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.     

The N-acetylglutamate synthase/N-acetylglutamate kinase metabolon of Saccharomyces cerevisiae allows co-ordinated feedback regulation of the first two steps in arginine biosynthesis.

In Saccharomyces cerevisiae, which uses the nonlinear pathway of arginine biosynthesis, the first two enzymes, N-acetylglutamate synthase (NAGS) and N-acetylglutamate kinase (NAGK), are controlled by feedback inhibition. We have previously shown that NAGS and NAGK associate in a complex, essential to synthase activity and protein level [Abadjieva, A., Pauwels, K., Hilven, P. & Crabeel, M. (2001) J. Biol. Chem.276, 42869-42880]. The NAGKs of ascomycetes possess, in addition to the catalytic domain that is shared by all other NAGKs and whose structure has been determined, a C-terminal domain of unknown function and structure. Exploring the role of these two domains in the synthase/kinase interaction, we demonstrate that the ascomycete-specific domain is required to maintain synthase activity and protein level. Previous results had suggested a participation of the third enzyme of the pathway, N-acetylglutamylphosphate reductase, in the metabolon. Here, genetic analyses conducted in yeast at physiological level, or in a heterologous background, clearly demonstrate that the reductase is dispensable for synthase activity and protein level. Most importantly, we show that the arginine feedback regulation of the NAGS and NAGK enzymes is mutually interdependent. First, the kinase becomes less sensitive to arginine feedback inhibition in the absence of the synthase. Second, and as in Neurospora crassa, in a yeast kinase mutant resistant to arginine feedback inhibition, the synthase becomes feedback resistant concomitantly. We conclude that the NAGS/NAGK metabolon promotes the co-ordination of the catalytic activities and feedback regulation of the first two, flux controlling, enzymes of the arginine pathway.     

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.     

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.     

Cloning and sequencing of arg3 and arg11 genes of Schizosaccharomyces pombe on a 10-kb DNA fragment. Heterologous expression and mitochondrial targeting of their translation products.

The Schizosaccharomyces pombe arginine anabolic genes encoding ornithine carbamoyltransferase (arg3) and acetylglutamate kinase/acetylglutamyl-phosphate reductase (arg11) were cloned by functional complementation of S. pombe arg3 and arg11 mutant strains from S. pombe DNA genomic libraries. Restriction analysis and sequencing of the two clones showed that both genes are located on a common DNA fragment. The arg3 gene encodes a 327-amino-acid polypeptide presenting a strong identity to Saccharomyces cerevisiae and human ornithine carbamoyltransferases. The arg11 gene encodes a 884-amino-acid polypeptide. The acetylglutamate kinase and acetylglutamate-phosphate reductase domains have been defined by their identity with the S. cerevisiae ARG5,6 protein. The cloned arg11 gene from S. pombe does not complement an arg5,6 mutation in S. cerevisiae, nor does the ARG5,6 gene complement the S. pombe arg11- mutation. In contrast, both ornithine-carbamoyltransferase-encoding genes function in S. pombe. However, the S. pombe arg3 gene complements only weakly an arg3 S. cerevisiae strain, which is in agreement with the low level of expression of the S. pombe gene in S. cerevisiae. The subcellular localization of both ornithine carbamoyltransferases in the two yeasts indicates that, in contrast to the S. pombe enzyme, more than 95% of the S. cerevisiae enzyme remains in the S. pombe cytoplasm. The low expression of S. pombe ornithine carbamoyltransferases in S. cerevisiae did not allow its localization. The promoters of S. pombe arg3 and arg11 genes do not present striking similarities among themselves nor with the promoters of the equivalent genes of S. cerevisiae.     

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.     

Insight on an arginine synthesis metabolon from the tetrameric structure of yeast acetylglutamate kinase

N-acetyl-L-glutamate kinase (NAGK) catalyzes the second, generally controlling, step of arginine biosynthesis. In yeasts, NAGK exists either alone or forming a metabolon with N-acetyl-L-glutamate synthase (NAGS), which catalyzes the first step and exists only within the metabolon. Yeast NAGK (yNAGK) has, in addition to the amino acid kinase (AAK) domain found in other NAGKs, a ~150-residue C-terminal domain of unclear significance belonging to the DUF619 domain family. We deleted this domain, proving that it stabilizes yNAGK, slows catalysis and modulates feed-back inhibition by arginine. We determined the crystal structures of both the DUF619 domain-lacking yNAGK, ligand-free as well as complexed with acetylglutamate or acetylglutamate and arginine, and of complete mature yNAGK. While all other known arginine-inhibitable NAGKs are doughnut-like hexameric trimers of dimers of AAK domains, yNAGK has as central structure a flat tetramer formed by two dimers of AAK domains. These dimers differ from canonical AAK dimers in the -110° rotation of one subunit with respect to the other. In the hexameric enzymes, an N-terminal extension, found in all arginine-inhibitable NAGKs, forms a protruding helix that interlaces the dimers. In yNAGK, however, it conforms a two-helix platform that mediates interdimeric interactions. Arginine appears to freeze an open inactive AAK domain conformation. In the complete yNAGK structure, two pairs of DUF619 domains flank the AAK domain tetramer, providing a mechanism for the DUF619 domain modulatory functions. The DUF619 domain exhibits the histone acetyltransferase fold, resembling the catalytic domain of bacterial NAGS. However, the putative acetyl CoA site is blocked, explaining the lack of NAGS activity of yNAGK. We conclude that the tetrameric architecture is an adaptation to metabolon formation and propose an organization for this metabolon, suggesting that yNAGK may be a good model also for yeast and human NAGSs.     

Site-directed mutagenesis studies of acetylglutamate synthase delineate the site for the arginine inhibitor

N-acetyl-l-glutamate synthase (NAGS), the first enzyme of bacterial/plant arginine biosynthesis and an essential activator of the urea cycle in animals, is, respectively, arginine-inhibited and activated. Site-directed mutagenesis of recombinant Pseudomonas aeruginosa NAGS (PaNAGS) delineates the arginine site in the PaNAGS acetylglutamate kinase-like domain, and, by extension, in human NAGS. Key residues for glutamate binding are identified in the acetyltransferase domain. However, the acetylglutamate kinase-like domain may modulate glutamate binding, since one mutation affecting this domain increases the K_m for glutamate. The effects on PaNAGS of two mutations found in human NAGS deficiency support the similarity of bacterial and human NAGSs despite their low sequence identity.     

N-acetyl-L-glutamate synthase of Neurospora crassa. Characteristics, localization, regulation, and genetic control

N-Acetylglutamate synthase, an early enzyme of the arginine pathway, provides acetylglutamate for ornithine synthesis in the so-called "acetylglutamate cycle." Because acetylglutamate is regenerated as ornithine is formed, the enzyme has only a catalytic or anaplerotic role in the pathway, maintaining "bound" acetyl groups during growth. We have detected this enzyme in crude extracts of Neurospora crassa and have localized it to the mitochondria along with other ornithine biosynthetic enzymes. The enzyme is bound to the mitochondrial membrane. The enzyme has a pH optimum of 9.0 and Km values for glutamate and CoASAc of 6.3 and 1.6 mM, respectively. It is feedback-inhibited by L-arginine (I0.5 = 0.16 mM), and its specific activity is augmented 2-3-fold by arginine starvation of the mycelium. Mutants of the newly recognized arg-14 locus lack activity for the enzyme. Because these mutants are complete auxotrophs, we conclude that N-acetylglutamate synthase is an indispensible enzyme of arginine biosynthesis in N. crassa. This work completes the assignment of enzymes of the arginine pathway of N. crassa to corresponding genetic loci. The membrane localization of the enzyme suggests a novel mechanism by which feedback inhibition might occur across a semipermeable membrane.     

Organization and control in the arginine biosynthetic pathway of Neurospora.

Eight enzymes involved in the conversion of acetylglutamate to arginine in Neurospora crassa were studied. The data indicate that of three enzymes early in the sequence, only the first, acetylglutamate kinase, is a nonorganellar enzyme. The next two, N-acetyl-gamma-glutamyl-phosphate reductase and acetylornithine aminotransferase, are in the mitochondrion, which was previously shown to contain the subsequent enzymes: acetylornithine-glutamate acetyltransferase, ornithine carbamyltransferase, and carbamyl-phosphate synthetase A (arginine specific). The last two enzymes of the pathway, argininosuccinate synthetase and argininosuccinate lyase, were previously shown to be cytosolic. All enzymes but one have low amplitudes or repression. Their levels respond little to arginine excess and are about twofold elevated (threefold for ornithine carbamyltransferase) as a result of arginine limitation in the arg-12-8 strain. No restriction of the incorporation of mitochondrial enzymes into mitochondria could be detected when the levels of these enzymes were elevated. Two enzymes, acetylglutamate kinase and carbamyl-phosphate synthetase A, which initiate the synthesis of the ornithine and guanidino moieties of arginine, respectively, show the lowest specific activities in crude extract. These enzymes display special regulatroy features. Acetylglutamate kinase, which has a typically low amplitude of repression, is subject to feedback inhibition. Carbamyl-phosphate synthetase A is wholly insensitive to arginine or citrulline in vitro or in vivo, but displays a very large amplitude of repression (about 60-fold). It is unique in that it can be almost completely repressed by growth of mycelia in excess arginine. These data suggest that mitochondrial localization may be incompatible with a mechanism of feedback inhibition by a cytosolic effector, arginine. Further, they suggest that the high repressibility of carbamyl-phosphate synthetase A compensates for its feedback insensitivity.     

Stimulatory effect of arginine on acetylglutamate synthesis in isolated mitochondria of mouse and rat liver.

N-Acetyl-L-glutamate synthetase (EC 2.3.1.1) catalyses the synthesis of N-acetyl-L-glutamate, an allosteric activator of carbamoyl-phosphate synthetase I in the liver of ureotelic animals, and the first enzyme is activated specifically by arginine. We have proposed that arginine can stimulate acetylglutamine synthetase in vivo and thereby increase the mitochondrial content of acetylglutamate. The effects of arginine on acetylglutamate synthesis in isolated mitochondria were investigated in detail in the present work. When rat liver mitochondria were isolated and incubated with [14C]glutamate and unlabelled acetate as substrates, acetyl[14C]glutamate synthesis in the mitochondria was more extensive in the presence than in the absence of L-arginine. There was no significant difference between the specific radioactivities of intramitochondrial [14C]glutamate in the presence and absence of arginine. When rat liver mitochondria were incubated with [14C]acetate and unlabelled glutamate as substrates, arginine also stimulated acetyl[14C]glutamate synthesis in the isolated mitochondria. L-Lysine or L-homoarginine, which does not activate acetylglutamate synthetase, had no effect on acetylglutamate synthesis, in the isolated mitochondria. The arginine concentration giving half-maximal synthesis of acetylglutamate in isolated mitochondria was about 50 microM, which is in the range of physiological concentrations of arginine in the liver. As we previously reported [Kawamoto, Ishida, Mori & Tatibana (1982) Eur. J. Biochem. 123, 637-641], the sensitivity of acetylglutamate synthetase to arginine activation undergoes marked changes after food ingestion. The extent of arginine activation of acetylglutamate synthesis in isolated mitochondria correlated well with the sensitivity of acetylglutamate synthetase extracted from the mitochondria to arginine activation. These data lend further support to the idea that arginine itself activates the mitochondrial synthesis of acetylglutamate.     

Modulation of the activity of rat liver acetylglutamate synthase by pH and arginine concentration

Acetylglutamate is known to modulate the activity of carbamyl phosphate synthetase, and thus probably to participate in regulation of the urea cycle. Therefore factors that regulate the activity of acetylglutamate synthase are relevant to control of urea synthesis and of systemic pH. An increase in the concentration of arginine increases both Vmax and S0.5 for glutamate of acetylglutamate synthase from rat liver. An increase in pH causes S0.5 for glutamate to decrease and does not affect Vmax. As a consequence of these effects, a rapid rate of synthesis of acetylglutamate requires a concentration of arginine of about 25 microM or higher and either relatively high glutamate concentrations or relatively high pH.     

The crystal structure of Pyrococcus furiosus UMP kinase provides insight into catalysis and regulation in microbial pyrimidine nucleotide biosynthesis

UMP kinase (UMPK), the enzyme responsible for microbial UMP phosphorylation, plays a key role in pyrimidine nucleotide biosynthesis, regulating this process via feed-back control and via gene repression of carbamoyl phosphate synthetase (the first enzyme of the pyrimidine biosynthesis pathway). We present crystal structures of Pyrococcus furiosus UMPK, free or complexed with AMPPNP or AMPPNP and UMP, at 2.4 A, 3 A and 2.55 A resolution, respectively, providing a true snapshot of the catalytically competent bisubstrate complex. The structure proves that UMPK does not resemble other nucleoside monophosphate kinases, including the UMP/CMP kinase found in animals, and thus UMPK may be a potential antimicrobial target. This enzyme has a homohexameric architecture centred around a hollow nucleus, and is organized as a trimer of dimers. The UMPK polypeptide exhibits the amino acid kinase family (AAKF) fold that has been reported in carbamate kinase and acetylglutamate kinase. Comparison with acetylglutamate kinase reveals that the substrates bind within each subunit at equivalent, adequately adapted sites. The UMPK structure contains two bound Mg ions, of which one helps stabilize the transition state, thus having the same catalytic role as one lysine residue found in acetylglutamate kinase, which is missing from P.furiosus UMPK. Relative to carbamate kinase and acetylglutamate kinase, UMPK presents a radically different dimer architecture, lacking the characteristic 16-stranded beta-sheet backbone that was considered a signature of AAKF enzymes. Its hexameric architecture, also a novel trait, results from equatorial contacts between the A and B subunits of adjacent dimers combined with polar contacts between A or B subunits, and may be required for the UMPK regulatory functions, such as gene regulation, proposed here to be mediated by hexamer-hexamer interactions with the DNA-binding protein PepA.     

Long-term ingestion of ammonium increases acetylglutamate and urea levels without affecting the amount of carbamoyl-phosphate synthase

Rats were fed the following diets: standard (20% protein), high-protein (80%), protein-free, standard plus ammonium and protein-free plus ammonium for six weeks. The standard plus ammonium diet was prepared to contain ammonia equivalent to that supplied by the high-protein diet. Addition of ammonium acetate (20% by mass) to the 20% protein or protein-free diets results in 2.3- and 10-fold increases of urea excretion respectively, without increase of carbamoyl-phosphate synthase. Supplementation of the standard diet with ammonium increases the mitochondrial content of acetylglutamate from 830 to 1590 pmol/mg protein, and of the protein-free diet from 130 to 1040 pmol/mg. However, ingestion of ammonium did not increase the activity of acetylglutamate synthase. Therefore the efflux of acetylglutamate from mitochondria was determined. After 30 min at 37 degrees C liver mitochondria from rats on standard diet released 61% of the initial acetylglutamate while mitochondria from animals on standard plus ammonium diet released only 20%. These results indicate that ingestion of ammonium increases the content of acetylglutamate in rat liver by decreasing its efflux from mitochondria. This effect is similar to that produced in mice by a high protein diet [Morita et al. (1982) J. Biochem. (Tokyo) 91, 563-569]. However, while the high-protein diet increases carbamoylphosphate synthase content, the ammonium diet does not.     

Molecular cloning and expression of the human deoxythymidylate kinase gene in yeast.

(Deoxy)thymidylate (dTMP) kinase is an enzyme which phosphorylates dTMP to dTDP in the presence of ATP and magnesium. This enzyme is important in cellular DNA synthesis because the synthesis of dTTP, either via the de novo pathway or through the exogenous supply of thymidine, requires the activity of this enzyme. It has been suggested that the activities of the enzymes involved in DNA precursor biosynthesis, such as thymidine kinase, thymidylate synthase, thymidylate kinase, and dihydrofolate reductase, are subjected to cell cycle regulation. Here we describe the cloning of a human dTMP kinase cDNA by functional complementation of a yeast dTMP kinase temperature-sensitive mutant at the non-permissive temperature. The nucleotide sequence of the cloned human cDNA is predicted to encode a 24 KD protein that shows considerable homology with the yeast and vaccinia virus dTMP kinase enzymes. The human enzyme activity has been investigated by expressing it in yeast. In this work, we demonstrate that the cloned human cDNA, when expressed in yeast, produces dTMP kinase activity.     

Glycogen synthase sensitivity to glucose-6-P is important for controlling glycogen accumulation in Saccharomyces cerevisiae

Glycogen is a storage form of glucose utilized as an energy reserve by many organisms. Glycogen synthase, which is essential for synthesizing this glucose polymer, is regulated by both covalent phosphorylation and the concentration of glucose-6-P. With the yeast glycogen synthase Gsy2p, we recently identified two mutants, R579A/R580A/R582A [corrected] and R586A/R588A/R591A, in which multiple arginine residues were mutated to alanine that were completely insensitive to activation by glucose-6-P in vitro (Pederson, B. A., Cheng, C., Wilson, W. A., and Roach, P. J. (2000) J. Biol. Chem. 275, 27753-27761). We report here the expression of these mutants in Saccharomyces cerevisiae and, as expected from our findings in vitro, they were not activated by glucose-6-P. The R579A/R580A/R582A [corrected] mutant, which is also resistant to inhibition by phosphorylation, caused hyperaccumulation of glycogen. In contrast, the mutant R586A/R588A/R591A, which retains the ability to be inactivated by phosphorylation, resulted in lower glycogen accumulation when compared with wild-type cells. When intracellular glucose-6-P levels were increased by mutating the PFK2 gene, glycogen storage due to the wild-type enzyme was increased, whereas that associated with R579A/R580A/R582A [corrected] was not greatly changed. This is the first direct demonstration that activation of glycogen synthase by glucose-6-P in vivo is necessary for normal glycogen accumulation.     

Acetylglutamate kinase. A mitochondrial feedback-sensitive enzyme of arginine biosynthesis in Neurospora crassa

The radioisotopic method used to assay acetylglutamate kinase (EC 2.7.2.8) of Neurospora crassa has been shown to detect two distinct enzymatically catalyzed reactions. The enzymes were separated by differential centrifugation into a cytosolic activity and an organellar activity. Both activities required ATP and were thermal-labile. The cytosolic activity was insensitive to inhibition by arginine and formed a stable reaction product in the absence of hydroxylamine. The organellar activity had an absolute requirement for hydroxylamine in order to form a stable reaction product. The product of the cytosolic activity was separated from acetylglutamate hydroxamate (the product of the organellar activity) and was identified as the cyclic amide pyroglutamate by cation exchange chromatography. The organellar activity has been implicated in arginine biosynthesis by the following criteria: it was completely and specifically inhibited by arginine concentrations as low as 200 microM; its level was elevated 2-fold in a mutant strain with derepressed levels of arginine biosynthetic enzymes; and it was absent in an arginine auxotrophic strain (the cytosolic activity was present). The organellar activity co-sedimented with mitochondria during isopycnic gradient centrifugation. The metabolic problems posed by a mitochondrial location of a feedback-sensitive enzyme and the cytosolic location of its effector are discussed.