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Porphyromonas gingivalis is an oral pathogenic bacterium that causes halitosis (bad breath) and periodontitis in humans. Halitosis manifests due to volatile sulfur compounds such as methylthiol, generated from methionine degradation by Methionine Gamma Lyase-Deaminase (Mgld). In this report, we cloned and expressed Porphyromonas gingivalis mgld using a bacterial expression system, and the purified homotetrameric enzyme was characterized by a novel isotope assay using L-[1-14C]-methionine as a substrate. Mgld is a PLP-dependent L-methionine C-S lyasedeaminase that cleaves the gamma-carbon-sulfur bond of L-methionine to methylthiol and forms the deaminated overall product α-ketobutyrate. We find that exogenous 3H-L-2-aminobutyrate is not deaminated into 3H-α-ketobutyrate under conditions of enzyme excess, supporting efficient transfer of bound intermediate(s) during L-methionine catabolism. The overall reaction to form α-ketobutyrate from L-methionine exhibits Km of 1.0 mM, Vmax of 5.27 μmol min-1 mg-1, and a monomeric kcat/Km of 3729.3 M-1 s-1. Mgld exhibits optimal activity above pH 8 and in a temperature range of 37°-50°C. Several compounds were tested for Mgld inhibition. The natural product DL-Propargylglycine stands out as the most effective Mgld inhibitor and thus may be useful for the control of halitosis. Mgld has almost no activity on N-formylmethionine, confirming the requirement of a free α-amino-nitrogen on the substrate to form a Schiff base with the enzyme. |
