We have extensively studied microbial metabolism of toxic compounds with a triple bond between carbon and nitrogen, such as nitriles [R–CN] and isonitriles [R–NC]. In the Pseudomonas chlororaphis B23 strain, whose nitrile hydratase enzyme was previously used for the industrial acrylamide production and is now used for the production of 5-cyanovaleramide, we clarified the enzyme gene organization composed of seven genes (including acsA).
We have studied several enzymes involved in cleavage and synthesis of a C-N single or triple bond (e.g., nitrilase, nitrile hydratase, amidase, isonitrile hydratase, N-substituted formamide deformylase, and aldoxime dehydratase).
During the nitrile studies, we found acyl-CoA synthetase (AcsA), which is the acsA product, plays an essential role in acid utilization in the nitrile-degradative pathway. AcsA ligates acid with CoA: a carbon-sulfur-bond formation. However, when L-cysteine was used as a substrate instead of CoA, N-acyl-L-cysteine was surprisingly detected as a reaction product. This finding demonstrates that AcsA synthesizes an amide bond comprising the amino group of cysteine and the carboxyl group of the acid. AcsA formed a variety of N-acyl-compounds, when various acids and cysteine-analogues were used as substrates. AcsA belongs to the adenylate-forming enzyme superfamily. Therefore, we investigated whether such superfamily enzymes show the similar activity or not. We found that luciferase involved in a monooxygenase reaction synthesized N-luciferyl-L-cysteine from luciferin and L-cysteine. We have investigated other members of this superfamily. Current efforts are focused on clarifying the reaction mechanism generating the unique C-N bond-synthetic activity of the thioester-bond-synthesizing enzymes.