Crystal structure and enzymatic properties of a bacterial family 19 chitinase reveal differences from plant enzymes

We describe the cloning, overexpression, purification, characterization and crystal structure of chitinase G, a single‐domain family 19 chitinase from the Gram‐positive bacterium Streptomyces coelicolor A3(2). Although chitinase G was not capable of releasing 4‐methylumbelliferyl from artificial chitooligosaccharide substrates, it was capable of degrading longer chitooligosaccharides at rates similar to those observed for other chitinases. The enzyme was also capable of degrading a colored colloidal chitin substrate (carboxymethyl‐chitin–remazol–brilliant violet) and a small, presumably amorphous, subfraction of α‐chitin and β‐chitin, but was not capable of degrading crystalline chitin completely. The crystal structures of chitinase G and a related Streptomyces chitinase, chitinase C [Kezuka Y, Ohishi M, Itoh Y, Watanabe J, Mitsutomi M, Watanabe T & Nonaka T (2006) J Mol Biol358, 472–484], showed that these bacterial family 19 chitinases lack several loops that extend the substrate‐binding grooves in family 19 chitinases from plants. In accordance with these structural features, detailed analysis of the degradation of chitooligosaccharides by chitinase G showed that the enzyme has only four subsites (− 2 to + 2), as opposed to six (− 3 to + 3) for plant enzymes. The most prominent structural difference leading to reduced size of the substrate‐binding groove is the deletion of a 13‐residue loop between the two putatively catalytic glutamates. The importance of these two residues for catalysis was confirmed by a site‐directed mutagenesis study.