Structural insights into flexible pyruvate binding in an (S)-selective ω-transaminase

(S)-selective ω-transaminases (S-ωTAs) are PLP-dependent enzymes widely employed in biocatalysis for the stereoselective amination of prochiral ketones, yielding enantiopure (S)-amines. Although their stereochemical preference is well established, the structural basis of keto-acceptor recognition and active-site flexibility remains poorly understood. Here, we present a 1.96 Å crystal structure of a marine S-ωTA OM-S25 in complex with PLP and pyruvate (PYR). The enzyme exhibits four protomer in one asymmetric unit, yet electron density reveals pronounced conformational heterogeneity in PYR binding across protomers, channel-proximal transitional states (chain C), deeply buried productive poses (chains A, B, D), and a channel-entrance pose salt-bridged to Lys166. These conformations may represent a potential substrate binding route for acidic acceptors. Molecular docking corroborates the final PYR position, and a conserved flipping arginine, which usual refer to an arginine switch, stabilizes the carboxylate moiety of PYR-like substrates. We propose a stepwise entry pathway for the acceptor PYR in the second half-reaction of S-ωTAs. This pathway involves initial capture of PYR by Lys166 within the access channel, followed by an ∼180° rotation facilitated by Phe22, Tyr153, and Tyr168, progressive relocation through positions C→ D→A→B, and ultimate in the position of chain B for further reaction. Supporting evidence includes activity assays with PYR derivatives, thermal shift assays revealing modest stability perturbations, and gate-region mutagenesis experiments that confirm the proposed entry route. Although PMP-bound structures are essential to fully resolve the second half-reaction, this study provides the most comprehensive structural framework to date for acceptor recognition and the overall transamination mechanism in S-ωTAs. These findings lay a strong foundation for future mechanistic studies and rational enzyme engineering to advance biocatalytic applications.