Identification and molecular characterization of vinyl phenol reductase from Dekkera bruxellensis CBS 4481

In enology \u201cBrett\u201d character refers to the wine spoilage caused by the yeast Dekkera/Brettanomyces bruxellensis and its production of volatile phenolic off-flavours. This peculiarity is due to the activity of the vinylphenol reductase enzyme, that catalyses the conversion from vinyl- to ethyl-phenols and it is both strain- and cultural condition dependent. Nineteen strains of D. bruxellensis were screened for vinyl phenol reductase (VPR) activity. Presence/absence of the precursors in the growth medium gave no significant differences in VPR activity, confirming that this enzyme is constitutively expressed. A purified protein with VPR activity was extracted from D. bruxellensis CBS4481 cells. The amino acidic sequence, achieved by trypsinolysis and mass spectrometry, revealed a high homology with a Cu/Zn superoxide dismutase (SOD) in the D. bruxellensis AWRI1499 genome. Actually, the isolated protein possessed both vinyl phenol reductase and superoxide dismutase activities. The bioinformatics analysis showed the presence of cofactor-binding sites, that are absent or severely altered in sequences of superoxide dismutases from other wine-relevant yeast species, which do not display vinyl phenol reduction. This moonlighting activity of D. bruxellensis SOD as a VPR is related to its capacity of catalyzing NADH-dependent reduction of vinyl phenols