Salila Pengthaisong

Structural Insights into Rice BGlu1 β-Glucosidase Oligosaccharide Hydrolysis and Transglycosylation

The structures of rice BGlu1 beta-glucosidase, a plant beta-glucosidase active in hydrolyzing cell wall-derived oligosaccharides, and its covalent intermediate with 2-deoxy-2-fluoroglucoside have been solved at 2.2 A and 1.55 A resolution, respectively. The structures were similar to the known structures of other glycosyl hydrolase family 1 (GH1) beta-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 beta-1,4-linked oligosaccharides. Though this active site structure is somewhat similar to that of the Paenibacillus polymyxa beta-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-beta-glucanase/beta-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/beta-II beta-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.

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 pH5, 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 1.90Å resolution, respectively. The Q176Nε 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.

Bacterial β-Glucosidase Reveals the Structural and Functional Basis of Genetic Defects in Human Glucocerebrosidase 2 (GBA2).

Human glucosylcerebrosidase 2 (GBA2) of the CAZy family GH116 is responsible for the breakdown of glycosphingolipids on the cytoplasmic face of the endoplasmic reticulum and Golgi apparatus. Genetic defects in GBA2 result in spastic paraplegia and cerebellar ataxia, while cross-talk between GBA2 and GBA1 glucosylceramidases may affect Gaucher disease. Here, we report the first three-dimensional structure for any GH116 enzyme, Thermoanaerobacterium xylanolyticum TxGH116 β-glucosidase, alone and in complex with diverse ligands. These structures allow identification of the glucoside binding and active site residues, which are shown to be conserved with GBA2. Mutagenic analysis of TxGH116 and structural modeling of GBA2 provide a detailed structural and functional rationale for pathogenic missense mutations of GBA2.

The role of the oligosaccharide binding cleft of rice BGlu1 in hydrolysis of cellooligosaccharides and in their synthesis by rice BGlu1 glycosynthase

Rice BGlu1 β‐glucosidase nucleophile mutant E386G is a glycosynthase that can synthesize p‐nitrophenyl (pNP)‐cellooligosaccharides of up to 11 residues. The X‐ray crystal structures of the E386G glycosynthase with and without α‐glucosyl fluoride were solved and the α‐glucosyl fluoride complex was found to contain an ordered water molecule near the position of the nucleophile of the BGlu1 native structure, which is likely to stabilize the departing fluoride. The structures of E386G glycosynthase in complexes with cellotetraose and cellopentaose confirmed that the side chains of N245, S334, and Y341 interact with glucosyl residues in cellooligosaccharide binding subsites +2, +3, and +4. Mutants in which these residues were replaced in BGlu1 β‐glucosidase hydrolyzed cellotetraose and cellopentaose with kcat/Km values similar to those of the wild type enzyme. However, the Y341A, Y341L, and N245V mutants of the E386G glycosynthase synthesize shorter pNP‐cellooligosaccharides than do the E386G glycosynthase and its S334A mutant, suggesting that Y341 and N245 play important roles in the synthesis of long oligosaccharides. X‐ray structural studies revealed that cellotetraose binds to the Y341A mutant of the glycosynthase in a very different, alternative mode not seen in complexes with the E386G glycosynthase, possibly explaining the similar hydrolysis, but poorer synthesis of longer oligosaccharides by Y341 mutants.

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 21.9kcal/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, 1.1kcal/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.

Rice BGlu1 glycosynthase and wild type transglycosylation activities distinguished by cyclophellitol inhibition

The rice BGlu1 β-D-glucosidase nucleophile mutant E386G is a glycosynthase that catalyzes the synthesis of cellooligosaccharides from α-d-glucopyranosyl fluoride (GlcF) donor and p-nitrophenyl (pNP) cellobioside (Glc2-pNP) or cello-oligosaccharide acceptors. When activity with other donors and acceptors was tested, the initial enzyme preparation cleaved pNP-β-D-glucopyranoside (Glc-pNP) and pNP-β-D-fucopyranoside (Fuc-pNP) to pNP and glucose and fucose, suggesting contamination with wild type BGlu1 β-glucosidase. The products from reaction of GlcF and Fuc-pNP included Fuc-β-(1→3)-Fuc-pNP, Glc-β-(1→3)-Fuc-pNP, and Fuc-β-(1→4)-Glc-β-(1→3)-Fuc-pNP, suggesting the presence of both wild type BGlu1 and its glycosynthase. Inhibition of the BGlu1 β-glucosidase activity within this preparation by cyclophellitol confirmed that the E386G glycosynthase preparation was contaminated with wild type BGlu1. Rice BGlu1 E386G-2, generated from a new construct designed to minimize back-mutation, showed glycosynthase activity without wild type hydrolytic or transglycosylation activity. E386G-2 catalyzed transfer of glycosyl residues from GlcF, α-L-arabinosyl fluoride, α-D-fucosyl fluoride, α-D-galactosyl fluoride, α-D-mannosyl fluoride, and α-D-xylosyl fluoride donors to Glc2-pNP acceptor. The synthetic products from the reactions of α-fucosyl fluoride and α-mannosyl fluoride donors were confirmed to result from addition of a β-(1→4)-linked glycosyl residue. Moreover, the E386G glycosynthase transferred glucose from GlcF donor to glucose, cellobiose, Glc-pNP, Fuc-pNP, pNP-β-D-galactopyranoside, and pNP-β-D-xylopyranoside acceptors, but little to pNP-β-D-mannopyranoside. Production of longer oligosaccharides occurred most readily on acceptors with an equatorial 4-OH. Elimination of wild type contamination thereby allowed a clear assessment of BGlu1 E386G glycosynthase catalytic abilities.

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.