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How lactobacilli synthesize inulinAnwar, Munir Ahmad

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Chapter 1

General Introduction:

Fructosyltransferase enzymes of lactic acid bacteria: current

advancements and emerging concepts

Munir A. Anwar, Slavko Kralj, Hans Leemhuis and Lubbert Dijkhuizen

Submitted for publication

Chapter 1

8

1. Lactic acid bacteria

Lactic acid bacteria (LAB) are a group of Gram positive bacteria that produce lactic

acid as a major product during carbohydrate fermentation. These organisms are

heterotrophic and generally have complex nutritional requirements because they lack many

biosynthetic capabilities. Most species have multiple requirements for amino acids and

vitamins, due to which lactic acid bacteria are generally abundant only in habitats where

these nutrients are available. Examples of such habitats are mucosal membranes of man and

animal (oral cavity, intestine and vagina), plants or material of plant origin, manure, and

man-made habitats such as sewage, spoiled food and fermenting food (131,195). The LAB

group encompasses the genera Lactobacillus, Streptococcus, Lactococcus, Leuconostoc,

Pediococcus, Aerococcus, Alloicoccus, Dolosigranulum, Enterococcus, Globicatella,

Lactospaera, Oenococcus, Carnobacterium, Tetragenococcus, Vagococcus and Weissella.

These microbes are widely used in the production of fermented food products, such as

yogurt (Streptococcus spp. and Lactobacillus spp.), cheeses (Lactococcus spp.), sauerkraut

(Leuconostoc spp.) and sausage (Lactobacillus curvatus, Lactobacillus sakei).

LAB are one of the industrially most important groups of bacteria. These

microorganisms are used in a variety of ways, such as food production, health improvement

and production of macromolecules, enzymes and metabolites (131). LAB maintain

remarkable importance in food industry due to their beneficial metabolic capabilities such as

carbohydrate and citrate metabolism, proteolysis, production of antimicrobials and the

production of an abundant variety of homo- and heteropolysaccharides

(35,36,42,69,116,180,193). These microorganisms play an important role in the fermentation

of food. Their growth lowers both the carbohydrate content of the foods that they ferment,

and the pH due to lactic acid production. This acidification process is one of the most

desirable side-effects of their growth and associated metabolic activities, which inhibits the

growth of most other microorganisms including most common human pathogens, thus

increasing the shelf life of foods. The acidity also changes the texture of the foods due to

precipitation of some proteins, and the biochemical conversions involved in growth enhance

the flavor and, frequently, the nutritional attributes of the products (7). Also LAB

antimicrobial peptides, called bacteriocins, have potential applications in food preservation

(31) and preventing pathogen infections (30). There is also a growing interest in LAB and

General Introduction

9

their polysaccharides because of the health benefits related to their probiotic and prebiotic

properties. A probiotic is as a viable microbial dietary supplement that beneficially affects

the host through its effects in the intestinal tract (138,172). Prebiotics are non-digestible food

ingredients that beneficially affect the host by selectively stimulating the growth and/or the

activity of one or a limited number of bacteria in the colon (44,131). LAB can also be used

to produce lactic acid (89) and B vitamins (15). To fully exploit their industrial potential,

LAB have been subjected to considerable research involving metabolic pathway engineering

and modeling (61,77,172). Comparative and functional genome analysis is used to obtain

global insights into physiological and metabolic capabilities of LAB (100,116,154,197). To

date, 25 LAB genomes have been sequenced and published (reviewed by Zhu et al. (197)),

while several more are in progress. Another recent comprehensive review encompasses

current knowledge about several interesting features of lactobacilli including genome-based

prediction of their extracellular proteome, comparative analysis of their genes and proteins,

and molecular interaction of their specific extracellular components with the host intestinal

system (76).

2. Exopolysaccharides of lactic acid bacteria

Some LAB, including species of Lactobacillus, are known to produce extracellular

polysaccharides often referred to as exopolysaccharides (EPS). These EPSs are large

molecules with molecular masses varying between 101 to 104 kDa, and may contain 50 to

50,000 glycosyl units. Depending on the composition of constituting glycosyl units, two

types of EPSs are produced by lactic acid bacteria, i.e. heteropolysaccharides, containing

different types of sugar moieties, and homopolysaccharides consisting of only one type of

glycopyranosyl residue.

Heteropolysaccharides are usually produced in low amounts and mostly glucose, galactose

and L-rhamnose are the main constituents (34). They can further be decorated with charged

groups like acetate, phosphate or glycerol phosphate. Heteropolysaccharides are produced by

many LAB such as Lactobacillus sakei (178), Lactobacillus casei (80), Lactobacillus

delbrueckii spp bulgaricus (21), Lactobacillus helveticus, Lactobacillus paracasei,

Lactobacillus acidophilus (139,140), Lactobacillus delbrueckii (51), Lactobacillus

Chapter 1

10

rhamnosus (98,99), Streptococcus salivarius ssp. thermophilus (1,22,50,103,162), and

different Lactococcus strains (20,78,111,176). Lactobacillus gasseri strain DSM 20243 was

also found to have a heteropolysaccharide gene cluster (2)(See also Chapter 3), but

heteropolysaccharide production by this strain has not been reported yet.

Homopolysaccharides of LAB consist of either glucose (polymer: α-glucan) or

fructose (polymer: β-fructan) residues with various binding types. They are synthesized by

extracellular enzymes of the sucrase type (see below), using a donor molecule, such as

sucrose, and an acceptor molecule, e.g. the growing polymer chain (109,182,197). The

enzymes responsible for the synthesis of glucose polymers are known as

glucosyltransferases (GTFs) or glucansucrases. The sucrase enzymes responsible for the

production of polymers consisting of fructose are referred to as fructosyltransferases (FTFs).

In the following paragraphs we mainly focus on fructans and FTF enzymes of lactic acid

bacteria and their (potential) applications.

3. Fructans

As mentioned above, fructans are homopolysaccharides comprising one type of

glycopyranosyl residues, i.e. fructose. Fructans are synthesized by microorganisms and

plants. Based on the type of bonds between the fructosyl residues, fructans can be

categorized as inulins, levans, mixed levans and neo-series inulins and levans (189). Inulins

and levans are linear or branched polymers of fructose, linked through β(2-1) and β(2-6)

bonds, respectively (Fig. 1). Occasionally, branches are attached to the main polymer chain

through β(2-1) linkages in levans and β(2-6) linkages in inulins (182). The fructans

synthesized from sucrose contain a terminal glucose molecule (40) and are designated as

GFn, where G refers to the terminal glucose unit, F refers to fructose units and n is the

number of fructosyl units per fructan molecule, also designated degree of polymerization

(DP). Short chain fructan oligosaccharides are usually labelled fructo-oligosaccharides

(FOS).

General Introduction

11

Fig.1. Structures of the fructan polysaccharides Inulin and Levan.

Inulin polymers from bacteria have a DP of 20-10,000 fructosyl units, while plant

inulins are relatively small polymers usually consisting of 30 to 150 fructosyl units per

fructan molecule (182). Similarly, bacterial levans have a DP > 100 fructosyl residues, while

plant levans usually have a DP < 100. In some cases, the molecular weight of the fructan

produced is dependent on bacterial growth/incubation conditions, e.g. the temperature,

salinity, and sucrose concentration used (12,58,167,168). Mixed levans, known as

gramminans, are produced by plants and have both β(2-1) and β(2-6) linked fructosyl

residues. Neo-series fructans are also produced by plants (189). In inulin and levan neo-

series, two inulin or levan type fructosyl chains are attached to the sucrose starter unit,

respectively. One chain is linked to the C1 and the other to the C6 of the glucose residue.

Fructans have a multitude of application for food and health, and in industry.

Possible applications of inulin and its oligosaccharides for food and health have been

Chapter 1

12

reviewed earlier (71). The probiotic role of several lactobacilli has been attributed to the

synthesis of prebiotic fructans from sucrose involving FTF enzymes (4,81,117). Inulin-type

fructans of higher degree of polymerization are of particular interest due to their

demonstrated pronounced in vitro prebiotic effects (177). In the food industry, inulin is also

used as fat replacer, and for providing texture and stability in several products, such as

desserts, bakery and fermented dairy products, as well as infant formula (138). Inulin

polymers also have potential application as surfactants. For instance, carbamoylated inulin

reduces the interfacial tension, thus providing a biodegradable surface-active agent (161).

Ievan was found to counteract inflammatory reactions to skin irritants in artificially

generated skin from human cell lines (3-D bio-artificial skin). Levan also exerted a cell-

proliferative effect in bio-artificial skin, thereby indicating its potential applicability as a

cosmeceutical agent (72). Levan also has found application as a biological glue

(http://www.polysaccharides.us/levanadhesive_summary.php). Populations of Paenibacillus

polymyxa are frequently found in the rhizosphere of various crop plants. High-molecular

weight levan produced by these bacteria is implicated in the aggregation of root-adhering

soil on wheat (14).

4. Physiological functions of fructans

Although the physiological roles of exopolysaccharides/fructans in bacteria have not

been clearly established yet, the available data suggest that these are related to the bacterial

ecological niches, providing protection against various environmental stresses, and serving

in energy storage. Most bacteria that synthesize fructans, interact with eukaryotic hosts and

their interactions with the environment are related to the presence of fructans (189). The

polymers may also render protection to microbial cells in their natural environment, against

desiccation, phagocytosis and phage attack, antibiotics or toxic compounds (e.g. toxic metal

ions, sulfur dioxide, ethanol), predation by protozoans, and osmotic stress. They may also

play a role in adhesion of cells to solid surfaces and biofilm formation, and also in cellular

recognition (20). Fructans (and glucans) may also contribute to the provision of reduced

oxygen tension, of strong relevance to facultatively anaerobic lactic acid bacteria, and

participate in the uptake of metal ions (20). Enhanced expression of FTF enzymes was

observed during growth of Lactobacillus reuteri under environmental conditions that affect

General Introduction

13

biophysical properties of the cytoplasmic membrane, indicating that fructans are involved in

resistance to certain stresses (148). Accordingly, it was shown in the same study that

fructans (inulin) protected cells of Lb. reuteri TMW1.106 during freezing and drying.

Streptococcus mutans and Streptococcus sanguis are commonly found in the oral

cavity, associated with dental plaque formation, and are therefore considered causal agents

of dental caries (9). Fructans (and glucans) may play a role in the adhesion and colonization

of cariogenic bacteria, thus contributing to the formation of dental plaque biofilm (143).

Other examples of fructans that function as adhesive agents and facilitate interactions

between bacteria and their hosts are (i) levan production by the sugar cane plant root

invading Gram-negative Gluconacetobacter diazotrophicus (164) and (ii) the persistence and

virulence of Actinomyces naeslundii and Actinomyces viscosus in the mammalian oral cavity

(13).

The physiological roles of microbial fructans also have been elucidated by analyzing

the effects of disruption of ftf genes. For instance, disruption of the ftf gene of Strep. mutans

strain V403 resulted in a decrease in virulence to about 70% of the cariogenic activity of the

wild-type strain (147). Erwinia amylovora is a pathogen of Pomoideae plants such as apple,

pear and rose. Studies involving disruption of the levansucrase gene of this bacterium have

suggested that levansucrase activity acts as a virulence factor in this pathogen (43,79).

Similarly, disruption of the levansucrase gene resulted in decrease in the severity of infection

of Pseudomonas syringae, which is a causal agent of blight disease in soybean leaves (94).

Lactobacillus spp. synthesizing glucan and fructan polymers are common inhabitants of the

mammalian and avian gastrointestinal tracts. Gene disruption studies have led to the

conclusion that the glucansucrase GtfA and inulosucrase Inu enzymes confer important

ecological attributes of Lb. reuteri TMW1.106 and contribute to colonization of the mouse

gastrointestinal tract (192).

In several Bacillus species and other bacterial strains within biofilms in oral cavities,

fructans also serve as extracellular carbohydrate storage compounds that can be metabolized

by bacteria during periods of nutrient deficiency (16,20,28,45). Also Lactobacillus paracasei

subsp. paracasei 8700:2 has been shown to metabolize inulin type

fructans/fructooligosaccharides producing lactic acid as the main end product (101). In

another study, Lb. gasseri CECT5714 and Lactobacillus fermentum CECT5716 were found

Chapter 1

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to metabolize 1-kestose (10). In our studies with Lb. johnsonii NCC 533 (3) (Chapter 2) and

Lb. gasseri strains DSM 20077, 20243, 20604, (2) (Chapter 3), we have not seen

degradation of inulin polymeric material.

A diverse number of naturally-occurring Lactobacillus species inhabit the

gastrointestinal (GI) tract of man and animals and are widely considered to exert a number of

beneficial effects on human health. Among these, Lactobacillus johnsonii strain NCC 533

belongs to the acidophilus group of intestinal lactobacilli that have been extensively studied

for their probiotic activities, pathogen inhibition, epithelial cell attachment, and

immunomodulation (32,47-49,64,74,115). The food company Nestlé uses this

microorganism in a yogurt-like dairy product called LC1, which, according to the company,

strengthens the body's natural defenses and keeps the bowel healthy (41). Lb. gasseri, which

is speculated to be an autochthonous human intestinal organism (8), constitutes the major

part of homofermentative Lactobacillus species occupying the human GI tract (88). Lb.

gasseri is associated with a variety of probiotic functions including reduction of activities of

fecal mutagenic enzymes (127), adherence to intestinal tissues (29,74) stimulation of

macrophages (73,171), and production of bacteriocins (65).

5. Fructan Synthesis

Fructans are synthesized by a wide variety of microorganisms and plants. Levans are

mainly synthesized by diverse types of Gram-positive and Gram-negative bacteria, whereas

inulin production is mainly limited to plants and a few species of lactic acid bacteria. Among

lactic acid bacteria, levan production has been reported for streptococci (19,54,155),

Leuconostoc mesenteroides (141), Lb. reuteri 121 (181,183), Lb. gasseri (2) (Chapter 3),

Lactobacillus sanfranciscensis (82), Lactobacillus panis (174,191). Also Lactobacillus

frumenti, Lactobacillus pontis and Weissella confusa were found to produce fructans, but the

linkage types in the fructan products have not been determined (102,174). Synthesis of β(2-

1) and/or β(2-6) linked fructooligosaccharides have also been reported in some fungi and

yeasts (60,67,91,136,187). Levan production has been studied in most detail in non-LAB, G.

diazotrophicus (56), Zymomonas mobilis (90,158), and Bacillus sp. (97,129,166). In contrast

to the wealth of information about levan synthesis, till recently only a few inulosucrase

enzymes were known, and no experimental evidence was available for in situ production of

General Introduction

15

inulin by lactic acid bacteria. We have identified Lb. johnsonii NCC 533 (Anwar et al.,

2008) and Lb. gasseri DSM 20604 (2) to produce inulin in situ (also see chapters 2 and 3).

6. Fructansucrase enzymes of bacterial origin

Whereas plants employ two consecutive FTF enzyme steps for fructan synthesis

(137,179), bacteria use a single FTF or fructansucrase (FS) enzyme for fructan synthesis.

Two types of fructan synthesizing enzymes are known, the inulosucrases, synthesizing β(2-

1) linked polymer inulin, and levansucrases, responsible for the synthesis of β(2-6) linked

polymer levan. Each of these enzymes is specific for the types of linkages that are

synthesized in their respective FOS and polymer products.

Levansucrase genes/enzymes have been found in a large variety of Gram-positive

(including LAB and non-LAB) and Gram-negative bacteria. Among LAB, levansucrase

enzymes have been characterized from Strep. salivarius (157), Strep. mutans (153), W.

confusa (102), Lb. reuteri (183,184), Lb. sanfranciscensis (82,173), Lb. panis (191), Leuc.

mesenteroides (70,110,121) and Lb. gasseri DSM 20077 (2) (chapter 3). Non-LAB with

levansucrase enzymes include Z. mobilis (53,158), Erwinia amylovora (43,52), G.

diazotrophicus (5), Bacillus polymyxa (14), Bacillus amyloliquefaciens (170), Geobacillus

stearothermophilus (97), Microbacterium laevaniformans (Park et al 2003), Bacillus

megaterium (62) and B. subtilis (160). In contrast to levansucrases, inulosucrases have been

characterized only from a few species of Gram-positive bacteria (all lactic acid bacteria

except Bacillus sp.) including Strep. mutans (142), Leuc. citreum CW28 (119,120), Lb.

reuteri 121 (186), Bacillus sp. (190), Lb. reuteri TMW 1.106 (149), Lb. johnsonii NCC 533

(3) and Lb. gasseri (2)(see also chapters 2 and 3). A phylogenetic tree of known FTF

proteins is shown in Fig. 2. Whereas FTFs of Gram-positive and Gram-negative bacteria are

clearly separate phylogenetically, no such clear separation is observed between inulosucrase

and levansucrase enzymes of Gram-positive bacteria. Nevertheless, the inulosucrases form a

small but distinct group indicating that there are small differences between these two groups

of closely related enzymes. Conversely, there should be additional similarities (or conserved

motifs) among these inulosucrase proteins that cause them to cluster together. Analysis of

such specific sequence features in inulosucrase enzymes may provide information about

their specific functional properties, i.e. ability to synthesize β(2,1) linkages.

Chapter 1

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Fig. 2. Phylogenetic tree of all bacterial fructosyltransferase (FTF) proteins (family GH68 proteins:

Lev, levansucrase; Inu, inulosucrase). FTF designation was used for proteins that have not been

characterized with respect to products synthesized. The FTFs characterized in this thesis are shown in

General Introduction

17

bold. Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 5

(165). Numbers at the nodes indicate confidence bootstrap percentages of 500 repeats. Scale bar

indicates number of changes per amino acid. Sequences are represented by the names of the

microorganisms followed by the GenBank accession number of the corresponding

fructosyltransferase protein. Branches represented by more than three proteins of the same species

were compressed and are composed of the following sequences: for Pseudomonas syringae

AAC36056, AAK49951, AAK49952, AAY35812, AAY37146, AAZ36063, AAZ38051,

AAC36063, AAZ37865, AAO54974, AAO55819, AAO59056; for Zymomonas mobilis AAA27702,

AAA27695, ACV75412, ACV75413, AAG29870; for Leuconostoc mesenteroides AAY19523,

AAT81165, ABJ62502, ABJ62503, ABJ62504. FTFs of various Burkholderia sp. that clustered

closely were also compressed to avoid complexity in the phylogenetic tree. These include:

Burkholderia cenocepacia ABF78985, ABK11005, CAR55261, Burkholderia mallei AAU46678,

ABN00553, ABO03647, ABM49108, Burkholderia pseudomallei ABN94007, ABA51542,

ABN86027, CAH38000; Burkholderia thailandensis ABC36154; Burkholderia vietnamiensis

ABO57793; Burkholderia phymatum AAC75109. FTF sequence of Lb. reuteri 100-23 was obtained

from http://genome.jgi-psf.org/lacro/lacro.home.html by “tblastn” using Lb. reuteri 121 levansucrase

as query sequence and Lb. panis levansucrase sequence was obtained from (191); no accession

numbers are available for these proteins.

7. Mechanism of fructan synthesis

Bacterial FTF enzymes belong to the Glycoside Hydrolase (GH) group of

carbohydrate active enzymes. Glycoside hydrolases have been classified into 115 families

(www.cazy.org) (18) on the basis of their amino acid sequence similarities, which imply

both structural and mechanistic relationships (55). The inulosucrase (E.C.2.4.1.9) and

levansucrase (E.C.2.4.1.10) enzymes belong to family 68 (GH 68) of carbohydrate active

enzymes. Family GH68 enzymes retain the configuration at the anomeric carbon in their

catalytic reactions. A ping-pong type reaction mechanism involving the formation of a

transient fructosyl-enzyme intermediate has been demonstrated for the B. subtilis (26), G.

diazotrophicus (56) and Strep. salivarius (156) FTF enzymes.

Chapter 1

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Fig 3. Schematic presentation of reactions catalyzed by FTF enzymes showing the involvement of

the three catalytic residues. This mechanism has been deduced taking into account the information

available at http://www.cazypedia.org/index.php/Glycoside_Hydrolase_Family_68, (133), (62),

(106) and (144).

In retaining enzymes, hydrolysis with net retention of configuration is most

commonly achieved via a two step, double-displacement mechanism involving a covalent

glycosyl-enzyme intermediate (Fig. 3). Each step passes through an oxocarbenium ion-like

transition state (http://www.cazypedia.org/index.php/Glycoside_Hydrolase_Family_68)

(133,144). Three invariant residues, including two aspartates and one glutamate, have been

identified essential for catalysis in family GH68 enzymes (see section 10.2.2 for details).

The reaction is catalyzed by one of the aspartates (acting as nucleophile) and the glutamate

General Introduction

19

(acid/base catalyst), while the second aspartate does not participate in the reaction but acts as

a transition state stabilizer. In the first step, the nucleophile attacks the anomeric carbon

while simultaneously the acid/base catalyst protonates the glycosidic oxygen forming an

oxocarbenium-ion like transition state, which subsequently ends in cleavage of the

glycosidic bond with the release of a glucose molecule and formation of fructosyl-enzyme

intermediate (Fig. 3). In the second step, the fuctosyl-enzyme is either hydrolyzed by water,

or the fructosyl unit is transferred to an acceptor molecule (e.g. sucrose) via oxocarbenium-

ion transition state. The transfer of a fructosyl unit to acceptors other than water (e.g.

sucrose, oligosaccharides, growing polymer chain etc.) is referred to as transglycosylation

activity, whereas its transfer to a water molecule results in hydrolytic activity. The sum of

transglycosylation and hydrolytic activities is called total activity. FTF enzyme activities are

measured by determining the amounts of glucose and fructose released from sucrose by the

enzyme. The amount of glucose (VG) formed reflects the total amount of sucrose utilized

during the reaction (total activity). The amount of fructose (VF) formed is a measure of the

hydrolytic activity. The transglycosylation activity is calculated by subtracting the amount of

free fructose from glucose (VG-VF).

Levansucrases also use various types of acceptor substrates other than sucrose and

water. For instance, B. subtilis levansucrase can use monosaccharides (D-glucose, D-

mannose, D-xylose, and D-galactose) as acceptor substrates to produce various di-, tri-, and

tetrasaccharides (with sucrose as donor substrate) (169). Another independent study with B.

subtilis levansucrase shows that, in addition to several monosaccharides, many disaccharides

such as maltose, isomaltose melibiose, cellobiose and lactose, can also be used as acceptor

substrates (150). Lb. sanfranciscensis levansucrase uses raffinose, maltotriose, maltose and

xylose as acceptors producing a range of heterooligosaccharides (175). Levansucrase from

Leuc. mesenteroides B-512 FMC can use maltose as an acceptor, producing erlose

(maltosylfructose: Glu-Glu-Fru) (70). This trisaccharide was also produced by B. subtilis

levansucrase using maltose as an acceptor (17,150). In addition, synthetic sucrose analogues

can be used as acceptor substrates in the FTF reaction to produce novel FOS, which have the

potential to be used as prebiotics or dietary fiber in food and health products, as well as

synthons to synthesize glycosylated drugs or pharmaceutical ingredients (84).

Chapter 1

20

8. Biochemical properties of bacterial FTF enzymes

A detailed survey of general kinetic properties of levansucrase and inulosucrase

enzymes has been presented recently (189). It appears that most levansucrases are

monomeric enzymes with a molecular weight of 46–73 kDa, with the exception of Rahnella

aquatilis JMC-1683 and Leuc. mesenteroides B-512 FMC levansucrases that form dimers

and have molecular weights of 120 kDa and 103 kDa, respectively (70,118). Optimum pH

values of levansucrases range from 5.0 to 6.2, while pI values range between 2.6 and 5.5. A

great variation is found in the sucrose Km values of levansucrases, which range from 4 to

160 mmol l−1 (189). The molecular weights of inulosucrases range from 83 to 170 kDa, their

pH optima range from 4.5 to 7.0 and pI values from 4.5 to 5.5 (2,3,120,186). The maximum

enzyme activity of most levansucrases lies between 28 and 55 ºC, except for Pseudomonas

syringae pv. phaseolicola and R. aquatilis enzymes that have temperature optima at 18 ºC

and 60 ºC, respectively (189). Inulosucrases display relatively high temperature optima, i.e.

from 45 ºC to 55 ºC. (2,189). No FTF enzymes have been characterized yet from

acidophilic, alkalophilic, or thermophilic bacteria.

A requirement for metal ions as cofactor has been reported for the Lb. reuteri, Leuc.

mesenteroides, B. subtilis, Strep. salivarius, Lb. panis and Lb. gasseri levansucrases, and Lb.

reuteri, Lb. johnsonii and Lb. gasseri inulosucrases, all requiring Ca2+ for optimum activity

(2,3,66,108,110,123,184). In the presence of Ca2+, Inu and Lev showed considerably

enhanced activities at high temperatures, and strongly increased thermostability. The activity

and stability of FTF enzymes of Gram-negative origin, such as the levansucrases of R.

aquatilis and P. syringae, apparently are not affected by the presence of calcium (58,118).

Available data shows that levansucrases are relatively more hydrolytic as compared

to inulosucrases: the Lb. reuteri and Lb. gasseri levansucrases for instance exhibited

transglycosylation/hydrolysis ratios of 2.6 and 0.2, respectively, whereas for the Lb. reuteri,

Lb. johnsonii and Lb. gasseri (InuG-BR) inulosucrases ratios of 4.3, 26.0 and 4.0 were

measured, respectively (2,3,124). The transglycosylation/hydrolysis ratio was also found to

be changed by immobilization of the enzyme on hydroxyapatite, mimicking in vivo

conditions where levansucrases are attached to the cell wall of bacteria or to tooth surfaces.

Immobilization of B. subtilis levansucrase shifted activity mainly to polymerization (25). For

several FTF enzymes it has been observed that higher sucrose substrate concentrations favor

General Introduction

21

transglycosylation activities (121,184,185). Also the presence of low molecular-weight

fructans was shown to accelerate the rate of polysaccharide formation and to increase the

fructan to free fructose product ratio (37,185).

Where studied, most FTF enzymes follow Michaelis-Menten type of kinetics for the

hydrolysis and transglycosylations reactions (173,184,191). Exceptions to this rule are the

Lb. reuteri inulosucrase and levansucrase enzymes, and the Lb. sanfranciscensis

levansucrase which cannot be saturated by their substrate sucrose (in case of the Lb. reuteri

levansucrase only at 50 °C) (173,184,185). This phenomenon has also been observed for

total and transfructosylation activities of the Lb. johnsonii and Lb. gasseri inulosucrases

(2,3)(See also chapters 2 and 3). The non-Michaelian behaviour of the Lb. reuteri 121 and

Lb. johnsonii inulosucrases has been attributed to the fructooligosaccharides that were

formed at the early stage of reaction (125) (3). FTF activity thus results in a range of

growing fructooligosaccharide chains and new fructan molecules, which in turn can be used

as acceptor substrates, complicating a detailed kinetic characterization.

9. Expression, secretion and folding of FTF proteins

Based on available data, induction of ftf gene expression seems to be strain

dependent and no uniform expression pattern has been observed among Gram-positive or

Gram-negative bacteria. For instance, whereas expression of B. subtilis (128), Lb. reuteri

(181) and Strep. mutans (96)levansucrases is reported to be induced by sucrose, the Strep.

salivarius FTF expression is not induced by sucrose; rather, sucrose stimulates release of the

FTF protein from the cell-wall (108), thus causing an increase in soluble FTF activity. A few

reports indicate that FTF expression was influenced by environmental pH, growth rate and

carbon source (63,75,96,194). A more recent study showed that expression of FTF was

negatively regulated by the covRS two-component regulatory system in Strep. mutans. It was

hypothesized that Strep. mutans senses the sucrose level in the environment, resulting in de-

repression of ftf transcription at low sucrose concentrations (93). Sugars like sucrose, D-

glucose, D-fructose, D-glucitol, D-mannitol and xylitol may enhance the expression level of

FTF enzyme in Strep. mutans (152). Induction of FTF enzyme expression may also be

independent of the carbohydrate source as in case of Act. naeslundii levansucrase (13).

Whereas the levansucrase gene of the Gram-negative G. diazotrophicus is expressed

Chapter 1

22

constitutively (56,57), a growth phase-dependent regulation of levansucrase expression has

been reported for another Gram-negative bacterium, R. aquatilis (151). Similarly, a more

recent transcriptional analysis study, using quantitative real time PCR, revealed that

expression of ftfA of Lb. reuteri LTH5448 was induced by sucrose, while sucrose had no

effect on gtfA and inu expression of Lb. reuteri strain TMW 1.106 (149).

Secretion of levansucrase proteins has most extensively been investigated in B.

subtilis and a two-step mechanism has been postulated for its translocation across the

membrane (Petit-Glatron et al., 1987). Two different transient forms of the levansucrase

protein, of 53,000-Mr and 50,000-Mr, were found to be located in the membrane

compartment of B. subtilis QB112 cells induced for synthesis of the extracellular form of the

levansucrase. The two step secretion mechanism postulates that the cellular events occur

according to the following sequence: (i) translation of the levansucrase mRNA into a

53,000-Mr protein which is attached to the cellular membrane, (ii) covalent modification

yielding a protein of 50,000-Mr, followed by (iii) secretion of the 50,000-Mr form.

Processing of the 53,000 Mr to the 50,000 Mr membrane-form of the protein involves

cleavage of signal peptide by signal peptidase. Pulse-labelling experiments indicated that the

second step of levansucrase translocation is an active transport step and it appears to be

directly linked to the influx of iron into the cell (23). Inside the cell, under cytosolic

conditions (pH 7, 37 ºC, and absence of free metals such as Ca2+ and Fe3+), the newly

synthesized protein remains unfolded. After spontaneous insertion into the membrane bilayer

and cleavage of the signal sequence, the protein is released extracellularly and refolded. Ca2+

and Fe3+ ions catalyze levansucrase folding outside the cell (130). By site-directed

mutagenesis, it was also shown in the same study that the residue G366 was involved in the

metal-dependent refolding of the protein after secretion by the organism.

The levansucrase (LsdA) of the Gram-negative bacterium G. diazotrophicus is also

secreted by a two-step mechanism, which is different from the B. subtilis levansucrase

translocation. This mechanism involves the removal of a 30-residue N-terminal signal

peptide during transport to the periplasm and transfer of the folded mature protein across the

outer membrane via a type II secretion system. The LsdA signal peptide has the essential

features of a Sec-dependent signal peptide (6). In contrast, other Gram-negative bacteria

secrete levansucrases by a signal-peptide-independent pathway (94,158,159,163).

General Introduction

23

10. Structural aspects of FTF enzymes

10.1. Three Dimensional Structures

Based on their common protein fold, some of the GH families are grouped at a higher

hierarchical level described as clans. Presently, 50 GH families have been grouped into 14

clans (A to N). Among these, the glycoside hydrolase family GH68 is grouped in Clan-J

together with the family GH32, which includes yeast and plant fructosyltransferases

(112,132), both families having 5-fold β-propeller topology. Whereas six 3D structures have

been resolved for different family GH32 proteins (reviewed by (92) and (27,188)) to date,

only two 3D structures have been published for family GH 68 members. Both of these are

levansucrase proteins: the B. subtilis levansucrase and the G. diazotrophicus levansucrase.

Initially, the high-resolution 3D structure for B. subtilis levansucrase (PDB ID:

1OYG) and a sucrose-bound inactive mutant (E342A) (PDB ID: 1PT2) of the same enzyme

were elucidated at a resolution of 1.5 Å and 2.1 Å, respectively (106). Recently, a raffinose

(Frc-Glc-Gal) bound complex with a mutated levansucrase (E342A) (PDB ID: 3BYN) has

also been resolved (107). The overall structure displays a rare five-fold β-propeller topology,

with the active site located at its center, in a deep, negatively charged pocket surrounded by

several conserved residues (Fig. 4 a). The fructosyl unit of sucrose is bound at the bottom of

this pocket (subsite -1, nomenclature according to Davies et al. (33)), with three absolutely

conserved acidic residues D86, D247, and E342 approaching it. The acid/base catalyst E342

forms a salt bridge with R246 and a strong hydrogen bond to the Tyr411 hydroxyl which, in

turn, forms a hydrogen bond with R360. R360 is located immediately adjacent to the central

pocket, in the vicinity of E342 and E340. Both R360 and E340 form tight H-bond with the

glucosyl moiety in the +1 subsite (Fig. 4 b). These specific contacts in the –1 and +1 subsite

lock the fructosyl and glycosyl moiety into a defined orientation, allowing catalysis. The –1

subsite is highly specific for accommodating fructose units, whereas the +1 binding site

might show more flexibility, allowing binding of glucose (sucrose or raffinose as donor

substrate) and fructose (sucrose or fructans as acceptor substrate) (107,124). The structure of

the mutant E342A complexed with raffinose revealed that the galactosyl moiety of raffinose

protruded out of the active site, while specificity-determining contacts were essentially

restricted to the sucrosyl (Frc-Glc) moiety (107).

Chapter 1

24

Fig. 4. The overall 3D structure of the B. subtilis levansucrase (1OYG), showing the three catalytic residues (a), and a closer view of the catalytic core with bound sucrose molecule (1PT2) showing +1/-1 subsite residues (b) (106).

The 3D structure of the levansucrase (LsdA) protein of the Gram-negative, non-LAB

G. diazotrophicus (PDB ID: 1W18) displays the same five-bladed β-propeller architecture in

which the catalytic residues are perfectly superimposable with their equivalents in the B.

subtilis levansucrase (104,105). It is worthy to mention that G. diazotrophicus levansucrase

contains E340 and R360, equivalent to N399 and H419, respectively, of B. subtilis

levansucrase.

The specific structural features in the levansucrase and inulosucrase enzymes

determing their glycosidic bond specificity remained to be determined. Only recently, a 3D

structure has become available for an inulosucrase protein. In collaboration with Tjaard

Pijning and Bauke Dijkstra (Biophysical Chemistry, University of Groningen) attempts to

crystallize available inulosucrase proteins were made. Properly diffracting crystals were

obtained with a truncated form of the Lb. johnsonii inulosucrase carrying a C-terminal His-

tag (InuJ145-708His), resulting in elucidation of a high (1.75 Å) resolution. The InuJ

inulosucrase structure shows the typical five-fold β-propeller topology of the catalytic core

domain (residues 210-662) present in levansucrases (Fig. 5) (see also chapter 4). Whereas

a b

General Introduction

25

the InuJ subsite -1 is virtually identical to that of SacB and LsdA, structural differences do

occur in the InuJ acceptor binding subsite architecture giving rise to an acceptor binding

pocket with different properties compared to levansucrase proteins. InuJ also contains a

unique helix (α7) and a unique 2-stranded antiparallel β-strand (β2-β3) packing against the

side of blade 1, which are absent in SacB and LsdA (for more details, see chapter 4).

Fig. 5. The overall 3D structure of the Lb. johnsonii inulosucrase (InuJ), showing the three catalytic

residues (see chapter 4 for more details).

10.2. Protein Assembly and structure-function Studies

Based on amino acid sequences, the microbial FTF enzymes can be divided into four

domains of secondary structure (Fig. 6): (i) a signal peptide, (ii) an N-terminal domain, (iii) a

catalytic core, and (iv) a C-terminal stretch, in some cases containing a cell-wall anchoring

binding domain. Characteristics for each domain are discussed below.

10.2.1. The signal peptide and N-terminal variable domain

Bacterial FTFs are extracellular enzymes, containing a secretion targeting signal

sequence of about 36 amino acids at the start of the N-terminus (Fig. 6). The signal-peptide

containing precursor is cleaved upon secretion of FTFs by Gram-positive bacteria

(13,14,97,134,145,160,170). The N-terminal domain of FTFs is variable in length and no

Chapter 1

26

function has yet been assigned to this domain. In Lb. sanfranciscensis TMW1.392

levansucrase, the N-terminal domain has a 16-amino-acid sequence

(DNATSGSTKQESSIAN) that is repeated seven times. However, truncation of these N-

terminal repeating units had no effects on the kinetic properties of the enzyme or on the

product spectrum produced from sucrose (173).

Fig. 6. Schematic presentation of lactic acid bacterial FTF proteins showing secondary structure

domain organization. The conserved amino acid sequence motifs with the 3 catalytic residues are

shown in sequence logos created by using WebLogo version 2.8.2 (www.weblogo.berkeley.edu).

Amino acid sequences of levansucrases of Strep. salivarius (AAA71925), Strep. mutans

(AAA88584), Lb. reuteri 121 (AAO14618), Lb. sanfranciscensis (CAD48195), Leuc. mesenteroides

(AAY19523), Lb. gasseri DSM 20077 (ACZ67287) and inulosucrase of Leuc. citreum CW28

(AAO25086), Lb. reuteri 121 (AAN05575), Lb. reuteri TMW 1.106 (CAL25302), Lb. johnsonii

NCC 533 (AAS08734), Lb. gasseri DSM 20243 (BK006921) and Lb. gasseri DSM 20604

(ACZ67286) were aligned with ClustalX (68) for creating sequence logos.

A signal peptide at the N-terminus domain of an FTF protein of a Gram-negative

bacterium has only been reported for the levansucrase of G. diazotrophicus. This signal

peptide is located in the first 30 amino acids, which are recognized by a type II secretion

system (6). With this exception, other FTF enzymes of Gram-negative origin apparently are

secreted by signal-peptide-independent pathways (6,43,59,158,159,163). FTFs of Gram-

General Introduction

27

negative bacteria have a conserved motif in the N-terminal domain, with the sequence

WT(R⁄I)ADA(L⁄M), which is also present in internal membrane-located proteins (163).

10.2.2. The core region

The catalytic domain of microbial FTFs consists of about 450 to 500 amino acids and

contains several highly conserved motifs. The functionally more important motifs of this

domain are shown in Table 1 (motifs I to IX). These motifs were deduced from amino acid

alignments made with ClustalX (68) and selected on the basis of available data depicting

their importance. Most FTF structure-function studies have focused on determining the roles

of amino acids in the catalytic domain. The 3D structure of the B. subtilis levansucrase has

provided important insights in functional roles of several conserved amino acid residues in

this core region (106). Firstly, all the GH68 family members contain an absolutely conserved

Asp residue in this domain (Table 1, motif I) corresponding to D86, which was identified as

the catalytic nucleophile based on the 3D structural analysis and site directed mutagenesis of

B. subtilis levansucrase and by the mutagenesis study of its corresponding residue (D272

Inu, D249 Lev) in Lb. reuteri FTFs mutants (106,126). Secondly, an RDP amino acid motif

(Table 1, motif V) is present in a conserved position in the core region of FTF enzymes.

Mutation of the aspartate amino acid residue in this motif of Strep. salivarius, G.

diazotrophicus, and Z. mobilis levansucrases resulted in a dramatic decrease in catalytic

activities while the affinities for their substrate sucrose remained almost unaltered

(11,156,196). Analysis of the 3D structure of B. subtilis SacB revealed that this D247

residue in the RDP motif, although required for catalysis, apparently is not directly involved

in the FTF reaction mechanism (i.e. bond making or bond breaking): It is involved in

stabilization of the transition state reaction intermediate (106). Thirdly, a drastic decrease in

catalytic activity of the Z. mobilis levansucrase was observed on mutation of E278 (196).

This amino acid residue is present in motif VI (glutamate adjacent to arginine) (Table 1).

Analysis of the sucrose-bound structure of a B. subtilis levansucrase inactive mutant

(E342A) revealed that the E342 residue, corresponding to the Z. mobilis E278 residue,

functions as a catalytic general acid. The nucleophile, general acid/base catalyst and

transition state stabilizer residues have also been identified in B. megaterium levansucrase by

site-directed mutagenesis (62).

Chapter 1

28

Table 1. Functionally important conserved motifs of selected bacterial FTFs

Two further highly conserved regions (Table 1, motifs II and IV) among FTFs have

been designated as “sucrose binding boxes” (146). In the E117Q mutant (motif II) of Z.

mobilis levansucrase the catalytic turnover was similar to that of the wild-type enzyme,

while greater transfructosylating activity was observed (196), indicating direct involvement

of motif II in catalysis of transfructosylation. Based on the analysis of the activity of site

directed mutants D312E/S/N/I/K located in motif III (Table 1) of Strep. salivarius

levansucrase, it has been suggested that this Asp residue is most likely involved in

determining acceptor recognition, or stabilization of a β-turn in the protein (156). The 3D

structure of the B. subtilis levansucrase shows that D312 indeed forms a 180º reverse β-turn

between motifs II and IV and is located on the surface of the protein, far from the active site

(106).

The coordination geometry and the protein-metal distances in the B. subtilis

levansucrase 3D structure suggest the presence of a Ca2+ site, as expected from biochemical

studies (106). The residue that is involved in Ca2+ binding in B. subtilis levansucrase has

been identified as D339, and equivalent residues have been proposed as D520 and D500 for

the Lb. reuteri Inu and Lev proteins, respectively (123). Sequence alignments of GH68

proteins revealed that residues involved in calcium binding are conserved in most enzymes

of Gram-positive bacteria, but are absent in proteins of Gram-negative bacteria (123,182). A

disulphide bridge in FTFs of Gram-negative bacteria may serve the same purpose, as has

been observed in the 3D structure of the levansucrase of G. diazotrophicus. It has been

suggested that these features have a role in the maintenance of protein fold stability (104).

General Introduction

29

Functional roles of some other conserved amino acids present in the catalytic core of

FTF enzymes have also been determined by site-directed mutagenesis. For instance, a

spontaneous mutation R331H in levansucrase of a mutant B. subtilis strain QB252 yielded

an enzyme with decreased polymerase activity relative to hydrolysis. Reverting this mutation

(H331R) by site-directed mutagenesis restored polymerase activity to the level observed for

the wild-type enzyme (24). Further, introducing the mutations R331K, R331S, and R331L at

this position resulted in the loss of ability of the enzyme to synthesize levan from sucrose as

a single substrate. It was only able to catalyze the first step of levan synthesis, i.e. production

of the trisaccharide kestose (GF2). These studies lead to the conclusion that the nature of the

amino acid at this position modulates the specificity and the efficiency of the

transfructosylation process (24). It should be mentioned that this R331 residue is equivalent

to R360 of B. subtilis levansucrase, whose 3D structure has been elucidated (section 10.1).

R360 is conserved in levansucrases from Gram-positive bacteria, whereas a histidine is

present at the equivalent position in levansucrases from Gram-negative bacteria. Replacing

the R360 counterpart in Z. mobilis levansucrase (H296) with arginine led to a reduction in

transfructosylation activity to 20% of the wild-type level and accumulation of a small

amount of oligosaccharides (196). On the basis of modeling and site-directed mutagenesis

experiments it was proposed that the corresponding H296 in Z. mobilis acts as an acceptor

substrate recognition and binding site (95). However, subsequently the Z. mobilis

levansucrase was found to exist in two active forms- micro-fibrils and dimer, depending

upon the pH and ionic strength. It was suggested that H296 might be a crucial amino acid for

generating enzyme micro-fibrils associated with the capacity for levan polymerization (46).

A H296Arg mutant indeed failed to form levanuscrase micro-fibrils and levan polymers.

Comparison of 3D structures of G. diazotrophicus (LsdA) and B. subtilis (SacB)

levansucrases revealed that both FTF enzymes possess identical active sites. Nevertheless,

LsdA synthesizes a large amount of FOS (1-kestotriose) while SacB produces mainly large

levan polymer (104). The mechanism underlying these differences has not been identified.

Subsequent site-directed mutagenesis studies with Inu of Lb. reuteri 121 showed that amino

acid residues (W271, W340 and R423) located at the (putative) -1 donor substrate sugar

binding subsite (selected on basis of the 3D structure of the B. subtilis levansucrase) play a

crucial role in determining size of the products synthesized (FOS versus inulin polymeric

Chapter 1

30

material) (124). Site-directed mutagenesis experiments with the B. megaterium levansucrase

indicated that N252 and R370, constituting the +2 subsite, are critical for the polymer versus

oligosaccharide product ratio of the enzyme (62). Recently, the crystal structure of the

S164A B. subtilis levansucrase mutant (PDB ID: 2VDT) has been elucidated. S164 was

found to be important in maintaining the position of the nucleophile in the active site.

Furthermore, site-directed mutagenesis experiments showed that R360, Y429, R433 (Table

1, motifs VII and VIII) have important roles in acceptor substrate specificity. These amino

acids have direct or indirect (through a water molecule) interaction with sucrose. The

mutants R360S, Y429N and R433A were clearly more hydrolytic than the wild-type

levansucrase and produced exclusively oligosaccharides (122). These mutations may have

reduced the affinity of +2, +3 and further subsites for the growing polymer chain, in line

with the proposal of Ozimek et al (125) that polymerization is a processive reaction

requiring a high enzyme affinity for the polymer at higher subsites.

A more detailed understanding of structure-function relationships in FTFs would

enable rational design of biocatalysts with increased specificity for the synthesis of tailored

polysaccharide and FOS products for a range of different applications. In this perspective, it

would be interesting to know what structural features in FTF enzymes determine a) their

specificity for synthesis of either β(2-6) or β(2-1) linkages, and b) the sizes of fructan

products synthesized. As levansucrase and inulosucrase enzymes of lactic acid bacteria are

closely related (>60 similarity), the difference in linkages of their fructan products moast

likely is based on a limited number of amino acid differences. At present, the structural and

mechanistic basis of product (β(2-6) vs β(2-1)) and reaction (transglycosylation/hydrolysis

ratio) specificities in these enzymes is still not known. In addition, only limited experimental

data about type and size distribution of FOS products synthesized by FTF enzymes from

sucrose and related substrates is available.

In analogy to FTF enzymes, glucansucrase (GTF) enzymes (belonging to family

GH70) catalyze hydrolysis and transglycosylation reactions on sucrose, leading to the

production of free glucose and fructose, glucooligosaccharides and glucose polymers (α-

glucans) with different linkage types between glucose units (83,109,182). Among various

types of lactobacterial glucan polymers, reuteran is mainly composed of α(1-4) glycosidic

bonds, whereas dextran is predominantly composed of α(1-6) linkages; these glucan

General Introduction

31

polymers are synthesized by the closely related reuteransucrase and dextransucrase

enzymes, respectively (85,86). Based on amino acid sequence alignments identifying

residues potentially involved in linkage type determination, and subsequent site-directed

mutagenesis, a reuteransucrase was transformed into a dextransucrases type of enzyme by

rational design (87). We adopted a similar strategy with Lb. reuteri 121 Inu to determine the

functional importance of amino acid residues which are well conserved in inulosucrases but

not in levansucrases. These residues have never been mutated before. Our data (chapter 5)

demonstrated that mutations in these Inu amino acid residues located in blade 4, including

the unique helix α7 (compared with the Lb. johnsonii NC 533 InuJ 3D structure, chapter 4),

had a clear effect on transglycosylation activity and the FOS product profile. Particularly,

mutation of N543, which forms a hydrogen bond to the adjacent arginine R544 (present near

subsites +1 and/or +2), synthesized less FOS compared to wild type (chapter 5). Introducing

mutation G416E, located at the rim of the active site pocket, increased hydrolytic activity 2-

fold, without significantly changing transglycosylation activity. Mutation A538S, located

behind the general acid/base, increased the total activity 2.5-fold. These results demonstrated

that the product specificity of Inu is easily altered by protein engineering, obtaining Inu

variants with higher transglycosylation specificity, higher catalytic rates and different FOS

oligosaccharide size distributions (chapter 5).

It has been speculated that addition of a fructosyl residue to sucrose by the action of a

levansucrase results in the formation of 6-kestose (O-β-D-fructofuranosyl-(2-6)-β-D-

fructofuranosyl-(2-1)-α-D-glucopyranoside), the first intermediate of levan biosynthesis

(189). However, B. megaterium levansucrase is the only FTF for which 6-kestose has been

characterized as one of the products synthesized (62), whereas 1-kestose (O-β-D-

fructofuranosyl-(2-1)-β-D-fructofuranosyl-(2-1)-α-D-glucopyranoside) has been shown to be

produced from sucrose by the levansucrase enzymes of Z. mobilis (Crittenden & Doelle

1993), B. subtilis C4 (38), Lb. sanfranciscensis LTH2590 (82) and Lb. sanfranciscensis

TMW 1.392 (173). Interestingly, the B. megaterium levansucrase data clearly shows the

consumption of 1-kestose and apparent accumulation of 6-kestose (62). Also the Lb. reuteri

levansucrase utilizes 1-kestose, producing long chain FOS (125). On the basis of this data, it

can be hypothesized that at least in the first step, levansucrases produce 1-kestose, which is

also synthesized by inulosucrases (2,3,125). The main structural differences between

Chapter 1

32

levansucrase and inulosucrases therefore maybe found in the +2, +3 or higher (if present,

remain to be identified) acceptor substrate subsites, which guide the subsequent binding of

fructosyl units to the growing polymer chain (FOS) in such a way that addition occurs either

with a β(2-6) or β(2-1) bond, producing levan or inulin, respectively. This kind of structural

difference between Inu and Lev has also been depicted schematically by Ozimek et al

(125)(Fig. 7), which shows that the -1 subsite is specific for binding of fructosyl residues

only whereas +1, +2 and +3 subsites are flexible to bind various types of acceptor substrates.

As families GH68 and GH32 share similar protein folds, it would be worthy to mention here

a recent report on the 3D structure of Aspergillus japonicus fructosyltransferase (a GH32

enzyme) with bound substrate molecules, where the residues Y404 and E405 have been

shown to contribute to the +2/+3 subsites and speculated to be responsible for the formation

of the inulin-type FOSs(27).

Fig. 7. Schematic representation of bound acceptor molecule in an FTF enzyme. The differences in

affinity between Inu and Lev at the +2 and +3 subsites are shown by a shallow cleft (dark grey; low

affinity), and a deep cleft (light grey; high affinity), respectively. Sugar binding subsites are either

shown in white (-1 subsite) reflecting specific and constant affinity for binding of fructosyl residues

only, or in light/dark grey (+1, +2, +3 subsites) reflecting their ability to bind either fructosyl,

glucosyl (with GFn substrate) or galactosyl (with raffinose) residues. Adopted from (125).

10.2.3. The C-terminal domain

The C-terminal region of FTF enzymes is variable in length and often contain a cell-

wall-binding domain. Although the function of the C-terminal region of FTFs has not been

studied extensively, suggestions for a possible function for this region have been obtained in

studies with the levansucrase gene of B. subtilis (23). For instance, read through of a stop

codon enabled a downstream region to enlarge the gene at the 3’-end. Changing this stop

codon into a glutamine codon yielded a mutant B. subtilis levansucrase protein that had

General Introduction

33

become enlarged by 3 kDa. This resulted in an enzyme that synthesized levan of molecular

weight higher than 50 × 106 Da, whereas the molecular weight of levan synthesized by the

native levansucrase was mainly lower than 5 × 105 Da. The increase in levan molecular

weight was mainly attributed to an increase in the number of branches in the levan.

Moreover, the extended enzyme was able to form an active dimer from two polypeptide

chains linked by an S-S bridge (23). In contrast, the truncation of the C-terminus of

inulosucrase of Lb. reuteri 121 showed no effect on product formation (186).

The cell-wall-binding domains in the C-terminal regions of cell-wall-anchored

proteins from Gram-positive bacteria typically contain (i) a spacer region (50 to 125 amino

acid residues) rich in P/G and / or T/S residues, (ii) an LPXTG sortase recognition motif, and

(iii) a stretch of hydrophobic amino acids containing PXX repeats (39,114,173), and (iv) two

to three positively charged amino acids (L, R, and H) at the C-terminus.

The motif LPXTG is well conserved in the cell wall associated proteins of Gram-

positive bacteria (39,184,186,191). Among FTF proteins it is mostly present in the enzymes

from lactobacilli (Table 1, motif IX). Although the LPXTG cell-wall anchoring motif was

found in both levansucrase and inulosucrase of Lb. reuteri 121 (184,186), neither the

presence of inulosucrase in culture supernatants nor actual cell-wall association of the

inulosucrase have been demonstrated in cultures grown in sucrose containing medium.

Contrarily, levansucrase was found cell-wall associated during growth of Lb. reuteri 121 on

glucose and was found predominantly in the supernatant when grown in sucrose containing

medium (181). The cell-wall associated Strep. salivarius FTF protein contains a C-terminal

region that resembles the cell-wall anchoring motif but lacks the actual LPXTG amino acids

(135). A slightly different motif (LPKAG) is found in the FTFs of Lb. johnsonii NCC 533

and Lb. gasseri DSM 20243 and 20077 (2,3). However, the inulosucrase protein of Lb.

gasseri DSM 20604 terminated abruptly and lacked the cell wall anchoring LPXTG/LPKAG

motif. The impact of lacking this motif was clearly apparent from the tendency of the

enzyme to be secreted into the growth medium, in contrast to the L. gasseri 20077

levansucrase, which was exclusively detected associated with the cell surface (2).

The stretch of hydrophobic residues acts as a membrane-spanning region with the

positively charged amino acid residues directed towards the cytosol. The protein is located

outside the cytoplasmic membrane with an N-terminal signal sequence attached to the

Chapter 1

34

membrane. After proteolytic cleavage of the signal sequence by a signal peptidase (SPase)

the remaining N-terminal part of the protein, spaced by the P/G and /or T/S rich region, is

directed outwards of the cell. Subsequently, the LPXTG motif is proteolytically cleaved

between the Thr and the Gly residues by a sortase enzyme and covalently linked to the

peptidoglycan layer (113,114).

An FTF with unusual structure is the inulosucrase (IslA) of Leuc. citreum CW28

(120). It is a naturally occurring chimeric enzyme resulting from the substitution of the

catalytic domain of alternansucrase with that of a fructosyltransferase. Its N-terminal region

therefore is similar to the variable region of GTF enzymes, its catalytic domain is similar to

the core region of FTFs from various bacteria, and its C-terminal domain presents similarity

to the glucan binding domain (GBD) from alternansucrase (119). Evidence concerning the

role of the glucan binding C-terminal region in determining enzyme activity and stability

was obtained from the characterization of the inulosucrase gene (islA) from Leuc. citreum

CW28. Truncation of the C-terminal domain resulted in an increase in total activity and

hydrolysis/transglycosylation ratio, and decrease in thermal stability of the enzyme (119).

11. Scope of this thesis

The studies described in this PhD thesis deal with fructansucrase or

fructosyltransferase (FTF) enzymes (family GH68; www.cazy.org) from probiotic

lactobacilli. At the start of these studies, only a few inulosucrase enzymes had been

characterized, compared to the relatively large number of levansucrase enzymes that were

known already. As a consequence, structural features responsible for the different linkage

types and (sizes of the) products synthesized by levansucrase and inulosucrase enzymes,

remained elusive. Moreover, a 3D structure had not been resolved for an inulosucrase

protein yet, whereas two high resolution 3D structures had become available for

levansucrase proteins from Gram-positive and Gram-negative bacteria, including structures

with bound substrate molecules (sucrose, raffinose). Previous structure-function studies with

FTF enzymes had focused on the functional roles of amino acid residues that are highly

conserved in both the levansucrase and inulosucrase enzymes. The aims of the research

described in this thesis therefore were (i) characterization of FTF proteins, and in particular

inulosucrases, from probiotic lactobacilli, (ii) identification of conserved residues in

General Introduction

35

inulosucrase proteins, differing in levansucrase proteins, constituting targets for site-directed

mutagenesis, (iii) introduction of point mutations in selected residues and analysis of the

effects on enzyme biochemical properties, (iv) over-expression, purification and

crystallization of inulosucrase proteins to resolve their 3D structures by X-ray

crystallographic studies.

Chapter 1 reviews current knowledge about fructansucrase enzymes, their biochemical

properties, domain organization and 3D protein architecture.

Chapter 2 describes the cloning of the inulosucrase gene of Lb. johnsonii NCC 533. A

detailed biochemical characterization of this enzyme (InuJ) is presented. Lb. johnsonii NCC

533 is the first Lactobacillus strain proven to produce inulin and FOS in situ.

In Chapter 3 we describe the isolation of 3 ftf genes from three different Lb. gasseri strains

and molecular and biochemical characterization of the FTF (inulosucrase and levansucrase)

enzymes encoded by these genes. Lb. gasseri DSM 20604 is the second Lactobacillus strain

shown to produce inulin polymer and FOS in situ, and is unique in its distribution of FOS

synthesized, ranging from DP2 to DP13.

Chapter 4 reports the first high resolution 3D structure of native and mutant Lb. johnsonii

NCC533 inulosucrase InuJ proteins.

Chapter 5 presents the characteristics of mutant Lb. reuteri 121 inulosucrase proteins

produced by introducing mutations in amino acid residues that are highly conserved in

inulosucrases but differing in levansucrases.

Chapter 1

36

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