期刊
BIOORGANIC & MEDICINAL CHEMISTRY
卷 22, 期 20, 页码 5569-5577出版社
PERGAMON-ELSEVIER SCIENCE LTD
DOI: 10.1016/j.bmc.2014.06.002
关键词
AurF; N-Oxygenation; Diiron monooxygenase; Biocatalysis; Electron transfer
资金
- United States Office of Naval Research [N00014-02-1-0725]
- Department of Chemical and Biomolecular Engineering at the University of Illinois at Urbana-Champaign
- NIH Molecular Biophysics Training Grant
AurF catalyzes the N-oxidation of p-aminobenzoic acid to p-nitrobenzoic acid in the biosynthesis of the antibiotic aureothin. Here we report the characterization of AurF under optimized conditions to explore its potential use in biocatalysis. The pH optimum of the enzyme was established to be 5.5 using phenazine methosulfate (PMS)/NADH as the enzyme mediator system, showing similar to 10-fold higher activity than previous reports in literature. Kinetic characterization at optimized conditions give a K-m of 14.7 +/- 1.1 mu M, a k(cat) of 47.5 +/- 5.4 min(-1) and a k(cat)/K-m of 3.2 +/- 0.4 mu M-1 min(-1.)PMS/NADH and the native electron transfer proteins showed significant formation of the p-hydroxylaminobenzoic acid intermediate, however H2O2 produced mostly p-nitrobenzoic acid. Alanine scanning identified the role of important active site residues. The substrate specificity of AurF was examined and rationalized based on the protein crystal structure. Kinetic studies indicate that the K-m is the main determinant of AurF activity toward alternative substrates. (C) 2014 Elsevier Ltd. All rights reserved.
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