Function and Structure Studies of GH Family 31 and 97 \Alpha

$Function and Structure Studies of GH Family 31 and 97 \Alpha$

110610 (RV-17)

Biosci. Biotechnol. Biochem., 75 (12), 110610-1–9, 2011 Award Review Function and Structure Studies of GH Family 31 and 97 -Glycosidases

Masayuki OKUYAMA

Research Faculty of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo 060-8589, Japan

Online Publication, December 7, 2011 [doi:10.1271/bbb.110610]

A huge number of glycoside hydrolases are classified an evolutionary relationship of proteins in the family, into the glycoside hydrolase family (GH family) based from which it is often possible to extract information on on their amino-acid sequence similarity. The glycoside function and structure.3) Classification in a GH family hydrolases acting on -glucosidic linkage are in GH has, accordingly, become indispensable for research on family 4, 13, 15, 31, 63, 97, and 122. This review deals glycoside hydrolases. Glycoside hydrolases are also mainly with findings on GH family 31 and 97 enzymes. divided into two mechanistic classes, inverting and Research on two GH family 31 enzymes is described: retaining enzymes: with inversion or net retention of clarification of the substrate recognition of Escherichia anomeric configuration during the catalytic reaction.4) coli -xylosidase, and glycosynthase derived from Schiz- The stereochemical outcome is generally conserved in a osaccharomyces pombe -glucosidase. GH family 97 is GH family. The inverting mechanism proceeds via a an aberrant GH family, containing inverting and simple single displacement. Two functional groups, retainingAdvance glycoside hydrolases. The inverting View enzyme usually carboxyl groups, act as general acid and general in GH family 97 displays significant similarity to base catalysts. The general acid catalyst donates a proton retaining -glycosidases, including GH family 97 retain- to the departure aglycon, and the general base catalyst ing -glycosidase, but the inverting enzyme has no simultaneously deprotonates the incoming water mole- catalytic nucleophile residue. It appears that a catalytic cule, which attacks the anomeric carbon (Fig. 1A). Most nucleophile has been eliminated during the molecular retaining glycosidases occur though a double-displace- evolution in the same way as a man-made nucleophile ment mechanism with two functional groups, which mutant enzyme, which catalyzes the inverting reaction, serve catalytic nucleophilic and catalytic acid/base. as in glycosynthase and chemical rescue. Although these functional groups mostly exist as carboxyl groups, sialidases use a phenolic hydroxyl Key words: glycoside hydrolase family 31; glycoside group of tyrosineProofs as a catalytic nucleophile.5) The hydrolase family 97; -xylosidase; -glyco- double-displace mechanism goes through two stages, synthase; divergence of catalytic residue glycosylation and deglycosylation (Fig. 1B). At the glycosylation step, the general acid/base catalyst proto- Carbohydrates are the most abundant organic mole- nates the glycosidic oxygen with bond cleavage, while cules in nature. It is traditionally thought that carbohy- the nucleophilic catalyst attacks the anomeric carbon drates are a significant fraction of the energy in the diet, from the opposite side of the glycosidic linkage and a storage form of energy, and structural components forms a covalent glycosyl-enzyme intermediate. At the such as cell walls of many organisms, the exoskeleton of deglycosylation step, the incoming water deprotonated insects, and the fibrous cellulose of plants. However, by the acid/base catalyst breaks down the intermediate carbohydrates, involved in glycoproteins and glycoli- by attacking the anomeric carbon from the opposite side pids, are now known to have essential roles in biological of the covalent bond of the intermediate. These inverting processes, including the mediation of some forms of and retaining mechanisms involve oxacarbenium-ion- intercellular communication. Glycoside hydrolases cat- like transition states. Recently, several interesting varia- alyze the hydrolysis of glycosidic linkages in such tions on the retaining mechanisms that go through a carbohydrates as glycosides, oligosaccharides, glycans, different transition state have been identified. Enzymes and glycoconjugates to obtain energy and to modify the that hydrolyze substrates containing an N-acetyl group at carbohydrates. the C2-position occasionally adopt the substrate-assisted A huge number of glycoside hydrolases have been mechanism, which goes through an oxazoline inter- discovered, and these are classified into glycoside mediate.6) These enzymes have no catalytic nucleophile hydrolase families (GH families) based on their residue, but the 2-acetamido group of the substrate acts amino-acid sequence similarity.1,2) Currently more than as an intramolecular nucleophile. Glycoside hydrolases 100 GH families are recognized. They are available that employ the aforementioned mechanisms catalyze through the website, Carbohydrate Active enZymes single or double substitution reactions. On the other (http://www.cazy.org/). Classifying glycoside hydrolases hand, the catalytic mechanism of GH family 4 and 109 is important because it indicates a common ancestor and glycoside hydrolases is distinctly different and includes

This review was written in response to the author’s receipt of the Japan Society for Bioscience, Biotechnology, and Agrochemistry Award for the Encouragement of Young Scientists in 2010. Correspondence: Fax: +81-11-706-2808; E-mail: [email protected] 110610-2 M. OKUYAMA

b Advance View

Fig. 1. General Mechanism of Inverting Glycosidases (a) and General Mechanism of Retaining Glycosidases (b). elimination and redox steps.7,8) This mechanism requires amylosucrase, oligo-1,6-glucosidase, and sucrose iso- NADþ, divalent metal cations, and reducing conditions, merase as well as -glucosidase. Although their catalytic and proceeds via anionic transition states. Bacillus activities are diverse, the tertiary structure and catalytic subtilis 6-phospho--glucosidase (GlvA), a member of residues are conserved in this family.11) The enzymes GH family 4, also hydrolyzes 6-phospho -glucosides possess a ð=Þ8Proofsbarrel fold as a catalytic domain and containing an activated leaving group with inversion.9) conserve catalytically significant residues at the rim of This unique specificity has been explained by a differ- the -barrel. The family employs the double-displace- ence in essential catalytic residues in GH family 4 ment mechanism to catalyze the reaction and preserves enzymes as compared with the typical glycosidases the catalytic nucleophile and the acid/base residues at that employ the standard oxocarbenium ion mecha- the ends of -strands 4 and 5 respectively. GH family 15 nism.9) While typical enzymes have essential catalytic includes glucan 1,4--glucosidase, so-called glucoamy- residues around the scissile bond, GH family 4 enzymes lase. The glucoamylase hydrolyzes terminal (1!4)- bear functional residues around positions, C2 and C3. linked -glucose residues from the non-reducing ends of The loose interaction of GlvA around the scissile bond the chains with release of -glucose; that is, the is responsible for GlvA being able to hydrolyze - glucoamylase is an inverting enzyme. The most of glycosides. glucoamylases are multi-domain enzymes consisting of Glycoside hydrolases acting on -glucose residues of a catalytic domain and a starch-binding domain.12) The the non-reducing end, so-called -glucosidases and catalytic domain folds as a ð=Þ6-barrel, while the glucoamylases, are found in GH family 4, 13, 15, 31, starch-binding domain folds as an antiparallel -barrel. 63, 97, and 122. Additionally, in a broad sense, GH A funnel-shaped active site is formed by long loops family 37 and 65 ,-trehalases are also included in this connecting the -helices. There has been considerable category (Table 1). Among them, GH family 31 and 97 research on the structure-function relationships of enzymes are featured in this review. A brief introduction glucoamylase, involving the substrate-binding and cata- to other -glycosidases is provided below. As mentioned lytic mechanisms.11) GH family 63 contains processing above, GH family 4 enzymes display the unusual -glucosidase I that hydrolyzes terminal (1!2)-linked mechanism to hydrolyze the -glucosidic linkage with -glucose residues of the N-glycan precursor of glyco- retention. The tertiary structure of GH family 4 enzymes proteins with inversion of the anomeric form. Recently, shows no similarity to that of any known glycoside a tertiary structure of a homolog of the processing hydrolases, but bears a resemblance to those of NAD- enzyme derived from E. coli (YgjK) was described.13) dependent dehydrogenases, such as lactate dehydrogen- The structure is composed of a -sandwich domain and ase and malate dehydrogenase.8,10) GH family 13 is one a catalytic domain. The catalytic domain displays an of the largest GH families, containing -amylase, ð=Þ6 barrel fold similar to those of clan GH-L, pullulanase, cyclodextrin glucanotransferase (CGTase), including GH family 15 glucoamylase and GH family 65 branching enzyme, neopullulanase, trehalose synthase, maltose phosphorylase. GH family 122 contains only Studies of GH 31 and 97 -Glycosidases 110610-3 Table 1. Glycoside Hydrolase Families Containing the Enzyme Acting on -Glucoside

Glycoside Catalytic hydrolase Fold Clan Known activities mechanism family Family 4 Retaining NAD(P)-binding — maltose-6-phosphate glucosidase, -glucosidase, -galactosidase Rossmann-fold 6-phospho--glucosidase, -glucuronidase Family 13 Retaining ð=Þ8 barrel GH-H -amylase, pullulanase, cyclomaltodextrin glucanotransferase, cyclomaltodextrinase, trehalose-6-phosphate hydrolase, oligo--glucosidase, maltogenic amylase, neopullulanase, -glucosidase, maltotetraose-forming -amylase, isoamylase, dextran glucosidase, maltohexaose-forming -amylase, maltotriose-forming -amylase, branching enzyme, trehalose synthase, 4--glucanotransferase, maltopentaose-forming -amylase, amylosucrase, sucrose phosphorylase, malto-oligosyltrehalose trehalohydrolase, isomaltulose synthase Family 15 Inverting ð=Þ6 toroid GH-L glucoamylase, glucodextranase, ,-trehalase Family 31 Retaining GH-D -glucosidase, -1,3-glucosidase, sucrase-isomaltase, -xylosidase, -glucan lyase, ð=Þ barrel 8 isomaltosyltransferase Family 37 Inverting ð=Þ6 toroid GH-G ,-trehalase Family 63 Inverting ð=Þ6 toroid GH-G processing -glucosidase, -1,3-glucosidase, -glucosidase Family 65 Inverting ð=Þ6 toroid GH-L ,-trehalase, maltose phosphorylase, trehalose phosphorylase, kojibiose phosphorylase, trehalose-6-phosphate phosphorylase, nigerose phosphorylase, kojibiose phosphorylase 1 Family 97 Inverting/Retaining ð=Þ8 barrel GH-D -glucosidase (inverting), -galactosidase (retaining) Family 122 Unknown Unknown — -glucosidase

1GH clan of GH family 97 is not defined in CAZy database, but GH family 97 enzymes, especially retaining enzymes, are significantly similar to clan GH-D enzymes Advancein their tertiary structure. View one characterized enzyme, derived from Pyrococcus furiosus.14) The -glucosidase displays broad substrate specificity for -glucosides, including panose, isomaltose, isopanose, maltotriose, turanose, and maltose. No structure-function relationships studies have been carried out on this family because it was described quite recently. Homologs of this -glucosidase seem to be distributed in hyperthermophilic archaea. Proofs I. Glycoside Hydrolase Family 31

1. Overview GH family 31 contains several enzymes, -glucosidase (EC 3.2.1.20), -xylosidase (EC 3.2.1.-), -glucan lyase (EC 4.2.2.13), and isomaltosyltransferase (EC 2.4.1.-) (Fig. 2). -Glucosidase belonging to GH family 31 is distributed in many organisms. Most of -glucosidases are associated with the degradation of malto-oligosaccharides to produce energy. For instance, plant -glucosidase is related to the hydrolysis of malto- oligosaccharides derived from starch in the germinating stage of seeds,15,16) and the sucrase-isomaltase complex (SUIS) and the maltase-glucoamylase complex (MGA), terminal enzymes degrading the carbon source, in the small intestine of mammals hydrolyze malto-oligosaccharides and sucrose into monosaccharides.17) Eukar- yotes possess -glucosidases that have significant roles besides carbohydrate metabolism. Endoplasmic reticulum -glucosidase, known as glucosidase II, trims the Fig. 2. Reactions Catalyzed by GH Family 31 Members, -Gluco- terminal (1!3)-linked -glucose residues of the N- sidase, -Xylosidase, -Glucan Lyase, and Isomaltosyl Transferase. glycan and participates in protein quality control in the endoplasmic reticulum.18) Lysosome -glucosidase is glycosidases able to modify the xyloglucan and/or concerned with the degradation of glycogen, and a xyloglucan oligosaccharides present in plant cell deficiency of the enzyme causes an accumulation of walls.19) Bacteria and archaea also possess -xylosidase, glycogen, resulting in serious glycogen storage disease but its role was not established. Recently, Larsbrink type II. -Xylosidase catalyzes the hydrolysis of - et al. found that -xylosidase from Cellvibrio japonicas xyloside residues at the non-reducing ends of xyloglucan (CjXyl31A) is involved in xyloglucan saccharifica- oligosaccharides. Plant -xylosidase is one of the exo- tion.20) -Glucan lyase (GLase) is a peculiar enzyme 110610-4 M. OKUYAMA of GH family 31 enzymes, because it catalyzes an elimination that produce 1,5-anhydrofructose from starch and maltodextrin.21) It is of interest that GLase displays significant similarity to the hydrolases. Lee et al. carried out a detailed kinetic analysis to elucidate the catalytic mechanism of GLase.22) They found that the catalytic reaction is divided into two steps through the glycosyl-enzyme intermediate as well as retaining glycoside hydrolases. At the first glycosylation step, oxocarbenium ion character of the transition state is developed, and aspartate residue, equivalent to nucleophile residue in GH family 31 -glucosidases, forms the covalent intermediate with the substrate. The intermediate is subsequently cleaved at the second step by a syn- elimination reaction. The key structural factor that discriminates between the hydrolase and the lyase remains obscure. Recently, the tertiary structure of the -glucan lyase was solved (unpublished; PDB ID code 2X2H, 2X2I, and 2X2J). The structures of the -glucan Fig. 3. Structural Factors Distinguishing -Xyloside and -Glucoside. lyase and the -glucosidases superpose well, and there The loop 1 mutant and the C307I/F308D mutant -xylosidase appears to be no significant difference in active sites. significantly hydrolyze -glucosides (see text for details). This Isomaltosyltransferase, the first transferase of GH family multiple sequence alignment compares ! loop 1 sequences as 31, catalyzes the transfer of the isomaltosyl unit to between -xylosidases (first and second lines) and -glucosidases. suitable acceptors.23) All GH family 31 enzymes were The UniProtKB/Swiss-Prot names of -xylosidase and -glucosi- Advance Viewdase are shown on the left. believed to recognize the monoglycosyl unit at the non- reducing end and the transferase is the first enzyme to recognize di- and tetra-glycosyl units. other hand, YicI evidently possesses -xylosidase Over the past several years, a number of three- activity: it efficiently hydrolyzes isoprimeverose (6-O- dimensional structures of GH family 31 enzymes have -xylosyl glucose) and xyloglucan oligosaccharide, and been described. The first tertiary structure solved was - releases xylose.32) xylosidase from Escherichia coli.24,25) The most recent solved structure is -xylosidase of Cellvibrio japonicas Glycon specificity of E. coli -xylosidase (CjXyl31A).20) In the meantime, the structures of four - E. coli -xylosidase displays approximately 30% glucosidases and one -glucan lyase were describ- identity with -glucosidases belonging to GH family ed.26–29) Each structure is composed of four domains, the 31, but -xylosidaseProofs hardly hydrolyzes -glucosidic N-terminal, catalytic, proximal, and distal C-terminal linkages.32) Hence we attempted to identify the struc- domains, except for CjXyl31A, which has two N- tural factor that discriminates between -xyloside and terminal domains, involving the PA 14 domain.20) The -glucoside.34) In order to select the target residue, we catalytic domain displays a ð=Þ8 barrel fold. The compared and contrasted the amino-acid residues at catalytically essential residues, catalytic nucleophile and subsite 1 in multiple sequence alignment of - acid/base, are at the ends of -strands 4 and 6 in the xylosidases and -glucosidases. Furthermore, we com- ð=Þ8 barrel fold, respectively. Based on the fold pared the tertiary structure of -xylosidase with that of similarity, GH family 31 is grouped into clan GH-D with GH family 27 -galactosidase to predict how GH family GH families 27 and 36. The clan is a group of families 31 -glucosidases accommodate C6 of glucopyranose. that are thought to have a common ancestry and are This is because no tertiary structure of GH family 31 recognized by significant similarities in tertiary structure -glucosidase had been available and we had no way of together with conservation of catalytic residues and the knowing the environment around C6 of GH family 31 catalytic mechanism.30) -glucosidase. GH family 27, belonging to the same clan with GH family 31, was the best family to predict 2. Substrate recognition of Escherichia coli -xylosi- the environment of C6. A site-directed mutagenesis dase study indicated that Cys307, Phe308, and the ! loop Two GH family 31 enzymes in E. coli K-12 1ofð=Þ8 barrel are important factors recognizing the The genome of E. coli K-12 encodes two GH family -xyloside (Fig. 3). The short loop 1-enzyme and 31 enzymes, YihQ and YicI.31) YihQ slightly but C307I/F308D mutant enzymes exhibited -glucosidase significantly hydrolyzes the susceptive substrate, - activity with 100–140 times higher hydrolytic activity glucopyranosyl fluoride.32) Hence, we tentatively defined than the wild-type enzyme. Hydrolyzing activity on YihQ as -glucosidase. However, the biological function p-nitrophenyl -xylopyranoside of the short loop 1- of YihQ was unclear because YihQ hardly hydrolyzes enzyme decreased by about 10-fold, and that of C307I/ natural substrates, such as malto-oligosaccharides and F308D to below the limit of detection. Hence, these glucobioses. Recently, Gibson et al. found that the mutant enzymes were not -xylosidase but -glucosi- yihQ mutant of Salmonella did not produce an O- dase. The ! loop 1, having three more amino-acid antigen capsular polysaccharide.33) YihQ is hence residues than -glucosidase, covers the active pocket believed to catalyze the transfer of the glucose residue and appears to prevent the hydroxymethyl substituent at onto the O-antigen capsular polysaccharide. On the C5 of glucopyranose from binding to the active site Studies of GH 31 and 97 -Glycosidases 110610-5 owing to steric hindrance, resulting in strict recognition border membrane, viz., sucrose, isomaltose, and maltose, of the -xylosyl moiety. Unlike long loop 1, the short raised the possibility that the transfer products can loop 1-enzyme having the -glucosidase-like sequence function as inhibitors to SUIS and MGA. -D-Xylp- might have enough space to accommodate the hydro- (1!6)-D-Manp inhibits maltase and isomaltase activ- xymethyl substituent of -glucoside. It is possible that ities, and -D-Xylp-(1!6)-D-Fruf inhibits sucrase ac- C307I/F308D mutant enzyme fix the O6 atom of tivity in the rat intestine extract.35) The concentrations -glucoside by a hydrogen bond from Asp308 in the giving 50% inhibition (IC50) toward maltose and same way as GH 27 -galactosidase, the Asp52, at the isomaltose hydrolysis by -D-Xylp-(1!6)-D-Manp end of -strand 2, has a hydrogen bond to the O6 atom were 18.1 mM and 8.62 mM respectively. For the sucrose 35) of galactose. Even though Phe308 is positioned substrate, -D-Xylp-(1!6)-D-Fruf showed IC50 at somewhat far from the active pocket, Ile307, substituted 6.47 mM. These IC50 values indicate that the trans- for Cys307, might put the carboxy group of Asp308 glycosylation products are weak inhibitors of intestinal close to the substrate, because spatially adjacent residues -glucosidases. Based on the effects of anti-diabetics affect the orientation of their side chains to each other.36) along with an absence of calories, these two novel sugars can displace sucrose and artificial sweeteners Aglycon specificity of E. coli -xylosidase after further detailed safety and taste tests. In general, the glycon specificity of exo-type glycosidases can be determined easily using various aryl The first -glycosynthase derived from Schizosac- glycoside substrates such as nitrophenyl glycosides, but charomyces pombe -glucosidase it is more difficult to determine aglycon specificity, Glycosynthases formed from retaining glycosidases because it is laborious and costly work to synthesize are mutant enzymes that have a nonfunctional group in various substrates having different aglycon moieties. place of catalytic nucleophile carboxy group. Even Hence we designed an efficient procedure to determine though the mutant enzymes are hydrolytically incom- aglycon specificity through the acceptor specificity of petent, they are able to transfer the glycosyl moiety to a Advance37) View transglycosylation. The retaining glycoside hydrolases suitable acceptor when the glycosyl fluoride substrate of can catalyze transglycosylation as well as hydrolysis. the anomeric configuration opposite to that of the natural When the covalent glycosyl-enzyme intermediate is substrate is provided (Fig. 4B). The enzymes recognize broken by nucleophile attack with a suitable acceptor in the glycosyl fluoride of opposite anomer as the homolog place of water, transglycosylation occurs. Using a of the glycosyl-enzyme intermediate, and catalyze part of substrate with a good leaving group as donor substrate, the deglycosylation step, in which the transfer reaction transglycosylation occurs mainly with accumulation mainly or completely occurs. This is because the kcat/Km of the intermediate, and the reaction velocity depends value for the transfer product is greater than that of the on the affinity between the acceptor and its binding hydrolysis product. Furthermore, the enzymes lose site. Characterization of transglycosylation gives infor- hydrolytic activity and the transfer product accumulates. mation on the interaction at the aglycon binding site of The first glycosynthaseProofs was reported in 1998 with GH the enzyme. family 1 Agrobacterium -glycosidase.39) Since then, The investigation, in which -xylopyranosyl fluoride several glycosynthases have been generated.40) Until was used as donor substrate, yielded information on recently, all glycosynthases were derived from retaining the aglycon-binding site, i.e., the plus subsite of the glycosidases, but Kitaoka et al. have reported a glyco- E. coli -xylosidase.37) (i) E. coli -xylosidase does not synthase derived from inverting enzymes, the reducing accommodate a sugar having axial OH-4 at subsite þ1. end xylose-releasing exo-oligoxylanase from Bacillus -Xylosidase is capable of transferring a xylosyl moiety halodurans and 1,2--L-fucosidase from Bifidobacterium to glucose, mannose, allose, and fructose, but not to bifidum.41,42) The catalytic base mutants of the inverting galactose, gulose, or talose, which have axial OH-4. enzymes, which lose their hydrolytic activity, catalyze (ii) -Xylosidase has an aglycon-binding site spatially only the condensation reaction by supplying the glycosyl large enough to accommodate at least trisaccharide. It fluoride of the opposite anomer. Recently, a novel is able to transfer the xylosyl moiety not only to glycosynthase derived from GH family 85, Mucor monosaccharides but also to oligosaccharides, including hiemalis endo--N-acetylglucosaminidase (Endo-M), maltose, maltotriose, cellobiose, and cellotriose. (iii) which proceeds via a substrate-assisted mechanism, has -Xylosidase has strict aglycon specificity. E. coli been published.43) The replacement of a conserved -xylosidase produces one transglycosylation product asparagine (Asn175 in Endo-M), which promotes the from each acceptor substrate. -Xylosidase of Sulfolo- internal nucleophile attack of 2-acetamide group to form bus solfataricus, a member of GH family 31, by the oxazoline inetermediate, by alanine diminishes the contrast, forms several regioisomers in tranglycosyla- hydrolytic activity. However, when supplied with an tion.38) (iv) The initial velocities for transglucosylation active sugar oxazoline that is an intermediate of the using various acceptors follow the order glucose > reaction, the mutant enzyme transfers this sugar oxazo- mannose > cellotriose > fructose > maltoriose > allose > line to GlcNAc-peptide. cellobiose > maltose, which shows the aglycon specific- All of the glycosynthases formed from retaining ity of -xylosidase. glycosidases were derived from -glycosidases until we Among the transfer products, -D-Xylp-(1!6)-D- derived glycosynthase from S. pombe -glucosidase.44) Manp, -D-Xylp-(1!6)-D-Fruf, and -D-Xylp-(1!3)- The catalytic nuclepophile residue of S. pombe - D-Frup are novel sugars. The structural similarity glucosidase is Asp481, as determined by chemical between these transfer products and the original sub- modification and site-directed mutagenesis studies.45) strates of SUIS and MGA in the small intestinal brush- Although the mutation Asp481!Gly leads to inactiva- 110610-6 M. OKUYAMA a

Fig.Advance 4. Catalytic Mechanisms of Wild-Type and Catalytic View Nucleophile-Eliminated Glycosidases. a, Wild-type; b, catalytic nucleophile mutant glycosidase with glycosyl ﬂuoride of opposite anomeric conﬁguration to that of the natural substrate (glycosynthase); c, catalytic nucleophile mutant glycosidase with azide ion (chemical rescue).

1 tion of the enzyme, the enzyme transfers the glucosyl one lone pair of O5 and trans to the other, and the C4 4 moiety to suitable acceptors, pNP -glucopyranoside conformation is thus more stable than the C1 con- (-1,6, 41%; -1,4, 29%), pNP -xylopyranoside (82%), formation. Compared with -glucopyranosyl fluoride, 1 pNP -mannopyranoside (10%), and pNP -glucopy- -xylopyranosyl fluoride have the C4 conformation, ranoside (13%), using -glucosyl fluoride as donor because the hydroxymethyl substituent at C5 is absent substrate (yield of transfer product given in parenthe- from -xylopyranosyl fluoride. It is also known that - ses). Compared with other glycosynthases, the yield of xylopyranosyl fluorideProofs triacetate is present mainly in the 1 transfer product is low. This can be explained by the C4 conformation, even though that conformation has lability of -glucosyl fluoride, which spontaneously the unfavorable 1,3-diaxial interaction of bulky ester decomposes as a result of the anomeric effect. pNP - group.47,48) Our NMR analysis of the -xylopyranosyl galactopyranoside cannot act as acceptor. This accords fluoride triacetate also revealed that it should be of the 1 with the aglycon specificity of E. coli -xylosidase, C4 conformation, because it shows 4.1 Hz of the vicinal discussed above. GH family 31 enzymes, at least - H1-H2 coupling (unpublished data). Accordingly, - glucosidase and -xylosidase, are perhaps unable to the xylopyranosyl fluoride, of which the 1,3-diaxial repul- accommodate galactosyl moiety at subsite þ1. sion of hydroxy group must be much weaker than the 1 We also investigated the glycosynthase derived from bulky acetyl group, can exit in the C4 conformation. E. coli -xylosidase (results unpublished). Unfortu- nately, the nucleophile mutant enzyme showed no II. Glycoside Hydrolase Family 97 glycosynthase activity. We believe that the instability of -xylopyranosyl fluoride is to be attributed in part to Members of a GH family share characteristic func- this failure. In order to achieve the glycosynthase tional features, such as mechanistic strategy in catalysis, reaction, -xylopyranosyl fluoride requires changing of because functionally and structurally significant residues 4 1 the pyranose ring conformation from C1 to S3. This is have been conserved within the family during molecular because the xylopyranosyl residue in the pseudo- evolution. However, we and others found an exception Michealis complex structure of the E. coli -xylosidase in GH family 97: one GH family contains both inverting 4 46) 49–51) displays the C1 conformation, and in the glycosyl- and retaining glycoside hydrolases. 1 enzyme intermediate shows a strained S3 conforma- 25) 4 tion. However, the C1 conformation of -xylopy- 1. Inverting -glucoside hydrolase ranosyl fluoride, the equatorially oriented fluorine of A GH family 97 enzyme was first found in Bacter- which is gauche to both lone pairs of the ring oxygen oides thetaiotaomicron. The enzyme, SusB, is a member atom (O5), is extremely unstable due to electronic of a starch utilization system (sus) operon in B. thetaio- repulsion between the fluorine and the lone pair, and taomicron. SusB was known only as -glucoside hydro- hence undergoes spontaneous hydrolysis. Additionally, lase.52) Hence we performed biochemical and structural it is possible that most -xylopyranosyl fluorides have analyses of recombinant SusB.49) SusB (hereafter 1 an incompatible C4 conformation, because the axially BtGH97a) catalyzes the hydrolysis of -glucosidic 1 orientated fluorine in the C4 conformation is gauche to linkages at the non-reducing terminal through an Studies of GH 31 and 97 -Glycosidases 110610-7 a b c

Fig. 5. Structures of GH Family 97 Enzymes. a, BtGH97a, inverting -glucoside hydrolase (PDB ID code, 2ZQ0). The catalytic residues and the conserved residues related to sugar-binding as well as the essential Ca2þ ion observed in the BtGH97a structure are shown in green, gold, and purple respectively. b, BtGH97b, retaining - glycoside hydrolase (PDB ID code, 3A24). The catalytic residues, the conserved residues related to sugar-binding, and the Ca2þ ion are shown in green, gold, and purple respectively. c, Superimposition of catalytic residues as between BtGH97a (green) and BtGH97b (yellow). The residue number of BtGH97b is indicated in italics. inverting mechanism. The tertiary structure is composed 2. Retaining -galactoside hydrolase of three domains: a ð=Þ8 barrel domain, flanked by Multiple sequence alignment indicated that about half two -sandwich domains at the N- and C-terminals of GH family 97 enzymes have an aspartate residue at (Fig. 5A). The complex structure with acarbose, pseudo- the position equivalent to -strand 4 in BtGH97a.49,51) In tetrasaccharide inhibitor, reveals that the loops connect- addition, the enzymes had no glutamate residues ingAdvance the -strand and the -helix in the ð= ViewÞ8 barrel corresponding to catalytic base residue. This finding domain contribute mainly to making the active-site raises the possibility that the asperatate residue acts as a pocket. Two loops from the N-terminal domain addi- catalytic nucleophile and that the enzymes are retaining tionally form a narrow plus-subsite groove. One calcium -glycosidase, that is, GH family 97 might contain ion is located at the active pocket in the structures inverting and retaining enzymes. Hence we character- of both the native and the acarbose complex. In the ized the enzyme that possesses aspertate residue at the BtGH97a-acarbose complex, the calcium ion is coordi- -strand 4.50) A protein having significant sequence nated by four oxygen atoms from glutamate residues, similarity to BtGH97a (identity 26.7%, similarity 42.4% including catalytic residues, and acarbose with a with 22.1% gaps), encoded by the BT 1871 gene in pentagonal bipyramid geometry. Removal of the cal- B. thetaiotaomicron was produced heterologously in cium ion by EDTA causes a considerable decrease in the E. coli and characterized. catalytic activity. The calcium ion must, accordingly, As expected, theProofs protein (hereafter BtGH97b) cata- play an important role in catalytic activity. For example, lyzed the hydrolysis of -galactoside with the retaining it accumulates functional groups and substrate and mechanism. The structure of BtGH97b proved to be very serves as a general Lewis acid, polarizing the anomeric similar to that of BtGH97a (Fig. 5B). The active-site carbon and rendering it a better electrophile. BtGH97a pocket was positioned at the C-terminal end of the barrel, has a suitable catalytic site to carry out inverting similarly to BtGH97a, and the residues forming the hydrolysis (Fig. 5C). The general acid catalyst, the active site and a calcium ion superposed well, except for carboxy group of Glu532, at ! loop 6, is closest to the catalytic residues at the -face. BtGH97b had no N4B of acarbose. A water molecule is positioned to glutamate residues at -strands 3 and 5, corresponding to attack an anomeric carbon from the opposite side of Glu439 and Glu508 respectively. Instead, it had an the general acid catalyst across the glycosidic bond (the asperatate residue, Asp415, at the end of -strand 4. -face). The water molecule is pinched by side chains Asp415 is ideally placed to attacking the anomeric of Glu508 and Glu439, which are at the C-terminals carbon of the substrate: the OD1 atom of Asp415 is of -strands 3 and 5 respectively. Accordingly, Glu439 poised at the same position as the catalytic water and Glu508 are the general base catalysts even for molecule of BtGH97a, which certainly nucleophilically the reduced activity of both the Glu439!Gln and the attacks the anomeric carbon (Fig. 5C). Thus Asp415 Glu508!Gln mutant enzyme. attacks the anomeric carbon from the -face of BtGH97b Even though BtGH97a is an inverting glycosidase, in place of the water molecule, forming a covalent the ð=Þ8 barrel domain of BtGH97a has significant intermediate. The site-directed mutation of Asp415! similarity to GH clan-D retaining glycosidases, in that Gly causes crucial inactivation of the enzyme. The the ð=Þ8 barrel domain is similar to GH family 27 - mutant enzyme was, however, reactivated by an external N-acetylgalactosaminidase and -galactosidase, to GH nucleophile, an azide ion, with inverted stereochemistry family 36 -galactosidase and to GH31 -glycosidase. (Fig. 4C). The inverted configuration compared with Structural superimposition of BtGH97a and GH clan-D the substrate made possible definitive identification of enzymes indicated that they share not only a similar fold Asp415 as the nucleophile residue of BtGH97b. but also several amino acid residues important for The slight difference in the position of the functional catalysis, even catalytic acid Glu532. On the other hand, residues at -face accounts for the difference in catalytic BtGH97a has no asparatate residue that GH clan-D mechanism (Fig. 5C). BtGH97a, the inverting enzyme, enzymes possess as catalytic nucleophile at the end of has catalytic base residues at -strands 3 and 5, and -strand 4. BtGH97b, the retaining enzyme, has a catalytic nucle- 110610-8 M. OKUYAMA ophile residue at -strand 4. The divergence of the At present GH family 97 is an extremely rare case, but catalytic residue appears to have hopped around on the it remains possible that other GH families containing active site during molecular evolution, a phenomenon different catalytic mechanisms exist. Even if such a GH called ‘‘hopping of functional groups.’’53) family is identified, it should be possible to predict the catalytic mechanism with careful consideration. Multi- 3. Molecular evolution of GH family 97 ple sequence alignment should indicate that the family It is generally accepted that important functional is unusual and should make it possible to predict residues and the catalytic mechanism used are conserved the catalytic mechanism by expanding the scope the within a GH family. This is an essential concept for the multiple sequence alignment into the GH clan, as has glycoside hydrolase family. A good example is GH been done for GH family 97. family 13.11) It contains hydrolases, such as -amylase, pullulanase, and -glucosidase, and transferases, such as Acknowledgments CGTase, trehalose synthase, amylosucrase, and sucrose isomerase. At first sight, these enzymes catalyze their I am grateful to Professor Emeritus Seiya Chiba reactions through different mechanisms, but each of and Professor Atsuo Kimura of Hokkaido University these enzymes conserves catalytic residues and employs for continuous encouragement. I sincerely thank Dr. the double-displacement mechanism. The divergence Haruhide Mori of Hokkaido University for many between the hydrolases and the transferases lie in valuable discussions. The structural studies were per- differences in the molecule that decomposes the glyco- formed with members of the Laboratory for X-Ray syl-enzyme intermediate. Additionally the variation in Structural Biology of the Faculty of Advanced Bio- the catalyzed reaction is due to the variety of subsite science, Hokkaido University, especially with Dr. Min structures. Of more than 100 GH families, several Yao and Ms. Momoyo Kitamura. The structure of the families in which a catalytic residue is not conserved -xylosides was determined in collaboration with Dr. exist.Advance Two examples are GH family 3 and 8. View GH family Katsuro Yaoi of the Institute for Biological Resources 3 enzymes divide into two broad subfamilies, multi- and Functions, Advanced Industrial Science and Tech- domain enzymes consisting of a ð=Þ8 barrel domain nology (AIST). MS and NMR analyses were performed and a / sandwich domain, and a single ð=Þ8 barrel in cooperation with Dr. Eri Fukushi of the GC-MS & enzymes. The former bear the catalytic acid/base NMR Laboratory, Graduate School of Agriculture, glutamate on the / sandwich domain,54) whereas, Hokkaido University. Most of these studies were the latter bear the catalytic acid/base residue on the cooperative efforts of a number of graduate students 55) ð=Þ8 barrel domain. Furthermore, the acid/base and researchers of the Laboratory of Molecular Enzy- residue of the latter is surprisingly histidine.55) Although mology, Research Faculty of Agriculture, Hokkaido this family possesses no conserved catalytic acid/base, University, and especially, Dr. Min-Sun Kang and Dr. the catalytic mechanism is conserved. GH family 8, Hironori Hondoh. I thank all my co-workers for their having an ð=Þ6 barrel fold, contains inverting chito- cooperation. TheseProofs studies were supported by a Grant- sanase, cellulase, and xylanase. Cellulase and xylanase in-Aid for Scientific Research from the Japan Society for have a catalytic base aspartate on a loop connecting 7 the Promotion of Science. and 8,56,57) whereas chitosanase has asparagine at the corresponding position. 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