Characterization and application of l-methionine γ-lyase Q349S mutant enzyme with an enhanced activity toward l-homocysteine.

l-Methionine γ-lyse (MGL), a pyridoxal 5'-phosphate-dependent enzyme, catalyzes the α,γ-elimination of l-methionine (l-Met) and l-homocysteine (l-Hcy) to produce α-keto acids, thiols, and ammonia. Previously, various mutant enzymes of Pseudomonas putida MGL (PpMGL) were prepared to identify a homocysteine (Hcy)-specific enzyme that would assist the diagnosis of homocystinuria. Among the mutat enzymes the Q349S mutant exhibited high degradation activity toward l-Hcy. In the present study, PpMGL Q349S was characterized; the results suggested that it could be applied to determine the amount of l-Hcy. Compared to the wild-type PpMGL, specific activities of the Q349S mutant with l-Hcy and l-Met were 1.5 and 0.7 times, respectively. Additionally, we confirmed that l-Hcy in plasma samples could be accurately detected using the Q349S mutant by preincubating it with cysteine desulfurase (CsdA). Furthermore, we determined the X-ray crystal structure of PpMGL Q349S l-Met or l-Hcy complexes Michaelis complex, germinal diamine, and external aldimine at 2.25-2.40 Å. These 3D structures showed that the interaction partner of the ß-hydroxyl group of Thr355 in the wild-type PpMGL was changed to the carboxyl group of the Hcy-PLP external aldimine in the Q349S mutant. The interaction of Ser349 and Arg375 was different between l-Met and l-Hcy recognition, indicating that it was important for the recognition of the carboxyl group of the substrate.

Structural basis for substrate specificity of l-methionine decarboxylase.

l -Methionine decarboxylase (MetDC) from Streptomyces sp. 590 is a vitamin B6 -dependent enzyme and catalyzes the non-oxidative decarboxylation of l -methionine to produce 3-methylthiopropylamine and carbon dioxide. We present here the crystal structures of the ligand-free form of MetDC and of several enzymatic reaction intermediates. Group II amino acid decarboxylases have many residues in common around the active site but the residues surrounding the side chain of the substrate differ. Based on information obtained from the crystal structure, and mutational and biochemical experiments, we propose a key role for Gln64 in determining the substrate specificity of MetDC, and for Tyr421 as the acid catalyst that participates in protonation after the decarboxylation reaction.