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Volume 270, Number 5, Issue of February 3, 1995 pp. 1983-1991
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A New Old Yellow Enzyme of Saccharomyces cerevisiae(*)

(Received for publication, August 26, 1994; and in revised form, October 18, 1994)

Yuko S. Niino (§), ,  Sumita Chakraborty,  Bette Jo Brown,  Vincent Massey (¶)

From the Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, Michigan 48109-0606

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In 1993, the first gene of Old Yellow Enzyme (OYE) of Saccharomyces cerevisiae was cloned (Stott, K., Saito, K., Thiele, D. J., and Massey, V.(1993) J. Biol. Chem. 268, 6097-6106) and named OYE2 to distinguish it from the first OYE gene cloned from Saccharomyces carlsbergenesis (Saito, K., Thiele, D. J., Davio, M., Lockridge, O., and Massey, V.(1991) J. Biol. Chem. 266, 20720-20724). The analysis of an OYE2 deletion mutant suggested that S. cerevisiae had at least two OYE genes. In the present study, we cloned a new OYE species named OYE3 and analyzed the OYE3 protein expressed in Escherichia coli. OYE3 consists of 400 amino acid residues and its molecular mass calculated by electrospray mass spectrometry is 44,788 daltons, in good agreement with the value of 44,920 daltons predicted from the amino acid sequence derived from the DNA sequence. In the downstream region of the OYE3 gene, the cytochrome oxidase (COX10) gene exists with a 426-base pair intermediate sequence. Some of the physicochemical and kinetic properties of OYE2 and OYE3 have been determined. Although the two enzymes are clearly closely related, they show differences in ligand binding properties and in their catalytic activities with oxygen and cyclohexen-2-one as acceptors.

INTRODUCTION

Old Yellow Enzyme (OYE) (^1)(NADPH oxidoreductase, EC. 1.6.99.1) was the first recognized flavoprotein and was purified from brewers' bottom yeast by Warburg and Christian in 1933(1) . Although the study of OYE has a long history, its physiological function is still unknown. OYE is known to catalyze the oxidation of beta-NADH, beta-NADPH, and alpha-NADPH and exists as a dimer with 1 molecule of FMN/subunit(2, 3, 4, 5, 6) .

Saito et al. cloned a gene encoding an isoform of OYE from Saccharomyces carlsbergenesis in 1991 (OYE1) (7) and from Saccharomyces cerevisiae in 1993 (OYE2) (8) . Both were composed of 400 amino acid residues and they have very similar molecular weights: 44890 and 44860, respectively. In order to help elucidate the function of OYE in S. cerevisiae, the OYE2 deletion strain DeltaOYE2 was constructed(8) . Surprisingly, DeltaOYE2 still had OYE activity(8) . Partial amino acid sequences of this new OYE-like protein have been reported (8, 9) . The amino acid sequences were very similar, but not identical, to those of OYE1 and OYE2. This suggested that S. cerevisiae has at least two OYE genes.

In the present study, we cloned and determined the DNA sequence of the new OYE gene (OYE3) from S. cerevisiae in order to help define the physiological function of OYE. The analysis of purified OYE3 expressed in E. coli showed many similarities with the properties of OYE2, but also distinctive differences.

EXPERIMENTAL PROCEDURES

Culture of Yeast and E. coli

The yeast strain S. cerevisiae, RZ49-1 (genotype: Mata, trp1, gal1-deletion, his3-532, ade3-52, CUP1R) was used throughout in this study. Yeast was grown in YPD medium (1% yeast extract, 2% Bacto-peptone, 2% dextrose) at 30 °C with constant shaking.

E. coli strains DH5alpha and XL-1 blue were used to maintain plasmids. Strain BL21 (DE3) was used for the expression of OYE. E. coli were grown in 2 times YT (1% yeast extract, 1.6% Bacto-tryptone, 0.5% NaCl) medium at 37 °C with constant shaking.

Polymerase Chain Reaction

To obtain a probe to screen for OYE3, we used the polymerase chain reaction (PCR) (10) with yeast genomic DNA, prepared from RZ49-1 (11) as a template. Two oligonucleotides based on published partial amino acid sequences(8, 9) were synthesized by the University of Michigan Molecular Biology Core Facility (see below for details). PCR was performed under the following conditions: 10 cycles of 94 °C, 1 min; 37 °C, 2 min; 72 °C, 3 min; and 25 cycles of 94 °C, 1 min; 50 °C, 2 min; 72 °C, 3 min. PCR products were cloned into plasmid pUC18, and DNA sequences were determined by the dideoxy chain termination method(12) .

Screening

A S. cerevisiae genomic library was constructed by size-fractionation of SacI-digested genomic DNA. Fractionated DNA (6-10 kb) was ligated to Zap II phage arms previously digested with SacI. Screening was performed by nucleic acid hybridization using probes prepared by PCR(13) . Probes were P-labeled by a random-primed DNA labeling kit (Boehringer Mannheim). Positive plaques were obtained by three rounds of screening and the insert DNA was subcloned from Zap phage to plasmid pBluescript SK(-) by in vivo excision with helper phage (Stratagene).

Southern Blot Analysis

Yeast genomic DNA was prepared by the method described by Sherman et al.(11) . DNA (4 µg) was digested by restriction enzymes and subjected to electrophoresis on 1% agarose gel. DNA was transferred to Hybond-N membrane (Amersham Corp.) by capillary transfer(13) . Hybridization was performed at 65 °C overnight with P-labeled probe DNA. A 1.2-kb DNA fragment of OYE2, which contains most of the coding region of OYE2, and the PCR product corresponding to OYE3 were used as probes. The final washing condition was 0.2 times SSC, 0.1% SDS, at 65 °C.

Expression Experiment

To construct the expression plasmid, we used pET3b, which is a plasmid having a T7 RNA polymerase promoter (14) . Since this plasmid has an NdeI site as a cloning site, we generated an NdeI site at the first methionine of the OYE3 gene with PCR. The following primer was synthesized by the University of Michigan Molecular Biology Core Facility: 5`-GTCGACGGTTTAAATTTAGCATATGCCATTTGTAAA-3`. The NdeI recognition site is underlined, and first methionine codon is in boldface font. After checking the DNA sequence of the PCR product, a 1.6-kb fragment which was partially digested by NdeI was ligated into pET3b (pETOYE3). OYE3 protein was induced by 0.4-1 mM IPTG in E. coli, BL21 (DE3).

SDS-PAGE

SDS-PAGE in a discontinuous Tris-glycine buffer system was used(15) . To determine the molecular mass, SDS-PAGE molecular size standards (high, low, and broad range) (Bio-Rad) were applied to the Laemmli system.

Materials

NADPH, cyclohex-2-enone, IPTG, PMSF, and xanthine were purchased from Sigma. Xanthine oxidase was prepared from cow's milk as described previously(16) .

Spectrophotometric Studies

Spectrophotometric analyses were carried out with Cary 219, Cary 3, or Hewlett-Packard diode array spectrophotometers, using temperature-controlled cuvette holders, typically in 0.1 M phosphate, pH 7.0, at 25 °C. Titrations of OYE with various ligands were carried out by recording the changes in the absorbance spectrum of the enzyme on addition of small volumes of ligand. Dissociation constants were determined from the ratio of bound to unbound ligand relative to the concentration of unbound enzyme, as determined from the relative extent of spectral perturbation.

Rapid-reaction Studies

The kinetics of reduction of enzyme by NADPH (the reductive half-reaction) and reoxidation (the oxidative half-reaction) were measured with a laboratory-made absorbance stopped flow spectrophotometer, interfaced to a Zenith data system computer, using a computer control and analysis system referred to as Program A (developed by Chung-Jen Chiu, Rong Chang, Joel Dinverno, and Dr. David P. Ballou, University of Michigan). This program allows the analyses of experimental data by exponential fits based on the Marquardt algorithm (17) .

Steady state kinetic data were also obtained in the stopped flow spectrophotometer by the enzyme monitored turnover method(18) .

Enzyme Purification

E. coli strain BL21(DE3) harboring the pET 3b expression vector was grown in 2 times YT medium at 37 °C for 11 h and induced with 0.4 mM IPTG. The cells were harvested 13 h after the induction and washed with buffer containing 40 mM TrisbulletHCl, pH 8.0, 10 mM MgCl(2), 10 mM dithiothreitol, 200 mM KCl, 10% glycerol, 1 mM PMSF and kept frozen at -20 °C. The cells were lysed by sonication. The rest of the purification was the same as that of the wild type enzyme isolated from brewers' bottom yeast(19) .

The concentration of the enzyme was determined from the UV-visible spectrum using = 10,600 M cm, and the enzyme was stored in 0.05 M KP(i), pH 7.0, employing 10 µM PMSF to minimize proteolysis.

RESULTS

Cloning and Sequencing Analysis of the OYE gene

Two primers which corresponded to amino acid sequences of OYE3 (RDTNLFEP and NLEHSIT) previously reported (8, 9) were used for PCR. The primers were 5`-CC GAATTCG(G,A,T,C)GA(T,C)AC(G,A,T,C)AA(T,C)TTATT(T,C)GA(G,A)CC-3`, which was sense to RDTNLFEP, and 5`-CGGAATTCTGT(G,A,T)AT(G,A)CT(G,A)TG(T,C)TC(G,A,T,C)AG(G,A)TT-3`, which was antisense to NLEHSIT. Both of them have an EcoRI linker in the 5`-ends (underlined). We got approximately 450-base pair PCR product using these primers. The PCR product was digested by EcoRI and subcloned into plasmid pUC18 to determine the DNA sequences. From the comparison between the amino acid sequence deduced from the DNA sequence of the PCR product and the partial amino acid sequences reported previously(8, 9) , it was shown that the PCR product was a part of the OYE3 gene. To evaluate the possibility of cross-hybridization of the gene of OYE2 and OYE3, we performed genomic Southern blot analysis using two different probes (Fig. 1). One probe was the HindIII-HpaI fragment of the OYE2 gene (1.2 kb) containing 98% of the coding region (8) and the other was the PCR product corresponding to OYE3. The pattern of genomic Southern blotting indicated that the genes of OYE2 and OYE3 did not cross-react with each other. We got the same data with low stringent washing conditions (45 °C) (data not shown).

Figure 1: Genomic Southern blot analysis. Four µg of yeast genomic DNA was digested by restriction enzymes as follows. Lane 1, BamHI; lane 2, EcoRI; lane 3, HindIII; lane 4, SacI; lane 5, SalI; lane 6, XbaI; lane 7, XhoI. The DNA probe used was the HindIII-HpaI fragment of the OYE2 gene (panel A) and the PCR product (panel B). Size markers are denoted at the right side.


Size-fractionated genomic DNA digested by SacI (6-10 kb) was ligated to ZapII phage previously digested by SacI to make a library. The library was screened with the PCR product as a probe. The restriction map, DNA sequence, and deduced amino acid sequence are shown in Fig. 2. The gene of OYE3 encoded 400 amino acid residues containing the first methionine (which is not present in the intact protein) like OYE1 and OYE2(7, 8) . Homology of the nucleotide sequence of the coding region between OYE1 and OYE2 was 85%, between OYE1 and OYE3 was 74%, and between OYE2 and OYE3 was 73%. Comparison of the DNA sequences of the coding region between OYE2 and OYE3 is shown in Fig. 3.


Figure 2: Restriction map, nucleotide sequence, and deduced amino acid sequences of OYE3 and COX10. A, restriction map of OYE3 and its flanking region. Openboxes indicate coding regions of OYE3 and a part of COX10. Restriction enzymes are: BI, BamHI; BII, BanII; E, EcoRI; HII, HincII; HIII, HindIII; K, KpnI; N, NdeI; P, PvuII; S, SacI; SBI, SnaBI. B, the DNA sequences between SnaBI and PvuII were determined (see panelA). The residue of the translational start codon (ATG) was positioned +1. The primers for PCR are indicated by doubleunderlines. The upperline is the nucleotide sequence, and the lowerline is the deduced amino acid sequence of OYE3 and a part of COX10. TATA and AATAAA sequences are singlyunderlined, and transcription termination signals are boxed. The first deduced amino acid sequence is that of OYE3, and the second is the N-terminal region of COX10(20) .


Figure 3: Comparison of DNA sequences of OYE2 and OYE3. The nucleotide sequences for the coding region of OYE2 and OYE3 are shown. The start methionine and translational stop codon are boxed. Asterisks indicate homologous nucleotides.


The DNA sequence of a short region upstream of the OYE3 open reading frame was determined. This sequence revealed the presence of two TATA sequences, which could be potential TATA boxes (Fig. 2B). In the sequence around the ATG start codon, there is an A at position -3 and a C at +5. They are conservative nucleotides in the leader region containing the sequence around the ATG start codon, which is relevant to translation in yeast(20, 21) . In the downstream region of the stop codon (TAG), consensus sequences for transcriptional termination in yeast (TAG . . . TAGT . . . TTT, TATGT . . . TTT, and TTTATA) have been detected(22, 23) . Furthermore, a common consensus sequence of the polyadenylation signal in eukaryotic cells, AATAAA, was found at +1211 and +1319 (Fig. 2B). However, it has been reported that the AATAAA sequence is not required for polyadenylation for some yeast genes(23) . The role of this sequence in the gene of OYE3 is unknown currently.

The yeast cytochrome oxidase (COX10) gene (24) was detected in the 3`-flanking sequence of OYE3 with a 426-base pair intermediate sequence.

Amino Acid Sequence of OYE

The amino acid sequences of the three cloned OYEs showed that OYE1 and OYE2 have 92% identity and 95% similarity, OYE1 and OYE3 have 80% identity and 87% similarity, and OYE2 and OYE3 have 82% identity and 89% similarity. The alignment of OYE1, OYE2, and OYE3 is shown in Fig. 4. The molecular masses determined by electrospray mass spectroscopy, performed by the University of Michigan Protein Sequencing Facility, were 44,890, 44,865, and 44,788 daltons, respectively.

Figure 4: Alignment of amino acid sequences of the three OYEs. Conserved amino acid residues among OYE1, OYE2, and OYE3 are indicated by shaded boxes.


In the present study, it was confirmed that there are at least two different OYEs in S. cerevisiae. However, the biological functions of these OYEs are still unknown. The amino acid composition of the three OYEs is shown in Table 1. Some interesting differences were found. Only OYE3 has cysteine residues. Although OYE1 and OYE2 have 26 lysines, OYE3 has only 18, and OYE3 has 21 threonine residues compared to OYE1 and OYE2, which have 16 each.

Expression of OYE3 in E. coli

To investigate the properties of OYE3, we constructed the OYE3 expression plasmid (pETOYE3) using the pET3b vector, which has a T7 RNA polymerase promoter. OYE3 induced by 1 mM IPTG in E. coli, BL21 (DE3), was loaded on SDS-PAGE with OYE2 (Fig. 5). The molecular masses calculated from amino acid sequences were 45010 daltons (OYE2) and 44,920 daltons (OYE3), in good agreement with those from mass spectroscopy: 44,865 daltons (OYE2) and 44,788 daltons (OYE3). Although SDS-PAGE is thought to separate proteins on the basis of molecular mass, the apparent molecular masses of OYE2 and OYE3 on SDS-PAGE were clearly different. Such anomalies have been reported for a change as small as a single amino acid substitution and could be a function of a change in the association of SDS with the protein(25) . Based on the deduced amino acid sequence and the electrospray mass spectroscopy results, the molecular masses of OYE2 and OYE3 are very similar.

Figure 5: SDS-PAGE of OYE1, OYE2, and OYE3. 1.2 µg of the expressed OYE1, 2 and 3 in E. coli was purified as described under ``Experimental Procedures'' and electrophoresed on 10% SDS-PAGE gel. Lane 1, OYE1; lane 2, OYE2; lane 3, OYE3; lane 4, molecular mass markers.


Charge Differences between OYE2 and OYE3

The FPLC elution profiles of OYE2 and OYE3 are quite different. While OYE2 elutes from an HR5/5 Mono-Q column at a relatively low NaCl concentration (105 ± 5 mM NaCl in the gradient system of (8) ), OYE3 requires a high NaCl concentration for elution (290 ± 10 mM). These results are similar to those reported previously for OYE2 and the protein DeltaOYE2, isolated from an OYE2 deletion mutant of S. cerevisiae strain R249-1 grown in YPD medium(8) . This is consistent with the isoelectric points calculated from the amino acid composition of 6.13 for OYE2 and 5.30 for OYE3.

Spectral Properties of OYE2 and OYE3

The absorbance spectra of the two OYE proteins differ slightly. OYE2 has wavelength maxima at 380 and 462 nm, while OYE3 has maxima at 384 and 464 nm. Both proteins exhibit the typical charge transfer spectra, characteristic of Old Yellow Enzyme on binding of phenolic compounds (Table 2). Again, small but distinctive differences are found between the two proteins, both with respect to the wavelength maximum of the charge transfer band and the K(d) for the association of ligand with the enzyme (Table 2). Consistently, the energy of the charge transfer transition, reflected in the wavelength maximum, is lower for OYE3 than it is for OYE2. In the case of OYE1, whose crystal structure has been determined, the phenolic ligands have been found to lie over the si-face of the flavin with the phenolate oxygen close to His-191(26) .

Steady State Turnover of OYE2 and OYE3 with beta-NADPH and Oxygen

Enzyme-monitored turnover experiments were done using a stopped flow spectrophotometer in which a known concentration of enzyme (typically 5 µM) in 20 mM KP(i), pH 7.0, was mixed with a limiting concentration of NADPH (typically 30 µM) in the same buffer, equilibrated with different concentrations of O(2) at 25 °C. The O(2) concentrations used were 0.160, 0.183, 0.256, 0.427, and 0.744 mM. The reaction was followed at 460 nm from approach to steady state (determined by the relative rates of reduction of the enzyme flavin by NADPH and reoxidation of the reduced enzyme by O(2)) until final reoxidation by the excess of O(2). Analysis of the data by the method of Gibson et al.(18) resulted in a series of Lineweaver-Burk plots, as found previously with wild type enzyme(4) . Replots of the intercepts versus 1/[oxygen] gave the kinetic constants listed in Table 3.

At most accessible concentrations of O(2), the observed NADPH-O(2) reductase activity of OYE3 is smaller than that of OYE2. This, however, is due to the very high K(m) for O(2) exhibited by OYE3. While the secondary plot of intercepts of Lineweaver-Burk plots for OYE2 give clearly defined values of k and K(m), those for OYE3 are almost directly proportional to the reciprocal of the oxygen concentration, making the exact determination of kinetic constants very difficult. The rationale for this phenomenon becomes obvious from data obtained by study of the separate reductive and oxidative half-reactions of the enzymes (see below).

Steady State Turnover of OYE2 and OYE3 with beta-NADPH and cyclohex-2-enone

In a previous paper(8) , OYE has been shown to employ cyclohexenone as an efficient electron acceptor in which the carbon-carbon double bond is reduced to form cyclohexanone. As is the case with O(2) as the acceptor, described in the previous section, there are also distinctive differences between OYE2 and OYE3 in the kinetics of the NADPH-cyclohexenone reductase activity. Cyclohexenone reoxidizes reduced enzyme very rapidly, making it difficult to perform enzyme-monitored turnover experiments of the type possible with O(2) as acceptor. Accordingly turnover data were collected mainly by reacting enzyme with mixtures of NADPH and cyclohexenone under anaerobic conditions in the stopped flow spectrophotometer, and monitoring NADPH oxidation at 340 nm. Linear Lineweaver-Burk plots of 1/vversus 1/NADPH were obtained, invariant with cyclohexenone concentration with OYE2, and giving a series of parallel lines with OYE3. Kinetic constants are summarized in Table 4.

Reductive Half-reaction of OYE2 and OYE3 with beta-NADPH

The kinetics of reduction of both enzymes by NADPH was followed under anaerobic conditions at pH 7.0, 25 °C, for comparison with steady state kinetic constants. Previous studies with wild type enzyme (4) had shown the existence of at least two oxidized flavin intermediates in the reduction of the enzyme flavin by NADPH. A minimal reaction sequence was determined.

In this sequence the initial complex between oxidized enzyme and NADPH was formed in the dead time of the stopped flow apparatus and was characterized by a perturbation of absorption spectrum, a phenomenon common with flavoproteins on binding ligands. The second oxidized enzymebulletNADPH complex is characterized by distinctive long wavelength absorbance and was formed at a rate of 21 s at 4 °C, with observed rate independent of NADPH concentration. The final observed phase was reduction of the flavin in this complex, accompanied by the major loss in absorbance in the 400-500 nm region, and in the loss of the long wavelength absorbance. The reduction was biphasic, with approximately 60% occurring at the rate of 1.2 s and the remainder at the rate of 0.28 s, both independent of the NADPH concentration, but showing a 9-12-fold H/D isotope effect with [4R-^2H]NADPH(4) .

The biphasic nature of the reduction step was indicative of the brewers' bottom yeast enzyme being a mixture of at least two proteins, as confirmed later by NMR studies(27) , high performance liquid chromatography studies(28) , and FPLC analysis and cloning and expression of one of the OYE genes of brewers' bottom yeast, OYE1(7) . Reductive half-reaction studies with recombinant enzyme, OYE1, carried out under the same conditions as in (4) , gave results quite consistent with this interpretation,^2 with formation of the long wavelength-absorbing intermediate occurring at a rate of 16 s and reduction at a single rate of 0.95 s.

Similar experiments carried out with OYE2 and OYE3, but at 25 °C, showed quite distinctive differences between the two proteins of S. cerevisiae. Both enzymes showed the formation of a Michaelis complex with NADPH preceding the formation of the long wavelength intermediate, as with the enzyme from brewers' bottom yeast. With OYE2, the first complex was formed extremely rapidly, as evident from the shift in absorbance spectrum found in the 3-ms dead time of the stopped flow instrument. The secondary formation of the long wavelength intermediate was experimentally detectable, but occurred at rates too fast to measure accurately (500-1000 s). The reduction of the enzyme flavin was quite slow, with an observed rate constant of 3.9 ± 0.1 s, independent of NADPH concentration (Fig. 6). With OYE3, on the other hand, there was no clearly defined dead time spectral change (Fig. 7A), and the formation of the long wavelength absorbing species was readily measurable, with k values changing with NADPH concentration. A double-reciprocal plot of these values gave a limiting rate of formation of this species of 200 s (Fig. 7B), with an initial slope-intercept value of 5 times 10M (Table 5). The subsequent reduction step, exemplified by the major absorbance change in the 400-500 nm region, and the loss of long wavelength absorbance, is significantly faster than with OYE2, with a limiting value of 18 ± 0.5 s. The observed pseudo-first order rate constant, k, for this step is slightly dependent on NADPH concentration, as shown in Fig. 7B. See ``Discussion'' for an interpretation of these results.

Figure 6: Reaction of OYE2 with NADPH. The line with solidcircles is that of 12.5 µM of enzyme, taken in the stopped flow spectrophotometer. The line with solidtriangles represents the absorbance at 10 ms after reaction with 100 µM NADPH. Greater than 90% of the absorbance change was obtained in the 3-ms dead time of the stopped flow instrument. The line marked by opencircles is that at the end of the reaction, obtained at all wavelengths with a rate constant of 3.9 ± 0.1 s. The same results were obtained with 50 and 200 µM NADPH. Conditions, 0.1 M phosphate, pH 7.0, 25 °C.


Figure 7: Reaction of OYE3 with NADPH. Panel A, spectra at various stages of the reaction carried out in 0.1 M phosphate, pH 7.0, 25 °C. Line without symbols, spectrum of oxidized enzyme (15.7 µM) before reaction. Solid triangles, estimated absorbance immediately after mixing with NADPH, obtained from the absorbance changes in the fast phase of the reaction, with the rate constants shown in panel B, adjusted for the 3-ms dead time of the stopped flow instrument. Open circles, absorbance at the end of the fast phase. Solid squares, absorbance at the end of the reaction, with the rate constants at all wavelengths those shown in panel B. Panel B, dependence of observed rate constants on the concentration of NADPH. Open circles, observed rate constants for the fast phase of the reaction (units shown on left-hand axis); solid circles, observed rate constants for the slow phase of reaction, the reduction of the enzyme-bound flavin (units shown on right-handaxis); crosses and opentriangles, rate constants for the fast and slow phases obtained by simulation of the experimental traces, employing the rate constants for k(1)-k(5) listed in Table 5.


Oxidative Half-reactions of Reduced OYE2 and OYE3 with O(2) and Cyclohexenone

Old Yellow Enzyme was reduced slowly in a tonometer under an atmosphere of argon using a xanthine/xanthine oxidase reducing system with a catalytic concentration of benzyl viologen as redox mediator (29) and loaded into the anaerobic stopped flow spectrophotometer. It was then reoxidized by mixing with buffer equilibrated with different concentrations of O(2), and the reaction monitored at different wavelengths. At all wavelengths in the range of 300-500 nm, approximately 90% of the reaction occurred in a single phase, and the observed pseudo-first order rate constant was found to be linearly proportional to the concentration of O(2). Thus there is no experimental evidence for a Michaelis complex of reduced enzyme and O(2); instead the reaction is adequately described by a slow second order rate constant, 2.4 times 10^3M s for OYE2 and 5.7 times 10^2M s for OYE3 (Table 5). The second slower phase was also dependent on oxygen concentration and appeared to be associated with the reoxidation of a small amount of the anionic flavin semiquinone form of the enzyme.

In similar experiments, carried out under anaerobic conditions, but with different concentrations of cyclohexenone, the observed rate of reoxidation showed clear saturation kinetics, indicating the formation of a Michaelis complex of reduced enzyme and cyclohexenone preceding oxidation. Analysis of the data according to Strickland et al.(30) yielded a limiting rate of 73 s and a K(d) of 1 times 10M for OYE2 and corresponding values of 20 s and 5 times 10M for OYE3. Thus it is clear that OYE3 is much more poorly equipped to employ cyclohexenone as an electron acceptor than is OYE2. The significance of the individual rate constants in the interpretation of steady state kinetics data will be considered further under ``Discussion.''

DISCUSSION

We report here the isolation and initial characterization of a new gene encoding an OYE isoform, named OYE3. It was confirmed that at least two OYEs (OYE2 and OYE3) existed in S. cerevisiae. Now we have the exact DNA and amino acid sequences of three OYEs: OYE1, OYE2, and OYE3. OYE1 was cloned from brewers' bottom yeast by Saito et al.(7) in 1991. Since brewers' bottom yeast is derived from yeast strains of S. cerevisiae, Saccharomyces bayanus, and Saccharomyces monacensis, it was not known which of the three strains was the origin of OYE1. OYE2 was then successfully cloned from S. cerevisiae by screening with OYE1 DNA as a probe(8) . In other words, the DNA sequences of OYE1 and OYE2 are similar enough to cross-hybridize each other. When OYE2 DNA was used as a probe, Southern blot analysis with the genomic DNA of the deletion OYE2 mutant showed that there was no positive band in the genomic DNA (data not shown). This indicates that while the first FPLC peak of OYE from brewers' bottom yeast is derived from S. cerevisiae, having an N-terminal amino acid sequence identical with that of OYE2(8) , the origin of OYE1 (corresponding to the third FPLC peak of brewers' bottom yeast) is not S. cerevisiae. Although the original strains from which they were cloned were not the same, the homology of DNA and amino acid sequences between OYE1 and OYE2 was higher than was that between OYE3 and OYE1 or OYE2. On the other hand, although the DNA sequence of OYE3 was 73% homologous to OYE2 (PCR products had 71% homology with OYE2), they did not cross-hybridize (Fig. 1). This indicates that while homology between the OYE2 and OYE3 genes is fairly high, it was not high enough to cause them to hybridize with each other. In their DNA sequences, there are some highly conserved sequences (550-668 and 982-1073 in Fig. 3) and non-conserved sequences (226-327 and 438-549 in Fig. 3). We speculate that those differences provide the reason why they could not cross-react.

Computer search of GenBank showed that the amino acid sequences of all three OYEs are highly homologous to the estrogen-binding protein of Candida albicans (EBP1). The EBP1 gene of this yeast was cloned recently by Madani et al.(31) . The derived amino acid sequence of EBP1 is 46% identical and 65% similar to OYE1, 47% identical and 66% similar to OYE2, and 47% identical and 66% similar to OYE3. It was reported that EBP1 overexpressed in yeast had oxidoreductase activity, particularly with cyclohexenone as electron acceptor(31) , which as already discussed was shown to be very reactive with OYE(8) . In their paper, an intense band and some less intense bands were found in the genomic Southern blotting with EBP probe. Since this result indicated that there were some related genes to the EBP1 gene in the genomic DNA, they named that gene EBP1. We suggest that there is a possibility that the less intense bands could be OYE genes in Candida albicans.

An estrogen-binding protein was found in S. cerevisiae by Feldman et al. in 1984(32) . Tanaka et al.(33) reported that in S. cerevisiae, estrogen stimulated recovery of growth of yeast cells from the G(0) phase and inhibited entry into the resting G(0) phase by increasing the intracellular cAMP level, suggesting that estrogen has a role in control of the cell cycle of yeast. These findings are made particularly intriguing by the discovery that OYE binds a wide variety of sterols, including estradiol, with considerable avidity ((34) , this paper). In addition, NADH oxidase(35) , bile acid-inducible operon protein C (36) and protein H(37) , and trimethylamine dehydrogenase (38) are also similar to OYE. At the amino acid level, NADH oxidase is 51% similar and 25% identical, protein C is 53% similar and 25% identical, protein H is 49% similar and 23% identical, and trimethylamine dehydrogenase is 49% similar and 25% identical to OYE3.

In the downstream region of the OYE3 locus, a cytochrome oxidase (COX10) gene was found. Nobrega et al.(24) have reported on the isolation and sequence of the COX10 gene. The intermediate DNA sequence between the OYE3 and COX10 genes is reported in the present study. This may be helpful in the study of transcriptional analysis of the COX10 gene.

The new member of the OYE family, OYE3, does not exist in S. cerevisiae in as great a quantity as OYE2. The main peak of OYE that we purified from wild type yeast on FPLC was OYE2. Although there was a small peak of heterodimer that seemed to consist of OYE2 and OYE3, there was no peak of OYE3(8) . To analyze the properties of OYE3, recombinant protein was prepared, and some of its physicochemical and kinetic properties are described in this paper and compared with those of OYE2. While the two proteins are quite similar, there are significant differences between them.

With respect to the traditional NADPH-oxygen reductase activity of OYE, under most attainable experimental conditions, OYE3 is less reactive than OYE2, despite the fact that it is reduced by NADPH faster than OYE2. The reason for this is the extremely slow reaction with O(2), 5.6 times 10^2M s, about 4 times slower than that with OYE2, 2.4 times 10^3M s. Previous studies with wild type enzyme from brewers' bottom yeast had shown that NADP dissociates rapidly from the reduced enzyme(4) . Hence the NADPH-oxygen oxidoreductase activity may be described by the following sequence, in which the species with the asterisk is the long wavelength-absorbing intermediate.

Using the method of net rate constants(39) , the initial rate equation for this sequence is easily solved, and yields the following expressions for the kinetic constants.

For both enzymes, k is approximately the same as k(5), which requires that k(4) be small, and k(3) > k(5), in agreement with the values of these rate constants derived from the stopped flow data (Table 5). Similarly, the K(m) for O(2) for both enzymes is fit well by the expression k / k. The values of K(m) for NADPH are harder to evaluate, since we have no valid measure of k(2)/k(1) in the case of OYE2. However, with OYE3, where we can measure the rate of formation of the long wavelength-absorbing species and its dependence on NADPH concentration (Fig. 7), we can obtain good fits of the reductive half-reaction data to simulations using the values of k(1)-k(5) shown in Table 5. These values would predict a K(m)(NADPH) of 7 µM, a value compatible with the observed results. The expression for K(m) (NADPH) requires that the K(d) of the primary binding step k(2)/k(1), be appreciably larger than the measured K(m) value. The low value of k has the consequence that this step becomes rate-limiting at most concentrations of O(2), thus accounting for the essentially linear dependence of rate on the O(2) concentration.

The NADPH-cyclohexenone reductase activity is described by the following sequence.

Solving for initial rate conditions(39) , the following expressions for the kinetic constants are obtained.

For both OYE2 and OYE3, the observed steady state kinetic constants are in good agreement with the individual rate constants obtained by stopped flow measurements, with a rapid equilibrium binding of cyclohexenone to the reduced enzyme, with the K(d) values shown in Table 5. The K(m) values for NADPH and cyclohexenone are lower than the corresponding K(d) values because the denominator is larger than the numerator for both constants. Again, the major difference between the two enzymes is the weaker affinity of cyclohexenone to OYE3 than to OYE2.

FOOTNOTES

*
This work was supported in part by Grant GM 11106 from the United States Public Health Service (to V. M.) and Grant M01RR00042 from the General Clinical Research Center at the University of Michigan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L29279[GenBank].

§
Present address: Division of Biochemistry and Cell Biology, National Institute of Neuroscience, NCNP, 4-1-1, Ogawahigashi, Kodaira-shi, Tokyo 187, Japan.

To whom correspondence should be addressed.

(^1)
The abbreviations used are: OYE, Old Yellow Enzyme; IPTG, isopropyl-1-thio-beta-D-galactopyranoside; PAGE, polyacrylamide gel electrophoresis; PMSF, phenylmethylsulfonyl fluoride; PCR, polymerase chain reaction; EBP, estrogen-binding protein; FPLC, fast protein liquid chromatography; kb, kilobase pair(s).

(^2)
K. Saito, S. Chakraborty, and V. Massey, unpublished results.

ACKNOWLEDGEMENTS

We are indebted to Dr. Alexander Tzagaloff for alerting us to the existence of the COX10 gene downstream of the OYE3 gene, and to Dr. Dennis J. Thiele for valuable advice and discussion.

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