Diversity in structure and substrate specificity of family 43 glycoside hydrolase enzymes

Abstract

There are strong drives to find a viable alternative to the use of petroleum as a transport fuel. Bioethanol presents an attractive option, but the long-term costs of producing this fuel from corn starch are now apparent. Second generation biofuels, derived from cellulosic plant cell walls, are a more acceptable alternative. A limiting factor in the economical utilisation of plant biomass is efficient saccharification of carbohydrates. The process is slowed by the chemical complexity of the substrate, notably the recalcitrant double substitution structures in arabinan and arabinoxylan. Reflecting this complex chemistry, microorganisms that degrade the wall synthesise an array of glycoside hydrolases. Several such organisms contain a large number of genes encoding family 43 (GH43) glycoside hydrolases. To better understand the biological rationale behind the expansion in this family, the biochemical properties of the GH43 enzymes of a human gut symbiont, Bacteroides thetaiotaomicron, were investigated. Through cloning experiments, soluble protein was obtained for 25 enzymes. Activity screens uncovered several enzymes with a weak xylanase activity, three arabinoxylan-specific arabinofuranosidases, two endo-arabinanases and a novel arabinofuranosidase with specificity for α-1,2 side chains of singly and doubly substituted backbone residues. The crystal structure of a close homologue of the novel arabinofuranosidase is reported here. These data show how B. thetaiotaomicron deploys a combination of endo-acting and side chain-cleaving hydrolases to metabolise arabinan polysaccharides. Two GH43 enzymes (designated AXHd3s) have been found to target the double substitution structure in arabinoxylan. The crystal structure of the Humicola insolens AXHd3 was sought to understand this specificity, and is presented in complex with reaction products. Structural and mutagenic data were used to identify the mechanism by which the enzyme houses the O3-linked arabinofuranose in the active site, while exploiting the O2 appended arabinofuranose and asymmetrical xylan backbone as specificity determinants. Analysis of these data showed that orientation of the backbone, mediated by interactions with a conserved Tryptophan, positions the O3 arabinose into the active site. Modification of the rim of the active site pocket generated an AXHd3 variant that displayed both endo-xylanase and AXHd3 arabinofuranosidase activities. The introduction of additional catalytic functions into a biotechnologically relevant glycoside hydrolase provides a platform for evolving further, industrially significant, activities into the AXHd3 scaffold.