YMR318C represents an open reading frame from Saccharomyces cerevisiae with unknown function. It possesses a conserved sequence motif, the zinc-containing alcohol dehydrogenase (ADH) signature, specific to the medium-chain zinc-containing ADHs. In the present study, the YMR318C gene product has been purified to homogeneity from overexpressing yeast cells, and found to be a homodimeric ADH, composed of 40kDa subunits and with a pI of 5.0–5.4. The enzyme was strictly specific for NADPH and was active with a wide variety of substrates, including aliphatic (linear and branched-chain) and aromatic primary alcohols and aldehydes. Aldehydes were processed with a 50-fold higher catalytic efficiency than that for the corresponding alcohols. The highest kcat/Km values were found with pentanal>veratraldehyde > hexanal > 3-methylbutanal >cinnamaldehyde. Taking into consideration the substrate specificity and sequence characteristics of the YMR318C gene product, we have proposed this gene to be called ADH6. The disruption of ADH6 was not lethal for the yeast under laboratory conditions. Although S. cerevisiae is considered a non lignin-degrading organism, the catalytic activity of ADHVI can direct veratraldehyde and anisaldehyde, arising from the oxidation of lignocellulose by fungal lignin peroxidases, to the lignin biodegradation pathway. ADHVI is the only S. cerevisiae enzyme able to significantly reduce veratraldehyde in vivo, and its overexpression allowed yeast to grow under toxic concentrations of this aldehyde. The enzyme may also be involved in the synthesis of fusel alcohols. To our knowledge this is the first NADPH-dependent medium-chain ADH to be characterized in S. cerevisiae.
Abbreviations used: ADH, alcohol dehydrogenase; AspADH, Acinetobacter sp. strain M-1 ADH; Ateli3-2, Arabidopsis thaliana ELI3; bcADH, branched-chain ADH; CAD, cinnamyl ADH; DTT, dithiothreitol; EgCAD2, Eucalyptus gunni CAD2; IEF, isoelectric focusing; MDR, medium-chain dehydrogenase/reductase; MM, minimal medium; MbADH, Myobacterium bovis BCG ADH; ORF, open reading frame; Pceli, Petroselinum crispum ELI3; ScADHI Saccharomyces cerevisiae ADHI; ZmCAD2, Zea mays CAD2.