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 β-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.