title: CmlI is anN-oxygenase in the biosynthesis of chloramphenicol.
abstract: The N-oxygenation of an amine group is one of the steps in the biosynthesis of the antibiotic chloramphenicol. The non-heme di-iron enzyme CmlI was identified as the enzyme catalyzing this reaction through bioinformatics studies and reconstitution of enzymatic activity. In vitro reconstitution was achieved using phenazine methosulfate and NADH as electron mediators, while in vivo activity was demonstrated inEscherichia coliusing two substrates. Kinetic analysis showed a biphasic behavior of the enzyme. Oxidized hydroxylamine and nitroso compounds in the reaction were detected both in vitro and in vivo based on LC-MS. The active site metal was confirmed to be iron based on a ferrozine assay. These findings provide new insights into the biosynthesis of chloramphenicol and could lead to further development of CmlI as a useful biocatalyst.
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA.
authors.forename: Haige
authors.initials: H
authors.lastname: Lu
authors.affiliation: Department of Chemistry, University of Illinois at Urbana-Champaign, USA.
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Chanco
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA ; Department of Chemistry, University of Illinois at Urbana-Champaign, USA.
authors.forename: Huimin
authors.initials: H
authors.lastname: Zhao
keywords: Arylamine oxygenation; Chloramphenicol biosynthesis; Di-iron oxygenase; N-Oxygenation