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Biomedical subjects

J R Guest

Publications and source records attributed to J R Guest.

At least 91 records · Page 5Linked to original sources
PubMed1992

Overproduction of the pyruvate dehydrogenase multienzyme complex of Escherichia coli and site-directed substitutions in the E1p and E2p subunits.

The aceEF-lpd operon of Escherichia coli encodes the pyruvate dehydrogenase (E1p), dihydrolipoamide acetyltransferase (E2p) and dihydrolipoamide dehydrogenase (E3) subunits of the pyruvate dehydrogenase multienzyme complex (PDH complex). An isopropyl beta-D-thiogalactopyranoside-inducible expression system was developed for amplifying fully lipoylated wild-type and mutant PDH complexes to over 30% of soluble protein. The extent of lipoylation was related to the degree of aeration during amplification. The specific activities of the isolated PDH complexes and the E1p component were 50-75% of the values normally observed for the unamplified complex. This could be due to altered stoichiometries of the overproduced complexes (higher E3 and lower E1p contents) or inactivation of E1p. The chaperonin, GroEL, was identified as a contaminant which copurifies with the complex. Site-directed substitutions of an invariant glycine residue (G231A, G231S and G231M) in the putative thiamine pyrophosphate-binding fold of the E1p component had no effect on the production of high-molecular-mass PDH complexes but their E1p and PDH complex activities were very low or undetectable, indicating that G231 is essential for the structural or catalytic integrity of E1p. A minor correction to the nucleotide sequence, which leads to the insertion of an isoleucine residue immediately after residue 273, was made. Substitution of the conserved histidine and arginine residues (H602 and R603) in the putative active-site motif of the E2p subunit confirmed that H602 of the E. coli E2p is essential, whereas R603 could be replaced without inactivating E2p. Deletions affecting putative secondary structural elements at the boundary of the E2p catalytic domain inhibited catalytic activity without affecting the assembly of the E2p core or its ability to bind E1p, indicating that the latter functions are determined elsewhere in the domain. The results further consolidate the view that chloramphenicol acetyltransferase serves as a useful structural and functional model for the catalytic domain of the lipoate acyltransferases.

Acetyltransferases↗
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↗
PubMed1992

The glutamate dehydrogenase gene of Clostridium symbiosum. Cloning by polymerase chain reaction, sequence analysis and over-expression in Escherichia coli.

The gene encoding the NAD(+)-dependent glutamate dehydrogenase (GDH) of Clostridium symbiosum was cloned using the polymerase chain reaction (PCR) because it could not be recovered by standard techniques. The nucleotide sequence of the gdh gene was determined and it was overexpressed from the controllable tac promoter in Escherichia coli so that active clostridial GDH represented 20% of total cell protein. The recombinant plasmid complemented the nutritional lesion of an E. coli glutamate auxotroph. There was a marked difference between the nucleotide compositions of the coding region (G + C = 52%) and the flanking sequences (G + C = 30% and 37%). The structural gene encoded a polypeptide of 450 amino acid residues and relative molecular mass (M(r) 49,295 which corresponds to a single subunit of the hexameric enzyme. The DNA-derived amino acid sequence was consistent with a partial sequence from tryptic and cyanogen bromide peptides of the clostridial enzyme. The N-terminal amino acid sequence matched that of the purified protein, indicating that the initiating methionine is removed post-translationally, as in the natural host. The amino acid sequence is similar to those of other bacterial GDHs although it has a Gly-Xaa-Gly-Xaa-Xaa-Ala motif in the NAD(+)-binding domain, which is more typical of the NADP(+)-dependent enzymes. The sequence data now permit a detailed interpretation of the X-ray crystallographic structure of the enzyme and the cloning and expression of the clostridial gene will facilitate site-directed mutagenesis.

Amino Acid Sequence↗
PubMed1992

Purification of HlyX, a potential regulator of haemolysin synthesis, and properties of HlyX:FNR hybrids.

The hlyX gene of the swine pathogen Actinobacillus pleuropneumoniae is homologous to FNR, an anaerobic transcriptional regulator of Escherichia coli. It endows a haemolytic phenotype upon E. coli, and will complement the anaerobic respiratory deficiencies of fnr mutants of E. coli. The coding region of the hlyX gene was expressed in E. coli and the HlyX protein was purified by using an assay based on its immunological cross-reactivity with anti-FNR antibodies. The HlyX protein had the predicted N-terminal sequence, and resembled the isolated FNR protein in size (Mr 29,000) and monomeric organization. It has no detectable haemolysin activity per se, and is therefore presumed to confer a haemolytic phenotype by activating a latent haemolysin gene in E. coli. Studies with gene fusions showed that HlyX, like FNR, can function as an anaerobic activator and repressor of FNR-regulated genes in vivo. Plasmids that express hybrid HlyX:FNR proteins in which the 189/190-residue N-terminal segments and the remaining 50/60-residue C-terminal segments are exchanged, retained their FNR-specific functions but failed to confer a haemolytic phenotype. This suggests that the specificity for activating the haemolytic response requires the participation of unique features in both the N- and C-terminal segments of HlyX.

Actinobacillus pleuropneumoniae↗
PubMed1992

The aconitase of Escherichia coli. Nucleotide sequence of the aconitase gene and amino acid sequence similarity with mitochondrial aconitases, the iron-responsive-element-binding protein and isopropylmalate isomerases.

The nucleotide sequence of the aconitase gene (acn) of Escherichia coli was determined and used to deduce the primary structure of the enzyme. The coding region comprises 2670 bp (890 codons excluding the start and stop codons) which define a product having a relative molecular mass of 97,513 and an N-terminal amino acid sequence consistent with those determined previously for the purified enzyme. The acn gene is flanked by the cysB gene and a putative riboflavin biosynthesis gene resembling the ribA gene of Bacillus subtilis. The 1004-bp cysB--acn intergenic region contains several potential promoter and regulatory sequences. The amino acid sequence of the E. coli aconitase is similar to the mitochondrial aconitases (27-29% identity) and the isopropylmalate isomerases (20-21% identity) but it is most similar to the human iron-responsive-element-binding protein (53% identity). The three cysteine residues involved in ligand binding to the [4Fe-4S] centre are conserved in all of these proteins. Of the remaining 17 active-site residues assigned for porcine aconitase, 16 are conserved in both the bacterial aconitase and the iron-responsive-element-binding protein and 14 in the isopropylmalate isomerases. It is concluded that the bacterial and mitochondrial aconitases, the isopropylmalate isomerases and the iron-responsive-element-binding protein form a family of structurally related proteins, which does not include the Fe-S-containing fumarases. These relationships raise the possibility that the iron-responsive-element-binding protein may be a cytoplasmic aconitase and that the E. coli aconitase may have an iron-responsive regulatory function.

Aconitate Hydratase↗
PubMed1991

Properties of lipoamide dehydrogenase altered by site-directed mutagenesis at a key residue (I184Y) in the pyridine nucleotide binding domain.

The binding of pyridine nucleotide to human erythrocyte glutathione reductase, an enzyme of known three-dimensional structure, requires some movement of the side chain of Tyr197. Moreover, this side chain lies very close to the isoalloxazine ring of the FAD cofactor. The analogous residue, Ile184, in the homologous enzyme Escherichia coli lipoamide dehydrogenase has been altered by site-directed mutagenesis to a tyrosine residue (I184Y) [Russell, G. C., Allison, N., Williams, C. H., Jr., & Guest, J.R. (1989) Ann. N.Y. Acad. Sci. 573, 429-431]. Characterization of the altered enzyme shows that the rate of the pyridine nucleotide half-reaction has been markedly reduced and that the spectral properties have been changed to mimic those of glutathione reductase. Therefore, Ile184 is shown to be an important residue in modulating the properties of the flavin in lipoamide dehydrogenase. Turnover in the dihydrolipoamide/NAD+ reaction is decreased by 10-fold and in the NADH/lipoamide reaction by 2-fold in I184Y lipoamide dehydrogenase. The oxidized form of I184Y shows remarkable changes in the fine structure of the visible absorption and circular dichroism spectra and also shows nearly complete quenching of FAD fluorescence. The spectral properties of the altered enzyme are thus similar to those of glutathione reductase and very different from those of wild-type lipoamide dehydrogenase. On the other hand, spectral evidence does not reveal any change in the amount of charge-transfer stabilization at the EH2 level. Stopped-flow data indicate that, in the reduction of I184Y by NADH, the first step, reduction of the flavin, is only slightly slowed but the subsequent two-electron transfer to the disulfide is markedly inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Evidence for two protein-lipoylation activities in Escherichia coli.

The lipoate acyltransferase subunits of the 2-oxo acid dehydrogenase complexes are post-translationally modified with one or more covalently-bound lipoyl cofactors. Two distinct lipoate-protein ligase activities, LPL-A and LPL-B, have been detected in E. coli by their ability to modify purified lipoyl apo-domains of the bacterial pyruvate dehydrogenase complex. Both enzymes require ATP and Mg2+, use L-lipoate, 8-methyllipoate, lipoyl adenylate and octanoyl adenylate as substrates, and both activate lipoyl-deficient pyruvate dehydrogenase complexes. In contrast, only LPL-B uses D-lipoate and octanoate and there are differences in the metal-ion and phosphate requirements. It is suggested that LPL-B may be responsible for the octanoylation of lipoyl domains observed previously under lipoate-deficient conditions.

Chromatography, Affinity↗
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

FNR activates and represses transcription in vitro.

FNR is an iron-binding transcriptional regulator for anaerobic gene expression in Escherichia coli. Footprinting studies with the purified protein have confirmed that it is a site-specific DNA-binding protein. Transcription tests with the positively-regulated FFmelR promoter and the negatively-regulated ndh promoter likewise demonstrated that FNR can activate or repress transcription in vitro. Reducing conditions were not required but activity was abolished by substituting an essential cysteine residue with alanine (C122A) and the affinity for DNA was reduced by iron-depletion. The start point(s) for transcription of the FNR-repressed NADH dehydrogenase II gene (ndh) were identified by transcript mapping and the corresponding promoter (-35 and -10 sequences) was located immediately downstream of the FNR-binding site.

Bacterial Proteins↗
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

Characterization of the FNR protein of Escherichia coli, an iron-binding transcriptional regulator.

FNR is a transcriptional regulator mediating the activation or repression of a variety of Escherichia coli genes in response to anoxia. The FNR protein resembles CRP (the cyclic-AMP receptor protein) except for the presence of a cysteine-rich N-terminal segment which may form part of an iron-binding redoxsensing domain. The FNR protein was purified by a new procedure. It was monomeric (Mr = 30,000) and contained as much as 1.1 mol of iron per monomer when purified in the presence of added iron. This iron was associated with cysteine residues, because there was an inverse relation between iron content and titratable sulphydryl groups. Other physical and chemical properties are reported including evidence for a potential disulphide group or analogous modification. The interaction between FNR protein and target DNA appeared weak and non-specific in gel-retardation assays, but specific binding to the proposed DNA-binding site was shown for the first time in footprinting studies. A role for iron in FNR-mediated gene expression was confirmed by using cultures in which FNR was inactivated by growth in the presence of the specific chelator, ferrozine, but protected by ferrous iron.

Bacterial Proteins↗
PubMed1991

A new family of bacterial regulatory proteins.

A new family of bacterial regulatory proteins has been identified by sequence similarity. The family contains the repressor of the Bacillus subtilis gluconate operon (GntR), the regulators for histidine utilization in Pseudomonas putida (HutCPp) and Klebsiella aerogenes (HutCKa), the repressor (FadR) of fatty acid degradation in Escherichia coli, a regulator involved in the conjugal transfer of the broad host range plasmid pIJ101 (KorA), and three proteins of unidentified function in E. coli (GenA, P30 and PhnF). The proteins share amino acid sequence similarities in a 69-residue N-terminal region. A helix-turn-helix motif is predicted in the most highly-conserved segment of each protein suggesting that they are members of a new family of helix-turn-helix DNA-binding proteins.

Amino Acid Sequence↗
PubMed1991

Site-directed mutagenesis of the lipoate acetyltransferase of Escherichia coli.

Remote but significant similarities between the primary and predicted secondary structures of the chloramphenicol acetyltransferases (CAT) and lipoate acyltransferase subunits (LAT, E2) of the 2-oxo acid dehydrogenase complexes, have suggested that both types of enzyme may use similar catalytic mechanisms. Multiple sequence alignments for CAT and LAT have highlighted two conserved motifs that contain the active-site histidine and serine residues of CAT. Site-directed replacement of Ser550 in the E2p subunit (LAT) of the pyruvate dehydrogenase complex of Escherichia coli, deemed to be equivalent to the active-site Ser148 of CAT, supported the CAT-based model of LAT catalysis. The effects of other substitutions were also consistent with the predicted similarity in catalytic mechanism although specific details of active-site geometry may not be conserved.

Acetyltransferases↗
PubMed1991

Sequence similarities within the family of dihydrolipoamide acyltransferases and discovery of a previously unidentified fungal enzyme.

A composite protein sequence database was searched for amino acid sequences similar to the C-terminal domain of the dihydrolipoamide acetyltransferase subunit (E2p) of the pyruvate dehydrogenase complex of Escherichia coli. Nine sequences with extensive similarity were found, of which eight were E2 subunits. The other was for a putative mitochondrial ribosomal protein, MRP3, from Neurospora crassa. Alignment of the MRP3 and E2 sequences showed that the similarity extends through the entire MRP3 sequence and that MRP3 is most closely related to the E2p subunit of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae, with 54% identical residues and a further 36% that are conservatively substituted. Other features of the MRP3 gene and protein are also consistent with it being the acyltransferase subunit of a 2-oxo acid dehydrogenase complex. A multiple alignment of 13 E2 sequences indicated that 120 (34%) of 353 equivalenced residues are identical or show some degree of conservation. It also identified residues that are potentially important for the structure, catalytic activity and substrate-specificity of the acyltransferases.

Acetyltransferases↗
PubMed1991

Functional consequences of substitution of the active site (phospho)histidine residue of Escherichia coli succinyl-CoA synthetase.

Succinyl-CoA synthetase (EC 6.2.1.5, succinate:CoA ligase (ADP-forming] of Escherichia coli is an alpha 2 beta 2 tetramer, with the active site believed to be located at the point of contact between the two subunit types. It has been previously established that the reaction involves the intermediate participation of a phosphorylated enzyme form in the process of catalysis. The site of phosphorylation (His-246) and the binding sites for the substrates ADP and ATP are located in the alpha subunit, and the succinate and CoA binding sites are in beta. A mutant form of this enzyme, with the active site histidine residue replaced by aspartate, has been produced in large quantities and purified to homogeneity. This form appears to be indistinguishable from the native enzyme with respect to its subunit assembly, but has no ability to catalyze the overall reaction. As expected, the His-246 alpha----Asp mutant is incapable of undergoing phosphorylation. We have developed an assay based upon the arsenolysis of succinyl-CoA that effectively isolates the partial reaction that occurs in the portion of the active site contributed by the beta subunit; this reaction does not involve covalent participation of His-246 alpha. We have found that the His-246 alpha----Asp mutant is also devoid of activity in this arsenolysis reaction, indicating that an intact His-246 alpha is required for the establishment of the microenvironment in this portion of the active site that is required for the corresponding step of the overall reaction.

Adenosine Diphosphate↗
PubMed1991

Adaptive responses to oxygen limitation in Escherichia coli.

Escherichia coli can grow under either aerobic or anaerobic conditions, deriving energy from a variety of respiratory of fermentative processes. The switch between different metabolic modes depends on the availability of oxygen or alternative electron acceptors, and it is controlled by regulatory mechanisms which ensure that the most energetically favourable metabolic mode is adopted in a specific environment. This article reviews the properties of two transcriptional regulators, ArcA and FNR, which control the expression of networks of genes in response to oxygen limitation.

Aerobiosis↗