An Enzymatic Pathway for the Biosynthesis of the Formylhydroxyornithine Required for Rhodochelin Iron Coordination

Abstract

Rhodochelin, a mixed catecholate–hydroxamate type siderophore isolated from <i>Rhodococcus jostii</i> RHA1, holds two l-δ-<i>N</i>-formyl-δ-<i>N</i>-hydroxyornithine (l-fhOrn) moieties essential for proper iron coordination. Previously, bioinformatic and genetic analysis proposed <i>rmo</i> and <i>rft</i> as the genes required for the tailoring of the l-ornithine (l-Orn) precursor [Bosello, M. (2011) <i>J. Am. Chem. Soc.</i> <i>133</i>, 4587–4595]. In order to investigate if both Rmo and Rft constitute a pathway for l-fhOrn biosynthesis, the enzymes were heterologously produced and assayed <i>in vitro</i>. In the presence of molecular oxygen, NADPH and FAD, Rmo monooxygenase was able to convert l-Orn into l-δ-<i>N</i>-hydroxyornithine (l-hOrn). As confirmed in a coupled reaction assay, this hydroxylated intermediate serves as a substrate for the subsequent <i>N</i>¹⁰-formyl-tetrahydrofolate-dependent (<i>N</i>¹⁰-fH₄F) Rtf-catalyzed formylation reaction, establishing a route for the l-fhOrn biosynthesis, prior to its incorporation by the NRPS assembly line. It is of particular interest that a major improvement to this study has been reached with the use of an alternative approach to the chemoenzymatic FolD-dependent <i>N</i>¹⁰-fH₄F conversion, also rescuing the previously inactive CchA, the Rft-homologue in coelichelin assembly line [Buchenau, B. (2004) <i>Arch. Microbiol.</i> <i>182</i>, 313–325; Pohlmann, V. (2008) <i>Org. Biomol. Chem.</i> <i>6</i>, 1843–1848]