Effect of Leu277 on Disproportionation and Hydrolysis Activity in < em > Bacillus stearothermophilus < /em > NO2 Cyclodextrin Glucosyltransferase

In this study, Leu277 of the CGTase from Bacillus stearothermophilus NO2, located near both the +2 subsite and the catalytic acid/base Glu253, was modified to assess the effect of side chain size at this position on disproportionation and hydrolysis activities. The best mutant, L277M, exhibited a reduced Km for the acceptor substrate maltose (0.48 mM versus 0.945 mM) and an increased k cat/Km (1175 s-1mM-1 versus 686.1 s-1mM-1), compared with those of the wild-type enzyme. The disproportionation to hydrolysis ratio of L277M was 2.4-fold greater than that of the wild-type. Existing structural data were combined with a multiple sequence alignment and Gly282 mutations to examine the mechanism behind the effects of the Leu277mutations. The Gly282 mutations were included to aid a molecular-dynamics (MD) analysis and the comparison of crystal structures. They reveal that changes to a hydrophobic cluster near Glu253 and the hydrophobicity of the +2 subsite combine to produce the observed effects.ImportanceIn this study, mutations that enhance the disproportionation to hydrolysis ratio of a CGTase have been discovered. For example, the disproportionation to hydrolysis ratio of the L277M mutant of Bacillus stearothermophilus NO2 CGTase was 2.4-fold greater than that of the wild-type. The mechanism behind the effects of these mutations is explained. This paper opens up other avenues for future research into the disproportionation and hydrolysis activities of CGTases. Productive mutatio...
Source: Applied and Environmental Microbiology - Category: Microbiology Authors: Source Type: research