Localization of carbamoylphosphate synthetase and aspartate carbamoyltransferase in chloroplasts

The localization of carbamoylphosphate synthetase (CPSase) and aspartate carbamoyltransferase (ACTase), the first two enzymes of the pyrimidine biosynthetic pathway, in chloroplasts was investigated. In dark-grown radish (Raphanus sativus) seedlings, light induced a prominent increase in CPSase activity, but had little effect on ACTase activity. Both enzymes were found in chloroplasts isolated from radish cotyledons and leaves of spinach (Spinacia oleracea), soybean (Glycine max), and corn (Zea mays). The higher activity of ACTase relative to CPSase is discussed in relation to the instability of carbamoylphosphate, the product of the CPSase, and to the control of pyrimidine synthesis. Based on these results, the function of CPSase and ACTase in chloroplasts is discussed.     

Repression of Escherichia coli carbamoylphosphate synthase: relationships with enzyme synthesis in the arginine and pyrimidine pathways.

Cumulative repression of Escherichia coli carbamoylphosphate synthase (CPSase; EC 2.7.2.9) by arginine and pyrimidine was analyzed in relation to control enzyme synthesis in the arginine and pyrimidine pathways. The expression of carA and carB, the adjacent genes that specify the two subunits of the enzyme, was estimated by means of an in vitro complementation assay. The synthesis of each gene product was found to be under repression control. Coordinate expression of the two genes was observed under most conditions investigated. They might thus form an operon. The preparation of strains blocked in the degradation of cytidine and harboring leaky mutations affecting several steps of pyrimidine nucleotide synthesis made it possible to distinguish between the effects of cytidine and uridine compounds in the repression of the pyrimidine pathway enzymes. The data obtained suggest that derivatives of both cytidine and uridine participate in the repression of CPSase. In addition, repression of CPSase by arginine did not appear to occur unless pyrimidines were present at a significant intracellular concentration. This observation, together with our previous report that argR mutations impair the cumulative repression of CPSase, suggests that this control is mediated through the concerted effects of regulatory elements specific for the arginine and pyrimidine pathways.     

Aquifex aeolicus aspartate transcarbamoylase, an enzyme specialized for the efficient utilization of unstable carbamoyl phosphate at elevated temperature

Aquifex aeolicus, an organism that flourishes at 95 degrees C, is one of the most thermophilic eubacteria thus far described. The A. aeolicus pyrB gene encoding aspartate transcarbamoylase (ATCase) was cloned, overexpressed in Escherichia coli, and purified by affinity chromatography to a homogeneous form that could be crystallized. Chemical cross-linking and size exclusion chromatography showed that the protein was a homotrimer of 34-kDa catalytic chains. The activity of A. aeolicus ATCase increased dramatically with increasing temperature due to an increase in kcat with little change in the Km for the substrates, carbamoyl phosphate and aspartate. The Km for both substrates was 30-40-fold lower than the corresponding values for the homologous E. coli ATCase catalytic subunit. Although rapidly degraded at high temperature, the carbamoyl phosphate generated in situ by A. aeolicus carbamoyl phosphate synthetase (CPSase) was channeled to ATCase. The transient time for carbamoyl aspartate formation was 26 s, compared with the much longer transient times observed when A. aeolicus CPSase was coupled to E. coli ATCase. Several other approaches provided strong evidence for channeling and transient complex formation between A. aeolicus ATCase and CPSase. The high affinity for substrates combined with channeling ensures the efficient transfer of carbamoyl phosphate from the active site of CPSase to that of ATCase, thus preserving it from degradation and preventing the formation of toxic cyanate.     

Structural and regulatory mutations allowing utilization of citrulline or carbamoylaspartate as a source of carbamoylphosphate in Escherichia coli K-12.

Escherichia coli mutants lacking carbamoylphosphate synthase require arginine and uracil for growth. It is, however, possible to obtain mutants in which carbamoylphosphate is obtained by phosphorolysis of citrulline or carbamyolaspartate. Citrulline utilizers are argG bradytrophs or strains in which the synthesis of ornithine carbamoyltransferase (either of the F or I type) is specifically depressed by unstable chromosomal rearrangements or stable mutations that presumably affect the operators of those genes. Carbamoylaspartate utilization as a source of carbamoylphosphate appears to require more than one mutation; the best-understood strains are pyrD pyrH or pyrC pyrH mutants in which aspartate carbamoyltransferase activity is high and the pool of cytidine triphosphate (feedback inhibitor of aspartate carbamoyl-transferase) is presumably low and in which channeling of carbamoylaspartate towards pyrimidine biosynthesis is considerably reduced. Selection of enzyme overproducers based on a metabolic dependency for a reversed enzymatic reaction can be regarded as a means for isolating regulatory mutants.     

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

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.     

Half of Saccharomyces cerevisiae carbamoyl phosphate synthetase produces and channels carbamoyl phosphate to the fused aspartate transcarbamoylase domain.

The first two steps of the de novo pyrimidine biosynthetic pathway in Saccharomyces cerevisiae are catalyzed by a 240-kDa bifunctional protein encoded by the ura2 locus. Although the constituent enzymes, carbamoyl phosphate synthetase (CPSase) and aspartate transcarbamoylase (ATCase) function independently, there are interdomain interactions uniquely associated with the multifunctional protein. Both CPSase and ATCase are feedback inhibited by UTP. Moreover, the intermediate carbamoyl phosphate is channeled from the CPSase domain where it is synthesized to the ATCase domain where it is used in the synthesis of carbamoyl aspartate. To better understand these processes, a recombinant plasmid was constructed that encoded a protein lacking the amidotransferase domain and the amino half of the CPSase domain, a 100-kDa chain segment. The truncated complex consisted of the carboxyl half of the CPSase domain fused to the ATCase domain via the pDHO domain, an inactive dihydroorotase homologue that bridges the two functional domains in the native molecule. Not only was the "half CPSase" catalytically active, but it was regulated by UTP to the same extent as the parent molecule. In contrast, the ATCase domain was no longer sensitive to the nucleotide, suggesting that the two catalytic activities are controlled by distinct mechanisms. Most remarkably, isotope dilution and transient time measurements showed that the truncated complex channels carbamoyl phosphate. The overall CPSase-ATCase reaction is much less sensitive than the parent molecule to the ATCase bisubstrate analogue, N-phosphonacetyl-L-aspartate (PALA), providing evidence that the endogenously produced carbamoyl phosphate is sequestered and channeled to the ATCase active site.     

Yeast carbamoyl-phosphate-synthetase--aspartate-transcarbamylase multidomain protein is phosphorylated in vitro by cAMP-dependent protein kinase

The first two steps of de novo pyrimidine synthesis in Saccharomyces cerevisiae are catalyzed by a multifunctional protein, coded by the URA2 gene and which has the carbamoyl-phosphate (CPSase) synthetase and aspartate transcarbamylase (ATCase) activities. The native enzyme purified from protease-B-deficient URA2-transformed cells, was phosphorylated in vitro using catalytic subunits of pure cAMP-dependent protein kinase. After electrophoresis under denaturing conditions, a single 240-kDa species was found to be phosphorylated. Trypsin digestion of this species gave a single, very acidic phosphopeptide upon isoelectric focussing. Purification by HPLC followed by amino acid sequencing of this peptide, showed a phosphoserine at the expected consensus sequence Arg-Arg-Phe-Ser. Knowledge of the URA2 gene sequence allowed the site to be located in the peptide link between dihydroorotase-like and ATCase domains. Such a location may explain why phosphorylation of the URA2 protein changed neither CPSase and ATCase activities nor their sensitivity to UTP, their common specific inhibitor.     

Arginine-sensitive phenotype of mutations in pyrA of Salmonella typhimurium: role of ornithine carbamyltransferase in the assembly of mutant carbamylphosphate synthetase.

The phenotype of certain mutations in pyrA, the gene encoding carbamylphosphate synthetase (CPSase), is expressed only in the presence od exogenous arginine. In unsupplemented media, synthesis of carbamylphosphate and growth was almost normal; in arginine-containing media, synthesis of carbamylphosphate stopped, as did growth, as a consequence of starvation for pyrimidine. Genetic and biochemical evidence suggests that arginine exerts this inhibition by repressing the synthesis of ornithine carbamyltransferase (OTCase), the intracellular presence of which is required for assembly of the unequal subunits and proper functioning of the mutant CPSase. After the addition of arginine to a culture of the mutant, CPSase activity (glutamine dependent) characteristic of the intact holoenzyme progressively decreased, whereas activity (ammonia dependent) characteristic of the free large (alpha) subunit increased. Extracts of mutant cells contain free small (beta) subunits, as demonstrated directly by in vitro complementation using purified alpha subunits from wild type. The mutant enzyme from cultures grown in the presence of arginine had a markedly decreased affinity for adenosine 5'-triphosphate. Mutations in argR that cause depressed synthesis of OTCase suppressed the phenotype, and a certain mutation in argI, the gene encoding OTCase, enhanced it. In vitro experiments using purified enzyme confirm the stimulatory effect of OTCase on the activity of mutant CPSase.     

Fine structural localization of aspartate carbamoyltransferase in rat liver

Summary A new procedure for demonstrating aspartate carbamoyltransferase (carbamoyl phosphate: L aspartate carbamoyltransferase, E. C.2.1,3.2) is described. The method is based on precipitating the released orthophosphate by lead ions. The resulting lead phosphate deposits serve for electron microscopic localization of aspartate carbamoyltransferase. There is a correlation of the morphological demonstration at rough endoplasmic reticulum and the biochemical determination in the microsomal fraction. Enzyme activity is found at the nucleus too.     

Escherichia coli ornithine carbamolytransferase isoenzymes: evolutionary significance and the isolation of lambdaargF and lambdaargI transducing bacteriophages.

Among the Enterobacteriaceae, Escherichia coli K-12 is the only strain known to have two structural genes (argF and argI) for ornithine carbamoyltransferase. The two gene products interact to form a family of four functional isoenzymes, respectively designated FFF, FFI, FII, and III. The FFF and III isoenzymes exhibit nearly identical kinetic parameters in the conditions applied. FFF is more thermolabile than III; this allows the straightforward characterization of new transducing phages carrying either argF or argI. The bearing of the available information regarding ornithine carbamoyltransferase isoenzymes on the evolution of the ancestral E. coli chromosome is reconsidered.     

Evolutionary divergence of genes for ornithine and aspartate carbamoyl-transferases--complete sequence and mode of regulation of the Escherichia coli argF gene; comparison of argF with argI and pyrB.

The complete nucleotide sequence of argF is presented, together with that of an operator-constitutive mutant. ArgF is compared with the other gene coding for ornithine carbamoyltransferase (OTCase) in E. coli K-12, argI, and with pyrB, encoding the catalytic monomer of aspartate carbamoyltransferase (ATCase). ArgF and argI appear very closely related having emerged from a relatively recent ancestor gene. The relationship between OTCase and ATCase appears more distant. Nevertheless, the homology observed between the two proteins (mainly in the polar domain) suggests a common origin.     

Regulation of arginine and pyrimidine biosynthesis in Pseudomonas putida.

Repression of biosynthetic enzyme synthesis in Pseudomonas putida is incomplete even when the bacteria are growing in a nutritionally complex environment. The synthesis of four of the enzymes of the arginine biosynthetic pathway (N-acetyl-alpha-glutamokinase/N-acetylglutamate-gamma-semialdehyde dehydrogenase, ornithine carbamoyltransferase and acetylornithine-delta-transaminase) could be repressed and derepressed, but the maximum difference observed between repressed and derepressed levels for any enzyme of the pathway was only 5-fold (for ornithine carbamoyltransferase). No repression of five enzymes of the pyrimidine biosynthetic pathway (aspartate carbamoyltransferase, dihydro-orotase, dihydro-orotate dehydrogenase, orotidine-5'-phosphate pyrophosphorylase and orotidine-5'-phosphate decarboxylase) could be detected on addition of pyrimidines to minimal asparagine cultures of P. putida A90, but a 1-5- to 2-fold degree of derepression was found following pyrimidine starvation of pyrimidine auxotrophic mutants of P. putida A90. Aspartate carbamoyltransferase in crude extracts of P. putida A90 was inhibited in vitro by (in order of efficiency) pyrophosphate, CTP, UTP and ATP, at limiting but not at saturating concentrations of carbamoyl phosphate.     

The human Rad9 checkpoint protein stimulates the carbamoyl phosphate synthetase activity of the multifunctional protein CAD

The human Rad9 checkpoint protein is a subunit of the heterotrimeric Rad9-Rad1-Hus1 (9-1-1) complex that plays a role as a damage sensor in the DNA damage checkpoint response. Rad9 has been found to interact with several other proteins outside the context of the 9-1-1 complex with no obvious checkpoint functions. During our studies on the 9-1-1 complex, we found that Rad9 immunoprecipitates contained a 240 kDa protein that was identified as carbamoyl phosphate synthetase/aspartate transcarbamoylase/dihydroorotase (CAD), a multienzymatic protein required for the de novo synthesis of pyrimidine nucleotides and cell growth. Further investigations revealed that only free Rad9, but not Rad9 within the 9-1-1 complex, bound to CAD. The rate-limiting step in de novo pyrimidine nucleotide synthesis is catalyzed by the carbamoyl phosphate synthetase II (CPSase) domain of CAD. We find that Rad9 binds to the CPSase domain, and, moreover, this binding results in a 2-fold stimulation of the CPSase activity of CAD. Similar results were also obtained with an N-terminal Rad9 fragment. These findings suggest that Rad9 may play a role in ribonucleotide biosynthesis.     

Urea cycle enzymes in human liver: ontogenesis and interaction with the synthesis of pyrimidines and polyamines

Summary All the five enzymes of urea synthesis and the formation of urea in vitro can already be demonstrated in human liver as early as the 9th week of fetal development. At this stage the activity of carbamoyl phosphate synthetase is the highest, whereas that of ornithine carbamoyltransferase is the lowest as compared to those in the adult. The kinetic parameters of the urea cycle enzymes are the same in fetal liver as in adult liver, except that the Km values of ornithine carbamoyltransferase for L-ornithine are 3.5 mM and 0.42 mM in the fetus and in adult liver, respectively.Urea formation in vivo seems to begin in the second half of fetal life, and a gradual increase can be detected in the activity of the enzymes of urea synthesis. The activity of ortnithine decarboxylase, the glutamine-dependent carbamoyl phosphate synthetase and aspartate carbamoyltransferase, however, changes in the opposite direction.The concentration of carbamoyl phosphate and aspartate remains constant, but that of ornithine gradually decreases during ontogenesis. The ornithine, carbamoyl phosphate and aspartate pools are probably utilized in the polyamine, pyrimidine and urea syntheses at varying rates.     

A specific requirement for biotin in the synthesis of ornithine carbamoyltransferase by yeast

1. Growth of a biotin-requiring strain of Saccharomyces cerevisiae in a medium containing a suboptimum concentration of biotin for growth caused a decreased synthesis of ornithine carbamoyltransferase as compared with yeast grown in a medium containing an optimum concentration of biotin. Inclusion of the biotin homologues norbiotin or homobiotin, but not bishomobiotin, in the biotin-deficient medium caused an appreciable increase in ornithine carbamoyltransferase synthesis without affecting growth or synthesis of total RNA and protein. The addition of norbiotin to biotin-deficient medium had no effect on the respiratory activity of the yeast or on the synthesis of aspartate carbamoyltransferase, acid phosphatase, beta-fructofuranosidase or malate dehydrogenase. 2. Synthesis of acetylornithine deacetylase and acetylornithine acetyltransferase was slightly diminished by the imposition of biotin deficiency, but the effect was not as great as on ornithine carbamoyltransferase synthesis. Incorporation of norbiotin in the biotin-deficient medium had no marked effect on the synthesis of any other arginine-pathway enzyme except ornithine carbamoyltransferase. 3. l-Ornithine induced synthesis of ornithine carbamoyltransferase in yeast grown in biotin-deficient medium, but in yeast grown in this medium supplemented with norbiotin it repressed synthesis of the enzyme. l-Arginine had no detectable effect on ornithine carbamoyltransferase synthesis by the yeast grown in biotin-deficient medium with or without norbiotin. l-Aspartate repressed synthesis of ornithine carbamoyltransferase in biotin-deficient yeast and completely nullified the stimulatory effect of norbiotin on synthesis of the enzyme in this yeast. 4. There was no increase in ornithine carbamoyltransferase synthesis in biotin-deficient yeast incubated in phosphate buffer, pH4.5, containing glucose and biotin or norbiotin. In biotin-deficient yeast suspended in complete medium containing an optimum concentration of biotin, there was an increase in ornithine carbamoyltransferase synthesis only after the onset of growth.     

Evidence that mammalian glutamine-dependent carbamyl phosphate synthetase arose through gene fusion

Summary On the basis of homology, the mammalian CAD (glutamine-dependent carbamyl phosphate synthetase-aspartate transcarbamylase-dihydroorotase) gene appears to have arisen from the fusion of four separate ancestral genes. Evidence for two of these precursor genes is found in the carbamyl phosphate synthetase (CPSase) domain of CAD. In prokaryotes, such as Escherichia coli CPSase is encoded by two distinct cistrons of the carAB operon. Whereas carA and carB are separated by a short noncoding intercistronic region, the homologous sequences of the CAD gene encode an amino acid bridge. This bridge connects the subdomains of the CAD CPSase. We constructed a bacterial carAB fusion gene in which the intercistronic region codes for a hamster bridgelike sequence. The fused carAB gene directs the synthesis of a stable bifunctional polypeptide whose glutamine-dependent CPSase activity is comparable to the E. coli CPSase holoenzyme. The fusion in E. coli of the single gene counterparts of CAD demonstrates a potential model system to study the genetic events that lead to gene fusion and the creation of multienzymatic proteins. Offprint requests to: J.N. Davidson     

Characterization of the aspartate carbamoyltransferase fragment generated by protease action on the pyrimidine-3 gene product of Neurospora crassa.

The molecular weight of the fragment of aspartate carbamoyltransferase (carbamoylphosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2) of Neurospora crassa following proteolysis was found to be 1.0-10(5) (aspartate carbamoyltransferase-L). It differs from the native form of the enzyme (aspartate carbamoyltransferase-N, 6.5-10(5)) in several respects. It has a lower V, has a much greater affinity (approx. 3-fold) for L-aspartate, and is strongly activated by glycine. Both forms of aspartate carbamyoltransferase have a pH optimum of approx. 9.5, and they exhibit similar affinities for carbamoyl phosphate.