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 phytopathogen Agrobacterium 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 commensal Bacteroides 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 the A. tumefaciens enzyme (beta). Findings identify a basic set of GUL‐encoded lyases for glucoside metabolism and assign physiological significance to GUL genetic diversity in bacteria.