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A gene encoding a eugenol oxidase was identified in the genome fromRho-dococcus 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. | ỊFEBS Journal Discovery of a eugenol oxidase from Rhodococcus sp. strain RHA1 Jianfeng Jin1 Hortense Mazon2 Robert H. H. van den Heuvel2 Dick B. Janssen1 and Marco W. Fraaije1 1 Laboratory of Biochemistry Groningen Biomolecular Sciences and Biotechnology Institute University of Groningen the Netherlands 2 Department of Biomolecular Mass Spectrometry Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences Utrecht University the Netherlands Keywords covalent flavinylation eugenol flavin oxidase Rhodococcus Correspondence M. W. Fraaije Laboratory of Biochemistry Groningen Biomolecular Sciences and Biotechnology Institute University of Groningen Nijenborgh 4 9747 AG Groningen The Netherlands Fax 31 50 3634165 Tel 31 50 3634345 E-mail m.w.fraaije@rug.nl Received 8 January 2007 revised 21 February 2007 accepted 2 March 2007 doi 10.1111 j.1742-4658.2007.05767.x A gene encoding a eugenol oxidase was identified in the genome from Rho-dococcus 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 pM kcat 3.1 s 1 . Vanillyl .
