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

• Published in: Journal of bioscience and bioengineering 2022-03, Vol.133 (3), p.213-221
• Main Authors: Okawa, Atsushi, Handa, Haruhisa, Yasuda, Eri, Murota, Masaki, Kudo, Daizo, Tamura, Takashi, Shiba, Tomoo, Inagaki, Kenji
• Format: Article
• Language: English
• Subjects: Carbon-Sulfur Lyases - chemistry; Carbon-Sulfur Lyases - genetics; Carbon-Sulfur Lyases - metabolism; Cysteine desulfurase (EC 2.8.1.7); Enzyme; Homocysteine; l-Homocysteine determination; l-Methionine γ-lyase; Methionine - metabolism; Modification of substrate specificity; Pseudomonas putida - metabolism; Pyridoxal 5’-phosphate; Pyridoxal Phosphate; Substrate recognition
• ISSN: 1389-1723; 1347-4421
• DOI: 10.1016/j.jbiosc.2021.11.008

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.