A novel bifunctional N-acetylglutamate synthase-kinase from Xanthomonas campestris that is closely related to mammalian N-acetylglutamate synthase

Abstract

Background: In microorganisms and plants, the first two reactions of arginine biosynthesis are catalyzed by N-acetylglutamate synthase (NAGS) and N-acetylglutamate kinase (NAGK). In mammals, NAGS produces an essential activator of carbamylphosphate synthetase I, the first enzyme of the urea cycle, and no functional NAGK homolog has been found. Unlike the other urea cycle enzymes, whose bacterial counterparts could be readily identified by their sequence conservation with arginine biosynthetic enzymes, mammalian NAGS gene was very divergent, making it the last urea cycle gene to be discovered. Limited sequence similarity between E. coli NAGS and fungal NAGK suggests that bacterial and eukaryotic NAGS, and fungal NAGK arose from the fusion of genes encoding an ancestral NAGK (argB) and an acetyltransferase. However, mammalian NAGS no longer retains any NAGK catalytic activity.

Results: We identified a novel bifunctional N-acetylglutamate synthase and kinase (NAGS-K) in the Xanthomonadales order of gamma-proteobacteria that appears to resemble this postulated primordial fusion protein. Phylogenetic analysis indicated that xanthomonad NAGS-K is more closely related to mammalian NAGS than to other bacterial NAGS. We cloned the NAGS-K gene from Xanthomonas campestis, and characterized the recombinant NAGS-K protein. Mammalian NAGS and its bacterial homolog have similar affinities for substrates acetyl coenzyme A and glutamate as well as for their allosteric regulator arginine.

Conclusion: The close phylogenetic relationship and similar biochemical properties of xanthomonad NAGS-K and mammalian NAGS suggest that we have identified a close relative to the bacterial antecedent of mammalian NAGS and that the enzyme from X. campestris could become a good model for mammalian NAGS in structural, biochemical and biophysical studies.

Figure 1

Arginine biosynthesis in microorganisms and plants. Most microbes and plants utilize a "cyclical" pathway in which ornithine acetyltransferase recycles the acetyl group from acetylornithine to glutamate. In these organisms, NAGS replenishes NAG lost in cell division and growth. Some bacteria, such as E. coli and Xanthomonadales, utilize a "linear" pathway in which NAGS catalyzes formation of the first intermediate of arginine biosynthesis. Abbreviations: NAGP, N-acetylglutamyl phosphate; NAGPR, N-acetylglutamyl phosphate reductase; AOAT, acetylornithine aminotransferase; OAT, ornithine acetyltransferase; AOD, acetylornithine deacetylase, OTC, ornithine transcarbamylase, ASS, argininosuccinate synthase; ASL, argininosuccinate lyase.

Figure 2

Phylogenetic tree of NAGS protein sequences from bacteria, fungi, algae, plants and vertebrates, and fungal NAGK. The tree was generated by the neighbor joining method using Phylip 3.6. Numbers indicate bootstrap values from 1000 replicas. Bootstrap values equal to 1000 are not indicated in the figure due to spatial constraints. NAGS, NAGSK and NAGS/K proteins are shown in blue, red and purple typeface, respectively. Proteins from M. maris, O. alexandrii and P. bermudensis are shown in black typeface because their ability to catalyze synthase reaction, kinase reaction, or both has not been examined experimentally. NAGS, NAGK and NAGS-K from the following organisms were included in the phylogenetic analysis: human – Homo sapiens, mouse – Mus musculus, rat – Ratus norvegicus, cow – Bos torus, dog – Canis familiaris, frog – Xenopus tropicalis, zebrafish – Danio rerio, pufferfish – Fugu rubripes, freshwater pufferfish – Tetraodon nigroviridis, soy – Glycine max, corn – Zea mayis, tomato – Solanum lycopersicum, rice – Oryza sativa, Arabidopsis – Arabidopsis thaliana, S. cerevisiae – Saccharomyces cerevisiae, S. pombe – Schizosaccharomyces pombe, C. albicans – Candida albicans, N. crassa – Neurospora crassa, D. discoideum – Dictiostelium discoideum, X. campestris – Xanthomonas campestris, X. axonopodis – Xanthomonas axonopodis, X. fastidiosa – Xylella fastidiosa, M. maris – Maricaulis maris, O. alexandrii – Oceanicaulis alexandrii, P. bermudensis – Parvulalcula bermudensis, P. aeruginosa – Pseudomonas aeruginosa, P. syringiae – Pseudomonas syringiae, N. gonorrhoeae – Neisseria gonorrhoeae, S. typhimurium – Salmonella typhimurioum, E. coli – Escherichia coli.

Figure 3

Organization of the arginine operon in X. campestris and amino acid sequence conservation between NAGS-K and both NAGS and NAGK. A. The arginine operon in X. campestris. ArgC, argD, argG and argH genes were identified based on their similarity with homologs in other bacteria. ArgF' encodes acetylornithine transcarbamylase (AOTCase) and argE gene product can catalyze deacetylation of acetylornithine as well as acetylcitrulline [7]. The argA gene was annotated based on its similarity with other acetyltransferases. Genes labeled with question marks encode hypothetical proteins of unknown function. ArgD gene is located elsewhere in the genome and not in the same cluster as other eight genes. B. Alignment of NAGS from mouse, S. cerevisiae and S. pombe, NAGS-K from X. campestris, and NAGK from T. maritima, E. coli, S. cerevisiae and S. pombe. Residues that are important for catalysis of NAGK are shown in red. Residues that are conserved in vertebrate and fungal NAGS are shown in green. Residues that are mutated in patients with NAGS deficiency are highlighted in grey. Conserved residues that are presumed to be part of AcCoA binding site are shown in blue.

Figure 4

Purification of recombinant XcNAGS-K. The XcNAGS-K with the N-terminal polyhistidine tag was overexpressed in E. coli and purified using nickel-affinity column. Lane 1 – cell lysate before induction of XcNAGS-K overexpression; lane 2 – cell lysate; lane 3 – purified XcNAGS-K with the polyhistidine tag; lane 4 – purified XcNAGS-K after removal of the tag.

Figure 5

Dependence of the synthase reaction of XcNAGS-K on the concentration of AcCoA and glutamate. A. AcCoA concentration was varied between 0 and 4 mM while glutamate concentration was fixed at 50 mM. B. Glutamate concentration was varied between 0 and 50 mM while AcCoA was fixed at 2 mM. The assays were performed either with XcNAGS-K fused with a polyhistidine tag (black) or without affinity tag (gray).

Figure 6

Effects of arginine on enzymatic activities of XcNAGS-K. Synthase (A) and kinase (B) activities of the XcNAGS-K with the polyhistidine tag (black) or without tag (gray) were measured in the presence of increasing arginine concentrations. Arginine concentration was varied between 0 and 2 mM in the synthase assay (A) and between 0 and 30 mM in the kinase assay (B).

Figure 7

Domain organization of NAGS and NAGK from bacteria and eukaryotes. Kinase domain is shown in solid black. Synthase domain is shown in solid gray. The N-terminal domain of fungal NAGS is shown as a hatched rectangle. Enzyme types (NAGS or NAGK or NAGS-K) and their origins are indicated to the right of each domain architecture. Names of the conserved domains are indicated below each domain. Multiple names are used for designation of some conserved domains because of the redundancy of the conserved domain database. They are separated by slash-marks.