An Alkaliphilic Chitinase Unveils Environment-Dependent Variation in the Canonical Catalytic Machinery of Family-18 Glycoside Hydrolases

Chitinases belonging to glycoside hydrolase family-18 (GH18) employ substrate-assisted catalysis and typically have neutral/acidic pH-optima. We describe the structural and functional analysis of <i>Ca</i>ChiA, a chitinase from the anaerobic alkaliphilic bacterium <i>Chitinivibrio alkaliphilus</i> with an alkaline pH optimum (8.8) and unique active site features, including a noncanonical catalytic H<i>xx</i>E<i>x</i>D<i>x</i>E motif, which is D<i>xx</i>D<i>x</i>D<i>x</i>E in other chitinases. Propka calculations indicated a significantly higher p<i>K</i>_a for the catalytic acid/base, Glu148, in <i>Ca</i>GH18, compared to other GH18 enzymes, aligning with its alkaline pH optimum. Both Propka calculations and functional studies of enzyme variants with mutations in the catalytic center suggested that not the change in the catalytic motif, but rather a unique glutamine, Gln57, modulating the properties of this motif, enables activity at alkaline pH. Further characterization of <i>Ca</i>ChiA unveiled additional peculiar enzyme properties, such as a unique ability to convert chitin to chitotriose. Thus, <i>Ca</i>ChiA adds novel catalytic capabilities to the widespread family of GH18 chitinases, made possible by adaptation of an intricate catalytic center.