Abstract
Enzymatic cleavage of the glycosidic bond yields products in which the anomeric configuration is either retained or inverted. Each mechanism reflects the dispositions of the enzyme functional groups; a facet of which is essentially conserved in 113 glycoside hydrolase (GH) families. We show that family GH97 has diverged significantly, as it contains both inverting and retaining α-glycosidases. This reflects evolution of the active center; a glutamate acts as a general base in inverting members, exemplified by
Bacteroides thetaiotaomicron
α-glucosidase
Bt
GH97a, whereas an aspartate likely acts as a nucleophile in retaining members. The structure of
Bt
GH97a and its complexes with inhibitors, coupled to kinetic analysis of active-site variants, reveals an unusual calcium ion dependence.
1
H NMR analysis shows an inversion mechanism for
Bt
GH97a, whereas another GH97 enzyme from
B. thetaiotaomicron
,
Bt
GH97b, functions as a retaining α-galactosidase.