Characterization of an isoeugenol monooxygenase (Iem) from Pseudomonas nitroreducens Jin1 that transforms isoeugenol to vanillin

Biosci Biotechnol Biochem. 2013;77(2):289-94. doi: 10.1271/bbb.120715. Epub 2013 Feb 7.

Abstract

The isoeugenol monooxygenase (iem) gene from Pseudomonas nitroreducens Jin1, responsible for the conversion of isoeugenol to vanillin, was cloned and overexpressed in Escherichia coli. The purified Iem had a predicted molecular mass of 54 kDa. The V(max), K(M), and k(cat) values for it, using isoeugenol as substrate, were 4.2 µmol vanillin min(-1) mg(-1) of protein, 120 µM, and 3.8 s(-1), respectively. Maximum substrate turnover for Iem occurred at 30 °C and pH 9.0. An (18)Oxygen-labeling experiment revealed that oxidative cleavage of isoeugenol by Iem was catalyzed via a monooxygenation reaction, and that incorporation of the oxygen atom from O(2) into vanillin was preferable to incorporation from water. While the catalytic activity of Iem, as prepared, did not require the addition of any organic or metal cofactor, ICP-MS analysis showed 0.7 mol of iron per mol of Iem. Moreover site-directed mutagenesis of Iem of four conserved histidine residues individually, His(167), His(218), His(282), and His(471), which appear to be involved in ligand bonding with Fe(2+), resulted in a loss of activity. Enzyme activity was not appreciably influenced by preincubation of Iem with high concentrations of chelators, suggesting that iron is tightly bound. Iem has an iron-mediated mechanism that is widely spread among the three domains of life.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Benzaldehydes / metabolism*
  • Biotransformation
  • Cloning, Molecular
  • Escherichia coli / genetics
  • Eugenol / analogs & derivatives*
  • Eugenol / metabolism
  • Gene Expression
  • Histidine / chemistry
  • Histidine / metabolism
  • Hydrogen-Ion Concentration
  • Iron / chemistry*
  • Iron Chelating Agents / chemistry
  • Kinetics
  • Mixed Function Oxygenases / chemistry
  • Mixed Function Oxygenases / genetics
  • Mixed Function Oxygenases / metabolism*
  • Molecular Weight
  • Mutagenesis, Site-Directed
  • Oxygen / chemistry
  • Oxygen / metabolism
  • Oxygen Isotopes
  • Pseudomonas / enzymology*
  • Pseudomonas / genetics
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism

Substances

  • Bacterial Proteins
  • Benzaldehydes
  • Iron Chelating Agents
  • Oxygen Isotopes
  • Recombinant Proteins
  • Eugenol
  • Histidine
  • isoeugenol
  • vanillin
  • Iron
  • Mixed Function Oxygenases
  • Oxygen