Trp residue at subsite - 5 plays a critical role in the substrate binding of two protistan GH26 β-mannanases from a termite hindgut.

Abstract

Symbiotic protists in the hindgut of termites provide a novel enzymatic resource for efficient lignocellulytic degradation of plant biomass. In this study, two β-mannanases, RsMan26A and RsMan26B, from a symbiotic protist community of the lower termite, Reticulitermes speratus, were successfully expressed in the methylotrophic yeast, Pichia pastoris. Biochemical characterization experiments demonstrated that both RsMan26A and RsMan26B are endo-acting enzymes and have a very similar substrate specificity, displaying a higher catalytic efficiency to galactomannan from locust bean gum (LBG) and glucomannan than to β-1,4-mannan and highly substituted galactomannan from guar gum. Homology modeling of RsMan26A and RsMan26B revealed that each enzyme displays a long open cleft harboring a unique hydrophobic platform (Trp79) that stacks against the sugar ring at subsite - 5. The Km values of W79A mutants of RsMan26A and RsMan26B to LBG increased by 4.8-fold and 3.6-fold, respectively, compared with those for the native enzymes, while the kcat remained unchanged or about 40% of that of the native enzyme, resulting in the decrease in the catalytic efficiency by 4.8-fold and 9.1-fold, respectively. The kinetic values for glucomannan also showed a similar result. These results demonstrate that the Trp residue present near the subsite - 5 has an important role in the recognition of the sugar ring in the substrate.