title: An enzymatic pathway for the biosynthesis of the formylhydroxyornithine required for rhodochelin iron coordination.
abstract: Rhodochelin, a mixed catecholate-hydroxamate type siderophore isolated from Rhodococcus jostii RHA1, holds two L-δ-N-formyl-δ-N-hydroxyornithine (L-fhOrn) moieties essential for proper iron coordination. Previously, bioinformatic and genetic analysis proposed rmo and rft as the genes required for the tailoring of the L-ornithine (L-Orn) precursor [Bosello, M. (2011) J. Am. Chem. Soc.133, 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 in vitro. In the presence of molecular oxygen, NADPH and FAD, Rmo monooxygenase was able to convert L-Orn into L-δ-N-hydroxyornithine (L-hOrn). As confirmed in a coupled reaction assay, this hydroxylated intermediate serves as a substrate for the subsequent N(10)-formyl-tetrahydrofolate-dependent (N(10)-fH(4)F) 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 N(10)-fH(4)F conversion, also rescuing the previously inactive CchA, the Rft-homologue in coelichelin assembly line [Buchenau, B. (2004) Arch. Microbiol.182, 313-325; Pohlmann, V. (2008) Org. Biomol. Chem.6, 1843-1848].
authors.affiliation: Biochemistry, Department of Chemistry, Philipps-University Marburg, Hans-Meerwein-Strasse, D-35043 Marburg, Germany.
authors.forename: Mattia
authors.initials: M
authors.lastname: Bosello
authors.forename: Andreas
authors.initials: A
authors.lastname: Mielcarek
authors.forename: Tobias W
authors.initials: TW
authors.lastname: Giessen
authors.forename: Mohamed A
authors.initials: MA
authors.lastname: Marahiel
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D006900:Hydroxylation; D019877:Hydroxymethyl and Formyl Transferases / Q000737:chemistry* / Q000235:genetics; D007501:Iron / Q000737:chemistry* / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism; D009952:Ornithine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012240:Rhodococcus / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D009842:Oligopeptides; D011994:Recombinant Proteins; C559051:rhodochelin; D007501:Iron; D009952:Ornithine; D006899:Mixed Function Oxygenases; D019877:Hydroxymethyl and Formyl Transferases