Biomedical subjects

E L Neidle

Publications and source records attributed to E L Neidle.

26 records · Page 2Linked to original sources

PubMed1990

DNA sequences of genes encoding Acinetobacter calcoaceticus protocatechuate 3,4-dioxygenase: evidence indicating shuffling of genes and of DNA sequences within genes during their evolutionary divergence.

The DNA sequence of a 2,391-base-pair HindIII restriction fragment of Acinetobacter calcoaceticus DNA containing the pcaCHG genes is reported. The DNA sequence reveals that A. calcoaceticus pca genes, encoding enzymes required for protocatechuate metabolism, are arranged in a single transcriptional unit, pcaEFDBCHG, whereas homologous genes are arranged differently in Pseudomonas putida. The pcaG and pcaH genes represent separate reading frames respectively encoding the alpha and beta subunits of protocatechuate 3,4-dioxygenase (EC 1.13.1.3); previously a single designation, pcaA, had been used to represent DNA encoding this enzyme. The alpha and beta protein subunits appear to share common ancestry with each other and with catechol 1,2-dioxygenases from A. calcoaceticus and P. putida. Marked conservation of amino acid sequence is observed in a region containing two histidyl residues and two tyrosyl residues that appear to ligate iron within each oxygenase. In some regions within the aligned oxygenase sequences, DNA sequences appear to be conserved at a level beyond the extent that might have been demanded by selection at the level of protein. In other regions, divergence of DNA sequences appears to have been achieved by substitution of DNA sequence from one genetic segment into another. The results are interpreted to be the consequence of sequence exchange by gene conversion between slipped strands of DNA during evolutionary divergence; mismatch repair between slipped strands may contribute to the maintenance of DNA sequence in divergent genes.

Acinetobacter↗

PubMed1989

Characterization of Acinetobacter calcoaceticus catM, a repressor gene homologous in sequence to transcriptional activator genes.

Two structural genes needed for catechol degradation, catA and catB, encode the respective enzymes catechol 1,2-dioxygenase (EC 1.13.11.1) and muconate cycloisomerase (EC 5.5.1.1). Catechol is an intermediate in benzoate degradation, and the catA and catB genes are clustered within a 17-kilobase-pair (kbp) region of Acinetobacter calcoaceticus chromosomal DNA containing all of the structural genes required for the conversion of benzoate to tricarboxylic acid cycle intermediates. catA and catB were transcribed in the same direction and were separated by 3.8 kbp of DNA. The 3.8-kbp sequence revealed that directly downstream from catA and potentially transcribed in the same direction were two open reading frames encoding polypeptides of 48 and 36 kilodaltons (kDa). Genetic disruption of these open reading frames did not discernably alter either catechol metabolism or its regulation. A third open reading frame, beginning 123 bp upstream from catB and transcribed divergently from this gene, was designated catM. This gene was found to encode a 28-kDa trans-acting repressor protein that, in the absence of cis,cis-muconate, prevented expression of the cat structural genes. Constitutive expression of the genes was caused by a mutation substituting Arg-156 with His-156 in the catM-encoded repressor. The repressor protein proved to be a member of a diverse family of procaryotic regulatory proteins which, with rare exception, are transcriptional activators. Repression mediated by catM was not the sole transcriptional control exercised over catA in A. calcoaceticus. Expression of catA was elicited by either benzoate or cis,cis-muconate in a genetic background from which catM had been deleted. This induction required DNA in a segment lying 1 kbp upstream from the catA gene. It is likely that an additional gene, lying outside the region containing the structural genes necessary for benzoate metabolism, contributes to this control.

Acinetobacter↗

PubMed1988

DNA sequence of the Acinetobacter calcoaceticus catechol 1,2-dioxygenase I structural gene catA: evidence for evolutionary divergence of intradiol dioxygenases by acquisition of DNA sequence repetitions.

The DNA sequence of a 1.6-kilobase-pair SalI-KpnI Acinetobacter calcoaceticus restriction fragment carrying catA, the structural gene for catechol 1,2-dioxygenase I, was determined. The 933-nucleotide gene encodes a protein product with a deduced molecular weight of 34,351. The similarly sized Pseudomonas clcA gene encodes catechol 1,2-dioxygenase II, an enzyme with relatively broad substrate specificity and relatively low catalytic efficiency. Comparison of the catA and clcA sequences demonstrated their common ancestry and suggested that acquisitions of direct and inverted sequence repetitions of 6 to 10 base pairs were frequent events in their evolutionary divergence. The catechol 1,2-dioxygenases proved to be evolutionarily homologous with the alpha and beta subunits of Pseudomonas protocatechuate 3,4-dioxygenase, and analysis of conserved residues in the intradiol dioxygenases revealed conserved histidyl and tyrosyl residues that are probably involved in the ligation of ferric ion in their active sites.

Acinetobacter↗

PubMed1987

Benzoate and muconate, structurally dissimilar metabolites, induce expression of catA in Acinetobacter calcoaceticus.

Biosynthetic regulation of catA, the gene encoding catechol 1,2-dioxygenase (EC 1.13.1.1), was studied in an Acinetobacter calcoaceticus mutant strain unable to metabolize benzoate. Benzoate and muconate independently induced the enzyme. In glucose-grown cells, benzoate yielded higher enzyme levels than did muconate, whereas muconate was the more effective inducer in succinate-grown cells.

Acinetobacter↗

PubMed1987

Cloning and expression in Escherichia coli of Acinetobacter calcoaceticus genes for benzoate degradation.

The catabolic genes necessary for the conversion of benzoate to catechol have been cloned from Acinetobacter calcoaceticus into Escherichia coli. The cloned genes, benABCD, encoded both a benzoate 1,2-dioxygenase system, composed of NADH-cytochrome c reductase and terminal oxygenase components, and a cis-diol dehydrogenase. The dioxygenase system appears to be encoded by three genes, benABC, whose products, 53-, 19-, and 38-kilodalton proteins, correspond in size to those of components in other bacterial dioxygenases. The cloned dioxygenase system is expressed at high level in E. coli, enabling the conversion of benzoate to a cis-diol, 2-hydro-1,2-dihydroxybenzoate, at a rate comparable to that of fully induced A. calcoaceticus cultures. A cis-diol dehydrogenase, the product of the A. calcoaceticus benD gene, when present in E. coli enables this organism to convert the cis-diol intermediate to catechol. The dehydrogenase has been partially purified and is a dimer with two identical 31-kilodalton subunits. The ben genes are clustered on the A. calcoaceticus chromosome with independently regulated genes needed for the dissimilation of catechol. In a 16-kilobase-pair region of the chromosome there are 10 genes for benzoate catabolism, organized in no fewer than three transcriptional units. This kind of arrangement, termed supraoperonic clustering, has been observed previously in pseudomonads.

Acinetobacter↗

PubMed1986

Cloning and expression of Acinetobacter calcoaceticus catBCDE genes in Pseudomonas putida and Escherichia coli.

This report describes the isolation and preliminary characterization of a 5.0-kilobase-pair (kbp) EcoRI DNA restriction fragment carrying the catBCDE genes from Acinetobacter calcoaceticus. The respective genes encode enzymes that catalyze four consecutive reactions in the catechol branch of the beta-ketoadipate pathway: catB, muconate lactonizing enzyme (EC 5.5.1.1); catC, muconolactone isomerase (EC 5.3.3.4); catD, beta-ketoadipate enol-lactone hydrolase (EC 3.1.1.24); and catE, beta-ketoadipate succinyl-coenzyme A transferase (EC 2.8.3.6). In A. calcoaceticus, pcaDE genes encode products with the same enzyme activities as those encoded by the respective catDE genes. In Pseudomonas putida, the requirements for both catDE and pcaDE genes are met by a single set of genes, designated pcaDE. A P. putida mutant with a dysfunctional pcaE gene was used to select a recombinant pKT230 plasmid carrying the 5.0-kbp EcoRI restriction fragment containing the A. calcoaceticus catE structural gene. The recombinant plasmid, pAN1, complemented P. putida mutants with lesions in catB, catC, pcaD, and pcaE genes; the complemented activities were expressed constitutively in the recombinant P. putida strains. After introduction into Escherichia coli, the pAN1 plasmid expressed the activities constitutively but at much lower levels that those found in the P. putida transformants or in fully induced cultures of A. calcoaceticus or P. putida. When placed under the control of a lac promoter on a recombinant pUC13 plasmid in E. coli, the A. calcoaceticus restriction fragment expressed catBCDE activities at levels severalfold higher than those found in fully induced cultures of A. calcoaceticus. Thus there is no translational barrier to expression of the A. calcoaceticus genes at high levels in E. coli. The genetic origin of the cloned catBCDE genes was demonstrated by the fact that the 5.0-kbp EcoRI restriction fragment hybridized with a corresponding fragment from wild-type A. calcoaceticus DNA. This fragment was missing in DNA from an A. calcoaceticus mutant in which the cat genes had been removed by deletion. The properties of the cloned fragment demonstrate physical linkage of the catBCDE genes and suggest that they are coordinately transcribed.

Acinetobacter↗

PubMed1986

Cloning and expression of Acinetobacter calcoaceticus catechol 1,2-dioxygenase structural gene catA in Escherichia coli.

Catechol 1,2-dioxygenase (EC 1.13.1.1), the product of the catA gene, catalyzes the first step in catechol utilization via the beta-ketoadipate pathway. Enzymes mediating subsequent steps in the pathway are encoded by the catBCDE genes which are carried on a 5-kilobase-pair (kbp) EcoRI restriction fragment isolated from Acinetobacter calcoaceticus. This DNA was used as a probe to identify Escherichia coli colonies carrying recombinant pUC19 plasmids with overlapping sequences. Repetition of the procedure yielded an A. calcoaceticus 6.7-kbp EcoRI restriction fragment which contained the catA gene and bordered the original 5-kbp EcoRI restriction fragment. When the catA-containing fragment was placed under the control of the lac promoter on pUC19 and induced with isopropylthiogalactopyranoside, catechol dioxygenase was formed in E. coli at twice the level found in fully induced cultures of A. calcoaceticus. A. calcoaceticus strains with mutations in the catA gene were transformed to wild type by DNA from lysates of E. coli strains carrying the catA gene on recombinant plasmids. Thus, A. calcoaceticus strains with a mutated gene can be used in a transformation assay to identify E. coli clones in which at least part of the wild-type gene is present but not necessarily expressed.

Acinetobacter↗

PubMed1983

An aminopeptidase from mouse brain cytosol that cleaves N-terminal acidic amino acid residues.

An aminopeptidase with specificity directed toward peptides with acidic N-terminal amino acid residues has been isolated from mouse brain cytosol. Purification by ion-exchange chromatography and gel filtration resulted in an enzyme that hydrolyzed aspartyl-phenylalanine methyl ester at a rate of 13.2 mumols/min/mg protein at pH 7.5, an increase in specific activity of 1000-fold over that of brain homogenate. Its apparent molecular weight, determined by gel filtration, is approximately 450,000. Dipeptides with N-terminal aspartyl residues are cleaved preferentially to glutamic-containing analogs, and a neutral amino acid (or histidine) is necessary in the adjacent position. For peptides of the form aspartyl-X, relative activity was 100, 81, 71, 66, 19, or 0, where X was alanine, serine, leucine, phenylalanine, histidine, or proline, respectively. Tripeptides were more rapidly hydrolyzed than dipeptides; however, activity tended to decline with increasing chain length. The acidic aminopeptidase can account for almost all of the activity of brain cytosol toward the N-terminal aspartyl residue of angiotensin II, aspartyl-phenylalanine methyl ester or aspartyl-alanine, and the N-terminal glutamyl residue of adrenocorticotropin(5-10). The enzyme was unaffected by bestatin or amastatin. It was inhibited by o-phenanthroline and EDTA. The latter effect could be reversed completely by Zn2+ and partially by Mn2+ or Mg2+; Co2+ and Fe2+ had no effect; Ca2+ was inhibitory. These properties distinguish the brain acidic aminopeptidase from aminopeptidase A isolated from human serum or pig kidney and the aspartyl aminopeptidase of dog kidney.

Amino Acids↗