Enzymology and folding of natural and engineered bacterial beta-glucanases studied by X-ray crystallography

1,3-1,4-beta-D-glucan 4-glucanohydrolases (beta-glucanases) are synthesized in both plants and bacteria. The enzymes specifically hydrolyze beta-1,4 glycosyl bonds that are adjacent to beta-1,3 linkages in beta-glucan, a linear polysaccharide containing these bonds in an approximate ratio of 2.5:1. Here we review structural studies by X-ray crystallography of natural Bacillus beta-glucanases and engineered variants characterized by hybrid sequences, single-site mutations and circular permutations. In combination with biochemical data and site-directed mutagenesis, the crystallographic evidence permits the formulation of a likely reaction mechanism for the retaining Bacillus beta-glucanases. In addition, the shape of the active site channel, the known binding mode of a cellobioside epoxyalkyl inhibitor and the energy profile of the beta-glucan substrate explain the specificity of the enzymes for beta-glucan and the requirement for a beta-1,3 glycosyl bond next to the scissile bond. beta-Glucanases with circularly permuted sequences retain conformational stability, enzymatic activity and the native fold. The jellyroll tertiary structure of Bacillus beta-glucanases is remarkably stable, resisting changes in amino acid sequence, chain topology, ligand binding and crystal packing.