Carbamoyl phosphate synthetase: a tunnel runs through it

The direct transfer of metabolites from one protein to another in a biochemical pathway or between one active site and another within a single enzyme has been described as substrate channeling. The first structural visualization of such a phenomenon was provided by the X-ray crystallographic analysis of tryptophan synthase, in which a tunnel of approximately 25 Å in length was observed. The recently determined three-dimensional structure of carbamoyl phosphate synthetase sets a new long distance record in that the three active sites are separated by nearly 100 Å.     

Carbamoyl phosphate synthetase subunit Cpa1 interacting with Dut1, controls development,arginine biosynthesis,and pathogenicity of Colletotrichum gloeosporioides

Carbamoyl phosphate synthetase is involved in arginine biosynthesis in many organisms. In this study, we investigate the biological function of Cpa1, a small subunit of carbamoyl phosphate synthetase of Colletotrichum gloeosporioides. The deletion of the CPA1 gene affected vegetative growth, arginine biosynthesis, and fungal pathogenicity. Genetic complementation with native CPA1 fully recovered all these defective phenotypes. We observed that Cpa1-RFP fusion protein is localized at the mitochondria, which is consistent with Cpa2, a large subunit of carbamoyl phosphate synthetase. We identified the proteins that interact with Cpa1 by using the two-hybrid screen approach, and we showed that Dut1 interacts with Cpa1 but without Cpa2 in vivo. Dut1 is dispensable for hyphal growth, appressorial formation, and fungal pathogenicity. Interestingly, the Dut1-Cpa1 complex is localized at the mitochondria. Further studies showed that Dut1 regulates Cpa1-Cpa2 interaction in response to arginine. In summary, our studies provide new insights into how Cpa1 interacts with its partner proteins to mediate arginine synthesis.     

Carbamoyl phosphate synthetase activity from the cotyledons of developing and germinating pea seeds

Carbamoyl phosphate synthetase activity was measured in partially purified extracts from cotyledons of developing and germinating seeds of Pisum sativum L. Some properties of the enzyme were established. During cotyledon development, the activity initially increased sharply but decreased during further development. The activity from germinating seeds was only one-tenth of the maximum activity at an early developmental phase. The results are discussed in relation to pea seed development and germination.     

Carbamoyl phosphate synthetase 1 deficiency in Italy: clinical and genetic findings in a heterogeneous cohort

Carbamoyl Phosphate Synthetase 1 deficiency (CPS1D) is a rare autosomal recessive urea cycle disorder, potentially leading to lethal hyperammonemia. Based on the age of onset, there are two distinct phenotypes: neonatal and late form. The CPS1 enzyme, located in the mitochondrial matrix of hepatocytes and epithelial cells of intestinal mucosa, is encoded by the CPS1 gene. At present more than 220 clear-cut genetic lesions leading to CPS1D have been reported. As most of them are private mutations diagnosis is complicated.Here we report an overview of the main clinical findings and biochemical and molecular data of 13 CPS1D Italian patients. In two of them, one with the neonatal form and one with the late form, cadaveric auxiliary liver transplant was performed. Mutation analysis in these patients identified 17 genetic lesions, 9 of which were new confirming their “private” nature. Seven of the newly identified mutations were missense/nonsense changes. In order to study their protein level effects, we performed an in silico analysis whose results indicate that the amino acid substitutions occur at evolutionary conserved positions and affect residues necessary for enzyme stability or function.     

Carbamoyl phosphate synthetase: closure of the B-domain as a result of nucleotide binding

Carbamoyl phosphate synthetase (CPS) catalyzes the production of carbamoyl phosphate which is subsequently employed in the metabolic pathways responsible for the synthesis of pyrimidine nucleotides or arginine. The catalytic mechanism of the enzyme occurs through three highly reactive intermediates: carboxyphosphate, ammonia, and carbamate. As isolated from Escherichia coli, CPS is an alpha, beta-heterodimeric protein with its three active sites separated by nearly 100 A. In addition, there are separate binding sites for the allosteric regulators, ornithine, and UMP. Given the sizable distances between the three active sites and the allosteric-binding pockets, it has been postulated that domain movements play key roles for intramolecular communication. Here we describe the structure of CPS from E. coli where, indeed, such a domain movement has occurred in response to nucleotide binding. Specifically, the protein was crystallized in the presence of a nonhydrolyzable analogue, AMPPNP, and its structure determined to 2.1 A resolution by X-ray crystallographic analysis. The B-domain of the carbamoyl phosphate synthetic component of the large subunit closes down over the active-site pocket such that some atoms move by more than 7 A relative to that observed in the original structure. The trigger for this movement resides in the hydrogen-bonding interactions between two backbone amide groups (Gly 721 and Gly 722) and the beta- and gamma-phosphate groups of the nucleotide triphosphate. Gly 721 and Gly 722 are located in a Type III' reverse turn, and this type of secondary structural motif is also observed in D-alanine:D-alanine ligase and glutathione synthetase, both of which belong to the "ATP-grasp" superfamily of proteins. Details concerning the geometries of the two active sites contained within the large subunit of CPS are described.     

Carbamoyl phosphate biosynthesis and partition in pyrimidine and arginine pathways of Escherichia coli. In situ properties of carbamoyl-phosphate synthase, ornithine transcarbamylase and aspartate transcarbamylase in permeabilized cells

A procedure for the permeabilization of Escherichia coli cells was adapted to the in situ determination of the catalytic and regulatory properties of the enzymes responsible for the biosynthesis of carbamoyl phosphate and its utilization in the pyrimidine and arginine pathways. Differences in enzyme sensitivity to effectors and changes in pH dependence were observed. Partition of carbamoyl phosphate in the two metabolic pathways could be measured under conditions of substrate saturation. The results obtained will allow to test experimentally the theoretical predictions made by A. Goldbeter (1973) PhD thesis, Université Libre de Bruxelles, on the distribution of carbamoyl phosphate and the oscillation of its intracellular concentration.     

Cell-cycle dependence of dolichyl phosphate biosynthesis

The cell-cycle dependence of dolichyl phosphate biosynthesis has been investigated in mouse L-1210 cells fractionated by centrifugal elutriation. Dolichyl phosphate levels increased linearly through the cell cycle, reaching a value in late S phase twice that of early G1. The cell-cycle dependences of four dolichyl phosphate metabolizing enzymes have been measured: cis-prenyltransferase, CTP-dependent dolichol kinase, dolichyl phosphatase, and dolichyl pyrophosphatase. The kinase, the cis-prenyltransferase, and the pyrophosphatase showed cell-cycle variations, increasing through G1 to a maximum in S phase while the monophosphatase activity was cell-cycle independent. The rate of accumulation of dolichyl phosphate was not affected by growing the cells in mevalonolactone-supplemented media. The evidence presented is consistent with models in which either the cis-prenyltransferase or the kinase/phosphatase couple (or both) regulates the levels of dolichyl phosphate in the cell.     

Carbamoyl phosphate synthetase (CPSase) in the PYR1-3 multigene of Dictyostelium discoideum.

We have isolated and sequenced the genomic DNA from the slime-mould Dictyostelium discoideum multi-gene (PYR1-3) encoding the carbamoyl phosphate synthetase II domain (CPSase, EC.6.3.5.5). We describe sequencing by oligo-walking directly on PCR product in the solid-phase, avoiding subcloning procedures. The 2.4 kb fragment completes the sequence of the PYR1-3 gene, has no introns, and has the same structure as the rudimentary gene of Drosophila melanogaster. Comparison with the carbamoyl phosphate synthetases (CPSase I and CPSase II) of other species supports the hypothesis that this gene has arisen by tandem duplication from a smaller common ancestral gene in the progenote.     

Regulation of nebramycin biosynthesis by inorganic phosphate

The effect of inorganic phosphate on the biosynthesis of nebramycin factors2, 4 and5′ was studied inStreptomyces tenebrarius strain A (forming2, 4 and5′ in natural ratios) and its mutants B (forming predominantly2), C (forming2 as the only major product) and D (forming predominantly5′). In phosphate-supplemented complex media, the production of2 in A, B and C was reduced by 20–70%, while the yields of5′ remained unchanged in A and decreased by 30–60% in B. The production of4 increased by 50–90% in A and was fully suppressed in B. In D the biosynthesis of the three factors was inhibited completely.     

Polyprenyl phosphate biosynthesis in Mycobacterium tuberculosis and Mycobacterium smegmatis

Mycobacterium smegmatis has been shown to contain two forms of polyprenyl phosphate (Pol-P), while Mycobacterium tuberculosis contains only one. Utilizing subcellular fractions from M. smegmatis and M. tuberculosis, we show that Pol-P synthesis is different in these species. The specific activities of the prenyl diphosphate synthases in M. tuberculosis are 10- to 100-fold lower than those in M. smegmatis. In M. smegmatis decaprenyl diphosphate and heptaprenyl diphosphate were the main products synthesized in vitro, whereas in M. tuberculosis only decaprenyl diphosphate was synthesized. The data from both organisms suggest that geranyl diphosphate is the allylic substrate for two distinct prenyl diphosphate synthases, one located in the cell membrane that synthesizes omega,E,Z-farnesyl diphosphate and the other present in the cytosol that synthesizes omega,E,E,E-geranylgeranyl diphosphate. In M. smegmatis, the omega,E, Z-farnesyl diphosphate is utilized by a membrane-associated prenyl diphosphate synthase activity to generate decaprenyl diphosphate, and the omega,E,E,E-geranylgeranyl diphosphate is utilized by a membrane-associated activity for the synthesis of the heptaprenyl diphosphate. In M. tuberculosis, however, omega,E,E,E-geranylgeranyl diphosphate is not utilized for the synthesis of heptaprenyl diphosphate. Thus, the difference in the compositions of the Pol-P of M. smegmatis and M. tuberculosis can be attributed to distinct enzymatic differences between these two organisms.