Biomaterial Database

Mammalian ferrochelatase. Overexpression in Escherichia coli as a soluble protein, purification and characterization. 1994

G C Ferreira

Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa 33612.

Ferrochelatase (protoheme ferrolyase, EC 4.99.1.1), a membrane-bound protein, catalyzes the terminal step of the heme biosynthesis in all living systems. A cDNA encoding the murine ferrochelatase (Taketani, S., Nakahashi, Y., Osumi, T., and Tokunaga, R. (1990) J. Biol. Chem. 265, 19377-19380) has been expressed in Escherichia coli, using the alkaline phosphatase promoter. Ferrochelatase was not only overexpressed in an active form, but more importantly, was produced as a "soluble protein" (i.e. associated with the soluble bacterial protein fraction). A simple purification from the ferrochelatase overproducing bacterial strain yielded approximately 50 mg of protein/2-3 liters of bacterial culture. Recombinant ferrochelatase exhibited identical physical and catalytic properties to those of mammalian ferrochelatases. Specifically, the recombinant ferrochelatase has iron and porphyrin as substrates, and N-methylprotoporphyrin and metal ions (e.g. Hg2+ and Mn2+), as strong inhibitors of its enzyme activity. The Km values are 112.5 microM for iron and 95 microM for deuteroporphyrin IX, which are in the same range of the Km values determined for the ferrochelatases isolated from natural sources. This report describes the overexpression of a mammalian ferrochelatase in E. coli, as a soluble protein, and its purification from an overproducing strain. The production of a functional and "soluble" ferrochelatase has significance for the pursuit of structural and functional studies of this enzyme.

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D008670	Metals	Electropositive chemical elements characterized by ductility, malleability, luster, and conductance of heat and electricity. They can replace the hydrogen of an acid and form bases with hydroxyl radicals. (Grant & Hackh's Chemical Dictionary, 5th ed)	Metal
D008969	Molecular Sequence Data	Descriptions of specific amino acid, carbohydrate, or nucleotide sequences which have appeared in the published literature and/or are deposited in and maintained by databanks such as GENBANK, European Molecular Biology Laboratory (EMBL), National Biomedical Research Foundation (NBRF), or other sequence repositories.	Sequence Data, Molecular,Molecular Sequencing Data,Data, Molecular Sequence,Data, Molecular Sequencing,Sequencing Data, Molecular
D008970	Molecular Weight	The sum of the weight of all the atoms in a molecule.	Molecular Weights,Weight, Molecular,Weights, Molecular
D011524	Protoporphyrins	Porphyrins with four methyl, two vinyl, and two propionic acid side chains attached to the pyrrole rings. Protoporphyrin IX occurs in hemoglobin, myoglobin, and most of the cytochromes.
D011994	Recombinant Proteins	Proteins prepared by recombinant DNA technology.	Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D002413	Cations, Divalent	Positively charged atoms, radicals or groups of atoms with a valence of plus 2, which travel to the cathode or negative pole during electrolysis.	Divalent Cations
D003001	Cloning, Molecular	The insertion of recombinant DNA molecules from prokaryotic and/or eukaryotic sources into a replicating vehicle, such as a plasmid or virus vector, and the introduction of the resultant hybrid molecules into recipient cells without altering the viability of those cells.	Molecular Cloning
D004926	Escherichia coli	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.	Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D005294	Ferrochelatase	A mitochondrial enzyme found in a wide variety of cells and tissues. It is the final enzyme in the 8-enzyme biosynthetic pathway of HEME. Ferrochelatase catalyzes ferrous insertion into protoporphyrin IX to form protoheme or heme. Deficiency in this enzyme results in ERYTHROPOIETIC PROTOPORPHYRIA.	Heme Synthetase,Porphyrin-Metal Chelatase,Protoheme Ferro-Lyase,Zinc Chelatase,Chelatase, Porphyrin-Metal,Chelatase, Zinc,Ferro-Lyase, Protoheme,Porphyrin Metal Chelatase,Protoheme Ferro Lyase,Synthetase, Heme