کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1178701 962713 2013 10 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
X-ray crystallography and QM/MM investigation on the oligosaccharide synthesis mechanism of rice BGlu1 glycosynthases
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
X-ray crystallography and QM/MM investigation on the oligosaccharide synthesis mechanism of rice BGlu1 glycosynthases
چکیده انگلیسی

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.

Figure optionsDownload high-quality image (301 K)Download as PowerPoint slideHighlights
► The mechanism of glycosynthase was explored by X-ray crystallographic and QM/MM methods.
► The catalytic reaction proceeds via a single-step mechanism.
► The energy barriers are sensitive to hindered interactions of the mutated residues.
► The incoming crystal water Wat1 was important to the reaction.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics - Volume 1834, Issue 2, February 2013, Pages 536–545
نویسندگان
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