title: Consecutive enzymatic modification of ornithine generates the hydroxamate moieties of the siderophore erythrochelin.
abstract: Biosynthesis of the hydroxamate-type siderophore erythrochelin requires the generation of δ-N-acetyl-δ-N-hydroxy-L-ornithine (L-haOrn), which is incorporated into the tetrapeptide at positions 1 and 4. Bioinformatic analysis revealed the FAD-dependent monooxygenase EtcB and the bifunctional malonyl-CoA decarboxylase/acetyltransferase Mcd to be putatively involved in the generation of L-haOrn. To investigate if EtcB and Mcd constitute a two-enzyme pathway for the biosynthesis of L-haOrn, they were produced in a recombinant manner and subjected to biochemical studies in vitro. Hydroxylation assays employing recombinant EtcB gave rise to δ-N-hydroxy-L-ornithine (L-hOrn) and confirmed the enzyme to be involved in building block assembly. Acetylation assays were carried out by incubating L-hOrn with recombinant Mcd and malonyl-CoA as the acetyl group donor. Substrate turnover was increased by substituting malonyl-CoA with acetyl-CoA, bypassing the decarboxylation reaction which represents the rate-limiting step. Consecutive enzymatic synthesis of L-haOrn was accomplished in coupled assays employing both the L-ornithine hydroxylase and Mcd. In summary, a biosynthetic route for the generation of δ-N-acetyl-δ-N-hydroxy-L-ornithine starting from L-ornithine has been established in vitro by tandem action of the FAD-dependent monooxygenase EtcB and the bifunctional malonyl-CoA decarboxylase/acetyltransferase Mcd.
authors.affiliation: Department of Chemistry/Biochemistry, Philipps-University Marburg, Hans-Meerwein-Strasse, D-35032 Marburg, Germany.
authors.forename: Lars
authors.initials: L
authors.lastname: Robbel
authors.forename: Verena
authors.initials: V
authors.lastname: Helmetag
authors.forename: Thomas A
authors.initials: TA
authors.lastname: Knappe
authors.forename: Mohamed A
authors.initials: MA
authors.lastname: Marahiel
mesh_terms: D000107:Acetylation; D000123:Acetyltransferases / Q000737:chemistry / Q000235:genetics; D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003653:Decarboxylation; D054659:Diketopiperazines; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D006877:Hydroxamic Acids / Q000737:chemistry / Q000378:metabolism*; D006900:Hydroxylation; D009097:Multienzyme Complexes / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D005810:Multigene Family; D009842:Oligopeptides / Q000096:biosynthesis* / Q000235:genetics; D009952:Ornithine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D010105:Oxygenases / Q000737:chemistry; D016930:Saccharopolyspora / Q000201:enzymology* / Q000235:genetics; D017262:Siderophores / Q000096:biosynthesis* / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D054659:Diketopiperazines; D006877:Hydroxamic Acids; D009097:Multienzyme Complexes; D009842:Oligopeptides; D017262:Siderophores; C549789:erythrochelin; D005182:Flavin-Adenine Dinucleotide; C029642:delta-N-acetyl-delta-N-hydroxy-L-ornithine; D009952:Ornithine; D010105:Oxygenases; D000123:Acetyltransferases; D002262:Carboxy-Lyases; C021532:malonyl-CoA decarboxylase