CmlI is an N-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 in Escherichia coli using 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.

A growing family of O2 activating dinuclear iron enzymes with key catalytic diiron(III)-peroxo intermediates: Biological systems and chemical models

“…The second diiron enzyme participating in the biosynthesis of chloramphenicol in Streptomyces venezuelae is the arylamine N-oxygenase CmlI [12]. UV-visible, EPR and Mössbauer spectroscopy analysis highlighted a diiron active site, which reactivity toward dioxygen differs significantly from previously described enzymes [109].…”

“…For example, the cyanobacterial aldehyde deformylase oxygenase converts fatty aldehydes into alkanes, with formic acid as by-product [11]. Other examples worth noting are the arylamine oxygenases (oxidizing amine functions to nitro functions) such as CmlI, involved in the biosynthesis of chloramphenicol [12], or AurF, converting p-aminobenzoate into p-nitrobenzoate in the biosynthesis of aureothin [13]. In all cases, these enzymes perform catalysis via the reductive activation of dioxygen to form a diiron(III)-peroxo intermediate, which appears to be a key intermediate for these different reactions.…”

Crystal structure of CmlI, the arylamine oxygenase from the chloramphenicol biosynthetic pathway

“…The primary route to the generation of biological nitroaromatics is by direct enzymatic nitration of substrates [1]. An alternative pathway involves oxygenase enzymes, which catalyze the direct incorporation of the O atoms of molecular dioxygen into the substrate to form the nitro group [6–8]. One of the most well-known and widely used natural nitroaromatic compounds is the antibiotic chloramphenicol (CAM), which is synthesized by Streptomyces venezuelae [4, 8–10].…”

“…An alternative pathway involves oxygenase enzymes, which catalyze the direct incorporation of the O atoms of molecular dioxygen into the substrate to form the nitro group [6–8]. One of the most well-known and widely used natural nitroaromatic compounds is the antibiotic chloramphenicol (CAM), which is synthesized by Streptomyces venezuelae [4, 8–10]. The biosynthesis of CAM employs a non-ribosomal peptide synthetase (NRPS) system and several tailoring enzymes to chemically modify a para-amino phenylalanine precursor to form the final product [8, 10–14].…”

Expression and characterization of an <i>N</i>-oxygenase from <i>Rhodococcus jostii</i> RHAI

“…These enzymes were initially characterized from S. thioluteus, in the case of AurF, and Pseudomonas fluorescens, in the case of PrnD. More recently, other putative N-oxygenases have been characterized, FrbG (Johannes et al, 2010), CmlI (Lu et al, 2012;Makris et al, 2010) and PsAAO (Platter et al, 2011). The few enzymes characterized thus far have demonstrated limited substrate specificities with a preference for paminobenzoates and o-aminophenols (Platter et al, 2011;Winkler et al, 2006).…”