Hommalai Greanggrai, Withers Stephen G, Chuenchor Watchalee, Cairns James R Ketudat, Svasti Jisnuson
Center for Protein Structure and Function, Mahidol University, Bangkok 10400, Thailand.
Glycobiology. 2007 Jul;17(7):744-53. doi: 10.1093/glycob/cwm039. Epub 2007 Apr 3.
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.
水稻BGlu1β-葡萄糖苷酶是一种糖苷水解酶家族1的酶，它对β-(1,4)-和短链β-(1,3)-连接的葡萄糖寡糖起外切葡聚糖酶的作用。其天然前体中谷氨酸残基414处的BGlu1β-葡萄糖苷酶突变破坏了该酶的催化活性，但在甲酸根和叠氮化物等阴离子亲核试剂存在的情况下该酶可以恢复活性，这证实该残基是催化亲核试剂。比较了三种候选突变体E414G、E414S和E414A在亲核试剂存在下的催化活性。E414G突变体的催化效率分别比E414A和E414S高约25倍和1400倍。当以α-葡萄糖基氟作为供体、对硝基苯基纤维二糖苷作为受体时，所有这三种突变体都可以通过转糖基作用催化混合长度寡糖的合成。E414G突变体的转糖基化速率最快，分别比E414S和E414A快约3倍和19倍，并且在供体-受体比例为5:1时产生高达70-80%的不溶性产物。碳-13核磁共振、甲基化分析和电喷雾电离质谱表明，不溶性产物是聚合度为5至至少11的β-(1,4)-连接的寡聚物。发现BGlu1 E414G糖基合酶更喜欢至少占据三个糖残基结合亚位点的较长链长度寡糖作为生产性转糖基化的受体。这是关于源自外切糖苷酶的β-葡聚糖合酶能够产生长链纤维寡糖的首次报道，这可能反映了水稻BGlu1β-葡萄糖苷酶延伸的寡糖结合位点。
