(Received for publication, November 28, 1994; and in revised form, January 31, 1995)
From the
Protoporphyrinogen oxidase (PPO; EC 1.3.3.4) is the enzyme that
catalyzes in the penultimate step in the heme biosynthetic pathway.
Hemes are essential components of redox enzymes, such as cytochromes.
Thus, a hemG mutant strain of Escherichia coli deficient in PPO is defective in aerobic respiration and grows
poorly even in rich medium. By complementation with a human placental
cDNA library, we were able to isolate a clone that enhanced the poor
growth of such a hemG mutant strain. The clone encoded the
gene for human PPO. Sequence analysis revealed that PPO consists of 477
amino acids with a calculated molecular mass of 50.8 kilodaltons. The
deduced protein exhibited a high degree of homology over its entire
length to the amino acid sequence of PPO encoded by the hemY gene of Bacillus subtilis. The NH-terminal
amino acid sequence of the deduced PPO contains a conserved amino acid
sequence that forms the dinucleotide-binding site in many
flavin-containing proteins. Northern blot analysis revealed the
synthesis of a 1.8-kilobase pair mRNA for PPO. A homogenate of the
monkey kidney COS-1 cells that had been transfected with the cDNA had
much higher PPO activity than an extract of control cells, and this
activity was inhibited by acifluorfen, a specific inhibitor of PPO.
Furthermore, the cDNA was expressed in vitro as 51-kilodalton
protein, and after incubation with isolated mitochondria the protein
was found to be located in the mitochondria, having just the same size
as before, an indication that PPO is a mitochondrial enzyme and has no
apparent transport-specific leader sequence.
Heme is essential as the prosthetic group of many respiratory
enzymes(1) , and it is also involved in the cell's
defense against the toxic effects of active species of
oxygen(2) . The pathway for the biosynthesis of heme is highly
conserved in a wide variety of organisms after the initial reactions
that lead to the synthesis of 5-aminolevulinic
acid(3, 4) . The enzyme protoporphyrinogen oxidase
(PPO) ()(EC 1.3.3.4) acts at the penultimate step in the
biosynthetic pathway to heme and catalyzes the six-electron oxidation
of protoporphyrinogen IX to protoporphyrin IX(5) . In
eukaryotes, PPO is located in the inner membrane of
mitochondria(6) , and it requires molecular oxygen for the
conversion of protoporphyrinogen IX to protoporphyrin IX. In
prokaryotes, by contrast, the oxidation is achieved by coupling to the
respiratory chain and some compounds that serve as terminal electron
acceptors.
PPO has been purified partially or to apparent homogeneity from yeast (7, 8) , barley(9) , rat(7) , bovine(10) , and mouse liver mitochondria (11) . The highly purified enzyme from bovine and mouse mitochondria is a monomer with a molecular mass of approximately 65 kDa. In prokaryote, PPO was purified only from Desulfovibrio gigas(12) and found to be composed of three dissimilar subunits. Recently, the hemY gene of Bacillus subtilis(13) was expressed in Escherichia coli(14) and shown to have PPO activity. Sequence data for PPO are available only from the hemG gene of E. coli(15) and the hemY gene of B. subtilis(13) . The nucleotide sequence and the length of the coding regions of hemG and hemY are different, probably because the electron acceptor for each PPO is different, although the actual reactions catalyzed by both enzymes are identical.
In humans, a deficiency in the activity of PPO has been associated with variegated porphyria(16) , one of the acute porphyrias that is classified as a hepatic porphyria. Skin lesions appear in areas exposed to light, and acute attacks are accompanied by abdominal pain. This enzymatic defect is inherited as an autosomal dominant trait. Human genes for the enzymes involved in the biosynthesis of heme have been characterized(17, 18, 19, 20, 21, 22, 23) with the exception of the gene for PPO. Isolation of PPO cDNA should facilitate studies on the nature of genetic mutations that lead to disease and should also aid in detection of carrier, prenatal diagnosis, and treatment.
In plants, PPO is the target molecule for acifluorfen-methyl(24) , one of the diphenyl ether herbicides. This herbicidal activity of this compound is light-dependent. The phytotoxicity is explained by the fact that accumulated protoporphyrinogen IX, the result of inhibition of PPO by acifluorfen, diffuses from the site of its synthesis and is then subjected to non-enzymatic oxidation to yield protoporphyrin IX, known as a powerful generator of singlet oxygen in light(25) . It is the singlet oxygen that is responsible for the harmful effects on plant cells, such as the peroxidation of membrane lipids.
We reported previously the
isolation of mutants of E. coli K-12 that were sensitive to
visible light(26) . This phenomenon was brought about by a
defect in the visA (hemH) gene that encodes
ferrochelatase(27, 28) , the enzyme that catalyzes the
final step in the heme biosynthetic pathway. A defect in this gene
causes the accumulation of protoporphyrin IX, the substrate of
ferrochelatase, which is a photosensitizer and produces an active
species of oxygen that is harmful to the cell. We isolated
photoresistant revertants from a visA-deleted strain and found
that the second mutations were located in other genes involved in the
heme biosynthetic pathway at steps prior to the reaction catalyzed by
ferrochelatase(29) . Thus, we obtained strains with mutations
in hemA, hemB, and the hemCD operon.
Furthermore, using hemA/hemA
diploid
bacteria, we isolated another set of photoresistant mutants and
obtained hemL, hemE, and hemG mutants (30) .
In spite of considerable efforts at purification of the protein and analysis of the enzymatic activity, no information relevant to the molecular characterization of PPO is available. In this study, as a first step toward a better understanding of the structure-function relationships of PPO and the mechanisms of regulation of PPO in mammalian cells, we isolated a cDNA clone for human PPO by in vivo complementation using one of the hemG mutants of E. coli, and then we characterized the encoded human PPO.
Figure 1: Nucleotide sequence of human PPO cDNA and the deduced amino acid sequence. The numbering of nucleotides is shown above the sequence. The single-letter code for amino acids is used. A putative polyadenylation signal is underlined.
Figure 2: Comparison of the deduced amino acid sequence of human PPO with that of the hemY gene product of B. subtilis PPO. Identical residues are indicated by asterisks, and quasi-identity, including conservative substitutions, is indicated by dots. Dashes indicate spaces that were introduced to allow maximum alignment of identical amino acids.
Figure 3:
Alignment of the amino-terminal amino acid
sequences of the FAD-binding domains of flavin-containing enzymes.
Numbers beside amino acid sequences indicate the starting points of
aligned sequences. The secondary structure of FAD- or NAD-binding
-folds is shown above the sequences. The length
of the loop can vary. Highly conserved glycine residues, characteristic
of the dinucleotide-binding domain of many flavin-containing enzymes,
are boxed and shadowed. Conserved amino acid residues
are also boxed. Entry names in the SWISS-PROT or PIR protein
data bases are included before the name of each
enzyme.
Total RNA obtained from K562 and HepG2 cells was subjected to Northern blot analysis using the insert cDNA of pBHPPO as probe. A band of an RNA of approximately 1.8 kilobase pairs was obtained with both samples. The intensity of the hybridization band of RNA from K562 cells was greater than that from HepG2 cells. The mRNA for PPO in erythroid cells appears to be the same as that in non-erythroid cells.
Figure 4:
Effects of acifluorfen on human PPO
activity in cells transfected with human PPO cDNA. COS-1 cells, 48 h
after transfection with pCDPPO-3 (sense, ) and pCDPPO-1
(antisense,
), were lysed, and PPO activity was measured in the
homogenates in the presence of acifluorfen.
Figure 5: Import of human PPO into isolated mitochondria from mouse liver. Human PPO was transcribed by T7 polymerase, and the synthesized RNA was translated in a rabbit reticulocyte lysate in vitro. A portion of the products of translation from the antisense plasmid (lane1) and from the sense plasmid (lane2) was subjected to electrophoresis on an SDS-polyacrylamide gel. Then the rest of the products was incubated with isolated mitochondria. Samples of protein were recovered from reisolated mitochondria and subjected to electrophoresis (lane3). Trypsin (lane4) and trypsin plus Triton X-100 (lane5) were added to the reisolated mitochondria before samples of protein were recovered and subjected to electrophoresis. Protein markers used were: phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), and carbonic anhydrase (30 kDa).
This is the first report of the isolation of a cDNA clone for mammalian PPO. The poor growth of a hemG mutant strain of E. coli enabled us to isolate cDNA for human PPO by in vivo complementation with cDNA in a human placental cDNA library. The length of the mRNA for PPO, as revealed by Northern blot analysis, was almost the same as that of the insert cDNA of HPPO18, suggesting that the insert was the full-length cDNA. The deduced polypeptide encoded by the cDNA consisted of 477 amino acids with a molecular mass of about 51 kDa, which is less than that of purified bovine and mouse PPOs, which were previously reported to have a molecular mass of approximately 65 kDa(11) . The reason for this disagreement is unclear. However, HPPO18 containing a single open reading frame for 477 amino acids complemented the hemG mutant of E. coli, and the deduced amino acid sequence was similar to that of the hemY gene product of B. subtilis. The length and molecular mass of human PPO are also the same as those of the enzyme from B. subtilis (471 amino acids, 51 kDa). It is not surprising that the amino acid sequence of human PPO and its length are completely different from those of PPO from E. coil, the product of the hemG gene(15) , since the electron acceptor for each PPO is different and, therefore, the structure of each enzyme would not be expected to be similar. For example, PPO of D. gigas is composed of three dissimilar subunits(12) , while mammalian PPO is a monomer(10, 11) .
The last three steps in the
biosynthesis of heme in mammalian cells occur in the
mitochondria(5) . Although mitochondrial ferrochelatase and
coproporphyrinogen oxidase are synthesized as precursor forms with a
presequence for targeting to and import into mitochondria, as is common
for mitochondrial proteins(31) , the PPO that becomes
associated with the inner membrane of mitochondria (6) is not
synthesized with a presequence. A so-called amphiphilic -helix,
with one highly charged and one hydrophobic face in the presequence,
was proposed previously to enable a protein precursor to interact with
the mitochondrial membrane (51, 52) . However, Allison
and Schatz (53) showed that such a sequence is not necessarily
essential for mitochondrial targeting and that the targeting function
may depend on the overall balance between basic, hydrophobic, and
hydroxylated amino acids. Thus, a small number of mitochondrial
proteins, including outer membrane proteins, are synthesized without a
presequence, and they are expected to have non-cleavable, but as yet
unidentified, mitochondrial targeting signals. To our knowledge,
mitochondrial 3-oxoacyl-CoA thiolase is the only known mitochondrial
matrix protein that does not have a presequence(41) . The
amino-terminal portion (14 amino acids) of 3-oxoacyl-CoA thiolase
contains 3 basic amino acid residues and no acidic residues, and it
shares common features with mitochondrial protein presequences. This
region might function as a mitochondrial targeting signal even though
it cannot form an amphiphilic
-helix (54) . In the case of
human PPO, the amino-terminal portion (28 amino acids) contains 3 basic
residues and no acidic residues, features characteristic of a
presequence. Although this portion can form an
-helix, as
suggested by the presence of the FAD-binding domain (Fig. 3), it
should not be referred to as an amphiphilic
-helix, because the
hydrophobic residues are not clustered on the opposite side of
positively charged amino acids and because hydroxylated amino acids are
scattered when this region is plotted on a helical wheel(55) .
It is possible that the amino-terminal portion of the human PPO has
some other type of mitochondrial targeting signal, as in the case of
3-oxoacyl-CoA thiolase. The amino-terminal residues 5-17 of human
PPO are highly conserved when compared with residues 8-20 of the
product of the hemY gene of B. subtilis(13) ,
and they play a role as the FAD-binding domain. Therefore, the amino
terminus of human PPO has features of both a mitochondrial targeting
signal and a catalytic domain.
Coproporphyrinogen oxidase has been shown to be loosely associated with the outside of the inner mitochondrial membrane, and ferrochelatase has been demonstrated to be the inner mitochondrial membrane with its active site present on the matrix side of that membrane(5) . Therefore, the question of porphyrin transport across the inner mitochondrial membrane arose. Ferreira et al.(50) proposed that PPO interacts with ferrochelatase to transport protoporphyrin. The hydropathy plot of PPO shows that this protein is a typical membrane protein (data not shown). However, it requires more study to determine whether or not this oxidase has any role in transport of PPO across membrane to reach the active site of ferrochelatase.
Yeast PPO has been purified to homogeneity, having a molecular mass of 55 kDa(8) . The enzyme is synthesized as a precursor with a molecular mass of 57 kDa, which is converted to the 55-kDa mature enzyme after import into mitochondria(8) . Yeast PPO is synthesized as a precursor and differs in this respect from human PPO. Indeed, the localization and regulation of the enzymes on the heme biosynthetic pathway in yeast are in general different from those of the mammalian enzymes. For example, coproporphyrinogen oxidase in yeast is located in the cytosol (8) . Sequence analysis of the gene for the yeast enzyme revealed that the enzyme does not have an obvious presequence for transport into mitochondria. It is possible that the transport pathway for newly synthesized PPO and its location in the mitochondrial membrane differ between mammalian cells and yeast.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D38537[GenBank].