Oxidation Of Protoporphyrinogen Research Articles

Design, Synthesis, and Biological Activity of Novel Phenyltriazolinone PPO Inhibitors Containing Five-Membered Heterocycles.

Protoporphyrinogen oxidase (PPO, EC 1.3.3.4) catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX, which is a key step in the synthesis of porphyrins in vivo. PPO inhibitors use protoporphyrinogen oxidase as the target and block the biosynthesis process of porphyrin by inhibiting the activity of the enzyme, eventually leading to plant death. In this paper, phenyl triazolinone was used as the parent structure, and the five-membered heterocycle with good herbicidal activity was introduced by using the principle of substructure splicing. According to the principle of bioisosterism, the sulfur atoms on the thiophene ring were replaced with oxygen atoms. Finally, 33 phenyl triazolinones and their derivatives were designed and synthesized, and their characterizations and biological activities were investigated. The in vitro PPO inhibitory activity and greenhouse herbicidal activity of 33 target compounds were determined, and compound D4 with better activity was screened out. The crop safety determination, field weeding effect determination, weeding spectrum determination, and crop metabolism study were carried out. The results showed that compound D4 showed good safety to corn, soybean, wheat, and peanut but poor selectivity to cotton. The field weeding effect of this compound is comparable to that of the commercial herbicide sulfentrazone. The herbicidal spectrum experiment showed that compound D4 had a wide herbicidal spectrum and a good growth inhibition effect on dicotyledonous weeds. Molecular docking results showed that compound D4 forms a hydrogen bond with amino acid residue Arg-98 in the tobacco mitochondria (mtPPO)-active pocket and forms two π-π stacking interactions with Phe-392. This indicates that compound D4 has stronger PPO inhibitory activity. This indicates that compound D4 has wide prospects for development.

An interaction network in Bacillus subtilis coproporphyrinogen oxidase is essential for the oxidation of protoporphyrinogen IX.

Coproporphyrinogen oxidase (CPO) plays important role in the biosynthesis of heme by catalyzing the coproporphyrinogen III to coproporphyrin III. However, in earlier research, it was regarded as the protoporphyrinogen oxidase (PPO) because it can also catalyze the oxidation of protoporphyrinogen IX to protoporphyrin IX. Identification of the commonalities in CPO and PPO would help us to get a further understanding of the enzyme function. In this work, we explored the role of a non-conserved residue, Asp65 in Bacillus subtilis CPO (bsCPO), whose corresponding residues in PPO from various species are neutral or positive residue (arginine in human PPO or asparagine in tobacco PPO, etc.). We found that Asp65 performs its function by forming a polar interaction network with its surrounding residues in bsCPO, which is important for enzymatic activity. This polar network maintains the substrate binding chamber and stabilizes the micro-environment of the isoalloxazine ring of FAD for the substrate-FAD interaction. Both the comparison of the crystal structures of bsCPO with PPO and our previous work showed that a similar polar interaction network is also present in PPOs. The results confirmed our conjecture that non-conserved residues can form a conserved element to maintain the function of CPO or PPO.

Chloroplast-Localized Protoporphyrinogen IX Oxidase1 Is Involved in the Mitotic Cell Cycle in Arabidopsis.

Protoporphyrinogen IX oxidase1 (PPO1) catalyzes the oxidation of protoporphyrinogen IX to form protoporphyrin IX in the plastid tetrapyrrole biosynthesis pathway and is also essential for plastid RNA editing in Arabidopsis thaliana. The Arabidopsis ppo1-1 mutation was previously shown to be seedling lethal; however, in this study, we showed that the heterozygous ppo1-1/+ mutant exhibited reproductive growth defects characterized by reduced silique length and seed set, as well as aborted pollen development. In this mutant, the second mitotic division was blocked during male gametogenesis, whereas female gametogenesis was impaired at the one-nucleate stage. Before perishing at the seedling stage, the homozygous ppo1-1 mutant displayed reduced hypocotyl and root length, increased levels of reactive oxygen species accumulation and elevated cell death, especially under light conditions. Wild-type seedlings treated with acifluorfen, a PPO1 inhibitor, showed similar phenotypes to the ppo1-1 mutants, and both plants possessed a high proportion of 2C nuclei and a low proportion of 8C nuclei compared with the untreated wild type. Genome-wide RNA-seq analysis showed that a number of genes, including cell cycle-related genes, were differentially regulated by PPO1. Consistently, PPO1 was highly expressed in the pollen, anther, pistil and root apical meristem cells actively undergoing cell division. Our study reveals a role for PPO1 involved in the mitotic cell cycle during gametogenesis and seedling development.

The cyanobacterial protoporphyrinogen oxidase HemJ is a new b-type heme protein functionally coupled with coproporphyrinogen III oxidase

Protoporphyrinogen IX oxidase (PPO), the last enzyme that is common to both chlorophyll and heme biosynthesis pathways, catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX. PPO has several isoforms, including the oxygen-dependent HemY and an oxygen-independent enzyme, HemG. However, most cyanobacteria encode HemJ, the least characterized PPO form. We have characterized HemJ from the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis 6803) as a bona fide PPO; HemJ down-regulation resulted in accumulation of tetrapyrrole precursors and in the depletion of chlorophyll precursors. The expression of FLAG-tagged Synechocystis 6803 HemJ protein (HemJ.f) and affinity isolation of HemJ.f under native conditions revealed that it binds heme b The most stable HemJ.f form was a dimer, and higher oligomeric forms were also observed. Using both oxygen and artificial electron acceptors, we detected no enzymatic activity with the purified HemJ.f, consistent with the hypothesis that the enzymatic mechanism for HemJ is distinct from those of other PPO isoforms. The heme absorption spectra and distant HemJ homology to several membrane oxidases indicated that the heme in HemJ is redox-active and involved in electron transfer. HemJ was conditionally complemented by another PPO, HemG from Escherichia coli. If grown photoautotrophically, the complemented strain accumulated tripropionic tetrapyrrole harderoporphyrin, suggesting a defect in enzymatic conversion of coproporphyrinogen III to protoporphyrinogen IX, catalyzed by coproporphyrinogen III oxidase (CPO). This observation supports the hypothesis that HemJ is functionally coupled with CPO and that this coupling is disrupted after replacement of HemJ by HemG.

Molecular Simulations Bring New Insights into Protoporphyrinogen IX Oxidase/Protoporphyrinogen IX Interaction Modes.

Protoporphyrinogen IX oxidase (PPO, EC 1.3.3.4) catalyzes the oxidation of protoporphyrinogen IX (protogen IX) to protoporphyrin IX (proto IX) in the haem/chlorophyll biosynthetic pathway. Although extensive studies of PPO have already afforded many insights into its biological function and its significance to agriculture and medicine, details of the enzymatic mechanism as well as the nature of the specific amino acids involved in substrate binding still remain largely unknown due to the lack of structural information about protogen IX binding to PPO. In this study, we carried out a detailed and systematic investigation on the binding mode of protogen IX in the Nicotiana tabacum PPO (mtPPO) by performing a computational docking followed by molecular simulations, quantum mechanics calculations, and an integrated analysis. The proposed binding mode was consistent with experimental studies, and several potential key residues that have not been investigated in previous studies, such as Thr70, Arg233, Ser235, Ser474 and Lys477, were identified. In addition, we compared the binding modes of protogen IX in mtPPO and Homo sapiens PPO, and found their differences. Considering the low sequence identity and the differences of biochemical properties among the PPOs from various species, the investigation could provide a valuable basis for the design of PPO inhibitors with high potency and species-selectivity.

Butafenacil: A positive control for identifying anemia- and variegate porphyria-inducing chemicals

Butafenacil is an herbicide that inhibits protoporphyrinogen oxidase (PPOX), an enzyme that catalyzes oxidation of protoporphyrinogen IX to protoporphyrin IX during chlorophyll and heme biosynthesis. Based on a high-content screen, we previously identified butafenacil as a potent inducer of anemia in zebrafish embryos. Therefore, the objective of this study was to begin investigating the utility of butafenacil as a positive control for identifying anemia- and variegate porphyria-inducing chemicals. Static exposure to butafenacil from 5 to 72h post-fertilization (hpf) in glass beakers resulted in a concentration-dependent decrease in arterial circulation at low micromolar concentrations. At 72hpf, the magnitude of butafenacil-induced anemia was similar when embryos were exposed in the presence or absence of light, whereas protoporphyrin accumulation and acute toxicity were significantly lower or absent when embryos were exposed under dark conditions. To identify sensitive developmental windows, we treated embryos to butafenacil from 5, 10, 24, or 48hpf to 72hpf in the presence of light, and found that anemia and protoporphyrin accumulation were present at 72hpf following initiation of exposure at 5 and 10hpf. On the contrary, protoporphyrin accumulation – but not anemia – was present following initiation of exposure at 24hpf. Lastly, protoporphyrin accumulation at 72hpf after exposure from 24 to 48hpf suggests that protoporphyrin was not eliminated over a 24-h recovery period. Collectively, our data suggests that butafenacil may be a reliable positive control for identifying anemia- and variegate porphyria-inducing chemicals.

Leishmania major possesses a unique HemG-type protoporphyrinogen IX oxidase.

Leishmania major was proposed to either utilize haem from its host or partially synthesize the tetrapyrrole from host provided precursors. However, only indirect evidence was available for this partial late haem biosynthetic pathway. Here, we demonstrate that the LMJF_06_1280 gene of L. major encodes a HemG-type PPO (protoporphyrinogen IX oxidase) catalysing the oxidation of protoporphyrinogen IX to protoporphyrin IX. Interestingly, trypanosomatids are currently the only known eukaryotes possessing HemG-type enzymes. The LMJF_06_1280 gene forms a potential transcriptional unit with LMJF_06_1270 encoding CPO (coproporphyrinogen III oxidase) and with LMJF_06_1290 for a cytochrome b5. In vivo function of the L. major hemG gene was shown by the functional complementation of the Escherichia coli ΔhemG strain LG285. Restored haem formation in E. coli was observed using HPLC analyses. Purified recombinant L. major HemG revealed PPO activity in vitro using different ubiquinones and triphenyltetrazolium as electron acceptors. FMN was identified as the L. major HemG cofactor. Active site residues were found to be essential for HemG catalysis. These data in combination with the solved crystal structures of L. major CPO and the physiological proof of a ferrochelatase activity provide clear-cut evidence for a partial haem biosynthetic pathway in L. major.

Identification of a gene essential for protoporphyrinogen IX oxidase activity in the cyanobacterium Synechocystis sp. PCC6803

Protoporphyrinogen oxidase (Protox) catalyses the oxidation of protoporphyrinogen IX to protoporphyrin IX during the synthesis of tetrapyrrole molecules. Protox is encoded by the hemY gene in eukaryotes and by the hemG gene in many γ-proteobacteria, including Escherichia coli. It has been suggested that other bacteria possess a yet unidentified type of Protox. To identify a unique bacterial gene encoding Protox, we first introduced the Arabidopsis hemY gene into the genome of the cyanobacterium, Synechocystis sp. PCC6803. We subsequently mutagenized the cells by transposon tagging and screened the tagged lines for mutants that were sensitive to acifluorfen, which is a specific inhibitor of the hemY-type Protox. Several cell lines containing the tagged slr1790 locus exhibited acifluorfen sensitivity. The slr1790 gene encodes a putative membrane-spanning protein that is distantly related to the M subunit of NADH dehydrogenase complex I. We attempted to disrupt this gene in the wild-type background of Synechocystis, but we were only able to obtain heteroplasmic disruptants. These cells accumulated a substantial amount of protoporphyrin IX, suggesting that the slr1790 gene is essential for growth and Protox activity of cells. We found that most cyanobacteria and many other bacteria possess slr1790 homologs. We overexpressed an slr1790 homolog of Rhodobacter sphaeroides in Escherichia coli and found that this recombinant protein possesses Protox activity in vitro. These results collectively demonstrate that slr1790 encodes a unique Protox enzyme and we propose naming the slr1790 gene "hemJ."

Identification of Escherichia coli HemG as a Novel, Menadione-Dependent Flavodoxin with Protoporphyrinogen Oxidase Activity

Protoporphyrinogen oxidase (PPO, EC 1.3.3.4) catalyzes the six-electron oxidation of protoporphyrinogen IX to the fully conjugated protoporphyrin IX. Eukaryotes and Gram-positive bacteria possess an oxygen-dependent, FAD-containing enzyme for this step, while the majority of Gram-negative bacteria lack this oxygen-dependent PPO. In Escherichia coli, PPO activity is known to be linked to respiration and the quinone pool. In E. coli SASX38, the knockout of hemG causes a loss of measurable PPO activity. HemG is a small soluble protein typical of long chain flavodoxins. Herein, purified recombinant HemG was shown to be capable of a menadione-dependent conversion of protoporphyrinogen IX to protoporphyrin IX. Electrochemical analysis of HemG revealed similarities to other flavodoxins. Interestingly, HemG, a member of a class of the long chain flavodoxin family that is unique to the gamma-proteobacteria, possesses a 22-residue sequence that, when transferred into E. coli flavodoxin A, produces a chimera that will complement an E. coli hemG mutant, indicating that this region confers PPO activity to the flavodoxin. These findings reveal a previously unidentified class of PPO enzymes that do not utilize oxygen as an electron acceptor, thereby allowing gamma-proteobacteria to synthesize heme in both aerobic and anaerobic environments.

Site-directed mutagenesis and computational study of the Y366 active site in Bacillus subtilis protoporphyrinogen oxidase

Protoporphyrinogen IX oxidase (PPO), the last common enzyme of heme and chlorophyll biosynthesis, catalyses the oxidation of protoporphyrinogen IX to protoporphyrin IX, with FAD as cofactor. Among PPO, Bacillus subtilis PPO (bsPPO) is unique because of its broad substrate specificity and resistance to inhibition by diphenylethers. Identification of the activity of bsPPO would help us to understand the catalysis and resistance mechanisms. Based on the modeling and docking studies, we found that Y366 site in bsPPO was adjacent to substrate and FAD. In order to evaluate the functional role of this site, three mutants Y366A Y366E and Y366H were cloned and kinetically characterized. The efficiency of catalysis for Y366A and Y366H reduced to 10% of the wild-type enzyme's activity, while Y366E just retained 1%. Y366E shows large resistance (K (i) = 153.94 microM) to acifluorfen. Molecular docking was carried out to understand the structure and functional relationship of PPO. The experimental results from the site-directed mutagenesis are consistent with the computational studies. The residue at position 366 is seemed to be responsible for substrate binding and catalysis and involved in herbicide resistance of bsPPO.

Complex formation between protoporphyrinogen IX oxidase and ferrochelatase during haem biosynthesis in Thermosynechococcus elongatus

During haem and chlorophyll biosynthesis, flavin-dependent protoporphyrinogen IX oxidase catalyses the six-electron oxidation of protoporphyrinogen IX to form protoporphyrin IX. In the following step, iron is inserted into protoporphyrin IX by ferrochelatase. Based on the solved crystal structures of these enzymes, an in silico model for a complex between these two enzymes was proposed to protect the highly photoreactive intermediate protoporphyrin IX. The existence of this complex was verified by two independent techniques. First, co-immunoprecipitation experiments using antibodies directed against recombinantly produced and purified Thermosynechococcus elongatus protoporphyrinogen IX oxidase and ferrochelatase demonstrated their physical interaction. Secondly, protein complex formation was visualized by in vivo immunogold labelling and electron microscopy with T. elongatus cells. Finally, oxygen-dependent coproporphyrinogen III oxidase, which catalyses the formation of protoporphyrinogen IX, was not found to be part of this complex when analysed with the same methodology.

Montalcino, A zebrafish model for variegate porphyria

Inherited or acquired mutations in the heme biosynthetic pathway leads to a debilitating class of diseases collectively known as porphyrias, with symptoms that can include anemia, cutaneous photosensitivity, and neurovisceral dysfunction. In a genetic screen for hematopoietic mutants, we isolated a zebrafish mutant, montalcino (mno), which displays hypochromic anemia and porphyria. The objective of this study was to identify the defective gene and characterize the phenotype of the zebrafish mutant. Genetic linkage analysis was utilized to identify the region harboring the mno mutation. Candidate gene analysis together with reverse transcriptase polymerase chain reaction was utilized to identify the genetic mutation, which was confirmed via allele-specific oligo hybridizations. Whole mount in situ hybridizations and o-dianisidine staining were used to characterize the phenotype of the mno mutant. mRNA and morpholino microinjections were performed to phenocopy and/or rescue the mutant phenotype. Homozygous mno mutant embryos have a defect in the protoporphyrinogen oxidase (ppox) gene, which encodes the enzyme that catalyzes the oxidation of protoporphyrinogen. Homozygous mutant embryos are deficient in hemoglobin, and by 36 hours post-fertilization are visibly anemic and porphyric. The hypochromic anemia of mno embryos was partially rescued by human ppox, providing evidence for the conservation of function between human and zebrafish ppox. In humans, mutations in ppox result in variegate porphyria. At present, effective treatment for acute attacks requires the administration intravenous hemin and/or glucose. Thus, mno represents a powerful model for investigation, and a tool for future screens aimed at identifying chemical modifiers of variegate porphyria.

Crystal structure of protoporphyrinogen oxidase from Myxococcus xanthus and its complex with the inhibitor acifluorfen.

Protoporphyrinogen IX oxidase, a monotopic membrane protein, which catalyzes the oxidation of protoporphyrinogen IX to protoporphyrin IX in the heme/chlorophyll biosynthetic pathway, is distributed widely throughout nature. Here we present the structure of protoporphyrinogen IX oxidase from Myxococcus xanthus, an enzyme with similar catalytic properties to human protoporphyrinogen IX oxidase that also binds the common plant herbicide, acifluorfen. In the native structure, the planar porphyrinogen substrate is mimicked by a Tween 20 molecule, tracing three sides of the macrocycle. In contrast, acifluorfen does not mimic the planarity of the substrate but is accommodated by the shape of the binding pocket and held in place by electrostatic and aromatic interactions. A hydrophobic patch surrounded by positively charged residues suggests the position of the membrane anchor, differing from the one proposed for the tobacco mitochondrial protoporphyrinogen oxidase. Interestingly, there is a discrepancy between the dimerization state of the protein in solution and in the crystal. Conserved structural features are discussed in relation to a number of South African variegate porphyria-causing mutations in the human enzyme.

GATA-1 Binding to Exon 1 Directs High Level Erythroid-Specific Expression of the Human Protoporphyrinogen Oxidase Gene.

Protoporphyrinogen oxidase (PPOX), the penultimate enzyme in the heme biosynthetic pathway, catalyzes the six-electron oxidation of protoporphyrinogen IX to protoporphyrin IX. Like other heme biosynthetic proteins, PPOX is involved in synthesizing heme for red cells (erythroid-specific expression) and as a cofactor for the respiratory cytochromes (housekeeping expression). To date, little is known about transcriptional regulation of the human PPOX gene (PPOX). We established the molecular basis for erythroid-specific expression of PPOX. Using transient in vitro transfection assays in human erythroleukemic K562 cells we studied tissue-specific expression of PPOX. We found that reporter constructs lacking exon 1 showed a 75% reduction in promoter strength in K562 cells (Figure, no 1 and 2). Hence, in vitro high-level erythroid-specific expression of PPOX is dependent on the presence of exon 1. Examination of erythroid-specific regulatory elements in exon 1 revealed two GATA-1 sites, one consensus (A/T)GATA(A/G) site (GATA-1_II AGATAA) and one non-consensus site, deviating at the first nucleotide (GATA-1_I, CGATAG). To study the relative contribution of these two GATA-1 sites to erythroid-specific transcriptional regulation, we performed in vitro transfections of wild-type and mutant (GATA → GTTA) reporter plasmids in K562 cells. We found that the highest level of transcription depended on the integrity of both sites (Figure, no 5). The consensus GATA-1_II site contributed the most to promoter strength (Figure, no 4). Subsequent electrophoretic mobility shift assay and supershift experiments using K562 nuclear extracts demonstrated that both GATA sites were able to bind GATA-1 in vitro. Our experiments showed that exon 1 was dispensable for PPOX promoter activity in human hepatoma HepG2 cells. Interestingly, in HeLa human cervical carcinoma cells the presence of exon 1 decreased promoter activity. Conclusively, exon 1 of the human PPOX gene contains two GATA-1 binding sites, which are required for high level erythroid-specific expression of PPOX and, in addition, bind GATA-1 in vitro. Our results contribute to a better understanding of the molecular mechanisms involved in differential regulation of the human PPOX promoter in erythroid and non-erythroid cells. [Display omitted]

Crystal structure of protoporphyrinogen IX oxidase: a key enzyme in haem and chlorophyll biosynthesis.

Protoporphyrinogen IX oxidase (PPO), the last common enzyme of haem and chlorophyll biosynthesis, catalyses the oxidation of protoporphyrinogen IX to protoporphyrin IX. The membrane-embedded flavoprotein is the target of a large class of herbicides. In humans, a defect in PPO is responsible for the dominantly inherited disease variegate porphyria. Here we present the crystal structure of mitochondrial PPO from tobacco complexed with a phenyl-pyrazol inhibitor. PPO forms a loosely associated dimer and folds into an FAD-binding domain of the p-hydroxybenzoate-hydrolase fold and a substrate-binding domain that enclose a narrow active site cavity beneath the FAD and an alpha-helical membrane-binding domain. The active site architecture suggests a specific substrate-binding mode compatible with the unusual six-electron oxidation. The membrane-binding domains can be docked onto the dimeric structure of human ferrochelatase, the next enzyme in haem biosynthesis, embedded in the opposite side of the membrane. This modelled transmembrane complex provides a structural explanation for the uncoupling of haem biosynthesis observed in variegate porphyria patients and in plants after inhibiting PPO.

Terminal steps of haem biosynthesis.

The terminal three steps in haem biosynthesis are the oxidative decarboxylation of coproporphyrinogen III to protoporphyrinogen IX, followed by the six-electron oxidation of protoporphyrinogen to protoporphyrin IX, and finally the insertion of ferrous iron to form haem. Interestingly, Nature has evolved distinct enzymic machinery to deal with the antepenultimate (coproporphyrinogen oxidase) and penultimate (protoporphyrinogen oxidase) steps for aerobic compared with anaerobic organisms. The terminal step is catalysed by the enzyme ferrochelatase. This enzyme is clearly conserved with regard to a small set of essential catalytic residues, but varies significantly with regard to size, subunit composition, cellular location and the presence or absence of a [2Fe-2S] cluster. Coproporphyrinogen oxidase and protoporphyrinogen oxidase are reviewed with regard to their enzymic and physical characteristics. Ferrochelatase, which is the best characterized of these three enzymes, will be described with particular emphasis paid to what has been learned from the crystal structure of the Bacillus subtilis and human enzymes.

Resistance of a soybean cell line to oxyfluorfen by overproduction of mitochondrial protoporphyrinogen oxidase.

The diphenyl ether herbicide oxyfluorfen (2-chloro-4-trifluoromethylphenyl 3-ethoxy-4-nitrophenyl ether) inhibits protoporphyrinogen oxidase (Protox) which catalyzes the oxidation of protoporphyrinogen IX (Protogen) to protoporphyrin IX (Proto IX), the last step of the common pathway to chlorophyll and haeme biosynthesis. We have selected an oxyfluorfen-resistant soybean cell line by stepwise selection methods, and the resistance mechanism has been investigated. No growth inhibition was observed in resistant cells at a concentration of 10(-7) M oxyfluorfen, a concentration at which normal cells did not survive. While the degree of inhibition of total extractable Protox by oxyfluorfen was the same in both cell types, the enzyme activity in the mitochondrial fraction from non-treated resistant cells was about nine-fold higher than that from normal cells. Northern analysis of mitochondrial Protox revealed that the concentration of mitochondrial Protox mRNA was much higher in resistant cells than that in normal cells. There were no differences in the absorption and metabolic breakdown of oxyfluorfen. The growth of resistant cells was also insensitive to oxadiazon [5-tert-butyl-3-(2,4-dichloro-5-isopropoxyphenyl)-1,3,4-oxadiazol-2-(3H)- one], the other chemical class of Protox inhibitor. Therefore, the resistance of the selected soybean cell line to oxyfluorfen is probably mainly due to the overproduction of mitochondrial Protox.

Oxidation of protoporphyrinogen IX in Escherichia coli is mediated by the aerobic coproporphyrinogen oxidase.

Protoporphyrinogen oxidase, the penultimate enzyme involved in the biosynthetic pathway for heme, catalyzes the removal of six electrons from protoporphyrinogen IX to generate protoporphyrin IX. In Escherichia coli, this enzyme is encoded by the hemG gene. In this study we examined possible alternate pathways for the oxidation of protoporphyrinogen IX to protoporphyrin IX, by isolating and investigating E. coli mutants that can still grow normally when the hemG gene is disrupted. One of these mutants was characterized in detail and had a mutation in the promoter region of the hemF gene, which encodes aerobic coproporphyrinogen oxidase, the enzyme involved in the step immediately before protoporphyrinogen oxidase. Measurement of the promoter activity of the hemF gene showed that the level of transcription was elevated by the mutation. Overexpression of a wild-type hemF gene cloned in a multicopy plasmid also restored the growth of deltahemG strain. Extracts from cells that overexpress hemF exhibited an increased ability to oxidize protoporphyrinogen IX to protoporphyrin IX. These findings suggest that the E. coli aerobic coproporphyrinogen oxidase has an intrinsic capacity to oxidize not only coproporphyrinogen III but also protoporphyrinogen IX.

Measurement of Protoporphyrinogen Oxidase Activity

Protoporphyrinogen oxidase catalyzes the oxidation of protoporphyrinogen to protophyrin. It is a membrane-bound mitochondrial enzyme and it is the target of photobleaching herbicides. The basic assay presented in this unit for measuring oxidase activity is based on oxidation of the colorless, nonfluorescent substrate, protoporphyrinogen, to the colored, fluorescent protophyrin. Alternate protocols are provided for the measuring the accumulation of protoporphyrinogen resulting from a decrease in oxidase activity due to treatment with diphenyl ether herbicides or oxidase inhibitor.

Identification of an FAD Superfamily Containing Protoporphyrinogen Oxidases, Monoamine Oxidases, and Phytoene Desaturase: EXPRESSION AND CHARACTERIZATION OF PHYTOENE DESATURASE OFMYXOCOCCUS XANTHUS

A large number of FAD-containing proteins have previously been shown to contain a signature sequence that is referred to as the dinucleotide binding motif. Protoporphyrinogen oxidase (PPO), the penultimate enzyme of the heme biosynthetic pathway, is an FAD-containing protein that catalyzes the six electron oxidation of protoporphyrinogen IX. Sequence analysis demonstrates the presence of the dinucleotide binding motif at the amino-terminal end of the protein. Analysis of the current data base reveals that PPO has significant sequence similarities to mammalian monoamine oxidases (MAO) A and B, as well as to bacterial and plant phytoene desaturases (PHD). Previously MAOs have been shown to contain FAD, but there are no publications demonstrating the presence of FAD in purified PHDs. We have carried out the expression and purification of PHD from the bacterium Myxococcus xanthus and demonstrate the presence of noncovalently bound FAD. Sequence analysis demonstrate that PPO is closely related to bacterial PHDs and more distantly to plant PHDs and animal MAOs. Interestingly bacterial MAOs are no more closely related to PPOs, PHDs, and animal MAO's than they are to the unrelated Pseudomonas phenyl hydroxylase. All of the related sequences contain not only the basic putative dinucleotide binding motif that is found frequently for FAD-binding proteins, but they also have high similarity in an approximately 60-residue long region that extends beyond the dinucleotide motif. This region is not found among any other proteins in the current data base and, therefore, we propose that this region is a signature motif for a superfamily of FAD-containing enzymes that is comprised of PPOs, animal MAOs, and PHDs.