Human ornithine transcarbamylase: crystallographic insights into substrate recognition and conformational changes

Shi, Dashuang; Morizono, Hiroki; Yu, Xiaolin; Tong, Liang; Allewell, Norma M.; Tuchman, Mendel

doi:10.1042/0264-6021:3540501

Cited by 44 publications

(75 citation statements)

References 58 publications

(55 reference statements)

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“…Our studies have identified that lysine 88 in OTC is acetylated. Interestingly, the OTC three-dimensional structure (17)(18)(19) shows that Lys 88 is not only localized near the carbamoyl phosphate-binding residues (residues 90 -93) but that it is also involved in the formation of a complex hydrogen- …”

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Lysine 88 Acetylation Negatively Regulates Ornithine Carbamoyltransferase Activity in Response to Nutrient Signals

Lin

Yao

et al. 2009

Journal of Biological Chemistry

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Ornithine carbamoyltransferase (OTC) is a key enzyme in the urea cycle to detoxify ammonium produced from amino acid catabolism. OTC deficiency is an X-linked genetic disorder ranging from fatal in newborns to hyperammonemia and anorexia in adults. Through affinity purification of acetylated peptides and mass spectrometry, we identified that OTC is acetylated on lysine residues, including Lys 88 , which is also mutated in OTC-deficient patients. OTC acetylation was confirmed to occur under physiological conditions. Biochemical characterizations revealed that OTC Lys 88 acetylation decreases the affinity for carbamoyl phosphate, one of the two OTC substrates, and the maximum velocity, whereas the K m for ornithine, the other OTC substrate, is not affected. Furthermore, Lys 88 acetylation is regulated by both extracellular glucose and amino acid availability, indicating that OTC activity may be regulated by cellular metabolic status. Our results provide an example of the novel mechanism of regulating metabolic enzyme activity through protein acetylation.

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Lysine 88 Acetylation Negatively Regulates Ornithine Carbamoyltransferase Activity in Response to Nutrient Signals

Lin

Yao

et al. 2009

Journal of Biological Chemistry

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“…Further analysis of the B. fragilis ArgFЈ protein sequence revealed that a conserved Ser-Met-Gly (SMG) motif, which is present in all OTCases, was absent. This motif is part of a flexible loop that moves to cradle L-ornithine when it binds to OTCase (17,19). This difference leads us to hypothesize that the B. fragilis ArgFЈ protein represents a new class of transcarbamylase.…”

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Acetylornithine Transcarbamylase: a Novel Enzyme in Arginine Biosynthesis

et al. 2006

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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...

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“…Studies with aspartate and ornithine transcarbamylases demonstrated that phosphonoacetyl-L-aspartate (7) and phosphonoacetyl-L-ornithine (35) are, respectively, highly potent inert bisubstrate inhibitors of these enzymes. Since these inhibitors have been successfully used in crystallization trials with these two enzymes (21,47) that led to the determination of their three-dimensional structures by X-ray diffraction, we reasoned that PAPU might be also a very potent and highly specific inhibitor of PTC and, if so, that it might help enzyme crystallization (see below). Although PAPU was synthesized previously (41), to our knowledge it has never been used with PTC.…”

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The Gene Cluster for Agmatine Catabolism ofEnterococcus faecalis: Study of Recombinant Putrescine Transcarbamylase and Agmatine Deiminase and a Snapshot of Agmatine Deiminase Catalyzing Its Reaction

et al. 2007

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Enterococcus faecalis makes ATP from agmatine in three steps catalyzed by agmatine deiminase (AgDI), putrescine transcarbamylase (PTC), and carbamate kinase (CK). An antiporter exchanges putrescine for agmatine. We have cloned the E. faecalis ef0732 and ef0734 genes of the reported gene cluster for agmatine catabolism, overexpressed them in Escherichia coli, purified the products, characterized them functionally as PTC and AgDI, and crystallized and X-ray diffracted them. The 1.65-Å-resolution structure of AgDI forming a covalent adduct with an agmatine-derived amidine reactional intermediate is described. We provide definitive identification of the gene cluster for agmatine catabolism and confirm that ornithine is a genuine but poor PTC substrate, suggesting that PTC (found here to be trimeric) evolved from ornithine transcarbamylase. N-(Phosphonoacetyl)-putrescine was prepared and shown to strongly (K i ‫؍‬ 10 nM) and selectively inhibit PTC and to improve PTC crystallization. We find that E. faecalis AgDI, which is committed to ATP generation, closely resembles the AgDIs involved in making polyamines, suggesting the recruitment of a polyamine-synthesizing AgDI into the AgDI pathway. The arginine deiminase (ADI) pathway of arginine catabolism probably supplied the genes for PTC and CK but not those for the agmatine/putrescine antiporter, and thus the AgDI and ADI pathways are not related by a single "en bloc" duplication event. The AgDI crystal structure reveals a tetramer with a five-blade propeller subunit fold, proves that AgDI closely resembles ADI despite a lack of sequence identity, and explains substrate affinity, selectivity, and Cys357-mediated-covalent catalysis. A three-tongued agmatine-triggered gating opens or blocks access to the active center.In addition to the fermentation of carbohydrates, Enterococcus faecalis (formerly Streptococcus faecalis) is able to use arginine and its decarboxylated derivative agmatine as energy sources for growth (8,10,45,48,49). Arginine and agmatine are metabolized via the arginine deiminase (ADI) and agmatine deiminase (AgDI) pathways, respectively. The two metabolic routes are very similar and include the sequential action of three enzymes (48, 49) and one antiporter (11), which are analogous in the two pathways. Arginine and agmatine are deiminated by ADI (EC 3.5.3.6) and AgDI (EC 3.5.3.12), respectively, yielding citrulline and carbamoyl putrescine, which are phosphorolyzed by ornithine transcarbamylase (OTC) (EC 2.1.3.3) and putrescine transcarbamylase (PTC) (EC 2.1.3.6). This generates carbamoyl phosphate for use in ADP phosphorylation by pathway-specific carbamate kinase (CK) (EC 2.7.2.2) isozymes, producing one ATP molecule (48, 49). The resulting ornithine and putrescine are exchanged with external arginine or agmatine by an arginine/ ornithine antiporter in one pathway and by an agmatine/putrescine antiporter in the other pathway (11).Possibly no microbial species has been more important for the biochemical characterization of the ADI and AgDI pathways tha...

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Human ornithine transcarbamylase: crystallographic insights into substrate recognition and conformational changes

Cited by 44 publications

References 58 publications

Lysine 88 Acetylation Negatively Regulates Ornithine Carbamoyltransferase Activity in Response to Nutrient Signals

Lysine 88 Acetylation Negatively Regulates Ornithine Carbamoyltransferase Activity in Response to Nutrient Signals

Acetylornithine Transcarbamylase: a Novel Enzyme in Arginine Biosynthesis

The Gene Cluster for Agmatine Catabolism ofEnterococcus faecalis: Study of Recombinant Putrescine Transcarbamylase and Agmatine Deiminase and a Snapshot of Agmatine Deiminase Catalyzing Its Reaction

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