Biomedical subjects

Ljubica Caldovic

Publications and source records attributed to Ljubica Caldovic.

10 recordsLinked to original sources

PubMed2006

Expression, crystallization and preliminary crystallographic studies of a novel bifunctional N-acetylglutamate synthase/kinase from Xanthomonas campestris homologous to vertebrate N-acetylglutamate synthase.

A novel N-acetylglutamate synthase/kinase bifunctional enzyme of arginine biosynthesis that was homologous to vertebrate N-acetylglutamate synthases was identified in Xanthomonas campestris. The protein was overexpressed, purified and crystallized. The crystals belong to the hexagonal space group P6(2)22, with unit-cell parameters a = b = 134.60, c = 192.11 A, and diffract to about 3.0 A resolution. Selenomethionine-substituted recombinant protein was produced and selenomethionine substitution was verified by mass spectroscopy. Multiple anomalous dispersion (MAD) data were collected at three wavelengths at SER-CAT, Advanced Photon Source, Argonne National Laboratory. Structure determination is under way using the MAD phasing method.

Amino-Acid N-Acetyltransferase↗

PubMed2006

Biochemical properties of recombinant human and mouse N-acetylglutamate synthase.

N-Acetylglutamate synthase (NAGS, EC 2.3.1.1) is a mitochondrial enzyme that catalyzes the formation of N-acetylglutamate (NAG) from glutamate and acetylcoenzyme A. NAG is an obligatory activator of carbamylphosphate I (CPSI), the first and a rate limiting enzyme of ureagenesis. The enzymatic activity of NAGS increases in the presence of arginine. Since the level of NAGS activity depends on the concentrations of two amino acids, glutamate and arginine, and it supplies the essential cofactor for CPSI, NAGS may play an important role in the regulation of ureagenesis. The amino acid sequences of human and mouse NAGS consist of three regions with different degrees of conservation: the mitochondrial targeting signal (MTS), the variable domain, and the conserved domain. Removal of the MTS results in mature NAGS (NAGS-M) while removal of the MTS and the variable domain results in conserved NAGS (NAGS-C). The biochemical properties of purified recombinant human and mouse NAGS-M and NAGS-C were determined in this study with the goal of better understanding the role of the variable domain in NAGS function. The activity of all four proteins doubled in the presence of arginine, while the affinities for substrates changed less than two fold. The turnover numbers of NAGS-C are double those of NAGS-M proteins. Processing of NAGS-M to form NAGS-C results in an enzyme with higher catalytic activity and could play a role in the regulation of NAG production, CPSI function, and urea synthesis.

Amino-Acid N-Acetyltransferase↗

PubMed2005

Crystal structure of N-acetylornithine transcarbamylase from Xanthomonas campestris: a novel enzyme in a new arginine biosynthetic pathway found in several eubacteria.

We have identified in Xanthomonas campestris a novel N-acetylornithine transcarbamylase that replaces ornithine transcarbamylase in the canonic arginine biosynthetic pathway of several Eubacteria. The crystal structures of the protein in the presence and absence of the reaction product, N-acetylcitrulline, were determined. This new family of transcarbamylases lacks the DxxSMG motif that is characteristic of all ornithine transcarbamylases (OTCases) and contains a novel proline-rich loop that forms part of the active site. The specificity for N-acetylornithine is conferred by hydrogen bonding with residues in the proline-rich loop via water molecules and by hydrophobic interactions with residues from the adjacent 80's, 120's, and proline-rich loops. This novel protein structure provides a starting point for rational design of specific analogs that may be useful in combating human and plant pathogens that utilize acetylornithine transcarbamylase rather than ornithine transcarbamylase.

Binding Sites↗

PubMed2005

Late onset N-acetylglutamate synthase deficiency caused by hypomorphic alleles.

N-acetylglutamate (NAG) is a unique cofactor that is essential for the conversion of ammonia to urea in the liver. N-acetylglutamate synthase (NAGS) catalyzes the formation of NAG. Deficiency of NAGS causes a block in ureagenesis resulting in hyperammonemia. Although a number of mutations have been identified in the NAGS gene, their effects on NAGS enzymatic activity have not been examined. We describe here three mutations in two families with NAGS deficiency. Studies of the purified recombinant mutant proteins revealed deleterious effects on NAGS affinity for substrates, and on the rate of catalysis. These studies provide a better understanding of the function of NAGS, and the mechanisms for deleterious effect of mutations causing inherited NAGS deficiency.

Adult↗

PubMed2004

Restoration of ureagenesis in N-acetylglutamate synthase deficiency by N-carbamylglutamate.

In a patient with N-acetylglutamate synthase (NAGS) deficiency, incorporation of an isotopic label from ammonium chloride into urea was markedly reduced before treatment with N-carbamyl-L-glutamate (NCLG) and completely normalized following treatment. Blood ammonia rose following ammonium tracer ingestion before treatment but remained low following treatment. Serum urea concentration doubled following the treatment.

Acetyltransferases↗

PubMed2004

Mammalian N-acetylglutamate synthase.

N-Acetylglutamate synthase (NAGS, E.C. 2.3.1.1) is a mitochondrial enzyme that catalyzes the formation of N-acetylglutamate (NAG), an essential allosteric activator of carbamylphosphate synthetase I (CPSI). The mouse and human NAGS genes have been identified based on similarity to regions of NAGS from Neurospora crassa and cloned from liver cDNA libraries. These genes were shown to complement an argA- (NAGS) deficient Escherichia coli strain, and enzymatic activity of the proteins was confirmed by a new stable isotope dilution assay. The deduced amino acid sequence of mammalian NAGS contains a putative mitochondrial-targeting signal at the N-terminus. The mouse NAGS preprotein was overexpressed in insect cells to determine post-translational modifications and two processed proteins with different N-terminal truncations have been identified. Sequence analysis using a hidden Markov model suggests that the vertebrate NAGS protein contains domains with a carbamate kinase fold and an acyl-CoA N-acyltransferase fold, and protein crystallization experiments are currently underway. Inherited NAGS deficiency results in hyperammonemia, presumably due to the loss of CPSI activity. We, and others, have recently identified mutations in families with neonatal and late-onset NAGS deficiency and the identification of the gene has now made carrier testing and prenatal diagnosis feasible. A structural analog of NAG, carbamylglutamate, has been shown to bind and activate CPSI, and several patients have been reported to respond favorably to this drug (Carbaglu).

Acetyltransferases↗

PubMed2003

N-acetylglutamate and its changing role through evolution.

N -Acetylglutamate (NAG) fulfils distinct biological roles in lower and higher organisms. In prokaryotes, lower eukaryotes and plants it is the first intermediate in the biosynthesis of arginine, whereas in ureotelic (excreting nitrogen mostly in the form of urea) vertebrates, it is an essential allosteric cofactor for carbamyl phosphate synthetase I (CPSI), the first enzyme of the urea cycle. The pathway that leads from glutamate to arginine in lower organisms employs eight steps, starting with the acetylation of glutamate to form NAG. In these species, NAG can be produced by two enzymic reactions: one catalysed by NAG synthase (NAGS) and the other by ornithine acetyltransferase (OAT). In ureotelic species, NAG is produced exclusively by NAGS. In lower organisms, NAGS is feedback-inhibited by L-arginine, whereas mammalian NAGS activity is significantly enhanced by this amino acid. The NAGS genes of bacteria, fungi and mammals are more diverse than other arginine-biosynthesis and urea-cycle genes. The evolutionary relationship between the distinctly different roles of NAG and its metabolism in lower and higher organisms remains to be determined. In humans, inherited NAGS deficiency is an autosomal recessive disorder causing hyperammonaemia and a phenotype similar to CPSI deficiency. Several mutations have been recently identified in the NAGS genes of families affected with this disorder.

Amino Acid Sequence↗

PubMed2003

Null mutations in the N-acetylglutamate synthase gene associated with acute neonatal disease and hyperammonemia.

N-acetylglutamate synthase (NAGS) is a mitochondrial enzyme that catalyzes the formation of N-acetylglutamate, an essential allosteric activator of carbamyl phosphate synthetase I, the first enzyme of the urea cycle. Liver NAGS deficiency has previously been found in a small number of patients with hyperammonemia. The mouse and human NAGS genes have recently been cloned and expressed in our laboratory. We searched for mutations in the NAGS gene of two families with presumed NAGS deficiency. The exons and exon/intron boundaries of the NAGS gene were sequenced from genomic DNA obtained from the parents of an infant from the Faroe Islands who died in the neonatal period and from two Hispanic sisters who presented with acute neonatal hyperammonemia. Both parents of the first patient were found to be heterozygous for a null mutation in exon 4 (TGG-->TAG, Trp324Ter). Both sisters from the second family were homozygous for a single base deletion in exon 4 (1025delG) causing a frameshift and premature termination of translation. The finding of deleterious mutations in the NAGS gene confirms the genetic origin of NAGS deficiency. This disorder can now be diagnosed by DNA testing allowing for carrier detection and prenatal diagnosis.

Acetyltransferases↗

PubMed2002

Cloning and expression of the human N-acetylglutamate synthase gene.

N-acetylglutamate synthase (NAGS, E.C. 2.3.1.1) is a mitochondrial enzyme catalyzing the formation of N-acetylglutamate (NAG), an essential allosteric activator of carbamylphosphate synthase I (CPSI), the first enzyme of the urea cycle. Patients with NAGS deficiency develop hyperammonemia because CPSI is inactive without NAG. The human NAGS cDNA was isolated from a liver library based on its similarity to mouse NAGS. The deduced amino acid sequence contains an N-terminal putative mitochondrial targeting signal of 49 amino acids (63% identity with mouse NAGS) followed by a "variable domain" of 45 amino acids (35% identity) and a "conserved domain" of 440 amino acids (92% identity). A cDNA sequence containing the "conserved domain" complements an NAGS-deficient Escherichia coli strain and the recombinant protein has arginine-responsive NAGS catalytic activity. The NAGS gene is expressed in the liver and small intestine; the intestinal transcript is smaller in size than liver transcript.

Acetyltransferases↗

PubMed2002

Identification, cloning and expression of the mouse N-acetylglutamate synthase gene.

In ureotelic animals, N-acetylglutamate (NAG) is an essential allosteric activator of carbamylphosphate synthetase I (CPSI), the first enzyme in the urea cycle. NAG synthase (NAGS; EC 2.3.1.1) catalyses the formation of NAG from glutamate and acetyl-CoA in liver and intestinal mitochondria. This enzyme is supposed to regulate ureagenesis by producing variable amounts of NAG, thus modulating CPSI activity. Moreover, inherited deficiencies in NAGS have been associated with hyperammonaemia, probably due to the loss of CPSI activity. Although the existence of the NAGS protein in mammals has been known for decades, the gene has remained elusive. We identified the mouse (Mus musculus) and human NAGS genes using their similarity to the respective Neurospora crassa gene. NAGS was cloned from a mouse liver cDNA library and was found to encode a 2.3 kb message, highly expressed in liver and small intestine with lower expression levels in kidney, spleen and testis. The deduced amino acid sequence contains a putative mitochondrial targeting signal at the N-terminus. The cDNA sequence complements an argA (NAGS)-deficient Escherichia coli strain, reversing its arginine auxotrophy. His-tagged versions of the pre-protein and two putative mature proteins were each overexpressed in E. coli, and purified to apparent homogeneity by using a nickel-affinity column. The pre-protein and the two putative mature proteins catalysed the NAGS reaction but one of the putative mature enzymes had significantly higher activity than the pre-protein. The addition of l-arginine increased the catalytic activity of the purified recombinant NAGS enzymes by approx. 2-6-fold.

Acetyltransferases↗