Juan Cabrera-Luque

Acetylornithine Transcarbamylase: a Novel Enzyme in Arginine Biosynthesis

Ornithine transcarbamylase is a highly conserved enzyme in arginine biosynthesis and the urea cycle. In Xanthomonas campestris, the protein annotated as ornithine transcarbamylase, and encoded by the argF gene, is unable to synthesize citrulline directly from ornithine. We cloned and overexpressed this X. campestris gene in Escherichia coli and show that it catalyzes the formation of N-acetyl-L-citrulline from N-acetyl-L-ornithine and carbamyl phosphate. We now designate this enzyme as an acetylornithine transcarbamylase. The K(m) values for N-acetylornithine and carbamyl phosphate were 1.05 mM and 0.01 mM, respectively. Additional putative transcarbamylases that might also be misannotated were found in the genomes of members of other xanthomonads, Cytophaga, and Bacteroidetes as well as in DNA sequences of bacteria from environmental isolates. It appears that these different paths for arginine biosynthesis arose very early in evolution and that the canonical ornithine transcarbamylase-dependent pathway became the prevalent form. A potent inhibitor, N(alpha)-acetyl-N(delta)-phosphonoacetyl-L-ornithine, was synthesized and showed a midpoint of inhibition at approximately 22 nM; this compound may prove to be a useful starting point for designing inhibitors specific to this novel family of transcarbamylases.

Inversion of allosteric effect of arginine on N-acetylglutamate synthase, a molecular marker for evolution of tetrapods

BackgroundThe efficient conversion of ammonia, a potent neurotoxin, into non-toxic metabolites was an essential adaptation that allowed animals to move from the aquatic to terrestrial biosphere. The urea cycle converts ammonia into urea in mammals, amphibians, turtles, snails, worms and many aquatic animals and requires N-acetylglutamate (NAG), an essential allosteric activator of carbamylphosphate synthetase I (CPSI) in mammals and amphibians, and carbamylphosphate synthetase III (CPSIII) in fish and invertebrates. NAG-dependent CPSI and CPSIII catalyze the formation of carbamylphosphate in the first and rate limiting step of ureagenesis. NAG is produced enzymatically by N-acetylglutamate synthase (NAGS), which is also found in bacteria and plants as the first enzyme of arginine biosynthesis. Arginine is an allosteric inhibitor of microbial and plant NAGS, and allosteric activator of mammalian NAGS.ResultsInformation from mutagenesis studies of E. coli and P. aeruginosa NAGS was combined with structural information from the related bacterial N-acetylglutamate kinases to identify four residues in mammalian NAGS that interact with arginine. Substitutions of these four residues were engineered in mouse NAGS and into the vertebrate-like N-acetylglutamate synthase-kinase (NAGS-K) of Xanthomonas campestris, which is inhibited by arginine. All mutations resulted in arginine losing the ability to activate mouse NAGS, and inhibit X. campestris NAGS-K. To examine at what point in evolution inversion of arginine effect on NAGS occur, we cloned NAGS from fish and frogs and examined the arginine response of their corresponding proteins. Fish NAGS were partially inhibited by arginine and frog NAGS were activated by arginine.ConclusionDifference in arginine effect on bacterial and mammalian NAGS most likely stems from the difference in the type of conformational change triggered by arginine binding to these proteins. The change from arginine inhibition of NAGS to activation was gradual, from complete inhibition of bacterial NAGS, to partial inhibition of fish NAGS, to activation of frog and mammalian NAGS. This change also coincided with the conquest of land by amphibians and mammals.

Structure and catalytic mechanism of a novel N-succinyl-L-ornithine transcarbamylase in arginine biosynthesis of Bacteroides fragilis.

A Bacteroides fragilis gene (argF′bf), the disruption of which renders the bacterium auxotrophic for arginine, was expressed and its recombinant protein purified and studied. The novel protein catalyzes the carbamylation of N-succinyl-l-ornithine but not l-ornithine or N-acetyl-l-ornithine, forming N-succinyl-l-citrulline. Crystal structures of this novel transcarbamylase complexed with carbamyl phosphate and N-succinyl-l-norvaline, as well as sulfate and N-succinyl-l-norvaline have been determined and refined to 2.9 and 2.8 Å resolution, respectively. They provide structural evidence that this protein is a novel N-succinyl-l-ornithine transcarbamylase. The data provided herein suggest that B. fragilis uses N-succinyl-l-ornithine rather than N-acetyl-l-ornithine for de novo arginine biosynthesis and therefore that this pathway in Bacteroides is different from the canonical arginine biosynthetic pathway of most organisms. Comparison of the structures of the new protein with those recently reported for N-acetyl-l-ornithine transcarbamylase indicates that amino acid residue 90 (B. fragilis numbering) plays an important role in conferring substrate specificity for N-succinyl-l-ornithine versus N-acetyl-l-ornithine. Movement of the 120 loop upon substrate binding occurs in N-succinyl-l-ornithine transcarbamylase, while movement of the 80 loop and significant domain closure take place as in other transcarbamylases. These findings provide new information on the putative role of succinylated intermediates in arginine biosynthesis and on the evolution of transcarbamylases.

A Novel N-acetylglutamate synthase architecture revealed by the crystal structure of the bifunctional enzyme from Maricaulis maris.

Novel bifunctional N-acetylglutamate synthase/kinases (NAGS/K) that catalyze the first two steps of arginine biosynthesis and are homologous to vertebrate N-acetylglutamate synthase (NAGS), an essential cofactor-producing enzyme in the urea cycle, were identified in Maricaulis maris and several other bacteria. Arginine is an allosteric inhibitor of NAGS but not NAGK activity. The crystal structure of M. maris NAGS/K (mmNAGS/K) at 2.7 Å resolution indicates that it is a tetramer, in contrast to the hexameric structure of Neisseria gonorrhoeae NAGS. The quaternary structure of crystalline NAGS/K from Xanthomonas campestris (xcNAGS/K) is similar, and cross-linking experiments indicate that both mmNAGS/K and xcNAGS are tetramers in solution. Each subunit has an amino acid kinase (AAK) domain, which is likely responsible for N-acetylglutamate kinase (NAGK) activity and has a putative arginine binding site, and an N-acetyltransferase (NAT) domain that contains the putative NAGS active site. These structures and sequence comparisons suggest that the linker residue 291 may determine whether arginine acts as an allosteric inhibitor or activator in homologous enzymes in microorganisms and vertebrates. In addition, the angle of rotation between AAK and NAT domains varies among crystal forms and subunits within the tetramer. A rotation of 26° is sufficient to close the predicted AcCoA binding site, thus reducing enzymatic activity. Since mmNAGS/K has the highest degree of sequence homology to vertebrate NAGS of NAGS and NAGK enzymes whose structures have been determined, the mmNAGS/K structure was used to develop a structural model of human NAGS that is fully consistent with the functional effects of the 14 missense mutations that were identified in NAGS-deficient patients.

A single mutation in the active site swaps the substrate specificity of N-acetyl-L-ornithine transcarbamylase and N-succinyl-L-ornithine transcarbamylase

Transcarbamylases catalyze the transfer of the carbamyl group from carbamyl phosphate (CP) to an amino group of a second substrate such as aspartate, ornithine, or putrescine. Previously, structural determination of a transcarbamylase from Xanthomonas campestris led to the discovery of a novel N‐acetylornithine transcarbamylase (AOTCase) that catalyzes the carbamylation of N‐acetylornithine. Recently, a novel N‐succinylornithine transcarbamylase (SOTCase) from Bacteroides fragilis was identified. Structural comparisons of AOTCase from X. campestris and SOTCase from B. fragilis revealed that residue Glu92 (X. campestris numbering) plays a critical role in distinguishing AOTCase from SOTCase. Enzymatic assays of E92P, E92S, E92V, and E92A mutants of AOTCase demonstrate that each of these mutations converts the AOTCase to an SOTCase. Similarly, the P90E mutation in B. fragilis SOTCase (equivalent to E92 in X. campestris AOTCase) converts the SOTCase to AOTCase. Hence, a single amino acid substitution is sufficient to swap the substrate specificities of AOTCase and SOTCase. X‐ray crystal structures of these mutants in complexes with CP and N‐acetyl‐L‐norvaline (an analog of N‐acetyl‐L‐ornithine) or N‐succinyl‐L‐norvaline (an analog of N‐succinyl‐L‐ornithine) substantiate this conversion. In addition to Glu92 (X. campestris numbering), other residues such as Asn185 and Lys30 in AOTCase, which are involved in binding substrates through bridging water molecules, help to define the substrate specificity of AOTCase. These results provide the correct annotation (AOTCase or SOTCase) for a set of the transcarbamylase‐like proteins that have been erroneously annotated as ornithine transcarbamylase (OTCase, EC 2.1.3.3).

A novel biochemically salvageable animal model of hyperammonemia devoid of N-acetylglutamate synthase

All knockout mouse models of urea cycle disorders die in the neonatal period or shortly thereafter. Since N-acetylglutamate synthase (NAGS) deficiency in humans can be effectively treated with N-carbamyl-l-glutamate (NCG), we sought to develop a mouse model of this disorder that could be rescued by biochemical intervention, reared to adulthood, reproduce, and become a novel animal model for hyperammonemia. Founder NAGS knockout heterozygous mice were obtained from the trans-NIH Knock-Out Mouse Project. Genotyping of the mice was performed by PCR and confirmed by Western blotting of liver and intestine. NCG and L-citrulline (Cit) were used to rescue the NAGS knockout homozygous (Nags(-/-)) pups and the rescued animals were characterized. We observed an 85% survival rate of Nags(-/-) mice when they were given intraperitoneal injections with NCG and Cit during the newborn period until weaning and supplemented subsequently with both compounds in their drinking water. This regimen has allowed for normal development, apparent health, and reproduction. Interruption of this rescue intervention resulted in the development of severe hyperammonemia and death within 48 h. In addition to hyperammonemia, interruption of rescue supplementation was associated with elevated plasma glutamine, glutamate, and lysine, and reduced citrulline, arginine, ornithine and proline levels. We conclude that NAGS deprived mouse model has been developed which can be rescued by NCG and Cit and reared to reproduction and beyond. This biochemically salvageable mouse model recapitulates the clinical phenotype of proximal urea cycle disorders and can be used as a reliable model of induced hyperammonemia by manipulating the administration of the rescue compounds.