Background: The 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.

Regenerative processes in brain pathologies require the production of distinct neural cell populations from endogenous progenitor cells. We have previously demonstrated that oligodendrocyte progenitor cell (OPC) proliferation is crucial for oligodendrocyte (OL) regeneration in a mouse model of neonatal hypoxia (HX) that reproduces diffuse white matter injury (DWMI) of premature infants. Here we identify the histone deacetylase Sirt1 as a Cdk2 regulator in OPC proliferation and response to HX. HX enhances Sirt1 and Sirt1/Cdk2 complex formation through HIF1α activation. Sirt1 deacetylates retinoblastoma (Rb) in the Rb/E2F1 complex, leading to dissociation of E2F1 and enhanced OPC proliferation. Sirt1 knockdown in culture and its targeted ablation in vivo suppresses basal and HX-induced OPC proliferation. Inhibition of Sirt1 also promotes OPC differentiation after HX. Our results indicate that Sirt1 is an essential regulator of OPC proliferation and OL regeneration after neonatal brain injury. Therefore, enhancing Sirt1 activity may promote OL recovery after DWMI.

The purpose of this study was to determine the efficacy of novel recombinant adeno-associated viral (AAV) vector constructs in correcting metabolic defects in the liver in two strains of ornithine transcarbamylase (OTC)-deficient mice (spf and spf-ash). AAV vectors expressing mouse OTC were produced with capsids from AAV2 and the novel serotypes AAV7, 8, and 9. OTC-deficient mice were infused with these vectors as well as a control AAV2/8 vector expressing LacZ. In vivo activity of OTC was assessed by measuring a surrogate marker, urine orotate. The novel vectors restored orotate levels to virtually normal 15 days after infusion, and each persisted to 1 year posttreatment. Liver OTC enzyme activity in spf mice was substantially higher in animals receiving novel vectors compared to those receiving AAV2 vectors. Animals receiving novel OTC-expressing vectors lived longer than those treated with AAV2 OTC or untreated controls, and they were tolerant to a challenge with NH3 at 21 days and beyond, which caused severe morbidity in control OTC-deficient animals. Numerous mice, representative of all treatment groups followed for +250 days, were observed to have either nodules or discrete tumors in the liver, the etiology of which is the subject of a companion paper.

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 ␣ -acetyl-N ␦ -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.The enzymatic biosynthesis of L-arginine is accomplished by a complex, highly interconnected pathway with at least eight steps to produce arginine from L-glutamate via a series of acetylated amino acid intermediates (Fig. 1). One of the essential enzymes in the pathway, ornithine transcarbamylase (OTCase) (EC 2.1.3.3), catalyzes the formation of citrulline from ornithine and carbamyl phosphate. Protein sequences within the OTCase family show strong conservation across diverse phylogenetic domains ranging from archaea to mammals (25,26). This suggests that the role of OTCase in arginine biosynthesis was established at a very early stage of evolution, an assertion that is further supported by phylogenetic comparison of the OTCase sequences to the paralogous aspartate transcarbamylases (ATCases) involved in pyrimidine synthesis. Thus, it appears that these two enzyme families already existed at the point of the last universal common ancestor (7).While exploring the arginine biosynthesis pathway in the anaerobe Bacteroides fragilis, we identified and solved the crystal structure of a transcarbamylase-like protein (the product of a gene denoted argFЈ [GI:22218874]) that is essential for arginine biosynthesis (16). However, repeated attempts to detect enzymatic carbamylation of a variety of substrates using the native or recombinant ArgFЈ protein were unsuccessful. The B. fragilis ArgFЈ protein shares only limited sequence homology with other OTCases (38% and 34% similarity to Escherichia coli ArgF and human OTCase, respectively) (16). By comparison, the amino acid sequence of the B. fragilis aspartate transcarbamylase (ATCase) catalytic subunit shares a much higher similarity (70.3%) with E. coli ATCase. Further analysis of t...

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- The canonical arginine biosynthetic pathway in microorganisms and plants involves N-acetylated intermediates in the first five steps. At the fifth step, N-acetyl-L-ornithine is deacetylated either hydrolytically to form ornithine in the linear pathway or by transfer of the acetyl group to glutamate to regenerate N-acetyl-L-glutamate in the cyclic pathway. Subsequently, ornithine is converted to arginine by three enzymes: ornithine transcarbamylase (OTCase), 3 argininosuccinate synthase and argininosuccinate lyase (1). We have recently shown that in some eubacteria (e.g. Xanthomonas), N-acetyl-L-ornithine rather than ornithine is carbamylated by a novel transcarbamylase that is essential for arginine biosynthesis (2, 3). In 2002, we reported the crystal structure of a transcarbamylase-like protein from Bacteroides fragilis with phosphate bound at 2.0 Å resolution (4). At the time, we postulated that it is a novel member of the transcarbamylase family, since no activity could be detected with ornithine or other amino acids that are known transcarbamylation substrates, and the crystal structure indicated that it has a unique second substrate binding pocket. We now provide evidence that N-succinyl-L-ornithine is a substrate for this novel transcarbamylase and that this protein is essential for de novo arginine biosynthesis in B. fragilis. These data imply that B. fragilis uses N-succinylated ornithine for arginine biosynthesis and is the first organism known to do so. EXPERIMENTAL PROCEDURESProduction of B. fragilis Arginine Auxotrophs and Identification of the argFЈ bf Gene-Several arginine requiring mutants were isolated after mutagenizing B. fragilis ADB77, a thyAϪ derivative of TM4000 (5) with transposon Tn4400Ј (6). Using the "clone out" protocol of Tang and Malamy (6), we were able to determine the chromosomal sequence on both sides of the transposon insertions. Strains JDP3a.3 and JDP3a.4 contained insertions in a gene with high similarity to the argF genes encoding OTCase in eubacteria; we designated this gene as argFЈ bf .