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W2000502765 abstract "The completion of theSaccharomyces cerevisiae genome project in 1996 showed that almost 60% of the potential open reading frames of the genome had no experimentally determined function. Using a conserved sequence motif present in the zinc-containing medium-chain alcohol dehydrogenases, we found several potential alcohol dehydrogenase genes with no defined function. One of these,YAL060W, was overexpressed using a multicopy inducible vector, and its protein product was purified to homogeneity. The enzyme was found to be a homodimer that, in the presence of NAD+, but not of NADP, could catalyze the stereospecific oxidation of (2R,3R)-2,3-butanediol (K m= 14 mm, k cat = 78,000 min−1) and meso-butanediol (K m = 65 mm,k cat = 46,000 min−1) to (3R)-acetoin and (3S)-acetoin, respectively. It was unable, however, to further oxidize these acetoins to diacetyl. In the presence of NADH, it could catalyze the stereospecific reduction of racemic acetoin ((3R/3S)- acetoin; K m = 4.5 mm, k cat = 98,000 min−1) to (2R,3R)-2,3-butanediol andmeso-butanediol, respectively. The substrate stereospecificity was determined by analysis of products by gas-liquid chromatography. The YAL060W gene product can therefore be classified as an NAD-dependent (2R,3R)-2,3-butanediol dehydrogenase (BDH).S. cerevisiae could grow on 2,3-butanediol as the sole carbon and energy source. Under these conditions, a 3.5-fold increase in (2R,3R)-2,3-butanediol dehydrogenase activity was observed in the total cell extracts. The isoelectric focusing pattern of the induced enzyme coincided with that of the pure BDH (pI 6.9). The disruption of the YAL060W gene was not lethal for the yeast under laboratory conditions. The disrupted strain could also grow on 2,3-butanediol, although attaining a lesser cell density than the wild-type strain. Taking into consideration the substrate specificity of the YAL060W gene product, we propose the name of BDH for this gene. The corresponding enzyme is the first eukaryotic (2R,3R)-2,3-butanediol dehydrogenase characterized of the medium-chain dehydrogenase/reductase family. The completion of theSaccharomyces cerevisiae genome project in 1996 showed that almost 60% of the potential open reading frames of the genome had no experimentally determined function. Using a conserved sequence motif present in the zinc-containing medium-chain alcohol dehydrogenases, we found several potential alcohol dehydrogenase genes with no defined function. One of these,YAL060W, was overexpressed using a multicopy inducible vector, and its protein product was purified to homogeneity. The enzyme was found to be a homodimer that, in the presence of NAD+, but not of NADP, could catalyze the stereospecific oxidation of (2R,3R)-2,3-butanediol (K m= 14 mm, k cat = 78,000 min−1) and meso-butanediol (K m = 65 mm,k cat = 46,000 min−1) to (3R)-acetoin and (3S)-acetoin, respectively. It was unable, however, to further oxidize these acetoins to diacetyl. In the presence of NADH, it could catalyze the stereospecific reduction of racemic acetoin ((3R/3S)- acetoin; K m = 4.5 mm, k cat = 98,000 min−1) to (2R,3R)-2,3-butanediol andmeso-butanediol, respectively. The substrate stereospecificity was determined by analysis of products by gas-liquid chromatography. The YAL060W gene product can therefore be classified as an NAD-dependent (2R,3R)-2,3-butanediol dehydrogenase (BDH).S. cerevisiae could grow on 2,3-butanediol as the sole carbon and energy source. Under these conditions, a 3.5-fold increase in (2R,3R)-2,3-butanediol dehydrogenase activity was observed in the total cell extracts. The isoelectric focusing pattern of the induced enzyme coincided with that of the pure BDH (pI 6.9). The disruption of the YAL060W gene was not lethal for the yeast under laboratory conditions. The disrupted strain could also grow on 2,3-butanediol, although attaining a lesser cell density than the wild-type strain. Taking into consideration the substrate specificity of the YAL060W gene product, we propose the name of BDH for this gene. The corresponding enzyme is the first eukaryotic (2R,3R)-2,3-butanediol dehydrogenase characterized of the medium-chain dehydrogenase/reductase family. alcohol dehydrogenase medium-chain dehydrogenase/reductase short-chain dehydrogenase/reductase polymerase chain reaction (2R,3R)-2,3-butanediol dehydrogenase gas-liquid chromatography One of the tasks left after the completion of the various genome projects is to ascertain the function of the sequenced genes. When theSaccharomyces cerevisiae genome project was finished, it was found that ∼60% of the potential open reading frames of the genome had no defined function (1Botstein D. Chervitz S.A. Cherry J.M. Science. 1997; 277: 1259-1260Crossref PubMed Scopus (301) Google Scholar). One way of finding the biological role of each gene is to study its pattern of expression. A systematic effort has been performed in S. cerevisiae by means of DNA microarrays. The study of the temporal program of the gene expression accompanying the metabolic shift from fermentation to respiration has yield useful information on virtually every gene of this yeast (2DeRisi J.L. Iyer V.R. Brown P.O. Science. 1997; 278: 680-686Crossref PubMed Scopus (3707) Google Scholar). Another approach is to use consensus sequences of well characterized protein families to reveal close relatives, previously uncharacterized, in the sequenced genomes. The alcohol dehydrogenase (ADH)1 superfamily catalyzes the reversible oxidation of alcohols to aldehydes or ketones and can be grouped in at least three enzyme families: medium-chain dehydrogenases/reductases (MDR), short-chain dehydrogenases/reductases (SDR), and iron-activated alcohol dehydrogenases (3Jörnvall H. Danielsson O. Eklund H. Helmqvist L. Höög J.-O. Parés X. Shafqat J. Weiner H. Crabb D.W. Flynn T.G. Enzymology and Molecular Biology of Carbonyl Metabolism. 4. Plenum Press, New York1993: 533-544Google Scholar, 4Persson B. Zigler Jr., J.S. Jörnvall H. Eur. J. Biochem. 1994; 226: 15-22Crossref PubMed Scopus (144) Google Scholar). Since most of the enzymes belonging to the MDR family contain one or two Zn2+ ions/subunit, they are also known as Zn2+-containing medium-chain ADHs. S. cerevisiae has seven genes coding for MDR enzymes with known function: ADH1 codes for the fermentative enzyme responsible for ethanol production from acetaldehyde and NADH, and it is produced in large amounts in glucose-grown cells (5Bennetzen J.L. Hall B.D. J. Biol. Chem. 1982; 257: 3018-3025Abstract Full Text PDF PubMed Google Scholar).ADH2 encodes the glucose-repressible isozyme (ADHII) that converts the ethanol accumulated under anaerobic conditions to acetaldehyde and allows the yeast to grow with ethanol as the carbon source (6Ciriacy M. Mol. Gen. Genet. 1975; 135: 157-164Crossref Scopus (132) Google Scholar, 7Wills C. Jörnvall H. Eur. J. Biochem. 1979; 99: 323-331Crossref PubMed Scopus (38) Google Scholar, 8Russell D.W. Smith M. Williamson V.M. Young E.T. J. Biol. Chem. 1983; 258: 2674-2682Abstract Full Text PDF PubMed Google Scholar). ADH3 codes for ADHIII, the mature form of which is located in mitochondria (9Young E.T. Pilgrim D. Mol. Cell. Biol. 1985; 5: 3024-3034Crossref PubMed Scopus (110) Google Scholar) and which is also repressed by glucose. ADH5 codes for an ADH with a 76% sequence identity to the ADHI isozyme (10Feldmann H. Aigle M. Aljinovic G. André B. Baclet M.C. Barthe C. Baur A. Becam A.M. Biteau N. Boles E. et al.EMBO J. 1994; 13: 5795-5809Crossref PubMed Scopus (216) Google Scholar). SFA1 encodes the glutathione-dependent formaldehyde dehydrogenase (class III alcohol dehydrogenase) (11Wehner E.P. Rao E. Brendel M. Mol. Gen. Genet. 1993; 237: 351-358Crossref PubMed Scopus (84) Google Scholar, 12Fernández M.R. Biosca J.A. Norin A. Jörnvall H. Parés X. FEBS Lett. 1995; 370: 23-26Crossref PubMed Scopus (36) Google Scholar, 13Fernández M.R. Biosca J.A. Torres D. Crossas B. Parés X. J. Biol. Chem. 1999; 274: 37869-37875Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar), which is a ubiquitous enzyme expressed in prokaryotes and eukaryotes with a formaldehyde detoxication role.SOR1 codes for a sorbitol dehydrogenase, which is induced in cells grown in the presence of sorbitol (14Sarthy A. Schopp C. Idler K.B. Gene (Amst.). 1994; 140: 121-126Crossref PubMed Scopus (44) Google Scholar). Finally,YLR070C has recently been shown to code for a xylitol dehydrogenase (15Richard P. Toirani M.H. Penttilä M. FEBS Lett. 1999; 457: 135-138Crossref PubMed Scopus (81) Google Scholar). The ADH4 gene (16Williamson V.M. Paquin C.E. Mol. Gen. Genet. 1987; 209: 374-381Crossref PubMed Scopus (105) Google Scholar) codes for ADHIV, which is considered a member of the “iron-activated” ADH family (17Drewke C. Ciriacy M. Biochim. Biophys. Acta. 1988; 950: 54-60Crossref PubMed Scopus (69) Google Scholar). In this work, we have used a conserved sequence motif found in the Zn2+-containing medium-chain ADH (the zinc-containing ADH signature) (18Persson B. Hallborn J. Walfridsson M. Hahn-Hägerdal B. Keränen S. Penttilä M. Jörnvall H. FEBS Lett. 1993; 324: 9-14Crossref PubMed Scopus (35) Google Scholar, 19Jörnvall H. Jansson B. Jörnvall H. Rydberg U. Terenius L. Vallee B.L. Toward a Molecular Basis of Alcohol Use and Abuse. Birkhäuser Verlag, Basel, Switzerland1994: 221-229Crossref Scopus (27) Google Scholar, 20Reid M.F. Fewson C.A. Crit. Rev. Microbiol. 1994; 20: 13-56Crossref PubMed Scopus (356) Google Scholar) to look for possible uncharacterized ADH genes in the yeast genome. One of the genes found with unknown function,YAL060W, was overexpressed in a yeast ADH-deficient strain, and the protein was purified to homogeneity and characterized. The enzyme was found to be a dimer that oxidized reversibly and stereospecifically (2R,3R)-2,3-butanediol andmeso-2,3-butanediol to (3R)-acetoin and (3S)-acetoin, respectively. Although other (2R,3R)-2,3-butanediol dehydrogenases have been described, this would be, to our knowledge, the first characterized eukaryotic protein with this specificity and known sequence belonging to the family of zinc-containing medium-chain ADHs. Restriction enzymes and T4 DNA ligase were from Roche Molecular Biochemicals (Mannheim, Germany). Vent polymerase was from New England Biolabs Inc. (Beverly, MA). DNA oligomers were synthesized and purified by Amersham Pharmacia Biotech (Uppsala, Sweden). Chemicals were purchased from Fluka (Buchs, Switzerland), Aldrich, or Sigma and were of the highest quality available. Hydroxylapatite Bio-Gel HT was from Bio-Rad; Cibacron blue 3GA-agarose was from Sigma; and the Superdex 200 HR 10/30 column was from Amersham Pharmacia Biotech. The consensus pattern GHEXXGXXXXX(GA)XX(IVAC), found in the zinc-containing medium-chain ADH family (18Persson B. Hallborn J. Walfridsson M. Hahn-Hägerdal B. Keränen S. Penttilä M. Jörnvall H. FEBS Lett. 1993; 324: 9-14Crossref PubMed Scopus (35) Google Scholar, 19Jörnvall H. Jansson B. Jörnvall H. Rydberg U. Terenius L. Vallee B.L. Toward a Molecular Basis of Alcohol Use and Abuse. Birkhäuser Verlag, Basel, Switzerland1994: 221-229Crossref Scopus (27) Google Scholar, 20Reid M.F. Fewson C.A. Crit. Rev. Microbiol. 1994; 20: 13-56Crossref PubMed Scopus (356) Google Scholar), was used as the input sequence in the BLAST program (NCBI, National Institutes of Health) to search for open reading frames in the Saccharomyces cerevisiae Genome Database. This sequence contains a histidine that is the second ligand of the catalytic zinc and several glycines that are important for structural reasons in the substrate-binding domain of these enzymes (21Sun H. Plapp B.V. J. Mol. Evol. 1992; 34: 522-535Crossref PubMed Scopus (134) Google Scholar). Multiple sequence alignments were generated using ClustalW Version 1.7 software (22Thompson J.D. Higgins D.G. Gibbson J.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (56002) Google Scholar) in combination with TreeView Version 1.6.1 (23Page R.D. Comput. Appl. Biosci. 1996; 12: 357-358PubMed Google Scholar) to study phylogenies. Escherichia coli XL1-Blue (Stratagene, La Jolla, CA) was used for cloning procedures. The yeast ADH-deficient strain WV36-405 (MAT a, ade2,ura3-52, trp1, adh1Δ,adh2Δ, adh3, adh4::TRP1) (24Atrian S. González-Duarte R. Fothergill-Gilmore L.A. Gene (Amst.). 1990; 93: 205-212Crossref PubMed Scopus (7) Google Scholar), constructed by Dr. Wolfgang Vogel (Institut fur Strahlenbiologie, Neuherberger, Germany) and generously provided by Dr. Silvia Atrian (Universitat de Barcelona), was used to search for the function of theYAL060W gene product. Because of its low background in alcohol oxidation reactions, this strain is useful in ascertaining potentially new ADH genes. The yeast strain FY834α (MATα, his3Δ200, ura3-52,leu2Δ1, lys2Δ202,trp1Δ63) (25Winston F. Dollard C. Ricupero-Hovasse S.L. Yeast. 1995; 11: 53-55Crossref PubMed Scopus (788) Google Scholar), used in the S. cerevisiae genome project, was used here to amplify theYAL060W gene by PCR. The cell growth in the presence of 2,3-butanediol, and the levels of 2,3-butanediol dehydrogenase activity in the homogenates were studied in both yeast strains (WV36-405 and FY834α). The protease-deficient yeast strain BJ5459 (MAT a, ura3-52,trp1, lys2-801,leu2Δ1, his3Δ200,pep4::HIS3,prb1Δ1.6R, can1, GAL) (26Jones E.W. Methods Enzymol. 1991; 194: 428-453Crossref PubMed Scopus (367) Google Scholar), generously provided by Dr. Benjamı́ Piña (Consejo Superior de Investigaciones Cientı́ficas, Barcelona, Spain), was used to overexpress and purify the YAL060W gene product. The inducible E. coli-yeast shuttle vector pYes2 (carrying the promoter and upstream activating sequences ofGAL1) from Invitrogen (Carlsbad, CA) was used to clone and overexpress the YAL060W gene in yeast strains WV36-405 and BJ5459. E. coli cells were grown at 37 °C in LB medium supplemented with 50 μg/ml ampicillin to select for the desired plasmid constructs. Yeast strains WV36-405 and BJ5459 were grown at 30 °C in synthetic complete medium lactriyourairl supplemented with 2% galactose to allow for the selection and induction of the yeast transformed with the pYes2 constructs. The medium used to grow the yeast in 2,3-butanediol contained 1% yeast extract (Difco), 2% peptone, and 0.5 or 3% 2,3-butanediol isomers (mixture of (2R,3R)-2,3-butanediol, (2S,3S)-2,3-butanediol, andmeso-2,3-butanediol). All DNA manipulations were performed under standard conditions as described (27Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Yeast genomic DNA was isolated from yeast strain FY834α by standard methods (28Ausubel F.H. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols of Molecular Biology. Greene Publishing/Wiley-Interscience, New York1991: 133.1-13.7.2Google Scholar), and theYAL060W gene was amplified by PCR using the oligonucleotide 5′-GGGGTACCAATTATGAGAGCTTTGGCATATTTC-3′, which hybridizes at the 5′-end of the gene and carries a KpnI restriction site, and the oligonucleotide 5′-GCGGAATTCTTACTTCATTTCACCGTGATTGTTAG-3′, which hybridizes at the 3′-end and carries an EcoRI restriction site. The amplification initiated with a “hot start,” which was followed by five cycles of 1 min at 95 °C, 1 min at 57 °C, and 90 s of extension at 72 °C. This initial phase was followed by 25 more cycles of 1 min at 95 °C, 1 min at 60 °C, and 90 s of extension at 72 °C at the end. The PCR mixture contained 1 unit of Vent DNA polymerase, 1 μmeach primer, 200 μm each dNTP, and 2 mmMgSO4. To construct theYAL060W expression vector under the control of theGAL1 promoter, the gel-purified PCR product obtained above was digested with KpnI and EcoRI and ligated to the pYes2 vector digested with the same restriction enzymes. Both chains of the plasmid construct, pYes2-YAL060W, were sequenced (Oswel Research Products, Southampton, UK) to verify that there were no mutations introduced by PCR and that the construct was correct. Yeast strains WV36-405 and BJ5459 were grown in rich medium and transformed with the pYes2 and pYes2-YAL060W vectors following the method of Ito et al. (29Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar), and the transformants were selected on SC-Ura plates (28Ausubel F.H. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols of Molecular Biology. Greene Publishing/Wiley-Interscience, New York1991: 133.1-13.7.2Google Scholar). The disruption of theYAL060W gene was carried out by one-step gene replacement (30Rothstein R.J. Methods Enzymol. 1983; 101: 202-211Crossref PubMed Scopus (2033) Google Scholar) with the TRP1 gene. The starting point was plasmid pYes2-YAL060W, containing the coding region ofYAL060W that was digested with MluNI. This digestion removed ∼360 base pairs of the YAL060W coding region and was followed by the insertion of the TRP1 gene. The TRP1 gene, which was obtained by digesting the YDp-W vector (31Berben G. Dumant J. Gilliquet V. Bolle P.A. Hilger F. Yeast. 1991; 7: 475-477Crossref PubMed Scopus (317) Google Scholar) with BamHI, was subcloned into theMluNI site mentioned above after making blunt ends. This construct was digested with KpnI and EcoRI, resulting in a linear fragment containing the TRP1 gene flanked by homologous regions of the YAL060W gene. This fragment was introduced into the yeast haploid strain FY834α by the lithium acetate method (29Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar), and after homologous recombination, the coding region of YAL060W was disrupted with theTRP1 gene. The yeast cells that had incorporated theTRP1 gene were selected by growing in Wikerham's medium supplemented for all auxotrophs except for tryptophan. The disruption of the YAL060W gene in several transformants was confirmed by PCR of the genomic DNA and by enzyme activity in the homogenates of the resulting yeast strains. Enzyme activities were determined spectrophotometrically by measuring the change in absorbance at 340 nm and 25 °C corresponding to the oxidation of NADH (ε340 = 6220m−1 cm−1) or the reduction of NAD+. The purification of theYAL060W gene product was followed by measuring the activity with 120 mm (2R,3R)-2,3-butanediol and 4 mm NAD+ in 33 mm sodium pyrophosphate (pH 8). To determine the steady-state kinetic constants, the enzyme assays were carried out with the pH 8 buffer and 5 mm NAD+ for the oxidation reactions, and with 33 mm sodium phosphate (pH 7) 0.2 mm NADH for the reduction reactions. After adding 20–50 μl of enzyme solution, the reaction was started by the addition of the substrate. One unit of activity corresponds to 1 μmol of NAD(H) formed/min. The initial velocities were measured in duplicate at eight different substrate concentrations, and the kinetic constants were calculated using the nonlinear regression program Enzfitter (Elsevier/Biosoft). All reported values are expressed as the mean ± S.E. of at least three separate experiments. (2R,3R)-2,3-Butanediol dehydrogenase (BDH) was purified from yeast strain BJ5459(pYes2-YAL060W), and all the purification steps were carried out at 4 °C. The cells (34 g) were suspended in 34 ml of buffer A (20 mm potassium phosphate (pH 6.8) containing 30% glycerol and 0.5 mmdithiothreitol) and broken up with glass beads 0.5 mm in diameter. The lysate was centrifuged at 29,000 × g for 1 h, and the supernatant was applied to a hydroxylapatite Bio-Gel HT column (10.5 × 2 cm) equilibrated with buffer A. After washing the column with 250 ml of the same buffer, the enzyme was eluted with a linear gradient of 20–600 mm potassium phosphate (pH 6.8) containing 30% glycerol and 0.5 mm dithiothreitol in a 400-ml total volume. The active fractions were pooled; concentrated in an Amicon concentrator, which also served for changing the buffer to buffer A; and applied to a Cibacron Blue 3GA-agarose column (11.5 × 2.4 cm) equilibrated with buffer A. After washing the column with buffer (330 ml), a linear gradient of NADH (0–250 μm in 200 ml) was applied. After washing again with buffer A (185 ml), the enzyme was eluted with a linear gradient of 0–2m NaCl in 700 ml of buffer. The active fractions were pooled and applied to a Superdex 200 HR 10/30 gel filtration column equilibrated with 50 mm sodium phosphate (pH 7), 0.15m NaCl, and 30% glycerol. The column was eluted at a flow rate of 0.2 ml/min with the equilibration buffer. The purified enzyme was concentrated and stored at −80 °C. The relative mass of the native enzyme was determined by size exclusion chromatography at 22 °C on a Superdex 200 HR 10/30 column. The column was connected to a high performance liquid chromatography apparatus (Waters) and was equilibrated with 2 volumes of 50 mm sodium phosphate (pH 7), 0.15 m NaCl, and 30% glycerol. The column was run at a flow rate of 0.2 ml/min. The molecular mass was estimated by comparison with the elution of protein standards. The molecular mass was also determined by native gradient polyacrylamide gel electrophoresis, in which proteins were visualized by silver staining. The submolecular structure of the enzyme was studied under denaturing conditions by SDS-polyacrylamide gel electrophoresis (32Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207537) Google Scholar) and silver staining. Isoelectric focusing was performed according to a reported method (33Robertson E.F. Dannelly H.K. Malloy P.J. Reeves H.C. Anal. Biochem. 1987; 167: 290-294Crossref PubMed Scopus (366) Google Scholar). The pI of the YAL060W gene product was determined by comparison with known standards (isoelectric focusing calibration kit, Amersham Pharmacia Biotech). The BDH activity was visualized by incubating the gel at pH 8.6 with 0.5 m(2R,3R)-2,3-butanediol, 0.6 mmNAD+, 5-methylphenazine methosulfate (0.1 mg/ml), and nitro blue tetrazolium chloride (0.2 mg/ml). GLC analyses were performed in a Shimadzu GC-14B gas chromatograph equipped with a chiral column (Supelco β-DEXTM 120, 30-m length, 0.25-mm inner diameter), helium as the carrier gas (2.4 ml/min), and a flame ionization detector (275 °C). The following temperature program was used: isotherm at 75 °C for 8 min, 2 °C/min ramp to 85 °C, and isotherm at 85 °C. A standard mixture of 40 mm(3R/3S)-acetoin, 7.5 mm(2S,3S)-2,3-butanediol, 9.5 mm(2R,3R)-2,3-butanediol, and 7 mm meso-2,3-butanediol was prepared by dissolving the pure compounds in 30 mm sodium phosphate buffer (pH 7). One volume of the standard mixture was extracted with 1 volume of ethyl acetate, and 1 μl of the organic phase was applied to the chiral column. This extraction protocol was repeated three times, showing that the recovery of the compounds was >70% after the first extraction and that the relative percentages of the recovered compounds were similar for each extraction step. The reagents and products of the enzymatic reaction mixtures were extracted with ethyl acetate before the analysis by GLC. The search performed with the BLAST program found the following genes of unknown function that could be members of the NAD+-dependent, zinc-containing medium-chain alcohol dehydrogenases: YDL246C, which codes for a protein nearly identical (>99% identity) to the product of theSOR1 gene (sorbitol dehydrogenase), and YAL060Wand YAL061W, two adjacent genes classified as genes coding for proteins with similarity to alcohol/sorbitol dehydrogenases. Two other genes, YMR318C and YCR105W, also belong to the yeast MDR family, although they have been found to code for NADP(H)-dependent enzymes (see below). A multiple sequence alignment performed with these five gene products, together with the seven dehydrogenases of known function (mentioned in the Introduction), was used to generate a phylogenetic tree based on the neighbor-joining method of Saitou and Nei (34Saitou N. Nei M. Mol. Biol. Evol. 1987; 4: 406-425PubMed Google Scholar) with 1000 bootstraps. The topology of the tree with the zinc-containing MDR enzymes from yeast (Fig. 1 B) shows three main groups. Yeast glutathione-dependent formaldehyde dehydrogenase is in the first group, whereas the second group (formed by enzymes active with polyol substrates) is composed of two subgroups: BDH (the product of the YAL060W gene; active with 2,3-butanediol) and Yal061p (with 51% identity to BDH and of unknown function) are in the first subgroup, whereas xylitol dehydrogenase, sorbitol dehydrogenase, and Ydl246p (active with sugars) are in the second. The third group is composed of two subgroups: the first one clusters the ethanol-active enzymes ADHI, ADHII, ADHIII, and ADHV, whereas the second is composed of two NADP(H)-dependent cinnamyl-alcohol dehydrogenases (Ycr105p and Ymr318p, showing 64% identity) recently characterized in our laboratory. 2C. Larroy, M. R. Fernández, E. González, X. Parés and J. A. Biosca, manuscript in preparation. Among the several previously uncharacterized MDR enzymes from yeast, we describe in this work the results found with YAL060W.Figure 1Yeast BDH, encoded by the geneYAL060W, is a member of the MDR family. A, multiple sequence alignment between BDH and other members of the MDR family. The alignment was obtained using the program ClustalW, except at position 174 (according to the numbering of horse liver ADH), which has been introduced manually. Blackand gray boxes indicate residues that are identical in at least five of the seven sequences aligned or similar, respectively. Thesolid arrows mark the residues that bind to the catalytic zinc, and the open arrows mark the residues involved in the binding of the structural zinc. ScBDH, S. cerevisiae BDH; PpBDH, P. putida2,3-butanediol dehydrogenase; EcTDH, E. colithreonine dehydrogenase; HsSDH, Homo sapienssorbitol dehydrogenase; ScADHI, S. cerevisiaeADHI; ScFALDH, S. cerevisiaeglutathione-dependent formaldehyde dehydrogenase;HLADH, horse liver alcohol dehydrogenase Class I subunit E.B, unrooted phylogenetic tree relating the zinc-containing MDR enzymes (discussed under “Results”) from the yeastS. cerevisiae. The tree was generated from a multiple sequence alignment with the ClustalW Version 1.7 and TreeView Version 1.6.1 programs. Numbers show results from bootstrap analyses (1000 bootstrap replicates). XDH, xylitol dehydrogenase.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Sequencing demonstrated that the construct for overexpressing the YAL060W gene was correct. To easily follow the purification of the corresponding enzyme, we needed to find a specific substrate. The sequence of the enzyme was similar (>30% sequence identity) to different alcohol and sorbitol dehydrogenases. Thus, to avoid interferences with related activities, we used an alcohol dehydrogenase-deficient strain (WV36-405) to transform with a multicopy inducible vector carrying the YAL060W gene. The activity toward several alcohols was measured in the corresponding homogenate, whereas the same strain transformed with the same plasmid without insert served as a control. The best substrate for the overexpressed enzyme was (2R,3R)-2,3-butanediol. The (2R,3R)-2,3-butanediol dehydrogenase-specific activity of the homogenate of the WV36-405 strain carrying the expression vector pYes2-YAL060W was 130 times higher than that of the homogenate of the control strain (10.9 versus0.08 units/mg). The product from the YAL060W gene was therefore a 2,3-butanediol dehydrogenase, which was later confirmed by kinetic analysis (see below). We designated the enzyme BDH. When BDH was overexpressed in yeast strain WV36-405, we found that 30% of the activity of BDH was lost from the lysate fraction after 2 h at 4 °C (in 20 mm potassium phosphate (pH 6.8) with 5 mm dithiothreitol). The initial activity was retained, however, in the presence of the same extraction buffer containing 30% glycerol. We decided therefore to overexpress and purify the enzyme from a protease-deficient yeast strain (BJ5459) using 30% glycerol in the buffers of all the purification steps. In addition, we had to develop a rapid protocol to purify the enzyme. Essentially, the homogenate was fractionated with a hydroxylapatite column, followed by dye-ligand chromatography and gel filtration chromatography. Table I shows the results of a typical purification experiment starting with 34 g of BJ5459(pYes2-YAL060W) cells. A major step in purification was the dye-ligand chromatography. The efficiency of this step was due, in part, to the strong binding of BDH to the column. An NADH gradient did not elute the enzyme, but eliminated other dehydrogenases. High ionic strength was needed to elute BDH. After the gel filtration step, the resulting enzyme was homogeneous, as detected by a single band upon SDS-polyacrylamide gel electrophoresis and native polyacrylamide gel electrophoresis (Fig.2 C). This last chromatographic step was also important to eliminate NADH, which otherwise would inhibit the oxidative reactions. The increase in specific activity after the gel filtration chromatography (Table I) is mostly a consequence of the NADH elimination since the preparation was already free from extraneous proteins after the dye-ligand chromatography (Fig.2 C). The pure enzyme was stable when kept at −80 °C with 30% glycerol for >1 month.Table IPurification of yeast (2R,3R)-2,3-butanediol dehydrogenaseStepProteinTotal activitySpecific activityPurificationYieldmgunitsunits/mg-fold%Crude extract2185427251100Hydroxylapatite1537992531070Cibacron blue 3GA-agarose0.76493.3647269Gel filtration0.15148968392.7Activity was measured with 120 mm(2R,3R)-2,3-butanediol and 5 mm" @default.
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W2000502765 date "2000-11-01" @default.
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W2000502765 title "Characterization of a (2R,3R)-2,3-Butanediol Dehydrogenase as theSaccharomyces cerevisiae YAL060W Gene Product" @default.
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W2000502765 doi "https://doi.org/10.1074/jbc.m003035200" @default.
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