of the side chain resulting in the formation of a

tertiary alcohol. This reaction fundamentally

differs from the O2-dependent hydroxylations

that occur in aerobic steroid metabolism. It is

well known that in anaerobic pathways

molybdenum-containing hydroxylases can be

regarded as a counterpart to oxygenases

functioning in aerobic metabolism. They use

water as source of the oxygen atom incorporated

into the product and require an electron acceptor;

in contrast, oxygenases use molecular oxygen as

source of oxygen and many require an electron

donor.

A paradigm for such an anaerobic

hydroxylase acting on a hydrocarbon side chain

is ethylbenzene dehydrogenase (12), and the

analogous cholesterol C25 hydroxylation was

proposed to be catalyzed by a similar

molybdenum enzyme (10). Ethylbenzene

dehydrogenase belongs to type II molybdenum

containing enzymes of the dimethylsulfoxide

(DMSO) reductase family and catalyzes the

hydroxylation of ethylbenzene to (S)-phenyl-

ethanol.

Here we purified and characterized

steroid C25 dehydrogenase (briefly C25 de-

hydrogenase). The molybdenum containing en-

zyme is membrane associated and genes coding

for the subunits of the heterotrimeric enzyme

were identified in the draft genome of the

organism. Furthermore, analysis of the genome

revealed the presence of at least seven further

proteins with high similarity to the molybdenum-

containing large subunit of ethylbenzene de-

hydrogenase and C25 dehydrogenase (hydroxyl-

lase). In contrast, no gene coding for an

oxygenase acting on steroids was found in the

genome. It appears indeed that several

molybdenum dependent anaerobic hydroxylases

take over the role of oxygenases in anaerobic

metabolism of steroids. In this organism, the

anaerobic strategy is even used for the aerobic

metabolism of steroids.

EXPERIMENTAL PROCEDURES

Materials and Bacterial Strain - The

chemicals used were of analytical grade and

were purchased from Sigma-Aldrich

(Heidelberg, Germany), Merck (Darmstadt,

Germany), Roth (Karlsruhe, Germany), or Santa

Cruz Biotechnology (Heidelberg, Germany).

Sterolibacterium denitrificans Chol-1ST (DSMZ

13999) was obtained from the Deutsche

Sammlung für Mikroorganismen und

Zellkulturen (Braunschweig, Germany).

Materials and equipment for protein purification

were obtained from Sigma-Aldrich (Heidelberg,

Germany) and GE Healthcare (München,

Germany).

Bacterial Cultures and Growth

Condition - S. denitrificans was grown on

cholesterol at 30 °C under oxic as well as under

anoxic, denitrifying conditions as described

(6,9). Cells were harvested by centrifugation in

the exponential growth phase at an optical

density (OD578 nm) of 1.0 to 1.6 (optical path 1

cm) and then stored at -70 °C. Large scale

fermenter cultures (200 liters) were set up as

described previously (10).

Preparation of Cell Extracts - Cell

extracts were prepared at 4 °C under anoxic

conditions. Frozen cells were suspended in two

volumes of 20 mM Tris/H3PO4 buffer (pH 7.0)

containing 0.1 mg of DNase I ml-1

. Cells were

broken by passing the cell suspension through a

French pressure cell (American Instruments,

Silver Spring, MD) twice at 137 MPa. The cell

lysate was fractionated by two steps of

centrifugation: at first, lysate was centrifuged for

30 min at 10,000 x g to get rid of the debris,

unbroken cells, and residual undissolved

cholesterol. Then, the supernatant (crude cell


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extract) was centrifuged at 150,000 x g for 2 h to

separate soluble proteins from membrane-bound

proteins. Extracts of aerobically grown cells

were prepared under oxic conditions. Separation of Subcellular Compart-

ments - Steroid C25 dehydrogenase localization

was studied under anoxic conditions. 50 ml of an

exponentionally growing culture (OD578 1) were

harvested by centrifugation at 2,800 x g for 15

min. To lyse the cells gently, the cell pellet was

resuspended in 10 ml buffer containing 20 mM

Tris/H3PO4 (pH 7.0), 1 M KCl, 10 mg lysozyme

ml-1

(100,000 Units mg-1

), 5 mg polymyxin B ml-

1, 1 mM dithioerythritol (DTE), and 0.1 mg

DNase I. The cell slurry was incubated on ice for

two hours and then centrifuged at 150.000 x g

for two hours. Supernatant containing the soluble

cell proteins was separated from the pellet

containing the membrane proteins. The pellet

was then resuspended in buffer containing 20

mM Tris/H3PO4 (pH 7.0) and 1 % Tween 20

(w/v). Both fractions were assayed for C25

dehydrogenase, cholest-4-en-3-one-∆1-

dehydrogenase, and malate dehydrogenase

activities.

To separate cytoplasmic membrane from

outer membrane, a sucrose gradient

centrifugation was carried out. 40 ml of an

exponentionally growing culture (OD578 1.4)

were harvested by centrifugation at 2,800 x g for

15 min. Cells were resuspended in two volumes

of buffer (20 mM Tris/H3PO4 (pH 7.0), 0.1 mg

DNase I ml-1

), and disrupted by passing through

the French press cell. The cell extract was

centrifuged at 10,000 x g for 20 min. The

supernatant (1.5 ml) was layered on top of a

sucrose bed, consisting of 0.6 M (8 ml), 0.9 M (8

ml), and 1.75 M (8 ml) sucrose in 20 mM

Tris/H3PO4 buffer (pH 7.0), and centrifuged at

38,000 x rpm (100,000 x g) for 16 h (rotor 60 Ti,

Beckman). Three fractions were carefully

collected by removal of material from top of the

gradient: (i) the soluble protein fraction (0.6 M

sucrose), (ii) the fraction containing the

cytoplasmic membrane (distinct layer between

0.6 M and 0.9 M sucrose) and (iii) the outer

membrane and unbroken cells (1.75 M sucrose).

The collected fractions were assayed for C25

dehydrogenase and malate dehydrogenase

activities.

In Vitro Assays - An HPLC-based C25

dehydrogenase assay was routinely performed

anaerobically under a nitrogen gas phase at 30

°C. The assay mixture (0.3 ml) contained 20 %

(w/v) (2-hydroxypropyl)-β-cyclodextrin, 100

mM potassium phosphate buffer (pH 7.5), 0.5

mM cholest-4-en-3-one (from 52 mM stock

dissolved in 1,4-dioxane), and 5 mM

K3[Fe(CN)6]. The reaction was started by

addition of enzyme, and the mixture was shaken

at 700 rpm. Samples of 80 µl were taken at

intervals, and the reaction was stopped by

addition of 20 µl of 25 % HCl. Samples were

centrifuged for 10 min at 20,000 x g, and 80 µl

were analyzed for substrate and products by

reverse-phase (RP) HPLC and UV detection at

240 nm . Note, that the calculated hydroxylation

rate based on UV detection of the product at 240

nm can be affected by further reactions of the

product in cell extract, which might result in

disappearance of the conjugated double bond

system and therefore in absorption decrease at

240 nm. When detergent was used instead of

cyclodextrin, the reaction mixture (0.3 ml)

contained 0.1 M potassium phosphate buffer (pH

7.0), 10 mM K3[Fe(CN)6], 2.6 mM cholest-4-en-

3-one, and 0.5 % detergent (e.g. Tween 20,

Triton X-100). The reaction was started by

addition of enzyme, and the mixture was shaken

at 700 rpm. The reaction was stopped by

extraction with two volumes of ethyl acetate.

The ethyl acetate soluble fraction was

concentrated under vacuum by vacuum

concentrator (Bachofer, Reutlingen, Germany)

and analysed by HPLC. For assaying C25

dehydrogenase in whole cells, 2 M sucrose was

added to the assay mixture to avoid cell lysis.

Alternatively, purified C25

dehydrogenase was assayed spectro-

photometrically with ferricenium tetrafluoro-

borate (FcBF4) as electron acceptor. The assay

mixture (0.5 ml) contained 28 % (w/v)

cyclodextrin, 100 mM potassium phosphate

buffer (pH 7.5), 0.5 mM cholest-4-en-3-one, and

0.5 mM FcBF4. The reaction was started by

addition of enzyme, and the decrease of

absorption caused by reduction of the

ferricenium ion was followed at 300 nm (∆ε =

3,587 M-1

cm-1

). To couple substrate

hydroxylation to cytochrome c reduction,

oxidized bovine heart cytochrome c was used in

a 40-fold molar excess to C25 dehydrogenase,

instead of ferricenium tetrafluoroborate. The

reaction was started by the addition of 0.5 mM

cholest-4-en-3-one to the reaction mixture and

the reduction of cytochrome c was followed at

550 nm ( ∆ε = 20,000 M-1

cm-1

) (13).

Malate dehydrogenase activity was measured at

30 °C in a reaction mixture (0.5 ml) containing

100 mM potassium phosphate buffer (pH 7.5),

0.25 mM NADH, and 0.2 mM oxaloacetate.

Oxidation of NADH was followed


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spectrophotometrically at 365 nm (∆ε = 3,400 M-

1 cm

-1) with a filter spectrophotometer.

Cholest-4-en-3-one-∆1-dehydrogenase was

assayed at 30 °C in 0.1 M potassium phosphate

buffer (pH 7.5) containing 5 mM 2,6-

dichlorophenol-indophenol, 0.5 mM cholest-4-

en-3-one, and 20 % (w/v) cyclodextrin. The

reaction was started by addition of enzyme

solution and the mixture (0.3 ml) was shaken at

700 rpm aerobically. Samples of 80 µl were

taken at intervals and the reaction was stopped

by addition of 20 µl of 25 % HCl. Samples were

centrifuged for 10 min at 20,000 x g, and 80 µl

were analyzed by reverse-phase HPLC.

HPLC Analysis - An analytical RP-C18

column (Luna 18(2), 5 µm, 150 by 4.6 mm;

Phenomenex, Aschaffenburg, Germany) was

used at a flow rate of 0.6 ml min-1

on a Waters

600 HPLC-system. The mobile phase comprised

a mixture of two solvents: A, 30 % (v/v)

acetonitrile; B, 80 % (v/v) 2-propanol. The

separation was performed with a linear gradient

of solvent B from 15 to 80 % within 20 min and

additionally from 80 to 95 % within 10 min. The

detection of UV absorbance was performed

routinely at 240 nm with a Waters 996

photodiode array detector.

Solubilization and Purification of the

Enzyme - All steps for solubilization and column

chromatography were performed in an anaerobic

glove box with an FPLC System (P500 pump

system, Amersham Pharmacia Biotech) at 8 °C.

The purification of C25 dehydrogenase started

with solubilization of the membrane-bound

protein fraction. The crude extract routinely

obtained from 40 g cells (wet mass) was

centrifuged at 150,000 x g, and the membrane-

bound protein fraction in the pellet was

resuspended in two volumes of buffer containing

20 mM Tris/H3PO4 (pH 7.0). The mixture was

stirred gently, and Tween 20 (70 % w/v in H2O)

was slowly added to a final concentration of 2

mg detergent (mg membrane protein)-1

. Then

glycerol was added to a final concentration of 10

% (w/v), and the solution was gently stirred for 2

h at 8 °C. After centrifugation at 150,000 x g for

2 h, the supernatant was applied onto a DEAE-

Sepharose column.

DEAE - Sepharose fast flow column (70 ml

volume) was equilibrated with 10 column

volumes of buffer A (20 mM Tris/H3PO4 (pH

7.0), 1 mM dithiothreitol (DTT), and 0.02 %

Tween 20). The solubilized membrane protein

fraction (672 mg protein) was applied onto the

DEAE column at a flow rate of 2 ml min-1

, and

the column was washed with 5 volumes of buffer

A, then with 5 volumes of buffer B (20 mM 2-

(N-morpholino)ethanesulfonic acid (MES)/Tris

(pH 6.0), 1 mM DTT, and 0.02 % Tween 20),

and additionally with 7 volumes of buffer B

containing 50 mM KCl. The active protein pool

was eluted with 1-2 volumes of buffer B

containing 100 mM KCl. Resource Q (Amersham Pharmacia Biotech)

column (6 ml volume) was equilibrated with 10

column volumes of buffer A. The active protein

pool after chromatography on DEAE-Sepharose

column (176 mg protein) was diluted with an

equal volume of buffer A and applied onto the

Resource Q column at a flow rate of 2 ml min-1

.

The column was washed with 3 volumes of

buffer A, then with 2 volumes of buffer B, and

additionally with 5 volumes of buffer B

containing 50 mM KCl. The active protein pool

was then eluted with 6-7 volumes of buffer B

containing 75 mM KCl.

Reactive Green 19 - Agarose (Sigma) column

(30 ml volume) was equilibrated with 10 column

volumes of buffer B. The active protein pool

after chromatography on the Resource Q column

(32 mg protein) was concentrated fourfold using

centrifugal devices (Pall, MicrosepTM

, 30 kDa),

diluted with a threefold volume of buffer B, and

concentrated fourfold again to reduce the salt

concentration. Then the concentrated protein

pool was diluted tenfold with buffer B, and

applied onto the column that was run at a flow

rate of 1 ml min-1

. The column was washed with

3 volumes of buffer B, then with 3 volumes of

buffer A, and the active protein pool was eluted

with 8-10 volumes of buffer A containing 2 mM

cholic acid.

Reactive Red 120 - Agarose (Sigma) column (10

ml volume) was equilibrated with 10 column

volumes of buffer B. The active protein pool

after chromatography on Reactive Green 19

column was concentrated fourfold by centrifugal

devices (Pall, MicrosepTM

, 30 kDa), diluted with

a ninefold volume of buffer B to reduce the

concentration of cholic acid, and applied onto the

column at a flow rate of 1 ml min-1

. The column

was washed with 2 volumes of buffer B, and the

enzyme was eluted with 1 volumes of 50 % of

buffer A. The fraction was concentrated by

centrifugal devices (Pall, MicrosepTM

, 30 kDa).

Preparation of cholest-4-en-3-one-25-ol

- C25 dehydrogenase assays (0.5 ml) were

performed for the synthesis of cholest-4-en-3-

one-25-ol. The reaction mixtures were incubated

for 14 h and were then extracted with three

volumes of ethyl acetate. The ethyl acetate

fractions were evaporated, the pellet was


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resuspended in 0.2 ml of 2-propanol, and applied

to a HPLC column. The peaks containing

product were collected, and the solution was

lyophilized. The obtained cholest-4-en-3-one-25-

ol was dissolved in 0.3 ml 1,4-dioxane, and the

concentration was determined by using a

calibration curve prepared with cholest-4-en-3-

one.

Characterization of the Enzyme -

Electron acceptor specificity of the C25

dehydrogenase was tested by using the following

electron acceptors: K3[Fe(CN)6], ferricenium

tetrafluoroborate, ferricenium hexafluoro-

phosphate, NAD+, NADP

+, phenazine metho-

sulphate, 2,6-dichlorophenol-indophenol,

methylene blue and duroquinone. HPLC-based

assays were set up with 5 mM each of the

electron acceptor. To assess the pH optimum,

C25 dehydrogenase was assayed in 100 mM

potassium phosphate buffer within a pH range of

6.0 – 8.0. Reversibility of the C25 hydroxylation

reaction was tested under anaerobic conditions in

100 mM potassium phosphate buffer (pH 7.5),

containing 10 mM methyl viologen, 5 mM

dithionite and 0.1 mM cholest-4-en-3-one-25-ol.

After adding the enzyme (4.5 µg), the reaction

mixture was incubated for 16 h and then

analyzed by HPLC. UV-visible spectra were

recorded with a Cary spectrophotometer (Cary

100 Bio, Varian) by using a gas-tight stoppered

quartz cuvette under anaerobic conditions. The

native molecular mass of C25 dehydrogenase

was determined by gel filtration on a Superdex

200 column (24 ml volume, Amersham

Pharmacia Biotech) at a flow rate 0.4 ml min-1

.

The column was equilibrated with buffer

containing 20 mM Tris/H3PO4 (pH 7.0), 1 mM

DTT, 500 mM KCl, and 0,02 % Tween 20. For

calibration thyroglobulin (669 kDa), ferritin (440

kDa), aldolase (158 kDa), bovine serum albumin

(69 kDa), chymotrypsinogen A (25 kDa), and

RNaseI (13.7 kDa) were used. N-terminal

labelling of native C25 dehydrogenase was

carried out by derivatization with ((N-

succinimidyloxycarbonylmethyl)tris(2,4,6-

trimethoxyphenyl) phosphonium bromide)

(TMPP-Ac-OSu) according to (14). MASCOT

MS/MS data searches were performed by using

the protein database derived from the genome

sequences of S. denitrificans (Dermer & Fuchs,

unpublished data).

Computational Analysis - The BLASTP

searches were performed via the NCBI BLAST

server (www.ncbi.nlm.nih.gov) (15,16). The

search was performed in September 2011. The

amino acid sequences of subunits of C25

dehydrogenase were used as queries for

BLASTP searches against assembled bacterial

genomes. Gene prediction and annotation of the

draft genome was carried out using the RAST

server (17) and Geneious package v5.4 for

manual correction (18). For construction of a

phylogenetic tree, the amino acid sequences

were aligned using ClustalW implemented

within MEGA4 (19). The multiple alignments

were performed using ClustalW implicated into

BioEdit package (version 7.0.9.0).

Other methods - Protein concentrations

were determined with a BCA protein

quantification kit (VWR, Darmstadt) with bovine

serum albumin as standard. Discontinuous SDS-

PAGE was performed in 12 % (w/v) polyacryl-

amide gels according to standard procedures

(20). Blue native PAGE was performed in 8 %

(w/v) polyacrylamide gels according to modified

standard procedures (21). Cathode buffer

contained 25 mM Tris/glycine (pH 8.4) and 0.02

% or 0.002 %, respectively, of Coomassie G250.

Anode buffer contained 25 mM Tris/glycine (pH

8.4). Sample buffer contained 0.25 M Tris/Cl

(pH 6.8), 20 % glycerol and traces of

bromophenol blue. The gel was run at 8 °C and

10 mA. Gels were stained by Coomassie

Brilliant Blue R-250. Image Lab software (Bio-

Rad version 2.0 build 8) was used for analysis of

subunits composition based on their relative

abundance in SDS-gel. MS analysis of excised

gel bands was carried out as described (22).

Photometric determinations of iron and inorganic

sulphide were performed by standard chemical

techniques (23). Additionally, a simultaneous

determination of 32 elements in the purified

enzyme was performed by inductively coupled

plasma optical emission spectroscopy (ICP-OES)

using a Jarrel Ash Plasma Comp 750 instrument

at the center of Complex Carbohydrate Research,

University of Georgia, USA. The identification

of the nucleotide moiety of the molybdenum

cofactor was performed by using a Lichrospher

100 RP-18 E column (5 μm particle size, 4 × 125

mm) as described (24).

Sequences - The sequence data of new

identified molybdoenzymes reported in this work

have been submitted to GenBank and have been

assigned the accession numbers: JQ292991

(S25dA); JQ292992 (S25dB); JQ292993

(S25dC); JQ292994 (S25dA2); JQ292995

(S25dA3); JQ292996 (S25dA4); JQ292997

(S25dB4); JQ292998 (S25dC4); JQ292999

(S25dD4); JQ293000 (S25dA5); JQ293001

(S25dB5); JQ293002 (S25dC5); JQ293003

(S25dA6); JQ293004 (S25dB6); JQ293005


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(S25dC6); JQ293006 (S25dA7); JQ293007

(S25dB7); JQ293008 (S25dC7); JQ293009

(EbdA-like); JQ293010 (EbdB-like); JQ293011

(EbdC-like); JQ293012 (EbdD-like).

RESULTS

C25 dehydrogenase (hydroxylase)

activity in Sterolibacterium denitrificans - An

HPLC-based assay with cholest-4-en-3-one as

substrate and potassium hexacyanoferrate (III) as

artificial electron acceptor was developed for

activity measurements in cell extracts and during

purification of the enzyme. Transformation of

the substrate to products was monitored routinely

at 240 nm (SI Fig. 1). With cell extract, 25-

hydroxy-cholest-4-en-3-one was formed as

product, but in addition cholesta-1,4-dien-3-one

and 25-hydroxy-cholesta-1,4-dien-3-one were

formed (SI Fig. 1B), which is due to the presence

of the initial enzymes of the pathway (see Fig.

1). These initial enzymes act also on compounds

I and II (Fig. 1). In contrast, purified C25

dehydrogenase produced only 25-hydroxy-

cholest-4-en-3-one (SI Fig. 1C, see below).

Coupling of substrate oxidation to the reduction

of the artificial electron acceptors 2,6-

dichlorophenol-indophenol or phenazine

methosulfate in a spectrophotometric test could

not be used with cell extracts due to unspecific

reactions occurring in extracts. A photometrical

assay with ferricenium cation as artificial

electron acceptor, however, was developed to

measure the activity of purified C25

dehydrogenase (see below). Activity was linearly

dependent on the amount of added protein in the

range 0 – 0.11 mg protein ml-1

and required the

presence of detergents like Triton X100, Tween

20, dodecyl β-D-maltoside, or 2-hydroxypropyl-

β-cyclodextrin (briefly cyclodextrin). Routinely,

20 % (w/v) cyclodextrin was added to dissolve

up to 0.7 mM of steroid substrates, and addition

of detergent or cyclodextrin resulted in a

thousand-fold activity increase. The pH optimum

of the reaction was between 7.0 and 7.5. Nearly

identical activities were obtained under oxic and

strictly anoxic conditions, indicating that

molecular oxygen is not required for the

hydroxylation of the side chain. The specific

hydroxylation rate in cell extract was 2 nmol

min-1

(mU) (mg protein)-1

. This value is close to

the calculated cholesterol degradation rate of 3

mU (mg protein)-1

, when cells were growing

under denitrifying conditions with a doubling

time of 44 h. Similar activities were measured in

cells that were grown on cholesterol under oxic

or anoxic (denitrifying) conditions. This

indicates that S. denitrificans may use the same

anaerobic degradation strategy both for

anaerobic and aerobic growth on cholesterol.

This conclusion is corroborated by genome

sequencing (see below).

Subcellular localization - When freshly

harvested cells were treated with polymyxin B

and lysozyme in hypotonic medium to gently

lyse the cells, formation of cell ghosts was

observed by microscopic examination. After

centrifugation at 150,000 x g, 60 % of C25

dehydrogenase activity and 83 % of cholest-4-

en-3-one-∆1-dehydrogenase activity were found

in the membrane fraction, whereas 90 % of

malate dehydrogenase activity was recovered in

the soluble protein fraction. Furthermore, after

centrifugation of cell extract on a sucrose

gradient, C25 dehydrogenase activity was

completely recovered in the cytoplasmic

membrane fraction, whereas 85 % of the

cytoplasmic marker protein malate

dehydrogenase was recovered in the soluble

protein fraction. To address, whether the electron

acceptor site of the enzyme faces the periplasmic

or cytoplasmic side, whole cell assays were

carried out. Note that the cytoplasmic membrane

is not permeable for charged ions like the

electron acceptors potassium hexacyanoferrate or

NAD+. Nevertheless, similar C25 dehydrogenase

activities were measured with whole cells and

with cell extract, whereas only residual activity

of the cytoplasmic marker enzyme malate

dehydrogenase (NAD+ dependent) was observed

when tested with whole cells (Fig. 2). Addition

of high concentrations (1 M) of the non-

chaotropic salt NaCl to the membrane fraction

did not solubilize the enzyme, whereas after

treatment with Tween 20, the C25 hydroxylation

activity was observed solely in the solubilized

protein fraction. These observations indicate that

C25 dehydrogenase is preferentially associated

with the cytoplasmic membrane with the electron

accepting site facing the periplasmic space.

Purification - The membrane-bound

protein fraction was used for purification

yielding a brownish enzyme preparation.

Routinely, the soluble protein fraction after

Tween 20 treatment was not used further; it

contained approximately 40 % of total C25

dehydrogenase activity and can be used for

enzyme purification following the same scheme.

The solubilization of membrane-bound protein

was performed with the weak detergent Tween

20, which is commonly used for solubilization of

peripheral membrane proteins. After


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solubilization, about 95 % of the activity was

obtained in the solubilized protein fraction,

whereas only residual activity could still be

observed in the membrane fraction. A nearly

homogeneous C25 dehydrogenase fraction

containing three subunits was obtained after four

chromatographic steps (Fig. 3A, SI Table 1).

Two chromatographic steps were carried out on

DEAE and Resource Q anion exchange columns,

followed by two affinity chromatographic steps

using Reactive Green 19 and Reactive Red 120.

C25 dehydrogenase was eluted from Reactive

Green 19 by the substrate analogue cholic acid,

whereas a pH-shift was used for its elution from

Reactive Red 120. Although the enzyme activity

was not affected by oxygen in cell extracts, after

the first chromatographic DEAE step the

enriched fraction quickly lost activity in air.

Therefore purification had to be carried out

under anoxic conditions.

Molecular properties - Steroid C25

dehydrogenase consists of three subunits of

approximately 108 kDa, 38 kDa, and 27 kDa, as

revealed by SDS-PAGE (Fig. 3A). The subunits

were present in an approximate 1:1:1 molar ratio.

The molecular mass of the native enzyme was

determined by gel filtration as 168 ± 12 kDa

indicating a αβγ-composition. Based on peptide

mass fingerprint analysis of gel bands the

corresponding genes were identified in a draft

genome of the organism (Dermer & Fuchs,

unpublished data) (Fig. 3B). The gene for the

alpha subunit was a single ORF related to the

large subunit of ethylbenzene dehydrogenase,

which contained a 5' sequence coding for an N-

terminal twin-arginine translocation (Tat) leader

peptide. In contrast, the genes for the beta and

gamma subunits were located 396 kbp away in a

cluster of three genes coding for α, β, and γ

subunits of another molybdenum-containing

enzyme also related to ethylbenzene

dehydrogenase. Apparently, the beta and gamma

subunits are shared by these two similar

molybdenum enzymes, one being C25

dehydrogenase, the function of the other being

unknown. The phenomenon of subunit sharing

may be common in this metabolic pathway (see

Discussion). The calculated sizes of the subunits

were 108 kDa (alpha), 38 kDa (beta), and 23

kDa (gamma). The reason for the apparent larger

size of the gamma subunit estimated by SDS-

PAGE may be due to hydrophobicity of this

putative membrane anchor protein.

Edman degradation for N-terminal

amino acid sequencing of the proteins after

blotting on a PVDF-membrane or electroelution

out of SDS-PAGE gels was not successful. This

failure could be caused by blocking of the α-

amino group. The N-terminal amino acid of the

alpha subunit could, however, be derivatized

with TMPP-Ac-OSu. After mass spectrometric

detection of labeled peptides, the TMPP-label

was identified on the methionine of the large

subunit corresponding to the first amino acid

coded by the gene. Surprisingly, in this enzyme

preparation the N-terminal leader peptide of the

α subunit apparently was not cleaved off. No

TMPP-label could be identified on the beta and

gamma subunits indicating that they were

probably not accessible for derivatization.

The molar contents of molybdenum,

iron, and acid-labile sulfur were determined. The

molybdenum content was 0.7 ± 0.1 mol (mol of

enzyme)-1

as determined by inductively coupled

plasma optical emission spectrometry (ICP-

OES), the iron content was 16 ± 5 mol (mol of

enzyme)-1

as determined by ICP-OES and

colorimetric assay, and the acid-labile sulfur

content was 18 ± 5 mol (mol of enzyme)-1

. These

values are consistent with the predicted presence

of 1 molybdenum atom, 4 [Fe4S4], and one

[Fe3S4]-clusters, and 1 heme. Analysis of the

nucleotide part of molybdenum cofactor revealed

the presence of GMP rather than AMP or CMP.

Spectral properties - The complex UV-

visible spectrum of the purified brownish

enzyme showed distinct absorption maxima at

424, 527, and 561 nm, which indicated the

presence of a reduced heme b cofactor, as well as

a broad shoulder around 400 nm. Anaerobic

oxidation of the enzyme with potassium

hexacyanoferrate (III) resulted in the

disappearance of α and β peaks of the heme at

561 and 527 nm and the shift of the Soret band at

424 nm to 416 nm (Fig. 4A). The difference

spectrum of reduced-minus-oxidized enzyme

indicated the presence of a heme b cofactor (Fig.

4B). The spectrum of the reduced enzyme could

be restored by addition of the substrate cholest-

4-en-3-one to the cyanoferrate-oxidized enzyme

(Fig. 4A). The substrate-reduced enzyme and the

enzyme as isolated showed identical heme b

spectra, suggesting that C25 dehydrogenase was

purified in the reduced heme form. Further

treatment of the enzyme with dithionite did not

result in further reduction of the heme cofactor,

but resulted in further bleaching of the

absorption between 400 and 500 nm (Fig. 4A),

which is indicative for the presence of iron-

sulfur clusters. Obviously, these clusters are not

fully reduced by the substrate and need a strong

chemical reductant such as dithionite for being


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entirely reduced. The heme content of the

enzyme was determined as 1.13 mol (mol

protein)-1

from dithionite-reduced enzyme, using

a molar absorption coefficient ε556 nm of 34,700

M-1

cm-1

for the α band (25).

Catalytic properties - The purified en-

zyme catalyzed the hydroxylation of the tertiary

C25 atom of the steroid side chain. The low

water solubility of the substrates, which required

the addition of detergents or cyclodextrin,

hampered the determination of kinetic properties

of C25 dehydrogenase. An HPLC based assay or

a photometrical assay containing ferricenium

cation as artificial electron acceptor was used to

measure activity of the purified enzyme in the

presence of cyclodextrin. The substrate routinely

used was cholest-4-en-3-one (SI Fig. 1A). Only

25-hydroxy-cholest-4-en-3-one was formed (SI

Fig. 1C). The stoichiometry was 1 mol C25

hydroxylated product formed per 1.8 mol

ferricenium added. Addition of water soluble

organic solvents like 1,4-dioxane or

dimethylsulfoxide to the reaction mixture to

increase the solubility of the substrates resulted

in strong decrease of the enzymatic activity.

Addition of detergents or cyclodextrin to the

assay resulted in thousand-fold increased

activity.

A specific activity of 220 nmol min-1

mg-

1 was found with cholest-4-en-3-one as substrate

at an optimal pH value of 7-7.5 and in the

presence of cyclodextrin; this activity is in the

same range as that reported for ethylbenzene

dehydrogenase (12). A slightly higher

hydroxylation rate was observed with cholesta-

4,6-dien-3-one, whereas lower activity was

observed with cholesterol, cholest-5-en-3β-ol-7-

one, and cholcalciferol (Table 1). No activity

could be measured with ergosterol, desmosterol,

or cholesterylbenzoate; the enzyme also did not

act on isoamylbenzoate, isoamyl alcohol or 2-

methyl-butane that mimic the branched side

chain of the steroids. The potential of C25

dehydrogenase to catalyze the reverse reaction

was tested by an anaerobic enzyme assay with

reduced methyl viologen as electron donor and

cholest-4-en-3-one-25-ol as substrate; no

reduction to cholest-4-en-3-one was detected.

The enzyme was not inhibited by sodium azide

or sodium cyanide (5 mM each).

The capability of bovine heart

cytochrome c to serve as an electron acceptor

was tested in a modified spectrophotometrical

assay. Cytochrome c was indeed reduced, the

specific enzyme activity being 20 nmol min-1

mg-1

with cholest-4-en-3-one as a substrate

which is 10-fold lower than with

hexacyanoferrate (III). The Km value for bovine

heart cytochrome c was estimated as 15 µM. Stability - The activity of C25 dehydro-

genase was not affected by aerobic preparation

of cell extracts, whereas the purified enzyme was

inactivated irreversibly by incubation in air with

a half-life of 14 min (SI Fig. 2). The inactivation

was largely prevented by addition of the artificial

electron acceptor potassium hexacyanoferrate

(III) to the enzyme preparation. In presence of

cyanoferrate, more than 90 % of the enzyme

activity was retained after two hours incubation

in air; still, the enzymatic activity was

completely lost after 24 h of incubation. The

enzyme can be stored for at least 3 months at 4

°C under anaerobic conditions.

Sequence analysis and phylogeny -

Analysis of the α subunit of C25 dehydrogenase

showed high similarity to the α subunit of

ethylbenzene dehydrogenase of Aromatoleum

aromaticum (35 % amino acid sequence identity,

accession number CAI07432), an unidentified

molybdopterin oxidoreductase of Desulfococcus

oleovorans strain Hxd3 (35 % amino acid

sequence identity, accession number

ABW66004), and further molybdopterin

containing oxidoreductases of type II group of

the DMSO reductase family. Examination of the

N-terminus of the α subunit revealed that it

contained a ‘twin arginine’ motif,

MQISRRQFIV (Fig. 5), indicative of proteins

that bind a prosthetic group and fold in the

cytoplasm, before the translocation via the Tat

system (26) . Furthermore a cysteine-rich motif,

GTHTRANCIGACSWDV-(26)-NPRGCQK,

was identified in the N-terminal part of the large

subunit (Fig. 5). This motif is characteristic of

type II enzymes with an aspartate molybdenum

ligand in the active site and is responsible for

coordination of a [Fe4S4]-cluster (27) . The β

subunit showed high similarity to the β subunit

of ethylbenzene dehydrogenase (55 % amino

acid sequence identity) and to further iron-sulfur

subunits of DMSO reductase like proteins.

Sequence analysis revealed conserved cysteines

(Fig. 6; SI Fig. 4), which are responsible for

coordination of four [Fe-S]-clusters (28).

Analysis of amino acid sequence of the γ subunit

showed similarity exclusively to the γ subunit of

ethylbenzene dehydrogenase and ethylbenzene

dehydrogenase-like proteins. Highly conserved

methionine (M111) and lysine (K203) residues

were identified (SI Fig. 5) which are the axial

ligands of the heme b iron in ethylbenzene

dehydrogenase (29). Furthermore, neither azide


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nor cyanide inhibit the C25-Hydroxylase. This

finding supports the assumption that the heme is

hexacoordinated.

Genes coding for steroid C25

dehydrogenase-like enzymes in the genome - S.

denitrificans harbors a set of genes coding for at

least seven further C25 dehydrogenase-like

enzymes (Fig. 7), which are scattered in the

genome. Genes of six C25 dehydrogenase-like

enzymes are organized in gene clusters coding

for α, β, and γ subunits, whereas an additional

gene for a single α subunit is located as a single

ORF, like the single gene for the alpha subunit of

steroid C25 dehydrogenase (Fig. 7).

Interestingly, only two genes encoding

maturation chaperones were identified in the

draft genome. Maturation of complex

molybdoenzymes requires enzyme-specific

chaperones for correct insertion of co-factors

before folding and translocation across the

membrane. The private chaperone genes often

occur in the gene clusters of respective

molybdoenzyme, as it was shown for all type II

enzymes (30-33). In contrast, the two maturation

chaperones identified in the genome of S.

denitrificans may be responsible for the

maturation of eight molybdoenzymes.

Interestingly, no gene resembling any

oxygenase gene from the known aerobic steroid

metabolism could be found. This is another

argument for the functioning of the O2

independent steroid pathway studied here, both

under anoxic and oxic conditions.

DISCUSSION

Function of C25 dehydrogenase -

Previous studies showed that under anoxic

conditions cholesterol is transformed to cholest-

4-en-3-one and cholesta-1,4-dien-3-one (Fig. 1)

(11). These transformations are similar to those

occurring in aerobic steroid degradation (5).

Here we described a novel molybdenum-

containing enzyme that catalyzes the subsequent

anaerobic hydroxylation of the C25 tertiary

carbon atom of the steroid side chain. It acts as

counterpart to C26 monooxygenase of aerobic

catabolism (34) that exploits the ability of

activated molecular oxygen to overcome the high

C-H bond stability. C25 dehydrogenase

resembles ethylbenzene dehydrogenase that

catalyzes a similar oxygen-independent

hydroxylation of a hydrocarbon, forming

secondary alcohols of a wide range of aromatic

and heterocyclic compounds with an ethyl or

propyl moiety (12). Hydroxylation may proceed

via formation of a carbocation, which is

stabilized by an adjacent aromatic ring (35).

Because of the high similarity of C25

dehydrogenase with ethylbenzene

dehydrogenase, we assume that C25

dehydrogenase employs a similar hydroxylation

mechanism. Owing to the higher stability of the

C25 carbocation compared to the terminal C26

or C27, its hydroxylation is probably favored.

S. denitrificans is a facultative anaerobic

cholesterol degrader. It appears that the same

anaerobic strategy is used under anoxic and oxic

conditions as indicated by the following

findings. (i) Comparable anaerobic C25

hydroxylation activities were measured in

extracts of anaerobically and aerobically grown

cells. (ii) Previous proteome analyses revealed

no differences in soluble protein patterns of

anaerobically and aerobically grown cells (9).

(iii) No oxygenase genes known from aerobic

cholesterol metabolism could be identified in the

draft genome of the organism. The usage of one

common anaerobic strategy is highly economic

for an organism growing on steroids both under

anoxic and oxic conditions. It reflects an

adaptation of a facultative anaerobic organism to

frequent periodical oxygen fluctuations allowing

the instantaneous usage of cholesterol, no matter

whether oxygen is available or not. Apparently,

the enzymes of the anaerobic strategy are

sufficiently oxygen-stabile in vivo and possibly

also in cell extracts, even though purified C25

dehydrogenase is oxygen labile.

Membrane association and catalysis at

lipid-water interface - C25 dehydrogenase

operates on highly hydrophobic substrates

which, owing to their low water solubility, likely

concentrate in the membrane bilayer rather than

in the aqueous phase of the cytoplasm. Thus, a

membrane associated C25 dehydrogenase can

easily reach the hydrophobic side chain of its

substrate (36), which may provide kinetic

advantages since both enzyme and its substrate

are concentrated within the membrane. A similar

situation was described for cytochrome P-450scc,

a mono-oxygenase cleaving the side chain of

cholesterol and forming pregnenolone. C25

dehydrogenase may partially immerse into the

membrane bilayer thus facilitating the binding of

cholesterol (37).

C25 dehydrogenase operates at the lipid-

water interface on lipophilic substrates; sequence

analysis does not predict transmembrane helices.

All attempts to establish an in vitro assay failed

when cholest-4-en-3-one was simply added to

the assay. However, the activity increased a


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thousand-fold when detergents or cyclodextrin

were added to the reaction mixture. The ability

of detergents to increase activity is well-known

for enzymes operating on hydrophobic

substrates. The phenomenon of interfacial

activation was described for lipases (38),

whereas the detergent dependent activation of

tyrosinase (39) or pyruvate oxidase (40) is

thought to be caused by protein-detergent

interaction. We suggest that the activation effect

on C25 dehydrogenase is caused by increased

substrate solubility rather than protein-detergent

interaction, as a comparable stimulation of C25

hydroxylation activity was achieved both with

detergents and cyclodextrin.

Electron acceptor - The estimated redox

potential of the C25-hydroxycholesterol/chole-

sterol pair is around + 30 mV, based on

theoretical calculations of related

alcohol/hydrocarbon couples. Steroid C25

dehydrogenase shows in vitro activity only with

artificial electron acceptors of high redox

potential, like cyanoferrate (E°’ = + 420 mV) or

ferricenium ion (E°’ = + 380 mV). The

difference in redox potential of the

substrate/product pair and that of the electron

acceptor may explain the irreversibility of the

reaction under experimental conditions. Due to

the accessibility of the electron acceptor site of

C25 dehydrogenase from the periplasmic space,

a periplasmic c type cytochrome of similar

positive redox potential may function as natural

electron acceptor, coupling C25 hydroxylation to

nitrate reduction or alternatively to O2 reduction.

The use of cytochrome c was also proposed for

the closely related ethylbenzene dehydrogenase

from A. aromaticum (12). Bovine heart

cytochrome c, tested here, is probably a poor

surrogate for the bacterial cytochrome c.

Substrate specifity - Steroid C25 dehydro-

genase catalyzes the hydroxylation of cholesterol

but also of intermediates II and III (Fig. 1).

Apparently, the first steps of anaerobic

degradation of cholesterol proceed randomly via

independent reaction steps operating in parallel

on the side chain and the ring system. A similar

situation is known for aerobic steroid

degradation (5). Thus, it appears to be a general

strategy of microbial steroid decomposition to

follow a branched route of reactions. The

hydroxylation rate of cholesterol was lower

compared to that of cholest-4-en-3-one or

cholesta-4,6-dien-3-one (Table 1), all substrates

having the same side chains but differing in the

ring systems (SI Fig. 3). Presence and

localization of double bonds have an impact on

the conformation of the molecule. Thus, the

more planar conformations of rings A and B of

cholest-4-en-3-one and cholesta-4,6-dien-3-one

may result in a better fitting into the active site of

the enzyme. No hydroxylation activity was

observed with stigmasterol and ergosterol, whose

side chains structures differ from that of

cholesterol (SI Fig. 3). They harbor additional

methyl or ethyl substituents at C24 which may

cause sterical hindrances in the active site.

Therefore, we assume that C25 dehydrogenase

operates selectively on the isooctane side chain

of steroids and requires a tertiary C25.

One may ask whether there is a

physiological reason for the preference of

cholest-4-en-3-one as substrate. It is well known

that cholesterol is an important constituent of

eukaryotic membranes. However, steroids can

affect the growth of some microorganisms,

damaging their cytoplasmic membranes, e. g. the

presence of cholest-4-en-3-one is toxic for

Mycobacterium tuberculosis (41). Therefore, a

higher C25 transformation rate of 3-ketosteroids

may reflect an adaptation of S. denitrificans to

remove toxic intermediates from the membrane.

Hydroxylation at C25 increases the water

solubility of the molecules, thus facilitating

migration out of the membrane (42).

C25 dehydrogenase, a new member of

the DMSO reductase family of molybdoenzymes

- Steroid C25 dehydrogenase is a molyb-

denum/iron-sulfur/heme containing enzyme and

belongs to the type II of DMSO reductase family

enzymes. Based on its high sequence similarity

to ethylbenzene dehydrogenase (29) and other

archetypal complex iron-sulfur-molybdo-

enzymes (28), the α subunit contains molyb-

denum coordinated by molybdo-bis(pyranopterin

guanine dinucleotide) cofactor and the FS0-

[Fe4S4]-cluster (Fig. 5B); the β subunit harbors

FSI - FSIII-[Fe4S4], and FSIV-[Fe3S4]-clusters

(Fig. 6), and the γ subunit contains the heme b

group with conserved methionine and lysine

axial ligands. Remarkably, the genome of S.

denitrificans harbors genes coding for at least

seven further C25 dehydrogenase-like enzymes

(Fig. 7). The large subunits of all of them

possess a signal peptide for Tat-dependent

translocation (Fig. 5A) and therefore may be

located in the periplasmic space. A phylogenetic

tree (Fig. 8) reveals that steroid C25

dehydrogenase and related enzymes form a

distinct clade, together with other heterotrimeric,

periplasmic enzymes like ethylbenzene dehydro-

genase, selenate reductase, dimethylsulfide

dehydrogenase, and chlorate reductase.


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A signature of all type II enzymes is the

presence of the FS0-[Fe4S4]-cluster in the

catalytic subunit, which is coordinated by an N-

terminal consensus sequence (CA/HA)-x2-3-CB-x3-

CC-x27-34-CD-x-K/R (27). So far, ethylbenzene

dehydrogenase represents an exception with a

HA-x3-CB-x5-CC-x34-CD-x-K consensus sequence.

Interestingly, the consensus sequence of C25

dehydrogenase and related enzymes of S.

denitrificans also differs from that of the other

type II enzymes (Fig. 5B). Ethylbenzene

dehydrogenase and C25 dehydrogenase act on

hydrophobic substrates, in contrast to other type

II enzymes that are involved in anaerobic

respiration. The identified differences in

consensus sequence may represent a signature of

the new clade of type II enzymes.

Molybdenum hydroxylases as

counterparts of oxygenases - Degradation of

cholesterol is challenging due to its complex

structure and requires, therefore, several

activation steps. Under aerobic conditions this

part is taken over by four oxygenases initiating

the degradation of the side chain and the

cleavage of the ring structures (5). In S.

denitrificans growing on cholesterol both under

anaerobic and aerobic conditions, molybdenum

hydroxylases obviously operate as oxygenase

counterparts. The presence of a set of eight C25

dehydrogenase-like enzymes supports this

suggestion. These paralogous enzymes may have

evolved by several gene duplication events.

Molybdenum hydroxylases probably represent a

general strategy of facultative microorganisms to

activate hydrophobic substrates containing a

sterane ring.

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34. Rosloniec, K. Z., Wilbrink, M. H., Capyk, J. K., Mohn, W. W., Ostendorf, M., van der Geize,

R., Dijkhuizen, L., and Eltis, L. D. (2009) Mol. Microbiol. 74, 1031-1043

35. Szaleniec, M., Hagel, C., Menke, M., Nowak, P., Witko, M., and Heider, J. (2007)

Biochemistry 46, 7637-7646

36. Rothman, J. E., and Engelman, D. M. (1972) Nat. New Biol. 237, 42-44

37. Seybert, D. W., Lancaster, J. R., Lambeth, J. D., and Kamin, H. (1979) J. Biol. Chem. 254,

2088-2098

38. Ferrato, F., Carriere, F., Sarda, L., and Verger, R. (1997) Methods Enzymol. 286, 327-347

39. Wittenberg, C., and Triplett, E. L. (1985) J. Biol. Chem. 260, 2535-2541

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FOOTNOTES

* This work was supported by Deutsche Forschungsgemeinschaft. We thank Michael Müller and

Volker Brecht, Freiburg, for mass spectrometry measurements, and Yin-Ru Chiang and Wael Ismail,

Freiburg, for their invaluable contributions during the early stages of this work.

FIGURE LEGENDS

Fig. 1. Initial steps of anaerobic cholesterol metabolism (acm) in S. denitrificans. AcmA -

cholesterol dehydrogenase/isomerase; AcmB - cholest-4-en-3-one-∆1-dehydrogenase; S25DH - steroid

C25 dehydrogenase. (I) - cholesterol, (II) - cholest-4-en-3-one, (III) - cholesta-1,4-dien-3-one, (IV) -

25-hydroxy-cholesterol, (V) - 25-hydroxy-cholest-4-en-3-one, (VI) - 25-hydroxy-cholesta-1,4-dien-3-

one. Conversion of intermediate (IV) to intermediate (V) was not experimentally proven (dashed

arrow).

Fig. 2. Cellular localization of C25 dehydrogenase. (A) C25 dehydrogenase assay with cell

suspension and cell extract. The assay mixture contained 0.1 M potassium phosphate buffer (pH 7.5),


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2 M sucrose, 7 % (w/v) (2-hydroxypropyl)-β-cyclodextrin, 0.7 mM cholest-4-en-3-one, 5 mM

K3[Fe(CN)6], and 0.1 ml cell suspension (0.3 mg protein) or cell extract (0.3 mg protein) in a total

volume of 0.3 ml. Solid line - assay with cell suspension, dashed line - assay with cell extract. (x) -

conversion of cholest-4-en-3-one; (●;○) - formation of cholesta-1,4-dien-3-one; (■;□) - formation of

25-hydroxy-cholesta-1,4-dien-3-one. (B) Relative activities of enzymes determined in cell suspension

and in cell extract. Solid bars - activities in cell extract: C25 dehydrogenase (2.2 mU mg-1

), cholest-4-

en-3-one-∆1-dehydrogenase (6 mU mg

-1), and malate dehydrogenase (1 U mg

-1); open bars - activities

measured with suspension of whole cells: C25 dehydrogenase (1.5 mU mg-1

), cholest-4-en-3-one-∆1-

dehydrogenase (6 mU mg-1

), and malate dehydrogenase (72 mU mg-1

).

Fig. 3. Purification and characterization of C25 dehydrogenase.

A. SDS-PAGE (11 %) of active pools during purification of C25 dehydrogenase (I) and blue

native-PAGE of the purified enzyme (II). I. Lane 1, cell extract (100 µg of protein); Lane 2,

solubilized membrane fraction (100 µg of protein); Lanes 3-5, active pools after chromatography on

DEAE (L3, 34 µg of protein); on Resource Q (L4, 20 µg of protein); on Reactive Red 120 (L5, 6 µg of

protein); Lane M, marker proteins (sizes given in right margin).

II. (A) Blue native-PAGE (8 %) of enzyme after Reactive Red 120 chromatography (9 µg). (B) The

visible band was cut out, treated with SDS-buffer, and analyzed by SDS-PAGE (11 %). Proteins were

stained with sensitive Coomassie blue.

B. Organization of genes encoding C25 dehydrogenase and C25 dehydrogenase-like enzyme in S.

denitrificans. (S25dA) alpha subunit of C25 dehydrogenase; (S25dB) beta subunit of C25

dehydrogenase; (S25dC) gamma subunit of C25 dehydrogenase; (S25dA3) molybdenum-containing

subunit of a C25 dehydrogenase-like protein of unknown function.

Fig. 4. UV-visible spectrum of C25 dehydrogenase. [A], spectrum of purified enzyme (0.9 mg ml

-

1). (a) Directly after purification under anaerobic conditions, (b) after anaerobic oxidation with 170

µM potassium hexacyanoferrate, (c) after re-reduction with 50 µM cholest-4-en-3-one dissolved in

dioxane, and (d) after vigorous reduction with dithionite. For better visibility, spectra b-d were offset

along the y axis (+ 0.01, + 0.02, + 0.03, respectively). [B], difference spectrum of the reduced

enzyme (a in panel A) minus potassium hexacyanoferrate-oxidized enzyme (b in A).

Fig. 5. Sequence alignment of the molybdenum-containing subunit of C25 dehydrogenase from

S. denitrificans, of ethylbenzene dehydrogenase from A. aromaticum, and of related enzymes.

(A) Alignment of the N-terminal sequences bearing a consensus motif for the Tat export way. The

conserved amino acids are highlighted.

(B) Alignment of the sequence surrounding the [Fe4S4]-cluster, which is characteristic of type II

enzymes of the DMSO reductase family. Highly conserved amino acids coordinating this FS0-[Fe4S4]-

cluster are highlighted. S25dA: alpha subunit of C25 dehydrogenase; EbdA: alpha subunit of

ethylbenzene dehydrogenase; S25dA2-S25dA7: molybdenum containing subunits of enzymes with

high similarity to C25 dehydrogenase, which were identified in the genome of S. denitrificans.

Fig. 6. Analysis of amino acid sequence of beta subunit of C25 dehydrogenase.

Coordination of the FS1-FS4 [Fe-S]-clusters in the beta subunit of C25 dehydrogenase, as proposed

based on the sequence similarity to ethylbenzene dehydrogenase beta subunit (EbdB) and the crystal

structure of ethylbenzene dehydrogenase.

Fig. 7. Genes coding for C25 dehydrogenase and C25 dehydrogenase-like enzymes in the genome

of S. denitrificans. Amino acid sequences of α, β, and γ subunits of C25 dehydrogenase were used as

queries for BLASTP searches against the genome of S. denitrificans. The BLASTP (2.2.14) search

tool implemented into RAST server was used. (S25dA) - molybdenum containing α subunit of steroid

C25 dehydrogenase; (S25dB) - [Fe-S]-clusters containing β subunit of steroid C25 dehydrogenase;

(S25dC) - heme b containing γ subunit of steroid C25 dehydrogenase; (α2-α7) - genes coding for

molybdenum containing subunits related to S25dA; (β4-β7) - genes coding for [Fe-S]-clusters

containing subunits related to S25dB; (γ4-γ7) - genes coding for heme b containing subunits related to

S25dB; (δ4, EbdD-like) - genes coding for proteins related to maturation chaperone of ethylbenzene

dehydrogenase. (EbdABC-like) - molybdenum containing enzyme related to S25dABC, but


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phylogenetically clustering with ethylbenzene dehydrogenase (Fig. 8). The sequence identities to the

corresponding subunits of C25 dehydrogenase are given in brackets.

Fig. 8. Phylogenetic tree of enzymes of the DMSO reductase family (type I, II, and III) based on

amino acid sequences of molybdenum-containing alpha subunits. C25 dehydrogenase and several

related enzymes in S. denitrificans belong to family II, as do ethylbenzene dehydrogenase from A.

aromaticum and related dehydrogenases. The tree was constructed using the neighbor-joining

algorithm. Bootstrap values higher than 75 % are marked with dots. The scale bar represents 0.1

changes per amino acid. GenBank accession numbers for sequences used to construct the tree are

listed in SI Table 2. Abbreviations for molybdenum-containing subunits of proteins: BisC, biotin

sulfoxide reductase; ClrA, chlorate reductase; DdhA, dimethylsulfide dehydrogenase;

DmsA/DorA/DsrA, dimethylsufoxide reductase; EbdA, ethylbenzene dehydrogenase; FdhG, formate

dehydrogenase; NarG, respiratory nitrate reductase; NapA, periplasmic nitrate reductase; NasA,

assimilatory nitrate reductase; PcrA, perchlorate reductase; PhsA, thiosulfate reductase; PsrA,

polysulfide reductase; SerA, selenate reductase; TorA, trimethylamine-N-oxide reductase.


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Table. 1: Relative activity of C25 dehydrogenase with steroid substrates. For structures of

cholesterol, cholest-4-en-3-one and cholesta-1,4-dien-3-one, see Fig. 1. Measurements of relative

activities were carried out in spectrophotometric assays. For steroid substrates also HPLC assays were

performed. No activity was observed with isoamylbenzoate, isoamylalcohol, 2-methyl-butane, ergosterol,

desmosterol, and stigmasterol. For MS-analysis the assay mixture (0.5 ml) was extracted with three

volumes of ethyl acetate. Ethyl acetate was evaporated, and the dry residue was dissolved in 100 µl of

chloroform. Electrospray ionization mass spectra (ESI-MS) were recorded with an Applied Biosystems

API 2000 triple quadrupole instrument running in positive ion mode.

n. d. - not determined

* - analysis with HPLC-ESI-MS

§ - analysis with HPLC-ESI-MS with atmospheric pressure photo ionization (APPI) source

# - formation of the product was observed using HPLC assay when the reaction mixture was incubated

for 12 h.

Substrate m/z

relative

activity

(%)

product m/z

cholest-4-en-3-one 385* (m+H) 100 401

* (m+H)

cholesta-4,6-dien-3-one 383* (m+H) 127 399

* (m+H)

5-cholesten-3β-ol-7-one 401* (m+H) 15 417

* (m+H)

cholesterol 369§ (m+H - H2O) 10 367

§ (m+H - 2 H2O)

cholecalciferol n. d. < 5# n. d.


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Fig. 1

OH

OH

OH

O

OH

O

O

OH

O

S25DH

S25DH

S25DH

AcmA

AcmB AcmB

AcmA

I

III

IV

V

VI

II


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Fig. 2

A

min

nm

ol

0

20

40

60

80

100

120

140

rela

tive

ac

tivit

y, %

B

50

100

150

200

250

300

350

0 50 100 150 200


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Fig. 3

A

B

201487 598131

( 396 kbp )S25dC S25dB S25dA3 S25dA


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Fig. 4

Data 1

400 450 500 550 6000.00

0.05

0.10

0.15

0.20

0.25

0.30

FeCN

Enzyme

S2

Dithionit

nm

Ab

s

Wavelength [nm]

Ab

so

rba

nce

∆A

A B

400 450 500 550 600

-0.020

0.005

0.030

0.055

424

561


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Fig. 5

10 20 30 40 50 60 70 80 90. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

C25dhA S.denitrificans - - - - - - - - - - - - - - - - - M Q I S R R Q F I V G S A V A A A G L G L Y S L R P K H Y V P A A P R P A D K - - - - - - - - - - - P G I P A K K V K Y N D Y S D I WR E KWKW

peg.1060 S.denitrificans - - - - - - - - - - - - - - - - - M QV S R R H F I V G T A A V A A GA G L Y S L R P K L T G P K - P G I T M P - - - - - - - - - - - P L V P A K K V K Y N D Y S D I WR E KWKW

peg.62 S.denitrificans - - - - - - - - - - M E S K P GM I GM D R R S F L K - A G GS A L A L S L C H L E L L P M N AM A Q A A GK D G N G N K A A A E L S P L E A A A E L E Y R S F E D L Y R K KWHW

peg.1309 S.denitrificans - - - - - - - - - - - - - - - - - M QV S R R N F I V G S A V A A A G L G L Y S L K P K T P A A I K A GP A L P - - - - - - - - - - - P L V S A K K I R Y N D Y S D I WR E KWKW

peg.1642 S.denitrificans - - - - - - - - M G I L A T S N L V S A S R R K F L V M A G - - - M A S A A GA A V G L F GC S R A P L Q H F K G T - - - T A S G R F D L GP R T T P K L GNWQ D L Y R Q RW T W

peg.481 S.denitrificans - - - - - - - - - M T T A S P A QP N P A R R R F L I L A G K T T V A G I A A A A T G L P GC N RM P L Q H F H G - - - - T V D G R F D L GP R T T P K L N NWQ D L Y R Q RW T W

peg.761 S.denitrificans - - - - - - - - - - - - - - M Q F M Q L T R R H F I M G S A A T V A G L A L Y S L R P R H Y V P A A P R P A D P - - - - - - - - - - - P T V P A K K V K Y N D Y S D I WR E KWKW

peg.1646 S.denitrificans - - - - - - M E R S S A S S T V G L S V S R R Q F L I K A G - - - L A S M A GG T L A L F GC H R A P L Q H F H G - - - - T M GG R F D L GP R T T P K L GNWQ D L Y R Q RW T W

EbdA-like D.oleovorans Hxd3 - - - - - - - - - - - - - - M K E V K I S R R T F L K G T S A T V A L L S L N S L G F L G GN T I A N A T E K I - - - - - - - - - - - - - - - F E DWK Y A GW E N L H R E EW T W

EbdhA A.aromaticum - M T R D EM I S V E P E A A E L Q D Q H R R D F L K R S G A A V L S L S L S S L A T G V V P G F L K D A QA G - - - - - - - - - - - - - - - T K A P G Y A S W E D I Y R K EWKW

EbdhA2 A.aromaticum M D D L K N T D A I R T GV S S A F D Q N R R G F L K R S G A G A L S L S L S S F A A G L V P G F V N A A QA G - - - - - - - - - - - - - - - K R G P T Y A T W E D V Y R N EWKW

EbdA-like gamma proteobachgter - - - - - M T L GA GM G I LWK Q K F D R R S F L K A S G - - - Y T V A A A A A V E L P - - - - - - S L H F K T - - - - - - - - A L A S D A A T P A P L K T W E D L Y R E RW T W

100 110 120 130 140 150 160 170 180. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

C25dhA S.denitrificans D R V V K G T H T R - A N C I G A C S WD V Y V K D G I AWR E E Q A A I Y E P H R P D I P D F N P R GC QK GA C Y T H L Q V N E S R I K Y P L K R V G E R G E GKWK R I T WD

peg.1060 S.denitrificans D K V V K G T H T R - A N C V A A C S WD V Y V R D G I AWR E E Q N T I Y E P P R P G I P D QN P R GC QK GA C Y T T L Q L S E T R V K Y P L K R V G E R G E GKWK R I T WD

peg.62 S.denitrificans D S V A K S T H F V N CW Y QR N C S WN V Y V K N G I AWR E E Q A A T Y E Q V D P N V P D Y N P R GC QK GA C Y S Q RM Y D A G R L T H P L R R V G A R G E GKWM R V S WD

peg.1309 S.denitrificans D K V V K G T H T R - A N C GD A C S WD V Y V R D G I AWR E E Q N A I Y E P H R A D V P DM N P R GC QK GA C Y T N L Q L S E A R L K Y P L K R V G E R G E GKWK R I S WD

peg.1642 S.denitrificans D K V A K G S H GW - A N C R S A C EWD L Y V K D GV V V R E E Q S A T Y E A S E P G I P D F N P R GC QK GA C Y T E V M Y G P S R T T V P L K R V G P R GS GKW E K I S W E

peg.481 S.denitrificans D K V A K G S H GW - A N C R S A C EWD L Y V K D GV V V R E E Q S A T Y E A S E P G I P D F N P R GC QK GA C Y T E V M Y G P S R T T V P L K R V G P R GS GKW E K I S W E

peg.761 S.denitrificans D R V V K G T H T R - A N C I A A C S WD V Y V R D G I AWR E E Q N A I Y E P H R P D I P D F N P R GC QK GA C Y T T L Q L S E A R L K Y P L K R I G K R G E GKWK R I T WD

peg.1646 S.denitrificans D K V A K G S H GW - A N C R S A C EWD L Y V K D G I V V R E E Q S A T Y E A S E P G V P D F N P R GC QK GA C Y T E V M Y G P S R T T V P L K R V G P R GS GKW E K I S W E

EbdA-like D.oleovorans Hxd3 D K V T Y G T H L V D C Y P - G N C L WR V Y S K D GV V F R E E Q A A K Y P V I D P S G P D F N P R GC QK GA S Y S L QM Y N P D R L K Y P M K Q V G GR GS GKWK R V S WD

EbdhA A.aromaticum D K V NWG S H L N I CWP QG S C K F Y V Y V R N G I V WR E E Q A A Q T P A C N V D Y V D Y N P L GC QK GS A F N N N L Y G D E R V K Y P L K R V G K R G E GKWK R V S WD

EbdhA2 A.aromaticum D K V TWG S H L N I CWP QG S C K F Y V Y V R N G I V WR E E Q A A Q T A A C N P D Y V D Y N P S GC QK GA A F N N N L Y G E E R L K Y P L K R V G K R G E GKWK R V S WD

EbdA-like gamma proteobachgter D R V V K S S H GW - L N C R S A C EWD I Y V K D GV V V R E E Q T A T Y E A S E P G I P D F N P R GC QK GA C Y T E V M Y G P S R L Y S P M K R V G E R GS GQW E K I S WD

190 200 210 220 230 240 250 260 270. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

C25dhA S.denitrificans E A L T E I A D K L I D A A V A E G T E S I I F D D G T T N A G Y G P E T A GD V R F A T S L Q T T K I D S WA G V S DM P M G L V Q T WGM Y N C E G T S D DW F R S D Y I V I W

peg.1060 S.denitrificans E A L N E I A D K L I D I S V E H G T E T I C F D D - L S N T G Y G P E T A GD F R F S T A L QV T R L D GWS G V G DM P L GV I Q T WGA F N C E G T S D DW F R S D Y I V I W

peg.62 S.denitrificans E A L A D I A D RM I D V M R T D G P G A I T WD P G T A N A G GG A S T A - P Y R L G F I L D T P M I D V N T E V G D H H Q GA QV T V GK I S F S GS M D D L F Y S D L I L V W

peg.1309 S.denitrificans E A L N E I C D K L I D V A I D QG T E S I I F D D G T T N GG F G P E T A GD V R F T E A L N C T QM D S WA G V S DM P M G L V Q T WGM F N S E GS A D DW YM S D F I V I W

peg.1642 S.denitrificans Q A L R E I A V K T V D A A E KWG T D T I Y Q D L GP N F D - F G A S T A GR F K F Q FM A GG I F A D NWA E I G D L N V GA S I T V GA A H L G GS A D EW F L S D F I V V W

peg.481 S.denitrificans Q A L R E I A V K T V D A A E KWG T D T I Y Q D L GP N F D - F G P S T G GR F K F Q F QV GG L F A D NWA E I G D L N I GA N I A L GA A H V G GS S D EW F L S D F I V V W

peg.761 S.denitrificans E A L T E I A D K L I D A A V A E G T E S I I F D D G T T N S G Y G P E T S GDWR F A D A I QA T K I D S WA G V S DM P M GA V Q T WGM Y N C E G T S D DW F R S D Y I V I W

peg.1646 S.denitrificans Q A L R E I A V K T V D A V E E Y G T D T V F Q D L GP N F D - F G P S T A GR F K F M Y QA S S L F S DMWG E I G D L N F GA T M A L GA A Q I G GS S D DW F L S D F I V V W

EbdA-like D.oleovorans Hxd3 Q C L A E I A E G I V D G L E A QG P E S I I F E S GP GN GG Y V H V M A - V H R L M V S L GA T V L D L D S T I G D F N R G I Y E T F GK F M FM D S V D GW Y F G K L L L I W

EbdhA A.aromaticum E A A G D I A D S I I D S F E A QG S D G F I L D A P H V H A G S I AWGA - G F RM T Y L M D G V S P D I N V D I G D T YM GA F H T F GKM HM G Y S A D N L L D A E L I F M T

EbdhA2 A.aromaticum E A T A D I A D A I I D G I E T E G T D S F I L D S P H V H A G S V A N S G - G Y RM T Y L L D G V S P D N N V D I G D T Y S GA F H T F GKM HM G Y S A D N L L D S E L I F M T

EbdA-like gamma proteobachgter Q A L G E I A E K I V D I S E K Y G T D Y I I H DM GP H H D - F G P T T A A R A R F F S M L GA S L A D DWA E I G D L N V A A T M T F G F P H V G GS S D EW F L S D Y L V V W

280 290 300 310 320 330 340 350 360. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

C25dhA S.denitrificans V GN P I Y T R I P E A H F L H E A R Y R G A K L V V I A P D L N P S T V H A D T W L K I N P E T D A A L G L A A A Q V M I T E N L I K K D Y V L E Q T DM P F L V R K D D K R F L

peg.1060 S.denitrificans L GN P N Y T R I P D A H F L H E A R Y R G A K L V V V S P D L N A S T V H A D RW I K V K P E T D A A L G L A C A Q V M I A E D L Y K K D Y V L E Q T D F P F L V R K D N QR F L

peg.62 S.denitrificans G A N P V Y T Q I P N A H F I N E A R Y N G A K V V S I A P D Y N A S S I H A D L W I G V N S GS D A A L G L S L A Q V I I E E K L H QP D F I R E Q T D L P L L V R E D N QQ Y L

peg.1309 S.denitrificans V GN P N Y T K I P E A H F F H E A R Y R G A KM C V I A P D L N P S S V H A DMWV K L R P E S D P A F G L A A A Q V I I E E K L Y K L D Y I L E Q T D F P F L V R K D N QR F L

peg.1642 S.denitrificans MM N P S V T Q I P D A H F L Y E A R Y N G T E L C V I D P Q Y S A T A I H A D QW L P L E S G T D A A L G L A V A R Y L L D T G A I D L P Y I R E Q T D L P L L A R L D T GR F L

peg.481 S.denitrificans MM N P S V T Q I P D A H F L Y E A R Y N G T E L C V V D P Q Y S A T A I H A D QW L P L E S G T D A A L G L A V A R H L F E V N A I D L P Y V R E Q T D L P L L A R L D T GR F L

peg.761 S.denitrificans V GN P I Y T R I P E A H F L H E A R Y R G A K L V V I A P D L N P S T V H A D T W I N I K P E T D A A L G L A A A Q V M I S E N L Y K Q D Y V L E Q T D F P F L V R K D N QR F L

peg.1646 S.denitrificans MM N P S V T Q I P D A H F L Y E A R Y N G T E L C V I D P Q Y S A T A I H A D QW L P I H T G T D A A L G L A V A R H L L E V G A I D L P F I R E Q T D L P L L V R I D S GR F L

EbdA-like D.oleovorans Hxd3 HM N P V Y T R I P S Y H F I S E A R Y N G A E I I S I A P D Y N P S CM H A D E Y I P V EM GS D A A L G L A V C Q V L M N K KWV D Y P F V K E Q S D L P L L V R K D T D R F L

EbdhA A.aromaticum C S NWS Y T Y P S S Y H F L S E A R Y K G A E V V V I A P D F N P T T P A A D L H V P V R V GS D A A F W L G L S Q V M I D E K L F D R Q F V C E Q T D L P L L V RM D T GK F L

EbdhA2 A.aromaticum C S NWS Y T Y P S S Y H F M T E A R Y K G A E V V V V A P D F N P T T P G A D L H V P V K V GR D A A F W L G L C Q V M I D E K L I D R Q F A S E Q T D L P L L V R T D N GK F L

EbdA-like gamma proteobachgter MM N P S V T Q I P D A H F L F E A K Y N G A T L T V I D P Q Y S A T A I H A D HWM P I E S G T D A A L AM Y V S R Y I W E N D R I D L P Y V K E Q T D L P M L V R I D N GR F L

370 380 390 400 410 420 430 440 450. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

C25dhA S.denitrificans R GA DM V K G GA D N A V Y I WD E A K Q A A V V A P GC E G D G E G GR S L K L N G I K P A L S G T F T V K L A N G E S V E V H T V F DM L K E K L D T E Y T P E L A E K V T G

peg.1060 S.denitrificans R T S D V V K G GV D N A F Y L WD E A K N A I V M A P GC E G D G N G GR S L K L GK L K P A L S G T R S V K L L D GS T V E C V T V F DM I R E R L D T E H T P E Q A A K I T G

peg.62 S.denitrificans R QR D L K K D GR E D V F Y V WD E K T R S L R Q A P - - - - - - - - QK S L A L GK L R P A L E G R Y QV T L P D GR K V F V Q T V F S R L R Q Q L D S RWK P E Q T A A T T G

peg.1309 S.denitrificans R A S D V V K G GD E N A L Y F WD E A T D KM V I A P GCM G D G D G GR S L K L GK L K P A L S G T R S V K L L D GS T V E C E T V F D K L K L K L D T E Y T P E Q S A K I A D

peg.1642 S.denitrificans R E S D L K A G GD E D Q L YMWH P Q K N A A V P A P GC L K N - - - T T R S L K L D F E P P I D G QWK I K L A N G E E V V V V P V G AM L K E H L - D P W T F E H A A QV T H

peg.481 S.denitrificans R E E D L K D G GS P D Q L YMWH P Q QN A P V P A P GC T G N - - - H T R K L T L D F E P P I D G QW T I K L K D G E E V A V V P I G AM L K E H L - E P W T F E H A A Q I T G

peg.761 S.denitrificans R A S D L V E G GA D N A L H V WD E A R N QA V I A P GC E G D G N G GR S L KM GG I K P A L S G T F S V K L V S G E T V E V Q T V F DM I K A R L D S E Y T P E Q A A R V T G

peg.1646 S.denitrificans R E E D L K D G GS P D Q L YMWH P Q K N A A V P A P GC T G N - - - T T R K L T L D F E P P I D G QW T I R L A N GQ E V L V A P V G A L L K E H L - E P W T F E H T A A V T K

EbdA-like D.oleovorans Hxd3 S A A D I E K G A R D D Q F C F WD S K N N K V V K A P - - - - - - - - L E T L K L - P C D P A L E G V Y K A T L L D GK T V E V E P V F N K L K A L L D S E Y T P E Q A S EM C R

EbdhA A.aromaticum S A E D V D - G G E A K Q F Y F F D E K A G S V R K A S - - - - - - - - R G T L K L - D FM P A L E G T F S A R L K N GK T I QV R T V F E G L R E H L - K D Y T P E K A S A K C G

EbdhA2 A.aromaticum S A A D V D - G GH A K Q F Y V I D E K S G A L R E A P - - - - - - - - R G T L R L - D G P V A L E G T F S A K L R D GG T V QV R P V F Q L M K D Q L D K E F T P E K A S A K S G

EbdA-like gamma proteobachgter T E Q D L Q K A GR T D V V Y YWD QN A G K P K L A S GS E G E L R H T R L H L D K D V D P A I E G I F QV QG A D GN V I H V T T V G S L I R E S L - E R Y T L D Y T A QV T K

S.denitrificans, S25dA3

S.denitrificans, EbdA-like

D.oleovorans, EbdA-like

S.denitrificans, S25dA






A.aromaticum, EbdA

strain HdN1, EbdA-like

A.aromaticum, EbdA2

HA CB CC CDB

10 20 30 40 50 60 70. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

C25dhA S.denitrificans - - - - - - - - - - - - - - - - - M Q I S R R Q F I V G S A V A A A G L G L Y S L R P K H Y V P A A P R P A D K - - - - - - - - - - - P G I

peg.1060 S.denitrificans - - - - - - - - - - - - - - - - - M Q V S R R H F I V G T A A V A A G A G L Y S L R P K L T G P K - P G I T M P - - - - - - - - - - - P L V

peg.62 S.denitrificans - - - - - - - - - - M E S K P GM I GM D R R S F L K - A GG S A L A L S L C H L E L L P M N AM A QA A GK D GN G N K A A A E L S P L E

peg.1309 S.denitrificans - - - - - - - - - - - - - - - - - M Q V S R R N F I V G S A V A A A G L G L Y S L K P K T P A A I K A G P A L P - - - - - - - - - - - P L V

peg.1642 S.denitrificans - - - - - - - - M G I L A T S N L V S A S R R K F L V M A G - - - M A S A A GA A V G L F G C S R A P L Q H F K G T - - - T A S G R F D L G

peg.481 S.denitrificans - - - - - - - - - M T T A S P A Q P N P A R R R F L I L A GK T T V A G I A A A A T G L P G C N RM P L Q H F H G - - - - T V D G R F D L G

peg.761 S.denitrificans - - - - - - - - - - - - - - M Q F M Q L T R R H F I M G S A A T V A G L A L Y S L R P R H Y V P A A P R P A D P - - - - - - - - - - - P T V

peg.1646 S.denitrificans - - - - - - M E R S S A S S T V G L S V S R R Q F L I K A G - - - L A S M A GG T L A L F G C H R A P L Q H F H G - - - - T M G G R F D L G

EbdA-like D.oleovorans Hxd3 - - - - - - - - - - - - - - M K E V K I S R R T F L K G T S A T V A L L S L N S L G F L GG N T I A N A T E K I - - - - - - - - - - - - - -

EbdhA A.aromaticum - M T R D EM I S V E P E A A E L QD Q H R R D F L K R S GA A V L S L S L S S L A T G V V P G F L K D A QA G - - - - - - - - - - - - - -

EbdhA2 A.aromaticum M D D L K N T D A I R T G V S S A F D Q N R R G F L K R S GA G A L S L S L S S F A A G L V P G F V N A A QA G - - - - - - - - - - - - - -

EbdA-like gamma proteobachgter - - - - - M T L GA GM G I L WK QK F D R R S F L K A S G - - - Y T V A A A A A V E L P - - - - - - S L H F K T - - - - - - - - A L A S D

80 90 100 110 120 130 140. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

C25dhA S.denitrificans P A K K V K Y N D Y S D I WR E KWKWD R V V K G T H T R - A N C I G A C S WD V Y V K D G I AWR E E QA A I Y E P H R P D I P D F N P

peg.1060 S.denitrificans P A K K V K Y N D Y S D I WR E KWKWD K V V K G T H T R - A N C V A A C S WD V Y V R D G I AWR E E QN T I Y E P P R P G I P D Q N P

peg.62 S.denitrificans A A A E L E Y R S F E D L Y R K KWHWD S V A K S T H F V N CW Y Q R N C S WN V Y V K N G I AWR E E QA A T Y E QV D P N V P D Y N P

peg.1309 S.denitrificans S A K K I R Y N D Y S D I WR E KWKWD K V V K G T H T R - A N C G D A C S WD V Y V R D G I AWR E E QN A I Y E P H R A D V P DM N P

peg.1642 S.denitrificans P R T T P K L G NWQ D L Y R Q RW T WD K V A K G S H GW - A N C R S A C EWD L Y V K D G V V V R E E QS A T Y E A S E P G I P D F N P

peg.481 S.denitrificans P R T T P K L N NWQ D L Y R Q RW T WD K V A K G S H GW - A N C R S A C EWD L Y V K D G V V V R E E QS A T Y E A S E P G I P D F N P

peg.761 S.denitrificans P A K K V K Y N D Y S D I WR E KWKWD R V V K G T H T R - A N C I A A C S WD V Y V R D G I AWR E E QN A I Y E P H R P D I P D F N P

peg.1646 S.denitrificans P R T T P K L G NWQ D L Y R Q RW T WD K V A K G S H GW - A N C R S A C EWD L Y V K D G I V V R E E QS A T Y E A S E P G V P D F N P

EbdA-like D.oleovorans Hxd3 - F E DWK Y A GW E N L H R E EW T WD K V T Y G T H L V D C Y P - G N C L WR V Y S K D G V V F R E E QA A K Y P V I D P S G P D F N P

EbdhA A.aromaticum - T K A P G Y A S W E D I Y R K EWKWD K V NWG S H L N I CWP Q G S C K F Y V Y V R N G I V WR E E QA A Q T P A C N V D Y V D Y N P

EbdhA2 A.aromaticum - K R G P T Y A TW E D V Y R N EWKWD K V T WG S H L N I CWP Q G S C K F Y V Y V R N G I V WR E E QA A Q T A A C N P D Y V D Y N P

EbdA-like gamma proteobachgter A A T P A P L K TW E D L Y R E RW T WD R V V K S S H GW - L N C R S A C EWD I Y V K D G V V V R E E Q T A T Y E A S E P G I P D F N P

150 160 170 180 190 200 210. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

C25dhA S.denitrificans R G C Q K GA C Y T H L Q V N E S R I K Y P L K R V G E R G E G KWK R I T WD E A L T E I A D K L I D A A V A E G T E S I I F D D G T T N

peg.1060 S.denitrificans R G C Q K GA C Y T T L Q L S E T R V K Y P L K R V G E R G E G KWK R I T WD E A L N E I A D K L I D I S V E H G T E T I C F D D - L S N

peg.62 S.denitrificans R G C Q K GA C Y S Q RM Y D A G R L T H P L R R V GA R G E G KWM R V S WD E A L A D I A D RM I D V M R T D G P GA I T WD P G T A N

peg.1309 S.denitrificans R G C Q K GA C Y T N L Q L S E A R L K Y P L K R V G E R G E G KWK R I S WD E A L N E I C D K L I D V A I D QG T E S I I F D D G T T N

peg.1642 S.denitrificans R G C Q K GA C Y T E V M Y GP S R T T V P L K R V GP R GS G KW E K I S W E Q A L R E I A V K T V D A A E KWG T D T I Y Q D L G P N F

peg.481 S.denitrificans R G C Q K GA C Y T E V M Y GP S R T T V P L K R V GP R GS G KW E K I S W E Q A L R E I A V K T V D A A E KWG T D T I Y Q D L G P N F

peg.761 S.denitrificans R G C Q K GA C Y T T L Q L S E A R L K Y P L K R I GK R G E G KWK R I T WD E A L T E I A D K L I D A A V A E G T E S I I F D D G T T N

peg.1646 S.denitrificans R G C Q K GA C Y T E V M Y GP S R T T V P L K R V GP R GS G KW E K I S W E Q A L R E I A V K T V D A V E E Y G T D T V F Q D L G P N F

EbdA-like D.oleovorans Hxd3 R G C Q K GA S Y S L QM Y N P D R L K Y P M K Q V GG R GS G KWK R V S WD Q C L A E I A E G I V D G L E A QG P E S I I F E S G P GN

EbdhA A.aromaticum L G C Q K GS A F N N N L Y GD E R V K Y P L K R V GK R G E G KWK R V S WD E A A G D I A D S I I D S F E A QG S D G F I L D A P H V H

EbdhA2 A.aromaticum S G C Q K GA A F N N N L Y G E E R L K Y P L K R V GK R G E G KWK R V S WD E A T A D I A D A I I D G I E T E G T D S F I L D S P H V H

EbdA-like gamma proteobachgter R G C Q K GA C Y T E V M Y GP S R L Y S P M K R V G E R GS G QW E K I S WD Q A L G E I A E K I V D I S E K Y G T D Y I I H DM G P H H

220 230 240 250 260 270 280. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

C25dhA S.denitrificans A G Y G P E T A GD V R F A T S L Q T T K I D S WA GV S DM P M G L V Q T WGM Y N C E G T S D DW F R S D Y I V I WV G N P I Y T R I P

peg.1060 S.denitrificans T G Y G P E T A GD F R F S T A L QV T R L D GWS GV G DM P L G V I Q T WG A F N C E G T S D DW F R S D Y I V I W L G N P N Y T R I P

peg.62 S.denitrificans A G GG A S T A - P Y R L G F I L D T P M I D V N T E V G D H H QG A Q V T V G K I S F S G S M D D L F Y S D L I L V WG A N P V Y T Q I P

peg.1309 S.denitrificans G G F G P E T A GD V R F T E A L N C T QM D S WA GV S DM P M G L V Q T WGM F N S E G S A D DW YM S D F I V I WV G N P N Y T K I P

peg.1642 S.denitrificans D - F G A S T A GR F K F Q F M A GG I F A D NWA E I G D L N V G A S I T V G A A H L GG S A D EW F L S D F I V V WMM N P S V T Q I P

peg.481 S.denitrificans D - F G P S T G GR F K F Q F Q V GG L F A D NWA E I G D L N I G A N I A L G A A H V GG S S D EW F L S D F I V V WMM N P S V T Q I P

peg.761 S.denitrificans S G Y G P E T S GDWR F A D A I QA T K I D S WA GV S DM P M G A V Q T WGM Y N C E G T S D DW F R S D Y I V I WV G N P I Y T R I P

peg.1646 S.denitrificans D - F G P S T A GR F K F M Y Q A S S L F S DMWG E I G D L N F G A TM A L G A A Q I GG S S D DW F L S D F I V V WMM N P S V T Q I P

EbdA-like D.oleovorans Hxd3 G G Y V H V M A - V H R L M V S L GA T V L D L D S T I G D F N R G I Y E T F G K F M F M D S V D GW Y F GK L L L I WHM N P V Y T R I P

EbdhA A.aromaticum A G S I AWG A - G F RM T Y L M D G V S P D I N V D I G D T YM G A F H T F G KM HM G Y S A D N L L D A E L I F M T C S NWS Y T Y P S

EbdhA2 A.aromaticum A G S V A N S G - G Y RM T Y L L D G V S P D N N V D I G D T Y S G A F H T F G KM HM G Y S A D N L L D S E L I F M T C S NWS Y T Y P S

EbdA-like gamma proteobachgter D - F G P T T A A R A R F F S M L GA S L A D DWA E I G D L N V A A TM T F G F P H V GG S S D EW F L S D Y L V V WMM N P S V T Q I P

290 300 310 320 330 340 350. . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . | . . . . |

C25dhA S.denitrificans E A H F L H E A R Y R G A K L V V I A P D L N P S T V H A D T W L K I N P E T D A A L G L A A A Q V M I T E N L I K K D Y V L E Q T DM P F

peg.1060 S.denitrificans D A H F L H E A R Y R G A K L V V V S P D L N A S T V H A D RW I K V K P E T D A A L G L A C A Q V M I A E D L Y K K D Y V L E Q T D F P F

peg.62 S.denitrificans N A H F I N E A R Y N G A K V V S I A P D Y N A S S I H A D L W I G V N S G S D A A L G L S L A Q V I I E E K L H Q P D F I R E Q T D L P L

peg.1309 S.denitrificans E A H F F H E A R Y R G A KM C V I A P D L N P S S V H A DMWV K L R P E S D P A F G L A A A Q V I I E E K L Y K L D Y I L E Q T D F P F

peg.1642 S.denitrificans D A H F L Y E A R Y N G T E L C V I D P Q Y S A T A I H A D QW L P L E S G T D A A L G L A V A R Y L L D T G A I D L P Y I R E Q T D L P L

peg.481 S.denitrificans D A H F L Y E A R Y N G T E L C V V D P Q Y S A T A I H A D QW L P L E S G T D A A L G L A V A R H L F E V N A I D L P Y V R E Q T D L P L

peg.761 S.denitrificans E A H F L H E A R Y R G A K L V V I A P D L N P S T V H A D T W I N I K P E T D A A L G L A A A Q V M I S E N L Y K Q D Y V L E Q T D F P F

peg.1646 S.denitrificans D A H F L Y E A R Y N G T E L C V I D P Q Y S A T A I H A D QW L P I H T G T D A A L G L A V A R H L L E V G A I D L P F I R E Q T D L P L

EbdA-like D.oleovorans Hxd3 S Y H F I S E A R Y N G A E I I S I A P D Y N P S CM H A D E Y I P V EM G S D A A L G L A V C Q V L M N K KWV D Y P F V K E Q S D L P L

EbdhA A.aromaticum S Y H F L S E A R Y K G A E V V V I A P D F N P T T P A A D L H V P V R V G S D A A F W L G L S Q V M I D E K L F D R Q F V C E Q T D L P L

EbdhA2 A.aromaticum S Y H F M T E A R Y K G A E V V V V A P D F N P T T P G A D L H V P V K V G R D A A F W L G L C Q V M I D E K L I D R Q F A S E Q T D L P L

EbdA-like gamma proteobachgter D A H F L F E A K Y N G A T L T V I D P Q Y S A T A I H A D HWM P I E S G T D A A L AM Y V S R Y I W E N D R I D L P Y V K E Q T D L P M

S.denitrificans, S25dA


S.denitrificans, EbdA-like






D.oleovorans, EbdA-like

A.aromaticum, EbdA

A.aromaticum, EbdA 2

strain HdN1, EbdA-like

A


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Fig. 6

FS IV

VACKTNKCIGCHTCS RICNHCTHPAC EACPR

MCFRFGRCRYPCAERACAQ-YCFICKQ GPCQR

FS IIIFS IIFS I


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Fig. 7

α3 (73%)S25dB (β)S25dC (γ)

S25dA (α)

α2 (82%)

γ7 (31%) β7 (59%) α7 (38%)

γ6 (31%) β6 (60%) α6 (38%)

γ5 (29%) β5 (59%) α5 (38%)

β4 (99%)γ4 (98%) α4 (71%) δ4

EbdC-like EbdB – like (59%) EbdA - like (41%) EbdD-like

(35%)


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Fig. 8

0.1

DmsA

BisC

TorA

DorA

TorA

PcrANarG NarG

ClrASerA

Type I

BisC

NapA / NasA FdnG

PhsA

PsrA

DdhA

γ-proteobacterium HdN1

S.denitrificans, S25dA2 Steroid C25

dehydrogenase

( like )

Ethylbenzene

dehydrogenase

( like )

Type III

Type II

DsrA


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Juri Dermer and Georg Fuchsatom in the side chain of cholesterol

Molybdoenzyme that catalyzes the anaerobic hydroxylation of tertiary carbon

published online September 1, 2012J. Biol. Chem.

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http://www.jbc.org/lookup/doi/10.1074/jbc.M112.407304

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;M112.407304v1&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2012/09/01/jbc.M112.407304

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=early/2012/09/01/jbc&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/early/2012/09/01/jbc.M112.407304

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/suppl/2012/09/01/M112.407304.DC1

http://www.jbc.org/

steroids represent a wide distributed and very diverse group of

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