Substrate specificity engineering of beta-mannosidase and beta-glucosidase from Pyrococcus by exchange of unique active site residues.
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 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 BglB 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.
Kaper
Thijs
T
Laboratory of Microbiology, Department of Agrotechnology and Food Sciences, Wageningen University, H. van Suchtelenweg 4, NL-6703 CT, Wageningen, The Netherlands.
van Heusden
Hester H
HH
van Loo
Bert
B
Vasella
Andrea
A
van der Oost
John
J
de Vos
Willem M
WM
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D006863:Hydrogen-Ion Concentration; D008361:Mannosidases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D015202:Protein Engineering; D019711:Pyrococcus / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000737:chemistry / Q000378:metabolism*; D044902:beta-Mannosidase
D017931:DNA Primers; D008361:Mannosidases; D001617:beta-Glucosidase; D044902:beta-Mannosidase