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.

Degradation of chloroanilines by toluene dioxygenase from Pseudomonas putida T57.

In this study, we investigated the ability of Pseudomonas putida toluene dioxygenase to oxidize chloroanilines. Toluene-induced P. putida T57 cells degraded 4-chloroaniline (4CA) more rapidly than toluene-non-induced cells, suggesting that toluene dioxygenase pathway was involved in 4CA degradation. Escherichia coli harboring P. putida T57 genes encoding toluene dioxygenase complex (todC1C2BA) showed 4CA degradation activity, demonstrating that toluene dioxygenase oxidizes 4CA. Thin-layer chromatography (TLC) and mass spectrometry (MS) analyses identified 4-chlorocatechol and 2-amino-5-chlorophenol as reaction products, suggesting that toluene dioxygenase catalyzes both 1,2- and 2,3-dioxygenation of 4CA. A plasmid containing the entire tod operon (todC1C2BADE) was introduced to P. putida T57 to enhance its ability to degrade 4CA. Resulting P. putida T57 (pHK-C1C2BADE) showed 250-fold higher 4CA degradation activity than P. putida T57 parental strain. P. putida T57 (pHK-C1C2BADE) degraded 2-chloroaniline (2CA), 3-chloroaniline (3CA), and 3,4-dichloroaniline (34DCA) as well as 4CA, but not 3,5-dichloroaniline (35DCA). The order of the degradation rate was: 4CA > 3CA > 2CA > 34DCA.