Anabolic ornithine carbamoyltransferase of Escherichia coli and catabolic ornithine carbamoyltransferase of Pseudomonas putida. Steady-state kinetic analysis.

The anabolic and catabolic ornithine carbamoyltransferases of Pseudomonas putida display an undirectional catalytic specialization: in citrulline synthesis for the anabolic enzyme, in citrulline phosphorolysis for the catabolic one. The irreversibility of the anabolic enzyme in vitro has been previously explained by its kinetic properties, whereas the irreversibility of the catabolic transferase in vivo was shown to be due to its allosteric behaviour. In this work a steady-state kinetic analysis has been carried out on the catabolic ornithine carbamoyltransferase at pH 6.8 in the presence of the allosteric activator, phosphate. The kinetic mechanism of Escherichia coli ornithine carbamoyltransferase serving as a reference was also determined. For the E. coli enzyme in the reverse direction, the initial velocity patterns converging on the abscissa were obtained with either citrulline or arsenate as variable substrate. The inhibition by the product ornithine was linear competitive with respect to citrulline and linear non-competitive with respect to arsenate. In the forward direction phosphate and its analogs induce an inhibition by ornithine which is partial and competitive with respect to carbamoylphosphate. Together with the results of thermo-inactivation studies in the presence of each reactant, this observation suggests a random kinetic mechanism, but with most of the reaction flux following the path where carbamoylphosphate adds before ornithine, when substrates are present at Km levels. The allosteric catabolic ornithine carbamoyltransferase of Pseudomonas displays qualitatively the same pattern as the E. coli enzyme.     

Genetic and physiological characterization of Pseudomonas aeruginosa mutants affected in the catabolic ornithine carbamoyltransferase.

In Pseudomonas aeruginosa arginine can be degraded by the arginine "dihydrolase" system, consisting of arginine deiminase, catabolic ornithine carbamoyltransferase, and carbamate kinase. Mutants of P. aeruginosa strain PAO affected in the structural gene (arcB) of the catabolic ornithine carbamoyltransferase were isolated. Firt, and argF mutation (i.e., a block in the anabolic ornithine carbamoyltransferase) was suppressed specifically by a mutationally altered catabolic ornithine carbamoyltransferase capable of functioning in the anabolic direction. The suppressor locus arcB (Su) was mapped by transduction between hisII and argA. Second, mutants having lost suppressor activity were obtained. The Su- mutations were very closely linked to arcB (Su) and caused strongly reduced ornithine carbamoyltransferase activities in vitro. Under aerobic conditions, a mutant (PA0630) which had less than 1% of the wild-type catabolic ornithine carbamoyltransferase activity grew on arginine as the only carbon and nitrogen source, at the wild-type growth rate. When oxygen was limiting, strain PA0630 grown on arginine excreted citrulline in the stationary growth phase. These observations suggest that during aerobic growth arginine is not degraded exclusively via the dihydrolase pathway.     

Molecular size and symmetry of Pseudomonas aeruginosa catabolic ornithine carbamoyltransferase. An X-ray crystallography analysis

The catabolic ornithine carbamoyltransferase (EC 2.1.3.3) from Pseudomonas aeruginosa, that shows allosteric behaviour, and a mutant version of this enzyme has been crystallized in several different crystal forms. All of these have been characterized by X-ray diffraction methods. A 4.5 A resolution data set has been collected on a triclinic crystal. Analysis of the data using the self-rotation function shows that 12 monomers associate to form a particle with cubic 23 point group symmetry.     

Immunological and structural relatedness of catabolic ornithine carbamoyltransferases and the anabolic enzymes of enterobacteria.

Purified catabolic ornithine carbamoyltransferase of Pseudomonas putida and anabolic ornithine carbamoyltransferase (argF product) of Escherichia coli K-12 were used to prepare antisera. The two specific antisera gave heterologous cross-reactions of various intensities with bacterial catabolic ornithine carbamoyltransferases formed by Pseudomonas and representative organisms of other bacterial genera. The immunological cross-reactivity observed only between the catabolic ornithine carbamoyltransferases and the anabolic enzymes of enterobacteria suggests that these proteins share some structural similarities. Indeed, the amino acid composition of the anabolic ornithine carbamoyltransferase of E. coli K-12 (argF and argI products) closely resembles the amino acid compositions of the catabolic enzymes of Pseudomonas putida, Aeromonas formicans, Streptococcus faecalis, and Bacillus licheniformis. Comparison of the N-terminal amino acid sequence of the E. coli anabolic ornithine carbamoyltransferase with that of the A. formicans and Pseudomonas putida catabolic enzymes shows, respectively, 45 and 28% identity between the compared positions; the A. formicans sequence reveals 53% identity with the Pseudomonas putida sequence. These results favor the conclusion that anabolic ornithine carbamoyltransferases of enterobacteria and catabolic ornithine carbamoyltransferases derive from a common ancestral gene.     

Linker insertion mutagenesis based on IS21 transposition: isolation of an AMP-insensitive variant of catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa

The bacterial insertion sequence IS21 when repeated in tandem efficiently promotes non-replicative cointegrate formation in Escherichia coli. An IS21-IS21 junction region which had been engineered to contain unique SalI and BglII sites close to the IS21 termini was not affected in the ability to form cointegrates with target plasmids. Based on this finding, a novel procedure of random linker insertion mutagenesis was devised. Suicide plasmids containing the engineered junction region (pME5 and pME6) formed cointegrates with target plasmids in an E.coli host strain expressing the IS21 transposition proteins in trans. Cointegrates were resolved in vitro by restriction with SalI or BglII and ligation; thus, insertions of four or 11 codons, respectively, were created in the target DNA, practically at random. The cloned Pseudomonas aeruginosa arcB gene encoding catabolic ornithine carbamoyltransferase was used as a target. Of 20 different four-codon insertions in arcB, 11 inactivated the enzyme. Among the remaining nine insertion mutants which retained enzyme activity, three enzyme variants had reduced affinity for the substrate ornithine and one had lost recognition of the allosteric activator AMP. The linker insertions obtained illustrate the usefulness of the method in the analysis of structure-function relationships of proteins.     

Methionine-321 in the C-terminal α-helix of catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa is important for positive homotropic cooperativity

Abstract Pseudomonas aeruginosa has a pair of distinct ornithine carbamoyltransferases. The anabolic ornithine carbamoyltransferase encoded by the argF gene catalyzes the formation of citrulline from ornithine and carbamoylphosphate. The catabolic ornithine carbamoyltransferase encoded by the arcB gene promotes the reverse reaction in vivo; although this enzyme can be assayed in vitro for citrulline synthesis, its unidirectionality in vivo is determined by its high concentration at half maximum velocity for carbamoylphosphate ([S]_0.5) and high cooperativity toward this substrate. We have mutant forms of catabolic ornithine carbamoyltransferase catalyzing the anabolic reaction in vivo. The corresponding arcB mutant alleles on a multicopy plasmid specifically suppressed an argF mutation of P. aeruginosa . Two new mutant enzymes were obtained. When methionine 321 was replaced by isoleucine, the mutant enzyme showed loss of homotropic cooperativity at physiological carbamoylphosphate concentrations. Substitution of glutamate 105 by lysine resulted in a partial loss of the sigmoidal response to increasing carbamoylphosphate concentrations. However, both mutant enzymes were still sensitive to the allosteric activator AMP and to the inhibitor spermidine. These results indicate that at least two residues of catabolic ornithine carbamoyltransferase are critically involved in positive carbamoylphisphate cooperativity: glutamate 105 (previously known to be important) and methionine 321. Mutational changes in either amino acid will affect the geometry of helix H2, which contains several residues required for carbamoylphosphate binding.     

Nucleotide sequence and expression in Escherichia coli of the Pseudomonas aeruginosa lasA gene.

Pseudomonas aeruginosa PAO-E64 is a mutant which produces parental levels of elastase antigen but has no elastolytic activity at 37 degrees C. The lesion (lasA1) in PAO-E64 is not a mutation in the structural gene for P. aeruginosa elastase (P.A. Schad, R.A. Bever, T.I. Nicas, F. Leduce, L.F. Hanne, and B.H. Iglewski, J. Bacteriol. 169: 2691-2696, 1987). A 1.7-kilobase segment of DNA that complements the lasA1 lesion was sequenced. Computer analysis of the DNA sequence showed that it contained an open reading frame which encoded a 41,111-dalton protein. The lasA gene was expressed under an inducible PT-7 promoter, and a 40,000-dalton protein was detected in Escherichia coli lysates. The lasA protein was localized in the outer membrane fraction of E. coli. This lasA protein produced in E. coli activated the extracellular elastase produced by the P. aeruginosa mutant, PAO-E64.     

Reconstitution of mitochondrial protein transport with purified ornithine carbamoyltransferase precursor expressed in Escherichia coli

Ornithine carbamoyltransferase (OTC; subunit, 36,000 Da) is initially synthesized as a precursor (pOTC) with a transient NH2-terminal presequence of 32 amino acid residues and imported posttranslationally into the mitochondrial matrix. The rat pOTC was synthesized in Escherichia coli using an expression vector containing a thermoinducible lambda pL promoter. The recombinant pOTC represented 5-10% of the total bacterial protein and was present in the precipitate of the disrupted bacteria. The precipitate was washed and pOTC was extracted with 8 M urea or 0.1% cetyltrimethylammonium bromide. The extracted pOTC was essentially homogeneous, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified pOTC was cleaved to the intermediate-sized product of 37,000 Da by a processing protease partially purified from the matrix fraction of rat liver mitochondria. The purified recombinant pOTC, but not the mature form of OTC synthesized in E. coli and purified, competed with the in vitro-synthesized, radiolabeled pOTC for uptake and processing by the isolated rat liver mitochondria. The radiolabeled and purified recombinant pOTC could be imported into the isolated mitochondria and processed to the mature form in an energy- and rabbit reticulocyte lysate-dependent manner. When the purified pOTC was subjected to sucrose gradient centrifugation, it sedimented as a large aggregate of greater than 60 S in the absence of reticulocyte lysate, whereas it sedimented as a complex of about 5 S in the presence of the lysate. These observations together with our previous results indicate that a protein factor(s) present in the lysate interacts with pOTC and holds it in an import-competent form.     

Characterization of two ornithine carbamoyltransferases from Pseudomonas syringae pv. phaseolicola,the producer of phaseolotoxin

Two ornithine carbamoyltransferases (OCT 1 and OCT 2) were isolated from Pseudomonas syringae pv. phaseolicola and purified by precipitation with ammonium sulfate, heat denaturation, chromatography on DEAE-Sephadex A-50 and Sephadex G-200. Molecular weights of both enzymes: 110,000; optimal activity: pH 8.5 to 9.5 (OCT 1), pH 8.4 (OCT 2); apparent K _m for ornithine: 7·10^-4 (both enzymes); apparent K _m for carbamoylphosphate: 7·10^-4 (OCT 1), 2.8·10^-3 (OCT 2). Both enzymes possess only an anabolic function. OCT 1 is highly inhibited by low concentrations of phaseolotoxin and Orn-P(O)(NH₂)-NH-SO₃H, OCT 2 is insensitive to both compounds. The inhibition of OCT 1 is reversible.Non-common abbreviation PNSOrn Ornithine--P(O)(NH₂)-NH-SO₃H     

Determination of the quaternary structure of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

A 2.5 A resolution data set has been collected for crystals of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa. Analysis of the data using the rotation function shows that the alpha 2 beta 2 tetramers associate to form a particle with cubic 23 (T) point group symmetry. Prior to this analysis it was believed that eight tetramers associated to form the holoenzyme. The symmetry of the crystalline holoenzyme also addresses questions concerning its iron content and substrate stoichiometry.     

Ornithine carbamoyltransferase from Escherichia coli W. Purification, structure and steady-state kinetic analysis.

Ornithine carbamoyltransferase from Escherichia coli W was purified to homogeneity. The enzyme has a molecular weight of 105000. It is composed of three apparently identical subunits with molecular weights of 35000. The mechanism of the ornithine carbamoyltransferase enzyme system from E. coli W was investigated kinetically by using the approach of product inhibition and dead-end inhibition of both forward and reverse reactions. On the basis of the kinetic data and binding studies it appears that the mechanism of the reaction involves a compulsory sequence of substrate binding to the enzyme, in which carbamoylphosphate is the first substrate to bind to the enzyme and phosphate the last product to be released. The same studies also indicate that the mechanism involves dead-end complexes. The reaction mechanism appears consistent with that proposed by Theorell and Chance. Values have been determined for the Michaelis and dissociation constants involved in the combination of each reactant with the enzyme. Comparison of the values for the kinetic constants which are common to both forward and reverse reaction have shown that they are always of a comparable magnitude.