Substrate specificity engineering of beta-mannosidase and beta-glucosidase from Pyrococcus by exchange of unique active site residues

Biochemistry
Thijs KaperW M de Vos

Abstract

A beta-mannosidase gene (PH0501) was identified in the Pyrococcus horikoshii genome and cloned and expressed in E. coli. The purified enzyme (BglB) was most specific for the hydrolysis of p-nitrophenyl-beta-D-mannopyranoside (pNP-Man) (Km: 0.44 mM) with a low turnover rate (kcat: 4.3 s(-1)). The beta-mannosidase has been classified as a member of family 1 of glycoside hydrolases. Sequence alignments and homology modeling showed an apparent conservation of its active site region with, remarkably, two unique active site residues, Gln77 and Asp206. These residues are an arginine and asparagine residue in all other known family 1 enzymes, which interact with the catalytic nucleophile and equatorial C2-hydroxyl group of substrates, respectively. The unique residues of P. horikoshii BglB were introduced in the highly active beta-glucosidase CelB of Pyrococcus furiosus and vice versa, yielding two single and one double mutant for each enzyme. In CelB, both substitutions R77Q and N206D increased the specificity for mannosides and reduced hydrolysis rates 10-fold. In contrast, BglB D206N showed 10-fold increased hydrolysis rates and 35-fold increased affinity for the hydrolysis of glucosides. In combination with inhibitor studies, it wa...Continue Reading

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Citations

Jul 2, 2003·Current Opinion in Microbiology·Dalia Shallom, Yuval Shoham
Oct 19, 2012·Protein Engineering, Design & Selection : PEDS·Hsiao-Lin LeePo-Huang Liang
Jan 1, 2008·Biotechnology & Genetic Engineering Reviews·Antonio TrinconeGiuseppina Andreotti
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Nov 15, 2018·Applied Microbiology and Biotechnology·Diandra Albuquerque Lopes Costa, Edivaldo Ximenes Ferreira Filho
Oct 10, 2012·Biochemistry·Somayesadat BadieyanChenming Zhang
Jun 11, 2003·Biochemistry·David L ZechelStephen G Withers

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