(Received for publication, August 26, 1994; and in revised form, October 18, 1994)
From the
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.
Old Yellow Enzyme (OYE) ()(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
-NADH,
-NADPH, and
-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 OYE2 was constructed(8) . Surprisingly,
OYE2 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.
E. coli strains DH5 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
YT (1% yeast extract,
1.6% Bacto-tryptone, 0.5% NaCl) medium at 37 °C with constant
shaking.
Steady state kinetic data were also obtained in the stopped flow spectrophotometer by the enzyme monitored turnover method(18) .
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
, pH 7.0, employing 10 µM PMSF to
minimize proteolysis.
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.
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.
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.
At most accessible concentrations
of O, the observed NADPH-O
reductase activity
of OYE3 is smaller than that of OYE2. This, however, is due to the very
high K
for O
exhibited by OYE3. While
the secondary plot of intercepts of Lineweaver-Burk plots for OYE2 give
clearly defined values of k
and K
, 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).
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
enzymeNADPH 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-
H]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, 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
10
M (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-k
listed in Table 5.
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
of 1
10
M for OYE2 and corresponding
values of 20 s
and 5
10
M 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.''
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 phase and inhibited entry into the resting G
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, 5.6
10
M
s
, about 4 times
slower than that with OYE2, 2.4
10
M
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
, which requires that k
be small, and k
> k
,
in agreement with the values of these rate constants derived from the
stopped flow data (Table 5). Similarly, the K
for O
for both enzymes is fit well by the expression k
/ k
. The values of K
for NADPH are harder to evaluate, since we have
no valid measure of k
/k
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
-k
shown in Table 5. These values would predict a K
(NADPH) of 7 µM, a value compatible
with the observed results. The expression for K
(NADPH) requires that the K
of the primary
binding step k
/k
, be
appreciably larger than the measured K
value. The
low value of k
has the consequence that this step
becomes rate-limiting at most concentrations of O
, thus
accounting for the essentially linear dependence of rate on the O
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 values shown in Table 5. The K
values
for NADPH and cyclohexenone are lower than the corresponding K
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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L29279[GenBank].