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

Ji Young Ryu, Jiyoung Seo, Sunhwa Park, Joong Hoon Ahn, Youhoon Chong, Michael J. Sadowsky, Hor Gil Hur

Research output: Contribution to journalArticle

15 Scopus citations

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 Vmax, KM, and kcat values for it, using isoeugenol as substrate, were 4.2μmol vanillin min-1 mg -1 of protein, 120μM, and 3.8 s-1, respectively. Maximum substrate turnover for Iem occurred at 30 °C and pH 9.0. An 18Oxygen-labeling experiment revealed that oxidative cleavage of isoeugenol by Iem was catalyzed via a monooxygenation reaction, and that incorporation of the oxygen atom from O2 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, His167, His 218, His282, and His471, which appear to be involved in ligand bonding with Fe2+, 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.

Original languageEnglish (US)
Pages (from-to)289-294
Number of pages6
JournalBioscience, Biotechnology and Biochemistry
Volume77
Issue number2
DOIs
StatePublished - 2013

Keywords

  • Isoeugenol
  • Monooxygenase
  • Phenylpropanoid
  • Pseudomonas
  • Vanillin

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