SEARCH · PubMed Health

Results for “Coproporphyrinogens”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources

PubMed1978

Factors determining the sequence of oxidative decarboxylation of the 2- and 4-propionate substituents of coproporphyrinogen III by coproporphyrinogen oxidase in rat liver.

Coproporphyrinogen oxidase (EC 1.3.3.3) catalyses the oxidative decarboxylation of the 2- and 4-propionate substituents of coproporphyrinogen III to form protoporphyrinogen IX. A 4-propionate-substituted porphyrinogen, harderoporphyrinogen, which is also a substrate for coproporphyrinogen oxidase, is formed during the reaction. Synthetic [(14)C]coproporphyrinogens III, specifically labelled in the carboxyl carbon atoms of either the 2- or 4-propionate substituents, were used to measure the rate of decarboxylation of each substituent by rat liver coproporphyrinogen oxidase. The experimental results, together with the recognition that in all known substrates of coproporphyrinogen oxidase only those propionate groups flanked by a specific arrangement of substituents are decarboxylated, indicate that the 4-propionate group of coproporphyrinogen III cannot be attacked until the 2-propionate group has been decarboxylated. Production of (14)CO(2) from the substrate labelled in the 2-propionate group therefore measures the formation of harderoporphyrinogen, whereas (14)CO(2) from the 4-propionate-labelled substrate measures protoporphyrinogen IX formation. The rate of harderoporphyrinogen formation is about twice that of protoporphyrinogen, and this ratio is unchanged by varying the concentration of coproporphyrinogen III or by competitive inhibition of the enzyme. When coproporphyrinogen III is present in an excess, two fractions of harderoporphyrinogen can be distinguished. One accumulates during the reaction, and the other, which is destined to become protoporphyrinogen IX, does not equilibrate with added harderoporphyrinogen. It is suggested that both decarboxylations take place at the same active centre, which becomes temporarily inaccessible to coproporphyrinogen III and added harderoporphyrinogen, and that the molecule rotates after the first decarboxylation to allow the second to take place.

Animals↗

PubMed1978

A radiochemical method for the measurement of coproporphyrinogen oxidase and the utilization of substrates other than coproporphyrinogen III by the enzyme from rat liver.

[14C2]Coproporphyrin III, 14C-labelled in the carboxyl carbon atoms of the 2- and 4-propionate substituents, was prepared by stepwise modification of the vinyl groups of protoporphyrin IX. The corresponding porphyrinogen was used as substrate in a specific sensitive assay for coproporphyrinogen oxidase (EC 1.3.3.3) in which the rate of production of 14CO2 is measured. With this method, the Km of the enzyme from rat liver for coproporphyrinogen III is 1.2 micron. Coproporphyrin III is a competitive inhibitor of the enzyme (Ki 7.6 micron). Apparent Km values for other substrates were measured by a mixed-substrate method: that for coproporphyrinogen IV is 0.9 micron and that for harderoporphyrinogen 1.6 micron. Rat liver mitochondria convert pentacarboxylate porphyrinogen III into dehydroisocoproporphyrinogen at a rate similar to that for the formation of protoporphyrinogen IX from coproporphyrinogen III. Mixed-substrate experiments indicate that this reaction is catalysed by coproporphyrinogen oxidase and that the Km for this substrate is 29 micron. It is suggested that the ratio of the concentration of pentacarboxylate porphyrinogen III to coproporphyrinogen III in the hepatocyte determines the relative rates of formation of dehydroisocoproporphyrinogen and protoporphyrinogen IX.

Animals↗

PubMed2001

Normal and abnormal heme biosynthesis. 3.(1)Synthesis and metabolism of tripropionate analogues of coproporphyrinogen-III: novel probes for the active site of coproporphyrinogen oxidase.

Coproporphyrinogen oxidase (copro'gen oxidase) catalyses the oxidative decarboxylation of two propionate side chains on coproporphyrinogen-III to produce protoporphyrinogen-IX. This process is very poorly understood at a molecular level, and copro'gen oxidase remains one of the least well-characterized enzymes in the heme biosynthetic pathway. To provide a rigorous test for a proposed model for substrate recognition and binding by this enzyme, two tripropionate analogues of copro'gen-III were prepared where an ethyl group replaced one of the usual propionate residues on positions 13 or 17. Although the required substrate probes are porphyrinogens (hexahydroporphyrins), the corresponding porphyrin methyl esters were initially synthesized via tripyrrene and a,c-biladiene intermediates. These were hydrolyzed and reduced with 3% sodium-amalgam to give the unstable porphyrinogens needed for the biochemical investigations. The modified structure with a 13-ethyl moiety was metabolized by avian preparations of copro'gen oxidase to give a monovinylic product, but the isomeric 17-ethylporphyrinogen afforded a divinylic product, albeit with poorer overall conversion. These results strongly support the proposed model for substrate binding at the active site of copro'gen oxidase.

Binding Sites↗

PubMed2002

Metabolism of analogues of coproporphyrinogen-III with modified side chains: implications for binding at the active site of coproporphyrinogen oxidase.

Porphyrinogens with modified propionate side chains bearing methyl substituents were found to be modest substrates for coproporphyrinogen oxidase; the results indicate that alteration of the substituents involved in secondary binding interactions has a comparable affect to modifying the side chain that undergoes degradation at the catalytic site.

Animals↗

PubMed1993

A soybean coproporphyrinogen oxidase gene is highly expressed in root nodules.

In plants the enzyme coproporphyrinogen oxidase catalyzes the oxidative decarboxylation of coproporphyrinogen III to protoporphyrinogen IX in the heme and chlorophyll biosynthesis pathway(s). We have isolated a soybean coproporphyrinogen oxidase cDNA from a cDNA library and determined the primary structure of the corresponding gene. The coproporphyrinogen oxidase gene encodes a polypeptide with a predicted molecular mass of 43 kDa. The derived amino acid sequence shows 50% similarity to the corresponding yeast amino acid sequence. The main difference is an extension of 67 amino acids at the N-terminus of the soybean polypeptide which may function as a transit peptide. A full-length coproporphyrinogen oxidase cDNA clone complements a yeast mutant deleted of the coproporphyrinogen oxidase gene, thus demonstrating the function of the soybean protein. The soybean coproporphyrinogen oxidase gene is highly expressed in nodules at the stage where several late nodulins including leghemoglobin appear. The coproporphyrinogen oxidase mRNA is also detectable in leaves but at a lower level than in nodules while no mRNA is detectable in roots. The high level of coproporphyrinogen oxidase mRNA in soybean nodules implies that the plant increases heme production in the nodules to meet the demand for additional heme required for hemoprotein formation.

Amino Acid Sequence↗

PubMed1995

Reduction of coproporphyrinogen oxidase level by antisense RNA synthesis leads to deregulated gene expression of plastid proteins and affects the oxidative defense system.

A full-length cDNA sequence encoding coproporphyrinogen oxidase was inserted in inverse orientation behind a CaMV promoter and transferred to tobacco (Nicotiana tabacum) by standard transformation techniques. Transformants showed reduced coproporphyrinogen oxidase activity and accumulation of photosensitive coproporphyrin(ogen), indicating antisense RNA expression. An inverse correlation was observed between the level of coproporphyrinogen oxidase and transformant phenotype. The latter is characterized by a broad range of growth retardation and necrosis, indicating oxidative leaf damage. Coproporphyrinogen is an apparent chromophore and its excitation finally leads to the production of reactive oxygen. Evidence is presented that indicates a direct correlation between the accumulation of non-metabolized coproporphyrinogen and oxidative damage to cellular structural components. Enzymatic and non-enzymatic antioxidants were investigated. Whereas superoxide dismutase activity increased in transgenic plants, catalase and ascorbate peroxidase activity remained constant. Tocopherol, rather than carotene or zeaxanthin, seemed to be involved in detoxification, indicating the putative localization and allocation of coproporphyrinogen. Expression of coproporphyrinogen oxidase antisense RNA did not significantly influence the level of other enzymes in the chlorophyll metabolic pathway, but deregulated gene expression of nuclear encoded plastid proteins. Accumulation of coproporphyrinogen and/or the resulting effects, such as oxidative stress, impairs a plastid/nuclear signal which may adapt gene expression to the plastid state.

Antioxidants↗

PubMed1993

Coproporphyrinogen oxidase. Purification, molecular cloning, and induction of mRNA during erythroid differentiation.

Coproporphyrinogen oxidase (EC 1.3.3.3), the enzyme involved in the sixth step of heme biosynthesis, was purified to apparent homogeneity from bovine liver; it has a molecular mass of 37,000 daltons. Partial amino acid sequences were determined. Two degenerate oligonucleotides based on the sequences of trypsin-digested peptides were used in a polymerase chain reaction to amplify a 198-base pair fragment of coproporphyrinogen oxidase DNA, using bovine kidney cells cDNA as a starting template. This fragment was used as a hybridization probe to isolate full-length coproporphyrinogen oxidase clones from a mouse erythroleukemia (MEL) cell cDNA library. Sequence analysis revealed that coproporphyrinogen oxidase comprises 354 amino acid residues (M(r) 40,647), with a putative leader sequence of 31 amino acid residues, the result being a mature protein of 323 amino acid residues (M(r) 37,255). RNA blot analysis revealed a 3.0-kilobase coproporphyrinogen oxidase mRNA in mouse liver and in MEL cells. Treatment of MEL cells with dimethyl sulfoxide led to an increase in coproporphyrinogen oxidase mRNA within 10 h, the induction reached a maximum at 24 h, and was in parallel with the induction of ferrochelatase mRNA. The cDNA allows for the expression of active coproporphyrinogen oxidase, the activity of which is mainly present in mitochondria of transfected cultured cells, thereby indicating that mammalian coproporphyrinogen oxidase is mitochondrial enzyme.

Amino Acid Sequence↗

PubMed1994

Molecular cloning, sequencing, and functional expression of a cDNA encoding human coproporphyrinogen oxidase.

Coproporphyrinogen oxidase (EC 1.3.3.3) catalyzes the sixth step in the heme biosynthetic pathway, the oxidation of coproporphyrinogen III to protoporphyrinogen IX. The activity of this enzyme is deficient in the disease hereditary coproporphyria. The sequence of the cDNA and predicted amino acid sequence of the human coproporphyrinogen oxidase are presented. The human protein sequence contains a region completely homologous to that we obtained by sequencing an 11-amino acid peptide fragment from purified murine liver coproporphyrinogen oxidase. Results of Southern blotting were consistent with the presence of a single human coproporphyrinogen oxidase gene, and Northern blotting demonstrated one transcript of similar size in erythroid and nonerythroid cell lines. Expression of the cDNA coding for the putative mature human coproporphyrinogen oxidase in Escherichia coli resulted in a 17-fold increase in coproporphyrinogen activity over endogenous activity.

Amino Acid Sequence↗

PubMed1985

Fluorometric assays for coproporphyrinogen oxidase and protoporphyrinogen oxidase.

We describe fluorometric assays for two enzymes of the heme pathway, coproporphyrinogen oxidase and protoporphyrinogen oxidase. Both assays are based on measurement of protoporphyrin IX fluorescence generated from coproporphyrinogen III by the two consecutive reactions catalyzed by coproporphyrinogen oxidase and protoporphyrinogen oxidase. Both enzymatic activities are measured by recording protoporphyrin IX fluorescence increase in air-saturated buffer in the presence of EDTA (to inhibit ferrochelatase that can further metabolize protoporphyrin IX) and in the presence of dithiothreitol (that prevents nonenzymatic oxidation of porphyrinogens to porphyrins). Coproporphyrinogen oxidase (limiting) activity is measured in the presence of a large excess of protoporphyrinogen oxidase provided by yeast mitochondrial membranes isolated from commercial baker's yeast. These membranes are easy to prepare and are stable for at least 1 year when kept at -80 degrees C. Moreover they ensure maximum fluorescence of the generated protoporphyrin (solubilization effect), avoiding use of a detergent in the incubation medium. The fluorometric protoporphyrinogen oxidase two-step assay is closely related to that already described (J.-M. Camadro, D. Urban-Grimal, and P. Labbe, 1982, Biochem. Biophys. Res. Commun. 106, 724-730). Protoporphyrinogen is enzymatically generated from coproporphyrinogen by partially purified yeast coproporphyrinogen oxidase. The protoporphyrinogen oxidase reaction is then initiated by addition of the membrane fraction to be tested. However, when very low amounts of membrane are used, low amounts of Tween 80 (less than 1 mg/ml) have to be added to the incubation mixture to solubilize protoporphyrin IX in order to ensure optimal fluorescence intensity. This detergent has no effect on the rate of the enzymatic reaction when used at concentrations less than 2 mg/ml. Activities ranging from 0.1 to 4-5 nmol protoporphyrin formed per hour per assay are easily and reproducibly measured in less than 30 min.

Animals↗

PubMed1994

Homozygous hereditary coproporphyria caused by an arginine to tryptophane substitution in coproporphyrinogen oxidase and common intragenic polymorphisms.

Coproporphyrinogen oxidase is a mitochondrial heme-biosynthetic enzyme that converts coproporphyrinogen to protoporphyrinogen. Inherited deficiency of this enzyme causes the human genetic disease hereditary coproporphyria. Recently, we isolated, sequenced and expressed the cDNA encoding human coproporphyrinogen oxidase. This allowed us to investigate the nature of the defect leading to a profound deficiency of coproporphyrinogen oxidase in a patient with homozygous hereditary coproporphyria. Using reverse-transcription, amplification of the cDNA and direct sequencing of the amplified products, we found a point mutation resulted in an arginine to tryptophane substitution (R231W). Expression studies of normal and mutated cDNAs in a bacterial system demonstrated that this substitution resulted in the synthesis of an unstable protein with a residual catalytic activity. This is the first mutation to be found at the coproporphyrinogen oxidase locus. Furthermore, three common polymorphisms within the coproporphyrinogen oxidase gene were detected. Two DNA polymorphisms resulted in amino acids changes (H172N and V194I) and the third one was silent (E230E).

Amino Acid Sequence↗

PubMed2000

Simple and rapid method for the determination of coproporphyrinogen oxidase activity.

Coproporphyrinogen oxidase, the sixth enzyme in the biosynthetic heme pathway, catalyzes the oxidative decarboxylation of coproporphyrinogen III to protoporphyrinogen IX. A reversed-phase high pressure liquid chromatography method was developed to measure coproporphyrinogen oxidase enzymatic activity in rat liver. With this method, the separation, identification and quantification of coproporphyrin III (oxidized substrate) and protoporphyrin IX (oxidized product) present in the assays could be carried out with no need of derivatization and in less than 15 min. Rat and human liver coproporphyrinogen oxidase basal activities determined using this method were 0.41+/-0.05 nmol of protoporphyrin IX/h per mg of hepatic protein and 0.87+/-0.06 protoporphyrin IX/h per mg of hepatic protein, respectively. Kinetic studies showed that optimum pH for rat CPGox is 7.3, and that its activity is linear in the range of protein concentrations and incubation times assayed. The present paper describes a sensitive, specific and rapid fluorometric high performance liquid chromatography method to measure coproporphyrinogen oxidase, which could be applied to the diagnosis of human coproporphyria, and which is also suitable for the study of lead and other metal poisoning that produce alterations in this enzymatic activity.

Adult↗

PubMed2006

The substrate radical of Escherichia coli oxygen-independent coproporphyrinogen III oxidase HemN.

During porphyrin biosynthesis the oxygen-independent coproporphyrinogen III oxidase (HemN) catalyzes the oxidative decarboxylation of the propionate side chains of rings A and B of coproporphyrinogen III to form protoporphyrinogen IX. The enzyme utilizes a 5'-deoxyadenosyl radical to initiate the decarboxylation reaction, and it has been proposed that this occurs by stereo-specific abstraction of the pro-S-hydrogen atom at the beta-position of the propionate side chains leading to a substrate radical. Here we provide EPR-spectroscopic evidence for intermediacy of the latter radical by observation of an organic radical EPR signal in reduced HemN upon addition of S-adenosyl-L-methionine and the substrate coproporphyrinogen III. This signal (g(av) = 2.0029) shows a complex pattern of well resolved hyperfine splittings from at least five different hydrogen atoms. The radical was characterized using regiospecifically labeled (deuterium or 15N) coproporphyrinogen III molecules. They had been generated from a multienzyme mixture and served as efficient substrates. Reaction of HemN with coproporphyrinogen III, perdeuterated except for the methyl groups, led to the complete loss of resolved proton hyperfine splittings. Substrates in which the hydrogens at both alpha- and beta-positions, or only at the beta-positions of the propionate side chains, or those of the methylene bridges, were deuterated showed that there is coupling with hydrogens at the alpha-, beta-, and methylene bridge positions. Deuterium or 15N labeling of the pyrrole nitrogens without labeling the side chains only led to a slight sharpening of the radical signal. Together, these observations clearly identified the radical signal as substrate-derived and indicated that, upon abstraction of the pro-S-hydrogen atom at the beta-position of the propionate side chain by the 5'-deoxyadenosyl radical, a comparatively stable delocalized substrate radical intermediate is formed in the absence of electron acceptors. The observed hyperfine constants and g values show that this coproporphyrinogenyl radical is allylic and encompasses carbon atoms 3', 3, and 4.

Bacillus megaterium↗

PubMed2002

Molecular, immunological, enzymatic and biochemical studies of coproporphyrinogen oxidase deficiency in a family with hereditary coproporphyria.

A 27-year-old woman who had recurrent pain in renal bed since 1998 with increasing character, was stationary admitted. The patient showed dark urine, complained of hair loss and took since 1994 a hormonal oral contraceptive. No photosensitivity was observed. Determinations of urinary porphyrin metabolites in 1998 revealed a porphyria cutanea tarda like excretion pattern with elevations of uro- (1767 nmol/24 hr, normal <29 nmol/24 hr) and heptacarboxyporphyrin (568 nmol/24 hr; normal <4 nmol/24 hr). Follow-up studies in feces showed the characteristics of a hereditary coproporphyria with dominance of coproporphyrin isomer III (total= 1470 nmol/g, isomer III= 93%), (normal: <37 nmol/g, isomer III = 25-35%). The excretion of porphyrin precursors (delta-aminolevulinic acid and porphobilinogen) was increased by taking an ethinylestradiol-cyproteronacetate-preparation, but acute and/or chronic manifestations were not observed. Coproporphyrinogen oxidase activity was decreased to 35% in the patient (normal=138+/-21 pkat/g protein; x+/-s), whereas the activity of red cell uroporphyrinogen decarboxylase was normal. Her mother and both sisters could be verified as heterozygous gene carriers of hereditary coproporphyria by their urinary and fecal excretion parameters and because of reduced coproporphyrinogen oxidase activity up to 50%. The father was normal with respect to his genotype. Molecular analysis revealed a hitherto unknown mutation with the transversion of a cytosine to thymine at nucleotide position 854 in exon 4 of the coproporphyrinogen oxidase gene. The gene defect was confirmed by DGGE in the mother and her three daughters. The investigation of the immunological nature of the defective coproporphyrinogen oxidase gene from the whole family revealed decreased concentrations of coproporphyrinogen oxidase protein in the patient, her mother and her two sisters.

Adult↗

PubMed1986

Negative control of yeast coproporphyrinogen oxidase synthesis by heme and oxygen.

Coproporphyrinogen oxidase (EC 1.3.3.3.) catalyzes the sixth enzymic step of the heme biosynthetic pathway. Coproporphyrinogen oxidase activity is increased in mutant cells of Saccharomyces cerevisiae deficient in heme synthesis and this effect can be partially reversed by the addition of exogenous hemin. A similar increase is found in wild type yeast cells grown anaerobically. The strain-dependent increase varies between 5- and 40-fold. The activity changes are paralleled by similar changes in the steady-state amounts of coproporphyrinogen oxidase protein determined by immunoblotting and the steady-state concentrations of coproporphyrinogen oxidase mRNA estimated by in vitro translation/immunoprecipitation. This demonstrates that coproporphyrinogen oxidase synthesis is regulated by heme and oxygen at a pretranslational level in a negative fashion.

Anaerobiosis↗

PubMed1993

In situ conversion of coproporphyrinogen to heme by murine mitochondria: terminal steps of the heme biosynthetic pathway.

Coproporphyrinogen oxidase (EC 1.3.3.3), protoporphyrinogen oxidase (EC 1.3.3.4), and ferrochelatase (EC 4.99.1.1) catalyze the terminal three steps of the heme biosynthetic pathway. All three are either bound to or associated with the inner mitochondrial membrane in higher eukaryotic cells. A current model proposes that these three enzymes may participate in some form of multienzyme complex with attendant substrate channeling (Grand-champ, B., Phung, N., & Nordmann, Y., 1978, Biochem. J. 176, 97-102; Ferreira, G.C., et al., 1988, J. Biol. Chem. 263, 3835-3839). In the present study we have examined this question in isolated mouse mitochondria using two experimental approaches: one that samples substrate and product levels during a timed incubation, and a second that follows dilution of radiolabeled substrate by pathway intermediates. When isolated mouse mitochondria are incubated with coproporphyrinogen alone there is an accumulation of free protoporphyrin. When Zn is added as a substrate for the terminal enzyme, ferrochelatase, along with coproporphyrinogen, there is formation of Zn protoporphyrin with little accumulation of free protoporphyrin. When EDTA is added to this incubation mixture with Zn, Zn protoporphyrin formation is eliminated and protoporphyrin is formed. We have examined the fate of radiolabeled substrates in vitro to determine if exogenously supplied pathway intermediates can compete with the endogenously produced compounds. The data demonstrate that while coproporphyrinogen is efficiently converted to heme in vitro when the pathway is operating below maximal capacity, exogenous protoporphyrinogen can compete with endogenously formed protoporphyrinogen in heme production.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Cloning, DNA sequence, and complementation analysis of the Salmonella typhimurium hemN gene encoding a putative oxygen-independent coproporphyrinogen III oxidase.

Coproporphyrinogen oxidation is a last step in heme biosynthesis. The biochemically characterized eukaryotic coproporphyrinogen III oxidases have an obligate requirement for molecular oxygen, and a similar enzyme is encoded by the hemF gene in Salmonella typhimurium. Anaerobic heme synthesis requires an oxygen-independent coproporphyrinogen oxidase, which is probably encoded by the hemN gene in S. typhimurium. The hemN gene has been cloned from an insertion mutant. The nucleotide sequence was obtained and used for PCR amplification of the wild-type gene. A single open reading frame was identified as the hemN gene on the basis of its interruption by the insertion mutation and plasmid complementation studies of hemF hemN double mutants. The predicted HemN protein has 38% amino acid sequence identity to a putative anaerobic Rhodobacter sphaeroides coproporphyrinogen oxidase. The hemN RNA 5' end and the inferred transcription initiation site were mapped by primer extension. The 52.8-kDa HemN protein is expressed from the second ATG codon of the hemN open reading frame. An open reading frame with an unknown function directly upstream of hemN has a striking amino acid sequence, including 11 acidic residues in a row.

Amino Acid Sequence↗

PubMed1995

Cloning and characterization of the Escherichia coli hemN gene encoding the oxygen-independent coproporphyrinogen III oxidase.

Coproporphyrinogen III oxidase, an enzyme involved in heme biosynthesis, catalyzes the oxidative decarboxylation of coproporphyrinogen III to form protoporphyrinogen IX. Genetic and biochemical studies suggested the presence of two different coproporphyrinogen III oxidases, one for aerobic (HemF) and one for anaerobic (HemN) conditions. Here we report the cloning of the hemN gene encoding the oxygen-independent coproporphyrinogen III oxidase from Escherichia coli by complementation of a Salmonella typhimurium hemF hemN double mutant. An open reading frame of 1,371 bp encoding a protein of 457 amino acids with a calculated molecular mass of 52.8 kDa was identified. Sequence comparisons revealed 92% amino acid sequence identity to the recently cloned S. typhimurium hemN gene and 35% identity to the Rhodobacter sphaeroides gene. The hemN gene was mapped to 87.3 min of the E. coli chromosome and found identical to open reading frame o459 previously discovered during the genome sequencing project. Complementation of S. typhimurium hemF hemN double mutants with the E. coli hemN gene was detected under aerobic and anaerobic conditions, indicating an aerobic function for HemN. The previously cloned E. coli hemF gene encoding the oxygen-dependent enzyme complemented exclusively under aerobic conditions. Primer extension experiments revealed a strong transcription initiation site 102 bp upstream of the translational start site. DNA sequences with homology to a sigma 70-dependent promoter were detected. Expression of the hemN gene in response to changing environmental conditions was evaluated by using lacZ reporter gene fusions. Under anaerobic conditions, hemN expression was threefold greater than under aerobic growth conditions. Removal of iron from the growth medium resulted in an approximately fourfold decrease of aerobic hemN expression. Subsequent addition of iron restored normal expression.

Aerobiosis↗

PubMed2006

Investigation of the catalytic and structural roles of conserved histidines of human coproporphyrinogen oxidase using site-directed mutagenesis.

BACKGROUND: The catalytic contribution of four conserved histidines of human coproporphyrinogen oxidase (CPO) has been investigated using site-directed mutagenesis to change histidine (H) into alanine (A). MATERIAL/METHODS: The wild-type and mutant enzyme forms were analyzed for their ability to utilize coproporphyrinogen-III, mesoporphyrinogen-VI, and harderoporphyrinogen as substrates. RESULTS: Wild-type CPO had specific activities of 4.9+/-0.9 nmole product/min/mg for coproporphyrinogen-III, 1.7+/-0.7 nmole product/min/mg for mesoporphyrinogen-VI, and 5.1+/-1.8 nmole product/min/mg for harderoporphyrinogen. The four mutant enzymes were catalytically competent with all three substrates, but to varying degrees. The most affected mutant was the H158A enzyme which exhibited approximately 50-fold lower activity than wild-type recombinant CPO. CONCLUSIONS: Thus, His158 of human CPO may have a role in the active site, but none of the conserved histidine residues of human coproporphyrinogen oxidase is essential for catalytic activity although changes in histidines have been implicated in the disease state hereditary coproporphyria.

Amino Acid Sequence↗