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S C Andrews

Publications and source records attributed to S C Andrews.

At least 37 records · Page 2Linked to original sources
PubMed1994

Iron incorporation into ferritins: evidence for the transfer of monomeric Fe(III) between ferritin molecules and for the formation of an unusual mineral in the ferritin of Escherichia coli.

Iron that has been oxidized by H-chain ferritin can be transferred into other ferritin molecules before it is incorporated into mature ferrihydrite iron cores. Iron(III) dimers are formed at the ferroxidase centres of ferritin H chains at an early stage of Fe(II) oxidation. Mössbauer spectroscopic data now show that the iron is transferred as monomeric species arising from dimer dissociation and that it binds to the iron core of the acceptor ferritin. Human H-chain ferritin variants containing altered threefold channels can act as acceptors, as can the ferritin of Escherichia coli (Ec-FTN). A human H-chain ferritin variant with a substituted tyrosine (rHuHF-Y34F) can act as a donor of Fe(III). Since an Fe(III)-tyrosinate (first identified in bullfrog H-chain ferritin) is absent from variant rHuHF-Y34F, the Fe(III) transferred is not derived from this tyrosinate complex. Mössbauer parameters of the small iron cores formed within Ec-FTN are significantly different from those of mammalian ferritins. Analysis of the spectra suggests that they are derived from both ferrihydrite and non-ferrihydrite components. This provides further evidence that the ferritin protein shell can influence the structure of its iron core.

Escherichia coli↗
PubMed1994

Direct observation of the iron binding sites in a ferritin.

X-Ray analysis of the ferritin of Escherichia coli (Ec-FTN) and of Ec-FTN crystals soaked in (NH4)2Fe(SO4)2 has revealed the presence of three iron-binding sites per subunit. Two of these form a di-iron site in the centre of the subunit as has been proposed for the 'ferroxidase centres' of human ferritin H chains. This di-iron site, lying within the 4-alpha-helix bundle, resemble those of ribonucleotide reductase, methane monoxygenase and haemerythrin. The third iron is bound by ligands unique to Ec-FTN on the inner surface of the protein shell. It is speculated that this state may represent the nucleation centre of a novel type of Fe(III) cluster, recently observed in Ec-FTN.

Bacterial Proteins↗
PubMed1994

Escherichia coli possesses two homologous anaerobic C4-dicarboxylate membrane transporters (DcuA and DcuB) distinct from the aerobic dicarboxylate transport system (Dct).

The nucleotide sequences of two Escherichia coli genes, dcuA and dcuB (formerly designated genA and genF), have been shown to encode highly homologous products, M(r) 45,751 and 47,935 (434 and 446 amino acid residues) with 36% sequence identity (63% similarity). These proteins have a high proportion (approximately 61%) of hydrophobic residues and are probably members of a new group of integral inner membrane proteins. The locations of the dcu genes, one upstream of the aspartase gene (dcuA-aspA) and the other downstream of the anaerobic fumarase gene (fumB-dcuB), suggested that they may function in the anaerobic transport of C4-dicarboxylic acids. Growth tests and transport studies with mutants containing insertionally inactivated chromosomal dcuA and dcuB genes show that their products perform analogous and mutually complementary roles as anaerobic dicarboxylate carriers. The anaerobic dicarboxylate transport systems (Dcu) are genetically and functionally distinct from the aerobic system (Dct).

Aerobiosis↗
PubMed1993

Overproduction, purification and characterization of the Escherichia coli ferritin.

Recent studies have indicated that Escherichia coli possesses at least two iron-storage proteins, the haem-containing bacterioferritin and ferritin. The ferritin protein has been amplified 600-fold to 11-14% of total cell protein in a bfr mutant and purified to homogeneity with an overall yield of 13%. The cellular ferritin content remained relatively constant throughout the growth cycle and amplification was accompanied by a 2.5-fold increase in cellular iron content. The isolated ferritin contained 5-20 non-haem iron atoms/holomer and resembled the eukaryotic ferritins rather than the prokaryotic bacterioferritins in containing no haem. The 24 subunits of this ferritin (M(r) 19,400) assemble into a spherical protein shell (12 +/- 1 nm diameter, M(r) 465,000) which sequesters at least 2000 iron atoms in vitro to form an electron-dense iron core of 7.9 +/- 1 nm diameter. Electron-microscopic and Mössbauer spectroscopic studies with iron-loaded ferritin showed that the core can be either crystalline (ferrihydrite) or amorphous, depending on the absence or presence of phosphate, respectively. Mössbauer spectroscopy with intact E. coli revealed a novel-high spin Fe(II) component which is enhanced in bacteria amplified for ferritin but not in the parental strain. Western blotting showed that ferritin and bacterioferritin are immunologically distinct proteins. E. coli is thus an organism containing both a ferritin and a bacterioferritin and the relative roles of the two iron-storage proteins are discussed in this study.

Amino Acid Sequence↗
PubMed1993

Kinetic and structural characterization of an intermediate in the biomineralization of bacterioferritin.

The mechanism by which iron-storage proteins take up and oxidise iron(II) is not understood. We show by rapid-kinetic and EPR measurements that iron uptake, in vitro, by a bacterial iron-storage protein, bacterioferritin, involves at least three kinetically distinguishable phases: phase 1, the binding of Fe(II) ions, probably at a dimeric iron ferroxidase centre; phase 2, oxidation of the Fe(II) dimer and production of mononuclear Fe(III); and phase 3, iron core formation.

Bacterial Proteins↗
PubMed1993

An EPR investigation of non-haem iron sites in Escherichia coli bacterioferritin and their interaction with phosphate. A study using nitric oxide as a spin probe.

EPR studies of bacterioferritin (BFR), an iron-storage protein of Escherichia coli [1993, Biochem. J. 292, 47-56], have revealed the presence of non-haem iron (III) (NHI) sites within the protein coat which may be involved in iron uptake and release. When nitric oxide was used as an EPR spin probe of the Fe(II) state of the NHI sites, two distinct mononuclear NHI species were found. Under certain conditions, an iron dimer was also observed. The reaction of phosphate with NHI species has been investigated. Results point to a function for this anion in core nucleation.

Bacterial Proteins↗
PubMed1993

Haem and non-haem iron sites in Escherichia coli bacterioferritin: spectroscopic and model building studies.

The bacterioferritin (BFR) of Escherichia coli is an iron-storage protein containing 24 identical subunits and between three and 11 protohaem IX groups per molecule. Titration with additional haem gave a maximum loading of 12-14 haems per molecule. The e.p.r. spectra and magnetic c.d. spectra of the protein-bound haem show it to be low-spin Fe(III), and coordinated by two methionine residues as previously reported for BFRs isolated from Pseudomonas aeruginosa and Azotobacter vinelandii [Cheesman, Thomson, Greenwood, Moore and Kadir, Nature (London) (1990) 346, 771-773]. A recent sequence alignment indicated that BFR may be structurally related to ferritin. The molecular model proposed for E. coli BFR has a four-alpha-helix-bundle subunit conformation and a quaternary structure similar to those of mammalian ferritins. In this model there are two types of hydrophobic pocket within which two methionine residues are correctly disposed to bind haem. The e.p.r. spectra also reveal a monomeric non-haem Fe(III) species with spin, S = 5/2. On the basis of sequence comparisons, a ferroxidase centre has recently been proposed to be present in BFR [Andrews, Smith, Yewdall, Guest and Harrison (1991) FEBS Lett. 293, 164-168] and the possibility that this Fe(III) ion may reside at or near the ferroxidase centre is discussed.

Amino Acid Sequence↗
PubMed1993

Overproduction, purification and characterization of the bacterioferritin of Escherichia coli and a C-terminally extended variant.

The bacterioferritin (BFR) of Escherichia coli is an iron-sequestering haemoprotein composed of 24 identical polypeptide chains forming an approximately spherical protein shell with a central iron-storage cavity. BFR and BFR-lambda, a variant with a 14-residue C-terminal extension, have been amplified (120-fold and 50-fold, respectively), purified by a new procedure and characterized. The overproduced BFR exhibited properties similar to those of natural BFR, but the iron content (25-75 non-haem Fe atoms/molecule) was 13-39-fold lower. Two major assembly states of BFR were detected, a 24-subunit protein (tetracosamer) and a novel haem-containing subunit dimer. BFR-lambda subunits assembled into tetracosamers having the same external-surface properties as BFR, presumably because their C-terminal extensions project into and occupy about 60% of the central cavity. As a result, BFR-lambda failed totake up iron under conditions that allowed incorporation into BFR in vitro. The haem content of BFR-lambda (1-2 haems/tetracosamer) was lower than that of BFR (3.5-10.5 haems/tetracosamer) and this, together with a difference in the visible spectra of the two haemoproteins, suggested that the C-terminal extensions in BFR-lambda perturb the haem-binding pockets. A subunit dimer form of BFR-lambda was not detected. A combination of Mössbauer spectroscopy and electron diffraction showed that the BFR loaded with iron in vitro has a ferrihydrite-like iron core, whereas the in-vivo loaded protein has an amorphous core.

Amino Acid Sequence↗
PubMed1992

Isolation of a ferritin from Bacteroides fragilis.

A ferritin was isolated from the obligate anaerobe Bacteroides fragilis. Estimated molecular masses were 400 kDa for the holomer and 16.7 kDa for the subunits. A 30-residue N-terminal amino acid sequence was determined and found to resemble the sequences of other ferritins (human H-chain ferritin, 43% identity; Escherichia coli gen-165 product, 37% identity) and to a lesser degree, bacterioferritins (E. coli bacterioferritin, 20% identity). The protein stained positively for iron, and incorporated 59Fe when B. fragilis was grown in the presence of [59Fe]citrate. However, the isolated protein contained only about three iron atoms per molecule, and contained no detectable haem. This represents the first isolation of a ferritin protein from bacteria. It may alleviate iron toxicity in the presence of oxygen.

Amino Acid Sequence↗
PubMed1992

The haemoglobin-like protein (HMP) of Escherichia coli has ferrisiderophore reductase activity and its C-terminal domain shares homology with ferredoxin NADP+ reductases.

Three soluble ferrisiderophore reductases (FsrA, FsrB and FsrC) were detected in Escherichia coli. FsrB was purified and identified as the haemoglobin-like protein (HMP) by size and N-terminal sequence analyses. HMP was previously isolated as a dihydropteridine reductase and is now shown to have ferrisiderophore reductase activity. Database searches revealed that the C-terminal region of HMP (FsrB) is homologous to members of a family of flavoprotein oxidoreductases which includes ferredoxin NADP+ reductase (FNR). The combination of FNR-like and haemoglobin-like regions in HMP (FsrB) represents a novel pairing of functionally and structurally distinct domains. Structure-function properties of other FNR-like proteins, including LuxG and VanB, are also discussed.

Bacterial Proteins↗
PubMed1991

Bacterioferritins and ferritins are distantly related in evolution. Conservation of ferroxidase-centre residues.

Iron-storage proteins can be divided into two classes; the bacterioferritins and ferritins. In spite of many apparent structural and functional analogies, no significant amino acid sequence similarity has been detected previously. This report now reveals a distant evolutionary relationship between bacterioferritins and ferritins derived by 'Profile Analysis'. Optimum alignment of bacterioferritin and ferritin sequences suggests that key residues of the ferroxidase centres of ferritins are conserved in bacterioferritins.

Amino Acid Sequence↗
PubMed1991

Physical, chemical and immunological properties of the bacterioferritins of Escherichia coli, Pseudomonas aeruginosa and Azotobacter vinelandii.

The 70-amino-acid-residue N-terminal sequence of the bacterioferritin (BFR) of Azotobacter vinelandii was determined and shown to be highly similar to the N-terminal sequences of the Escherichia coli and Nitrobacter winogradskyi bacterioferritins. Electrophoretic and immunological analyses further indicate that the bacterioferritins of E. coli, A. vinelandii and Pseudomonas aeruginosa are closely related. A novel, two-subunit assembly state that predominates over the 24-subunit form of BFR at low pH was demonstrated. The results indicate that the bacterioferritins form a family of proteins that are distinct from the ferritins of plants and animals.

Amino Acid Sequence↗
PubMed1991

A molecular analysis of the 53.3 minute region of the Escherichia coli linkage map.

The coding characteristics of four plasmids expressing a protein (BCP) which comigrates with bacterioferritin were examined and the nucleotide sequence of a common 1985 bp segment from the 53 min region of the Escherichia coli linkage map was determined. Three open reading-frames (orf1, orf2 and orf3) were detected, and orf2 (bcp, 156 amino acid codons) appeared to encode the bacterioferritin comigratory protein, BCP. The translation product of orf3 (205 amino acid codons) resembled the iron-sulphur protein component (DMS B subunit) of the anaerobic dimethylsulphoxide reductase complex of E. coli.

Amino Acid Sequence↗
PubMed1989

Amino acid sequence of the bacterioferritin (cytochrome b1) of Escherichia coli-K12.

The complete amino acid sequence of bacterioferritin (cytochrome b1) from Escherichia coli-K12 has been derived from the nucleotide sequence of the cloned gene. It comprises 158 amino acid residues giving an Mr of 18,495. The identity of the gene product was confirmed by an 87 residue N-terminal sequence obtained from the purified protein, but it differs significantly from much of the previously published partial amino acid sequence (1). Secondary structure prediction indicates a high alpha-helical content consistent with a 4-helix-bundle conformation. The fully assembled bacterioferritin molecule comprising 24 identical subunits and 12 haem moieties is a tetracosamer with an Mr of approximately 452,000.

Amino Acid Sequence↗
PubMed1989

Ferritin subunits in livers of siderotic mice.

The major ferritin species of mouse liver has been resolved by SDS-PAGE into two bands similar to the H and L subunits of rat liver ferritin with the L subunit predominating. Amino acid sequencing has confirmed the major, faster-migrating component as L chain. An additional, electrophoretically fast, minor ferritin was isolated from siderosome-containing subcellular fractions. In denaturing gels it gave a single 'F' subunit band of about 17 kDa, significantly smaller than the L and H subunits (about 20 and 21 kDa respectively). A small fragment isolated from the fast ferritin was sequenced. It corresponds to a 19-residue C-terminal peptide cleaved from L subunits in the assembled molecules. The F subunit must be derived from L subunits by loss of this peptide, and is not the expression product of a different gene. 'Fast' ferritins of siderotic mice and rats are thus analogous.

Amino Acid Sequence↗