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P M Jordan

Publications and source records attributed to P M Jordan.

At least 37 records · Page 2Linked to original sources
PubMed1992

A putative anaerobic coproporphyrinogen III oxidase in Rhodobacter sphaeroides. I. Molecular cloning, transposon mutagenesis and sequence analysis of the gene.

A mutant of Rhodobacter sphaeroides, N1, has been isolated which is incapable of photosynthetic growth and, instead of synthesizing bacteriochlorophyll, N1 excretes coproporphyrin III into the growth medium. Using conjugative gene transfer, several clones were isolated from a R. sphaeroides gene library which restored normal pigment synthesis and photosynthetic growth to N1. Using transposon Tn5 mutagenesis, the gene was located to a 1.05 kb EcoRI fragment. Sequence and transcription analysis defined the position and expression of an open reading frame of approximately 920 bp, which is proposed as the anaerobic coproporphyrinogen III oxidase dedicated to bacteriochlorophyll biosynthesis.

Amino Acid Sequence↗
PubMed1991

Mutagenesis of arginine residues in the catalytic cleft of Escherichia coli porphobilinogen deaminase that affects dipyrromethane cofactor assembly and tetrapyrrole chain initiation and elongation.

Substitutions of conserved arginine residues in the catalytic cleft of Escherichia coli porphobilinogen deaminase were constructed by site-specific mutagenesis of the hemC gene. Mutant proteins exhibited a range of defects including the failure to assemble the dipyrromethane cofactor and the inability to initiate and propagate the tetrapolymerization reaction. Mutations of arginine residues at positions 11, 131, 132 and 155, all of which interact with the carboxylic acid side chains of the dipyrromethane cofactor, were the most disruptive.

Amino Acid Sequence↗
PubMed1989

Purification and properties of uroporphyrinogen III synthase (co-synthase) from an overproducing recombinant strain of Escherichia coli K-12.

The Escherichia coli hemD gene, encoding the enzyme uroporphyrinogen III synthase (co-synthase), was cloned into multi-copy plasmids in E. coli cells that were used to generate strains producing up to 1000 times the concentration of the synthase in the wild-type. The enzyme was purified to homogeneity from these strains in milligram amounts. The enzyme is a monomer of Mr 28,000 with an isoelectric point of 5.2 and a pH optimum of 7.8. The specific activity of the purified synthase is 1500 units/mg and the Km for the substrate, pre-uroporphyrinogen, is 5 microM. The N-terminal sequence of the enzyme is Ser-Ile-Leu-Val-Thr-Arg-Pro-Ser-Pro-Ala-Gly-, in agreement with the gene-derived protein sequence. The enzyme contains four 5,5'-dithiobis-(2-nitrobenzoic acid)-titratable groups, one reacting rapidly with the reagent and three further groups having lower reactivity. The enzyme is heat-sensitive, and during heat inactivation all four thiol groups become equally available for reaction.

Amino Acid Sequence↗
PubMed1988

Investigation into the nature of substrate binding to the dipyrromethane cofactor of Escherichia coli porphobilinogen deaminase.

The formation of the dipyrromethane cofactor of Escherichia coli porphobilinogen deaminase was shown to depend on the presence of 5-aminolevulinic acid. A hemA- mutant formed inactive deaminase when grown in the absence of 5-aminolevulinic acid since this strain was unable to biosynthesize the dipyrromethane cofactor. The mutant formed normal levels of deaminase, however, when grown in the presence of 5-aminolevulinic acid. Porphobilinogen, the substrate, interacts with the free alpha-position of the dipyrromethane cofactor to give stable enzyme-intermediate complexes. Experiments with regiospecifically labeled intermediate complexes have shown that, in the absence of further substrate molecules, the complexes are interconvertible by the exchange of the terminal pyrrole ring of each complex. The formation of enzyme-intermediate complexes is accompanied by the exposure of a cysteine residue, suggesting that substantial conformational changes occur on binding substrate. Specific labeling of the dipyrromethane cofactor by growth of the E. coli in the presence of 5-amino[5-14C]levulinic acid has confirmed that the cofactor is not subject to catalytic turnover. Experiments with the alpha-substituted substrate analogue alpha-bromoporphobilinogen have provided further evidence that the cofactor is responsible for the covalent binding of the substrate at the catalytic site. On the basis of these cumulative findings, it has been possible to construct a mechanistic scheme for the deaminase reaction involving a single catalytic site which is able to catalyze the addition or removal of either NH3 or H2O. The role of the cofactor both as a primer and as a means for regulating the number of substrates bound in each catalytic cycle is discussed.

Aminolevulinic Acid↗
PubMed1988

Purification, crystallization and properties of porphobilinogen deaminase from a recombinant strain of Escherichia coli K12.

Porphobilinogen deaminase has been purified and crystallized from an overproducing recombinant strain of Escherichia coli harbouring a hemC-containing plasmid which has permitted the purification of milligram quantities of the enzyme. Determination of the Mr of the enzyme by SDS/polyacrylamide-gel electrophoresis (35,000) and gel filtration (32,000) agrees with the gene-derived Mr of 33,857. The enzyme has a Km of 19 +/- 7 microM, an isoelectric point of 4.5 and an N-terminal sequence NH2-MLDNVLRIAT. The substrate, porphobilinogen, binds to the active-site dipyrromethane cofactor to form three intermediate complexes: ES, ES2 and ES3. The gene-derived primary structure of the E. coli deaminase is compared with that derived from the cDNA of the human enzyme.

Amino Acid Sequence↗
PubMed1988

Identification of a cysteine residue as the binding site for the dipyrromethane cofactor at the active site of Escherichia coli porphobilinogen deaminase.

The dipyrromethane cofactor of Escherichia coli porphobilinogen deaminase was specifically labelled with 13C by growth of the bacteria in the presence of 5-amino[5-13C]levulinic acid. Using 13C-NMR spectroscopy, the structure of the cofactor was confirmed as a dipyrromethane made up of two linked pyrrole rings each derived from porphobilinogen. The chemical shift data indicate that one of the pyrrole rings of the cofactor is covalently linked to the deaminase enzyme through a cysteine residue. Evidence from protein chemistry studies suggest that cysteine-242 is the covalent binding site for the cofactor.

Aminolevulinic Acid↗
PubMed1988

Nucleotide sequence for the hemD gene of Escherichia coli encoding uroporphyrinogen III synthase and initial evidence for a hem operon.

1. The hemD gene, encoding uroporphyrinogen III synthase, has been located adjacent to the hemC gene at 85 min on the Escherichia coli chromosome. 2. The entire nucleotide sequence (741 base pairs) of the hemD gene is reported. 3. E. coli strains harbouring plasmics containing the hemD gene produce greatly elevated levels of uroporphyrinogen III synthase. 4. Purified uroporphyrinogen III synthase, isolated from the hemD-containing strain ST1046, has an Mr of 29,000, in close agreement with that predicted from the nucleotide sequence. 5. The existence of a hem operon is suggested.

Base Sequence↗
PubMed1987

Evidence for a dipyrromethane cofactor at the catalytic site of E. coli porphobilinogen deaminase.

Porphobilinogen deaminase isolated from Escherichia coli is shown to contain a dipyrromethane cofactor (DPMC) linked covalently to the enzyme. The structure of the cofactor is proposed on the basis of its reaction with Ehrlich's reagent and from its chemical properties. The cofactor is involved in the binding of intermediates during the catalytic reaction but is not incorporated into the product preuroporphyrinogen, E. coli strains containing the cloned porphobilinogen deaminase gene (hemC) when grown on 5-amino[14C]-levulinic acid incorporate 14C radioactivity specifically into the dipyrromethane cofactor of porphobilinogen deaminase.

Ammonia-Lyases↗
PubMed1986

Nucleotide sequence of the hemC locus encoding porphobilinogen deaminase of Escherichia coli K12.

Porphobilinogen deaminase, the product of the hemC locus in Escherichia coli K12, catalyses the tetrapolymerisation of porphobilinogen (PBG) into the hydroxymethylbilane, preuroporphyrinogen. The hemC locus has been subcloned from the Clarke and Carbon plasmid pLC41-4. The sequence of the hemC structural gene and flanking DNA was determined by the dideoxy chain termination method of Sanger. The structural gene for hemC is located within a 942bp sequence encoding the monomeric PBG deaminase, molecular weight 33,857. The extent of the coding region was confirmed by sequencing the N-terminus of the purified enzyme and by determination of the molecular weight. The hemC locus is closely linked to the cyaA locus, the genes being transcribed in a divergent manner. Upstream of the hemC coding region, a possible promoter and three repeated GGATG sequences were identified. This is the first report of a complete DNA sequence for a structural gene specifying an enzyme of the heme biosynthetic pathway in prokaryotes.

Ammonia-Lyases↗
PubMed1986

Identification of lysine at the active site of human 5-aminolaevulinate dehydratase.

Reduction of human 5-aminolaevulinate dehydratase with NaBH4 in the presence of 14C-labelled substrate led to complete loss of catalytic activity and to incorporation of label into the enzyme protein. By comparison with authentic lysyl-aminolaevulinic acid, prepared chemically, the modified active-site amino acid obtained by acid hydrolysis was shown to be lysine. Sequencing of a CNBr-cleavage peptide isolated from the inactivated 14C-labelled enzyme revealed that the lysine was present within the sequence M-V-K-P-G-M.

Amino Acid Sequence↗
PubMed1985

Purification and properties of 5-aminolaevulinate dehydratase from human erythrocytes.

A new procedure for the isolation of homogeneous human 5-aminolaevulinate dehydratase (porphobilinogen synthase, EC 4.2.1.24) is described in which the enzyme is purified 35000-fold and in 65-74% yield. The specific activity of the purified enzyme, 24 units/mg, is the highest yet reported. An efficient stage for the removal of haemoglobin is incorporated in the method, which has general application to the purification of other erythrocyte enzymes. The erythrocyte dehydratase (Mr 285 000) is made up of eight apparently identical subunits of Mr 35 000. The enzyme is sensitive to oxygen, and its activity is maintained by the presence of thiols such as dithioerythritol. Zn2+ is obligatory for enzyme activity, the apoenzyme being essentially inactive (approximately equal to 12% of control) when assayed in buffers devoid of Zn2+. Addition of Zn2+ to the apoenzyme restores activity as long as the sensitive thiol groups are fully reduced; optimal stimulation occurs between 100 and 300 microM-Zn2+. The human enzyme is inhibited by Pb2+ in a non-competitive fashion [KiI (dissociation constant for E X S X Pb2+ complex) = 25.3 +/- 3.0 microM; KiS (dissociation constant for E X Pb2+ complex) = 9.0 +/- 2.0 microM]. Modification of thiol groups, inactivation by oxidation, alkylation or reaction with thiophilic reagents demonstrates the importance of sensitive thiol groups for full enzymic activity.

Chromatography, Gel↗
PubMed1985

Mechanism of action of 5-aminolaevulinate dehydratase from human erythrocytes.

Purified 5-aminolaevulinate dehydratase (porphobilinogen synthase, EC 4.2.1.24) from human erythrocytes was incubated initially with limiting amounts of 5-amino [5-14C]laevulinate in a rapid-mixing apparatus. The single-turnover reaction with respect to the bound labelled 5-aminolaevulinate was completed by the addition of unlabelled 5-aminolaevulinate and the resulting radioactive porphobilinogen was isolated and degraded. The 14C label was found to be located predominantly at C-2 of the product, demonstrating that, of the two substrate molecules participating in the reaction, the 5-aminolaevulinate molecule initially bound to the enzyme provides the propionic acid 'side' of the porphobilinogen. The same enzyme-[14C]substrate species that yields regiospecific porphobilinogen may be trapped by reaction with NaBH4, showing that the substrate molecule initially bound to the enzyme does so in the form of a Schiff base. A conventional incubation with 5-amino[5-14C]laevulinate yielded porphobilinogen with an equal distribution of the label between C-2 and C-11. The reaction mechanism of the human erythrocyte 5-aminolaevulinate dehydratase thus follows the same course as that of other dehydratases studied in our laboratory by using single-turnover techniques.

Aminolevulinic Acid↗
PubMed1985

Investigation of the effect of metal ions on the reactivity of thiol groups in human 5-aminolaevulinate dehydratase.

The reaction of human 5-aminolaevulinate dehydratase with 5,5'-dithiobis-(2-nitrobenzoic acid) (Nbs2) results in the release of 4 molar equivalents of 5-mercapto-2-nitrobenzoic acid (Nbs) per subunit. Two of the thiol groups reacted very rapidly (groups I and II), and their rate constants were determined by stopped-flow spectrophotometry; the other two thiol groups (groups III and IV) were observed by conventional spectroscopy. Titration of the enzyme with a 1 molar equivalent concentration of Nbs2 resulted in the release of 2 molar equivalents of Nbs and the concomitant formation of an intramolecular disulphide bond between groups I and II. Removal of zinc from the holoenzyme increased the reactivity of groups I and II without significantly affecting the rate of reaction of the other groups. The reactions of the thiol groups in both the holoenzyme and apoenzyme were little affected by the presence of Pb2+ ions at concentrations that strongly inhibit the enzyme, suggesting that Zn2+ and Pb2+ ions may have independent binding sites. Protein fluorescence studies with Pb2+ and Zn2+ have shown that the binding of both metal ions results in perturbation of the protein fluorescence.

Apoenzymes↗
PubMed1983

Mechanism of action of beta-oxoacyl-CoA thiolase from rat liver cytosol. Direct evidence for the order of addition of the two acetyl-CoA molecules during the formation of acetoacetyl-CoA.

Single-turnover enzyme reactions were employed with beta-oxoacyl-CoA thiolase purified from rat liver cytosol to determine the order of binding of the two acetyl-CoA molecules to the enzyme during the formation of acetoacetyl-CoA. Equimolar quantities of [1-14C]acetyl-CoA and enzyme were mixed initially in a rapid mixing device and the reaction was quenched by addition of an excess of unlabelled acetyl-CoA. Degradation of the resulting acetoacetyl-CoA revealed that the larger proportion of the radioactivity was in C-3. In the converse experiment, in which unlabelled acetyl-CoA was mixed with enzyme and the reaction was quenched with [1-14C]acetyl-CoA, radioactivity was incorporated preferentially into C-1. Similar results were obtained when [14C]acetyl-enzyme complex isolated by gel filtration was reacted with unlabelled acetyl-CoA, the radioactivity appearing largely in C-3. These findings lead to the conclusion that of the two molecules of acetyl-CoA that are bound by the enzyme and converted into acetoacetyl-CoA, it is the one giving rise to C-3 and -4 that is bound initially to the enzyme in the form of the acetyl-enzyme intermediate complex.

Acetyl Coenzyme A↗
PubMed1983

Anaerobic and aerobic coproporphyrinogen III oxidases of Rhodopseudomonas spheroides. Mechanism and stereochemistry of vinyl group formation.

An improved method for the preparation of various species of porphobilinogen stereospecifically labelled with 3H in the side chains (at C-6, C-7 and C-8) is described. These labelled samples were used to study the mechanism and stereochemistry of anaerobic as well as aerobic coproporphyrinogen III oxidase of light-grown Rhodopseudomonas spheroides. It was shown that both the oxidases catalyse the conversion of the propionate side chains of coproporphyrinogen III into the vinyl groups of protoporphyrinogen IX, (formula; see text) with the labilization of the pro-S-hydrogen atom at the beta-position. These results are similar to those previously recorded for such conversions in animal and plant systems. In the light of the cumulative information available to date, mechanisms for the conversion, (formula; see text) are discussed and doubt is cast on the participation of hydroxylated intermediates in the process.

Aerobiosis↗