scholarly journals The coproporphyrin ferrochelatase of Staphylococcus aureus: mechanistic insights into a regulatory iron-binding site

2017 ◽  
Vol 474 (20) ◽  
pp. 3513-3522 ◽  
Author(s):  
Charlie Hobbs ◽  
James D. Reid ◽  
Mark Shepherd
Keyword(s):  
Staphylococcus Aureus ◽  
Ferrous Iron ◽  
Substrate Inhibition ◽  
Iron Concentration ◽  
Protoporphyrin Ix ◽  
Site Directed Mutagenesis ◽  
Bacterial Phyla ◽  
Haem Biosynthesis ◽  
And Staphylococcus Aureus ◽  
Dependent Pathway

The majority of characterised ferrochelatase enzymes catalyse the final step of classical haem synthesis, inserting ferrous iron into protoporphyrin IX. However, for the recently discovered coproporphyrin-dependent pathway, ferrochelatase catalyses the penultimate reaction where ferrous iron is inserted into coproporphyrin III. Ferrochelatase enzymes from the bacterial phyla Firmicutes and Actinobacteria have previously been shown to insert iron into coproporphyrin, and those from Bacillus subtilis and Staphylococcus aureus are known to be inhibited by elevated iron concentrations. The work herein reports a Km (coproporphyrin III) for S. aureus ferrochelatase of 1.5 µM and it is shown that elevating the iron concentration increases the Km for coproporphyrin III, providing a potential explanation for the observed iron-mediated substrate inhibition. Together, structural modelling, site-directed mutagenesis, and kinetic analyses confirm residue Glu271 as being essential for the binding of iron to the inhibitory regulatory site on S. aureus ferrochelatase, providing a molecular explanation for the observed substrate inhibition patterns. This work therefore has implications for how haem biosynthesis in S. aureus is regulated by iron availability.

Measurement of ferrochelatase activity using a novel assay suggests that plastids are the major site of haem biosynthesis in both photosynthetic and non-photosynthetic cells of pea (Pisum sativum L.)

2002 ◽  
Vol 362 (2) ◽  
pp. 423-432 ◽  
Author(s):  
Johanna E. CORNAH ◽  
Jennifer M. ROPER ◽  
Davinder Pal SINGH ◽  
Alison G. SMITH
Keyword(s):  
Ferrous Iron ◽  
Specific Activity ◽  
Higher Plants ◽  
Protoporphyrin Ix ◽  
Chlorophyll Synthesis ◽  
Marker Enzyme ◽  
Hexane Extraction ◽  
Higher Plant ◽  
Major Site ◽  
Haem Biosynthesis

Ferrochelatase is the terminal enzyme of haem biosynthesis, catalysing the insertion of ferrous iron into the macrocycle of protoporphyrin IX, the last common intermediate of haem and chlorophyll synthesis. Its activity has been reported in both plastids and mitochondria of higher plants, but the relative amounts of the enzyme in the two organelles are unknown. Ferrochelatase is difficult to assay since ferrous iron requires strict anaerobic conditions to prevent oxidation, and in photosynthetic tissues chlorophyll interferes with the quantification of the product. Accordingly, we developed a sensitive fluorimetric assay for ferrochelatase that employs Co2+ and deuteroporphyrin in place of the natural substrates, and measures the decrease in deuteroporphyrin fluorescence. A hexane-extraction step to remove chlorophyll is included for green tissue. The assay is linear over a range of chloroplast protein concentrations, with an average specific activity of 0.68nmol·min−1·mg of protein−1, the highest yet reported. The corresponding value for mitochondria is 0.19nmol·min−1·mg of protein−1. The enzyme is inhibited by N-methylprotoporphyrin, with an estimated IC50 value of ≈ 1nM. Using this assay we have quantified ferrochelatase activity in plastids and mitochondria from green pea leaves, etiolated pea leaves and pea roots to determine the relative amounts in the two organelles. We found that, in all three tissues, greater than 90% of the activity was associated with plastids, but ferrochelatase was reproducibly detected in mitochondria, at levels greater than the contaminating plastid marker enzyme, and was latent. Our results indicate that plastids are the major site of haem biosynthesis in higher plant cells, but that mitochondria also have the capacity for haem production.

Terminal steps of haem biosynthesis

2002 ◽  
Vol 30 (4) ◽  
pp. 590-595 ◽  
Author(s):  
H. A. Dailey
Keyword(s):  
Crystal Structure ◽  
Bacillus Subtilis ◽  
Ferrous Iron ◽  
Protoporphyrin Ix ◽  
Oxidative Decarboxylation ◽  
Protoporphyrinogen Oxidase ◽  
Coproporphyrinogen Oxidase ◽  
Cellular Location ◽  
Haem Biosynthesis ◽  
Small Set

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 (co-proporphyrinogen 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.

scholarly journals A new class of [2Fe-2S]-cluster-containing protoporphyrin (IX) ferrochelatases

2006 ◽  
Vol 397 (1) ◽  
pp. 47-52 ◽  
Author(s):  
Mark Shepherd ◽  
Tamara A. Dailey ◽  
Harry A. Dailey
Keyword(s):  
Convergent Evolution ◽  
Ferrous Iron ◽  
Myxococcus Xanthus ◽  
Protoporphyrin Ix ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
New Class ◽  
Micro Organisms ◽  
Amino Acid Segment

Protoporphyrin (IX) ferrochelatase catalyses the insertion of ferrous iron into protoporphyrin IX to form haem. These ferrochelatases exist as monomers and dimers, both with and without [2Fe-2S] clusters. The motifs for [2Fe-2S] cluster co-ordination are varied, but in all cases previously reported, three of the four cysteine ligands are present in the 30 C-terminal residues and the fourth ligand is internal. In the present study, we demonstrate that a group of micro-organisms exist which possess protoporphyrin (IX) ferrochelatases containing [2Fe-2S] clusters that are co-ordinated by a group of four cysteine residues contained in an internal amino acid segment of approx. 20 residues in length. This suggests that these ferrochelatases have evolved along a different lineage than other bacterial protoporphyrin (IX) ferrochelatases. For example, Myxococcus xanthus protoporphyrin (IX) ferrochelatase ligates a [2Fe-2S] cluster via cysteine residues present in an internal segment. Site-directed mutagenesis of this ferrochelatase demonstrates that changing one cysteine ligand into serine results in loss of the cluster, but unlike eukaryotic protoporphyrin (IX) ferrochelatases, this enzyme retains its activity. These data support a role for the [2Fe-2S] cluster in iron affinity, and strongly suggest convergent evolution of this feature in prokaryotes.

DECONTAMINATION OF VETERINARY SURVEILLANCE OBJECTS BY NEW GENERATION MEDICINE

1970 ◽  
pp. 64-67
Author(s):  
М. S. Saypullaev ◽  
А. U. Koychuev ◽  
Т. B. Mirzoeva
Keyword(s):  
Staphylococcus Aureus ◽  
Inhalation Exposure ◽  
Gram Negative Bacteria ◽  
Hazard Class ◽  
E Coli ◽  
Highly Efficient ◽  
Mucous Membranes ◽  
Environmentally Safe ◽  
New Generation ◽  
And Staphylococcus Aureus

The successful conduct of disinfection measures largely depends on the availability of veterinary practice a highly efficient, environmentally safe disinfectants. In this regard, finding new highly efficient disinfectant remains relevant. Studies found that the "Polied" (OOO "Razvitie XXI Vek, Russia) can be attributed to the highly efficient and environmentally friendly means. Solutions "Polied" have a high disinfectant activity against smooth and rough surfaces in the laboratory against gram-positive, gram-negative bacteria, mycobacteria and spores of microorganisms. Studies have established that solutions should be "Polied" obezzarajivatmi E. coli (EA 1257) concentrations of 0.1% on smooth surfaces and Staphylococcus aureus concentration of 0.05% in 1 hour from the calculation of 0.25-0.3 litres/m2. Disinfection of rough test surfaces against Escherichia coli and Staphylococcus aureus occurred after treatment with 0,3% solution of 3-hour exposure, at a rate of 0.5 l/m2. It was also found that 1.0% solution "Polied" fully obezzarazhivatel test the surface of mycobacteria (PCs-5) and at double the 0.6% concentration for 24 hours. Disinfection of rough test surfaces contaminated with spores of B. cereus (PCs 96) was achieved with a 4.0% solution at twice the irrigation rate of 0.5 l/m2 at an exposure of 24 hours. Toxicity solutions of the drug "Polied" refer to "moderate" threat (hazard class 3) and low-hazard substances (4 hazard class) when applied to the skin, mucous membranes of the eyes, and inhalation exposure on the respiratory system.

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