Consecutive enzymatic modification of ornithine generates the hydroxamate moieties of the siderophore erythrochelin

Biochemistry. 2011 Jul 12;50(27):6073-80. doi: 10.1021/bi200699x. Epub 2011 Jun 15.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylation
  • Acetyltransferases / chemistry
  • Acetyltransferases / genetics
  • Carboxy-Lyases / chemistry
  • Carboxy-Lyases / genetics
  • Carboxy-Lyases / metabolism
  • Decarboxylation
  • Diketopiperazines
  • Flavin-Adenine Dinucleotide / chemistry
  • Hydroxamic Acids / chemistry
  • Hydroxamic Acids / metabolism*
  • Hydroxylation
  • Multienzyme Complexes / biosynthesis*
  • Multienzyme Complexes / chemistry
  • Multienzyme Complexes / genetics
  • Multigene Family
  • Oligopeptides / biosynthesis*
  • Oligopeptides / genetics
  • Ornithine / analogs & derivatives*
  • Ornithine / biosynthesis
  • Ornithine / genetics
  • Ornithine / metabolism
  • Oxygenases / chemistry
  • Saccharopolyspora / enzymology*
  • Saccharopolyspora / genetics
  • Siderophores / biosynthesis*
  • Siderophores / genetics
  • Substrate Specificity / genetics

Substances

  • Diketopiperazines
  • Hydroxamic Acids
  • Multienzyme Complexes
  • Oligopeptides
  • Siderophores
  • erythrochelin
  • Flavin-Adenine Dinucleotide
  • delta-N-acetyl-delta-N-hydroxy-L-ornithine
  • Ornithine
  • Oxygenases
  • Acetyltransferases
  • Carboxy-Lyases
  • malonyl-CoA decarboxylase