Biologia 63/6: 1020—1027, 2008 Section Cellular and Molecular Biology DOI: 10.2478/s11756-008-0166-0 Review

Bacterial : properties and structural studies

Moez Rhimi,RichardHaser & Nushin Aghajari*

Laboratoire de BioCristallographie, Institut de Biologie et Chimie des Protéines, UMR 5086–CNRS/Université de Lyon, IFR128 “BioSciences Gerland – Lyon Sud”, 7 Passage du Vercors, F-69367 Lyon cedex 07, France; e-mail: [email protected]

Abstract: Due to their significant role in food industry, sucrose isomerases are good candidates for rational protein engineering. Hence, specific modifications in order to modify affinity and selectivity, specificity but also to adapt their catalytic properties to particular industrial process conditions, is interesting. Our work on the structural studies of the sucrose , MutB, which presents the first structural data available on a synthase and the first experimental data on complexed forms of sucrose isomerases represents a significant advance in the understanding of these . In this review we give an overview of what is known on biochemical properties and structural aspects of different sucrose isomerases in particular those reported from bacteria. Key words: sucrose isomerases; trehalulose; ; immobilized cells; structural studies.

Introduction (Fig. 1) are structural of sucrose and are natu- rally present in in very low quantities (Low & Sucrose, α-d-glucopyranosyl-(1,2)-β-d-fructofuranosi- Sporns 1988). These natural sugars display a sweet- de, is the most widespread translocated ening power, bulk, organoleptic and physical proper- by plants (Bieniawska et al. 2007). High amounts of ties similar to those of sucrose (Takazoe 1989; Hamada this sugar are stored in the edible parts of several 2002). Furthermore, several characteristics make these plants, making it the mainly abundant natural sweet- two isomers advantageous over sucrose for some ap- ener (Salvucci 2003). Currently, sucrose is widely used plications in food industry. In fact, isomaltulose (also in many agro-alimentary products. However, this dis- known as palatinose) and trehalulose are non-cariogenic accharide is efficiently metabolized, which highly influ- and have low glycaemic indexes by decreasing the rate ences the glycaemia of the body and promotes den- with which monosaccharides and insulin are released tal caries (Sissons et al. 2007). Such effects are un- in blood, promoting their use for diabetics (Yamada et desirable for health and especially for those suffering al. 1985). These sugars were reported to induce a se- from obesity and diabetes (Saris 2003; Schulze et al. lective promotion of growth of beneficial bifidobacteria 2004). Nowadays we assist in an increasing consumer in human intestinal microflora and to be highly stable demand for low caloric food products especially in the in foods and beverages (Birch & Wu 2007). The safety case of diabetes and low caloric diets, the actual chal- of isomaltulose and trehalulose was also checked, and lenge is to set out sweeteners having a low caloric con- these natural sugars were without side effects and ap- tent combined with intense taste. Artificial sweeteners proved as being safe for use in human food (Goda et as, e.g., aspartame, cyclamate and saccharine were re- al. 1988). Thus, these can be used as a ported to be efficient sucrose substitutes (Renwick 2006; sucrose substitute in foods, soft drinks and medicines Cardoso & Bolini 2007). While these artificial com- (Takazoe 1989). Due to its reducing properties, iso- pounds have a high sweetening power, they are among maltulose is attractive as an industrial precursor for the most controversial food additives due to allegations the manufacturing of biosurfactants and biocompatible of adverse health effects including dermatological prob- polymers (Lichtenthaler & Peters 2004). lems, headaches, mood variations, cardiopulmonary dif- In nature, trehalulose and isomaltulose, which act ficulties, seizures, and cancer (Hertelendy et al. 1993; as a reserve material during periods of low carbon avail- Ravaud et al. 2007). Thus, industrials focused on the ability in many bacteria, are present in low quantities use of natural and hypo-caloric sweeteners having a and their chemical synthesis is not easy (Ravaud et al. high sweetness value. 2007; and references therein). Fortunately these disac- Isomaltulose (6-o-α-d-glucopyranosyl-d-) charides can be produced at an industrial scale through and trehalulose (1-o-α-d-glucopyranosyl-d-fructose) isomerization of sucrose using sucrose isomerases which

* Corresponding author

c 2008 Institute of Molecular Biology, Slovak Academy of Sciences Bacterial sucrose isomerases: properties and structural studies 1021

Fig. 1. Isomerization and hydrolysis of sucrose as catalyzed by sucrose isomerases. bio-convert sucrose into both isomaltulose and trehalu- less rapidly cleaved in the small intestine than sucrose. lose. Numerous microorganisms have been recognized This causes an attenuated blood and insulin for their ability to produce sucrose isomerase with dif- response, a property which is very attractive for dia- ferent levels of isomaltulose and trehalulose production. betics (Kawai et al. 1985; Yamada et al. 1985). It is Sucrose isomerases, also called sucrose or α- worth noting that pH-telemetry studies, which are the glucosyltransferases (EC 5.4.99.11), belong to the gly- internationally recognized standard for tooth-friendly coside (GH) family 13 of retaining enzymes claims, proved the non-cariogenicity of palatinose (iso- (Henrissat 1991). These family 13 proteins display a maltulose) (Lina et al. 2002); trehalulose being also re- number of conserved residues (described ported to be “tooth friendly” (Hamada 2002). More- later in the text), and act by a retaining double dis- over, also prebiotic (bifidogenic) properties of isomal- placement mechanism via a covalent glycosyl- tulose on bifidobacteria were reported (Mattes et al. intermediate, and yielding the α-anomeric products. 1998). For these reasons palatinose and trehalulose ap- Structural studies of sucrose isomerases have been pear as ideal substitutes for sucrose in several agro- reported for the PalI from Kleb- alimentary and pharmaceutical applications. siella sp. LX3 (Zhang et al. 2003a,b), for the Pseu- Sucrose isomerases from Agrobacterium radiobac- domonas mesoacidophila MX-45 trehalulose synthase, ter MX-232 and Pseudomonas mesoacidophila MX- MutB (Ravaud et al. 2005, 2007) and for the isomal- 45 produce primarily trehalulose with yields evalu- tulose synthase SmuA from Protaminobacter rubrum ated to 91% (Miyata et al. 1992; Nagai-Miyata et al. (Ravaud et al. 2006). In order to gain detailed in- 1993), whereas enzymes from Pantoea dispersa, Er- sights into the catalytic mechanism, crystal structures winia rhapontici, Serratia plymuthica, Klebsiella sp. of MutB (native and mutant enzymes) in complex with LX3 and Enterobacter sp. FMB1 produced essen- substrates, substrate analogues and inhibitors have tially isomaltulose with percentages around 75–85% been solved and reported (Ravaud et al. 2007). (Cheetham 1984; Veronese & Perlot 1999; Zhang et al. 2002; Wu & Birch 2005). Due to difficulties in the chem- Properties and production of isomaltulose and ical synthesis of these sugars, their microbial production trehalulose is of commercial interest. Many bacterial strains can convert sucrose to isomaltulose and/or trehalulose in Isomaltulose and trehalulose are occurring naturally in different ratios by using their sucrose isomerases. The honey and sugar cane juice. The taste and appearance enzymes from Protaminobacter rubrum (Yamada et al. of these disaccharides are similar to sucrose, while their 1985), Serratia plymuthica (Krastanov & Yoshida 2003; sweetening power is around 30% and 70%, when com- Krastanov et al. 2006), Erwinia rhapontici (Krastanov pared to that of sucrose, for palatinose and trehalu- & Yoshida 2003) and Enterobacter sp. FMB1 (Cho et al. lose, respectively (Cheetham et al. 1982; Salvucci 2003). 2007) produce essentially isomaltulose (75–90%), minor Viscosities of aqueous solutions and organoleptic prop- quantities of trehalulose and trace amounts of fructose erties of these sugars are quite similar to that of su- and glucose. Also several Klebsiella species are known crose (Mattes et al. 1998). In addition hereto, toxic- to produce 60–70% palatinose (Krastanov & Yoshida ity analysis revealed that isomaltulose and trehalulose, 2003), while Pseudomonas mesoacidophila MX-45 and as sucrose, are non-toxic (Kawai et al. 1985; Jonker Agrobacterium radiobacter MX-332 strains convert su- et al. 2002). As concerns their physicochemical prop- crose mainly to trehalulose (∼90%), small amounts of erties, these sucrose isomers appear adequate for sub- isomaltulose and trace amounts of the monosaccharides stitution of sucrose in mainly sweet foods. Interestingly, (Cheetham et al. 1982). isomaltulose and trehalulose are hardly fermented and Production of palatinose from sucrose by using im- 1022 M. Rhimi et al.

Table 1. Biochemical properties of sucrose isomerase reported in the literature.

Organisms Topt pHopt pI Km Iso- Tre- Specific activity Reference ( ◦C) (mM) maltulosea halulosea (U/mg)

Klebsiella pneumoniae NK33-98-8 30 6.0 NR 42.7 77 33 2362 Aroonnual et al. (2007) Pantoea dispersa UQ68J 30–35 5.0 7.0 40 91 3 562 Wu & Birch (2005) Klebsiella planticola UQ14S 35 6.0 NR 76 66 15 351 Wu & Birch (2005) Klebsiella sp. LX3 35 6.0 6.6 55 83 21 328 Zhang et al. (2002) Klebsiella sp. 35 6.0 NR 120 86 NR NR Park et al. (1996) Serratia plymuthica ATCC15928 30 6.2 9.0 65 73 9 NR Veronese & Perlot (1999) Erwinia rhaponthici NCPPB 1578 30 7.0 NR 280 85 15 4 Cheetham (1984) Protaminobacter rubrum CBS574.77 30 5.5 9.9 140 86 9 NR Nakajima (1984) Pseudomonas mesoacidophila MX-45 40 5.8 5.4 19 8 91 14 Nagai et al. (1994) a Percentage of sucrose conversion into the given di-saccharide (%). NR: not reported. mobilized Protaminobacter rubrum cells, with a palati- UQ68J, Klebsiella sp. LX3, Protaminobacter rubrum nose:trehalulose:monosaccharides ratio of 86:9:5 was CBS574.77, Erwinia rhapontici NCPPB 1578 and Ser- reported (Weiden Hagen & Lorenz 1957). More recently ratia plymuthica ATCC15928. In this context, it was re- it was reported that when using immobilized Serratia ported that temperature influences the ratio of formed plymuthica cells in chitosan, a nearly complete conver- monosaccharides, but did not change the final amount sion (99%) of a 40% sucrose solution into palatinose was of formed sugars. Moreover, higher temperatures favor achieved in 4 h in a batch-type enzyme reactor (Kras- the hydrolytic reaction generating glucose and fructose, tanov et al. 2006). Using free and/or immobilized cells whereas the lower temperatures increase the amount of had no impact on the composition of the end-products trehalulose production (Nagai et al. 1994; Veronese & solution, which contained 80% isomaltulose and 7% tre- Perlot 1999; Zhang et al. 2002). The optimal pHs of the halulose (Krastanov et al. 2006). With these immobi- majority of the sucrose isomerases characterized to date lized cells, Krastanov et al. (2006) reported the effect of are comprised between 6.0 and 7.0 (Table 1). Zhang et the inlet sucrose concentration (30–70%), the reaction al (2003a) reported that the reaction end-products were temperature of 30–60 ◦C and the residence time on the clearly related to the charge of the catalytic pocket. specific volumetric productivity. Whereas a residence Thus, operating at pHs higher than the enzymes opti- time of 1.7–3.0 h was sufficient for 98–100% conversion mal pH allow the formation of trehalulose, whereas at of sucrose at substrate concentrations of 30–50%, the lower pH the production of glucose and fructose was highest palatinose content was achieved (94%), when a promoted (Nagai et al. 1994; Zhang et al. 2002; Wu 40% inlet sucrose solution was used. & Birch 2005). The kinetic parameters of various su- Furthermore, when Serratia plymuthica cells were crose isomerases are presented in Table 1. Interestingly, immobilized in a hollow-fibre membrane reactor, a com- analysis of the various enzymes affinities for sucrose in- plete conversion of concentrated sucrose solutions into dicates that substrate recognition was clearly variable. palatinose was achieved (Krastanov et al. 2007). By us- For example, the Km value reported for the sucrose ing these immobilized cells a bioconversion rate of over isomerase from Pseudomonas mesoacidophila MX-45, 90% was reached in 36–48 h for all concentrations (40– MutB, was 14-fold higher than that of the Erwinia 60%) of the substrate solution. Microbiological tests of rhapontici NCPPB 1578 enzyme (Cheetham 1984; Na- the produced palatinose syrup showed that the product gai et al. 1994). meets standard requirements for this kind of products Until now very little has been reported concern- in order to be used as an additive in different food prod- ing the enhancement of thermoactivity, thermostability ucts (Krastanov et al. 2007). and catalytic efficiency of sucrose isomerases (Zhang et As concerns the production of trehalulose on an al. 2002). For industrial applications it is of interest to industrial scale, immobilized cells of the bacteria Pseu- operate with biocatalysts being thermoactive and ther- domonas mesoacidophila MX-45 was used (Nagai et al. mostable. Taking advantage of the quite recent knowl- 2003). When a 40% sucrose solution was supplied to edge obtained from structural studies of sucrose iso- the reactor, approximately 83% of the substrate was merases, use of protein engineering appears to be a converted into trehalulose (Nagai et al. 2003). powerful tool to gain further insights on the structure- function relationships of these enzymes with the aim of Biochemical properties of sucrose isomerases creating biocatalysts more suitable for diverse biotech- nological applications. For efficient production of isomaltulose and trehalu- lose using sucrose isomerases, the reaction conditions Inhibitions studies of sucrose isomerases such as pH and temperature should be optimal. In Ta- ble 1 the optimal reaction conditions of reported bac- Ravaud et al. (2007) reported the effect of several terial sucrose isomerases are listed. The optimal tem- α-glucosidase inhibitors such as castanospermine, de- perature of sucrose conversion is ranged between 30– oxynojirimycin and acarbose on the MutB activity. Ow- 40 ◦C, such as those derived from Pantoea dispersa ing to this study castanospermine was a more potent in- Bacterial sucrose isomerases: properties and structural studies 1023

Fig. 2. Primary structure alignment of a selection of sucrose isomerases: Erwinia rhapontici (Erwinia), Protaminobacter rubrum (SmuA), Klebsiella sp. LX3 (PalI), Klebsiella planticola (Klebsiella) which are all isomaltulose synthases, and Pseudomonas mesoaci- dophila MX-45 (MutB) which is a trehalulose synthase. Secondary structure elements deduced from the three-dimensional structures of PalI and MutB, respectively, are indicated above the alignment. hibitor than deoxynojirimycin, while acarbose did not it was also tested and found to be more weakly bound affect the activity. Concerning Tris, which inhibits some (Ravaud et al. 2007) than castanospermine and de- GH13 enzymes (Aghajari et al. 1998; Skov et al. 2001), oxynojirimycin. 1024 M. Rhimi et al.

terminal domain in a solvent accessible loop and is sit- uated approximately 22 A˚ from the active site (Ravaud et al. 2007). The structure of PalI lacks this ion (Zhang et al. 2003a) as does the native SmuA structure (S. Ravaud et al., manuscript in preparation), whereas a calcium ion was observed in the crystal structure of a complex with deoxynojirymycin (S. Ravaud et al., man- uscript in preparation). Besides the above mentioned crystal structures, three-dimensional structures of MutB have been deter- mined in complex with the competitive inhibitors Tris, deoxynojirimycin and castanospermine (Ravaud et al. 2005, 2007). Furthermore, the three-dimensional struc- tures of MutB inactive mutants, D200A and E254Q, were determined in complex with d-glucose and with its natural substrate sucrose, respectively (Ravaud et al. 2007). Therefore description of the structural studies of sucrose isomerases will be done on the basis of this enzyme hereafter. The active site of MutB is pocket- shaped and implies 14 residues, among which Asp200, Glu254, Asp327, Tyr64, His104 and His326 are con- served in the active sites of other GH13 enzymes. Five additional residues (Asp61, Phe145, Phe164, Gln168 and Arg414) are conserved in Neisseria polysaccharea amylosucrase, in Bacillus cereus oligo-1,6-glucosidase and in the isomaltulose synthases PalI and SmuA, all members of the so-called oligo-1,6-glucosidase sub- family (Oslancova & Janecek 2002). Finally, Phe144, Asn328 and Glu386 are specific for the sucrose iso- merases PalI, SmuA and MutB. This topology, which Fig. 3. Overall structure of sucrose isomerases: superimposition also is characteristic of amylosucrase, PalI and oligo- ofMutBinredonPalIinblue. 1,6-glucosidase, was considered as a crucial determinant of the substrate specificity of amylosucrase for sucrose (Skov et al. 2001). The orientations of the catalytic Structural studies of sucrose isomerases residues are guided by Arg198, Asp384 and Arg284 also present in the pocket and which stabilize the active site The sucrose isomerases PalI from Klebsiella sp. LX3 (Ravaud et al. 2007). (Zhang et al. 2003a), MutB from Pseudomonas meso- At the entrance of the pocket a pair of pheny- acidophila MX45 (Ravaud et al. 2005) and SmuA from lalanines, Phe256 and Phe280 presenting an “aromatic Protaminobacter rubrum (Ravaud et al. 2006) have clamp”, are situated which forms a gateway approx- been crystallized and their three-dimensional structures imately 11 A˚ above the bottom of the pocket, and solved (Zhang et al. 2003a; Ravaud et al. 2005, 2007). thereby limits the reducing end of the substrate bind- All three-dimensional structures solved are highly sim- ing site to sub-site +1. The bottom of the pocket is ilar monomers of 557 amino acids for MutB, 598 amino formed by Tyr64 and a salt bridge (Asp200-Arg198), acids for PalI and 573 amino acids for SmuA. With a and the non-reducing end is thus delimited to a sin- topology being similar to that observed in the vast ma- gle sub-site, −1. Sub-site +1 is directly accessible to jority of GH13 enzyme structures solved to date, they the solvent through the active site entrance channel display three domains (Fig. 3) with the N-terminal do- and is presumed to serve as exit. Further details on main which also is the major and central domain having the three-dimensional structures of sucrose isomerases a(β/α)8-barrel super secondary structure (domain A can be found in Ravaud et al. (2007). in other GH13 proteins). This N-terminal domain dif- fers from the classical TIM-barrel (Banner et al. 1975); Reaction mechanism see Ravaud et al. (2007) for further details. A short loop rich sub-domain (domain B in other GH13 en- All GH13 enzymes proceed using a two-step double- zyme structures) also comprises an α-helix, a 310-helix displacement mechanism which includes formation and and three anti-parallel β-strands, and protrudes from breakdown of a covalent glucosyl-enzyme intermediate the (β/α)8-barrel between Nβ3andNα3. Domain C is (Davies & Henrissat 1995; Jensen et al. 2004; Mosi et made up of two antiparallel β-sheets as in other three- al. 1997). Both steps are suggested to be carried out dimensional structures of GH13 enzymes. In all MutB via oxocarbenium ion transition states (Ravaud et al. structures, a calcium ion is bound to the catalytic N- 2007). Bacterial sucrose isomerases: properties and structural studies 1025

The first studies concerning the sucrose isomerase ization rate of sucrose during the reaction. Ravaud et reaction mechanism were reported by Cheetham (1984) al. (2007) proposed that the glucose moiety of tre- and Veronese & Perlot (1998, 1999) who proposed a re- halulose most probably will bind in a chair conforma- action mechanism for the Erwinia rhapontici sucrose tion in subsite −1 as seen in the tertiary structures isomerase and similar enzymes from Serratia plymuth- of the reported MutB complexes with the exception of ica and Protaminobacter rubrum. The suggested mech- MutB/castanospermine, and that the differences in the anism was based on the intramolecular rearrangement interaction pattern between the enzyme and sucrose of sucrose that simultaneously produces the two differ- isomers are rather to be found in the acceptor sub- ent isomerisation products (isomaltulose and trehalu- site. Zhang et al. (2003a) reported that a unique motif lose). By studying the way in which the glucose moiety “RLDRD” (residues 325–329, PalI numbering) found from sucrose was transferred to an exogenous acceptor, in the isomaltulose synthase (PalI), and other sucrose the authors deduced that a free hydroxyl on the C6 isomerases producing predominantly isomaltulose, was of glucose is required for activity since the crucial for determining whether isomaltulose or trehalu- 6-chloro and 6-deoxy-analogs have efficient inhibitory lose was formed. This sequence which is rich in charged effects (Veronese & Perlot 1999). Furthermore, it was residues and situated in the vicinity of the active site demonstrated that the 6-fluoro-deoxyglucose inhibited was subjected to site-directed mutagenesis studies, and both the Erwinia rhapontici isomaltulose synthase and the authors found that the PalI mutants D327N and a whitefly (Bemisia argentifolii) trehalulose synthase, R329N in particular decreased the percentage of isomal- indicating a common requirement for a free hydroxyl tulose by 43–52% and increased the trehalulose content on the glucose moiety (Salvucci 2003). In the reac- by 41–54% (Zhang et al. 2003b). tion mechanism proposed by Veronese & Perlot (1998), Based on the mutagenesis studies of the F256A/ once the sucrose is cleaved the glucose moi- F280A double-mutant (Ravaud et al. 2007), which re- ety remains tightly bound to the enzyme active site sulted in an enzyme displaying entirely hydrolytic activ- while fructose is only loosely bound. Consistent with ity, it was proposed that these mutations which destroy this proposal, incubation of the enzyme with equimo- the aromatic clamp (described earlier), would result in lar concentrations of free fructose and sucrose increased a catalytic pocket being continuously open, thus allow- the proportion of trehalulose synthesized by the enzyme ing the hydrolyzed glucose to diffuse rapidly away as (Veronese & Perlot 1999). Recently, due to the solution opposed to the clamp, which locks the glucose moiety of crystal structures of MutB, producing in majority in the enzyme intermediate and prevents release of the trehalulose, in complex with the substrate sucrose, sub- fructosyl ring. Moreover, it was proposed (Ravaud et strate analogs and inhibitors (Ravaud et al. 2007), de- al. 2007) that while mutants of these aromatic residues tailed information on the reaction mechanism was pro- were active, the nature and position of the aromatic vided, in particular of the first step in the isomerisation residues modulate the reaction specificity by altering mechanism. In the E254Q/sucrose structure where the the volume of the pocket and hence the space for tau- catalytic acid/base Glu254 was mutated, thus resulting tomerization. Therefore, the authors concluded that the in an inactive enzyme, an image on how the substrate is aromatic clamp phenylalanines are implicated both in bound in the active site is given. The three-dimensional substrate recognition and partially in controlling the structure of this complex furthermore illustrated the reaction specificity (Ravaud et al. 2007). An additional role of this glutamate as a proton donor; see Ravaud role attributed to this aromatic clamp was its implica- et al. (2007) for details. A complex between MutB and tion in entry as well as release to/from the active site the inhibitor castanospermine was thought to give an and thus in isomerase versus hydrolytic activity of the image of the precursor of the transition state in that enzyme. castanospermine adopted a half-chair conformation as Finally it was reported (Ravaud et al. 2007), based reported in previous studies (Strynadka & James 1991). on the solved structures of the Pseudomonas mesoaci- A mimic of the oxocarbenium ion transition state was dophila MX-45 trehalulose synthase, that mobile re- revealed by the MutB/deoxynojirimycin complex, and gions probably have important role during the enzy- finally information on the glycosyl-enzyme intermedi- matic reaction. The concerned regions were the loops ate was illustrated by a three-dimensional structure of between Nβ4andNα4, between Nβ6andNα6(which the inactive mutant of the catalytic nucleophile, D200A, contains the so-called “isomerization motif” described in complex with glucose; for details, see Ravaud et al. for PalI and discussed earlier), and between Nβ8and (2007). Nα8’, which includes η8 (Ravaud et al. 2007). Interest- Reaction steps succeeding the covalent glucosyl- ingly, examination of MutB, SmuA and PalI surfaces enzyme intermediate are more speculative. As a con- presented as a function of charge revealed that MutB sequence of the low number of interactions between is more negatively charged on the face displaying the the fructosyl ring and the enzyme active site, it was entrance of the catalytic pocket. Furthermore, analysis proposed that this feature may allow this sugar moi- of the surface distribution of invariant residues between ety to be displaced, to rotate, and/or to tautomerize MutB, SmuA and PalI revealed that most of the non- generating the two isomers, isomaltulose and trehalu- conserved residues are located at the surface of the en- lose (Ravaud et al. 2007). It should be noticed that zyme on this side, which may contribute to explain the trehalulose formation highly depends on the tautomer- distinct charge distributions between the two enzymes 1026 M. Rhimi et al.

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Received February 17, 2008 Accepted May 26, 2008