J. Appl. Glycosci., 59, 51‒62 (2012) doi: 10.5458/jag.jag.JAG-2011_022 ©2012 The Japanese Society of Applied Glycoscience Review Protein-carbohydrate Interactions Leading to Hydrolysis and Transglycosylation in Plant Glycoside Family 1 (Received December 7, 2011; Accepted January 30, 2012) (J-STAGE Advance Published Date: February 11, 2012) James R. Ketudat Cairns,1,＊ Salila Pengthaisong,1 Sukanya Luang,1 Sompong Sansenya, 1 Anupong Tankrathok1 and Jisnuson Svasti2 1Schools of Biochemistry and Chemistry, Institute of Science, Suranaree University of Technology (Muang District, Nakhon Ratchasima 30000, Thailand) 2Department of Biochemistry and Centre for Protein Structure and Engineering, Faculty of Science, Mahidol University (Phayathai, Bangkok 10400, Thailand)

Abstract: family 1 (GH1) includes enzymes with a wide range of specifi cities in terms of reactions, substrates and products, with plant GH1 enzymes covering a particularly wide range of hy- drolases and transglycosylases. In plants, in addition to β-D-, β-D-mannosidases, disacchari- dases, thioglucosidases and hydroxyisourate hydrolase, GH1 has recently been found to include galactosyl and glucosyl that utilize galactolipid and acyl donors, respectively. The amino acids binding to the nonreducing residue of glycosides and oligosaccharides in subsite －1 are largely conserved in GH1 glycoside , despite their different glycon specifi cities, and residues outside this subsite contribute to specifi city. The conserved subsite －1 residues form extensive hy- drogen bonding and aromatic stacking interactions to the glycon to distort it toward the transition state, so they must make different interactions with different . Aglycon specifi city is largely determined by interactions with the cleft leading into the , but different enzymes appear to interact with their substrates via different residues. The most extended aglycon binding interactions that have been studied extensively are those for cellooligosaccharides. Rice Os3BGlu7 (BGlu1) β-D-glucosidase, which binds cel- looligosaccharides residues in subsites +1 to +4 primarily by water-mediated hydrogen bonds and a few aromatic-sugar stacking interactions, appears to show remarkable plasticity in this binding. Although mutations that change the mechanism of the hydrolases, such as glycosynthases and thioglycoligases cre- ate transglycosylases, the structural basis for natural transglycosylase vs. glycoside hydrolase activities in GH1 enzymes remains to be determined.

Key words: glycoside hydrolase, transglycosylase, β-glucosidase, specifi city, glycosides, oligosac- charides

Glycoside hydrolases (GH, glycosidases) have been grouped into families related by sequence similarity and ＊ Corresponding author (Tel +66‒44‒22‒4304, Fax +66‒44‒22‒4185, larger clans that show structural and mechanistic similari- Email: [email protected]). ty,1‒3) as catalogued in the Carbohydrate-Active enZYmes Abbreviations: AA5GT, anthocyanin 5-O-glucosyltransferase; AA7GT, (CAZy) website (http://www.cazy.org).4) GH family 1 (GH1) anthocyanin 7-O-glucosyltransferase; CAZY, Carbohydrate-Active en- zymes; DGDG, digalactosyl diacylglyceride; DIBOA, 2,4-dihydroxy-1,4- is one of these families with diverse functional properties, benzoxazin-3-one; DIMBOA, 2,4-dihydroxy-7-methoxy-1,4-benzoxazin- which belongs to GH Clan A.5,6) GH1 includes retaining 3-one; DIMBOA-Glc, 2-O-β-D-glucopyranosyl-4-hydroxy-7-methoxy-1, glycosidases from archaea, eubacteria and eukaryotes, with 4-benzoxazin-3-one; DP, degree of polymerization; GGGT, galactolipid: a wide range of substrate and reaction specifi cities, as well galactolipid galactosyl ; GH, glycoside hydrolase; GH1, as proteins with other biological and enzymatic functions. glycoside hydrolase family 1; GT; glycosyltransferases; HIUH, purine hydrolase hydroxyisourate hydrolase; HvBII, Hordeum vulgare β-D- These include broad spectrum β-D-glycosidases, as well as glucosidase isoenzyme βII; MGDG; monogalactosyl diacylglyceride; β-D-6-phosphoglycosidases (EC 3.2.1.85 and EC 3.2.1.86), NMR, nuclear magnetic resonance; Os3BGlu6, Oryza sativa β-D-glucosidases (EC 3.2.1.21), β-D- (EC β-glucosidase isoenzyme Os3BGlu6; Os3BGlu7, O. sativa β-glucosidase 3.2.1.23) and β-D-mannosidases (EC 3.2.1.25) in archaea isoenzyme Os3BGlu7; Os4BGlu12, O. sativa β-glucosidase isoenzyme Os4BGlu12; Os9BGlu31, O. sativa β-transglucosylase isoenzyme Os- and eubacteria, insect (β-D-thioglucosidases, 9BGlu31; Os7BGlu26, O. sativa β-glucosidase isoenzyme Os7BGlu26; EC 3.2.1.147), digestive β-glycosidases, cytoplasmic Os3BGlu8, O. sativa β-glucosidase isoenzyme Os8BGlu8; pNP, para- β-D-glucosidase and the signaling protein and its nitrophenol; QM/MM, quantum mechanics/molecular mechanics; relatives in animals, and hydroxyisourate hydrolase (EC SbDhr1, Sorghum bicolor dhurrinase isoenzyme 1; SFR2, Sensitive-to- 3.5.2.17), as well as β-D-glycosidases with various specifi c- Freezing-2; ZmGlu1, Zea mays β-glucosidase isoenzyme Glu1; 7) UDP-glucose, uridine diphosphate-α-D-glucose; XET, xyloglucan endo- ities in plants. transferases. Although GH1 proteins from all domains and phyla of 52 J. Appl. Glycosci., Vol. 59, No. 2 (2012) living organisms have fascinating and potentially useful Activities of plant glycoside hydrolase family 1 enzymes. activities, it is in plants that these proteins have expanded the While archaea, bacteria and fungi tend to have one to a most to acquire a great variety of catalytic activities and this few GH1 genes and humans (Homo sapiens) have fi ve GH1 review will focus on the plant GH1 enzymes. Bacterial and genes, plants have many more.4,7) Arabidopsis (Arabidopsis animal enzymes will be mentioned when they have been thaliana) has 48 GH1 genes, while 40 GH1 genes have been used to demonstrate catalytic mechanisms, while the basis of identifi ed in rice genome databases, 34 of which are thought substrate binding and reaction specifi city will primarily be to encode translated rice proteins.8,9) With the exception of illustrated with plant enzymes, particularly those from rice Sensitive-to-Freezing-2 (SFR2) and its orthologues, plant (Oryza sativa). First, we will describe the types of activities GH1 enzymes fall into a relatively closely related phyloge- found in plants, then the catalytic mechanism and substrate netic cluster within GH1, as shown in Fig. 1. binding of GH1, and fi nally the engineering of GH1 enzymes As noted above, GH1 members have a wide variety of to produce transglycosylase activities and the recently activities, some of which may not even involve glycoside discovered naturally occurring plant GH1 transglycosylases. hydrolase activities, although the plant GH1 members that The roles of carbohydrate binding in these activities will be have been described to date are all hydrolases or transferas- emphasized. es. The GH1 glycosidases in plants include β-D-glucosidases,

Fig. 1. Phylogenetic tree of plant and other glycoside hydrolase family 1 proteins. The tree was produced from GH1 sequences, including all rice and Arabidopsis genes with sequences including the catalytic amino acids in the genomic databases named as in Xu et al.8); Opassiri et al.,9) as well as all available GH1 sequences of the moss Phycomitrella patens ssp. patens, plant GH1 with known 3D structures and plant sequences previously compared in references 8 and 9 (designated by genus and species initials and Genbank accession), and a selection of archaeal, bacterial, animal and fungal sequences. The tree was generated by the neighbor-joining meth- od79) with MEGA580) after protein sequence alignment by MUSCLE.81) The phylogenetic clusters that include both rice and Arabidopsis sequences (At/Os1‒8) and the Brassicales-specifi c clusters (At I and II) are labeled as designated by Opassiri et al.9) The analysis of animal, fungal, archaeal and fungal sequences is not extensive, but includes those with known 3D structural models and those with high similarity to SFR2. The analysis involved 146 amino acid sequences. All positions with less than 95% site coverage were eliminated. There were a total of 372 positions in the fi nal dataset. Percent reproducibility in 1,000 bootstrap trees are listed on branches with >50% reproducibility, while branches with <50% reproducibility are not marked. It may be noted that in this analysis, clusters At/Os1, At/Os2 and At/Os4 form a reliably clustered group, as do At/Os7, At I, At II and the monocot plastid β-glucosidases. At/Os8, consisting of SFR2 galactosyl transferase homologues, clusters with bacterial and archaeal β-galactosidases/β-glycosidases and is distant from other plant, animal, fungal and certain bacterial and archaeal β-glucosidases. Ketudat Cairns et al.: Plant Glycoside Hydrolase Family 1 Specifi city 53

β-D-mannosidases, more general β-glycosidases, β-D- feruloyl glucose, p-coumaroyl glucose, vanillyl glucose and thioglucosidases and disaccharidases.10‒12) Many of the similar glucose esters, as was seen for the anthocyanidin β-D-glucosidases in fact have higher activities toward 3-O-glucoside glucosyl transferases, but Os9BGlu31 β-D-fucoside, but due to the paucity of β-D-fucosides in recognizes a different set of acceptor substrates. nature, are presumed to act primarily as β-D-glucosidases.7) Many of these enzymes also have lesser activities toward Catalytic mechanism of family 1 glycoside hydrolases. para-nitrophenyl (pNP)-β-D-galactoside, pNP-β-D- As with other GH Clan A families, GH1 enzymes act xyloside and pNP-α-L-arabinoside, which vary from through a retaining mechanism, utilizing one conserved enzyme to enzyme, despite the conservation of the glycon- glutamate residue as a catalytic acid/base and a second as a described below. Only a subset of plant GH1 nucleophile or base.22) Although there has been some debate enzymes can hydrolyze β-D-mannosides, with the ones in the fi eld, the production of covalent intermediates of having been described falling in At/Os cluster 4 in the 2-F-glucosides23) and of natural glucoside with acid/base phylogenetic tree shown in Fig. 1. Certain GH1 enzymes mutants24) support a two-step mechanism with a covalent also hydrolyze 6-O-modifi ed glucosides, such as the acetyl enzyme-α-glucoside intermediate (Fig. 3). In the glycosyla- and malonyl derivatives of diadzin and genestin found in tion step, the catalytic nucleophile of the enzyme displaces legumes,12‒14) and 1,6-linked glycosides, where the aglycon with general acid-assistance from the catalytic the glycon, such as primeverose or acuminose, is removed acid/base to form the intermediate. The deglycosylation step from the aglycon as a disaccharide (Fig. 2).10‒13) In addition is the reverse of the fi rst step, with water or another nucleo- to the O-glycosidases, plants in the order Brassicales produce phile attacking from the opposite side, with basic assistance β-D-thioglucosidases, or myrosinases, which are found in from the catalytic acid/base, to displace the enzyme from the the two Arabidopsis-specifi c clusters in Fig. 1 (At I and At glycon. If the displacing nucleophile is water, hydrolysis II). A few GH1 β-D-glucosidases have been shown to have results; whereas with other nucleophiles, transglycosylation low levels of thioglucosidase activities, particularly the results. defense-related enzymes of phylogenetic cluster At/Os It is thought that both steps of the reaction should pass 7.15,16) through an oxocarbenium ion-like transition state, where the Aside from glycoside hydrolases, the purine hydrolase leaving group electrons are being withdrawn and the develop- hydroxyisourate hydrolase (HIUH) from soybean (Glycine ing cation on the anomeric carbon is stabilized by donation max) has also been identifi ed as a GH1 enzyme.17) As shown of electrons from the cyclic 5 oxygen.22,25) This requires that at the bottom right of Fig. 2, this enzyme cuts an amide-like the atomic orbitals C1 and O5 be sp2 hybridized so that they bond between N1 and C6 of hydroxyisourate by insertion of can share a double bond, which requires that C5, O5, C1 and water with the same catalytic groups used in glycoside C2 all fall within the same plane. The shapes of a pyranosyl 4 3 hydrolases, suggesting the catalytic mechanism is residue that can fulfi ll this requirement are the H3 and H4 17,18) 5,2 similar. half chairs and the B and B5,2 boats, and β-glucosidases Recently, family 1 transglycosylases (TG) have been have been proposed to go through a sugar conformational 1 4 4 identifi ed. SFR2, which is a chloroplast enzyme necessary trajectory of S3 skew boat to H3 half chair to C1 relaxed for freezing resistance, was previously thought to be a chair.25) The structures of various bacterial gluco-substrate β-D-glucosidase, but was recently shown to be a galactolip- hydrolyzing enzymes in Michaelis complexes that resemble 19,20) 1 4 id: galactolipid galactosyl transferase (GGGT). SFR2 S3, complexes with transition state analogues with H3-like was shown to transfer galactosyl moieties from monogalac- shapes and covalent intermediates in relaxed chair confi gu- tosyl diacylglyceride (MGDG) to other galactolipids to form rations support this confi gurational trajectory.25‒29) digalactosyl diacylglyceride (DGDG), trigalactosyl diacylg- For plant GH1 enzymes, the covalent intermediate with lyceride and diacylglycerides with longer galactosyl chains.20) 2-deoxy-2-fl uoroglucoside was fi rst identifi ed in the relaxed 4 30) The subsequent removal of the free diacylglyceride aglycon C1 chair in Sinapis alba and was subsequently is thought to allow membrane contraction and prevent shown to have the same conformation in rice Os3BGlu7 freezing damage. (also called BGlu1),31) Os3BGlu6 32) and Os4BGlu12 A similar phenomenon has been shown to occur in the β-glucosidases16) and wheat (Triticum aestivum) and rye conversion of anthocyanidin 3-O-glucoside (Fig. 2) to 3,5- (Secale cereale) hydroxaminic acid β-glucosidases.33) and 3,7-diglucosides by transfer of glucose from acyl Among plant GH1, the structures of the weakly inhibited S. glucosides by GH1 enzymes: anthocyanin 5-O-glucosyl- alba myrosinase with the putative transition state inhibitors transferase (AA5GT) from carnation (Dianthus caryophyl- glucohydroximino lactam (Ki = 0.6 mM) and glucotetrazole lus) and anthocyanin 7-O-glucosyltransferase (AA7GT) (Ki = 0.7 mM),34) and inactive E191D mutant of maize (Zea from delphinium (Delphinium grandifl orum), respectively.21) mays) ZmGlu1 β-glucosidase with glucotetrazole have been 4 AA5GT and AA7GT require glucose esters, such as feruloyl reported, which was described as taking a H3 conforma- 35) 4 glucose and vanillyl glucose as donors and transfer with tion. A similar H3 conformation has been observed for retention of anomeric confi guration, as expected for GH1 glucoimidazole, which binds with >50-fold greater affi nity enzymes. The carnation and delphidium glucosyl transferas- than glucotetrazole, in the rice Os7BGlu26 β- / es belong to the At/Os 6 phylogenetic cluster, as described β-glucosidase active site, confi rming the transition state may by Opassiri et al.,9) and shown in Fig. 1. Our lab has recently be close to this structure (Tankrathok et al., unpublished found that the rice GH1 cluster At/Os6 enzyme Os9BGlu31 results). Numerous reports of substrates bound in inactive has similar transglycosylase activity (Luang et al., mutants of plant GH1 β-glucosidases or substrate-like inhibi- unpublished). For Os9BGlu31, the best donor substrates are tors in active GH1 enzyme active sites have shown the 54 J. Appl. Glycosci., Vol. 59, No. 2 (2012)

Fig. 2. Structures of substrates for GH1 enzymes, including examples of those discussed in this paper. In addition to β-glucosidase substrates, the disaccharidase substrates phenylethyl primeveroside and dalpatein β-acuminoside, the transglycosylase substrates feruroyl glucose (donor), cyanidin 3-O-glucoside (acceptor) and monogalac- tosyl diacyl glyceride, the hydroxyisourate hydrolase substrate are shown. An arrow points to the bond that is cleaved in hydroxyisourate. The common synthetic substrate pNPGlc is shown for comparison. Ketudat Cairns et al.: Plant Glycoside Hydrolase Family 1 Specifi city 55

Fig. 3. Double displacement mechanism proposed for retaining β-glycosidases, such as those in glycoside hydrolase family 1. The fi rst step of glycosylation is shown in the top row, which forms a semistable glycosyl enzyme intermediate. The deglycosylation step in the bottom row is split into hydrolysis to the right and transglycosylation to the left, depending on whether water or another nucleophile acts as the acceptor substrate.

1 4 46) nonreducing terminal glucosyl residue is distorted into a S3 distortion from the relaxed C1 chair. Similarly, QM/MM skew boat or similar structure.16,32,33,35－38) Notably, the rice simulations of glycoside binding to Os3BGlu7 and an HvBII Os3BGlu7 E176Q mutant structures in complex with model based on the Os3BGlu7 crystal structure supported 1 oligosaccharides showed poor density for the C1 and O1 and binding of both glucosides and mannosides in a S3 confor- 1 the fi nal refi ned structures were shown to fall between a S3 mation, but with the caveat that the strain energy was much 4 3 O,3 and E structure, suggesting the sugars were moving toward lower for the S5 ( B) structure. However, preliminary 4 38) the H3 transition state. structural studies show that the closely related Os7BGlu26 Although most of the sugars acting as glycons in GH1 β-mannosidase binds mannoimidazole in a structure similar substrates may follow a similar conformational path through to the B2,5 boat, suggesting these enzymes may hydrolyze catalysis, mannose, with its axial rather than equatorial OH2, β-D-mannosides via the prescribed conformational pathway 1 is thought to follow a path from a S5 skew boat to a B2,5 to (Tankratok et al., unpublished). Since all the glycon-binding O an S2 skew boat conformation, as shown in GH family 2 and residues are conserved in Os3BGlu7, Os7BGlu26 and barley 26 enzymes.25,27,39) It has been suggested that this refl ects the HvBII, the basis for Os3BGlu7 hydrolyzing β-D-glucoside evolution of GH to bind catalytic trajectories that follow the faster than β-D-mannoside, while HvBII and Os7BGlu26 low energy conformations of the pyranose ring that move show the opposite tendencies remains to be determined. toward the bond lengths and angles of the lowest energy The glycon-binding amino acid residues in the －1 subsite transition states, based on free-energy surface analysis.40) are highly conserved among GH1 glycoside hydrolases, Since GH1 contains enzymes with both β-mannosidase and though they are absent in hydroxyisourate hydrolase. The β-glucosidase activities, we have explored the basis for this residues were fi rst identifi ed in the 2-deoxy-2-fl uorogluco- and whether the same enzymes follow different conforma- side derivative of S. alba myrosinase (Fig. 4), where tional reaction pathways with different substrates or they hydrogen bonds were found between Asn186 and the 2-fl uoro drive substrates through noncanonical reaction pathways. group, His139 and Gln39 and the Glc 3-hydroxyl (OH3), Closely related enzymes in phylogenetic cluster At/Os4 (Fig. Gln39 and Glu434 and the Glc 4-hydroxyl (OH4), and 1) have both β-mannosidase and β-glucosidase activities, Glu434 and the 6-hydroxyl (OH6).30) Other interactions were with β-mannosidase activity predominating in barley noted with aromatic groups including Tyr330, Trp457, (Hordeum vulgare) HvBII, tomato (Lycopersicon esculen- Phe465 and Phe473, with Trp457 serving as the platform for tum) β-mannosidase, Arabidopsis AtGlu45 and rice stacking the sugar ring. These residues and interactions are Os7BGlu26, while β-glucosidase activity is higher in largely conserved in plant GH1 β-D-O-glycosidase covalent Os3BGlu7 and Os3BGlu8.8,41－45) Saturation transfer NMR intermediates, inhibitor, and inactive mutant/substrate experiments with thioglucoside and thiomannoside showed complexes.16,31‒33,35,37,38,47) For instance, the rice Os3BGlu7 1 3 O,3 that barley HvBII could bind in either S3 or S5 ( B) confi g- E176Q acid/base mutant binds cellooligosaccharides with urations, while thiomannoside could not bind well in the subsite －1 interactions including Trp433 serving as the active site of either HvBII or rice Os3BGlu7 and showed no platform to stack the sugar, Asn175 and Glu386 hydrogen 56 J. Appl. Glycosci., Vol. 59, No. 2 (2012)

Aglycon binding. Since the function of most plant β-D-glucosidases is likely to be largely determined by their substrate aglycon specifi c- ity, much work has been done over the past 12 years to investigate aglycon binding specifi city. Although synthetic colorimetric substrates, like oNP and pNP-glycosides, are convenient and have been used to investigate aglycon binding residues,50,51) natural substrates offer a wider structur- al range of more relevance to enzyme function in the plant. Natural substrates that have been used in mutagenic and structural studies of plant GH1 aglycon specifi city include the hydroxaminic acids DIMBOA (2,4-dihydroxy-7- methoxy-1,4-benzoxazin-3-one) and DIBOA (2,4-dihydroxy- 1,4-benzoxazin-3-one), cyanogenic glycosides (dhurrin and linamarin), isofl avonoids and oligosaccharides (Fig. 2). The β-1,4-linked oligosaccharides provide the longest aglycone, Fig. 4. Covalent intermediate of S. alba myrosinase with gluco- the binding cleft for which can be divided into binding syl-2-fl uoride. subsites for successive glycosyl residues, starting from the The amino acid residues (stick representation) making interac- sugar residue bound to the glycon via the hydrolyzed tions with the G2F ligand (ball and stick representation) are shown with dashed lines representing hydrogen bonds. In the on- glycosidic bond (subsite +1) to the reducing end (subsite line version, the ligand carbons are in yellow, the carbons of the +(n－1)), where n is the number of monosaccharide residues catalytic nucleophile and the Gln corresponding to the catalytic in the oligosaccharide.52) These subsites may be defi ned acid-base in β-O-glucosidases are in pink, carbons of other amino either kinetically53) or structurally,52) but these two defi ni- acid residues are in gray, oxygens are red, nitrogens blue and tions are only roughly correlated, due to the possibility of fl uoride green. binding in more than one productive confi guration.54) The determinants for aglycon specifi city have been most bonding to the Glc1 (nonreducing terminal glucosyl residue) thoroughly investigated in monocot chloroplastic OH2, Gln29, His130 and Trp441 hydrogen bonding to Glc1 β-glucosidases. Esen and colleagues made a comparison of OH3, Gln29 and Glu440 hydrogen-bonding to Glc1 OH4, maize ZmGlu1 β-glucosidase and sorghum (Sorghum Glu440 hydrogen bonding to OH6 and Tyr315 binding to O5 bicolor) dhurrinase 1 (SbDhr1), which share 70% amino of the Glc1 pyranose ring (Fig. 5).38) Although the pyranose acid sequence identity, but do not hydrolyze each other’s 1 ring is distorted to a conformation between a S3 skew boat substrates. ZmGlu1 has rather broad specifi city, hydrolyzing and a 4E envelope in these Michaelis complex structures, the the hydroxaminic acid glucoside 2-β-D-glucosyl 2,4- same groups are hydrogen bonded in the covalent complexes dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOAGlc, of the wild type and E176Q mutant with 2-deoxy-2-fl uorog- Fig. 2)55) and cytokinin glucosides,56) among others, but does 4 lucoside, where the pyranose takes the C1 relaxed chair not hydrolyze the cyanogenic glucoside dhurrin, for which conformation (Fig. 5).31,38) SbDhr1 is very specifi c. When chimeras were made to switch As noted previously, plant GH1 glycosidases show a range segments of these two enzymes, the regions conveying the of glycon specifi cities, in terms of accepting β-D-glucose ability to hydrolyze each substrate appeared to be different and other pyranoses, such as β-D-fucose, β-D-, for the two enzymes.55) The subsequent structure of the maize β-D-xylose, α-L-arabinose, and, in some cases, β-D-mannose. enzyme and its inactive E191D (acid-base Glu changed to Despite this variation, all the glycon-binding residues Asp) mutant in complex with DIMBOA, DIMBOAGlc, and mentioned above are generally conserved in GH1 enzymes dhurrin showed that the aglycon is sandwiched between a with different glycon preferences. Sanz-Aparacio et al.48) Trp (Trp378) on one side and three Phe residues (Phe198, noted that the conserved Glu residue straddling OH4 and Phe205 and Phe466) on the other.36,47) Of these residues, only OH6 is able to bind either equatorial or axial OH4 groups, Trp378 is conserved with SbDhr1 (Trp376) and most other accounting for the ability of bacterial GH1 to show both plant GH1 enzymes. In sorghum, the Phe residues were β-D-glucosidase and β-D-galactosidase activities, which replaced by Val196, Leu203 and Ser462, respectively, of apparently applies to plant GH1 enzymes, as well. Investi- which Ser462 appeared most critical for binding by forming gating an insect GH1 β-D-glucosidase, Mendonça and a water-mediated hydrogen bond to the dhurrin phenolic Marana49) showed that mutation of residues outside the active hydroxyl.35) site that are in interaction networks with active-site surface Mutations of these residues in ZmGlu1 (or its isoform Zm residues can change relative levels of β-D-fucosidase and 60.1) generally led to decreased hydrolysis of both synthetic β-D-glucosidase activities. Thus, it appears that subtle and natural substrates.50,51) However, it was conversion of the changes in the shape of the active site and positioning of the neighboring Tyr473 residue that hydrogen bonds to the －1 subsite amino acid residues are responsible for differ- conserved Trp376 to the SbDhr1 Phe (Y473F) that caused ences in glycon sugar specifi city. We are still investigating the greatest acquisition of dhurrinase activity by ZmGlu1, how this applies to β-D-mannosidases, where the enzyme giving it 10% of the relative effi ciency for dhurrin hydroly- may have to stabilize a completely different set of reaction sis of SbDhr1. This suggested that the orientation of the intermediate and transition state conformations compared to conserved Trp is important for substrate specifi city, an β-D-glucosidases.25) observation that has also been made for strictosidine Ketudat Cairns et al.: Plant Glycoside Hydrolase Family 1 Specifi city 57

Fig. 5. Substrate binding by rice Os3BGlu7 (BGlu1) β-glucosidase. (A) Binding of cellopentaose in the active site of rice Os3BGlu7 E176Q. (B) Binding of the nonreducing glucosyl residue of cellopentaose in the －1 subsite by strong hydrogen bonds and aromatic interactions. (C) Binding of 2-deoxy-2-fl uoro- glucoside in the active site of its covalent intermediate with Os3BGlu7 for comparison of binding between the covalent intermediate and the substrate. Note that the hydrogen bonding (dashed lines) is the same, despite the differences in the shape of the glucosyl moiety in the substrate Michaelis complex (A and B) and the covalent intermediate (C).

β-glucosidase.37) Surprisingly, the wheat and rye occur without acid-assistance, and a small nucleophile, like β-glucosidases that hydrolyze DIMBOAGlc and its azide, which can remove the sugar from the enzyme without 7-demethoxy analogue (DIBOAGlc) show a lack of conser- basic assistance.61,62) So, binding could be quantifi ed by vation with maize in these aglycon-binding residues other oligosaccharide inhibition of pNPGlc hydrolysis, which than the conserved Trp.33,57) gave results consistent with the binding of at least 6 Glc The most extensive aglycon binding interactions observed residues, as seen in the kinetic subsite analysis.38) in GH1 are those of the oligosaccharide-hydrolyzing The structure of rice Os3BGlu7 E176Q bound to cellopen- β-glucosidases/β-mannosidases from rice and barley. The taose showed that only the nonreducing glucosyl residue in barley HvBII and rice Os3BGlu7 have been shown by kinetic the －1 subsite is bound by strong direct hydrogen bonds, subsite mapping by the method of Hiromi53,58) to have interac- while the remaining residues appear to be bound primarily tions for at least 6 β-1,4-linked glucosyl residues, where the by aromatic stacking interactions and water-mediated fi rst Glc (Glc1) refers to the nonreducing end residue that is hydrogen bonds, as shown in Fig. 5.38) The only direct cut off in the hydrolysis reaction and its binding site is hydrogen bonds were observed between Asn245 and the referred to as subsite －1.59,60) These analyses were limited Glc3 residue in the +2 subsite. This is consistent with the by the solubility of cellooligosaccharides with degree of strong binding at the +2 subsite in kinetic subsite analysis.60) polymerization (DP) >6, whereas cellopentaose (DP 5) was In contrast, Asn245 makes a direct hydrogen bond to the the longest cellooligosaccharide that could be bound in the Glc2 residue OH1 in the +1 subsite in the Os3BGlu7 complex active site in complexes with the Os3BGlu7 E176Q acid-base with laminaribiose. Interestingly, when cellotetraose and mutant.38) This acid-base mutant is rescued by substrates cellopentaose were soaked into the Os3BGlu7 E386G with good leaving groups, which allow the glycosylation to mutant, the Asn245 moved to form hydrogen bonds between 58 J. Appl. Glycosci., Vol. 59, No. 2 (2012) the subsite +1 Glc2 OH6 and the subsite +2 OH2, although glucosyl residue along its length, in addition to stacking of the oligosaccharides bound in nearly the same positions in the second, third and fi fth glucosyl residues onto Trp78, the two mutants.54) Evidently, the almost imperceptible shift Trp129 and Phe128, respectively.63) This likely refl ects the in the cellooligosaccharide binding was enough to make the need of the Os3BGlu7 enzyme to release or adjust the difference in the binding modes. position of the oligosaccharide upon hydrolysis of the The plasticity in oligosaccharide binding was even more glycosidic bond, in order to allow release of the resulting evident when the glucosyl moiety binding residues were glucose residue before further oligosaccharide hydrolysis, mutated.54) The mutation of the Tyr341 that serves as an whereas the CBM has no such requirement. aromatic platform for stacking of the subsite +3 and +4 Another way to look at the oligosaccharide binding sites glucosyl residues in the Os3BGlu7 E176Q cellopentaose is to compare enzymes with different activities toward complex to Leu or Ala resulted in very small effects on oligosaccharides. Although Os3BGlu7 hydrolyzes cellooli- cellooligosaccharide hydrolysis (Fig. 6), though the same gosaccharides with increasing effi ciency (kcat/Km) as the DP mutations in the Os3BGlu7 E386G glycosynthase mutant increases from 2 to 6, as do other enzymes in the phyloge- appeared to decrease synthesis of long oligosaccharides (see netic cluster At/Os4,45,60) Os4BGlu12 shows a big jump from below). When the structure of the Os3BGlu7 E386G/Y341A DP 2 to 3, but only marginal increase in kcat/Km as the DP complex with cellotetraose was solved, it showed that the increases from 3 to 5,64) while Os3BGlu6 hydrolyzes all nonreducing residue was still in the same position in the －1 cellooligosaccharides very poorly.32) In contrast, all three subsite, but the other residues were in a completely different enzymes have relatively high activities to laminaribiose, but binding mode. The latter three residues had fl ipped nearly not longer β-(1,3)-linked oligosaccharides. The crystal 180° and now the direct hydrogen bonds were between structure of Os3BGlu6 suggested it could not bind cellooli- Glc4 and the Gln187 sidechain on the opposite side of the gosaccharides in the same position as Os3BGlu7 due to the active site cleft. Since this mutation gave only a small effect replacement of Asn245 with Met251, which not only cannot on cellotetraose hydrolysis (kcat/Km of 58 mM－1s－1 vs. 64 provide hydrogen bonding but also imposes a steric block to mM－1s－1 for wild type), the binding in this alternate mode is binding in the same position. When Os3BGlu6 Met251 was likely of similar energy to the previously identifi ed mode. replaced with Asn, the enzyme showed increased binding of This suggests that though the lowest energy form is seen in oligosaccharides and appeared to have gained a +2 subsite crystal structures, different binding modes may coexist in for β-(1,4)-linked oligosaccharides.65) Correspondingly, solution. when Os3BGlu7 Asn245 was replaced with Val31) or Met, The binding of the celloligosaccharides in different modes there was a large decrease in effi ciency (kcat/Km) for hydroly- may increase the effi ciency of their hydrolysis and perhaps sis of β-(1,4)-linked oligosaccharides, with the Met having a allow these oligosaccharides to be retained for further larger effect, supporting a steric effect (Fig. 6). Mutation of hydrolysis upon removal of the nonreducing glucosyl the corresponding His243 of Os4BGlu12 to Met caused a residue, although Os3BGlu7 is not known to be processive. smaller decrease in cellooligosaccharide hydrolysis, suggest- The presence of two or more binding modes suggests that ing this residue is not as important for oligosaccharide- the subsites identifi ed by kinetic subsite or inhibition studies binding as Os3BGlu7 Asn245. It was observed that hydroly- do not correspond to a single binding position in the 3D sis of longer oligosaccharides (cellotetraose and structure, but rather to the combination of the binding of the cellopentaose) was less affected by the Os3BGlu7 N245M corresponding glycosyl residue in all productive binding mutation than that of cellotriose, possibly due to binding in modes. The apparently weak and plastic interactions of the the second mode noted above, in which Asn245 plays a active-site residues with aglycon glucose residues is in smaller role. contrast to the strong interactions seen in many carbohy- drate-binding module (CBM) domains. For example, the Transglycosylase activity. Clostridium josui Cel5A endoglucanse CBM28 domain The processes of hydrolysis and glycosyl transfer are binds cellopentaose with strong hydrogen bonds to each interrelated and indeed retaining glycosidases can generally catalyze glycosyl transfer reactions, as shown in Fig. 3. Although glycosyltransferases that have been found in nature are mainly those that transfer from a nucleotidyl sugar, such as uridine diphosphate-α-D-glucose (UDP-glucose), to an acceptor, and it has been suggested that the GT designation be reserved for enzymes that use this class of donors,66) transglycosylases (TG) that use other sugar donors have been identifi ed, and these are generally related to glycoside hydrolases. For instance, xyloglucan endotransferases (XET), which cross-link the cell wall of plants by cutting xyloglucans from one strand and transferring the nonreduc- ing end half onto another xyloglucan strand, fall in GH family 16, which also includes xyloglucan hydrolases.67) GH1 TG have also been identifi ed recently, as described above. The determinants that differentiate an enzyme that is Fig. 6. Comparison of the kcat/Km values of Os3BGlu7 and its ac- tive site mutants for hydrolysis of cellooligosaccharides. primarily a hydrolase from one that is primarily a transferase are still under investigation. Ketudat Cairns et al.: Plant Glycoside Hydrolase Family 1 Specifi city 59

As noted above, two types of TG were recently identifi ed as GH1 enzymes, the SFR2 GGGT20) and acyl glucose- dependent anthocyanin O-glucoside glucosyl tranferases.21) Although SFR2 is the most phylogenetically divergent plant GH1 enzyme, its GGGT activity appears to be similar to the transglycosylation seen with other GH1 enzymes in vitro, although, evidently, without hydrolysis. The fact that SFR2 is localized to the chloroplast outer membrane and acts on substrates within that membrane is likely to play a role in excluding water from the active site and promoting transgly- cosylation rather than hydrolysis.20) The conversion of cyanidin 3-O-glucoside to 3,5- and 3,7-diglucosides in carnation and delphinium petals by AA5GT and AA7GT, on the other hand, apparently occurs in the vacuole lumen.21) Interestingly, both the acyl glucoside/anthocyanidin glucosyl Fig. 7. Superposition of Os9BGlu31 homology model with Os- transferases, Os9BGlu31 and HIUH have the sequence 3BGlu6 covalent complex with 2-deoxy-2-fl urogluco- “HENG” surrounding their catalytic nucleophiles, while side. most GH1 β-glycosidases that have been characterized have Os3BGlu31 (green carbons) is a transglucosylase while Os3B- the sequence “T/SENG,” except for other cluster At/Os6 Glu6 (light grey carbons) is a β-D-glucosidase with very little gly- enzymes, which have a range of amino acids in the position cosyl transferase activity. Os3BGlu6 was used as the template for modeling Os9BGlu31 in the SwissModel server.82) The protein before the catalytic nucleophile. Although the GH1 TGs residues within approximately 10 Å of the water binding site above transfer glycons from unusual donors, it should be noted that the anomeric carbon that are different between the two proteins are in each case, the initial glycoconjugate is most likely shown in stick representations, while other catalytically important produced by nucleotide-sugar dependent GTs. residues and the Cα backbones are shown as lines. It is yet unclear what exactly differentiates a GH1 TG from a GH, but it is conjectured that water is excluded from be low due to their ability to hydrolyze the substrates and/or the acceptor site. Many GH1 hydrolases in fact appear to products. prefer transglycosylation when substrate concentrations are In the interest of glycoside and oligosaccharide synthesis, taken into account. For instance, rice Os3BGlu7 makes GH mutants that catalyze transfer reactions with little signifi cant amounts of pNP-oligosaccharides at pNPGlc hydrolysis have been developed, primarily by mutating the concentrations of 5‒10 mM, despite the 5,000‒10,000-fold catalytic groups to change the enzyme mechanism. Mutation excess of water (56 M) as acceptor.60) In GH16, it has been of the nucleophile of retaining glycosidases to a small shown that shortening of an active site single loop that is nonnucleophilic residue like Ala, Gly or Ser creates a elongated in naturtium (Tropaeolum majus) xyloglucan nonhydrolytic “glycosynthase” enzyme, which can transfer a hydrolase by 5 residues to more closely match that of XET sugar from a donor of the opposite anomeric confi guration to increased the ratio of transglycosylation to hydrolysis by 40 a sugar or other donor without hydrolysis of the product.74,75) fold, although it could not produce an exclusive XET.68) Kitaoka and colleagues have also shown that a similar activi- Indeed, homology modeling of Os9BGlu31 (Fig. 7), AA5GT, ty can be achieved from an inverting glycosidase by changing and AA7GT show differences in the loops around the active the catalytic base to another residue, such as Cys76) or site and somewhat more hydrophobic amino acids compared mutating a conserved Tyr that helps position the nucleophilic to the known GH1 hydrolase structures (Luang et al., water molecule.77) Another strategy is to mutate the catalytic unpublished), but a clear understanding of the differences acid-base in a retaining GH and use donor and acceptor may await structure determination and mutational studies. substrates that do not require acidic and basic assistance, GH1 glycosidases have been used to synthesize oligosac- respectively, thereby creating so called “thioglycoligase” charides and glycosides, either by reversal of hydrolysis or mutants.61,62) Such mutants can be used to create thio-linked by transglycosylation, the pathways for both of which are glycosides and oligosaccharides. This is in fact the case of shown in Fig. 3.69－71) Cassava linamarinase was found to be the BGlu1 E176Q mutant described above. Although plant particularly useful for production of glycosides, due to its thioglucosidase are similar to the thioglycoligases, in that ability to transglycosylate secondary and tertiary alcohols.72) they lack an acid-base and can be rescued by an ascorbate Recently, conversion of Thai rosewood (Dalbergia ,34) they are still hydrolytic enzymes, and GH1 cochinchinensis) β-glucosidase active site residues to those enzymes acting as transglycosylases do not appear to use of cassava linamarinase has explored to improve transglyco- similar mechanisms to achieve transfer instead of hydroly- sylation of secondary and tertiary alcohols by the Thai sis. rosewood enzyme, which resulted in increased activity It may be noted that the determinants for TG and GH toward secondary, but not tertiary alcohols.73) Interestingly, activities in the same or comparable enzymes do not always the Ile185 to Ala mutation, which resulted in the highest correlate completely. The Os3BGlu7 E386G glycosynthase increase in production of transglycosylation products, also can produce long β-1,4-linked oligosaccharides when primed resulted in the largest decrease in the hydrolytic effi ciency. with a pNP-cellobiose (C2) or cellooligosaccharide with DP Despite the marginal success in improving the transglycosy- of 3 or greater.78) However, although the Os3BGlu7 Y341A lation by mutagenesis, a weakness in the use of GH for mutation mentioned above has little effect on hydrolysis of glycoside and oligosaccharide synthesis is that yields tend to cellooligosaccharides, it does decrease the production of 60 J. Appl. Glycosci., Vol. 59, No. 2 (2012) long oligosaccharides by the Os3BGlu7 E386G/Y341A and two barley glycanases form a superfamily of glycosynthase.54) It is likely that the second binding mode is enzymes with 8-fold β/α architecture and with two conserved glutamates near the carboxy-terminal ends of β-strands four and either less stable without a glucosyl residue in the 1 subsite － seven. FEBS Lett., 362, 281‒285 (1995). or does not select for production of the β-1,4-linkages needed 6 ) B. Henrissat, I. Callebaut, S. Fabrega, P. Lehn, J.P. Mornon and to form long straight polymers. Thus, the requirements for G. Davies: Conserved catalytic machinery and the prediction of a binding of the transferase acceptor may be different from common fold for several families of glycosyl hydrolases. Proc. those for hydrolytic substrates. Natl. Acad. Sci. USA, 92, 7090‒7094 (1995). 7 ) J.R. Ketudat Cairns and A. Esen: β-Glucosidases. Cell. Mol. Life Sci., 67, 3389‒3405 (2010). Conclusions. 8 ) Z. Xu, L.L. Escamilla-Treviño, L. Zeng, M. Lalgondar, D.R. GH1 is a family of proteins with very diverse functional- Bevan, B.S.J. Winkel, A. Mohamed, C. Cheng, M. Shih, J.E. ities, especially in plants. Plant GH1 enzymes are known to Poulton and A. Esen: Functional genomic analysis of Arabidopsis have a broad range of hydrolase activities. Although many of thaliana glycoside hydrolase family 1. Plant Mol. Biol., 55, 343‒367 (2004). these enzymes have been known to have transglycosylase 9 ) R. Opassiri, B. Pomthong, T. Okoksoong, T. Akiyama, A. Esen activities for years, only recently has it been realized that and J.R. Ketudat Cairns: Analysis of rice glycosyl hydrolase family includes transglycosylases that use galactolipid or family 1 and expression of Os4bglu12 β-glucosidase. BMC Plant glucosyl ester donors in nature. The interactions of GH1 Biol., 6, 33 (2006). 10) M. Mizutani, H. Nakanishi, J. Ema, S.-J. Ma, E. Noguchi, M. enzymes with glycoside and oligosaccharide substrates Inohara-Ochiai, M. Fukuchi-Mizutani, M. Nakao and K. Sakata: display remarkable plasticity, both in terms of the evolution- Cloning of β-primeverosidase from tea leaves, a key enzyme in ary adaptation to select for a broad range of substrates, and tea aroma formation. Plant Physiol., 130, 2164‒2176 (2002). in terms of the presence of different binding modes in the 11) Y.O. Ahn, M. Mizutani, H. Saino and K. Sakata: Furcatin hydro- same enzymes. Although structural and mutagenic work has lase from Viburnum furcatum Blume is a novel disaccharide- specifi c acuminosidase in glycosyl hydrolase family 1. J. Biol. identifi ed important glycon and aglycon-binding residues in Chem., 279, 23405‒23414 (2004). a number of GH1 glycosidases, the specifi city differences 12) P. Chuankhayan, Y. Hua, J. Svasti J, S. Sakdarat, P.A. Sullivan often appear to be driven by active site shape differences that and J.R. Ketudat Cairns: Purifi cation of an isofl avonoid must refl ect underlying and distant amino acid residues. 7-O-β-apiosyl-glucoside β-glycosidase and its substrates from Thus, much work remains for the characterization of the Dalbergia nigrescens Kurz. Phytochemistry, 66, 1880‒1889 (2005). determinants of substrate specifi city in GH1 hydrolases, 13 ) P. Chuankhayan, T. Rimlumduan, W. Tantanuch, N. Mothong, while the determinants of GH1 transglycosylase activities P.T. Kongsaeree, P. Metheenukul, J. Svasti, O.N. Jensen and J.R. are just beginning. Work on GH1 hydrolase mutants that act Ketudat Cairns: Functional and structural differences between as transferases suggests that the requirements for transglyco- isofl avonoid β-glycosidases from Dalbergia sp. Arch. Biochem. Biophys., 468, 205 216 (2007). sylation specifi city may be different from those for hydrolase ‒ 14 ) H. Suzuki, S. Takahasi, R. Watanabe, Y. Fukushima, N. Fujita, A. specifi city. Given the important functions of many GH1 Noguchi, R. Yokoyama, K. Nishitani, T. Nishino and T. Nakaya- substrates and products, the interactions between GH1 ma: An isofl avone conjugate-hydrolyzing β-glucosidse from the enzymes and their substrates is likely to be a fruitful area of roots of soybean (Glycine max) seedlings. J. Biol. Chem., 281, study for years to come. 30251‒30259 (2006). 15 ) H. Shen and L.D. Byers: Thioglycoside hydrolysis by β-glucosidase. Biochem. Biophys. Res. Comm., 362, 717‒720 ACKNOWLEDGEMENTS (2007). 16 ) S. Sansenya, R. Opassiri, B. Kuaprasert, C.-J. Chen and J.R. We are grateful to many participants in the Japan Applied Glycosci- Ketudat Cairns: The crystal structure of rice (Oryza sativa L.) ence Society Meeting on α- and Related Enzymes at Hokkai- Os4BGlu12, an oligosaccharide and tuberonic acid glucoside- do University, September, 2011, for insightful questions and comments, hydrolyzing β-glucosidase with signifi cant thioglucohydrolase including Atsuo Kimura, Shinya Fushinobu, Hironori Hondoh and activity. Arch. Biochem. Biophys., 510, 62‒72 (2011). Ken Tokuyasu. Ayako Suzuki and Yasuhito Takeda are thanked for 17 ) A. Raychaudhuri and P.A. Tipton: Cloning and expression of the supporting our participation in the JAG meeting. JRKC is funded by gene for soybean hydroxyisourate hydrolase. Localization and the Thailand Research Fund grant BRG5380017 and the Suranaree implications for function and mechanism. Plant Physiol., 130, University of Technology National Research University Project of the 2061‒2068 (2002). 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