Identification and molecular characterization of the eugenol hydroxylase genes (ehyA/ehyB) of Pseudomonas sp. strain HR199.

The gene loci ehyA and ehyB, which are involved in the bioconversion of eugenol to coniferyl alcohol by Pseudomonas sp. strain HR199 (DSM 7063), were identified as the structural genes of a eugenol hydroxylase that represents an enzyme of the flavocytochrome c class. These genes were localized downstream of the eugenol catabolism genes vanA and vanB, encoding vanillate-O-demethylase, on an EcoRI fragment (E230) that has recently been cloned from a Pseudomonas sp. strain HR199 genomic library. The gene encoding the cytochrome c subunit (ehyA) was identified on a subfragment (K18) of E230 by complementation of a nitrosoguanidine-induced, eugenol-negative mutant of strain HR199. The nucleotide sequences of fragment K18 and adjacent regions were determined, revealing open reading frames of 354 and 1,554 bp that represent ehyA and ehyB, respectively. These genes are most probably organized in one operon together with a third open reading frame (ORF2) of 687 bp that was located between ehyA and ehyB. The deduced amino acid sequences of ehyA and ehyB exhibited up to 29 and 55% amino acid identity to the corresponding subunits of p-cresol methylhydroxylase from Pseudomonas putida. Moreover, the amino-terminal sequences of the alpha- and beta-subunits reported recently for a eugenol dehydrogenase of Pseudomonas fluorescens E118 corresponded well with appropriate regions of ehyA and ehyB. The sequence of ORF2 and the deduced amino acid sequence exhibited no significant similarities to any DNA or amino acid sequence from the databases. The eugenol hydroxylase genes were amplified by PCR, cloned in pBluescript SK(-), and functionally expressed in Escherichia coli. Transfer of a DNA fragment comprising ehyA and ehyB to various strains of Pseudomonas species that were unable to utilize eugenol as a carbon source conferred to these bacteria the ability to grow on this substrate.