Acetylglutamyl-phosphate Reductase Research Articles

A new yeast metabolon involving at least the two first enzymes of arginine biosynthesis: acetylglutamate synthase activity requires complex formation with acetylglutamate kinase.

Open reading frame YJL071W of Saccharomyces cerevisiae was shown to be ARG2 and identified as the structural gene for acetylglutamate synthase, first step in arginine biosynthesis. The three Ascomycete acetylglutamate synthases characterized to date appear homologous, but unlike the other enzymes of the yeast arginine biosynthesis pathway, they showed no significant similarity to their prokaryotic equivalents. The measured synthase activity did not increase with the number of ARG2 gene copies unless the number of ARG5,6 gene copies was increased similarly. ARG5,6 encodes a precursor that is maturated in the mitochondria into acetylglutamate kinase and acetylglutamyl-phosphate reductase, catalyzing the second and third steps in the pathway. The results imply that the synthase must interact stoichiometrically in vivo with the kinase, the reductase, or both to be active. Results obtained with synthetic ARG5 and ARG6 genes suggested that both the kinase and the reductase could be needed. This situation, which has completely escaped notice in yeast until now, is reminiscent of the observation in Neurospora crassa that nonsense arg-6 kinase/reductase mutants lack synthase activity (Hinde, R. W., Jacobson, J. A., Weiss, R. L., and Davis, R. H. (1986) J. Biol. Chem. 261, 5848-5852). In immunoprecipitation experiments, hemagglutinin-tagged synthase coprecipitated with a protein proven by microsequencing to be the kinase. Western blot analyses showed that the synthase has reduced stability in the absence of the kinase/reductase. Our data demonstrate the existence of a new yeast arginine metabolon involving at least the first two, and possibly the first three, enzymes of the pathway. Hypotheses regarding the biological significance of this interaction are discussed.

Cloning, analysis and one-step disruption of the ARG5,6 gene of Candida albicans.

The ARG5,6 gene from the dimorphic fungus Candida albicans was cloned by functional complementation of the arginine auxotrophy present in strain EL2 (Arg-) using a gene library constructed in the double autonomously replicating sequence vector pRM1. Sequence analysis revealed a putative 857 amino acid polypeptide (95 kDa) which showed high homology (63% protein identity) to the Saccharomyces cerevisiae ARG5,6 gene. Similarly to the S. cerevisiae gene, the C. albicans ARG5,6 gene is responsible for both the acetylglutamate kinase and acetylglutamyl-phosphate reductase activities, the second and third steps of arginine biosynthesis at the mitochondria. The C. albicans ARG5,6 gene complemented the arg6 mutation present in S. cerevisiae (strain D160-4D) on a yeast episomal plasmid using its own regulatory signals. A set of non-integrative high-efficiency plasmid vectors based on this gene marker was constructed and a null C. albicans arg5,6 delta strain was obtained using the common URA3-blaster strategy. In addition, we generated an arg5,6 delta null mutant in a single transformation event, thus improving the basic strategy for generating gene deletions in C. albicans.

A single precursor protein for two separable mitochondrial enzymes in Neurospora crassa.

The arg-6 locus of Neurospora crassa encodes two early enzymes of the arginine biosynthetic pathway, acetylglutamate kinase and acetylglutamyl-phosphate reductase. Previous genetic and biochemical analyses of this locus and its products showed that: 1) strains carrying polar nonsense mutations in the acetylglutamate kinase gene lacked both enzyme activities (Davis, R.H., and Weiss, R.L. (1983) Mol. Gen. Genet. 192, 46-50), and 2) the proteins isolated from mitochondria were completely separable (Wandinger-Ness, A., Wolf, E.C., Weiss, R.L., and Davis, R.H. (1985) J. Biol. Chem. 260,5974-5978). These data suggested that the two enzymes were initially synthesized as a single precursor which was subsequently cleaved into two distinct polypeptides. We report here the identification of a high molecular weight protein, synthesized in vitro from isolated N. crassa RNA, that contains sequences corresponding to acetylglutamate kinase as well as acetylglutamyl-phosphate reductase. An analogous precursor was identified in vivo by pulse-labeling experiments. The precursor was similar to other mitochondrial precursors in that its uptake and processing in vivo was rapid and required an intact mitochondrial electrochemical gradient. This represents the first report of a bifunctional protein precursor which gives rise to two mitochondrial enzymes.

Simultaneous purification of three mitochondrial enzymes. Acetylglutamate kinase, acetylglutamyl-phosphate reductase and carbamoyl-phosphate synthetase from Neurospora crassa.

The early enzymes of arginine biosynthesis in Neurospora crassa are localized in the mitochondrion and catalyze the conversion of glutamate to citrulline. The final conversion of citrulline to arginine occurs via two enzymatic steps in the cytoplasm. We have devised a method for the isolation and purification of three of the mitochondrial arginine biosynthetic enzymes from a single extract. Acetylglutamate kinase and acetylglutamyl-phosphate reductase (both products of the complex arg-6 locus) were purified to homogeneity and near homogeneity, respectively. The large catalytic subunit of carbamoyl-phosphate synthetase was also purified to homogeneity. The three enzymes were resolved into separate fractions by chromatography on three dye-ligand affinity resins, which are specific for nucleotide binding enzymes and have a high protein binding capacity. High performance liquid chromatography was employed in the final stages of purification and was extremely effective in fractionating both acetylglutamate kinase and acetylglutamyl-phosphate reductase from proteins with very similar properties, which were not removed by other techniques. The purified proteins were used to raise specific antisera against these proteins. Acetylglutamate kinase and acetylglutamyl-phosphate reductase were shown to be immunologically unrelated. This finding suggests that the arg-6 locus encompasses two nonoverlapping cistrons. The antisera raised against carbamoyl-phosphate synthetase has been shown to cross-react with related enzymes in Saccharomyces cerevisiae, Escherichia coli, and rat liver (Ness, S. A., and Weiss, R. L. (1985) J. Biol. Chem. 260, 14355-14362). Acetylglutamate kinase is a regulatory enzyme and has been shown to be feedback-inhibited by arginine. We have determined the submitochondrial localization of acetylglutamate kinase and the second arg-6 product, acetylglutamyl-phosphate reductase. Both enzymes were shown to be soluble matrix enzymes. We discuss the relevance of this finding with respect to possible mechanisms for end product inhibition of a mitochondrial enzyme by a cytoplasmic effector.

Genetical evidence of carbamoylphosphate compartmentation in Schizosaccharomyces pombe and Schizosaccharomyces japonicus

Mutants defective in carbamoylphosphate synthetase have been isolated in the fission yeasts Schizosaccharomyces pombe and Schizosaccharomyces japonicus. Their growth properties indicate a compartmentation of the carbamoylphosphate pools between arginine and pyrimidine biosyntheses, as in Neurospora crassa but unlike Saccharomyces cerevisiae. Ornithine carbamoyltransferase-minus mutants, arg3−, were also isolated in both Schizosaccharomyces species. In Schiz. pombe, a very close linkage was observed between arg3 and arg11, a gene putatively coding for acetylglutamylphosphate reductase. Arg4 and arg5, the two genes encoding the carbamoylphosphate synthetase of the arginine pathway, are also closely linked but not adjacent.

Acetylglutamate kinase-acetylglutamyl-phosphate reductase complex of Neurospora crassa. Evidence for two polypeptides.

Mutations at the arg-6 locus in Neurospora crassa are divided into two complementation groups (A and B) and a third noncomplementing group. There are many suppressible nonsense mutations among mutants in complementation group B and one in the noncomplementing group; no nonsense mutations exist among mutants in complementation group A (Davis, R. H., and Weiss, R. L. (1983) Mol. Gen. Genet. 192, 46-50). We show here that the mutants are defective in either or both of two enzymes of arginine biosynthesis, acetylglutamate kinase and/or acetylglutamyl-phosphate reductase. Mutants in complementation group A lack acetylglutamate kinase, those in complementation group B lack acetylglutamyl-phosphate reductase, and those in the noncomplementing group lack both activities. Mutants in group B also have reduced levels of acetylglutamate kinase. The enzymes from purified mitochondria are readily separable by gel filtration and by Blue A dye affinity chromatography. Acetylglutamate kinase appears to be an octamer with a molecular weight of 400,000, whereas acetylglutamyl-phosphate reductase appears to be a dimer with a molecular weight of 93,000. This suggests that the two activities reside on distinct polypeptides. These results are best accommodated by the following model: the arg-6 locus encodes a single mRNA which is translated into a single polypeptide; the latter is then cleaved post-translationally to yield two physically separable enzymes.

Identification of nonsense mutations in Neurospora: Application to the complex arg-6 locus

The arg-6 locus of Neurospora crassa encodes two enzymes of arginine synthesis, acetylglutamate kinase and acetylglutamyl phosphate reductase. Mutants lacking one or the other enzyme fall into two different complementation groups; a large non-complementing group lacks both enzymes. We wished to survey over 50 alleles for suppressibility by a nonsense suppressor. We compared two methods of assessing suppressibility. One, based on trans-action of a nonsense suppressor, was simple, but not efficient in detecting all suppressible alleles. The other, based on crosses involving a marker linked to the locus surveyed was very efficient in methodology and is suited to all cases, such as the arg-6 locus, in which allelic crosses are sterile. The data indicate that the arg-6 locus encodes a bifunctional protein.

Arginine metabolism in Saccharomyces cerevisiae: subcellular localization of the enzymes

Subcellular localization of enzymes of arginine metabolism in Saccharomyces cerevisiae was studied by partial fractionation and stepwise homogenization of spheroplast lysates. These enzymes could clearly be divided into two groups. The first group comprised the five enzymes of the acetylated compound cycle, i.e., acetylglutamate synthase, acetylglutamate kinase, acetylglutamyl-phosphate reductase, acetylornithine aminotransferase, and acetylornithine-glutamate acetyltransferase. These enzymes were exclusively particulate. Comparison with citrate synthase and cytochrome oxidase, and results from isopycnic gradient analysis, suggested that these enzymes were associated with the mitochondria. By contrast, enzymatic activities going from ornithine to arginine, i.e., arginine pathway-specific carbamoylphosphate synthetase, ornithine carbamoyltransferase, argininosuccinate synthetase, and argininosuccinate lyase, and the two first catabolic enzymes, arginase and ornithine aminotransferase, were in the "soluble" fraction of the cell.