Hydroxamate formation in the lipopeptide containing siderophore taiwachelin

Cupriavidus taiwanensis is a nitrogen-fixing bacterium found in root nodules of the invasive tropical weed Mimosa pudica. C. taiwanensis has been shown to grow in environments contaminated with heavy metals such as Pb, Cu, and Cd. Taiwachelin, a recently identified siderophore produced by C. taiwanensis, is an iron chelator that contains a hydroxamate functional group for metal binding. In its biosynthesis, a flavin-dependent N-monooxygenase (NMO) was predicted to hydroxylate l-ornithine, leading to formation of the metal binding hydroxamate moiety. Here, we report the cloning, expression, and biochemical characterization of this enzyme, herein referred to as CtNMO. CtNMO was isolated with bound FAD and catalyzed the hydroxylation of l-ornithine with a k _cat of 0.72 ± 0.04 s^-1 and a K _M of 156 ± 26 μM. A ∼25-fold preference for NADPH over NADH was observed. Expression of CtNMO as a fusion to maltose-binding protein resulted in enhanced enzymatic activity. These results validate CtNMO as an efficient l-ornithine hydroxylase and establish its potential utility in future applications, including bioremediation.