title: Characterization of the N-oxygenase AurF from Streptomyces thioletus.
abstract: 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 ~10-fold higher activity than previous reports in literature. Kinetic characterization at optimized conditions give a Km of 14.7 ± 1.1 μM, a kcat of 47.5 ± 5.4 min(-1) and a kcat/Km of 3.2 ± 0.4 μM(-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 Km is the main determinant of AurF activity toward alternative substrates.
authors: Department of Chemistry, University of Illinois at Urbana-Champaign, 600 S. Mathews Avenue, Urbana, IL 61801, USA.
authors: Emmanuel
authors: E
authors: Chanco
authors: Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 S. Mathews Avenue, Urbana, IL 61801, USA.
authors: Yoo Seong
authors: YS
authors: Choi
authors: Department of Biochemistry, University of Illinois at Urbana-Champaign, 600 S. Mathews Avenue, Urbana, IL 61801, USA.
authors: Ning
authors: N
authors: Sun
authors: Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 S. Mathews Avenue, Urbana, IL 61801, USA.
authors: Michael
authors: M
authors: Vu
authors: Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 S. Mathews Avenue, Urbana, IL 61801, USA. Electronic address: zhao5@illinois.edu.
authors: Huimin
authors: H
authors: Zhao
mesh_terms: D010129:4-Aminobenzoic Acid / Q000737:chemistry / Q000378:metabolism; D055162:Biocatalysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D009579:Nitrobenzoates / Q000737:chemistry / Q000378:metabolism; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013302:Streptomyces / Q000201:enzymology* / Q000378:metabolism
chemical_list: D009579:Nitrobenzoates; D010105:Oxygenases; C008629:4-nitrobenzoic acid; D010129:4-Aminobenzoic Acid
keywords: AurF; Biocatalysis; Diiron monooxygenase; Electron transfer; N-Oxygenation