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

Abstract

Rice BGlu1 b-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 a-glucosyl fluoride were solved and the a-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 12, 13, and 14. Mutants in which these residues were replaced in BGlu1 b-glucosidase hydrolyzed cellotetraose and cellopentaose with k cat /K m values similar to those of the wild type enzyme. However, the Y341A, Y341L, and N245V mutants of the E386G glycosynthase synthesize shorter pNPcellooligosaccharides 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

“…Diffraction data parameters are shown in Table S1 and model parameters for the structures of the glycosynthases are summarized in Table 1. The resolution limits of 1.85 Å for BGlu E386S and 2.10 Å for E386A and free residual (R free ) values of 20.5% for E386S and 20.6% for E386A in complexes with α-GlcF are comparable to the previously reported values for the corresponding complex of E386G of 1.95 Å and 20.7% [29]. However, the temperature (B) factors reported for the E386G protein (16.6 Å 2 ) and α-GlcF (14.0 Å 2 ) in the E386G complex are significantly higher than those in the E386S (9.5 and 5.7 Å 2 , respectively) and E386A (10.8 Å 2 and 8.2 Å 2 , respectively) complexes.…”

“…Data were processed and scaled with the HKL-2000 package [31]. Structures were solved by molecular replacement with wild type BGlu1 (PDB code: 2RGL), refined, and validated as previously described for BGlu1 E386G and its complex with α-GlcF [29].…”

“…Recently, we published the structure of the E386G mutant and its complex with α-GlcF (PDB code: 3SCO) [29]. Since the crystal of rice BGlu1 E386G mutant was only complexed with the donor α-GlcF, and the transglucosylation activities were detected under the acceptor pNPC2, the pNPC2 was docked into the binding pocket using the Autodock 4.0 program [32].…”

“…An active site water molecule is seen in the wild type covalent intermediate as well as in all three glycosynthase mutant complexes with α-GlcF. This water occupies a very similar position in BGlu1 E386G[29], BGlu1 E386A (B) and BGlu1 E386S (C), with the superimposition of all three of these rice BGlu1 glycosynthases being shown in (D). The α-GlcF is represented as balls and sticks, the surrounding amino acid residues are shown as sticks, and the water molecule is represented as a ball.…”

“…Recently, the structure of the BGlu1 E386G glycosynthase mutant alone and in complexes with α-GlcF, cellotetraose or cellopentaose were reported [29]. However, those structures could not explain the relative activities of the BGlu1 E386G, E386S and E386A glycosynthases.…”