Crystal structure of the transcription factor sc-mtTFB offers insights into mitochondrial transcription.

Although it is commonly accepted that binding of mitochondrial transcription factor sc-mtTFB to the mitochondrial RNA polymerase is required for specific transcription initiation in Saccharomyces cerevisiae, its precise role has remained undefined. In the present work, the crystal structure of sc-mtTFB has been determined to 2.6 A resolution. The protein consists of two domains, an N-terminal alpha/beta-domain and a smaller domain made up of four alpha-helices. Contrary to previous predictions, sc-mtTFB does not resemble Escherichia coli sigma-factors but rather is structurally homologous to rRNA methyltransferase ErmC'. This suggests that sc-mtTFB functions as an RNA-binding protein, an observation standing in contradiction to the existing model, which proposed a direct interaction of sc-mtTFB with the mitochondrial DNA promoter. Based on the structure, we propose that the promoter specificity region is located on the mitochondrial RNA polymerase and that binding of sc-mtTFB indirectly mediates interaction of the core enzyme with the promoter site.

Human ferrochelatase: characterization of substrate-iron binding and proton-abstracting residues.

The terminal step in heme biosynthesis, the insertion of ferrous iron into protoporphyrin IX to form protoheme, is catalyzed by the enzyme ferrochelatase (EC 4.99.1.1). A number of highly conserved residues identified from the crystal structure of human ferrochelatase as being in the active site were examined by site-directed mutagenesis. The mutants Y123F, Y165F, Y191H, and R164L each had an increased K(m) for iron without an altered K(m) for porphyrin. The double mutant R164L/Y165F had a 6-fold increased K(m) for iron and a 10-fold decreased V(max). The double mutant Y123F/Y191F had low activity with an elevated K(m) for iron, and Y123F/Y165F had no measurable activity. The mutants H263A/C/N, D340N, E343Q, E343H, and E343K had no measurable enzyme activity, while E343D, E347Q, and H341C had decreased V(max)s without significant alteration of the K(m)s for either substrate. D340E had near-normal kinetic parameters, while D383A and H231A had increased K(m)s for iron. On the basis of these data and the crystal structure of human ferrochelatase, it is proposed that residues E343, H341, and D340 form a conduit from H263 in the active site to the protein exterior and function in proton extraction from the porphyrin macrocycle. The role of H263 as the porphyrin proton-accepting residue is central to catalysis since metalation only occurs in conjunction with proton abstraction. It is suggested that iron is transported from the exterior of the enzyme at D383/H231 via residues W227 and Y191 to the site of metalation at residues R164 and Y165 which are on the opposite side of the active site pocket from H263. This model should be general for mitochondrial membrane-associated eucaryotic ferrochelatases but may differ for bacterial ferrochelatases since the spatial orientation of the enzyme within prokaryotic cells may differ.

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.

Ferrochelatase at the millennium: structures, mechanisms and [2Fe-2S] clusters.

Ferrochelatase (E.C. 4.99.1.1, protoheme ferrolyase) catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme (heme). In the past 2 years, the crystal structures of ferrochelatases from the bacterium Bacillus subtilis and human have been determined. These structures along with years of biophysical and kinetic studies have led to a better understanding of the catalytic mechanism of ferrochelatase. At present, the complete DNA sequences of 45 ferrochelatases from procaryotes and eucaryotes are available. These sequences along with direct protein studies reveal that ferrochelatases, while related, vary significantly in amino acid sequence, molecular size, subunit composition, solubility, and the presence or absence of nitric-oxide-sensitive [2Fe-2S] cluster.

Human ferrochelatase: crystallization, characterization of the [2Fe-2S] cluster and determination that the enzyme is a homodimer.

Ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) catalyzes the terminal step in the heme biosynthetic pathway, the insertion of ferrous iron into protoporphyrin IX to form protoheme IX. Previously we have demonstrated that the mammalian enzyme is associated with the inner surface of the inner mitochondrial membrane and contains a nitric oxide sensitive [2Fe-2S] cluster that is coordinated by four Cys residues whose spacing in the primary sequence is unique to animal ferrochelatase. We report here the characterization and crystallization of recombinant human ferrochelatase with an intact [2Fe-2S] cluster. Gel filtration chromatography and dynamic light scattering measurements revealed that the purified recombinant human ferrochelatase in detergent solution is a homodimer. EPR redox titrations of the enzyme yield a midpoint potential of -453+/-10 mV for the [2Fe-2S] cluster. The form of the protein that was crystallized has a single Arg to Leu substitution. This mutation has no detectable effect on enzyme activity but is critical for crystallization. The crystals belong to the space group P2(1)2(1)2(1) and have unit cell constants of a=93.5 A, b=87.7 A, and c=110.2 A. There are two molecules in the asymmetric unit and the crystals diffract to better than 2.0 A resolution. The Fe to Fe distance of the [2Fe-2S] cluster is calculated to be 2.7 A based upon the Bijvoet difference Patterson map.

Molecular characterization of homozygous variegate porphyria.

Variegate porphyria (VP) is a low penetrance, autosomal dominant disorder that results from partial deficiency of protoporphyrinogen oxidase (PPOX) activity caused by mutation in the PPOX gene. The rare homozygous variant of VP is characterized by severe PPOX deficiency, onset of photosensitization by porphyrins in early childhood, skeletal abnormalities of the hand and, less constantly, short stature, mental retardation and convulsions. We have identified PPOX mutations on both alleles of five of the 11 unrelated patients with homozygous VP reported to date. Two patients were homoallelic for missense mutations (D349A and A433P), while three were heteroallelic. Functional analysis by prokaryotic expression showed that the D349A and A433P and one missense mutation in each of the three heteroallelic patients (G358R in two patients and A219KANA) preserved some PPOX activity (9.5-25% of wild-type). Mutations on the other allele of the heteroallelic patients abolished or markedly decreased activity. There was no relation between genotype assessed by functional analysis and the presence or severity of non-cutaneous manifestations. The mutations were absent from 104 unrelated patients with autosomal dominant VP. Our findings define the molecular pathology of homozygous VP and suggest that mild PPOX mutations occur in the general population but have very low or no clinical penetrance in heterozygotes.

Purification of and kinetic studies on a cloned protoporphyrinogen oxidase from the aerobic bacterium Bacillus subtilis.

The previously cloned and expressed protoporphyrinogen oxidase from Bacillus subtilis has been purified to homogeneity by Ni2+ affinity chromatography using a His6 tag and characterized. The enzyme has a molecular weight of approximately 56,000 daltons, a pI of 7.5, a pH optimum (protoporphyrinogen) of 8.7, and a noncovalently bound flavine adenine dinucleotide cofactor. The Michaelis constants (Km) for protoporphyrinogen-IX, coproporphyrinogen-III, and mesoporphyrinogen-IX are 1.0, 5.29, and 4.92 microM, respectively. Polyclonal antibody to B. subtilis protoporphyrinogen oxidase demonstrated weak cross-reactivity with both human and Myxococcus xanthus protoporphyrinogen oxidase. B. subtilis protoporphyrinogen oxidase is not inhibited by the diphenyl ether herbicide acifluorfen at 100 microM and is weakly inhibited by methylacifluorfen at the same concentration. Bilirubin, biliverdin, and hemin are all competitive inhibitors of this enzyme.

Identification of an FAD superfamily containing protoporphyrinogen oxidases, monoamine oxidases, and phytoene desaturase. Expression and characterization of phytoene desaturase of Myxococcus 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.

Examination of ferrochelatase mutations that cause erythropoietic protoporphyria.

Ferrochelatase (E.C. 4.99.1.1), the enzyme that catalyzes the terminal step in the heme biosynthetic pathway, is the site of defect in the human inherited disease erythropoietic protoporphyria (EPP). Previously it has been demonstrated that patients with EPP may have missense mutations leading to amino acid substitutions, early chain termination, or exon deletions. While it has been clearly demonstrated that two missense mutations result in lowered enzyme activity, it has never been shown what effect specific exon deletions may have. In the current work, recombinant human ferrochelatase has been engineered to have individual exon deletions corresponding to exons 3 through 11. When expressed in Escherichia coli, none of these possesses significant enzyme activity and all lack the [2Fe-2S] cluster. One of the human missense mutations, F417S, and a series of amino acid replacements at this site (ie, F417W, F417Y, and F417L) were examined. With the exception of F417L, all lacked enzyme activity and did not contain the [2Fe-2S] cluster in vivo or as isolated in vitro.

Characteristics of human protoporphyrinogen oxidase in controls and variegate porphyrias.

Protoporphyrinogen oxidase (E.C.1.3.3.4) (PPO) catalyzes the penultimate step in the heme biosynthetic pathway. Deficiency in activity of this enzyme results in the human genetic disease variegate porphyria. Herein we detail the cloning, expression, purification and characterization of the normal and variegate porphyria forms of human PPO. The cDNA sequence for human ppo is approximately 1.8 kb in length and codes for a protein of 477 amino acids. This protein, which does not contain a typical cleavable mitochondrial targeting sequence, is approximately 51 kDa and contains a putative dinucleotide binding motif near the amino terminus. The active enzyme is a homodimer and contains an FAD. Attachment of a six his amino terminal tag allows for the rapid and efficient purification of approximately 10 mg of enzyme from one liter of E. coli culture. Three variegate porphyria mutant PPO enzymes were expressed and characterized. These mutations, R59W, R168C and A433P, result in decreased enzyme activity by causing a decrease in kcat without a significant change in Km for the substrate protoporphyrinogen IX. Purified R59W lacks the FAD cofactor which may be explained by the fact that this mutation resides within the dinucleotide binding motif of PPO.

Expression and purification of mammalian 5-aminolevulinate synthase.

We have described a procedure for production and purification of recombinant, mature-length mouse ALAS-2. The fact that E. coli utilizes the C5 path for ALA production means that there is no problem with contamination of the recombinant ALAS-2 by host cell enzyme, such as one may have with a yeast expression system. While the detailed procedure produces enzyme in good yield with relatively common protein purification techniques, future expression systems may be developed to take advantage of the rapid purification achieved by the use of a 6-histidine (His6) aminoterminal tag and metal chelate chromatography. Such approaches in this laboratory with protoporphyrinogen oxidase, coproporphyrinogen oxidase, and uroporphyrinogen decarboxylase have resulted in the production and purification of enzymes whose kinetic and physical parameters are essentially identical to those of proteins lacking the His6 tag.

A R59W mutation in human protoporphyrinogen oxidase results in decreased enzyme activity and is prevalent in South Africans with variegate porphyria.

Variegate porphyria (VP), a low-penetrant autosomal dominant inherited disorder of haem metabolism, is characterised by photosensitivity (Fig. 1) and a propensity to develop acute neuropsychiatric attacks with abdominal pain, vomiting, constipation, tachycardia, hypertension, psychiatric symptoms and, in the worst cases, quadriplegia. Acute attacks, often precipitated by inappropriate drug therapy, are potentially fatal. While earlier workers thought the distal haem biosynthetic enzyme ferrochelatase may be involved in the genesis of VP, it was shown in the early 1980's, and is now accepted, that VP is associated with decreased protoporphyrinogen oxidase activity (PPO) (E.C.1.3.3.4). VP prevalence is much higher in South Africa than elsewhere; probably due to a founder effect with patients descending from a 17th century Dutch immigrant. PPO cDNAs from Bacillus subtilis, Myxococcus xanthus, human placenta and mouse liver have been cloned, sequenced and expressed. Human and mouse cDNAs consist of open reading frames 1431 nucleotides long, encoding a 477 amino acid protein. The human PPO gene contains thirteen exons, spanning approximately 4.5 kb. We have identified a C to T transition in codon 59 (in exon 3) resulting in an arginine to tryptophan substitution (R59W). A protein expressed from an in vitro-mutagenized PPO construct exhibits substantially less activity than the wild type. The R59W mutation was present in 43 of 45 patients with VP from 26 of 27 South African families investigated, but not in 34 unaffected relatives or 9 unrelated British patients with PPO deficiency. Since at least one of these families is descended from the founder of South African VP, this defect may represent the founder gene defect associated causally with VP in South Africa.

Protoporphyrinogen oxidase of Myxococcus xanthus. Expression, purification, and characterization of the cloned enzyme.

Protoporphyrinogen oxidase (EC 1.3.3.4) catalyzes the six electron oxidation of protoporphyrinogen IX to protoporphyrin IX. The enzyme from the bacterium Myxococcus xanthus has been cloned, expressed, purified, and characterized. The protein has been expressed in Escherichia coli using a Tac promoter-driven expression plasmid and purified to apparent homogeneity in a rapid procedure that yields approximately 10 mg of purified protein per liter of culture. Based upon the deduced amino acid sequence the molecular weight of a single subunit is 49,387. Gel permeation chromatography in the presence of 0.2% n-octyl-beta-D-glucopyranoside yields a molecular weight of approximately 100,000 while SDS gel electrophoresis shows a single band at 50,000. The native enzyme is, thus, a homodimer. The purified protein contains a non-covalently bound FAD but no detectable redox active metal. The M. xanthus enzyme utilizes protoporphyrinogen IX, but not coproporphyrinogen III, as substrate and produces 3 mol of H2O2/mol of protoporphyrin. The apparent Km and kcat for protoporphyrinogen in assays under atmospheric concentrations of oxygen are 1.6 microM and 5.2 min-1, respectively. The diphenyl ether herbicide acifluorfen at 1 microM strongly inhibits the enzyme's activity.

Human protoporphyrinogen oxidase: expression, purification, and characterization of the cloned enzyme.

Protoporphyrinogen oxidase (E.C.1.3.3.4) catalyzes the oxygen-dependent oxidation of protoporphyrinogen IX to protoporphyrin IX. The enzyme from human placenta has been cloned, sequenced, expressed in Escherichia coli, purified to homogeneity, and characterized. Northern blot analysis of eight different human tissues show evidence for only a single transcript in all tissue types and the size of this transcript is approximately 1.8 kb. The human cDNA has been inserted into an expression vector for E. coli and the protein produced at high levels in these cells. The protein is found in both membrane and cytoplasmic fractions. The enzyme was purified to homogeneity in the presence of detergents using a metal chelate affinity column. The purified protein is a homodimer composed of subunits of molecular weight of 51,000. The enzyme contains one noncovalently bound FAD per dimer, has a monomer extinction coefficient of 48,000 at 270 nm and contains no detectable redox active metals. The apparent K(m) and Kcat for protoporphyrinogen IX are 1.7 microM and 10.5 min-1, respectively. The enzyme does not use coproporphyrinogen III as a substrate and is inhibited by micromolar concentrations of the herbicide acifluorfen. Protein database searches reveal significant homology between protoporphyrinogen oxidase and monoamine oxidase.

Cloning, sequence, and expression of mouse protoporphyrinogen oxidase.

Protoporphyrinogen oxidase (EC 1.3.3.4) is the penultimate enzyme in the heme biosynthetic pathway, catalyzing the six-electron oxidation of protoporphyrinogen to protoporphyrin. A dominantly inherited genetic deficiency in this enzyme results in the disease variegate porphyria. We now report the cloning, sequence, and expression of mouse protoporphyrinogen oxidase. The cDNA for mouse protoporphyrinogen oxidase was obtained by complementation of Escherichia coli SASX38, a protoporphyrinogen oxidase-deficient strain, with a mouse erythroleukemia (MEL) cell expression library. The sequence of this cDNA along with 5' untranslated sequence obtained by 5' rapid amplification of cDNA ends of MEL cell mRNA is 1814 bp in length and contains an open reading frame of 1431 bp. This encodes a protein of 477 amino acid residues with a calculated molecular weight of 50,870. The protein as expressed in E. coli is sensitive to inhibition by the diphenyl ether herbicide acifluorfen. Northern blot analyses of RNA from uninduced and induced MEL cells as well as mouse hepatoma cells all show two major mRNA species of 1.8 and 3.6 kb.

A new mutation of the ALAS2 gene in a large family with X-linked sideroblastic anemia.

X-linked sideroblastic anemia is a genetic disorder characterized by a hypochromic microcytic anemia of variable intensity with the presence of ring sideroblasts in the bone marrow of the patients. Two different mutations have been reported in the ALAS2 gene in patients with this disease. We have studied a large kindred with a pyridoxine-sensitive form of X-linked sideroblastic anemia. Sequencing amplified cDNA of the proband revealed a guanine-to-adenine change at nucleotide 871 of the coding sequence (exon 7 of the gene). This results in a glycine to serine substitution that is responsible for a marked decrease in the enzymatic activity of the mutated protein. A polymerase chain reaction assay demonstrated the presence of the same mutation in three affected males and two female carriers in the kindred. The carrier status was excluded in eight females at risk. Early detection of the mutant allele in family members may thus be important for the prevention of anemia in males and of iron overload both in affected males and carrier females.

Mammalian ferrochelatase. Expression and characterization of normal and two human protoporphyric ferrochelatases.

Ferrochelatase (EC 4.99.1.1) catalyzes the terminal step in the heme biosynthetic pathway, the insertion of ferrous iron into protoporphyrin IX. Herein we report the expression, purification, and characterization of the mature processed form of human and mouse ferrochelatase in Escherichia coli JM109. Metal analysis of the recombinant normal human ferrochelatase reveals that there are approximately 2 iron atoms/molecule of enzyme. This, along with the presence of spectral absorbance near 320 nm, is strongly suggestive that recombinant mammalian ferrochelatase as expressed in E. coli may contain an iron sulfur cluster. Two human protoporphyric ferrochelatases, F417S and M267I, were also expressed and characterized. The M267I mutant possesses the same Km and Vmax as the normal enzyme but exhibits increased thermolability when compared with normal human ferrochelatase. The F417S mutant has less than 2% of the normal activity. Since the Phe-->Ser substitution in this mutation is both chemically and structurally significant, three single amino acid substitutions (Lys, Tyr, and Trp) were engineered and characterized. None of these resulted in a protein with wild type activity. Additionally the carboxyl-terminal 10-amino acid segment, which contains Phe-417, from the yeast sequence was substituted, but this construct had no activity. Elimination of the carboxyl-terminal 30 amino acid residues (which include Phe-417) results in a protein the same length as the bacterial ferrochelatases, but it is an inactive enzyme.

Expression of a cloned protoporphyrinogen oxidase.

The previously cloned hem Y gene of Bacillus subtilis (Hansson, M., and Hederstedt, L. (1992) J. Bacteriol. 174, 8081-8093) has been expressed in Escherichia coli. The expressed protein has been shown to be the penultimate enzyme of the heme biosynthetic pathway, protoporphyrinogen oxidase (EC 1.3.3.4) and, thus, the gene designation should be hem G. This represents the first report of the expression of a cloned protoporphyrinogen oxidase from any source. The enzyme is present in the soluble cytoplasmic fraction and is, thus, unlike all previously reported eukaryotic or prokaryotic protoporphyrinogen oxidases, which are membrane-bound. It utilizes molecular oxygen as a terminal electron acceptor, and protoporphyrinogen IX, mesoporphyrinogen IX, and coproporphyrinogen III serve as substrates. The diphenyl ether herbicide acifluorfen, which is a strong inhibitor of the eukaryotic enzyme, is only weakly inhibitory. The enzyme has a predicted molecular weight of 51,200, which corresponds well with molecular weight determination via high performance liquid chromatography and SDS-polyacrylamide gel electrophoresis. In addition the enzyme contains a putative dinucleotide binding region at the amino terminus, which is consistent with the previously demonstrated presence of a flavin moiety in the characterized mammalian enzymes.