Characterization of a Saccharomyces cerevisiae NADP(H)‐dependent alcohol dehydrogenase (ADHVII), a member of the cinnamyl alcohol dehydrogenase family

Abstract

A new NADP(H)-dependent alcohol dehydrogenase (the YCR105W gene product, ADHVII) has been identified in Saccharomyces cerevisiae. The enzyme has been purified to homogeneity and found to be a homodimer of 40 kDa subunits and a pI of 6.2-6.4. ADHVII shows a broad substrate specificity similar to the recently characterized ADHVI (64% identity), although they show some differences in kinetic properties. ADHVI and ADHVII are the only members of the cinnamyl alcohol dehydrogenase family in yeast. Simultaneous deletion of ADH6 and ADH7 was not lethal for the yeast. Both enzymes could participate in the synthesis of fusel alcohols, ligninolysis and NADP(H) homeostasis.Keywords: cinnamyl alcohol dehydrogenase; fusel alcohols; NADP(H) homeostasis; ligninolysis.The current version of the Yeast Proteome Database (http://www.proteome.com) lists approximately 260 oxidoreductases (160 of them have been characterized experimentally, and the rest predicted by sequence similarity or by other analysis) [1]. Our group is interested in the identification and characterization of novel alcohol dehydrogenase (ADH) gene products from Saccharomyces cerevisiae [2,3]. ADHs are oxidoreductases that catalyze the reversible oxidation of alcohols to aldehydes or ketones, with the corresponding reduction of NAD or NADP. ADHs constitute a large group of enzymes that can be subdivided into at least three distinct enzyme superfamilies: medium-chain and short-chain dehydrogenases/reductases, and iron-activated alcohol dehydrogenases [4,5]. The medium-chain dehydrogenase/ reductase (MDR) superfamily consists of enzymes with a subunit size of approximately 350 residues, dimeric or tetrameric, with two domains in each subunit: one catalytic and one responsible for the binding of the nucleotide (NAD or NADP). Many enzymes of the MDR family have zinc in their active site, and have a sequence motif known as the zinc-containing ADH signature: GHEX 2 GX 5 (G,A)X 2 (I,V,A,C,S) [6]. According to the Pfam and COG databases [7,8], the S. cerevisiae genome codes for 21 potential MDR enzymes, with 12 of them showing the zinc ADH signature described above. These 12 zinc-containing yeast MDR include ADH1, ADH2, ADH3, ADH5, ADH6, SFA1, SOR1 and its 99% identical YDL246C, XYL2, BDH1, YAL061W and YCR105W. All these yeast MDRs, except YCR105W and YAL061W, have enzymatic activities experimentally determined. In the present study, we report the characterization of the YCR105W gene from S. cerevisiae as a new alcohol dehydrogenase. The gene was overexpressed in yeast cells and the corresponding protein product purified to homogeneity. The enzyme showed a wide substrate specificity, using NADP(H) as coenzyme. Given the similar substrate specificities and the sequence identity (64%) between the Ycr105p and the recently characterized ADHVI [3], we propose the name of ADH7, for the YCR105W gene and ADHVII for its coded protein. A null adh7 yeast strain and a double mutant adh6D adh7D were constructed and their growths compared with a wild-type strain. M A T E R I A L S A N ...

Characterization of an Alcohol Dehydrogenase from the Cyanobacterium Synechocystis sp. Strain PCC 6803 That Responds to Environmental Stress Conditions via the Hik34-Rre1 Two-Component System

“…In Synechocystis, salt stress has been reported to enhance PSI-dependent cyclic electron flow (58) that generates ATP but not NADPH (36), thus participating in determining the ATP/NADPH ratio when photosynthesis operates under changing environmental conditions (45). The fact that AdhA from Synechocystis oxidizes NADPH much more efficiently than it reduces NADP ϩ supports a possible role for this enzyme in oxidizing NADPH under such conditions and thus contributing to the maintenance of the ATP/NADPH balance, as was also previously proposed for two members of the CADH family in Saccharomyces (27,28).…”

Resistance of Saccharomyces cerevisiae to High Concentrations of Furfural Is Based on NADPH-Dependent Reduction by at Least Two Oxireductases

“…Among the most upregulated genes in both strains upon furfural increase from 15 to 25 mM were the oxireductases ADH7, GRE2, OYE3, AAD4, YML131W, YDL124W, OYE2, and GCY1 (see Table S1 in the supplemental material). Six of them prefer NADPH as the cofactor (6,7,21,26,33,44), while for AAD4 and YML131W, the cofactor preference is not known (Saccharomyces Genome Database at http://www.yeastgenome.org). This is consistent with our finding that central carbon metabolism doubles the NADPH-generating pentose phosphate pathway fluxes upon 25 mM furfural challenge.…”

“…ADH7 encodes a NADPH-dependent, medium-chain-length alcohol dehydrogenase with a broad substrate specificity that includes aromatic compounds. It is a member of the cinnamyl family of alcohol dehydrogenases and is suspected to be involved in the buildup of fusel alcohols (26). Nothing is known about the molecular function or the regulation of ADH7.…”

Comparison of the Gene Expression Patterns of Alcohol Dehydrogenase Isozymes in the Thermotolerant YeastKluyveromyces marxianusand Their Physiological Functions

“…7) ScADH5 encodes for ScAdh5, and has 76% sequence identity to the ScAdh1 isozyme. 8) ScADH6 and ScADH7 encode for ScAdh6 and ScAdh7 respectively, which show broad substrate specificity 9,10) and are assumed to contribute the maintenance of a proper NADP/NADPH balance.…”