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J. Biol. Chem., Vol. 275, Issue 46, 35876-35885, November 17, 2000

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Characterization of a (2R,3R)-2,3-Butanediol Dehydrogenase as the Saccharomyces cerevisiae YAL060W Gene Product

DISRUPTION AND INDUCTION OF THE GENE^*

Eva González, M. Rosario Fernández, Carol Larroy, Lluís Solà^§, Miquel A. Pericàs^§, Xavier Parés^¶, and Josep A. Biosca

From the  Department of Biochemistry and Molecular Biology, Faculty of Sciences, Universitat Autònoma de Barcelona, E-08193 Bellaterra (Barcelona) and the ^§ Unitat de Recerca en Síntesi Asimètrica, Departament de Química Orgànica, Universitat de Barcelona, E-08028 Barcelona, Spain

Received for publication, April 11, 2000, and in revised form, July 28, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The completion of the Saccharomyces 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 min¹) and meso-butanediol (K_m = 65 mM, k_cat = 46,000 min¹) 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 min¹) to (2R,3R)-2,3-butanediol and meso-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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

One of the tasks left after the completion of the various genome projects is to ascertain the function of the sequenced genes. When the Saccharomyces cerevisiae genome project was finished, it was found that ~60% of the potential open reading frames of the genome had no defined function (1). 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 (2). 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 (3, 4). 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 (5). 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 (6-8). ADH3 codes for ADHIII, the mature form of which is located in mitochondria (9) and which is also repressed by glucose. ADH5 codes for an ADH with a 76% sequence identity to the ADHI isozyme (10). SFA1 encodes the glutathione-dependent formaldehyde dehydrogenase (class III alcohol dehydrogenase) (11-13), 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 (14). Finally, YLR070C has recently been shown to code for a xylitol dehydrogenase (15). The ADH4 gene (16) codes for ADHIV, which is considered a member of the "iron-activated" ADH family (17).

In this work, we have used a conserved sequence motif found in the Zn²⁺-containing medium-chain ADH (the zinc-containing ADH signature) (18-20) 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 and meso-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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials

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.

Search for Zinc-containing Alcohol Dehydrogenases in S. cerevisiae

The consensus pattern GHEXXGXXXXX(GA)XX(IVAC), found in the zinc-containing medium-chain ADH family (18-20), 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 (21). Multiple sequence alignments were generated using ClustalW Version 1.7 software (22) in combination with TreeView Version 1.6.1 (23) to study phylogenies.

Yeast and Bacterial Strains, Plasmids, and Media

Escherichia coli XL1-Blue (Stratagene, La Jolla, CA) was used for cloning procedures. The yeast ADH-deficient strain WV36-405 (MATa, ade2, ura3-52, trp1, adh1 , adh2, adh3, adh4::TRP1) (24), 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 the YAL060W gene product. Because of its low background in alcohol oxidation reactions, this strain is useful in ascertaining potentially new ADH genes. The yeast strain FY834 (MAT , his3200, ura3-52, leu21, lys2202, trp163) (25), used in the S. cerevisiae genome project, was used here to amplify the YAL060W 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 FY834). The protease-deficient yeast strain BJ5459 (MATa, ura3-52, trp1, lys2-801, leu21, his3200, pep4::HIS3, prb11.6R, can1, GAL) (26), generously provided by Dr. Benjamí Piña (Consejo Superior de Investigaciones Científicas, 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 of GAL1) 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, and meso-2,3-butanediol).

Subcloning Methods

All DNA manipulations were performed under standard conditions as described (27).

Amplification of YAL060W-- Yeast genomic DNA was isolated from yeast strain FY834 by standard methods (28), and the YAL060W 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 µM each primer, 200 µM each dNTP, and 2 mM MgSO₄.

Construction of pYes2-YAL060W-- To construct the YAL060W expression vector under the control of the GAL1 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.

Construction of Yeast Strains WV36-405(pYes2), WV36-405(pYes2-YAL060W), BJ5459(pYes2), and BJ5459(pYes2-YAL060W)-- 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. (29), and the transformants were selected on SC-Ura plates (28).

Disruption of the YAL060W Gene-- The disruption of the YAL060W gene was carried out by one-step gene replacement (30) with the TRP1 gene. The starting point was plasmid pYes2-YAL060W, containing the coding region of YAL060W 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 (31) with BamHI, was subcloned into the MluNI 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 FY834 by the lithium acetate method (29), and after homologous recombination, the coding region of YAL060W was disrupted with the TRP1 gene. The yeast cells that had incorporated the TRP1 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 Assays

Enzyme activities were determined spectrophotometrically by measuring the change in absorbance at 340 nm and 25 °C corresponding to the oxidation of NADH (₃₄₀ = 6220 M¹ cm¹) or the reduction of NAD⁺. The purification of the YAL060W 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.

Enzyme Purification

(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 mM dithiothreitol) 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 0-2 M 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), 0.15 M 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.

Molecular Mass Determinations

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 (32) and silver staining.

Other Electrophoretic Methods

Isoelectric focusing was performed according to a reported method (33). 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 mM NAD⁺, 5-methylphenazine methosulfate (0.1 mg/ml), and nitro blue tetrazolium chloride (0.2 mg/ml).

Gas-Liquid Chromatography

GLC analyses were performed in a Shimadzu GC-14B gas chromatograph equipped with a chiral column (Supelco -DEX^TM 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.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Search for Zinc-containing Alcohol Dehydrogenases in S. cerevisiae-- 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 the SOR1 gene (sorbitol dehydrogenase), and YAL060W and 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 (34) with 1000 bootstraps. The topology of the tree with the zinc-containing MDR enzymes from yeast (Fig. 1B) 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.² Among the several previously uncharacterized MDR enzymes from yeast, we describe in this work the results found with YAL060W.

View larger version (105K): [in this window] [in a new window]   Fig. 1.   Yeast BDH, encoded by the gene YAL060W, 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. Black and gray boxes indicate residues that are identical in at least five of the seven sequences aligned or similar, respectively. The solid 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. putida 2,3-butanediol dehydrogenase; EcTDH, E. coli threonine dehydrogenase; HsSDH, Homo sapiens sorbitol dehydrogenase; ScADHI, S. cerevisiae ADHI; ScFALDH, S. cerevisiae glutathione-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 yeast S. 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.

Homologous Overexpression and Purification of the YAL060W Gene Product-- 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 versus 0.08 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. 2C). 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. 2C). The pure enzyme was stable when kept at 80 °C with 30% glycerol for >1 month.

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Determination of the molecular properties of yeast (2R,3R)-2,3-butanediol dehydrogenase. A, size exclusion chromatography on a Superdex 200 HR 10/30 column. a, cytochrome c (12.4 kDa); b, carbonic anhydrase (29 kDa); c, bovine serum albumin (66 kDa); d, yeast alcohol dehydrogenase (150 kDa); e, -amylase (200 kDa); p, BDH. B, native gradient polyacrylamide gel (8-25%) stained with silver salts. Lane 1, 0.4 µg of catalase (220 kDa); lane 2, 0.4 µg of yeast glutathione-dependent formaldehyde dehydrogenase (80 kDa); lane 3, 0.4 µg of ovalbumin (43 kDa); lanes 4 and 5, 0.07 µg of BDH. C, SDS-polyacrylamide gel electrophoresis of the active fractions obtained after each purification step of yeast BDH. The proteins were revealed by silver staining. Lanes 1 and 5, molecular mass standards; lane 2, crude extract (10 µg of protein); lane 3, hydroxylapatite chromatography (5 µg); lane 4, Cibacron blue 3GA-agarose chromatography (1 µg).

Molecular Mass of BDH-- Gel filtration of the pure enzyme on a Superdex 200 HR 10/30 column showed a molecular mass of ~81,800 Da (Fig. 2A). When a sample of the pure enzyme was loaded on a native polyacrylamide gel and subjected to electrophoresis, a mobility pattern very similar to the one shown by pure yeast glutathione-dependent formaldehyde dehydrogenase was obtained (Fig. 2B). Given the close isoelectric points of BDH (pI 6.9) and yeast glutathione-dependent formaldehyde dehydrogenase (pI 6.7) (35) and the known molecular mass (80,000 Da) of yeast glutathione-dependent formaldehyde dehydrogenase, this technique also supports a molecular mass of ~80,000 Da for the native BDH. Analysis by SDS-polyacrylamide gel electrophoresis showed a protein band of ~41,000 Da (Fig. 2C), consistent with the predicted molecular mass of the BDH protein sequence (41,530 Da). We conclude that the native BDH is a homodimer composed of two subunits with molecular masses of 41,000 Da.

Substrate Specificity and Kinetic Properties-- BDH catalyzed the oxidation of several 1,2- and 2,3-diols (Table II), showing a higher activity for secondary alcohols (such as 2,3-butanediol) than for primary alcohols (such as 1,2-butanediol), with a maximum of activity toward (2R,3R)-2,3-butanediol. (2S,3S)-2,3-Butanediol was neither a substrate of the enzyme nor an inhibitor for the oxidation of (2R,3R)-2,3-butanediol. (100 mM (2S,3S)-2,3-butanediol did not inhibit the oxidation of 15 mM (2R,3R)-2,3-butanediol.) BDH could oxidize meso-2,3-butanediol, although showing less activity than with the 2R,3R-isomer. The enzyme could not oxidize (3R/3S)-acetoin (a racemic mixture of (3R)- and (3S)-3-hydroxy-2-butanone), glycerol, sorbitol, or xylitol.

Either -diketo groups or vicinal hydroxyketo functions were required as structural elements of the compounds that were substrates for the reduction reaction. Thus, (3R/3S)-acetoin was the best substrate in the reduction reaction, followed by diacetyl (2,3-butanedione). Other substrates were hydroxyacetone, methylglyoxal, dihydroxyacetone, and 2,3-pentanedione. As in the oxidation reactions, the four-carbon length substrates were also preferred in the reduction reactions.

The enzyme specifically required NAD(H), which could not be substituted by NADP(H). Thus, the activity displayed with NADP(H) was <1% of the activity with NAD(H) (at 5 mM oxidized cofactors and 1 mM reduced cofactors) with (2R,3R)-2,3-butanediol and (3R/3S)-acetoin, respectively.

The rate of the enzymatic reactions was affected by the pH of the assay buffer. The pH optima for the oxidation of 10 mM (2R,3R)-2,3-butanediol and for the reduction of 10 mM (3R/3S)-acetoin were 8 and 7, respectively. We tried to determine the kinetic parameters for all the best substrates assayed for BDH (Table II). However, only (2R,3R)-2,3-butanediol, meso-2,3-butanediol, and 1,2-butanediol for the oxidation and (3R/3S)-acetoin for the reduction saturated the enzyme (Table III). The catalytic efficiency constant (k_cat/K_m) was greater for the reduction of (3R/3S)-acetoin than for the oxidation of (2R,3R)-2,3-butanediol. Moreover, the K_m for NADH was 10-fold lower than that for NAD. Therefore, the enzyme would preferentially function as a reductase rather than as a dehydrogenase.

Stereospecificity of BDH-- Some experiments were carried out to unambiguously demonstrate the specificity of the enzyme in the oxidation-reduction processes of the 2,3-butanediol/acetoin interconversion. In the first place, we developed a GLC analytical system able to efficiently separate substrates and products of the reaction. The retention times of the substrates and products were as follows: (3R)-acetoin, 9.9 min; (3S)-acetoin, 10.6 min; (2S,3S)-2,3-butanediol, 26.6 min; (2R,3R)-2,3-butanediol, 27.9 min; and meso-2,3-butanediol, 31.1 min (Fig. 3A). With respect to the oxidation reaction, when meso-2,3-butanediol was incubated in the presence of BDH and NAD⁺ (Fig. 3C), (3S)-acetoin was the only product detected. This acetoin resulted from the selective oxidation of the alcohol function at the R-carbon of the meso-diol. No (3R)-acetoin was detected, which would arise from the oxidation of the S-carbon, confirming the extremely high specificity (>99.9% enantiomeric excess) of the enzyme for carbons possessing an R-configuration. On the other hand, when (2R,3R)-2,3-butanediol was treated under the same reaction conditions (Fig. 3B), (3R)-acetoin was, as expected, the only product obtained. Moreover, the absence of (3S)-acetoin among the products of this reaction provides additional information: no racemization occurred at all under the employed experimental conditions. Concerning the reduction process, when a racemic mixture of (3R/3S)-acetoin was incubated in the presence of BDH and NADH (Fig. 3D), a completely enantioselective reduction of the carbonyl groups leading to an R-alcohol took place. Thus, a product mixture of (2R,3R)-2,3-butanediol (arising from (3R)-acetoin) and meso-2,3-butanediol (arising from (3S)-acetoin) was obtained. Although both acetoins were substrates for the enzyme, the R-isomer was considerably more reactive since the final reaction mixture was enriched in the (3S)-acetoin (Fig. 3D) as compared with the initially identical concentrations of both isomers. Controls, performed for all experiments at identical conditions, but without BDH, showed no significant conversion (<3%) to the corresponding products.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   GLC analyses of the substrates and products of reactions catalyzed by (2R,3R)-2,3-butanediol dehydrogenase. A, 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 in 100 mM sodium phosphate (pH 7) was extracted with ethyl acetate, and 1 µl of the organic phase was inoculated in the GLC apparatus. B-D, shown are the products after the reaction catalyzed by 2 units of BDH with the following initial mixtures prepared at 40 mM concentrations: (2R,3R)-2,3-butanediol and NAD+ (B), meso-2,3-butanediol and NAD+ (C), and (3R/3S)-acetoin and NADH (D). All starting mixtures, including the standard, were dissolved in 100 mM sodium phosphate (pH 7) and were allowed to proceed for 24 h at room temperature. The final mixtures were extracted with ethyl acetate and analyzed by GLC as described under "Experimental Procedures."

Growth on 2,3-Butanediol-- The wild-type FY834 yeast strain could grow on 2,3-butanediol (mixture of 2R,3R-, 2S,3S-, and meso-isomers) as the sole energy and carbon source, with growth rates of 0.06 and 0.05 h¹ at 3 and 0.5% 2,3-butanediol, respectively. The extract of FY834 cells harvested at its late exponential phase had specific activities for the oxidation of (2R,3R)-2,3-butanediol of 0.25 units/mg of protein for a culture grown on 2% glucose and of 0.93 units/mg for a culture grown on 3% 2,3-butanediol. When these extracts were analyzed on an isoelectric focusing gel and stained by activity with (2R,3R)-2,3-butanediol and NAD⁺, a clear induction of the enzyme was observed (Fig. 4). The maximum cell densities reached with the FY834 yeast strain were 1.2 × 10⁸ cells/ml when it was grown on 2% glucose and 1.2 × 10⁷ cells/ml when it was grown on 2,3-butanediol.

View larger version (97K): [in this window] [in a new window]   Fig. 4.   Isoelectric focusing analysis (pH 3-9) of purified BDH and extracts of yeast strains grown on different carbon sources. Lane 1, homogenate of FY834 cells (38 µg of protein) grown on 2% glucose (0.25 units/mg of protein); lanes 2 and 3, homogenates of FY834 cells (48 µg of protein) grown on 3% 2,3-butanediol (mixture of isomers; 0.93 units/mg of protein); lane 4, 0.02 µg of purified BDH (968 units/mg of protein); lane 5, homogenate of yeast cells overexpressing BDH (WV36-405(pYes2-YAL060W); 48.3 µg of protein) grown on 2% galactose (10.9 units/mg of protein). The cells were harvested in the mid-exponential phase, and the crude extracts were obtained by agitation with glass beads. Activity staining was performed with (2R,3R)-2,3-butanediol.

Disruption of the YAL060W Gene-- The disruption of the YAL060W gene was confirmed by PCR of genomic DNA from the transformed strains (Fig. 5) and by isoelectric focusing analysis and measurement of the (2R,3R)-2,3-butanediol dehydrogenase activity of the corresponding yeast extracts. Thus, the size of the PCR band resulting from the selective amplification of the genomic YAL060W locus with two primers that hybridize in the coding region of YAL060W was longer in the disrupted strain than in the wild type. This is due to the fact that the size of the TRP1 gene introduced is longer than the 360-base pair fragment eliminated from the coding region of YAL060W (Fig. 5). Moreover, isoelectric focusing analysis showed no (2R,3R)-2,3-butanediol dehydrogenase activity band in the disrupted strains as opposed to the one shown by the wild-type strain (data not shown). The disruption of the gene was not lethal for S. cerevisiae, although the cell density attained in the stationary phase by the disrupted strain grown on 2,3-butanediol (mixture of isomers) was half of the density attained by the wild-type strain.

View larger version (106K): [in this window] [in a new window]   Fig. 5.   Analysis of the disruption of the YAL060W gene from S. cerevisiae. Shown are the results from the agarose gel electrophoresis (0.7%) of genomic DNA from the FY834, EG2, and EG3 yeast strains (isogenic to FY834, except with yal060w::TRP1) amplified with two oligonucleotides that hybridize to YAL060W. Lane 1, molecular size standards (shown in kilobases (kb)); lane 2, PCR fragment obtained by the amplification of the YAL060W gene from FY834; lanes 3 and 4, PCR fragments obtained by the amplification of the yal060w-disrupted gene (yal060w::TRP1) from EG2 and EG3.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The YAL060W gene product, BDH, exhibits the characteristic properties of an MDR enzyme (Fig. 1). Thus, as indicated in the multiple alignment of Fig. 1A, it shows a suitable chain length and has 13 amino acids highly conserved in the MDR family. These are distributed in three clusters: one in the substrate-binding domain (Gly⁶⁶, Gly⁷¹, Gly⁷⁷, Gly⁸⁶, and Val⁸⁰); another in the coenzyme-binding domain (Gly¹⁹², Gly²⁰¹, Gly²⁰⁴, and Gly²³⁶), with numbering according to the numbering of horse liver ADH); and a third involved in the binding of the catalytic zinc (Cys⁴⁶ and His⁶⁷, putative ligands of the catalytic zinc, and Asp⁴⁹ and Glu⁶⁸, which are considered "second sphere ligands," interacting with the zinc ligands themselves). We believe that the third ligand of the catalytic zinc in BDH would be Glu¹⁷⁴. Thus, it can be aligned with Glu¹⁷⁴ from mammalian sorbitol dehydrogenase (which is known to be a ligand of the catalytic zinc) (36) and also with Glu¹⁷⁴ from the Pseudomonas putida 2,3-butanediol dehydrogenase, where it has also been assigned as the third ligand for the catalytic zinc (37). It is also aligned with Asp¹⁷⁴ in E. coli threonine dehydrogenase (38) and with Cys¹⁷⁴ in S. cerevisiae ADHI, S. cerevisiae FALDH, and horse liver ADH, which are the known ligands occupying the third position in the coordination sphere of the catalytic zinc in these enzymes (20).

There are two more relevant residues conserved in the alignment: Ser⁴⁸ and Glu²²³. Ser⁴⁸ is aligned with Ser/Thr⁴⁸ in the other medium-chain ADHs and could function in a proton relay system to facilitate removal of the proton from the alcohol (39). Glu²²³, like Asp²²³ of the other members of the medium-chain ADH family, would interact with the 2'- and 3'-hydroxyl groups of the adenosine ribose of the coenzyme and would importantly contribute to the discrimination between NAD(H) and NADP(H) (40-42). The present BDH and P. putida BDH are the first examples of NAD(H)-dependent medium-chain ADHs with Glu²²³ instead of Asp²²³. The Glu²²³ change, a residue bigger than Asp and therefore imposing additional steric hindrance to an extra phosphate, could contribute to the stronger NAD⁺ specificity of BDH (practically without activity with NADP) as compared with other MDR enzymes that, in general, have some activity with NADP(H).

Whereas several members of the zinc-containing medium-chain ADH family have two Zn²⁺ ions/subunit, others, such as mammalian sorbitol dehydrogenase, have only one (36). Although the alignment of BDH with the members of the medium-chain ADH family is reasonably good for the ligands of the catalytic zinc, it is more speculative in regard to the ligands for the so-called "structural zinc." The ligands for the structural zinc in the medium-chain ADHs are four cysteines located within a short-segment region. In the case of BDH, there are four cysteines aligned with the cysteines that typically act as the zinc ligands in medium-chain ADHs (Cys⁹⁷, Cys¹⁰⁰, Cys¹⁰³, and Cys¹¹¹). It is possible, then, that BDH could bind a second zinc, although the long insertion existing between Cys⁹⁷ and Cys¹⁰⁰ may interfere with the binding.

The phylogenetic tree generated from a multiple sequence alignment of 12 MDR enzymes from S. cerevisiae containing the "zinc ADH signature" resulted in five subgroups. Whereas the clustering of the polyol MDR enzymes (xylitol dehydrogenase, sorbitol dehydrogenase, and its close homolog Yal246p in one subgroup and BDH and Yal061p in the other) is well supported (>95% bootstraps), the grouping of the two other clusters (the one comprising the ethanol-active enzymes (ADHI, ADHII, ADHIII, and ADHV) and the one with the putative cinnamyl-alcohol dehydrogenases (Ycr105p and Ymr318p)) is supported by only 63% of the bootstraps. Ycr105p has already been described as an MDR enzyme in a recent report (43).

The S. cerevisiae YAL060W gene product, BDH, catalyzes the NAD⁺-dependent oxidation of (2R,3R)-2,3-butanediol and meso-2,3-butanediol to (3R)-acetoin and (3S)-acetoin, respectively, as well as the corresponding reverse reactions. A chiral column that could resolve the three 2,3-butanediol isomers and the two acetoin isomers was used to demonstrate the stereospecificity of the enzyme. The GLC profiles indicated that the oxidation of meso-2,3-butanediol yielded (3S)-acetoin (showing the specificity toward the secondary alcohol in R-configuration), whereas the reduction of (3R/3S)-acetoin yielded (2R,3R)-2,3-butanediol (from (3R)-acetoin) and meso-2,3-butanediol (from (3S)-acetoin). This demonstrated that the reduction of the carbonyl from acetoin yielded also the R-configuration of the corresponding alcohol. It has also been shown that the rate of the reduction of the carbonyl depends on the configuration of the vicinal alcohol, being greater if it is in R-configuration than in S-configuration. This is also true for the oxidation reaction since the k_cat value for the oxidation of meso-2,3-butanediol is approximately two-thirds of the value found for the oxidation of (2R,3R)-2,3-butanediol. As many other 2,3-butanediol dehydrogenases, BDH can also reduce diacetyl to acetoin.

Other 2,3-butanediol dehydrogenases have been partially characterized in S. cerevisiae (44), Candida utilis (45, 46), and Candida salmanticensis (45), although their properties are different from those of the present enzyme. Thus, the molecular mass of the enzyme from S. cerevisiae is 140,000 Da, with a subunit molecular mass of 35,000 Da (44), whereas the molecular mass of the enzyme studied here is 81,800 Da, with a subunit molecular mass of 41,000 Da. Also, the specific activity given by the pure (2R,3R)-2,3-butanediol dehydrogenase previously isolated from S. cerevisiae was 20.25 units/mg, but was >900 units/mg in the present work (measured under similar conditions). Also, the present kinetic constants differ from those in the previous report. The specific activities and the kinetic constants of the enzymes with 2,3-butanediol dehydrogenase activity from C. utilis (45, 46) were also different from those of our enzyme. Moreover, the lack of sequence information for those enzymes precludes a complete comparison with the present BDH.

Most prokaryotic 2,3-butanediol dehydrogenases characterized so far in terms of their stereospecificity toward the different 2,3-butanediol isomers and corresponding acetoins (such as the ones from Brevibacterium saccharolyticum, Klebsiella terrigena, and Klebsiella pneumoniae (47-49)) have characteristics of the SDR family of enzymes. Only one known example in prokaryotes, the BDH from P. putida, corresponds to an MDR enzyme (37). It shows 36.5% identity (and 45.6% similarity) to BDH (Fig. 1 A), which represents the highest homology found for BDH to any related enzyme. Little is known of enzymes with BDH specificity in eukaryotes. The only reported eukaryotic BDH, a bovine butanediol dehydrogenase similar to that from K. terrigena, also belongs to the SDR family (50). Therefore, the present enzyme from S. cerevisiae, BDH, would be the first characterized eukaryotic MDR with demonstrated stereospecificity toward the 2,3-butanediol isomers (and corresponding acetoins).

During normal alcoholic fermentation from glucose, yeast produces 2,3-butanediol at a concentration of ~1 mM (51). This compound derives from the reduction of the physiological intermediates acetoin (3-hydroxy-2-butanone) and diacetyl (2,3-butanedione) (52) and constitutes a mixture of ~67% (2R,3R)-()-2,3-butanediol and 33% meso-2,3-butanediol (51), although a small percentage of (2S,3S)-2,3-butanediol cannot be excluded. There is general agreement that acetoin, the precursor of 2,3-butanediol, is produced by yeast at least by three different mechanisms (53-56) (Fig. 6): 1) by the action of pyruvate decarboxylase, which catalyzes an aldol-type condensation reaction between two molecules of acetaldehyde; 2) by the addition of acetaldehyde to an intermediate formed between pyruvate and thiamin pyrophosphate in a reaction also catalyzed by pyruvate decarboxylase; and 3) by the action of -acetolactate synthase on pyruvate, yielding -acetolactate, which is decarboxylated to diacetyl and reduced to acetoin. Although it has been reported that S. cerevisiae does not have -acetolactate decarboxylase, it still can produce diacetyl from -acetolactate by spontaneous oxidative decarboxylation (57, 58). Moreover, the yeast has at least two NADPH-dependent diacetyl reductases, which can reduce diacetyl to (3R)-acetoin and (3S)-acetoin (59).

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Metabolic pathway for the formation of acetoin and 2,3-butanediol in yeast. Acetoin can be produced from acetaldehyde and pyruvate by the action of the thiamin pyrophosphate-dependent enzyme, pyruvate decarboxylase (PDC), located in the cytosol. It can also be formed by the reduction of diacetyl, arising from the spontaneous decarboxylation (broken arrow) of -acetolactate produced by the mitochondrial enzyme -acetolactate synthase (ALS). The diacetyl reduction reaction can be catalyzed by the NAD(H)-dependent enzyme BDH and by two NADP(H)-dependent diacetyl reductases (not shown in the pathway). 2,3-Butanediol arises from the reduction of acetoin by BDH.

In P. putida, the gene coding for 2,3-butanediol dehydrogenase is part of the acetoin dehydrogenase operon that allows this bacterium to grow on 2,3-butanediol (37). S. cerevisiae can also grow on 2,3-butanediol as the sole carbon and energy source, and the yeast shows a >3-fold increase in BDH content when it grows in the presence of this compound. These data suggest that the enzyme reported here is needed in both, the fermentation giving 2,3-butanediol from acetoin, and under oxidation conditions allowing the yeast to use 2,3-butanediol as a carbon and energy source.

The fact that the strain with the YAL060W gene disrupted could still grow on 2,3-butanediol (mixture of isomers) indicates that S. cerevisiae has other enzymes that can metabolize 2,3-butanediol. We are currently using the YAL060W-disrupted yeast strain to study the induction of other genes by 2,3-butanediol.

A potential use of BDH would be in the synthesis of chiral acetoinic pure compounds (the three 2,3-butanediol and two acetoin isomers) and other related compounds. An enzyme already reported in this regard is (2S,3S)-2,3-butanediol dehydrogenase from Bacillus stearothermophilus (60, 61), which has been used for the interconversion between (2S,3S)-2,3-butanediol and (3S)-acetoin and in the preparation of enantiomerically pure bicyclic octenols/octenones and heptenols/heptenones.

	FOOTNOTES

^* This work was supported by Grants PB98-0855 and PB96-1167 from the Dirección General de Enseñanza Superior e Investigación Científica and Grant BIO4-CT97-2123 from the Commission of the European Union.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel.: 34-93-581-3026; Fax: 34-93-581-1264; E-mail: xavier.pares@uab.es.

Published, JBC Papers in Press, August 9, 2000, DOI 10.1074/jbc.M003035200

² C. Larroy, M. R. Fernández, E. González, X. Parés and J. A. Biosca, manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: ADH, alcohol dehydrogenase; MDR, medium-chain dehydrogenase/reductase; SDR, short-chain dehydrogenase/reductase; PCR, polymerase chain reaction; BDH, (2R,3R)-2,3-butanediol dehydrogenase; GLC, gas-liquid chromatography.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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