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Enzymatic Synthesis of alpha‐Ketobutyrate from Methionine: Characterization of Methionine gamma lyase deaminase (Mgld) from Porphyromonas gingivalis

Formation of alpha‐ketobutyrate (KB) from methionine involves catalysis that is facilitated by covalently bound coenzyme pyridoxal phosphate of methionine gamma lyase deaminase (Mgld). In this study we used molecularly cloned, overexpressed, and purified Mgld from Porphyromonas gingivalis. Sequence comparison of Mgld amino acids between organism revealed conserved residues of active site pockets. Based on pH and temperature responses of the purified enzyme we speculated that up until the release of the final product KB, the participation of active site residues on catalysis must be present. Deuterium labeled methionine substrates at alpha and beta position showed significant decrease in the reaction rate. This revealed that there are multiple rate‐limiting steps, with abstraction of alpha‐proton and gamma cleavage being the most rate limiting steps. From overall steady‐state catalytic reactions monitored by UV‐VIS spectroscopy, the heavily populated 478 nm absorbing species of L‐Met, L‐ethionine, L‐methionine sulfone and L‐homoserine was assigned to a late crotonate intermediate. In addition, this is the first report to show the more red‐shifted (498 nm) species observed during L‐homoserine‐lactone reaction with Mgld, which we assigned to a quinonoid species from DFT and time‐dependent self‐consistent field (TD‐SCF) calculations. The kinetic parameters differed significantly for the various substrates. Highest catalytic efficiency was observed for L‐vinylglycine (kcat/Km = 6455 s‑1 M‑1), exceeding that of L‐Met (kcat/Km = 4211 s‑1 M‑1). L‐Met sulfone and L‐Hse were markedly weaker binders (Km » 30 mM) while L‐Met sulfone displayed the largest turnover number (kcat = 1638 min‐1). A linear semi‐logarithmic correlation between experimental kcat values for substrates and DFT‐calculated g‐cleavage Gibbs activation energies was determined. Lower calculated activation energies for gamma elimination were associated with larger kcat values, suggesting that the gamma cleavage step in the reaction pathway is strongly rate‐determining.