Andre Pierard - Academia.edu (original) (raw)

Papers by Andre Pierard

Research paper thumbnail of Primary structure, partial purification and regulation of key enzymes of the acetyl cycle of arginine biosynthesis in Bacillus stearothermophilus: dual function of ornithine acetyltransferase.

J Gen Microbiol, Mar 1993

A 3.4 kb EcoRI fragment, cloned in E. coli, that carries part of a cluster of genes encoding argi... more A 3.4 kb EcoRI fragment, cloned in E. coli, that carries part of a cluster of genes encoding arginine biosynthetic functions of the thermophilic bacterium Bacillus stearothermophilus, was sequenced on both strands. The sequence consists of a truncated argC gene, an argJ region encoding a polypeptide with both N-acetylglutamate synthase and ornithine acetyltransferase activities, the argB gene and the N-terminal part of argD. The argB gene encodes a 258-amino-acid polypeptide with a deduced M(r) of 26918. A very high and thermostable N-acetylglutamate 5-phosphotransferase activity was detected in extracts of E. coli arg B mutants transformed with the 3.4 kb fragment on a plasmid. A polypeptide band of M(r) 27,000 was detected by SDS-PAGE of heat-treated extract from such a strain. Both N-acetylglutamate synthase and ornithine acetyltransferase are encoded by the same 1290 bp open reading frame. The deduced sequence of 410 amino acids corresponds to a peptide of M(r) 43,349. The subcloned B. stearothermophilus argJ can complement a double argA argE E. coli mutant to prototrophy. Gel-filtration of a heat-treated extract of the complemented double mutant E. coli host showed that N-acetylglutamate synthase and ornithine acetyltransferase activities co-elute in a single peak corresponding to M(r) 110,000. Both activities were also heat-inactivated at the same temperature and strongly inhibited by ornithine. These results suggest that both activities can be ascribed to a single protein.

Research paper thumbnail of Co-operative interactions between the catalytic sites in Escherichia coli aspartate transcarbamylase

Journal of Molecular Biology, 1990

In aspartate transcarbamylase (ATCase) each regulatory chain interacts with two catalytic chains ... more In aspartate transcarbamylase (ATCase) each regulatory chain interacts with two catalytic chains each one belonging to a different trimeric catalytic subunit (R1-C1 and R1-C4 types of interactions as defined in ). In order to investigate the interchain contacts that are involved in the co-operative interactions between the catalytic sites, a series of modified forms of the enzyme was prepared by site-directed mutagenesis. The amino acid replacements were devised on the basis of the previously described properties of an altered form of ATCase (pAR5-ATCase) which lacks the homotropic co-operative interactions between the catalytic sites. The results obtained (enzyme kinetics, bisubstrate analog influence and pH studies) show that the R1-C4 interaction is essential for the establishment of the enzyme conformation that has a low affinity for aspartate (T state), and consequently for the existence of co-operativity between the catalytic sites. This interaction involves the 236-250 region of the aspartate binding domain of the catalytic chain (240s loop) and the 143-149 region of the regulatory chain which comprises helix H3'.

Research paper thumbnail of Primary structure, partial purification and regulation of key enzymes of the acetyl cycle of arginine biosynthesis in Bacillus stearothermophilus: dual function of ornithine acetyltransferase

Journal of general microbiology, 1993

A 3.4 kb EcoRI fragment, cloned in E. coli, that carries part of a cluster of genes encoding argi... more A 3.4 kb EcoRI fragment, cloned in E. coli, that carries part of a cluster of genes encoding arginine biosynthetic functions of the thermophilic bacterium Bacillus stearothermophilus, was sequenced on both strands. The sequence consists of a truncated argC gene, an argJ region encoding a polypeptide with both N-acetylglutamate synthase and ornithine acetyltransferase activities, the argB gene and the N-terminal part of argD. The argB gene encodes a 258-amino-acid polypeptide with a deduced M(r) of 26918. A very high and thermostable N-acetylglutamate 5-phosphotransferase activity was detected in extracts of E. coli arg B mutants transformed with the 3.4 kb fragment on a plasmid. A polypeptide band of M(r) 27,000 was detected by SDS-PAGE of heat-treated extract from such a strain. Both N-acetylglutamate synthase and ornithine acetyltransferase are encoded by the same 1290 bp open reading frame. The deduced sequence of 410 amino acids corresponds to a peptide of M(r) 43,349. The subcl...

Research paper thumbnail of Structure-function relationship in allosteric aspartate carbamoyltransferase from Escherichia coli *1I. Primary structure of a pyrI gene encoding a modified regulatory subunit

Journal of Molecular Biology, 1985

In a previous article, we have identified a lambda bacteriophage directing the synthesis of a mod... more In a previous article, we have identified a lambda bacteriophage directing the synthesis of a modified aspartate carbamoyltransferase lacking substrate-co-operative interactions and insensitive to the feedback inhibitor CTP. These abnormal properties were ascribed to a mutation in the gene pyrl encoding the regulatory polypeptide chain of the enzyme. N'e now report the sequence of the mutated pyrl and show that, during the generation of this pyrB1-bearing phage, six codons from lambda DNA have been substituted for the eight terminal codons of the wild-type gene. A model is presented for the formation of this modified pyrI gene during the integrative recombination of the parental lambda phage with the Esch,erichia coli chromosome. An accompanying paper emphasizes the importance of t'hr carboxy-terminal end of the regulatory chain for the homotropic and heterotropica interactions of aspartate carbamoyltransferase.

Research paper thumbnail of A re-examination of the pathway for ornithine biosynthesis in a thermophilic and two mesophilic Bacillus species

Journal of General Microbiology, 1992

The expression of Buciffus steurothermophifus genes complementing arginine auxotrophs of Escheric... more The expression of Buciffus steurothermophifus genes complementing arginine auxotrophs of Escherichiu coli was studied. The activity responsible for the formation of ornithine in B. steurothermophifus was identified as a repressible ornithine acetyltransferase (genetic symbol urgJ) encoded by the same DNA fragment as the argC, urgA and urgB genes. Buciffus subtilis and Buciffus ficheniformis displayed the same pattern of enzyme activities as B. steurothermophifus. In contrast to previous reports, these organisms consequently use the cyclic pathway of ornithine biosynthesis. B. steurothermophifus also pssesses a broad specificity aminoacylase which exhibits low affinity towards NZ-acetyl-L-ornithine.

Research paper thumbnail of CarP, a novel gene regulating the transcription of the carbamoylphosphate synthetase operon of Escherichia coli

Journal of Molecular Biology, 1988

The carAB operon, encoding carbamoylphosphate synthetase (CPSase; EC 6.3.5.5) is transcribed from... more The carAB operon, encoding carbamoylphosphate synthetase (CPSase; EC 6.3.5.5) is transcribed from two tandem promoters. The upstream promoter (P1) is controlled by pyrimidines and the downstream promoter (P2) is controlled by arginine. We have isolated a new type of constitutive mutation (carP) that specifically affects the control of the pyrimidine-sensitive promoter but does not appear to influence other genes of the pyrimidine pathway. The carP mutation acts in trans and is dominant, which suggests that the carP product is an activator of car transcription. The downstream promoter P2, which is repressed by arginine, overlaps two operator modules characteristic of the arginine regulon. We have isolated two operator-constitutive mutations that specifically affect P2; both map in the upstream ARG box at a strongly conserved position.

Research paper thumbnail of DNA sequence of the carA gene and the control region of carAB: tandem promoters, respectively controlled by arginine and the pyrimidines, regulate the synthesis of carbamoyl-phosphate synthetase in Escherichia coli K-12

Proceedings of the National Academy of Sciences, 1984

The carAB operon of Escherichia coli K-12, which encodes the two subunits of carbamoyl-phosphate ... more The carAB operon of Escherichia coli K-12, which encodes the two subunits of carbamoyl-phosphate synthetase (glutamine hydrolyzing) [carbon-dioxide: L-glutamine amido-ligase (ADP-forming, carbamate-phosphorylating); EC 6.3.5.5], is cumulatively repressed by arginine and the pyrimidines. We describe the structure of the control region of carAB and the sequence of the carA gene. Nuclease S1 mapping experiments show that two adjacent tandem promoters within the carAB control region serve as initiation sites. The upstream promoter P1 is controlled by pyrimidines; the downstream promoter P2 is regulated by arginine. Attenuation control does not appear to be involved in the expression of carAB. A possible mechanism by which control at these promoters concurs to produce a cumulative pattern of repression is discussed. The translational start of carA is atypical; it consists of a UUG or AUU codon.

Research paper thumbnail of Structure-function relationship in allosteric aspartate carbamoyltransferase from Escherichia coli

Journal of Molecular Biology, 1985

In a previous article, we have identified a lambda bacteriophage directing the synthesis of a mod... more In a previous article, we have identified a lambda bacteriophage directing the synthesis of a modified aspartate carbamoyltransferase lacking substrate-co-operative interactions and insensitive to the feedback inhibitor CTP. These abnormal properties were ascribed to a mutation in the gene pyrI encoding the regulatory polypeptide chain of the enzyme. We now report the sequence of the mutated pyrI and show that, during the generation of this pyrBI-bearing phage, six codons from lambda DNA have been substituted for the eight terminal codons of the wild-type gene. A model is presented for the formation of this modified pyrI gene during the integrative recombination of the parental lambda phage with the Escherichia coli chromosome. An accompanying paper emphasizes the importance of the carboxy-terminal end of the regulatory chain for the homotropic and heterotropic interactions of aspartate carbamoyltransferase.

Research paper thumbnail of Enzyme systems in the mycobacteria

Archives of Biochemistry and Biophysics, 1963

Research paper thumbnail of Structure-function relationships in the arginine pathway carbamoylphosphate synthase of Saccharomyces cerevisiae

Journal of Bacteriology, Apr 1, 1978

The arginine pathway carbamoylphosphate synthase (CPSase A) from Saccharomyces cerevisiae was sho... more The arginine pathway carbamoylphosphate synthase (CPSase A) from Saccharomyces cerevisiae was shown to be highly unstable and could not be substantially purified. In spite of this instability, a number of important properties of this enzyme were determined with crude preparations. A molecular weight of 140,000 (7.9S) was estimated for the native enzyme by sucrose gradient centrifugation; a significantly higher value, 175,000, was obtained by gel filtration on Sephadex. The enzyme is an aggregate consisting of two protein components, coded for by the unlinked genes cpaI and cpaII. These components were separated by diethylaminoethyl-cellulose chromatography. Their molecular weights, estimated by Sephadex gel filtration, were 36,000 and 130,000. The large component catalyzed the synthesis of carbamoylphosphate from ammonia. The small component was required in addition to the large one for the physiologically functional glutaminedependent activity. Apparent Michaelis constants at pH 7.5 of 1.25 mM for glutamine and 75 mM for NH4Cl were measured with the native enzyme. The use of various glutamine analogs, including 2-amino-4-oxo-5-chloropentanoic acid, indicated that binding of glutamine to a site located on the small component was followed by transfer of its amide nitrogen to the ammonia site on the heavy component. This ammonia site was able to function independently of the utilization of glutamine. However, binding of glutamine was conjectured to cause a conformational change in the heavy component that greatly increased the rate of synthesis of carbamoylphosphate from ammonia. Glutamine, which was also shown to stabilize the aggregation of the two components, appeared to be a major effector of the catalytic and structural properties of CPSase A. In view of these observations, the CPSase A of yeast appears to share a number of structural and catalytic properties with the Escherichia coli enzyme. Obviously, the unlinked cpaI and cpalI genes of yeast are homologous to the adjacent carA and carB genes that code for the two subunits of the bacterial enzyme.

Research paper thumbnail of Control of the activity of Escherichia coli carbamoyl phosphate synthetase by antagonistic allosteric effectors

Science (New York, N.Y.), Jan 23, 1966

The synthesis of carbamoyl phosphate required in both arginine and pyrimidine biosyntheses is car... more The synthesis of carbamoyl phosphate required in both arginine and pyrimidine biosyntheses is carried out by a single enzyme in Escherichia coli. Opposed effects of pyrimidine nucleotides and of ornithine on the activity of the enzyme ensure a proper supply of carbamoyl phosphate according to the needs of the two biosynthetic sequences.

Research paper thumbnail of Regulation and mutation affecting a glutamine dependent formation of carbamyl phosphate in

Biochemical and Biophysical Research Communications, 1964

Research paper thumbnail of Comparative studies on the degradation of guanidino and ureido compounds by Pseudomonas

Journal of General Microbiology, Nov 1, 1990

The utilization of guanidino and ureido compounds was studied in several Pseudomonas species. Mul... more The utilization of guanidino and ureido compounds was studied in several Pseudomonas species. Multiple routes of agmatine catabolism were found. All members of the homology group I of Pseudomonas use the initial deamination of agmatine to carbamoylputrescine which is subsequently converted to putrescine. In Pseudomonas indigofera, the catabolism of agmatine can also occur via an initial hydrolysis of the amidino group to putrescine catalyzed by an agmatine amidinohydrolase. A third pathway was found in Pseudomonas cepacia, namely oxidative deamination producing guanidinobutyraldehyde catalyzed by agmatine dehydrogenase, followed by formation of guanidinobutyrate and removal of urea by guanidinobutyrate amidinohydrolase to produce 4-aminobutyrate. Novel amidinohydrolases were characterized in P.putida for the utilization of arcaine and audouine, and in P. cepacia for arcaine, homoarginine and guanidinovalerate. Guanidinovalerate amidinohydrolase was also detected in P. doudorofii Some of these amidinohydrolases accept more than one substrate, e.g. guanidinobutyrate and guanidinovalerate utilization by P. doudorofii and P. cepacia, the catabolism of arcaine and audouine by P. putida, and the degradation of arcaine and homoarginine by P. cepacia.

Research paper thumbnail of Pathways of arginine biosynthesis in extreme thermophilic archaeo- and eubacteria

Journal of General Microbiology, 1990

The pathway of arginine biosynthesis was investigated in two thermophilic eubacteria, Thermus qua... more The pathway of arginine biosynthesis was investigated in two thermophilic eubacteria, Thermus quaticus and Thermotoga maritima, and in two thermophilic archaeobacteria, SulfoIobus solfataricus and Pyrococcus furiosus. In the first three organisms, arginine biosynthesis proceeds via N-acetylated intermediates as in mesophilic microorganisms. Only the enzymes catalysing the three last steps of the pathway could be detected in P. furiusus. The two eubacterial strains possess an ornithine acetyltransferase and are thus able to recycle the acetyl group from acetylornithine to glutamate. The archaeobacterium, S. solfataricus, uses the linear pathway in which the formation of ornithine is mediated by the hydrolytic enzyme acetylornithinase. Repression of enzyme synthesis by arginine was observed for most of the enzymes tested in T. aquaticus and S. solfataricus. Feedback inhibition by arginine was shown only on the ornithine acetyltransferase from T. aquaticus. This inhibition pattern is of interest since it would be the first example of control of arginine biosynthesis at this particular step. Data concerning the thermal stability of the arginine biosynthetic enzymes are presented. 0001-6018 0 1990 SGM

Research paper thumbnail of Mutations affecting uridine monophosphate pyrophosphorylase or the argR gene in Escherichia coli

MGG Molecular & General Genetics, 1972

In the course of experiments directed towards the isolation of mutants of Escherichia coli K12 wi... more In the course of experiments directed towards the isolation of mutants of Escherichia coli K12 with altered regulation of the synthesis of carbamoylphosphate synthetase, two types of mutations were found to affect the cumulative repression of this enzyme by arginine and uracil. Alteraction of the arginine pathway regulatory gene, argR, was shown to reduce the repressibility of the enzyme by both end products while mutations affecting uridine monophosphate pyrophosphorylase (upp) besides affecting uracil uptake preclude enzyme repression by uracil or cytosine in the biosynthesis of carbamoylphosphate and the pyrimidines. The upp mutations were located on the chromosome near the gua operon. Mutations previously designated as uraP are shown to belong to this class.

Research paper thumbnail of A comparative analysis of extreme thermophilic bacteria belonging to the genus Thermus

Archives of Microbiology, 1978

Several extreme thermophilic Gram negative bacteria found in a thermally polluted river in Belgiu... more Several extreme thermophilic Gram negative bacteria found in a thermally polluted river in Belgium have been compared with Thermus strains isolated from widely distant geographical areas. This analysis has become possible after the design of a new culture medium (162). All strains examined (including the isolate successively denominated Flavobacterium thermophilum and Thermus thermophilus) were found to be morphologically identical with strain YT-1 of Thermus aquaticus. The cells are immotile, rod-like, strictly aerobic, catalase and oxidase positive. They produce amylase, hydrolyze gelatin and are confirmed to be highly sensitive towards penicillin. The nutritional pattern of all strains has been analysed extensively, by testing a broad spectrum of possible substrates. The strains display a uniform response to the microbiological tests applied and most probably belong to the same species: Thermus aquaticus.

Research paper thumbnail of On the role of the Shine-Dalgarno sequence in determining the efficiency of translation initiation at a weak start codon in the car operon of Escherichia coli K12

Journal of Molecular Biology, Dec 20, 1988

Translation of the carA gene is efficiently initiated at the intrinsically weak UUG codon. A sing... more Translation of the carA gene is efficiently initiated at the intrinsically weak UUG codon. A single nucleotide substitution changing the Shine-Dalgarno box of carA (GGAGG) into the sequence TGAGG reduces translation of carA sevenfold. This result supports the view that extensive complementarity between the Shine-Dalgarno sequence and 16S RNA contributes significantly to the efficiency of translation when the latter starts at a weak initiation triplet.

Research paper thumbnail of Control-mechanisms acting at the transcriptional and post-transcriptional levels are involved in the synthesis of the arginine pathway carbamoylphosphate synthase of yeast

The Embo Journal, Feb 1, 1983

In Saccharomyces cerevisiae, the synthesis of the arginine pathway enzyme carbamoylphosphate synt... more In Saccharomyces cerevisiae, the synthesis of the arginine pathway enzyme carbamoylphosphate synthase (CPSase A) is subject to two control mechanisms. One mechanism, the general control of amino acid biosynthesis, influences the expression of both CPA1 and CPA2 genes, the structural genes for the two subunits of the enzyme. The second mechanism, the specific control of arginine biosynthesis, only affects the expression of CPA1. To study these mechanisms in more detail, we have cloned the CPA1 and CPA2 genes and used their DNA to measure the CPA1 and CPA2 mRNA content of cells grown under various conditions. A close coordination was observed in the variation of the levels of CPA1 and CPA2 mRNAs and polypeptide products under conditions where the general control of amino acid biosynthesis operates. In contrast, little correlation was found between the levels of CPA1 mRNA and the corresponding protein for conditions affecting repression by arginine: the total amplitude of variation was 6-fold higher for the CPA1 protein than for the CPA1 messenger transcript. Such findings are consistent with the conclusion that the general control operates at the transcriptional level and that the specific arginine control acts primarily at a post-transcriptional level.

Research paper thumbnail of Serine 948 and threonine 1042 are crucial residues for allosteric regulation of Escherichia coli carbamoylphosphate synthetase and illustrate coupling effects of activation and inhibition pathways 1 1 Edited by A. R. Fersht

J Mol Biol, 1999

Escherichia coli carbamoylphosphate synthetase (CPSase) is a key enzyme in the pyrimidine nucleot... more Escherichia coli carbamoylphosphate synthetase (CPSase) is a key enzyme in the pyrimidine nucleotides and arginine biosynthetic pathways. The enzyme harbors a complex regulation, being activated by ornithine and inosine 5′-monophosphate (IMP), and inhibited by UMP. CPSase mutants obtained by in vivo mutagenesis and selected on the basis of particular phenotypes have been characterized kinetically. Two residues, serine 948 and threonine 1042, appear crucial for allosteric regulation of CPSase. When threonine 1042 is replaced by an isoleucine residue, the enzyme displays a greatly reduced activation by ornithine. The T1042I mutated enzyme is still sensitive to UMP and IMP, although the effects of both regulators are reduced. When serine 948 is replaced by phenylalanine, the enzyme becomes insensitive to UMP and IMP, but is still activated by ornithine, although to a reduced extent. When correlating these observations to the structural data recently reported, it becomes clear that both mutations, which are located in spatially distinct regions corresponding respectively to the ornithine and the UMP/IMP binding sites, have coupled effects on the enzyme regulation. These results provide an illustration that coupling of regulatory pathways occurs within the allosteric subunit of E. coli CPSase.In addition, other mutants have been characterized, which display altered affinities for the different CPSase substrates and also slightly modified properties towards the allosteric effectors: P165S, P170L, A182V, P360L, S743N, T800F and G824D. Kinetic properties of these modified enzymes are also presented here and correlated to the crystal structure of E. coli CPSase and to the phenotype of the mutants.

Research paper thumbnail of In Vitro Synthesis of Escherichia coli Carbamoylphosphate Synthase: Evidence for Participation of the Arginine Repressor in Cumulative Repression

Journal of Bacteriology, 1980

A deoxyribonucleic acid-directed in vitro system for the synthesis of Escherichia coli carbamoylp... more A deoxyribonucleic acid-directed in vitro system for the synthesis of Escherichia coli carbamoylphosphate synthase has been developed, and its properties have been studied. The system uses the deoxyribonucleic acid of a lambda phage carrying the car genes (AdcarAB) as template and mediates the synthesis of both subunits of the enzyme. This newly synthesized enzyme exhibits the properties of native carbamoylphosphate synthase. A study of the in vitro synthetic capacities of S-30 extracts from strains containing either a mutated or the wild-type allele of gene argR supports earlier suggestions, based on in vivo evidence, that the argR product is involved in cumulative repression of carbamoylphosphate synthase by arginine and the pyrimidines. Repression in vitro is as efficient as in vivo. In keeping with such observation it is shown that in vitro synthesis of carbamoylphosphate synthase is repressed by partially purified arginine repressor.

Research paper thumbnail of Primary structure, partial purification and regulation of key enzymes of the acetyl cycle of arginine biosynthesis in Bacillus stearothermophilus: dual function of ornithine acetyltransferase.

J Gen Microbiol, Mar 1993

A 3.4 kb EcoRI fragment, cloned in E. coli, that carries part of a cluster of genes encoding argi... more A 3.4 kb EcoRI fragment, cloned in E. coli, that carries part of a cluster of genes encoding arginine biosynthetic functions of the thermophilic bacterium Bacillus stearothermophilus, was sequenced on both strands. The sequence consists of a truncated argC gene, an argJ region encoding a polypeptide with both N-acetylglutamate synthase and ornithine acetyltransferase activities, the argB gene and the N-terminal part of argD. The argB gene encodes a 258-amino-acid polypeptide with a deduced M(r) of 26918. A very high and thermostable N-acetylglutamate 5-phosphotransferase activity was detected in extracts of E. coli arg B mutants transformed with the 3.4 kb fragment on a plasmid. A polypeptide band of M(r) 27,000 was detected by SDS-PAGE of heat-treated extract from such a strain. Both N-acetylglutamate synthase and ornithine acetyltransferase are encoded by the same 1290 bp open reading frame. The deduced sequence of 410 amino acids corresponds to a peptide of M(r) 43,349. The subcloned B. stearothermophilus argJ can complement a double argA argE E. coli mutant to prototrophy. Gel-filtration of a heat-treated extract of the complemented double mutant E. coli host showed that N-acetylglutamate synthase and ornithine acetyltransferase activities co-elute in a single peak corresponding to M(r) 110,000. Both activities were also heat-inactivated at the same temperature and strongly inhibited by ornithine. These results suggest that both activities can be ascribed to a single protein.

Research paper thumbnail of Co-operative interactions between the catalytic sites in Escherichia coli aspartate transcarbamylase

Journal of Molecular Biology, 1990

In aspartate transcarbamylase (ATCase) each regulatory chain interacts with two catalytic chains ... more In aspartate transcarbamylase (ATCase) each regulatory chain interacts with two catalytic chains each one belonging to a different trimeric catalytic subunit (R1-C1 and R1-C4 types of interactions as defined in ). In order to investigate the interchain contacts that are involved in the co-operative interactions between the catalytic sites, a series of modified forms of the enzyme was prepared by site-directed mutagenesis. The amino acid replacements were devised on the basis of the previously described properties of an altered form of ATCase (pAR5-ATCase) which lacks the homotropic co-operative interactions between the catalytic sites. The results obtained (enzyme kinetics, bisubstrate analog influence and pH studies) show that the R1-C4 interaction is essential for the establishment of the enzyme conformation that has a low affinity for aspartate (T state), and consequently for the existence of co-operativity between the catalytic sites. This interaction involves the 236-250 region of the aspartate binding domain of the catalytic chain (240s loop) and the 143-149 region of the regulatory chain which comprises helix H3'.

Research paper thumbnail of Primary structure, partial purification and regulation of key enzymes of the acetyl cycle of arginine biosynthesis in Bacillus stearothermophilus: dual function of ornithine acetyltransferase

Journal of general microbiology, 1993

A 3.4 kb EcoRI fragment, cloned in E. coli, that carries part of a cluster of genes encoding argi... more A 3.4 kb EcoRI fragment, cloned in E. coli, that carries part of a cluster of genes encoding arginine biosynthetic functions of the thermophilic bacterium Bacillus stearothermophilus, was sequenced on both strands. The sequence consists of a truncated argC gene, an argJ region encoding a polypeptide with both N-acetylglutamate synthase and ornithine acetyltransferase activities, the argB gene and the N-terminal part of argD. The argB gene encodes a 258-amino-acid polypeptide with a deduced M(r) of 26918. A very high and thermostable N-acetylglutamate 5-phosphotransferase activity was detected in extracts of E. coli arg B mutants transformed with the 3.4 kb fragment on a plasmid. A polypeptide band of M(r) 27,000 was detected by SDS-PAGE of heat-treated extract from such a strain. Both N-acetylglutamate synthase and ornithine acetyltransferase are encoded by the same 1290 bp open reading frame. The deduced sequence of 410 amino acids corresponds to a peptide of M(r) 43,349. The subcl...

Research paper thumbnail of Structure-function relationship in allosteric aspartate carbamoyltransferase from Escherichia coli *1I. Primary structure of a pyrI gene encoding a modified regulatory subunit

Journal of Molecular Biology, 1985

In a previous article, we have identified a lambda bacteriophage directing the synthesis of a mod... more In a previous article, we have identified a lambda bacteriophage directing the synthesis of a modified aspartate carbamoyltransferase lacking substrate-co-operative interactions and insensitive to the feedback inhibitor CTP. These abnormal properties were ascribed to a mutation in the gene pyrl encoding the regulatory polypeptide chain of the enzyme. N'e now report the sequence of the mutated pyrl and show that, during the generation of this pyrB1-bearing phage, six codons from lambda DNA have been substituted for the eight terminal codons of the wild-type gene. A model is presented for the formation of this modified pyrI gene during the integrative recombination of the parental lambda phage with the Esch,erichia coli chromosome. An accompanying paper emphasizes the importance of t'hr carboxy-terminal end of the regulatory chain for the homotropic and heterotropica interactions of aspartate carbamoyltransferase.

Research paper thumbnail of A re-examination of the pathway for ornithine biosynthesis in a thermophilic and two mesophilic Bacillus species

Journal of General Microbiology, 1992

The expression of Buciffus steurothermophifus genes complementing arginine auxotrophs of Escheric... more The expression of Buciffus steurothermophifus genes complementing arginine auxotrophs of Escherichiu coli was studied. The activity responsible for the formation of ornithine in B. steurothermophifus was identified as a repressible ornithine acetyltransferase (genetic symbol urgJ) encoded by the same DNA fragment as the argC, urgA and urgB genes. Buciffus subtilis and Buciffus ficheniformis displayed the same pattern of enzyme activities as B. steurothermophifus. In contrast to previous reports, these organisms consequently use the cyclic pathway of ornithine biosynthesis. B. steurothermophifus also pssesses a broad specificity aminoacylase which exhibits low affinity towards NZ-acetyl-L-ornithine.

Research paper thumbnail of CarP, a novel gene regulating the transcription of the carbamoylphosphate synthetase operon of Escherichia coli

Journal of Molecular Biology, 1988

The carAB operon, encoding carbamoylphosphate synthetase (CPSase; EC 6.3.5.5) is transcribed from... more The carAB operon, encoding carbamoylphosphate synthetase (CPSase; EC 6.3.5.5) is transcribed from two tandem promoters. The upstream promoter (P1) is controlled by pyrimidines and the downstream promoter (P2) is controlled by arginine. We have isolated a new type of constitutive mutation (carP) that specifically affects the control of the pyrimidine-sensitive promoter but does not appear to influence other genes of the pyrimidine pathway. The carP mutation acts in trans and is dominant, which suggests that the carP product is an activator of car transcription. The downstream promoter P2, which is repressed by arginine, overlaps two operator modules characteristic of the arginine regulon. We have isolated two operator-constitutive mutations that specifically affect P2; both map in the upstream ARG box at a strongly conserved position.

Research paper thumbnail of DNA sequence of the carA gene and the control region of carAB: tandem promoters, respectively controlled by arginine and the pyrimidines, regulate the synthesis of carbamoyl-phosphate synthetase in Escherichia coli K-12

Proceedings of the National Academy of Sciences, 1984

The carAB operon of Escherichia coli K-12, which encodes the two subunits of carbamoyl-phosphate ... more The carAB operon of Escherichia coli K-12, which encodes the two subunits of carbamoyl-phosphate synthetase (glutamine hydrolyzing) [carbon-dioxide: L-glutamine amido-ligase (ADP-forming, carbamate-phosphorylating); EC 6.3.5.5], is cumulatively repressed by arginine and the pyrimidines. We describe the structure of the control region of carAB and the sequence of the carA gene. Nuclease S1 mapping experiments show that two adjacent tandem promoters within the carAB control region serve as initiation sites. The upstream promoter P1 is controlled by pyrimidines; the downstream promoter P2 is regulated by arginine. Attenuation control does not appear to be involved in the expression of carAB. A possible mechanism by which control at these promoters concurs to produce a cumulative pattern of repression is discussed. The translational start of carA is atypical; it consists of a UUG or AUU codon.

Research paper thumbnail of Structure-function relationship in allosteric aspartate carbamoyltransferase from Escherichia coli

Journal of Molecular Biology, 1985

In a previous article, we have identified a lambda bacteriophage directing the synthesis of a mod... more In a previous article, we have identified a lambda bacteriophage directing the synthesis of a modified aspartate carbamoyltransferase lacking substrate-co-operative interactions and insensitive to the feedback inhibitor CTP. These abnormal properties were ascribed to a mutation in the gene pyrI encoding the regulatory polypeptide chain of the enzyme. We now report the sequence of the mutated pyrI and show that, during the generation of this pyrBI-bearing phage, six codons from lambda DNA have been substituted for the eight terminal codons of the wild-type gene. A model is presented for the formation of this modified pyrI gene during the integrative recombination of the parental lambda phage with the Escherichia coli chromosome. An accompanying paper emphasizes the importance of the carboxy-terminal end of the regulatory chain for the homotropic and heterotropic interactions of aspartate carbamoyltransferase.

Research paper thumbnail of Enzyme systems in the mycobacteria

Archives of Biochemistry and Biophysics, 1963

Research paper thumbnail of Structure-function relationships in the arginine pathway carbamoylphosphate synthase of Saccharomyces cerevisiae

Journal of Bacteriology, Apr 1, 1978

The arginine pathway carbamoylphosphate synthase (CPSase A) from Saccharomyces cerevisiae was sho... more The arginine pathway carbamoylphosphate synthase (CPSase A) from Saccharomyces cerevisiae was shown to be highly unstable and could not be substantially purified. In spite of this instability, a number of important properties of this enzyme were determined with crude preparations. A molecular weight of 140,000 (7.9S) was estimated for the native enzyme by sucrose gradient centrifugation; a significantly higher value, 175,000, was obtained by gel filtration on Sephadex. The enzyme is an aggregate consisting of two protein components, coded for by the unlinked genes cpaI and cpaII. These components were separated by diethylaminoethyl-cellulose chromatography. Their molecular weights, estimated by Sephadex gel filtration, were 36,000 and 130,000. The large component catalyzed the synthesis of carbamoylphosphate from ammonia. The small component was required in addition to the large one for the physiologically functional glutaminedependent activity. Apparent Michaelis constants at pH 7.5 of 1.25 mM for glutamine and 75 mM for NH4Cl were measured with the native enzyme. The use of various glutamine analogs, including 2-amino-4-oxo-5-chloropentanoic acid, indicated that binding of glutamine to a site located on the small component was followed by transfer of its amide nitrogen to the ammonia site on the heavy component. This ammonia site was able to function independently of the utilization of glutamine. However, binding of glutamine was conjectured to cause a conformational change in the heavy component that greatly increased the rate of synthesis of carbamoylphosphate from ammonia. Glutamine, which was also shown to stabilize the aggregation of the two components, appeared to be a major effector of the catalytic and structural properties of CPSase A. In view of these observations, the CPSase A of yeast appears to share a number of structural and catalytic properties with the Escherichia coli enzyme. Obviously, the unlinked cpaI and cpalI genes of yeast are homologous to the adjacent carA and carB genes that code for the two subunits of the bacterial enzyme.

Research paper thumbnail of Control of the activity of Escherichia coli carbamoyl phosphate synthetase by antagonistic allosteric effectors

Science (New York, N.Y.), Jan 23, 1966

The synthesis of carbamoyl phosphate required in both arginine and pyrimidine biosyntheses is car... more The synthesis of carbamoyl phosphate required in both arginine and pyrimidine biosyntheses is carried out by a single enzyme in Escherichia coli. Opposed effects of pyrimidine nucleotides and of ornithine on the activity of the enzyme ensure a proper supply of carbamoyl phosphate according to the needs of the two biosynthetic sequences.

Research paper thumbnail of Regulation and mutation affecting a glutamine dependent formation of carbamyl phosphate in

Biochemical and Biophysical Research Communications, 1964

Research paper thumbnail of Comparative studies on the degradation of guanidino and ureido compounds by Pseudomonas

Journal of General Microbiology, Nov 1, 1990

The utilization of guanidino and ureido compounds was studied in several Pseudomonas species. Mul... more The utilization of guanidino and ureido compounds was studied in several Pseudomonas species. Multiple routes of agmatine catabolism were found. All members of the homology group I of Pseudomonas use the initial deamination of agmatine to carbamoylputrescine which is subsequently converted to putrescine. In Pseudomonas indigofera, the catabolism of agmatine can also occur via an initial hydrolysis of the amidino group to putrescine catalyzed by an agmatine amidinohydrolase. A third pathway was found in Pseudomonas cepacia, namely oxidative deamination producing guanidinobutyraldehyde catalyzed by agmatine dehydrogenase, followed by formation of guanidinobutyrate and removal of urea by guanidinobutyrate amidinohydrolase to produce 4-aminobutyrate. Novel amidinohydrolases were characterized in P.putida for the utilization of arcaine and audouine, and in P. cepacia for arcaine, homoarginine and guanidinovalerate. Guanidinovalerate amidinohydrolase was also detected in P. doudorofii Some of these amidinohydrolases accept more than one substrate, e.g. guanidinobutyrate and guanidinovalerate utilization by P. doudorofii and P. cepacia, the catabolism of arcaine and audouine by P. putida, and the degradation of arcaine and homoarginine by P. cepacia.

Research paper thumbnail of Pathways of arginine biosynthesis in extreme thermophilic archaeo- and eubacteria

Journal of General Microbiology, 1990

The pathway of arginine biosynthesis was investigated in two thermophilic eubacteria, Thermus qua... more The pathway of arginine biosynthesis was investigated in two thermophilic eubacteria, Thermus quaticus and Thermotoga maritima, and in two thermophilic archaeobacteria, SulfoIobus solfataricus and Pyrococcus furiosus. In the first three organisms, arginine biosynthesis proceeds via N-acetylated intermediates as in mesophilic microorganisms. Only the enzymes catalysing the three last steps of the pathway could be detected in P. furiusus. The two eubacterial strains possess an ornithine acetyltransferase and are thus able to recycle the acetyl group from acetylornithine to glutamate. The archaeobacterium, S. solfataricus, uses the linear pathway in which the formation of ornithine is mediated by the hydrolytic enzyme acetylornithinase. Repression of enzyme synthesis by arginine was observed for most of the enzymes tested in T. aquaticus and S. solfataricus. Feedback inhibition by arginine was shown only on the ornithine acetyltransferase from T. aquaticus. This inhibition pattern is of interest since it would be the first example of control of arginine biosynthesis at this particular step. Data concerning the thermal stability of the arginine biosynthetic enzymes are presented. 0001-6018 0 1990 SGM

Research paper thumbnail of Mutations affecting uridine monophosphate pyrophosphorylase or the argR gene in Escherichia coli

MGG Molecular & General Genetics, 1972

In the course of experiments directed towards the isolation of mutants of Escherichia coli K12 wi... more In the course of experiments directed towards the isolation of mutants of Escherichia coli K12 with altered regulation of the synthesis of carbamoylphosphate synthetase, two types of mutations were found to affect the cumulative repression of this enzyme by arginine and uracil. Alteraction of the arginine pathway regulatory gene, argR, was shown to reduce the repressibility of the enzyme by both end products while mutations affecting uridine monophosphate pyrophosphorylase (upp) besides affecting uracil uptake preclude enzyme repression by uracil or cytosine in the biosynthesis of carbamoylphosphate and the pyrimidines. The upp mutations were located on the chromosome near the gua operon. Mutations previously designated as uraP are shown to belong to this class.

Research paper thumbnail of A comparative analysis of extreme thermophilic bacteria belonging to the genus Thermus

Archives of Microbiology, 1978

Several extreme thermophilic Gram negative bacteria found in a thermally polluted river in Belgiu... more Several extreme thermophilic Gram negative bacteria found in a thermally polluted river in Belgium have been compared with Thermus strains isolated from widely distant geographical areas. This analysis has become possible after the design of a new culture medium (162). All strains examined (including the isolate successively denominated Flavobacterium thermophilum and Thermus thermophilus) were found to be morphologically identical with strain YT-1 of Thermus aquaticus. The cells are immotile, rod-like, strictly aerobic, catalase and oxidase positive. They produce amylase, hydrolyze gelatin and are confirmed to be highly sensitive towards penicillin. The nutritional pattern of all strains has been analysed extensively, by testing a broad spectrum of possible substrates. The strains display a uniform response to the microbiological tests applied and most probably belong to the same species: Thermus aquaticus.

Research paper thumbnail of On the role of the Shine-Dalgarno sequence in determining the efficiency of translation initiation at a weak start codon in the car operon of Escherichia coli K12

Journal of Molecular Biology, Dec 20, 1988

Translation of the carA gene is efficiently initiated at the intrinsically weak UUG codon. A sing... more Translation of the carA gene is efficiently initiated at the intrinsically weak UUG codon. A single nucleotide substitution changing the Shine-Dalgarno box of carA (GGAGG) into the sequence TGAGG reduces translation of carA sevenfold. This result supports the view that extensive complementarity between the Shine-Dalgarno sequence and 16S RNA contributes significantly to the efficiency of translation when the latter starts at a weak initiation triplet.

Research paper thumbnail of Control-mechanisms acting at the transcriptional and post-transcriptional levels are involved in the synthesis of the arginine pathway carbamoylphosphate synthase of yeast

The Embo Journal, Feb 1, 1983

In Saccharomyces cerevisiae, the synthesis of the arginine pathway enzyme carbamoylphosphate synt... more In Saccharomyces cerevisiae, the synthesis of the arginine pathway enzyme carbamoylphosphate synthase (CPSase A) is subject to two control mechanisms. One mechanism, the general control of amino acid biosynthesis, influences the expression of both CPA1 and CPA2 genes, the structural genes for the two subunits of the enzyme. The second mechanism, the specific control of arginine biosynthesis, only affects the expression of CPA1. To study these mechanisms in more detail, we have cloned the CPA1 and CPA2 genes and used their DNA to measure the CPA1 and CPA2 mRNA content of cells grown under various conditions. A close coordination was observed in the variation of the levels of CPA1 and CPA2 mRNAs and polypeptide products under conditions where the general control of amino acid biosynthesis operates. In contrast, little correlation was found between the levels of CPA1 mRNA and the corresponding protein for conditions affecting repression by arginine: the total amplitude of variation was 6-fold higher for the CPA1 protein than for the CPA1 messenger transcript. Such findings are consistent with the conclusion that the general control operates at the transcriptional level and that the specific arginine control acts primarily at a post-transcriptional level.

Research paper thumbnail of Serine 948 and threonine 1042 are crucial residues for allosteric regulation of Escherichia coli carbamoylphosphate synthetase and illustrate coupling effects of activation and inhibition pathways 1 1 Edited by A. R. Fersht

J Mol Biol, 1999

Escherichia coli carbamoylphosphate synthetase (CPSase) is a key enzyme in the pyrimidine nucleot... more Escherichia coli carbamoylphosphate synthetase (CPSase) is a key enzyme in the pyrimidine nucleotides and arginine biosynthetic pathways. The enzyme harbors a complex regulation, being activated by ornithine and inosine 5′-monophosphate (IMP), and inhibited by UMP. CPSase mutants obtained by in vivo mutagenesis and selected on the basis of particular phenotypes have been characterized kinetically. Two residues, serine 948 and threonine 1042, appear crucial for allosteric regulation of CPSase. When threonine 1042 is replaced by an isoleucine residue, the enzyme displays a greatly reduced activation by ornithine. The T1042I mutated enzyme is still sensitive to UMP and IMP, although the effects of both regulators are reduced. When serine 948 is replaced by phenylalanine, the enzyme becomes insensitive to UMP and IMP, but is still activated by ornithine, although to a reduced extent. When correlating these observations to the structural data recently reported, it becomes clear that both mutations, which are located in spatially distinct regions corresponding respectively to the ornithine and the UMP/IMP binding sites, have coupled effects on the enzyme regulation. These results provide an illustration that coupling of regulatory pathways occurs within the allosteric subunit of E. coli CPSase.In addition, other mutants have been characterized, which display altered affinities for the different CPSase substrates and also slightly modified properties towards the allosteric effectors: P165S, P170L, A182V, P360L, S743N, T800F and G824D. Kinetic properties of these modified enzymes are also presented here and correlated to the crystal structure of E. coli CPSase and to the phenotype of the mutants.

Research paper thumbnail of In Vitro Synthesis of Escherichia coli Carbamoylphosphate Synthase: Evidence for Participation of the Arginine Repressor in Cumulative Repression

Journal of Bacteriology, 1980

A deoxyribonucleic acid-directed in vitro system for the synthesis of Escherichia coli carbamoylp... more A deoxyribonucleic acid-directed in vitro system for the synthesis of Escherichia coli carbamoylphosphate synthase has been developed, and its properties have been studied. The system uses the deoxyribonucleic acid of a lambda phage carrying the car genes (AdcarAB) as template and mediates the synthesis of both subunits of the enzyme. This newly synthesized enzyme exhibits the properties of native carbamoylphosphate synthase. A study of the in vitro synthetic capacities of S-30 extracts from strains containing either a mutated or the wild-type allele of gene argR supports earlier suggestions, based on in vivo evidence, that the argR product is involved in cumulative repression of carbamoylphosphate synthase by arginine and the pyrimidines. Repression in vitro is as efficient as in vivo. In keeping with such observation it is shown that in vitro synthesis of carbamoylphosphate synthase is repressed by partially purified arginine repressor.