Effect of Leu²⁷⁷ on Disproportionation and Hydrolysis Activity in Bacillus stearothermophilus NO2 Cyclodextrin...

The disproportionation activity of cyclodextrin glucosyltransferase (CGTase; EC 2.4.1.19) can be used to convert small molecules into glycosides, thereby enhancing their solubility and stability. However, CGTases also exhibit a competing hydrolysis activity. The +2 subsite of the substrate binding cleft plays an important role in both the disproportionation and hydrolysis activities, but almost all known mutations at this site decrease disproportionation activity. 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 K for the acceptor substrate maltose (0.48 mM versus 0.945 mM) and an increased k_cat/K (1,175 s^-1 mM^-1 versus 686.1 s^-1 mM^-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. IMPORTANCE In 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 mutations are no longer limited to the acceptor subsite, since mutations that indirectly affect the acceptor subsite also enhance enzymatic activity.