The synthesis of murine ferrochelatase in vitro and in vivo

Woodruff

2005

Biochemical Journal

The initial and the terminal three enzymes of the mammalian haem biosynthetic pathway are nuclear encoded, cytoplasmically synthesized and post-translationally translocated into the mitochondrion. The first enzyme, ALAS (5-aminolaevulinate synthase), occurs as an isoenzyme encoded on different chromosomes and is synthesized either as a housekeeping protein (ALAS-1) in all nonerythroid cell types, or only in differentiating erythroid precursor cells (ALAS-2). Both ALAS proteins possess mitochondrial targeting sequences that have putative haem-binding motifs. In the present study, evidence is presented demonstrating that two haembinding motifs in the leader sequence, as well as one present in the N-terminus of the mature ALAS-1 function in vivo in the haemregulated translocation of ALAS-1. Coproporphyrinogen oxidase, the antepenultimate pathway enzyme, possesses a leader sequence that is approx. 120 residues long. In contrast with an earlier report suggesting that only 30 residues were required for translocation of the coproporphyrinogen oxidase, we report that the complete leader is necessary for translocation and that this process is not haem-sensitive in vivo. PPO (protoporphyrinogen oxidase) lacks a typical mitochondrial targeting leader sequence and was found to be effectively targeted by just 17 N-terminal residues. Bacillus subtilis PPO, which is very similar to human PPO at its N-terminal end, is not targeted to the mitochondrion when expressed in mammalian cells, demonstrating that the translocation is highly specific with regard to both the length and spacing of charged residues in this targeting region. Ferrochelatase, the terminal enzyme, possesses a typical N-terminal leader sequence and no evidence of a role for the C-terminus was found in mitochondrial targeting.

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Examination of mitochondrial protein targeting of haem synthetic enzymes: in vivo identification of three functional haem-responsive motifs in 5-aminolaevulinate synthase

Woodruff

2005

Biochemical Journal

“…13 The nuclear-encoded and cytoplasmically synthesized precursor of the enzyme is translocated, proteolytically processed, and bound to the matrix side of the inner mitochondrial membrane. 1,15,16 Assuming an equivalent level of transcription and translation of normal and EPP ferrochelatase alleles, it would be expected that the ratio of homodimeric normal to heterodimeric normal/EPP to homodimeric EPP ferrochelatases would be 1:2:1.…”

Section: Introductionmentioning

confidence: 99%

Production and characterization of erythropoietic protoporphyric heterodimeric ferrochelatases

Najahi‐Missaoui

2005

Blood

Mutations resulting in diminished activity of the dimeric enzyme ferrochelatase are a prerequisite for the inherited disorder erythropoietic protoporphyria (EPP). Patients with clinical EPP have only 10% to 30% of normal levels of ferrochelatase activity, and although many patients with EPP have one mutant allele and one "low-expression" normal allele, the possibility remains that, for some, low ferrochelatase activity may result from an EPP mutation that has an impact on both subunits of the wild-type/mutant heterodimer. Here we present data for 12 ferrochelatase wild-type/EPP mutant heterodimers showing that some mutations result in heterodimers with the residual activity anticipated from individual constituents, whereas others result in heterodimers with significantly lower activity than would be predicted. Although the data do not allow an a priori prediction of heterodimeric residual activity based solely on the in vitro activity of EPP homodimers or the position of the mutated residue within ferrochelatase, mutations that affect the dimer interface or [2Fe-2S] cluster have a significantly greater impact on residual activity than would be predicted. These data suggest that some EPP mutations may result in clinically overt EPP in the absence of a low-expression, wild-type allele; this is of potential significance for genetic counseling of patients with EPP. (Blood. 2005;106:1098-1104)

“…A comparison of all currently known ferrochelatase sequences reveals the existence of three distinct segments or domains (6). The first segment (I) is present in all eukaryotes and is identifiable as an amino-terminal organelle-targeting motif that is proteolytically removed after organelle targeting (12,16). The second (II) represents the core 330 amino acid residues of the enzyme and is present in all ferrochelatases.…”

mentioning

confidence: 99%

Identification of [2Fe-2S] Clusters in Microbial Ferrochelatases

2002

J Bacteriol

The enzyme ferrochelatase (EC 4.99.1.1) catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme IX (heme) (4, 6). This enzyme is present in essentially all eukaryotes and prokaryotes with the exception of a few obligate pathogens and some anaerobic prokaryotes. Recently a gene sequence that appears by homology to be ferrochelatase was reported for the archaeon, Thermoplasma acidophilum (17). A comparison of all currently known ferrochelatase sequences reveals the existence of three distinct segments or domains (6). The first segment (I) is present in all eukaryotes and is identifiable as an amino-terminal organelle-targeting motif that is proteolytically removed after organelle targeting (12, 16). The second (II) represents the core 330 amino acid residues of the enzyme and is present in all ferrochelatases. The third (III) is a 30-to 50-amino-acid-residue sequence found at the carboxyl terminus of some ferrochelatases. Region III of animal ferrochelatases, which is involved in the dimerization motif for these enzymes (21), contains three of the four cysteinyl ligands for a [2Fe-2S] cluster with the fourth cluster ligand present in region II (3,5,19,21). Region III of plant ferrochelatases is approximately 50 residues in length, and its role is currently unknown (6). Plant ferrochelatases do not possess the cysteinyl residues in region III and do not contain [2Fe-2S] clusters. The ferrochelatase of the yeast Saccharomyces cerevisiae, which has been cloned, purified, and characterized (9, 10, 13), possesses region III, does not contain any cysteinyl residues in this region, and does not possess a [2Fe-2S] cluster. The gene for ferrochelatase of the yeast Schizosaccharomyces pombe has been sequenced (GenBank accession number AL022245) and, interestingly, revealed the presence of four cysteinyl residues in the same sequence position as is found in animal ferrochelatases. Previously, as part of a study on the carboxyl termini of eukaryotic ferrochelatases, it was determined that S. pombe ferrochelatase contains a [2Fe-2S] cluster (14).All bacterial ferrochelatases previously biochemically characterized possessed only region II (see references 6 and 11). However, with the plethora of genome sequencing projects, there are now several putative ferrochelatases in the bacterial genome databases which also possess a region III in their derived amino acid sequences. Of the region III-possessing bacterial ferrochelatases identified to date, all contain cysteinyl residues, but none have the same cluster-ligating cysteinyl residue motif as is found in animal ferrochelatases or other [2Fe-2S]-containing proteins.In the present work we have expressed, purified, and characterized ferrochelatases from the bacteria Caulobacter crescentus and Mycobacterium tuberculosis. Both of these possess region III. The C. crescentus enzyme is homodimeric and membrane-associated and contains [2Fe-2S] clusters. However, although the M. tuberculosis ferrochelatase contains region III and a [2Fe-2S] cluster, it is monomeri...