M. Rosario Fernández

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

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.

Enhanced Formaldehyde Detoxification by Overexpression of Glutathione-Dependent Formaldehyde Dehydrogenase from Arabidopsis

The ADH2 gene codes for the Arabidopsis glutathione-dependent formaldehyde dehydrogenase (FALDH), an enzyme involved in formaldehyde metabolism in eukaryotes. In the present work, we have investigated the potential role of FALDH in detoxification of exogenous formaldehyde. We have generated a yeast (Saccharomyces cerevisiae) mutant strain (sfa1Δ) by in vivo deletion of the SFA1 gene that codes for the endogenous FALDH. Overexpression of Arabidopsis FALDH in this mutant confers high resistance to formaldehyde added exogenously, which demonstrates the functional conservation of the enzyme through evolution and supports its essential role in formaldehyde metabolism. To investigate the role of the enzyme in plants, we have generated Arabidopsis transgenic lines with modified levels of FALDH. Plants overexpressing the enzyme show a 25% increase in their efficiency to take up exogenous formaldehyde, whereas plants with reduced levels of FALDH (due to either a cosuppression phenotype or to the expression of an antisense construct) show a marked slower rate and reduced ability for formaldehyde detoxification as compared with the wild-type Arabidopsis. These results show that the capacity to take up and detoxify high concentrations of formaldehyde is proportionally related to the FALDH activity in the plant, revealing the essential role of this enzyme in formaldehyde detoxification.

Class III alcohol dehydrogenase from Saccharomyces cerevisiae: Structural and enzymatic features differ toward the human/mammalian forms in a manner consistent with functional needs in formaldehyde detoxication

Alcohol dehydrogenase class III (glutathione‐dependent formaldehyde dehydrogenase) from Saccharomyces cerevisiae was purified and analyzed structurally and enzymatically. The corresponding gene was also analyzed after cloning from a yeast genome library by screening with a probe prepared through PCR amplification. As with class III alcohol dehydrogenase from other sources, the yeast protein was obtained in two active forms, deduced to reflect different adducts/modifications. Protein analysis established N‐terminal and C‐terminal positions, showing different and specific patterns in protein start positions between the human/mammalian, yeast, and prokaryotic forms. K m values with formaldehyde differ consistently, being about 10‐fold higher in the yeast than the human/mammalian enzymes, but compensated for by similar changes in κ cat values. This is compatible with the different functional needs, emphasizing low formaldehyde concentration in the animal cells but efficient formaldehyde elimination in the microorganisms. This supports a general role of the enzyme in formaldehyde detoxication rather than in long‐chain alcohol turnover.

Properties and functional significance of Saccharomyces cerevisiae ADHVI

The completion of the Saccharomyces cerevisiae genome project has provided the opportunity to explore for new genes of the medium-chain dehydrogenase/reductase enzyme superfamily. Our group has recently identified a new gene, the YMR318C open reading frame, which coded for a Zn-containing NADP(H)-dependent alcohol dehydrogenase (ADHVI). ADHVI has been purified to homogeneity from over expressing yeast cells, and found to be a homodimer of 40 kDa subunits. The enzyme showed a strict specificity for NADP(H) and high activity with a variety of long chain aliphatic and bulky substrates. Aldehydes exhibited 50-12000 times higher catalytic efficiency than the corresponding alcohols. Substrates with high k(cat)/K(m) were: pentanal, veratraldehyde and cinnamaldehyde. The ADHVI expression was strongly induced when galactose was the sole carbon source in the culture medium. Phylogenetic trees include ADHVI in the cinnamyl alcohol dehydrogenase (CADH) family. In contrast to the plant CADH, involved in lignin biosynthesis, this is not the function for ADHVI, since yeast does not synthesize lignin. ADHVI may be physiologically involved in several steps of the lignin degradation pathway, initiated by other microorganisms, in the synthesis of fusel alcohols, products derived from the aminoacidic metabolism, and in the homeostasis of NADP(H). Disruption of ADH6 was not lethal for the yeast, under laboratory conditions.

Role of Saccharomyces cerevisiae oxidoreductases Bdh1p and Ara1p in the metabolism of acetoin and 2,3-butanediol.

ABSTRACT NAD-dependent butanediol dehydrogenase (Bdh1p) from Saccharomyces cerevisiae reversibly transforms acetoin to 2,3-butanediol in a stereospecific manner. Deletion of BDH1 resulted in an accumulation of acetoin and a diminution of 2,3-butanediol in two S. cerevisiae strains under two different growth conditions. The concentrations of (2R,3R)-2,3-butanediol are mostly dependent on Bdh1p activity, while those of (meso)-2,3-butanediol are also influenced by the activity of NADP(H)-dependent oxidoreductases. One of them has been purified and shown to be d-arabinose dehydrogenase (Ara1p), which converts (R/S)-acetoin to meso-2,3-butanediol and (2S,3S)-2,3-butanediol. Deletion of BDH2, a gene adjacent to BDH1, whose encoded protein is 51% identical to Bdh1p, does not significantly alter the levels of acetoin or 2,3-butanediol in comparison to the wild-type strain. Furthermore, we have expressed Bdh2p with a histidine tag and have shown it to be inactive toward 2,3-butanediol. A whole-genome expression analysis with microarrays demonstrates that BDH1 and BDH2 are reciprocally regulated.

Characterization and functional role of Saccharomyces cerevisiae 2,3-butanediol dehydrogenase

Using a conserved sequence motif, a new gene (YAL060W) of the MDR family has been identified in Saccharomyces cerevisiae. The expressed protein was a stereoespecific (2R,3R)-2,3-butanediol dehydrogenase (BDH). The best substrates were (2R,3R)-2,3-butanediol for the oxidation and (3R/3S)-acetoin and 1-hydroxy-2-propanone for the reduction reactions. The enzyme is extremely specific for NAD(H) as cofactor, probably because the presence of Glu223 in the cofactor binding site, instead of the highly conserved Asp223. BDH is inhibited competitively by 4-methylpyrazole with a K(i) of 34 microM. Yeast could grow on 2,3-butanediol or acetoin as a sole energy and carbon sources, and a 3.6-fold increase in BDH activity was observed when cells were grown in 2,3-butanediol, suggesting a role of the enzyme in 2,3-butanediol metabolism. However, the disruption of the YAL060W gene was not lethal for the yeast under laboratory conditions, and the disrupted strain could also grow in 2,3-butanediol and acetoin. This suggests that other enzymes, in addition to BDH, can also metabolize 2,3-butanediol in yeast.

A Double Residue Substitution in the Coenzyme-binding Site Accounts for the Different Kinetic Properties between Yeast and Human Formaldehyde Dehydrogenases

Glutathione-dependent formaldehyde dehydrogenase (FALDH) is the main enzymatic system for formaldehyde detoxification in all eukaryotic and many prokaryotic organisms. The enzyme of yeasts and some bacteria exhibits about 10-fold higherk cat and K m values than those of the enzyme from animals and plants. Typically Thr-269 and Glu-267 are found in the coenzyme-binding site of yeast FALDH, but Ile-269 and Asp-267 are present in the FALDH of animals. By site-directed mutagenesis we have prepared the T269I and the D267E mutants and the D267E/T269I double mutant of Saccharomyces cerevisiae FALDH with the aim of investigating the role of these residues in the kinetics. The T269I and the D267E mutants have identical kinetic properties as compared with the wild-type enzyme, although T269I is highly unstable. In contrast, the D267E/T269I double mutant is stable and shows low K m (2.5 μm) and low k cat (285 min−1) values with S-hydroxymethylglutathione, similar to those of the human enzyme. Therefore, the simultaneous exchange at both residues is the structural basis of the two distinct FALDH kinetic types. The local structural perturbations imposed by the substitutions are suggested by molecular modeling studies. Finally, we have studied the effect of FALDH deletion and overexpression on the growth of S. cerevisiae. It is concluded that the FALDH gene is not essential but enhances the resistance against formaldehyde (0.3–1 mm). Moreover, the wild-type enzyme (with highk cat and K m ) provides more resistance than the double mutant (with lowk cat and K m ).

Biocatalytic production of alpha-hydroxy ketones and vicinal diols by yeast and human aldo–keto reductases

The α-hydroxy ketones are used as building blocks for compounds of pharmaceutical interest (such as antidepressants, HIV-protease inhibitors and antitumorals). They can be obtained by the action of enzymes or whole cells on selected substrates, such as diketones. We have studied the enantiospecificities of several fungal (AKR3C1, AKR5F and AKR5G) and human (AKR1B1 and AKR1B10) aldo-keto reductases in the production of α-hydroxy ketones and diols from vicinal diketones. The reactions have been carried out with pure enzymes and with an NADPH-regenerating system consisting of glucose-6-phosphate and glucose-6-phosphate dehydrogenase. To ascertain the regio and stereoselectivity of the reduction reactions catalyzed by the AKRs, we have separated and characterized the reaction products by means of a gas chromatograph equipped with a chiral column and coupled to a mass spectrometer as a detector. According to the regioselectivity and stereoselectivity, the AKRs studied can be divided in two groups: one of them showed preference for the reduction of the proximal keto group, resulting in the S-enantiomer of the corresponding α-hydroxy ketones. The other group favored the reduction of the distal keto group and yielded the corresponding R-enantiomer. Three of the AKRs used (AKR1B1, AKR1B10 and AKR3C1) could produce 2,3-butanediol from acetoin. We have explored the structure/function relationships in the reactivity between several yeast and human AKRs and various diketones and acetoin. In addition, we have demonstrated the utility of these AKRs in the synthesis of selected α-hydroxy ketones and diols.

MDR quinone oxidoreductases: The human and yeast ζ-crystallins

The medium-chain dehydrogenase/reductase (MDR) superfamily can be divided into Zn-containing and Zn-lacking proteins. Zn-containing MDRs are generally well-known enzymes, mostly acting as dehydrogenases. The non-Zn MDR are much less studied, and classified in several families of NADP(H)-dependent reductases, including quinone oxidoreductases (QOR). zeta-Crystallins are the best studied group of QOR, have a structural function in the lens of several mammals, exhibit ortho-quinone reductase activity, and bind to specific adenine-uracil-rich elements (ARE) in RNA. In the present work, we have further characterized human zeta-crystallin and Saccharomyces cerevisiae Zta1p, the only QOR in yeast. Subcellular localization using a fluorescent protein tag indicates that zeta-crystallin is distributed in the cytoplasm but not in nucleus. The protein may also be present in mitochondria. Zta1p localizes in both cytoplasm and nucleus. NADPH, but not NADH, competitively prevents binding of zeta-crystallin to RNA, suggesting that the cofactor-binding site is involved in RNA binding. Interference of NADPH on Zta1p binding to RNA is much lower, consistent with a weaker binding of NADPH to the yeast enzyme. Disruption of the yeast ZTA1 gene does not affect cell growth under standard conditions but makes yeast more sensitive to oxidative stress agents. Sequence alignments, phylogenetic tree analysis and kinetic properties reveal a close relationship between zeta-crystallin and Zta1p. Amino acid conservation, between the substrate-binding sites of the two proteins and that of an E. coli QOR, indicates that zeta-crystallins maintained their kinetic function throughout evolution. Quinones are toxic compounds and a relevant step in their detoxification is reduction to their corresponding hydroquinones. Many enzymes of several superfamilies can reduce quinones, including NAD(P)H:quinone oxidoreductase 1 (NQO1 or DT-diaphorase), aldo-keto reductases and short-chain dehydrogenases/reductases. In this context, the physiological role of zeta-crystallins is discussed.

Novel alkenal/one reductase activity of yeast NADPH:quinone reductase Zta1p. Prospect of the functional role for the ζ-crystallin family

ζ-Crystallins are a Zn(2+)-lacking enzyme group with quinone reductase activity, which belongs to the medium-chain dehydrogenase/reductase superfamily. It has been recently observed that human ζ-crystallin is capable of reducing the α,β-double bond of alkenals and alkenones. Here we report that this activity is also shared by the homologous Zta1p enzyme from Saccharomyces cerevisiae. While the two enzymes show similar substrate specificity, human ζ-crystallin exhibits higher activity with lipid peroxidation products and Zta1p is more active with cinnamaldehyde. The presence of Zta1p has an in vivo protective effect on yeast strains exposed to the toxic substrate 3-penten-2-one. Analysis of ZTA1 gene expression indicates an induction under different types of cellular stress, including ethanol and dimethylsulfoxide exposure and by reaching the stationary growth phase. The role of Zta1p in the yeast adaptation to some stress types and the general functional significance of ζ-crystallins are discussed.