Catalytic mechanism of Escherichia coli isopentenyl diphosphate isomerase involves Cys-67, Glu-116, and Tyr-104 as suggested by crystal structures of complexes with transition state analogues and irreversible inhibitors.

Isopentenyl diphosphate (IPP):dimethylallyl diphosphate (DMAPP) isomerase is a key enzyme in the biosynthesis of isoprenoids. The reaction involves protonation and deprotonation of the isoprenoid unit and proceeds through a carbocationic transition state. Analysis of the crystal structures (2 A) of complexes of Escherichia coli IPP.DMAPPs isomerase with a transition state analogue (N,N-dimethyl-2-amino-1-ethyl diphosphate) and a covalently attached irreversible inhibitor (3,4-epoxy-3-methyl-1-butyl diphosphate) indicates that Glu-116, Tyr-104, and Cys-67 are involved in the antarafacial addition/elimination of protons during isomerization. This work provides a new perspective about the mechanism of the reaction.

Resistance profile and cross-resistance of HIV-1 among patients failing a non-nucleoside reverse transcriptase inhibitor-containing regimen.

The objectives were to determine the resistance profile and the rate of cross-resistance in HIV-1 infected patients failing an efavirenz or a nevirapine or a nevirapine then efavirenz containing regimens, and to investigate if zidovudine and more generally thymidine analog nucleosides lead to a particular genotypic pattern in nevirapine failing patients. A study was conducted in 104 patients with virological rebound to a non-nucleoside reverse transcriptase inhibitors (NNRTI) regimen (efavirenz n = 39, nevirapine n = 46 and nevirapine then efavirenz n = 19). Genotypic resistance testing was carried out of detectable plasma HIV-1 RNA (> 200 copies/ml). Among the 104 patients studied, only two patients failed to respond to the nevirapine regimen without selection of a NNRTI resistance mutation. All patients failing an efavirenz regimen harboured mutations conferring cross-resistance to nevirapine (K103N, Y188L, G190S). Among patients failing the nevirapine regimen and presenting with NNRTI mutations, 35 (80%) harboured mutations conferring cross-resistance to efavirenz (K101E, K103N, Y188L) and 9 (20%) harboured mutations conferring resistance to nevirapine alone (V106A and Y181C). In patients failing nevirapine then efavirenz therapy, all NNRTI resistance profile led to cross-resistance to all available NNRTIs. Among patients receiving nevirapine, the selection of mutations associated with a cross-resistance to efavirenz was more frequent statistically when a thymidine nucleoside analog (zidovudine or stavudine) was used in the regimen (P = 0.02). In conclusion, 100% of patients developed cross-resistance to nevirapine and efavirenz after treatment by efavirenz and 80% after treatment by nevirapine. The use of a thymidine analog concomitantly with nevirapine leads to the preferential selection of cross-resistance NNRTI mutations.

Probing the role of oligomerization in the high thermal stability of Pyrococcus furiosus ornithine carbamoyltransferase by site-specific mutants.

The Pyrococcus furiosus ornithine carbamoyltransferase (OTCase) is extremely heat stable and maintains 50% of its catalytic activity after 60 min at 100 degrees C. The enzyme has an unusual quaternary structure when compared to anabolic OTCases from mesophilic organisms. It is built up of four trimers arranged in a tetrahedral manner, while other anabolic enzymes are single trimers. Residues Trp21, Glu25, Met29 and Trp33 are located in the main interfaces that occur between the catalytic trimers within the dodecamer. They participate in either hydrophobic clusters or ionic interactions. In order to elucidate the role played by the oligomerization in the enzyme stability at very high temperatures, we performed mutagenesis studies of these residues. All the variants show similar catalytic activities and kinetic properties when compared to the wild-type enzyme, allowing the interpretation of the mutations solely on heat stability and quaternary structure. The W21A variant has only a slight decrease in its stability, and is a dodecamer. The variants E25Q, M29A, W33A, W21A/W33A and E25Q/W33A show that altering more drastically the interfaces results in a proportional decrease in heat stability, correlated with a gradual dissociation of dodecamers into trimers. Finally, the E25Q/M29A/W33A variant shows a very large decrease in heat stability and is a trimer. These results suggest that extreme thermal stabilization of this OTCase is achieved in part through oligomerization.

Crystal structure of isopentenyl diphosphate:dimethylallyl diphosphate isomerase.

Isopentenyl diphosphate:dimethylallyl diphosphate (IPP:DMAPP) isomerase catalyses a crucial activation step in the isoprenoid biosynthesis pathway. This enzyme is responsible for the isomerization of the carbon-carbon double bond of IPP to create the potent electrophile DMAPP. DMAPP then alkylates other molecules, including IPP, to initiate the extraordinary variety of isoprenoid compounds found in nature. The crystal structures of free and metal-bound Escherichia coli IPP isomerase reveal critical active site features underlying its catalytic mechanism. The enzyme requires one Mn(2+) or Mg(2+) ion to fold in its active conformation, forming a distorted octahedral metal coordination site composed of three histidines and two glutamates and located in the active site. Two critical residues, C67 and E116, face each other within the active site, close to the metal-binding site. The structures are compatible with a mechanism in which the cysteine initiates the reaction by protonating the carbon-carbon double bond, with the antarafacial rearrangement ultimately achieved by one of the glutamates involved in the metal coordination sphere. W161 may stabilize the highly reactive carbocation generated during the reaction through quadrupole- charge interaction.

Preliminary structural studies of Escherichia coli isopentenyl diphosphate isomerase.

Escherichia coli isopentenyl diphosphate isomerase, an enzyme catalyzing a key step in isoprenoid biosynthesis, has been produced in selenomethionyl form. The protein was purified and crystallized by the hanging-drop vapour-diffusion method. Crystals display trigonal symmetry, with unit-cell parameters a = b = 71.3, c = 61.7 A, and diffract to 1.45 A resolution.

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.

Purification, crystallization and preliminary X-ray analysis of catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa.

The catabolic ornithine carbamoyltransferase (OTCase) from Pseudomonas aeruginosa exhibits allosteric behaviour, with two conformational states of the molecule: an active R form and an inactive T form. The enzyme is a dodecamer with a molecular mass of 455700 Da. Three crystal forms have been obtained. Crystals of allosteric state T are rhombohedral, belonging to the R3 space group, with hexagonal unit-cell parameters a = b = 180.6, c = 122.0 A. They diffract to a resolution of 4.5 A. Two crystal forms for allosteric state R have been obtained, with hexagonal and cubic symmetries. Hexagonal crystals, which diffract to a resolution of 3. 4 A, belong to the space group P6(3) with unit-cell parameters a = b = 140.8, c = 145.6 A. The cubic crystals belong to space group I23, with unit-cell parameter a = 134.32 A and diffract to a resolution better than 2.5 A. In all crystal forms, the dodecamer exhibits a 23 point-group symmetry.

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.

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.

Allosteric regulation in Pseudomonas aeruginosa catabolic ornithine carbamoyltransferase revisited: association of concerted homotropic cooperative interactions and local heterotropic effects.

The allosteric catabolic ornithine carbamoyltransferase (OTCase) from Pseudomonas aeruginosa, a dodecamer build up of four trimers of identical subunits, shows strong carbamoylphosphate homotropic co-operativity. Its activity is allosterically inhibited by spermidine and activated by AMP. Modified forms of the enzyme exhibiting substantial alterations in both homotropic and heterotropic interactions were recently obtained. We report here the first detailed kinetic characterization of homotropic and heterotropic modulations in allosteric wild-type and in engineered OTCases. Homotropic co-operativity for the saturation either by citrulline or arsenate was also observed when arsenate was utilised as an alternate substrate of the reverse reaction. Amino acid substitution of glutamate 105 by a glycine produces an enzyme devoid of homotropic interactions between the catalytic sites for carbamoylphosphate. This mutant, which is blocked in an active conformation, is still sensitive to the allosteric effector AMP, which increases affinity with respect to the substrate, carbamoylphosphate. It is also observed that homotropic co-operative interactions do not reappear in the E105G enzyme upon strong inhibition by the allosteric inhibitor of the wild-type enzyme, spermidine.Replacement of residues 34 to 101 of the native enzyme by the homologous amino acids of anabolic Escherichia coli OTCase produces a trimeric enzyme which retains reduced homotropic co-operativity. Activation by AMP and inhibition by spermidine of this chimaeric OTCase do not affect carbamoylphosphate homotropic co-operativity. AMP acts by reducing the concentration of substrate at half maximum velocity while spermidine acts in the inverse way. These observations indicate that in the two mutant forms of OTCase, homotropic and heterotropic interactions can be uncoupled and therefore must involve different molecular mechanisms. Furthermore, the results of stimulation of enzyme activity by phosphate, arsenate, pyrophosphate and phosphonoacetyl-l-ornithine on wild-type and mutant OTCases suggest that the physiological substrate phosphate, besides acting at the catalytic site, may act at an allosteric site. On the other hand, pyrophosphate and phosphonoacetyl-l-ornithine activation results exclusively from interactions of this effector with the active site residues.

-Cerebral air embolism after removal of an internal jugular vein catheter-. / Embolie gazeuse cérébrale après retrait d'un cathéter jugulaire interne.

Central venous catheters are usually inserted and manipulated by anaesthetists-intensivists and others familiar with their use under surgical conditions, yet they are often removed on the wards by junior doctors or nurses insufficiently trained in the removal procedure. In order to illustrate the risks presented by such a practice, we report a case of cerebral air embolism following the withdrawal of an internal jugular catheter in a sitting patient. The mechanisms of air entry into the venous and systemic circulation are considered, as well as the preventive and therapeutic measures.

The crystal structure of Pyrococcus furiosus ornithine carbamoyltransferase reveals a key role for oligomerization in enzyme stability at extremely high temperatures.

The Pyrococcus furiosus (PF) ornithine carbamoyltransferase (OTCase; EC 2.1.3.3) is an extremely heat-stable enzyme that maintains about 50% of its activity after heat treatment for 60 min at 100 degrees C. To understand the molecular basis of thermostability of this enzyme, we have determined its three-dimensional structure at a resolution of 2.7 A and compared it with the previously reported structures of OTCases isolated from mesophilic bacteria. Most OTCases investigated up to now are homotrimeric and devoid of allosteric properties. A striking exception is the catabolic OTCase from Pseudomonas aeruginosa, which is allosterically regulated and built up of four trimers disposed in a tetrahedral manner, an architecture that actually underlies the allostery of the enzyme. We now report that the thermostable PF OTCase (420 kDa) presents the same 23-point group symmetry. The enzyme displays Michaelis-Menten kinetics. A detailed comparison of the two enzymes suggests that, in OTCases, not only allostery but also thermophily was achieved through oligomerization of a trimer as a common catalytic motif. Thermal stabilization of the PF OTCase dodecamer is mainly the result of hydrophobic interfaces between trimers, at positions where allosteric binding sites have been identified in the allosteric enzyme. The present crystallographic analysis of PF OTCase provides a structural illustration that oligomerization can play a major role in extreme thermal stabilization.

Kinetic studies of allosteric catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa.

The pseudo-reverse reaction of Pseudomonas aeruginosa catabolic ornithine carbamoyltransferase in which arsenate is first coupled to citrulline followed by elimination of carbamylarsenate has been studied. Arsenate and citrulline saturation curves are sigmoidal. The different responsiveness of the transcarbamoylase to isosteric and allosteric ligands was examined both in the forward reaction, the carbamoylation of ornithine, and in the pseudo-reverse reaction, the arsenolytic cleavage of citrulline. Nucleoside monophosphates and polyamines that act as allosteric activators and inhibitors, respectively, on the carbamoylation reaction have the same effect on the rate of the arsenolytic cleavage of citrulline. ATP and other nucleoside triphosphates were found to stimulate enzyme activity at low carbamoylphosphate concentration with little influence on the carbamoylphosphate concentration at half-maximum velocity as well as on the cooperative index. When measuring the initial rate of the reverse reaction, the arsenolytic cleavage of citrulline, ATP was found to be a weak inhibitor, whereas CTP still stimulates the reaction and UTP was without influence. This unidirectional inhibition or activation phenomenon is likely apparent since initial studies were conducted and no consideration was given to equilibrium conditions. Regulation of catabolic OTCase by nucleoside triphosphates is without physiological meaning. In contrast, stimulation by nucleoside monophosphates may indicate that energy limitation could promote the synthesis and activity of the catabolic enzyme.

The arginine deiminase pathway in Rhizobium etli: DNA sequence analysis and functional study of the arcABC genes.

Sequence analysis upstream of the Rhizobium etli fixLJ homologous genes revealed the presence of three open reading frames homologous to the arcABC genes of Pseudomonas aeruginosa. The P. aeruginosa arcABC genes code for the enzymes of the arginine deiminase pathway: arginine deiminase, catabolic ornithine carbamoyltransferase (cOTCase), and carbamate kinase. OTCase activities were measured in free-living R. etli cells and in bacteroids isolated from bean nodules. OTCase activity in free-living cells was observed at a different pH optimum than OTCase activity in bacteroids, suggesting the presence of two enzymes with different characteristics and different expression patterns of the corresponding genes. The characteristics of the OTCase isolated from the bacteroids were studied in further detail and were shown to be similar to the properties of the cOTCase of P. aeruginosa. The enzyme has a pH optimum of 6.8 and a molecular mass of approximately 450 kDa, is characterized by a sigmoidal carbamoyl phosphate saturation curve, and exhibits a cooperativity for carbamoyl phosphate. R. etli arcA mutants, with polar effects on arcB and arcC, were constructed by insertion mutagenesis. Bean nodules induced by arcA mutants were still able to fix nitrogen but showed a significantly lower acetylene reduction activity than nodules induced by the wild type. No significant differences in nodule dry weight, plant dry weight, and number of nodules were found between the wild type and the mutants. Determination of the OTCase activity in extracts from bacteroids revealed a strong decrease in activity of this enzyme in the arcA mutant compared to the wild-type strain. Finally, we observed that expression of an R. etli arcA-gusA fusion was strongly induced under anaerobic conditions.

[Does laparoscopy increase the bacteriological risk of appendectomy? Results of a randomized prospective study]. / La coelioscopie augmente-t-elle le risque bactériologique de l'appendicectomie? Résultats d'une étude prospective randomisée.

OBJECTIVE: The authors compare the risk of bacteraemia in open and laparoscopic appendectomy in a prospective randomized study. METHODS: 35 patients with a presumptive diagnosis of acute appendicitis were randomized to have conventional open or laparoscopic surgical procedures. Before randomization, patients signed a consent form to participate in the study. Patients who were converted from laparoscopic to open appendectomy (3 cases), HIV+, allergic to Augmentin or who had contraindications to laparoscopic surgery were excluded from the study. A total of 32 patients were randomized: 17 to open (group I) and 15 to laparoscopic surgery (group II). There were no significant differences with regard to age, ASA score, symptoms or macroscopic aspect of the appendix. Two patients had a normal appendix, 12 had acute appendicitis, 14 gangrenous appendicitis and 4 ruptured or abscessed appendicitis. All patients received preoperative antibiotic prophylaxis (Augmentin) after blood cultures (H1) were drawn. Five other blood cultures were performed in standard medium and medium neutralizing Augmentin: at the time of opening the peritoneum (H2), after appendectomy (H3), after closure of the abdomen (H4), and at 6 (H5) and 12 hours (H6) after the operation. Bacterial cultures from the appendix site were performed before (P1) and after (P2) appendectomy. RESULTS: The operative mortality rate after conventional or laparoscopic appendectomy was nil. The incidence of post-operative morbidity was 4 cases in group I and 2 cases in group II. No positive bacterial culture was obtained in 17 patients. The distribution of these patients was similar in groups I and II. Samples P1 and P2 were positive in 5 cases. Nine of 27 cases with negative P1 became positive in P2 (33%). There was no significant difference between the two groups with regard to the appearance of the appendix. Only two patients had positive blood cultures at H1. One of them had blood cultures at H3, H4 positive for a second germ. CONCLUSION: A low risk of bacteraemia exists for both open and laparoscopic appendectomy. This risk did not appear to increase for laparoscopy. Conventional and laparoscopic surgical procedures led to positive peritoneal bacterial cultures after appendectomy in 33% of cases.

Use of a designed fusion protein dissociates allosteric properties from the dodecameric state of Pseudomonas aeruginosa catabolic ornithine carbamoyltransferase.

The catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa, an enzyme consisting of 12 identical 38-kDa subunits, displays allosteric properties, namely carbamoylphosphate homotropic cooperativity and heterotropic activation by AMP and other nucleoside monophosphates and inhibition by polyamines. To shed light on the effect of the oligomeric organization on the enzyme's activity and/or allosteric behavior, a hybrid ornithine carbamoyltransferase/glutathione S-transferase (OTCase-GST) molecule was constructed by fusing the 3' end of the P. aeruginosa arcB gene (OTCase) to the 5' end of the cDNA encoding Musca domestica GST by using a polyglycine encoding sequence as a linker. The fusion protein was overexpressed in Escherichia coli and purified from cell extracts by affinity chromatography, making use of the GST domain. It was found to exist as a trimer and to retain both the homotropic and heterotropic characteristic interactions of the wild-type catabolic OTCase but to a lower extent as compared with the wild-type OTCase. The dodecameric organization of catabolic P. aeruginosa OTCase may therefore be related to an enhancement of the substrate cooperativity already present in its trimers (and perhaps also to the thermostability of the enzyme).

Catabolic ornithine carbamoyltransferase of Pseudomonas aeruginosa. Importance of the N-terminal region for dodecameric structure and homotropic carbamoylphosphate cooperativity.

Pseudomonas aeruginosa has an anabolic (ArgF) and a catabolic (ArcB) ornithine carbamoyltransferase (OTCase). Despite extensive sequence similarities, these enzymes function unidirectionally in vivo. In the dodecameric catabolic OTCase, homotropic cooperativity for carbamoylphosphate strongly depresses the anabolic reaction; the residue Glu1O5 and the C-terminus are known to be essential for this cooperativity. When Glu1O5 and nine C-terminal amino acids of the catabolic OTCase were introduced, by in vitro genetic manipulation, into the closely related, trimeric, anabolic (ArgF) OTCase of Escherichia coli, the enzyme displayed Michaelis-Menten kinetics and no cooperativity was observed. This indicates that additional amino acid residues are required to produce homotropic cooperativity and a dodecameric assembly. To localize these residues, we constructed several hybrid enzymes by fusing, in vivo or in vitro, the E. coli argF gene to the P. aeruginosa arcB gene. A hybrid enzyme consisting of 101 N-terminal ArgF amino acids fused to 233 C-terminal ArcB residues and the reciprocal ArcB-ArgF hybrid were both trimers with little or no cooperativity. Replacing the seven N-terminal residues of the ArcB enzyme by the corresponding six residues of E. coli ArgF enzyme produced a dodecameric enzyme which showed a reduced affinity for carbamoylphosphate and an increase in homotropic cooperativity. Thus, the N-terminal amino acids of catabolic OTCase are important for interaction with carbamoylphosphate, but do not alone determine dodecameric assembly. Hybrid enzymes consisting of either 26 or 42 N-terminal ArgF amino acids and the corresponding C-terminal ArcB residues were both trimeric, yet they retained some homotropic cooperativity. Within the N-terminal ArcB region, a replacement of motif 28-33 by the corresponding ArgF segment destabilized the dodecameric structure and the enzyme existed in trimeric and dodecameric states, indicating that this region is important for dodecameric assembly. These findings were interpreted in the light of the three-dimensional structure of catabolic OTCase, which allows predictions about trimer-trimer interactions. Dodecameric assembly appears to require at least three regions: the N- and C-termini (which are close to each other in a monomer), residues 28-33 and residues 147-154. Dodecameric structure correlates with high carbamoylphosphate cooperativity and thermal stability, but some trimeric hybrid enzymes retain cooperativity, and the dodecameric Glu1O5-->Ala mutant gives hyperbolic carbamoylphosphate saturation, indicating that dodecameric structure is neither necessary nor sufficient to ensure cooperativity.

Crystal structure of Pseudomonas aeruginosa catabolic ornithine transcarbamoylase at 3.0-A resolution: a different oligomeric organization in the transcarbamoylase family.

The crystal structure of the Glu-105-->Gly mutant of catabolic ornithine transcarbamoylase (OTCase; carbamoyl phosphate + L-ornithine = orthophosphate + L-citrulline, EC 2.1.3.3) from Pseudomonas aeruginosa has been determined at 3.0-A resolution. This mutant is blocked in the active R (relaxed) state. The structure was solved by the molecular replacement method, starting from a crude molecular model built from a trimer of the catalytic subunit of another transcarbamoylase, the extensively studied aspartate transcarbamoylase (ATCase) from Escherichia coli. This model was used to generate initial low-resolution phases at 8-A resolution, which were extended to 3-A by noncrystallographic symmetry averaging. Four phase extensions were required to obtain an electron density map of very high quality from which the final model was built. The structure, including 4020 residues, has been refined to 3-A, and the current crystallographic R value is 0.216. No solvent molecules have been added to the model. The catabolic OTCase is a dodecamer composed of four trimers organized in a tetrahedral manner. Each monomer is composed of two domains. The carbamoyl phosphate binding domain shows a strong structural homology with the equivalent ATCase part. In contrast, the other domain, mainly implicated in the binding of the second substrate (ornithine for OTCase and aspartate for ATCase) is poorly conserved. The quaternary structures of these two allosteric transcarbamoylases are quite divergent: the E. coli ATCase has pseudo-32 point-group symmetry, with six catalytic and six regulatory chains; the catabolic OTCase has 23 point-group symmetry and only catalytic chains. However, both enzymes display homotropic and heterotropic cooperativity.

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

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 isolated 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 carbamoylphosphate 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.

Purification and properties of a succinyltransferase from Pseudomonas aeruginosa specific for both arginine and ornithine.

The arginine and ornithine succinyltransferase from Pseudomonas aeruginosa, a bifunctional enzyme involved in the aerobic utilization of arginine and ornithine, has been purified to homogeneity. The apparent molecular mass of the native enzyme was 150 kDa by gel filtration and 140 kDa by polyacrylamide gel electrophoresis under non-denaturing conditions. After SDS/PAGE two subunits of 35 kDa and 37 kDa were evident, indicating that the enzyme is a heterotetramer. Microsequence analysis of the electroblotted protein bands gave two different but well-conserved N-terminal amino acid sequences. The L-arginine saturation curve followed Henri-Michaelis kinetics with an apparent Km value of 0.5 mM. The sigmoidal saturation curve for L-ornithine indicated allosteric behaviour. D-Arginine, a competitive inhibitor with respect to L-arginine, reduced L-ornithine cooperativity. In the presence of spermidine, the L-ornithine saturation curve became increasingly sigmoidal, the Hill coefficient shifting from 2.5 in the absence of the inhibitor, to 3.5 in the presence of 20 mM spermidine. The L-arginine analog, L-homoarginine, was also a substrate of the succinyltransferase, and the saturation of the enzyme by this substrate was also cooperative. All these data confirmed the allosteric nature of the enzyme. Moreover, a mutant growing faster on L-ornithine than the parent strain had a modified succinyltransferase with a reduced L-ornithine cooperativity. The fate of L-homoarginine was different depending on whether the succinyltransferase was induced or not; excreted succinylhomoarginine was found in cultures induced for the transferase activity whereas guanidinovalerate was excreted in non-induced cultures. The 'waste' of succinyl CoA, which could not be regenerated from the excreted succinylhomoarginine, explained the inhibition exerted by L-homoarginine on growth when ornithine or arginine was used as the growth medium.