(Received for publication, December 18, 1995)
From the
Protoporphyrinogen oxidase, which catalyzes the oxygen-dependent
aromatization of protoporphyrinogen IX to protoporphyrin IX, is the
molecular target of diphenyl ether type herbicides. The structural gene
for the yeast protoporphyrinogen oxidase, HEM14, was isolated
by functional complementation of a hem14-1 protoporphyrinogen
oxidase-deficient yeast mutant, using a novel one-step colored
screening procedure to identify heme-synthesizing cells. The hem14-1 mutation was genetically linked to URA3, a
marker on chromosome V, and HEM14 was physically mapped on the
right arm of this chromosome, between PRP22 and FAA2.
Disruption of the HEM14 gene leads to protoporphyrinogen
oxidase deficiency in vivo (heme deficiency and accumulation
of heme precursors), and in vitro (lack of immunodetectable
protein or enzyme activity). The HEM14 gene encodes a
539-amino acid protein (59,665 Da; pI 9.3) containing an
ADP--binding fold similar to those of several other
flavoproteins. Yeast protoporphyrinogen oxidase was somewhat similar to
the HemY gene product of Bacillus subtilis and to the
human and mouse protoporphyrinogen oxidases. Studies on
protoporphyrinogen oxidase overexpressed in yeast and purified as
wild-type enzyme showed that (i) the NH
-terminal
mitochondrial targeting sequence of protoporphyrinogen oxidase is not
cleaved during importation; (ii) the enzyme, as purified, had a typical
flavin semiquinone absorption spectrum; and (iii) the enzyme was
strongly inhibited by diphenyl ether-type herbicides and readily
photolabeled by a diazoketone derivative of tritiated acifluorfen. The
mutant allele hem14-1 contains two mutations, L422P and K424E,
responsible for the inactive enzyme. Both mutations introduced
independently in the wild-type HEM14 gene completely
inactivated the protein when analyzed in an Escherichia coli expression system.
Protoporphyrinogen oxidase (EC 1.3.3.4) is the penultimate
enzyme of the heme biosynthetic pathway. This membrane-bound enzyme
catalyzes the oxidative O-dependent aromatization of the
colorless protoporphyrinogen IX to the highly conjugated protoporphyrin
IX. The existence of an enzyme catalyzing this oxidative step has long
been controversial, since porphyrinogens are rapidly oxidized to their
corresponding porphyrins in the presence of air via a light-sensitive,
autocatalytic reaction(1) . However, biochemical and genetic
evidences now indicates the enzymatic nature of protoporphyrinogen
oxidation in living cells. Protoporphyrinogen oxidase deficiency has
been described in bacteria(2, 3, 4) ,
yeast(5) , and man, where a lack of protoporphyrinogen oxidase
activity is responsible for the inherited disease porphyria
variegata(6) . Studies on the structure and function of
protoporphyrinogen oxidase were recently stimulated by the discovery
that diphenyl ether-type herbicides are very potent inhibitors of the
protoporphyrinogen oxidase activities of yeast, mammalian, and plant
mitochondria and plant chloroplasts in
vitro(7, 8) . The phytotoxicity of diphenyl
ether-type herbicides is light-dependent and involves intracellular
peroxidations promoted by protoporphyrin IX, the heme and chlorophyll
precursor, leading to cell damage and lysis(9) , mimicking the
symptoms of human patients with porphyria variegata. Protoporphyrin IX
toxicity is due to the nonenzymatic oxidation of accumulated
protoporphyrinogen that diffused away from its site of synthesis and is
thus not further metabolized to metalloporphyrins by iron or magnesium
chelatases. However, the mechanism of protoporphyrin accumulation in
diphenyl ether-treated plants may also involve some nonspecific
porphyrinogen-oxidizing enzymes, such as
peroxidases(10, 11, 12) , that can oxidize
protoporphyrinogen and also coproporphyrinogen or uroporphyrinogen to
their corresponding porphyrins. These activities are insensitive to
diphenyl ether inhibition, but are markedly inhibited by reducing
agents such as dithiothreitol(11) . These nonspecific
protoporphyrinogen-oxidizing activities may give rise to some
discrepancies in the biochemical characterization of protoporphyrinogen
oxidase in various organisms that are quite surprising, since the
biochemical and structural properties of the enzymes of the heme
biosynthesis pathway are remarkably well conserved through evolution
(for a revue, see (13) ). In contrast, protoporphyrinogen
oxidases purified from various sources appear to be rather different in
terms of molecular mass, cofactor, and kinetic
properties(14, 15, 16, 17) ,
electron
acceptor(18, 19, 20, 21, 22) ,
and even subunit composition(20) . This biochemical diversity
seems to be also found at the genetic level. The HemG gene of Escherichia coli, located at 87 min on the genetic map of E. coli K12(4) , has been cloned and
sequenced(23) . This gene encodes a 181-amino acid protein (M
21,200) and complements a mutation affecting
the oxidation of protoporphyrinogen and heme synthesis in E. coli. It restores the protoporphyrinogen oxidase activity to 7-25
times that of a nontransformed wild-type strain when introduced into
the original mutant on a high copy number plasmid. However, a second
genetic locus, HemK, has been recently described as being involved in
protoporphyrinogen oxidation in E. coli(24) . This
gene is part of a HemA-PrfA-HemK operon and encodes a 225-amino acid
protein without homology to the HemG gene product, that may be a
component of an alternative protoporphyrinogen oxidation pathway. The
hemG mutant of E. coli was recently used to clone a human cDNA (25) and its murine homologue (26) that complement the
mutation. These cDNAs encode a 477-amino acid protein with
protoporphyrinogen oxidase activity and deduced sequences identical to
that of peptides obtained from partially purified bovine
protoporphyrinogen oxidase(26) . These mammalian
protoporphyrinogen oxidases have substantial domains that are similar
to the HemY gene product of Bacillus subtilis. HemY was
isolated by complementation of a mutation affecting heme synthesis in vivo by altering either coproporphyrinogen oxidase, or
protoporphyrinogen oxidase activities, or both(27) . This gene
encodes a 472-amino acid extrinsic membrane-bound polypeptide that,
when overexpressed in E. coli, is able to oxidize
protoporphyrinogen IX to protoporphyrin IX in vitro and, more
efficiently, coproporphyrinogen III to coproporphyrin
III(28, 29) , unlike any eukaryotic protoporphyrinogen
oxidase. The HemY gene product is not inhibited by diphenyl ether-type
herbicides (29) and thus resembles more the nonspecific
oxidases recently found in higher plants that may be involved in the
rapid accumulation of protoporphyrin in diphenyl ether-treated plants.
As part of our efforts to understand the relationship between structure and function of eukaryotic protoporphyrinogen oxidase and eventually to elucidate the molecular basis of protoporphyrinogen oxidase and hence the mechanisms underlying the pathophysiology associated with the enzyme defect (human porphyria) or inhibition (herbicidal effects of diphenyl ethers), we recently purified protoporphyrinogen oxidase from the yeast Saccharomyces cerevisiae. This integral protein of the inner mitochondrial membrane is a 55-kDa cationic FAD-containing flavoprotein synthesized as a high molecular mass precursor (58 kDa) that is rapidly converted to the mature membrane-bound form in vivo(30) . Antibodies raised against purified yeast protoporphyrinogen oxidase were used to characterize the enzyme in a heme-deficient mutant lacking protoporphyrinogen oxidase activity (5) carrying a single nuclear mutation hem14-1(31) . This mutant strain was shown to synthesize normal amounts of an inactive protein that did not bind a tritiated inhibitor(32) . We therefore undertook the cloning of the yeast gene by functional complementation of the hem14-1 gene defect. Like any heme-deficient yeast strain, the mutant cannot grow aerobically on nonfermentative carbon sources (ethanol, glycerol, or lactate), but grows via fermentative metabolism, provided the growth medium contains Tween 80 and ergosterol as precursors of unsatured fatty acids and sterols, whose syntheses are dependent on specific hemoproteins. The present study describes the isolation and nucleotide sequence of the protoporphyrinogen oxidase gene from the yeast S. cerevisiae, the phenotypic effects of its disruption and further characterization of the protoporphyrinogen oxidase protein overexpressed in yeast cells. The mutant hem14-1 allele was also sequenced, and normal and mutated protoporphyrinogen oxidases were analyzed in a E. coli expression system.
Plasmid pools of partial Sau3A digests of yeast chromosomal DNA ligated at the BamHI site of the high copy number vector YEpEMBL23 (34) or partial HindIII digests of yeast chromosomal DNA ligated at the HindIII site of the low copy number vector pRS316 (35) were kindly provided by Dr. D. Thomas (Laboratoire d'Enzymologie, CNRS, Gif sur Yvette, France).
The E. coli strains used were (i) DH5 (from Life Technologies, Inc.) for
cloning, maintenance, and propagation of plasmids; (ii) BL21(DE3) (from
Novagen) for expression studies; and (iii) XLmutS (from Stratagene) for
selection of altered plasmids in site-directed mutagenesis experiments.
They were grown in LB medium containing 100 µg/ml ampicillin when
necessary to maintain all of the Amp
-based plasmids.
The pBluescript KS(+) phagemid vector (Stratagene) was the cloning vehicle for all HEM14 containing fragments used for sequencing reactions. The pT7-5 vector was used for expression studies of HEM14 in E. coli and as the template for in vitro site-directed mutagenesis of the cloned HEM14 gene.
The overlaps of the HEM14 clone with
the PRP22 and ACS1 clones were recognized initially
by sequencing the 5` and 3` ends of the 4.9-kb ()insert of
plasmid pBS19-1 that complemented the hem14-1 mutation. The
restriction site patterns of the cloned insert matched those published
and prompted us to delete a 1.9-kb SalI-XhoI fragment
yielding the plasmid pBS19-1XS9 that complemented the hem14-1 mutation. This plasmid was used for expression studies of the
cloned protoporphyrinogen oxidase in yeast. The 3-kb XhoI-SmaI fragment of pBS19-1 was cloned at the XhoI-SmaI sites of the phagemid
pBluescript(KS
). This fragment was sequenced on
subclones obtained by (i) internal deletions in the
KS
BS19-1XhSm plasmid taking into account unique
restriction sites in the insert and adequate sites of the polylinker of
pKS
(NruI/SmaI, EcoR5/SmaI, EcoR1-Klenow/SmaI, XbaI/XbaI); and (ii) one-directional deletions
generated by exonuclease III digestions (38) in plasmid
KS
BS19-1XhSm cut by SacII and SmaI
using the Erase-a-Base kit (Promega, Madison, WI). The double-stranded
plasmid DNA was sequenced by the chain termination method using the
modified T7 DNA polymerase (39) from the Sequanase v. 2.0
sequencing kit (U. S. Biochemical Corp. and
-
S-dATP
(1000Ci/mmol, Amersham Corp.). The sequencing primers used were
universal(-40) and reverse primers available for pBluescript and
various 17-mer oligonucleotides complementary to the HEM14 sequence already determined. The plasmid DNA (2-3 µg)
was first denaturated in 0.4 N NaOH for 10 min at room
temperature, precipitated with cold ethanol and annealed to the primer
for 30 min at room temperature at a molar ratio of 1:1 when using
pBluescript primers, and 1:20 when using synthetic HEM14-specific oligonucleotides. The oligonucleotides used as
sequencing primers were synthesized on an Applied Biosystems DNA
synthesizer and were used without further purification. To sequence the
mutant allele hem14-1, the HEM14 locus was amplified
by PCR from genomic DNA of the strains CC750-3D and W303-1A using the
primers 5`-CGCGGATCCCTTTGTTGCTTCGGAGTCGC-3` (HindIII; (+)
strand) and 5` CCCAAGCTTATAGTTCCAACCCATCATCG-3` (BamHI;(-) strand). The PCR products were purified from
agarose gels (JetSorb, Genomed Inc.) digested by HindIII and BamHI and the repurified DNA (JetSorb, Genomed Inc.) was
ligated into the dephosphorylated phagemid BlusScript KS+
linearized with HindIII and BamHI. The nucleotide
sequence of the wild-type (control processed under the same conditions)
and mutant alleles were obtained on DNAs pooled from seven independent
clones. The nature of the mutations was confirmed on both strands. The
DNA for PCR amplification of genomic sequences was prepared by the
technique of Hoffman and Winston(40) .
The L422P mutation was introduced using the oligonucleotide
5`-AACCCAAACGCCCCCAACAAATAAACAAAAGTG-3`. The K424E mutation was
introduced using the oligonucleotide
5`-CGCCCTCAACGAATATACAAAAGTGACTGCGATG-3`. Enrichment in mutated plasmid
was done by eliminating the single ScaI restriction site in the
ampicillin resistance gene of pT7-5. The plasmids were then transformed
into the BL21(DE3) strain of E. coli. The T7 RNA polymerase
gene was chemically induced by adding 0.2 mM isopropyl-1-thio--D-galactopyranoside to the cell
cultures grown to an OD
of 0.6-1 and incubation for
3 h at 25 °C. The cells were collected by centrifugation,
resuspended in the lysis buffer (0.1 M potassium phosphate
buffer, pH 7.2, containing 0.1 M KCl, 1% n-octyl
glucoside, 1 mM EDTA, and 20 µg/ml PMSF), sonicated three
times for 5 s each, and centrifuged. The protoporphyrinogen oxidase
activity in the resulting cell-free extracts was measured. The
homogenates were diluted twice in Laemmli sample buffer and processed
for electrophoresis and immunodecoration as described below.
Protoporphyrinogen oxidase was assayed by measuring the rate of appearance of protoporphyrin fluorescence(46) . Enzyme assays were carried out at 30 °C. The incubation mixture was 0.1 M potassium phosphate buffer, pH 7.2, saturated with air, containing 2 µM protoporphyrinogen IX, 3 mM palmitic acid (in dimethyl sulfoxide, 0.5%, v/v, final concentration), 5 mM DTT, 1 mM EDTA, and 0.3 mg/ml (final concentration) Tween 80 to ensure a maximum fluorescence signal of protoporphyrin IX. Protoporphyrinogen was prepared by reducing protoporphyrin IX hydrochloride dissolved in KOH/EtOH (0.04 N, 20%) with 3% sodium amalgam(21) .
The plasmid carrying the shortest insert (4.9 kb) was further analyzed. Subcloning and deletion analysis of the insert indicated that the complementing activity was restricted to a 2.5-kb fragment (Fig. 1). This fragment was sequenced (see ``Experimental Procedures'') and found to contain a 1.6-kb open reading frame encoding a 539-amino acid protein (Fig. 2). The phenotype of a cell lacking this gene was determined by removing a 439-bp NruI-EcoRV fragment containing the initiation codon and the 120 amino-terminal residues from the cloned insert and replacing it with a 820-bp fragment containing the TRP1 gene. A 2.7-kb linear BglI fragment, now containing the TRP1 substitution, was transformed into haploid trp1 cells. Tryptophan prototroph transformants were selected on a synthetic medium supplemented with Tween 80, ergosterol, and hemin. The transformants had a phenotype closely related to that of the original hem14-1 mutants. They were respiratory deficient (no growth on glycerol or ethanol as sole source of carbon), they accumulated porphyrins (Fig. 3) and had no detectable protoporphyrinogen oxidase activity in vitro. However, they were strictly auxotroph for Tween 80 and ergosterol (Te), while the original mutant could sustain some growth without Te. The cells disrupted for the HEM14 gene also lacked any immunodetectable protoporphyrinogen oxidase protein (Fig. 4). Similar phenotypes were obtained by deleting most of the open reading frame by replacing the 1493 bp NruI-EcoRI fragment of HEM14 (Fig. 2) by the 820-bp fragment containing the TRP1 gene.
Figure 1: Restriction map of the yeast genomic DNA fragment complementing the hem14-1 mutation and subcloning analysis. Upper maps, restriction sites from the cloned fragments; lower maps, restriction sites from the vectors. Protox, protoporphyrinogen oxidase.
Figure 2: Nucleotide sequence of the HEM14 gene and deduced amino acid sequence of protoporphyrinogen oxidase. Putative TATA box (&cjs0808;&cjs0808;) and transcription termination signals (-) are underlined. The NruI, EcoRV, and EcoRI recognition sequences are in boldface type. The stop codon of PRP22 and initiation codon of FAA2 are boxed.
Figure 3:
Low temperature spectra of yeast cells
(480-640 nm). All the strains were grown on YPG-Te. The cells
were collected during the stationary phase of growth. A,
S150-2B; B, CC750-3D; C, CC750-3D transformed by
plasmid pBS19-1; D, S150-2Bhem14::TRP1; E,
S150-2B
hem14::TRP1 transformed by plasmid
pBS19XS9.
Figure 4:
Immunocharacterization of yeast
protoporphyrinogen oxidase after SDS-polyacrylamide gel electrophoresis
of total protein extracts from yeast cells collected during the
stationary phase of growth on YPG-Te. 1, S150-2B (10 µg of
protein); 2, S150-2Bhem14::TRP1 (20 µg protein); 3, S150-2B
hem14::TRP1 transformed by plasmid pBS19XS9 (3
µg of protein). Panel A, Ponceau S staining of total
proteins; Panel B, immunodetection of yeast protoporphyrinogen
oxidase.
Figure 5: Hydropathy plot of yeast protoporphyrinogen oxidase, according to Kyte and Doolittle, and distribution of acidic (A) and basic (B) amino acid residues in the protein.
Figure 6:
Alignment (Clustal-w) of yeast
(SCHEM14), B. subtilis (BS
HEMY), human (HS
POX), and
mouse (MM
POX) protoporphyrinogen oxidases. Residues
identical in three or four sequences are boxed. Symbols over
the yeast sequence are defining a putative ADP-
-binding
domain according to Wierenga et al.(55) . (
),
basic or hydrophylic; (
), small or hydrophobic; (
),
glycine; (
), acid.
Figure 7: Protoporphyrinogen oxidase activity in cell-free extracts from cells harvested at various stages of aerobic growth on glucose, galactose or ethanol as source of carbon.
The physicochemical and kinetic properties of
overexpressed protoporphyrinogen oxidase were further analyzed. The
membrane bound overexpressed protoporphyrinogen oxidase was inhibited
by diphenyl ether type herbicides, acifluorfen-methyl
(methyl-5-[-2-chloro-4-(trifluoromethyl)-phenoxy-]-2-nitrobenzoic
acid) and oxadiazon
(5-tert-butyl-3-(2,4-dichloro-5-isopropoxyphenyl)-1,3,4-oxadiazol-2-one)
at concentrations comparable that inhibit wild-type yeast; the
IC were 2 and 30 nM, respectively (Fig. 8)(30) . Protoporphyrinogen oxidase was purified
to homogeneity (200-fold, 60% recovery) from the mitochondrial
membranes of the transformed cells. The specific activity of the enzyme
was 43,600 nmol of protoporphyrinogen
oxidized
h
mg
protein.
The Michaelis constant for protoporphyrinogen was 0.02 µM.
The enzyme was specific for protoporphyrinogen IX and did not oxidize
coproporphyrinogen III to coproporphyrin III. The absorption spectrum
of the enzyme as purified (Fig. 9) was typical of a flavoprotein
with a flavin semiquinone. Preliminary electron spin resonance
spectroscopy experiments did not detect the stable free radical,
expected in such a redox state of the flavin (data not shown). The
reactivity of purified protoporphyrinogen oxidase toward a
photoaffinity probe (32) was identical to that of the wild-type
enzyme. The protoporphyrinogen oxidase polypeptide appeared to be
specifically labeled by diazo-[
H]acifluorfen (Fig. 10, lane B). The labeling of the purified protein
was completely blocked by 10 µM acifluorfen-methyl, an
efficient inhibitor of protoporphyrinogen oxidase (Fig. 10, lane C). The NH
-terminal peptide sequence of the
protein was MLLPLTKLKPRAKVAVV. This sequence is identical to that
deduced from the first ATG (Met) of the open reading frame of the HEM14 gene.
Figure 8:
Inhibition of protoporphyrinogen oxidase
activity from cells overproducing the enzyme, by the diphenyl
ether-type herbicides acifluorfen-methyl () and oxadiazon
(
).
Figure 9:
Absorption spectrum of purified
overexpressed yeast protoporphyrinogen oxidase (230-650 nm). Insert, 5 magnification of the 350-650-nm
region.
Figure 10:
Photoaffinity labeling of purified
overexpressed yeast protoporphyrinogen oxidase. Lane A, C-radiolabeled molecular mass markers; lane B, 10
ng of photolabeled enzyme; lane C, 10 ng of enzyme
photolabeled in the presence of 10 µM acifluorfen-methyl
as unlabeled competitor.
We have isolated the HEM14 gene that encodes yeast
protoporphyrinogen oxidase by functional complementation of a
heme-deficient hem14-1 strain. It has been recently
demonstrated that several oxidases are able to oxidize
protoporphyrinogen to protoporphyrin in
vitro(10, 11, 12) . Such oxidases may
complement our mutant, depending on their intracellular location and
concentration. It was therefore essential to establish nonambiguously
that HEM14 is the structural gene of yeast protoporphyrinogen
oxidase. This was done by genetic and biochemical studies. First, the hem14-1 mutation was complemented by the cloned gene on both
single and multiple-copy plasmids. Second, yeast strains with a
disrupted HEM14 open reading frame were constructed. These
strains had the phenotypes of a protoporphyrinogen deficiency,
producing a respiratory deficiency, accumulation of porphyrins and lack
of immunodetectable protoporphyrinogen oxidase protein. Third, the HEM14 gene product (characterized by the
NH-terminal protein sequence) was overexpressed in yeast.
The product was recognized by anti-yeast protoporphyrinogen oxidase
antibodies and was purified as authentic protoporphyrinogen oxidase.
The protein was fully inhibited by a variety of diphenyl ether-type
herbicides and labeled by photoaffinity using a highly specific probe.
Fourth, two mutations in the hem14-1 allele were
characterized, and the mutations were further analyzed by site-directed
mutagenesis of the wild-type allele in an expression system for the HEM14 gene in E. coli.
The mapping of the HEM14 gene on chromosome V was demonstrated by both genetic
linkage to URA3, a known marker of this chromosome and
sequence specific in vitro amplification of the HEM14 locus on selected fragments of an ordered library of chromosome
V(51) . The genes flanking HEM14 are PRP22 at
the 5` end of HEM14 and FAA2 already mapped on
chromosome V(50) , at the 3` end of HEM14. The
approximate genetic distance between URA3 and HEM14 (17 cm, 16 tetrades analyzed) is compatible with the physical
distance calculated from Isono's and Rile's maps (60 kb)
and from the complete sequence of a set of ordered cosmids covering the
entire chromosome V, as the contribution led by Dr. D. Bostein to the
Yeast Genome Sequencing Project. Our results allow the ``putative
59.5-kDa open reading frame'' between PRP22 and FAA2 to be assigned to HEM14. HEM14 lies in a quite compact
region of chromosome V, since the distance between the TAA stop codon
of PRP22 and the ATG initiation codon of HEM14 is
only 322 bp, and the distance between the TAA stop codon of HEM14 and the ATG initiation codon of FAA2 is only 321 bp. A
single plasmid complementing the hem14 mutation was obtained
by screening a yeast DNA library generated by partial HindIII
digestion of genomic DNA. The insert was very long (>15 kb). This
result is consistent with the fact that the PRP22-HEM14-FAA2 locus lies in a 20-kb HindIII fragment.
Several
features of yeast protoporphyrinogen oxidase were revealed by the amino
acid sequence of the protein deduced from the nucleotide sequence of
the HEM14 gene which were not predicted by the biochemical
characteristics of the enzyme. The predicted molecular mass of the HEM14 gene product is 59 kDa, close to the 58-kDa
determined for the precursor form of the protein, with an isoelectric
point of 9.3 comparable to that of the purified protein (pI >8.5).
Yeast protoporphyrinogen oxidase was described as an integral protein
of the inner mitochondrial membrane(30) . The predicted
sequence of protoporphyrinogen oxidase should therefore contain those
amino acid sequences that target the protein to the mitochondria and
anchor it to the lipid bilayer. The first 13 amino acids of the open
reading frame of the HEM14 gene contains all the information
typical of a protein imported to the mitochondria, without acidic
residues, but with high contents of Leu, Arg, Lys and Thr, and a
propensity to form an amphiphilic helix. This sequence is immediately
followed by a putative
-dinucleotide binding fold,
suggesting that the 13 amino-terminal residues may act as a cleaved
presequence. We therefore determined the NH
-terminal
sequence of purified protoporphyrinogen oxidase. The terminal 17
residues are identical to the deduced NH
-terminal sequence
of the HEM14 gene product, and starts with the initiation
methionine, as expected from the known specificity of the yeast
methionine aminopeptidase that do not cleave the Met-Leu
bond(56) . The lack of cleavage of the mitochondrial targeting
sequence of the protein during the importation of the protein into the
mitochondria is not usual for an inner membrane-bound protein, but a
sequence as short as 11 residues has been characterized as the
uncleaved targeting sequence of the mitochondrial isoform of the tRNA N
-adenosine isopentyl transferase in
yeast(57) . Although the sequence of protoporphyrinogen oxidase
contains several hydrophobic regions, none of those is longer than 15
uncharged residues and they are therefore unlikely to form membrane
spanning segments. However, shorter helical domains could be
responsible for insertion of the protoporphyrinogen oxidase into the
inner mitochondrial membrane, as described for prostaglandin H
synthase-1, a monotopic membrane-bound protein of the endoplasmic
reticulum(58) . Another possibility, compatible with a
post-translational modification of the protein leading to the shift in
electrophoretic mobility initially attributed to the proteolytic
cleavage of a putative presequence previously described(30) ,
is that protoporphyrinogen oxidase is anchored to the inner
mitochondrial membrane by a different mechanism, such as acylation.
Preliminary experiments tend to support this hypothesis. (
)
One of our goals is to obtain enough yeast protoporphyrinogen oxidase for structural studies on the protein. Commercially available yeast cells contain slightly more protoporphyrinogen oxidase activity and protein than laboratory grown cells. The industrial strains are usually grown on molasses and harvested in the late stationary phase of growth. This indicated that protoporphyrinogen oxidase seems to be fairly stable under such conditions, but the enzyme accounts for less than 0.001% of the membrane-bound proteins. We further optimized the production of the enzyme in our transformed laboratory strains by measuring protoporphyrinogen oxidase activity during the aerobic growth of yeast cells in media containing various carbon sources. Protoporphyrinogen oxidase activity was maximum during the stationary phase of growth of cells up to 8000 units (0.2 mg)/g (wet weight) of transformed cell in all cases. The activity during the exponential phase of growth was 2-3 times higher in cells grown on galactose or ethanol than in cells grown on glucose. Detailed analysis of the promotor of HEM14 will be needed to find out more about the mechanism and physiological relevance of HEM14 expression regulation in yeast. The overexpressed protein is associated with the membrane fraction. Both the membrane-bound and the purified enzyme are inhibited by diphenyl ether type herbicides and specifically photolabeled with an affinity probe, as is the enzyme from a wild-type strain.
The
primary sequences of yeast protoporphyrinogen oxidase and related
proteins (mammalian protoporphyrinogen oxidases and HemYp) are not easy
to compare because of the overall low level of similarity of the
proteins; the best conserved domain is the -ADP binding
fold in the NH
terminus of these proteins. If the targeting
sequence of yeast protoporphyrinogen oxidase is short and not cleaved,
the mammalian protoporphyrinogen oxidases are synthesized without such
mitochondrial targeting sequences and therefore have a NH
terminus starting with the
-ADP binding fold as do
monoamine oxidases, proteins associated with the outer membrane of
mitochondria. As mentioned previously, this motif is found in the
NH
-terminal domain of several well characterized
flavoproteins (monoamine oxidases, amino-acids oxidases, and so forth)
and, in this area, of some proteins of unknown function. A search in
the S. cerevisiae genome data base (at
www.genome-stanford.edu) with ``protoporphyrinogen'' as a
keyword identified the YHR009c open reading frame. Careful examination
of this sequence showed that this gene product has a
NH
-terminal sequence very similar to that of HemYp (and
therefore to the yeast protoporphyrinogen oxidase) but has no other
domain similar to either HemYp or HEM14p. This unknown protein may,
however, be a flavoprotein. The central question is to determine
whether the conserved residues in the alignment of the yeast mammalian
and bacterial proteins are involved in the substrate specificity of the
enzymes and/or the catalytic activity. The B. subtilis enzyme
has a poor substrate specificity, while the eukaryotic enzymes are more
specific for protoporphyrinogen IX. However, the mammalian
protoporphyrinogen oxidases are more closely related to HemYp than to
the yeast enzyme, especially in their COOH-terminal domain. The two
similar blocks in the mammalian and yeast enzymes are in poorly
structured domains. These domains may, however, be important for the
specificity of the enzyme.
The characterization of mutations
affecting protoporphyrinogen oxidase activity should help determine the
structure/function relationships. The short deletion affecting the
HemYp function recently described (28) is located close to the
end of the -ADP binding fold. The present paper,
describes the first mutations affecting an eukaryotic
protoporphyrinogen oxidase. The hem14-1 mutant allele carries
two point mutations that introduce two changes in amino acids in a
relatively COOH-terminal part of the protein, without significant
similarity to the mammalian or bacterial protoporphyrinogen oxidases.
The two mutations, L422P and K424E, are located in a domain close to a
cysteine residue (Cys-435) in a Gly-Gly-Cys motif that may, by analogy
to bovine monoamine oxidase(59) , be involved in the covalent
binding of FAD to the enzyme. The two mutations are rather different in
nature. A proline may drastically affect secondary structure in the
protein, while the lysine to glutamic acid change may affect the
catalytic properties of the enzyme. One model of how the enzyme acts
involves the removal of hydrogen from protoporphyrinogen as at least
one hydride involving the flavin, and one proton involving a basic
residue in the protein. It is thus tempting to speculate that Lys-422
may be this basic residue. Alternatively, the lysine may help stabilize
the flavin in an anionic semiquinone form necessary for activity, as in
other oxidases (for a review, see (60) ). In a preliminary
attempt to better characterize each mutation, we introduced the two
mutations independently into the wild-type gene cloned in an expression
system in E. coli. Both mutations led to the production (under
standard conditions) of an inactive protein in E. coli. These
mutated genes will now be introduced into the yeast strain lacking a
functional HEM14 gene to detect residual activity through
functional complementation or the involvement of some
post-translational modification affecting the functioning of the
mutated enzymes in vivo and in vitro.
The availability of transformed cells overexpressing protoporphyrinogen oxidase (yeast and E. coli) overcomes the basic barriers to investigating the structural properties of the enzyme and the topology of its active site, due to the small amounts of the protein in wild-type yeast strains. This approach will also help the characterization of the molecular changes involved in the mutation in the protoporphyrinogen oxidase-deficient yeast strain that provided the basis for site-directed mutagenesis of the yeast protoporphyrinogen oxidase gene. Our present results provide evidence that yeast protoporphyrinogen oxidase is an adequate model to elucidate the molecular basis of protoporphyrinogen oxidase function, and hence the mechanisms underlying the pathophysiology associated with the enzyme defect (human porphyria) and inhibition (herbicidal effects of diphenyl esters).