Two structures of an N-hydroxylating flavoprotein monooxygenase: ornithine hydroxylase from Pseudomonas aeruginosa

J Biol Chem. 2011 Sep 9;286(36):31789-98. doi: 10.1074/jbc.M111.265876. Epub 2011 Jul 13.

Abstract

The ornithine hydroxylase from Pseudomonas aeruginosa (PvdA) catalyzes the FAD-dependent hydroxylation of the side chain amine of ornithine, which is subsequently formylated to generate the iron-chelating hydroxamates of the siderophore pyoverdin. PvdA belongs to the class B flavoprotein monooxygenases, which catalyze the oxidation of substrates using NADPH as the electron donor and molecular oxygen. Class B enzymes include the well studied flavin-containing monooxygenases and Baeyer-Villiger monooxygenases. The first two structures of a class B N-hydroxylating monooxygenase were determined with FAD in oxidized (1.9 Å resolution) and reduced (3.03 Å resolution) states. PvdA has the two expected Rossmann-like dinucleotide-binding domains for FAD and NADPH and also a substrate-binding domain, with the active site at the interface between the three domains. The structures have NADP(H) and (hydroxy)ornithine bound in a solvent-exposed active site, providing structural evidence for substrate and co-substrate specificity and the inability of PvdA to bind FAD tightly. Structural and biochemical evidence indicates that NADP(+) remains bound throughout the oxidative half-reaction, which is proposed to shelter the flavin intermediates from solvent and thereby prevent uncoupling of NADPH oxidation from hydroxylated product formation.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Catalysis
  • Catalytic Domain
  • Crystallography, X-Ray
  • Flavin-Adenine Dinucleotide
  • Mixed Function Oxygenases / chemistry*
  • NADP
  • Ornithine
  • Oxidation-Reduction
  • Oxygenases / chemistry*
  • Protein Conformation
  • Pseudomonas aeruginosa / enzymology*
  • Substrate Specificity

Substances

  • Flavin-Adenine Dinucleotide
  • NADP
  • Ornithine
  • Mixed Function Oxygenases
  • Oxygenases
  • ornithine hydroxylase, Pseudomonas aeruginosa
  • dimethylaniline monooxygenase (N-oxide forming)

Associated data

  • PDB/3S5W
  • PDB/3S61