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

Thijs Kaper, Hester H. Van Heusden, Bert Van Loo, Andrea Vasella, John Van der Oost*, Willem M. De Vos

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

37 Citations (Scopus)

Abstract

A β-mannosidase gene (PH0501) was identified in the Pyrococcus horikoshii genome and cloned and expressed in E. coli. The purified enzyme (Bg1B) was most specific for the hydrolysis of p-nitrophenyl-β-D-mannopyranoside (pNP-Man) (Km: 0.44 mM) with a low turnover rate (kcat: 4.3 s-1). The β-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, G1n77 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 Bg1B were introduced in the highly active β-glucosidase Ce1B of Pyrococcus furiosus and vice versa, yielding two single and one double mutant for each enzyme. In Ce1B, both substitutions R77Q and N206D increased the specificity for mannosides and reduced hydrolysis rates 10-fold. In contrast, Bg1B D206N showed 10-fold increased hydrolysis rates and 35-fold increased affinity for the hydrolysis of glucosides. In combination with inhibitor studies, it was concluded that the substituted residues participate in the ground-state binding of substrates with an equatorial C2-hydroxyl group, but contribute most to transition-state stabilization. The unique activity profile of Bg1B seems to be caused by an altered interaction between the enzyme and C2-hydroxyl of the substrate and a specifically increased affinity for mannose that results from Asp206.

Original languageEnglish
Pages (from-to)4147-4155
Number of pages9
JournalBiochemistry
Volume41
Issue number12
Early online date3 Mar 2002
DOIs
Publication statusPublished - Mar 2002
Externally publishedYes

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