Expression of Two Old Yellow Enzyme Homologues from Gluconobacter oxydans and Identification of their Citral Hydrogenation Abilities

Abstract

Two genes that encode proteins which share 30-35% sequence identity with yeast OYE (Old Yellow Enzyme, an NAD(P)H FMN-oxidoreductase), the well-studied archetype of the OYE protein family, have been identified in Gluconobacter oxydans M5. The two genes are localized in the chromosome and plasmid, respectively. Comparison of the deduced amino acid sequences of the enzymes with database entries revealed 75.1% similarity and 64.9% identity to that of the Pseudomonas syringae pv. glycinea NAD(P)H-dependent 2-cyclohexen-1-one reductase. The two proteins were expressed as His-tag fusion proteins in Escherichia coli and purified. The ability of the purified proteins to hydrogenate citral was identified. The results showed that the alpha,beta-double bond of citral cis-isomer 'neral' could be stereoselectively reduced to produce citronellal by the purified OYE homologues.