title: Discovery of a eugenol oxidase from Rhodococcus sp. strain RHA1.
abstract: A gene encoding a eugenol oxidase was identified in the genome from Rhodococcus sp. strain RHA1. The bacterial FAD-containing oxidase shares 45% amino acid sequence identity with vanillyl alcohol oxidase from the fungus Penicillium simplicissimum. Eugenol oxidase could be expressed at high levels in Escherichia coli, which allowed purification of 160 mg of eugenol oxidase from 1 L of culture. Gel permeation experiments and macromolecular MS revealed that the enzyme forms homodimers. Eugenol oxidase is partly expressed in the apo form, but can be fully flavinylated by the addition of FAD. Cofactor incorporation involves the formation of a covalent protein-FAD linkage, which is formed autocatalytically. Modeling using the vanillyl alcohol oxidase structure indicates that the FAD cofactor is tethered to His390 in eugenol oxidase. The model also provides a structural explanation for the observation that eugenol oxidase is dimeric whereas vanillyl alcohol oxidase is octameric. The bacterial oxidase efficiently oxidizes eugenol into coniferyl alcohol (KM=1.0 microM, kcat=3.1 s-1). Vanillyl alcohol and 5-indanol are also readily accepted as substrates, whereas other phenolic compounds (vanillylamine, 4-ethylguaiacol) are converted with relatively poor catalytic efficiencies. The catalytic efficiencies with the identified substrates are strikingly different when compared with vanillyl alcohol oxidase. The ability to efficiently convert eugenol may facilitate biotechnological valorization of this natural aromatic compound.
authors.affiliation: Laboratory of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, The Netherlands.
authors.forename: Jianfeng
authors.initials: J
authors.lastname: Jin
authors.forename: Hortense
authors.initials: H
authors.lastname: Mazon
authors.forename: Robert H H
authors.initials: RH
authors.lastname: van den Heuvel
authors.forename: Dick B
authors.initials: DB
authors.lastname: Janssen
authors.forename: Marco W
authors.initials: MW
authors.lastname: Fraaije
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D002384:Catalysis; D004926:Escherichia coli / Q000235:genetics; D005054:Eugenol / Q000378:metabolism*; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D005656:Fungal Proteins; D005182:Flavin-Adenine Dinucleotide; D005054:Eugenol; D006899:Mixed Function Oxygenases; C520320:eugenol oxidase, Rhodococcus; D000429:Alcohol Oxidoreductases; C076865:vanillyl-alcohol oxidase