Enzyme Machinery for Bacterial Glucoside Metabolism through a Conserved Non-hydrolytic Pathway.

Abstract

Flexible acquisition of substrates from nutrient pools is critical for microbes to prevail in competitive environments. To acquire glucose from diverse glycoside and disaccharide substrates, many free-living and symbiotic bacteria have developed, alongside hydrolysis, a non-hydrolytic pathway comprised of four biochemical steps and conferred from a single glycoside utilization gene locus (GUL). Mechanistically, this pathway integrates within the framework of oxidation and reduction at the glucosyl/glucose C3, the eliminative cleavage of the glycosidic bond and the addition of water in two consecutive lyase-catalyzed reactions. Here, based on study of enzymes from the phytopathogenAgrobacterium tumefaciens, we reveal a conserved Mn2+metallocenter active site in both lyases and identify the structural requirements for specific catalysis to elimination of 3-keto-glucosides and water addition to the resulting 2-hydroxy-3-keto-glycal product, yielding 3-keto-glucose. Extending our search of GUL-encoded putative lyases to the human gut commensalBacteroides thetaiotaomicron, we discover a Ca2+metallocenter active site in a putative glycoside hydrolase-like protein and demonstrate its catalytic function in the eliminative cleavage of 3-keto-glucosides of opposite (alpha) anomeric configuration as preferred by theA. tumefaciensenzyme (beta). Findings identify a basic set of GUL-encoded lyases for glucoside metabolism and assign physiological significance to GUL genetic diversity in bacteria.