Location of the catalytic site of the respiratory fumarate reductase of Escherichia coli

The location of the catalytic site of the membrane-bound respiratory fumarate reductase of Escherichia coli was investigated using mutants and inhibitors of dicarboxylic acid transport. Comparison of apparent Km and Vmax values for fumarate in intact cells and in inverted membrane vesicles showed that externally added fumarate was required to be transported across the cytoplasmic membrane prior to reduction. The catalytic site of fumarate reductase must therefore be located on the cytoplasmic face of the membrane.     

Investigation of Escherichia coli fumarate reductase subunit function using transposon Tn5

Seventy two Tn5 transposon insertions were isolated in the frd operon carried on the multicopy plasmid pFRD79. The polar nature of these mutations permitted examination of the expression and localization of the frd polypeptides in novel subunit combinations. The minimal catalytic unit is the FRDA plus B dimer. A transposon within frdB (frdB::Tn5) produces inactive, soluble FRDA polypeptide which has covalently attached 8 alpha(N3-histidyl)flavin adenine dinucleotide cofactor. A transposon mutation within frdC (frdC::Tn5) produces soluble, catalytically active dimer. An insertion in frdD (frdD::Tn5) produces both a soluble trimer composed of FRDABC, and a tetramer of FRDABC and truncated FRDD bound to the inner membrane. Eighty percent of the activity is in the soluble form. Using this mutant, the requirement for FRDD both for optimal activity of the catalytic domain and for proper anchorage in the cytoplasmic membrane was demonstrated.     

The nucleotide sequence of the gene encoding the K99 subunit of enterotoxigenic Escherichia coli

Abstract The nucleotide sequence of the gene encoding the K99 fimbrial subunit of enterotoxigenic Escherichia coli was determined. It appeared that the subunit is composed of 159 amino acid residues preceded by a N-terminal signal sequence of 22 amino acid residues. The secondary structure of the mature K99 polypeptide and the location of potential antigenic determinants were predicted. A comparison was made between the amino acid sequence of the K99 subunit and the subunits of other fimbrial adhesins.     

Nucleotide sequence of genes coding for dicyclohexylcarbodiimide-binding protein and the alpha subunit of proton-translocating ATPase of Escherichia coli

A liquid membrane electrode has been made which is selective for ethidium ion. The membrane is formed in a capillary by a 3-nitro-o-xylene solution of an ethidium-tetraphenyl borate complex. The electrode emf (vs saturated KCl-calomel reference) has a linear dependence upon the logarithm of ethidium concentration from 2 μM to 0.5 mM. The electrode is used here to measure free ethidium ion in mixtures with calf thymus DNA. The binding isotherms obtained are in general agreement with a control photometric titration and with literature results. Direct measurement of free ethidium concentration by convenient potentiometric methods is useful in the study of ligand binding to nucleic acids and to related compounds.     

The nucleotide sequence of the Escherichia coli rts gene

The nucleotide sequence of rts, an essential Escherichia coli gene, has been determined. Transformation of an rts mutant with the plasmid, pJAF1, containing the rts gene resulted in rescue of the defect. The transformation experiments indicate that the rts gene is distinct from the flanking birA, tRNA and tufB genes.     

Assembly of Escherichia coli fumarate reductase holoenzyme

The production and assembly of the four fumarate reductase polypeptides into holoenzyme was studied in vivo in a T7-promoter-conditional expression system. No posttranslational modification of any of the subunits was detected, although the ratio of polypeptides produced varied with the temperature at which expression occurred. FrdC and FrdD, the membrane anchor polypeptides, assembled rapidly into the membrane and then were capped with FrdA and FrdB in separate events. Truncation of the C-terminal domain of FrdD by insertion of transposon Tn5 into the frdD cistron interfered with membrane insertion of the anchor polypeptides and assembly of the holoenzyme. Proteolytic degradation of truncated FrdD was implicated in the production of a soluble FrdABC trimer.     

DNA sequence of the gene coding for Escherichia coli ribonuclease H

The gene for Escherichia coli ribonuclease H has been studied by use of a plasmid which contains a segment of the E. coli chromosome. The genomic DNA was subcloned from pLC28-22 to pBR322 by use of various restriction enzymes. Such subcloning limited the RNase H gene to a piece of DNA no longer than 760 base pairs. Cells bearing plasmids containing the RNase H gene produce as much as 10-15 times the normal amount of RNase H without any drastic effect on maintenance of the plasmid or cell growth. DNA sequence analysis has permitted the prediction of a protein whose molecular weight is 17,559 (155 amino acid residues). The predicted sequence was confirmed by amino acid analysis, NH2-terminal amino acid sequence, and size determination of highly purified RNase H.     

Succinate dehydrogenase and fumarate reductase from Escherichia coli

Succinate-ubiquinone oxidoreductase (SQR) as part of the trichloroacetic acid cycle and menaquinol-fumarate oxidoreductase (QFR) used for anaerobic respiration by Escherichia coli are structurally and functionally related membrane-bound enzyme complexes. Each enzyme complex is composed of four distinct subunits. The recent solution of the X-ray structure of QFR has provided new insights into the function of these enzymes. Both enzyme complexes contain a catalytic domain composed of a subunit with a covalently bound flavin cofactor, the dicarboxylate binding site, and an iron–sulfur subunit which contains three distinct iron–sulfur clusters. The catalytic domain is bound to the cytoplasmic membrane by two hydrophobic membrane anchor subunits that also form the site(s) for interaction with quinones. The membrane domain of E. coli SQR is also the site where the heme b₅₅₆ is located. The structure and function of SQR and QFR are briefly summarized in this communication and the similarities and differences in the membrane domain of the two enzymes are discussed.     

Interactions of oxaloacetate with Escherichia coli fumarate reductase

Fumarate reductase of Escherichia coli is converted to a deactivated state when tightly bound by oxaloacetate (OAA). Incubation of the inhibited enzyme with anions or reduction of the enzyme by substrate restores both the activity of the enzyme and its sensitivity to thiol reagents. In these respects the enzyme behaves like cardiac succinate dehydrogenase. Close to an order of magnitude difference was found to exist between the affinities of OAA for the oxidized (KD approximately 0.12 microM) and reduced (KD approximately 0.9 microM) forms of fumarate reductase. Redox titrations of deactivated fumarate reductase preparations have confirmed that reductive activation, as in cardiac succinate dehydrogenase (B. A. C. Ackrell, E. B. Kearney, and D. Edmondson (1975) J. Biol. Chem. 250, 7114-7119), is the result of reduction of the covalently bound FAD moiety and not the non-heme iron clusters of the enzyme. However, the processes differed for the two enzymes; activation of fumarate reductase involved 2e- and 1H+, consistent with reduction of the flavin to the anionic hydroquinone form, whereas the process requires 2e- and 2H+ in cardiac succinate dehydrogenase. The reason for the difference is not known. The redox potential of the FAD/FADH2 couple in FRD (Em approximately -55 mV) was also slightly more positive than that in cardiac succinate dehydrogenase (-90 mV).     

Complete nucleotide sequence of the Escherichia coli recB gene.

The complete nucleotide sequence of the Escherichia coli recB gene which encodes a subunit of the ATP-dependent DNase, Exonuclease V, has been determined. The proposed coding region for the RecB protein is 3543 nucleotides long and would encode a polypeptide of 1180 amino acids with a calculated molecular weight of 133,973. The start of the recB coding sequence overlaps the 3' end of the upstream ptr gene, and the recB termination codon overlaps the initiation codon of the downstream recD gene, suggesting that these genes may form an operon. No sequences which reasonably fit the consensus for an E. coli promoter could be identified upstream of the proposed recB translational start. The predicted RecB amino acid sequence contains regions of homology with ATPases, DNA binding proteins and DNA repair enzymes.     

Activated conformers of Escherichia coli sulfite reductase heme protein subunit

The heme protein subunit of Escherichia coli sulfite reductase shows enhanced reactivity with its substrate and a number of other ligands after a cycle of reduction and reoxidation at alkaline pH. At pH 9.5 this variant of the enzyme possesses at least four EPR-detectable, chloride-sensitive high-spin conformers, in contrast to the single chloride-insensitive species observed in the oxidized, resting enzyme at pH 7.7. Quantitative reversal of the spectral and ligand-binding properties of the "activated" enzyme to those of the resting enzyme is observed on reacidification to pH 7.7. At intermediate pH values, there occurs an acid-catalyzed relaxation of the activated enzyme to the resting form. This reaction is distinct from the one responsible for the accelerated ligand binding and production of multiple EPR conformers, which appears to be regulated by a process with a pK of 8.5.