Mutation of a pH-modulating residue in a GH51 伪-l-arabinofuranosidase leads to a severe reduction of the secondary hydrolysis of transfuranosylation products

详细信息    查看全文

• 作者：Bastien Bissaro ; Olivier Saurel ; Faten Arab-Jaziri ; Luc Saulnier ; Alain Milon ; Maija Tenkanen ; Pierre Monsan ; Michael J. O'Donohue ; R茅gis Faur茅
• 关键词：Abfs ; 伪-l-arabinofuranosidases ; AXOS ; arabinoxylo-oligosaccharides ; d-Xylp ; d-xylopyranosyl ; FH ; furanoside hydrolase ; GH ; glycoside hydrolase ; KIE ; kinetic isotope effects ; l-Araf ; l-arabinofuranosyl ; 4NTC-伪-l-Araf ; 4-nitrochatecol 伪-l-arabinofuranoside ; mNP ; meta-nitrophenol ; oNP ; ortho-nitrophenol ; pNP ; para-nitrophenol ; pNP-伪-l-Araf ; para-nitrophenyl 伪-l-arabinofuranoside ; RT ; transfer rate ; STD NMR ; Saturation Transfer Difference Nuclear Magnetic Resonance ; T/H ratio ; transglycosylatio
• 刊名：Biochimica et Biophysica Acta (BBA)/General Subjects
• 出版年：January, 2014
• 年：2014
• 卷：1840
• 期：1
• 页码：626-636
• 全文大小：1908 K

文摘

lass="h4">Background

The development of enzyme-mediated glycosynthesis using glycoside hydrolases is still an inexact science, because the underlying molecular determinants of transglycosylation are not well understood. In the framework of this challenge, this study focused on the family GH51 伪-l-arabinofuranosidase from Thermobacillus xylanilyticus, with the aim to understand why the mutation of position 344 provokes a significant modification of the transglycosylation/hydrolysis partition.

lass="h4">Methods

Detailed kinetic analysis (k_cat, K_M, pK_a determination and time-course NMR kinetics) and saturation transfer difference nuclear magnetic resonance spectroscopy was employed to determine the synthetic and hydrolytic ability modification induced by the redundant N344 mutation disclosed in libraries from directed evolution.

lass="h4">Results

The mutants N344P and N344Y displayed crippled hydrolytic abilities, and thus procured improved transglycosylation yields. This behavior was correlated with an increased pK_a of the catalytic nucleophile (E298), the pK_a of the acid/base catalyst remaining unaffected. Finally, mutations at position 344 provoked a pH-dependent product inhibition phenomenon, which is likely to be the result of a significant modification of the proton sharing network in the mutants.

lass="h4">Conclusions and general significance

Using a combination of biochemical and biophysical methods, we have studied TxAbf-N344 mutants, thus revealing some fundamental details concerning pH modulation. Although these results concern a GH51 伪-l-arabinofuranosidase, it is likely that the general lessons that can be drawn from them will be applicable to other glycoside hydrolases. Moreover, the effects of mutations at position 344 on the transglycosylation/hydrolysis partition provide clues as to how TxAbf can be further engineered to obtain an efficient transfuranosidase.