Functional analysis of the hemK gene product involvement in protoporphyrinogen oxidase activity in yeast

The Escherichia coli hemK gene has been described as being involved in protoporphyrinogen oxidase activity; however, there is no biochemical evidence for this. In the context of characterizing the mechanisms of protoporphyrinogen oxidation in the yeast Saccharomyces cerevisiae, we investigated the yeast homolog of HemK, which is encoded by the ORF YNL063w, to find out whether it has any protoporphyrinogen oxidase activity and/or whether it modulates protoporphyrinogen oxidase activity. Phenotype analysis and enzyme activity measurements indicated that the yeast HemK homolog is not involved in protoporphyrinogen oxidase activity. Complementation assays in which the yeast HemK homolog is overproduced do not restore wild-type phenotypes in a yeast strain with deficient protoporphyrinogen oxidase activity. Protein sequence analysis of HemK-related proteins revealed consensus motif for S-adenosyl-methionine-dependent methyltransferase.     

Characterisation of the flavin adenine dinucleotide binding region of Myxococcus xanthus protoporphyrinogen oxidase

Protoporphyrinogen oxidase (PPOX), the penultimate enzyme in the haem biosynthetic pathway catalysers the six electron oxidation of protoporphyrinogen-IX to protoporphyrin-IX, in the presence of flavin adenine dinucleotide (FAD) and oxygen. In humans, partial defects in PPOX result in variegate porphyria. In this study, the FAD binding region in Myxococcus xanthus PPOX was analysed by engineering and characterising a selection of mutant proteins. Amino acid residues which interact with FAD via their side chains were selected for study. Mutants were characterised and compared with wild type protein. Characterisation included FAD quantitation, analysis of FAD spectra and kinetic assay. Results revealed that Serine 20 mutants could still bind FAD, but polarity in this position is favourable, yet not essential for the integrity of FAD binding. Study of Glutamate 39 mutants suggest that a negative charge at position 39 is clearly favoured for interaction with the ribose ring of FAD, as all non-conservative replacements could not bind sufficient FAD. Asparagine 441 appears not to be directly involved in FAD binding but rather in stabilizing the FAD, and polarity in this position appears important. Tryptophan 408 may play a role in orientating or stabilizing the bound substrate during catalysis, and a non-polar (or slightly polar) residue is favoured at this position; however, aromaticity in this position appears not to be critical. Overall this study sheds further light on how M. xanthus PPOX interacts with FAD.     

A capillary electrophoresis assay for recombinant Bacillus subtilis protoporphyrinogen oxidase

Protoporphyrinogen oxidase (PPO) is a flavin adenine dinucleotide (FAD)-containing enzyme in the tetrapyrrole biosynthetic pathway that leads to the formation of both heme and chlorophylls, which has been identified as one of the most important action targets of commercial herbicides. The literature reports gave different PPO-catalytic kinetic parameters for the substrate protoporphyrinogen IX (K_m of 0.1 to 10.4 μM) with different sources of PPO using fluorescent or HPLC methods. Herein we assayed the enzymatic activity of recombinant Bacillus subtilis PPO by using capillary electrophoresis (CE), a method with high separation efficiency, easy automation, and low sample consumption. The Michaelis constant and maximum reaction velocity were determined as 7.0 ± 0.6 μM and 0.38 ± 0.02 μmol min^-1 μg⁻¹, respectively. The interaction between PPO and acifluorfen, a commercial PPO-inhibiting herbicide, was measured as the inhibition constant 186.9 ± 9.3 μМ. The relationship between cofactor FAD and PPO activity can also be quantitatively studied by this CE method. The CE method used here should also be a convenient, reliable method for PPO study.     

Mitochondrial targeting of normal and mutant protoporphyrinogen oxidase

We have investigated the signal sequence for mitochondrial transport of mutants (I12T, 78insC, IVS2-2a-->c, 338G-->C, R152C, 470A-->C, and L401F) and the wild type protoporphyrinogen oxidase (PPOX), which is the penultimate enzyme in the heme biosynthesis. We constructed the corresponding green fluorescent protein fusion proteins and studied their intracellular localization in COS-1 cells. We showed that 28 amino acids in the amino terminus of PPOX contain an independently functioning signal for mitochondrial targeting. The experiments with amino-terminally truncated green fluorescent protein fusion proteins revealed that amino acids 25-477 of PPOX contained an additional mitochondrial targeting signal(s). We constructed a structural model for the interaction between the amino-terminal end of PPOX and the putative mitochondrial receptor protein Tom20. The model suggests that leucine and isoleucine residues Leu-8, Ile-12, and Leu-15 forming an alpha-helical hydrophobic motif, LXXXIXXL, were crucial for the recognition of the targeting signal. The validity of the model was tested using mutants L8Q, I12T, and L15Q disrupting the hydrophobic surface of the LXXXIXXL helix. The results from in vitro expression studies and molecular modeling were in accordance supporting the hypothesis that the recognition of the mitochondrial targeting signal is dependent on hydrophobic interactions between the targeting signal and the mitochondrial receptor.     

Dual targeting of Myxococcus xanthus protoporphyrinogen oxidase into chloroplasts and mitochondria and high level oxyfluorfen resistance

Much attention has been paid to the signal sequences of eukaryotic protoporphyrinogen oxidases (protoxes); both the organelles targeted by protoxes and the role of protoxes in conferring resistance against protox‐inhibiting herbicides, such as oxyfluorfen, have been examined. However, there have been no reports on the translocation of prokaryotic protoxes. This study investigated the targeting ability of Myxococcus xanthus protox in vitro and in vivo. In an in vitro translocation assay using a dual import system, M. xanthus protein was detected in chloroplasts and mitochondria, suggesting that the M. xanthus protox protein was targeted into both organelles. In order to confirm the in vitro dual targeting ability of M. xanthus, we used a stable transgenic strategy to investigate dual targeting in vivo. In transgenic rice plants overexpressing M. xanthus protox, M. xanthus protox antibody cross‐reacted with proteins with predicted molecular masses of 50 kDa from both chloroplasts and mitochondria, and this in vivo transgene expression corresponded to a prominent increase in chloroplastic and mitochondrial protox activity. Seeds from the transgenic lines M4 and M7 germinated in solid Murashige and Skoog media of up to 500 µm of oxyfluorfen, whereas wild‐type seeds did not germinate in 1 µm . After 4‐week‐old‐rice plants were treated with oxyfluorfen for 3 d, lines M4 and M7 exhibited normal growth, whereas the wild‐type line was severely bleached and necrotized. The herbicidal resistance is attributed to the insignificant accumulation of photodynamic protoporphyrin IX in cytosol because the high chloroplastic and mitochondrial protox activity in oxyfluorfen‐treated transgenic lines, compared with that in oxyfluorfen‐treated and untreated wild‐type plants, metabolizes protoporphyrinogen IX to chlorophyll and heme. A practical application of the dual targeting of M. xanthus protox for obtaining outstanding resistance to peroxidizing herbicides is discussed.     

A continuous fluorimetric assay for protoporphyrinogen oxidase by monitoring porphyrin accumulation

A continuous spectrofluorimetric assay for protoporphyrinogen oxidase (PPO, EC 1.3.3.4) activity has been developed using a 96-well plate reader. Protoporphyrinogen IX, the tetrapyrrole substrate, is a colorless nonfluorescent compound. The evolution of the fluorescent tetrapyrrole product, protoporphyrin IX, was detected using a fluorescence plate reader. The apparent Km (Kapp) values for protoporphyrinogen IX were measured as 3.8+/-0.3, 3.6+/-0.5, and 1.0+/-0.1 microM for the enzymes from human, Myxococcus xanthus, and Aquifex aeolicus, respectively. The Ki for acifluorfen, a diphenylether herbicide, was measured as 0.53 microM for the human enzyme. Also, the specific activity of mouse liver mitochondrial PPO was measured as 0.043 nmol h-1/mg mitochondria, demonstrating that this technique is useful for monitoring low-enzyme activities. This method can be used to accurately measure activities as low as 0.5 nM min-1, representing a 50-fold increase in sensitivity over the currently used discontinuous assay. Furthermore, this continuous assay may be used to monitor up to 96 samples simultaneously. These obvious advantages over the discontinuous assay will be of importance for both the kinetic characterization of recombinant PPOs and the detection of low concentrations of this enzyme in biological samples.     

Expression of recombinant protoporphyrinogen oxidase influences growth and morphological characteristics in transgenic rice

Transgenic rice plants expressing a Bacillus subtilis protoporphyrinogen oxidase (Protox), the last shared enzyme of the porphyrin pathway, in the cytoplasm (C89) or the plastids (P72) were compared with wild-type rice plants in their growth characteristics. Production of tiller buds 18 d after seeding was more profuse in transgenic plants than in wild-type plants, especially in plastid-targeted plants. Transgenic plants had 12–27% increase in tiller number and 17–33% increase in above-ground biomass compared with wild-type plants 4 and 8 weeks after transplanting of 2-week-old rice seedlings, demonstrating that tiller production and above-ground biomass correlate with each other. Cytoplasm-expressed and plastid-targeted transgenic plants also had a distinct phenotypic characteristic of narrower and more horizontal leaves than wild-type plants. Phenotypic and anatomical characteristics of the transgenic plants were clearly different from wild-type plants, indicating that regulation of porphyrin biosynthesis by expression of B. subtilis Protox in rice influences morphological characteristics of plant growth as well as biomass.     

Design and synthesis of 1-(benzothiazol-5-yl)-1H-1,2,4-triazol-5-ones as protoporphyrinogen oxidase inhibitors

Protoporphyrinogen oxidase (PPO, E.C. 1.3.3.4) is the action target for several structurally diverse herbicides. A series of novel 4-(difluoromethyl)-1-(6-halo-2-substituted-benzothiazol-5-yl)-3-methyl-1H-1,2,4-triazol-5(4H)-ones 2a–z were designed and synthesized via the ring-closure of two ortho-substituents. The in vitro bioassay results indicated that the 26 newly synthesized compounds exhibited good PPO inhibition effects with K_i values ranging from 0.06 to 17.79 μM. Compound 2e, ethyl 2-{[5-(4-(difluoromethyl)-3-methyl-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)-6-fluorobenzo-thiazol-2-yl]thio}acetate, was the most potent inhibitor with K_i value of 0.06 μM against mtPPO, comparable to (K_i = 0.03 μM) sulfentrazone. Further green house assays showed that compound 2f (K_i = 0.24 μM, mtPPO), ethyl 2-{[5-(4-(difluoromethyl)-3-methyl-5-oxo-4,5-dihydro-1H-1,2,4-triazol-1-yl)-6-fluorobenzothiazol-2-yl]thio}propanoate, showed the most promising post-emergence herbicidal activity with broad spectrum even at concentrations as low as 37.5 g ai/ha. Soybean exhibited tolerance to compound 2f at the dosages of 150 g ai/ha, whereas they are susceptible to sulfentrazone even at 75 g ai/ha. Thus, compound 2f might be a potential candidate as a new herbicide for soybean fields.     

Characteristics of murine protoporphyrinogen oxidase.

Protoporphyrinogen oxidase (EC 1.3.3.4) (PPO) is the penultimate enzyme of the heme biosynthetic pathway. Mouse PPO has been purified in low yield and kinetically characterized by this laboratory previously. A new more rapid purification procedure is described herein, and with this protein we detect a noncovalently bound flavin moiety. This flavin is present at approximately stoichiometric amounts in the purified enzyme and has been identified by its fluorescence spectrum and high performance liquid chromatography as flavin mononucleotide (FMN). Fluorescence quenching studies on the flavin yielded a Stern-Volmer quenching constant of 12.08 M-1 for iodide and 1.1 M-1 for acrylamide. Quenching of enzyme tryptophan fluorescence resulted in quenching constants of 6 M-1 and 10 M-1 for iodide and acrylamide, respectively. Plasma scans performed on purified enzyme preparations did not reveal the presence of stoichiometric amounts of protein-bound metal ions, and we were unable to detect any protein-associated pyrroloquinoline quinone (PQQ). Data from circular dichroism studies predict a secondary structure of the native protein consisting of 30.5% alpha helix, 40.5% beta sheet, 13.7% turn, and 15.3% random coil. Denaturation of PPO with urea resulted in a biphasic curve when ellipticity is plotted against urea concentration, typical of amphipathic proteins.     

Expression and characterization of the terminal heme synthetic enzymes from the hyperthermophile Aquifex aeolicus

The terminal two heme biosynthetic pathway enzymes, protoporphyrinogen oxidase and ferrochelatase, of the hyperthermophilic bacterium Aquifex aeolicus have been expressed in Escherichia coli, purified to homogeneity, and biochemically characterized. Ferrochelatase and protoporphyrinogen oxidase of this organism are both monomeric, as was found for the corresponding enzymes of Bacillus subtilis. However, unlike the B. subtilis proteins, both A. aeolicus enzymes are membrane-associated. Both proteins have temperature optima over 60 degrees C. This is the first demonstration of functional heme biosynthetic enzymes in an extreme thermophilic bacterium.     

A point mutation of valine-311 to methionine in Bacillus subtilis protoporphyrinogen oxidase does not greatly increase resistance to the diphenyl ether herbicide oxyfluorfen

In an effort to asses the effect of Val311Met point mutation of Bacillus subtilis protoporphyrinogen oxidase on the resistance to diphenyl ether herbicides, a Val311Met point mutant of B. subtilis protoporphyrinogen oxidase was prepared, heterologously expressed in Escherichia coli, and the purified recombinant Val311Met mutant protoporphyrinogen oxidase was kinetically characterized. The mutant protoporphyrinogen oxidase showed very similar kinetic patterns to wild type protoporphyrinogen oxidase, with slightly decreased activity dependent on pH and the concentrations of NaCl, Tween 20, and imidazole. When oxyfluorfen was used as a competitive inhibitor, the Val311Met mutant protoporphyrinogen oxidase showed an increased inhibition constant about 1.5 times that of wild type protoporphyrinogen oxidase. The marginal increase of the inhibition constant indicates that the Val311Met point mutation in B. subtilis protoporphyrinogen oxidase may not be an important determinant in the mechanism that protects protoporphyrinogen oxidase against diphenyl ether herbicides.     

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.     

Mitochondrial import of human and yeast fumarase in live mammalian cells: retrograde translocation of the yeast enzyme is mainly caused by its poor targeting sequence

Studies on yeast fumarase provide the main evidence for dual localization of a protein in mitochondria and cytosol by means of retrograde translocation. We have examined the subcellular targeting of yeast and human fumarase in live cells to identify factors responsible for this. The cDNAs for mature yeast or human fumarase were fused to the gene for enhanced green fluorescent protein (eGFP) and they contained, at their N-terminus, a mitochondrial targeting sequence (MTS) derived from either yeast fumarase, human fumarase, or cytochrome c oxidase subunit VIII (COX) protein. Two nuclear localization sequences (2x NLS) were also added to these constructs to facilitate detection of any cytosolic protein by its targeting to nucleus. In Cos-1 cells transfected with these constructs, human fumarase with either the native or COX MTSs was detected exclusively in mitochondria in >98% of the cells, while the remainder 1-2% of the cells showed varying amounts of nuclear labeling. In contrast, when human fumarase was fused to the yeast MTS, >50% of the cells showed nuclear labeling. Similar studies with yeast fumarase showed that with its native MTS, nuclear labeling was seen in 80-85% of the cells, but upon fusion to either human or COX MTS, nuclear labeling was observed in only 10-15% of the cells. These results provide evidence that extramitochondrial presence of yeast fumarase is mainly caused by the poor mitochondrial targeting characteristics of its MTS (but also affected by its primary sequence), and that the retrograde translocation mechanism does not play a significant role in the extramitochondrial presence of mammalian fumarase.     

A specific isoform of poly(ADP-ribose) glycohydrolase is targeted to the mitochondrial matrix by a N-terminal mitochondrial targeting sequence

Poly(ADP-ribose) polymerases (PARPs) convert NAD to polymers of ADP-ribose that are converted to free ADP-ribose by poly(ADP-ribose) glycohydrolase (PARG). The activation of the nuclear enzyme PARP-1 following genotoxic stress has been linked to release of apoptosis inducing factor from the mitochondria, but the mechanisms by which signals are transmitted between nuclear and mitochondrial compartments are not well understood. The study reported here has examined the relationship between PARG and mitochondria in HeLa cells. Endogenous PARG associated with the mitochondrial fraction migrated in the range of 60 kDa. Transient transfection of cells with PARG expression constructs with amino acids encoded by exon 4 at the N-terminus was targeted to the mitochondria as demonstrated by subcellular fractionation and immunofluorescence microscopy of whole cells. Deletion and missense mutants allowed identification of a canonical N-terminal mitochondrial targeting sequence consisting of the first 16 amino acids encoded by PARG exon 4. Sub-mitochondrial localization experiments indicate that this mitochondrial PARG isoform is targeted to the mitochondrial matrix. The identification of a PARG isoform as a component of the mitochondrial matrix raises several interesting possibilities concerning mechanisms of nuclear-mitochondrial cross talk involved in regulation of cell death pathways.     

Comparison of the Properties of Semipurified Mitochondrial and Cytosolic Monoamine Oxidases from Rat Brain

Mitochondrial and cytosolic monoamine oxidases were purified 220- and 129-fold, respectively, from rat brain. The purification procedure involved extraction (without the use of detergents for mitochondrial monoamine oxidase), ammonium sulfate precipitation, and chromatography on Sephadex G-25 and a DEAE-cellulose column. The properties of both enzymes with kynuramine as substrate, including Km values and pH optima at different kynuramine concentrations; the Rf values on polyacrylamide gel electrophoresis; and the thermal inactivation patterns were different. 2-Mercaptoethanol, together with heat treatment, released the flavin and decreased the enzyme activity differentially for the two enzymes. The absorption spectrum showed a "Red shift" in the absorption maxima when the spectra of the non-Triton-treated purified preparations were compared with those of the Triton-treated ones, thus possibly revealing that the mitochondrial and the cytosolic monoamine oxidases may be two different enzyme entities.     

Mitochondrial targeting signal in human neuropeptide Y gene

Neuropeptide Y (NPY) is universally expressed in many different neuronal and non-neuronal cells. Human NPY gene has two in-frame kozak sequences and thus, has potentially two translation initiation sites producing two NPY peptides with different molecular weights. In the present study, the intracellular location of NPY was studied in endothelial cells endogenously expressing NPY, and in neuronal (SK-N-BE) and non-neuronal (CHO-K1) cells transfected with NPY-GFP-constructs. By mutating kozak sequences we discovered that kozak-1 directs the NPY peptide to secretory vesicles, and kozak-2 is a prerequisite for mitochondrial targeting. If both kozak sequences are present, non-neuronal cells seem to benefit leaky scanning to initiate translation at both initiation sites, in contrast to neuronal cells, which prefer the kozak-1. This finding suggests that both the kozak sequences of NPY mRNA can be used in the translation depending on the cell type. The size and the function of the novel NPY fragment routed to mitochondria remains to be determined.     

ZFN‐mediated gene targeting of the Arabidopsis protoporphyrinogen oxidase gene through Agrobacterium‐mediated floral dip transformation

Previously, we showed that ZFN‐mediated induction of double‐strand breaks (DSBs) at the intended recombination site enhanced the frequency of gene targeting (GT) at an artificial target locus using Agrobacterium‐mediated floral dip transformation. Here, we designed zinc finger nucleases (ZFNs) for induction of DSBs in the natural protoporphyrinogen oxidase (PPO) gene, which can be conveniently utilized for GT experiments. Wild‐type Arabidopsis plants and plants expressing the ZFNs were transformed via floral dip transformation with a repair T‐DNA with an incomplete PPO gene, missing the 5′ coding region but containing two mutations rendering the enzyme insensitive to the herbicide butafenacil as well as an extra KpnI site for molecular analysis of GT events. Selection on butafenacil yielded 2 GT events for the wild type with a frequency of 0.8 × 10^?3 per transformation event and 8 GT events for the ZFNs expressing plant line with a frequency of 3.1 × 10^?3 per transformation event. Molecular analysis using PCR and Southern blot analysis showed that 9 of the GT events were so‐called true GT events, repaired via homologous recombination (HR) at the 5′ and the 3′ end of the gene. One plant line contained a PPO gene repaired only at the 5′ end via HR. Most plant lines contained extra randomly integrated T‐DNA copies. Two plant lines did not contain extra T‐DNAs, and the repaired PPO genes in these lines were transmitted to the next generation in a Mendelian fashion.     

Structural basis of pyrrole polymerization in human porphobilinogen deaminase

Human porphobilinogen deaminase (PBGD), the third enzyme in the heme pathway, catalyzes four times a single reaction to convert porphobilinogen into hydroxymethylbilane. Remarkably, PBGD employs a single active site during the process, with a distinct yet chemically equivalent bond formed each time. The four intermediate complexes of the enzyme have been biochemically validated and they can be isolated but they have never been structurally characterized other than the apo- and holo-enzyme bound to the cofactor. We present crystal structures for two human PBGD intermediates: PBGD loaded with the cofactor and with the reaction intermediate containing two additional substrate pyrrole rings. These results, combined with SAXS and NMR experiments, allow us to propose a mechanism for the reaction progression that requires less structural rearrangements than previously suggested: the enzyme slides a flexible loop over the growing-product active site cavity. The structures and the mechanism proposed for this essential reaction explain how a set of missense mutations result in acute intermittent porphyria.