Characterization of the Saccharomyces cerevisiae YMR318C (ADH6) gene product as a broad specificity NADPH-dependent alcohol dehydrogenase: relevance in aldehyde reduction

Abstract

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 40 kDa 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 k(cat)/K(m) 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.

Retro-biosynthetic screening of a modular pathway design achieves selective route for microbial synthesis of 4-methyl-pentanol

“…PuuC Ec and FeaB Ec were tested in strains expressing ADH6 Sc . Like FeaB Ec , Adh6p Sc has reported K m values for relevant substrates in the 100-200 mM range 53 . Adh6p Sc was observed to have k cat values (B100 s À 1 ) an order of magnitude higher than values observed for FeaB Ec and PuuC Ec (B10 s À 1 ) for related aliphatic aldehydes.…”

“…The wide array of identified alcohol dehydrogenases created a high probability that an alcohol dehydrogenase could be found for conversion to the final alcohol 13,58,59 . S. cerevisiae Adh6p Sc was initially selected because it was previously found to be a broad specificity alcohol dehydrogenase with high activity on medium-and branchedchain aliphatic aldehydes 53 .…”

Influences of cinnamic aldehydes on H+ extrusion activity and ultrastructure of Candida

“…Cinnamaldehyde inhibits the growth of bacteria, yeast and filamentous moulds and is thought to act by inhibiting ATPases (Shreaz et al, 2010;Usta et al, 2003;Gill & Holley, 2004) and cell-wall biosynthesis (Bang et al, 2000) and by changing membrane structure and integrity (Giordani et al, 2006;Xie et al, 2004;Di Pasqua et al, 2006, 2007. Derivatives of cinnamaldehyde, such as 4-hydroxy-3-methoxycinnamaldehyde (coniferyl aldehyde) and 3,5-dimethoxy-4-hydroxycinnamaldehyde (sinapaldehyde), have also been shown to affect fungal growth and metabolism (Larsson et al, 2001;Larroy et al, 2002;Li et al, 2000). Previously, we reported that cinnamaldehyde and its synthesized derivative affect proton extrusion and ergosterol biosynthesis (Shreaz et al, 2010.…”

Crystallization and preliminary X-ray analysis of NADP(H)-dependent alcohol dehydrogenases fromSaccharomyces cerevisiaeandRana perezi

“…In plants, CAD enzymes participate in the synthesis of cinnamyl alcohol derivatives in the last steps of lignin biosynthesis (Luderitz & Grisebach, 1981). Since S. cerevisiae does not synthesize lignin, we hypothesized that the yeast ADHVI could contribute to lignin degradation and the last step of the synthesis of fusel alcohols derived from amino-acid and -ketoacid metabolism (Larroy et al, 2002). No crystallographic structure has been reported for any member of the CAD family.…”

“…We have recently reported the characterization of a new yeast ADH, named ADHVI, presumably a dimer of molecular weight 80 kDa, with only 26% sequence identity to the tetrameric enzyme family (Larroy et al, 2002). Primary structure analysis shows that ADHVI belongs to a different family within the MDRs, the cinnamyl alcohol dehydrogenases (CADs; 37% identity).…”