©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning of a Human cDNA for Protoporphyrinogen Oxidase by Complementation in Vivo of a hemG Mutant of Escherichia coli(*)

(Received for publication, November 28, 1994; and in revised form, January 31, 1995)

Koichi Nishimura Shigeru Taketani (1) Hachiro Inokuchi (§)

From the Department of Biophysics, Faculty of Science, Kyoto University, Sakyo-ku, Kyoto 606-01, Japan and the Department of Hygiene, Kansai Medical University, Moriguchi, Osaka 570, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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(2)-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.


INTRODUCTION

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) (^1)(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.


EXPERIMENTAL PROCEDURES

Materials

Restriction endonucleases and other nucleic acid-modifying enzymes were purchased from Toyobo Co. (Osaka, Japan) and Takara Co. (Osaka, Japan). [alpha-P]dCTP (3,000 Ci/mmol) and [S]methionine (>1,000 Ci/mmol) were obtained from Amersham Corp. The riboprobe transcription kit and rabbit reticulocyte lysates transcription system were from Promega (Madison, WI). Acifluorfen was provided from Rhône Poulenc Agrochimie. Human erythroleukemia K562 cells and hepatoma HepG2 cells were cultured as described elsewhere(23) . Monkey kidney COS-1 cells were obtained from the Japan Cell Bank and were grown as described previously(31) . Protoporphyrin IX was from Porphyrin Products Co. Protoporphyrinogen was generated by the reduction of free protoporphyrin IX solution with a 3% (w/v) sodium amalgam in darkness by the method of Jacobs and Jacobs(32) .

Bacterial Strains

DH5alpha and LE392 (33) were used for transformation and manipulation of phage. As a hemG mutant of E. coli, VSR800 was used. VSR800 was a visA (hemH) lysogen (26) of VSR751, which was isolated as a photoresistant revertant from a photosensitive visA-deleted strain(30) . It was shown to have a mutation in the hemG gene. (^2)As a control for complementation, clone 549 of Kohara's library (34) and hemG^2 that contained only the hemG gene of E. coli(15) of the pmaCI site of inin5 (35) were used.

Complementation

Mutants defective in the heme biosynthetic pathway do not synthesize cytochromes and lack aerobic respiratory ability. Therefore, even in rich medium they grow very poorly. Complementation was monitored in terms of the restoration of a normal growth rate upon infection by phage or introduction of plasmid.

Isolation of a cDNA Clone for PPO by Complementation

A human placental cDNA library (23) was amplified and used to infect VSR800 cells. After incubation to allow the expression of PPO, the bacterial cells were spread on LB plates. Two days later, several larger colonies were visible among numerous dwarf colonies. The phage responsible for the restoration of the normal growth of cells was recovered by inducing each revertant with UV irradiation. Then each phage was plaque-purified. The phage containing the longest insert was named HPPO18 and selected for further experiments.

DNA Sequencing

The insert of HPPO18 was recloned to the BlueScript plasmid vector (36) to yield pHPPO18. Sequencing was carried out, after stepwise deletions(37) , by the dideoxy chain termination method (38) using the Sequenase version 2 kit (U. S. Biochemical Corp.).

Northern Blots

Total RNA was isolated from human erythroleukemia K526 cells and hepatoma HerG2 cells by the guanidinium isothiocyanate method(23) . Twenty µg of RNA were applied to a 1% agarose/formaldehyde gel, subjected to electrophoresis, and then transferred to a nylon membrane (Hybond N; Amersham Corp.). Conditions of hybridization and washing were as described elsewhere(23) .

Expression of Cloned Human PPO

The insert of pHPPO18 was ligated into the pCD vector(39) . The resulting plasmid (pCDPPO) was used to transfect COS-1 cells by the DEAE-dextran method(33) . After a 48-h incubation, the cells were collected and washed twice with phosphate-buffered saline. The cells were resuspended in 25 mM Tris-HCl buffer, pH 7.4, that contained 10 mM 2-mercaptoethanol and disrupted by sonication for 1 min. A supernatant was obtained after centrifugation at 900 times g for 10 min and used for the assay of PPO activity.

Assay of PPO Activity

The activity of PPO was measured in duplicate by the method of Jacobs and Jacobs(32) . The reaction mixture contained 100 mM Tris-HCl buffer, pH 8.7, 10 mM 2-mercaptoethanol, 0.3% Tween 20, 5 mM EDTA, 5 µM protoporphyrinogen, and the supernatant of a cell extract, as described above (500 µg of protein), in a final volume of 1.0 ml. The incubation was carried out in darkness at 37 °C for 30 min. The formation of protoporphyrin was monitored fluorimetrically with excitation and emission wavelengths of 410 and 633 nm(32) , respectively. Autoxidation of protoporphyrinogen was also monitored to ensure that the results were not attributable to a non-enzymatic reaction.

Production of PPO Protein

Plasmid pBHPPO18, in which the entire cDNA for human PPO was cloned downstream of the T7 promoter, was used to produce protein in vitro. The plasmid was transcribed in vitro by T7 polymerase in the presence of m^7GpppG and a Riboprobe kit from Promega. The RNA formed was precipitated with ethanol, washed, and dissolved in autoclaved H(2)O. Five µg of transcript were translated in 40 µl of a rabbit reticulocyte lysate in the presence of 5 µCi of [S]methionine.

Isolation of Mouse Mitochondria and Import Studies

Mitochondria were prepared from mouse liver as described elsewhere (40) and used for import studies in vitro essentially as described by Mori et al.(41) . Mitochondria (0.5 mg of protein) were incubated with the translation mixture in a total volume of 200 µl at 30 °C for 30 min. After incubation, mitochondria were reisolated by centrifugation at 15,000 times g for 5 min and, where indicated, treated with trypsin (150 µg/ml) for 30 min on ice. After the reaction was stopped by the addition of trypsin inhibitor (final concentration, 0.4 mg/ml), mitochondria were washed twice with 25 mM Tris-HCl buffer, pH 7.4, that contained 0.25 M sucrose. Proteins in samples were precipitated by the addition of trichloroacetic acid at a final concentration of 10% (v/v), and the precipitates were washed with 5% trichloroacetic acid, with ethanol, and finally with diethyl ether. Samples were dissolved with Laemmli's sample buffer(42) , heated, and then analyzed on an SDS-polyacrylamide gel (10%) by the method of Laemmli (42) and subsequently by fluorography.


RESULTS

Isolation of cDNA for Human PPO by Complementation in Vivo of a hemG Mutant of E. coli

Mutants defective in the heme biosynthetic pathway lack aerobic respiratory ability, and even in rich medium they grow very poorly. A complementation test exploiting this phenomenon enabled us to isolate cDNA from a human placental cDNA library that could overcome the poor growth of VSR800, a strain of E. coli with a mutation in the hemG gene. VSR800 cells were spread on LB plates after infection with the amplified human placental cDNA library. Two days later, several larger colonies became visible among numerous dwarf colonies. The phage responsible for the restoration of the normal growth of the hemG mutant were recovered from the large colonies. Each phage clone had an insert of 1.8 kilobase pairs or a shorter one. The clone containing the longest insert was selected and named HPPO18. We then reconfirmed that HPPO18 complemented the mutation responsible for the poor growth of VSR800.

Nucleotide Sequence of the cDNA and the Deduced Amino Acid Sequence of PPO

The nucleotide sequence of cDNA in HPPO18 was determined on both strands. Sequence analysis revealed that the HPPO18 clone contained an insert of 1,767 base pairs with a single long open reading frame that started with an ATG codon at position 278 and ended at position 1,708 (Fig. 1). The open reading frame encoded a protein of 477 amino acids with a calculated molecular mass of 50.8 kDa. The consensus site for polyadenylation, 5`-AATAAA-3`, is present 22 base pairs after a TGA termination codon. A computer-assisted search revealed that the deduced amino acid sequence of human PPO displays a high degree of similarity (27.6%) to that encoded by the hemY gene (13) for the PPO of B. subtilis (Fig. 2). In particular, the portion corresponding to amino acid residues 5-17 is highly conserved between the human protein and that from B. subtilis. The amino-terminal amino acid sequence of PPO can be configured as a betaalphabeta dinucleotide-binding fold, a common structural feature found within FAD- or NAD-binding domains(43) . This finding is supported by the presence of the characteristic motif GXGXXG. This arrangement of glycine residues is typical of the dinucleotide-binding domain of many flavin-containing proteins (Fig. 3), such as amine oxidases(44, 45) , methoxyneurosporene dehydrogenase(46) , flavocytochrome c(47) , 6-hydroxynicotine oxidase(48) , and dimethylglycine dehydrogenase(49) . These results strongly suggest that PPO is a flavin-containing enzyme(11) .


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 betaalphabeta-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.

Expression of Human PPO in COS-1 Cells

We constructed a plasmid (pCDPPO-3) that carried the insert cDNA derived from pBHPPO and transfected COS-1 cells with the plasmid. After 48 h, cells were lysed, and the enzymatic activity was measured. As shown in Fig. 4, the homogenate of cells transfected with pCDPPO-3 yielded enzymatic activity that was 3.6-fold higher than that of cells transfected with pCDPPO-1 (antisense). We next examined the effects of acifluorfen, a specific inhibitor of PPO, on the enzymatic activity. The PPO activity was inhibited to an increasing extent by increasing concentrations of acifluorfen. The effect of acifluorfen on the enzymatic activity in control cells (pCDPPO-1) was similar to that in cells transfected with pCDPPO-3.


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, bullet) and pCDPPO-1 (antisense, circle), were lysed, and PPO activity was measured in the homogenates in the presence of acifluorfen.



Import of Human PPO into Mitochondria in Vitro

Transcription of pBHPPO by T7 RNA polymerase in vitro, followed by translation in the rabbit reticulocyte lysate system in the presence of [S]methionine, produced a major polypeptide of approximately 51 kDa (Fig. 5, lane2), a size consistent with the molecular mass calculated from the deduced amino acid composition of the human PPO. The products of translation were then incubated with isolated mouse mitochondria, and the 51-kDa protein was found in the reisolated mitochondria (Fig. 5, lane3). When the mitochondria were treated with trypsin, the 51-kDa protein was retained (Fig. 5, lane4), an indication that the protein was protected from proteolysis by the mitochondrial membrane. When 0.3% Triton X-100 was included during the treatment with trypsin, the protein was completely digested (Fig. 5, lane5).


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).




DISCUSSION

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 alpha-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 alpha-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 alpha-helix, as suggested by the presence of the FAD-binding domain (Fig. 3), it should not be referred to as an amphiphilic alpha-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.


FOOTNOTES

*
This research was supported by Grant-in-aid for Scientific Research on Priority Areas 04266105 from the Ministry of Education, Science, and Culture of Japan. 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) D38537[GenBank].

§
To whom correspondence should be addressed. Tel.: 81-75-753-4200; Fax: 81-75-791-0271.

(^1)
The abbreviation used is: PPO, protoporphyrinogen oxidase.

(^2)
K. Nishimura, T. Nakayashiki, and H. Inokuchi, unpublished data.


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