Effects of active site cleft residues on oligosaccharide binding,hydrolysis, and glycosynthase activities of rice BGlu1 and its mutants

Rice BGlu1 (Os3BGlu7) is a glycoside hydrolase family 1 β‐glucosidase that hydrolyzes cellooligosaccharides with increasing efficiency as the degree of polymerization (DP) increases from 2 to 6, indicating six subsites for glucosyl residue binding. Five subsites have been identified in X‐ray crystal structures of cellooligosaccharide complexes with its E176Q acid‐base and E386G nucleophile mutants. X‐ray crystal structures indicate that cellotetraose binds in a similar mode in BGlu1 E176Q and E386G, but in a different mode in the BGlu1 E386G/Y341A variant, in which glucosyl residue 4 (Glc4) interacts with Q187 instead of the eliminated phenolic group of Y341. Here, we found that the Q187A mutation has little effect on BGlu1 cellooligosaccharide hydrolysis activity or oligosaccharide binding in BGlu1 E176Q, and only slight effects on BGlu1 E386G glycosynthase activity. X‐ray crystal structures showed that cellotetraose binds in a different position in BGlu1 E176Q/Y341A, in which it interacts directly with R178 and W337, and the Q187A mutation had little effect on cellotetraose binding. Mutations of R178 and W337 to A had significant and nonadditive effects on oligosaccharide hydrolysis by BGlu1, pNPGlc cleavage and cellooligosaccharide inhibition of BGlu1 E176Q and BGlu1 E386G glycosynthase activity. Hydrolysis activity was partially rescued by Y341 for longer substrates, suggesting stacking of Glc4 on Y341 stabilizes binding of cellooligosaccharides in the optimal position for hydrolysis. This analysis indicates that complex interactions between active site cleft residues modulate substrate binding and hydrolysis.     

The structural basis of oligosaccharide binding by rice BGlu1 beta-glucosidase

Rice BGlu1 β-glucosidase is an oligosaccharide exoglucosidase that binds to six β-(1→4)-linked glucosyl residues in its active site cleft. Here, we demonstrate that a BGlu1 E176Q active site mutant can be effectively rescued by small nucleophiles, such as acetate, azide and ascorbate, for hydrolysis of aryl glycosides in a pH-independent manner above pH 5, consistent with the role of E176 as the catalytic acid–base. Cellotriose, cellotetraose, cellopentaose, cellohexaose and laminaribiose are not hydrolyzed by the mutant and instead exhibit competitive inhibition. The structures of the BGlu1 E176Q, its complexes with cellotetraose, cellopentaose and laminaribiose, and its covalent intermediate with 2-deoxy-2-fluoroglucoside were determined at 1.65, 1.95, 1.80, 2.80, and 1.90 Å resolution, respectively. The Q176 Nε was found to hydrogen bond to the glycosidic oxygen of the scissile bond, thereby explaining its high activity. The enzyme interacts with cellooligosaccharides through direct hydrogen bonds to the nonreducing terminal glucosyl residue. However, interaction with the other glucosyl residues is predominantly mediated through water molecules, with the exception of a direct hydrogen bond from N245 to glucosyl residue 3, consistent with the apparent high binding energy at this residue. Hydrophobic interactions with the aromatic sidechain of W358 appear to orient glucosyl residues 2 and 3, while Y341 orients glucosyl residues 4 and 5. In contrast, laminaribiose has its second glucosyl residue positioned to allow direct hydrogen bonding between its O2 and Q176 Oε and O1 and N245. These are the first GH1 glycoside hydrolase family structures to show oligosaccharide binding in the hydrolytic configuration.     

Enzymatic synthesis of cello-oligosaccharides by rice BGlu1 {beta}-glucosidase glycosynthase mutants

Rice BGlu1 beta-glucosidase is a glycosyl hydrolase family 1 enzyme that acts as an exoglucanase on beta-(1,4)- and short beta-(1,3)-linked gluco-oligosaccharides. Mutations of BGlu1 beta-glucosidase at glutamate residue 414 of its natural precursor destroyed the enzyme's catalytic activity, but the enzyme could be rescued in the presence of the anionic nucleophiles such as formate and azide, which verifies that this residue is the catalytic nucleophile. The catalytic activities of three candidate mutants, E414G, E414S, and E414A, in the presence of the nucleophiles were compared. The E414G mutant had approximately 25- and 1400-fold higher catalytic efficiency than E414A and E414S, respectively. All three mutants could catalyze the synthesis of mixed length oligosaccharides by transglucosylation, when alpha-glucosyl fluoride was used as donor and pNP-cellobioside as acceptor. The E414G mutant gave the fastest transglucosylation rate, which was approximately 3- and 19-fold faster than that of E414S and E414A, respectively, and gave yields of up to 70-80% insoluble products with a donor-acceptor ratio of 5:1. (13)C-NMR, methylation analysis, and electrospray ionization-mass spectrometry showed that the insoluble products were beta-(1,4)-linked oligomers with a degree of polymerization of 5 to at least 11. The BGlu1 E414G glycosynthase was found to prefer longer chain length oligosaccharides that occupy at least three sugar residue-binding subsites as acceptors for productive transglucosylation. This is the first report of a beta-glucansynthase derived from an exoglycosidase that can produce long-chain cello-oligosaccharides, which likely reflects the extended oligosaccharide-binding site of rice BGlu1 beta-glucosidase.     

Structural insights into rice BGlu1 beta-glucosidase oligosaccharide hydrolysis and transglycosylation

The structures of rice BGlu1 β-glucosidase, a plant β-glucosidase active in hydrolyzing cell wall-derived oligosaccharides, and its covalent intermediate with 2-deoxy-2-fluoroglucoside have been solved at 2.2 Å and 1.55 Å resolution, respectively. The structures were similar to the known structures of other glycosyl hydrolase family 1 (GH1) β-glucosidases, but showed several differences in the loops around the active site, which lead to an open active site with a narrow slot at the bottom, compatible with the hydrolysis of long β-1,4-linked oligosaccharides. Though this active site structure is somewhat similar to that of the Paenibacillus polymyxa β-glucosidase B, which hydrolyzes similar oligosaccharides, molecular docking studies indicate that the residues interacting with the substrate beyond the conserved -1 site are completely different, reflecting the independent evolution of plant and microbial GH1 exo-β-glucanase/β-glucosidases. The complex with the 2-fluoroglucoside included a glycerol molecule, which appears to be in a position to make a nucleophilic attack on the anomeric carbon in a transglycosylation reaction. The coordination of the hydroxyl groups suggests that sugars are positioned as acceptors for transglycosylation by their interactions with E176, the catalytic acid/base, and Y131, which is conserved in barley BGQ60/β-II β-glucosidase, that has oligosaccharide hydrolysis and transglycosylation activity similar to rice BGlu1. As the rice and barley enzymes have different preferences for cellobiose and cellotriose, residues that appeared to interact with docked oligosaccharides were mutated to those of the barley enzyme to see if the relative activities of rice BGlu1 toward these substrates could be changed to those of BGQ60. Although no single residue appeared to be responsible for these differences, I179, N190 and N245 did appear to interact with the substrates.     

X-ray crystallography and QM/MM investigation on the oligosaccharide synthesis mechanism of rice BGlu1 glycosynthases

Nucleophile mutants of retaining β-glycosidase can act as glycosynthases to efficiently catalyze the synthesis of oligosaccharides. Previous studies proved that rice BGlu1 mutants E386G, E386S and E386A catalyze the oligosaccharide synthesis with different rates. The E386G mutant gave the fastest transglucosylation rate, which was approximately 3- and 19-fold faster than those of E386S and E386A. To account for the differences of their activities, in this paper, the X-ray crystal structures of BGlu1 mutants E386S and E386A were solved and compared with that of E386G mutant. However, they show quite similar active sites, which implies that their activities cannot be elucidated from the crystal structures alone. Therefore, a combined quantum mechanical/molecular mechanical (QM/MM) calculations were further performed. Our calculations reveal that the catalytic reaction follows a single-step mechanism, i.e., the extraction of proton by the acid/base, E176, and the formation of glycosidic bond are concerted. The energy barriers are calculated to be 19.9, 21.5 and 21.9 kcal/mol for the mutants of E386G, E386S and E386A, respectively, which is consistent with the order of their experimental relative activities. But based on the calculated activation energies, 1.1 kcal/mol energy difference may translate to nearly 100 fold rate difference. Although the rate limiting step in these mutants has not been established, considering the size of the product and the nature of the active site, it is likely that the product release, rather than chemistry, is rate limiting in these oligosaccharides synthesis catalyzed by BGlu1 mutants.     

A β-(1,2)-glycosynthase and an attempted selection method for the directed evolution of glycosynthases

Understanding how enzymes mediate catalysis is a key to their reprogramming for biotechnological applications. The family 3 retaining glycosidase postulated to be involved in erythromycin self-resistance was cloned, recombinantly expressed in Escherichia coli, purified, and characterized. Bioinformatics analysis allowed the identification of the acid/base and nucleophile residues, and mutation of these residues resulted in hydrolytically inactive proteins. One mutant was able to synthesize a glycosidic linkage using α-glucosyl fluoride as a donor and macrolide antibiotics as acceptors. This shows an unprecedented application of glycosynthase technology in accomplishing a challenging β-(1,2)-glycosylation of an amino sugar. This work also provides the first biochemical characterization of the EryBI protein and supports its role in the self-resistance mechanism involved in erythromycin biosynthesis. An in vivo selection approach was used in an attempt to spur evolution of the glycosynthase, and the results from the attempted selection method provide insight into the requirements for in vivo directed evolution of glycosynthases.     

The E358S mutant of Agrobacterium sp. beta-glucosidase is a greatly improved glycosynthase

Glycosynthases are nucleophile mutants of retaining glycosidases that catalyze the glycosylation of sugar acceptors using glycosyl fluoride donors, thereby synthesizing oligosaccharides. The 'original' glycosynthase, derived from Agrobacterium sp. beta-glucosidase (Abg) by mutating the nucleophile glutamate to alanine (E358A), synthesizes oligosaccharides in yields exceeding 90% [Mackenzie, L.F., Wang, Q., Warren, R.A.J. and Withers, S.G. (1998) J. Am. Chem. Soc. 120, 5583-5584]. This mutant has now been re-cloned with a His(6)-tag into a pET-29b(+) vector, allowing gram scale production and single step chromatographic purification. A dramatic, 24-fold, improvement in synthetic rates has also been achieved by substituting the nucleophile with serine, resulting in improved product yields, reduced reaction times and an enhanced synthetic repertoire. Thus poor acceptors for Abg E358A, such as PNP-GlcNAc, are successfully glycosylated by E358S, allowing the synthesis of PNP-beta-LacNAc. The increased glycosylation activity of Abg E358S likely originates from a stabilizing interaction between the Ser hydroxyl group and the departing anomeric fluorine of the alpha-glycosyl fluoride.     

Re-engineering specificity in 1,3-1, 4-β-glucanase to accept branched xyloglucan substrates

Family 16 carbohydrate active enzyme members Bacillus licheniformis 1,3-1,4-β-glucanase and Populus tremula x tremuloides xyloglucan endotransglycosylase (XET16-34) are highly structurally related but display different substrate specificities. Although the first binds linear gluco-oligosaccharides, the second binds branched xylogluco-oligosaccharides. Prior engineered nucleophile mutants of both enzymes are glycosynthases that catalyze the condensation between a glycosyl fluoride donor and a glycoside acceptor. With the aim of expanding the glycosynthase technology to produce designer oligosaccharides consisting of hybrids between branched xylogluco- and linear gluco-oligosaccharides, enzyme engineering on the negative subsites of 1,3-1,4-β-glucanase to accept branched substrates has been undertaken. Removal of the 1,3-1,4-β-glucanase major loop and replacement with that of XET16-34 to open the binding cleft resulted in a folded protein, which still maintained some β-glucan hydrolase activity, but the corresponding nucleophile mutant did not display glycosynthase activity with either linear or branched glycosyl donors. Next, point mutations of the 1,3-1,4-β-glucanase β-sheets forming the binding site cleft were mutated to resemble XET16-34 residues. The final chimeric protein acquired binding affinity for xyloglucan and did not bind β-glucan. Therefore, binding specificity has been re-engineered, but affinity was low and the nucleophile mutant of the chimeric enzyme did not show glycosynthase activity to produce the target hybrid oligosaccharides. Structural analysis by X-ray crystallography explains these results in terms of changes in the protein structure and highlights further engineering approaches toward introducing the desired activity.     

Rice Os9BGlu31 Is a Transglucosidase with the Capacity to Equilibrate Phenylpropanoid,Flavonoid, and Phytohormone Glycoconjugates

Glycosylation is an important mechanism of controlling the reactivities and bioactivities of plant secondary metabolites and phytohormones. Rice (Oryza sativa) Os9BGlu31 is a glycoside hydrolase family GH1 transglycosidase that acts to transfer glucose between phenolic acids, phytohormones, and flavonoids. The highest activity was observed with the donors feruloyl-glucose, 4-coumaroyl-glucose, and sinapoyl-glucose, which are known to serve as donors in acyl and glucosyl transfer reactions in the vacuole, where Os9BGlu31 is localized. The free acids of these compounds also served as the best acceptors, suggesting that Os9BGlu31 may equilibrate the levels of phenolic acids and carboxylated phytohormones and their glucoconjugates. The Os9BGlu31 gene is most highly expressed in senescing flag leaf and developing seed and is induced in rice seedlings in response to drought stress and treatment with phytohormones, including abscisic acid, ethephon, methyljasmonate, 2,4-dichlorophenoxyacetic acid, and kinetin. Although site-directed mutagenesis of Os9BGlu31 indicated a function for the putative catalytic acid/base (Glu¹⁶⁹), catalytic nucleophile residues (Glu³⁸⁷), and His³⁸⁶, the wild type enzyme displays an unusual lack of inhibition by mechanism-based inhibitors of GH1 β-glucosidases that utilize a double displacement retaining mechanism.     

Hydrolase and glycosynthase activity of endo-1,3-beta-glucanase from the thermophile Pyrococcus furiosus

Pyrococcus furiosus laminarinase (LamA, PF0076) is an endo-glycosidase that hydrolyzes beta-1,3-glucooligosaccharides, but not beta-1,4-gluco-oligosaccharides. We studied the specificity of LamA towards small saccharides by using 4-methylumbelliferyl beta-glucosides with different linkages. Besides endo-activity, wild-type LamA has some exo-activity, and catalyzes the hydrolysis of mixed-linked oligosaccharides (Glcbeta4Glcbeta3Glcbeta-MU (Glc = glucosyl, MU = 4-methylumbelliferyl)) with both beta-1,4 and beta-1,3 specificities. The LamA mutant E170A had severely reduced hydrolytic activity, which is consistent with Glu170 being the catalytic nucleophile. The E170A mutant was active as a glycosynthase, catalyzing the condensation of alpha-laminaribiosyl fluoride to different acceptors. The best condensation yields were found at pH 6.5 and 50 degrees C, but did not exceed 30%. Depending on the acceptor, the synthase generated either a beta-1,3 or a beta-1,4 linkage.     

The crystal structure of rice (Oryza sativa L.) Os4BGlu12, an oligosaccharide and tuberonic acid glucoside-hydrolyzing β-glucosidase with significant thioglucohydrolase activity

Rice Os4BGlu12, a glycoside hydrolase family 1 (GH1) β-glucosidase, hydrolyzes β-(1,4)-linked oligosaccharides of 3–6 glucosyl residues and the β-(1,3)-linked disaccharide laminaribiose, as well as certain glycosides. The crystal structures of apo Os4BGlu12, and its complexes with 2,4-dinitrophenyl-2-deoxyl-2-fluoroglucoside (DNP2FG) and 2-deoxy-2-fluoroglucose (G2F) were solved at 2.50, 2.45 and 2.40 Å resolution, respectively. The overall structure of rice Os4BGlu12 is typical of GH1 enzymes, but it contains an extra disulfide bridge in the loop B region. The glucose ring of the G2F in the covalent intermediate was found in a ⁴C₁ chair conformation, while that of the noncovalently bound DNP2FG had a ¹S₃ skew boat, consistent with hydrolysis via a ⁴H₃ half-chair transition state. The position of the catalytic nucleophile (Glu393) in the G2F structure was more similar to that of the Sinapsis alba myrosinase G2F complex than to that in covalent intermediates of other O-glucosidases, such as rice Os3BGlu6 and Os3BGlu7 β-glucosidases. This correlated with a significant thioglucosidase activity for Os4BGlu12, although with 200- to 1200-fold lower k_cat/K_m values for S-glucosides than the comparable O-glucosides, while hydrolysis of S-glucosides was undetectable for Os3BGlu6 and Os3BGlu7.     

Structural and biochemical characterization of glycoside hydrolase family 79 β-glucuronidase from Acidobacterium capsulatum

We present the first structure of a glycoside hydrolase family 79 β-glucuronidase from Acidobacterium capsulatum, both as a product complex with β-D-glucuronic acid (GlcA) and as its trapped covalent 2-fluoroglucuronyl intermediate. This enzyme consists of a catalytic (β/α)(8)-barrel domain and a β-domain with irregular Greek key motifs that is of unknown function. The enzyme showed β-glucuronidase activity and trace levels of β-glucosidase and β-xylosidase activities. In conjunction with mutagenesis studies, these structures identify the catalytic residues as Glu(173) (acid base) and Glu(287) (nucleophile), consistent with the retaining mechanism demonstrated by (1)H NMR analysis. Glu(45), Tyr(243), Tyr(292)-Gly(294), and Tyr(334) form the catalytic pocket and provide substrate discrimination. Consistent with this, the Y292A mutation, which affects the interaction between the main chains of Gln(293) and Gly(294) and the GlcA carboxyl group, resulted in significant loss of β-glucuronidase activity while retaining the side activities at wild-type levels. Likewise, although the β-glucuronidase activity of the Y334F mutant is ~200-fold lower (k(cat)/K(m)) than that of the wild-type enzyme, the β-glucosidase activity is actually 3 times higher and the β-xylosidase activity is only 2.5-fold lower than the equivalent parameters for wild type, consistent with a role for Tyr(334) in recognition of the C6 position of GlcA. The involvement of Glu(45) in discriminating against binding of the O-methyl group at the C4 position of GlcA is revealed in the fact that the E45D mutant hydrolyzes PNP-β-GlcA approximately 300-fold slower (k(cat)/K(m)) than does the wild-type enzyme, whereas 4-O-methyl-GlcA-containing oligosaccharides are hydrolyzed only 7-fold slower.     

The A-kinase-anchoring protein AKAP-Lbc facilitates cardioprotective PKA phosphorylation of Hsp20 on Ser16

BcChi-A, a GH19 chitinase from the moss Bryum coronatum, is an endo-acting enzyme that hydrolyses the glycosidic bonds of chitin, (GlcNAc)(n) [a β-1,4-linked polysaccharide of GlcNAc (N-acetylglucosamine) with a polymerization degree of n], through an inverting mechanism. When the wild-type enzyme was incubated with α-(GlcNAc)2-F [α-(GlcNAc)(2) fluoride] in the absence or presence of (GlcNAc)(2), (GlcNAc)(2) and hydrogen fluoride were found to be produced through the Hehre resynthesis-hydrolysis mechanism. To convert BcChi-A into a glycosynthase, we employed the strategy reported by Honda et al. [(2006) J. Biol. Chem. 281, 1426-1431; (2008) Glycobiology 18, 325-330] of mutating Ser(102), which holds a nucleophilic water molecule, and Glu(70), which acts as a catalytic base, producing S102A, S102C, S102D, S102G, S102H, S102T, E70G and E70Q. In all of the mutated enzymes, except S102T, hydrolytic activity towards (GlcNAc)(6) was not detected under the conditions we used. Among the inactive BcChi-A mutants, S102A, S102C, S102G and E70G were found to successfully synthesize (GlcNAc)(4) as a major product from α-(GlcNAc)(2)-F in the presence of (GlcNAc)(2). The S102A mutant showed the greatest glycosynthase activity owing to its enhanced F(-) releasing activity and its suppressed hydrolytic activity. This is the first report on a glycosynthase that employs amino sugar fluoride as a donor substrate.     

Mutation of fungal endoglucanases into glycosynthases and characterization of their acceptor substrate specificity

Humicola insolens mutant Cel7B E197A is a powerful endo-glycosynthase displaying an acceptor substrate specificity restricted to β-d-glucosyl, β-d-xylosyl, β-d-mannosyl and β-d-glucosaminyl in +1 subsite. Our aim was to extend this substrate specificity to β-d-N-acetylglucosaminyl, in order to get access to a wider array of oligosaccharidic structures obtained through glycosynthase assisted synthesis. In a first approach a trisaccharide bearing a β-d-N-acetylglucosaminyl residue was docked at the +1 subsite of H. insolens Cel7B, indicating that the mutation of only one residue, His209, could lead to the expected wider acceptor specificity. Three H. insolens Cel7B glycosynthase mutants (H209A, H209G and H209A/A211T) were produced and expressed in Aspergillus oryzae. In parallel, sequence alignment investigations showed that several cellulases from family GH7 display an alanine residue instead of histidine at position 209. Amongst them, Trichoderma reesei Cel7B, an endoglucanase sharing the highest degree of sequence identity with Humicola Cel7B, was found to naturally accept a β-d-N-acetylglucosaminyl residue at +1 subsite. The T. reesei Cel7B mutant nucleophile E196A was produced and expressed in Saccharomyces cerevisiae, and its activity as glycosynthase, together with the H. insolens glycosynthase mutants, was evaluated toward various glycosidic acceptors.     

Diverse substrate recognition mechanism revealed by Thermotoga maritima Cel5A structures in complex with cellotetraose, cellobiose and mannotriose

The hyperthermophilic endoglucanase Cel5A from Thermotoga maritima can find applications in lignocellulosic biofuel production, because it catalyzes the hydrolysis of glucan- and mannan-based polysaccharides. Here, we report the crystal structures in apo-form and in complex with three ligands, cellotetraose, cellobiose and mannotriose, at 1.29? to 2.40? resolution. The open carbohydrate-binding cavity which can accommodate oligosaccharide substrates with extensively branched chains explained the dual specificity of the enzyme. Combining our structural information and the previous kinetic data, it is suggested that this enzyme prefers β-glucosyl and β-mannosyl moieties at the reducing end and uses two conserved catalytic residues, E253 (nucleophile) and E136 (general acid/base), to hydrolyze the glycosidic bonds. Moreover, our results also suggest that the wide spectrum of Tm_Cel5A substrates might be due to the lack of steric hindrance around the C2-hydroxyl group of the glucose or mannose unit from active-site residues.     

Site-directed mutation of noncatalytic residues of Thermobifida fusca exocellulase Cel6B.

Fifteen mutant genes in six loop residues and eight mutant genes in five conserved noncatalytic active site residues of Thermobifida fusca Cel6B were constructed, cloned and expressed in Escherichia coli or Streptomyces lividans. The mutant enzymes were assayed for catalytic activity on carboxymethyl cellulose (CMC), swollen cellulose (SC), filter paper (FP), and bacterial microcrystalline cellulose (BMCC) as well as cellotetraose, cellopentaose, and 2, 4-dinitrophenyl-beta-D-cellobioside. They were also assayed for ligand binding, enzyme processivity, thermostability, and cellobiose feedback inhibition. Two double Cys mutations that formed disulfide bonds across two tunnel forming loops were found to significantly weaken binding to ligands, lower all activities, and processivity, demonstrating that the movement of these loops is important but not essential for Cel6B function. Two single mutant enzymes, G234S and G284P, had higher activity on SC and FP, and the double mutant enzyme had threefold and twofold higher activity on these substrates, respectively. However, synergism with endocellulase T. fusca Cel5A was not increased with these mutant enzymes. All mutant enzymes with lower activity on filter paper, BMCC, and SC had lower processivity. This trend was not true for CMC, suggesting that processivity in Cel6B is a key factor in the hydrolysis of insoluble and crystalline cellulose. Three mutations (E495D, H326A and W329C) located near putative glycosyl substrate subsites -2, +1 and +2, were found to significantly increase resistance to cellobiose feedback inhibition. Both the A229V and L230C mutations specifically decreased activity on BMCC, suggesting that BMCC hydrolysis has a different rate limiting step than the other substrates. Most of the mutant enzymes had reduced thermostability although Cel6B G234S maintained wild-type thermostability. The properties of the different mutant enzymes provide insight into the catalytic mechanism of Cel6B.     

1,2-alpha-l-Fucosynthase: a glycosynthase derived from an inverting alpha-glycosidase with an unusual reaction mechanism

Fucosyloligosaccharides have great therapeutic potential. Here we present a new route for synthesizing a Fucalpha1,2Gal linkage by introducing glycosynthase technology into 1,2-alpha-l-fucosidase. The enzyme adopts a unique reaction mechanism, in which asparagine-423 activated by aspartic acid-766 acts as a base while asparagine-421 fixes both a catalytic water and glutamic acid-566 (an acid) in the proper orientations. Glycosynthase activity of N421G, N423G, and D766G mutants was examined using beta-fucosyl fluoride and lactose, and among them, the D766G mutant most effectively synthesized 2'-fucosyllactose. 1,2-alpha-l-Fucosynthase is the first glycosynthase derived from an inverting alpha-glycosidase and from a glycosidase with an unusual reaction mechanism.     

Artificial mixed-linked β-glucans produced by glycosynthase-catalyzed polymerization: tuning morphology and degree of polymerization

The glycosynthase derived from Bacillus licheniformis 1,3-1,4-β-glucanase was able to polymerize glycosyl fluoride donors (G4)(m)G3GαF (m = 0-2, G = Glcβ) leading to artificial mixed-linked β-glucans with regular sequences and variable β1,3 to β1,4 linkage ratios. With the E134A glycosynthase mutant, polymers had average molecular masses (M(w)) of 10-15 kDa. Whereas polymer 2 ([4G4G3G](n)) was an amorphous precipitate, the water-insoluble polymers 1 ([4G3G](n)) and 3 ([4G4G4G3G](n)) formed spherulites of 10-20 μm diameter. With the more active E134S glycosynthase mutant, polymerization led to high molecular mass polysaccharides, where M(w) was linearly dependent on enzyme concentration. Remarkably, a homo-polysaccharide [4G4G4G3G](n) with M(w) as high as 30.5 kDa (n ≈ 47) was obtained, which contained a small fraction of products up to 70 kDa, a value that is in the range of the molecular masses of low viscosity cereal 1,3-1,4-β-glucans, and among the largest products produced by a glycosynthase. Access to a range of novel tailor-made β-glucans through the glycosynthase technology will allow to evaluate the implications of polysaccharide fine structures in their physicochemical properties and their applications as biomaterials, as well as to provide valuable tools for biochemical characterization of β-glucan degrading enzymes and binding modules.     

A specific tryptophan in the I-II linker is a key determinant of beta-subunit binding and modulation in Ca(V)2.3 calcium channels

The ancillary beta subunits modulate the activation and inactivation properties of high-voltage activated (HVA) Ca(2+) channels in an isoform-specific manner. The beta subunits bind to a high-affinity interaction site, alpha-interaction domain (AID), located in the I-II linker of HVA alpha1 subunits. Nine residues in the AID motif are absolutely conserved in all HVA channels (QQxExxLxGYxxWIxxxE), but their contribution to beta-subunit binding and modulation remains to be established in Ca(V)2.3. Mutations of W386 to either A, G, Q, R, E, F, or Y in Ca(V)2.3 disrupted [(35)S]beta3-subunit overlay binding to glutathione S-transferase fusion proteins containing the mutated I-II linker, whereas mutations (single or multiple) of nonconserved residues did not affect the protein-protein interaction with beta3. The tryptophan residue at position 386 appears to be an essential determinant as substitutions with hydrophobic (A and G), hydrophilic (Q, R, and E), or aromatic (F and Y) residues yielded the same results. beta-Subunit modulation of W386 (A, G, Q, R, E, F, and Y) and Y383 (A and S) mutants was investigated after heterologous expression in Xenopus oocytes. All mutant channels expressed large inward Ba(2+) currents with typical current-voltage properties. Nonetheless, the typical hallmarks of beta-subunit modulation, namely the increase in peak currents, the hyperpolarization of peak voltages, and the modulation of the kinetics and voltage dependence of inactivation, were eliminated in all W386 mutants, although they were preserved in part in Y383 (A and S) mutants. Altogether these results suggest that W386 is critical for beta-subunit binding and modulation of HVA Ca(2+) channels.     

Function and Structure Studies of GH Family 31 and 97 α-Glycosidases

A huge number of glycoside hydrolases are classified into the glycoside hydrolase family (GH family) based on their amino-acid sequence similarity. The glycoside hydrolases acting on α-glucosidic linkage are in GH family 4, 13, 15, 31, 63, 97, and 122. This review deals mainly with findings on GH family 31 and 97 enzymes. Research on two GH family 31 enzymes is described: clarification of the substrate recognition of Escherichia coli α-xylosidase, and glycosynthase derived from Schizosaccharomyces pombe α-glucosidase. GH family 97 is an aberrant GH family, containing inverting and retaining glycoside hydrolases. The inverting enzyme in GH family 97 displays significant similarity to retaining α-glycosidases, including GH family 97 retaining α-glycosidase, but the inverting enzyme has no catalytic nucleophile residue. It appears that a catalytic nucleophile has been eliminated during the molecular evolution in the same way as a man-made nucleophile mutant enzyme, which catalyzes the inverting reaction, as in glycosynthase and chemical rescue.