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

Abstract

Rice BGlu1 (Os3BGlu7) is a glycoside hydrolase family 1 b-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.

Intrinsic dynamic behavior of enzyme:substrate complexes govern the catalytic action of β-galactosidases across clan GH-A

“…In family GH1, deep active site pockets have already been experimentally described for the interaction of rice β-glucosidase (PDB: 3F5K, 3SCT, 4QLK, etc.) with cellotetraose 46 . By analogy, we explored by docking the interaction of xyloglucan-derived oligosaccharides with SsLacS, as done above with CjBgl35A.…”

When substrate inhibits and inhibitor activates: implications of β-glucosidases

“…2). Regardless of other plausible alternatives, it is tempting to speculate that alterations in the nonproductive binding of substrate upon mutations are at least partly responsible for the effects of mutations far from the catalytic residues observed with many BGs [55, 63, 70–73]. Contrarily to H. insolens BG in the structures of BG Td2F2 [30], and BG of Paenibacillus polymyxa [66] glucose is found in the binding site −1.…”

Structural Basis of Specific Glucoimidazole and Mannoimidazole Binding by Os3BGlu7

“…The 4 E conformation of GIm has previously been found in crystal structures of rice Os7BGlu26 β-glycosidase [ 29 ] and Thermotoga maritima ( Tm GH1) β-glycosidase [ 59 ]. A standard relaxed 4 C 1 chair conformation was observed in the structures of rice Os3BGlu7 and its E176Q mutant in covalent complexes with 2-deoxy-2-fluoro-glucoside inhibitor [ 11 , 19 ] and a 1 S 3 screw boat conformation was detected in the −1 subsite of non-reducing glucosyl residue in the structures of Os3BGlu7 E176Q and E386G mutants with oligosaccharides [ 11 , 60 ]. The conformations of the GIm is consistent with the 1 S 3 → 4 E / 4 H 3 ⧧ → 4 C 1 conformational itinerary proposed for β- d -glucoside hydrolysis by Os3BGlu7 and other retaining β-glucosidases [ 11 , 15 , 59 ].…”