Affiliation:
1. Institute of Applied Biochemistry and Graduate School of Life and Environmental Sciences, The University of Tsukuba, Tennodai, Tsukuba, Ibaraki, Japan
Abstract
ABSTRACT
Previously, we isolated a new enzyme,
N
-substituted formamide deformylase, that catalyzes the hydrolysis of
N
-substituted formamide to the corresponding amine and formate (H. Fukatsu, Y. Hashimoto, M. Goda, H. Higashibata, and M. Kobayashi, Proc. Natl. Acad. Sci. U. S. A. 101:13726–13731, 2004,
doi:10.1073/pnas.0405082101
). Here, we discovered that this enzyme catalyzed the reverse reaction, synthesizing
N
-benzylformamide (NBFA) from benzylamine and formate. The reverse reaction proceeded only in the presence of high substrate concentrations. The effects of pH and inhibitors on the reverse reaction were almost the same as those on the forward reaction, suggesting that the forward and reverse reactions are both catalyzed at the same catalytic site. Bisubstrate kinetic analysis using formate and benzylamine and dead-end inhibition studies using a benzylamine analogue, aniline, revealed that the reverse reaction of this enzyme proceeds via an ordered two-substrate, two-product (bi-bi) mechanism in which formate binds first to the enzyme active site, followed by benzylamine binding and the subsequent release of NBFA. To our knowledge, this is the first report of the reverse reaction of an amine-forming deformylase. Surprisingly, analysis of the substrate specificity for acids demonstrated that not only formate, but also acetate and propionate (namely, acids with numbers of carbon atoms ranging from C
1
to C
3
), were active as acid substrates for the reverse reaction. Through this reaction,
N
-substituted carboxamides, such as NBFA,
N
-benzylacetamide, and
N
-benzylpropionamide, were synthesized from benzylamine and the corresponding acid substrates.
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
Cited by
1 articles.
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