Acetylornithine Transcarbamylase: a Novel Enzyme in Arginine Biosynthesis

Abstract

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...

Surprising Arginine Biosynthesis: a Reappraisal of the Enzymology and Evolution of the Pathway in Microorganisms

“…Our textbook vision of arginine biosynthesis beyond acetylornithine was shattered by the recent discovery of acetylornithine carbamoyltransferase (AOTC) (ArgFЈ) in the ␥-proteobacterium Xanthomonas campestris (50,65). AOTC catalyzes the formation of acetylcitrulline from acetylornithine but not that of citrulline from ornithine (Fig.…”

“…ArgE then allows the process to go directly to citrulline (step 6Ј) and subsequently to arginine. Box 1 underlines this dual activity of ArgE (EC 3.5.1.16), which can deacetylate either N-acetyl-ornithine or N-acetyl-citrulline (50).…”

Intricate Knots in Proteins: Function and Evolution

“…N-acetylornithine transcarbamylase (AOTCase [17]) is essential for the arginine biosynthesis in several major pathogens. In other bacteria, animals, and humans, a homologous enzyme (OTCase) processes L-ornithine instead [30]. Both proteins have two active sites.…”

Systems and synthetic biology perspective of the versatile plant-pathogenic and polysaccharide-producing bacterium Xanthomonas campestris

“…This included information relating to the uptake and utilization of nutrients from the environment [79,[120][121][122][123][124]. Further important metabolic studies considered in the iterative enhancement of functional genome annotation [74] focused on the biosynthesis of amino acids [107,125] and a novel, yeast-like phosphatidylcholine synthesis route [126]. A common theme of these studies was the discovery of novel biosynthetic routes that differ from the respective metabolic pathways of other bacterial model organisms.…”

“…Following its automated generation, the XccCyc database indicated unclear amino acid biosynthetic pathways. While the biosynthetic pathway for arginine was available from a previous experimental study [125], biosynthetic pathways for 16 proteinogenic amino acids were determined by employing systematic and thorough interpretation of results from the bioinformatic tools (Table 1). For three remaining amino acids, 13 C NMR measurements were required to resolve those metabolic routes actually used for their synthesis (Fig.…”