Understanding the structure and mechanism of sugar nucleotide processing enzymes is invaluable in the generation of designer enzymes for biotransformation, for instance, in connection with engineering antibiotic glycosylation. In this study, homology modelling and mechanistic comparison to the structurally related RmlC epimerase family has been used to identify and assign functions to active-site residues in the Tyl1a-catalysed keto-sugar nucleotide isomerisation process. Tyl1a His63 is implicated as the base that initiates the isomerisation process by substrate C-3 deprotonation, with Arg109 stabilising the resulting enolate. Subsequent O-3 deprotonation (potentially by His65) and C-4 protonation (potentially by Tyr49) complete the isomerisation process.