1.85-Å Resolution Crystal Structure of Human Ornithine Transcarbamoylase Complexed withN-Phosphonacetyl-l-ornithine

The crystal structure of human ornithine transcarbamoylase complexed with the bisubstrate analogN-phosphonacetyl-l-ornithine has been solved at 1.85-Å resolution by molecular replacement. Deleterious mutations produce clinical hyperammonia that, if untreated, results in neurological symptoms or death (ornithine transcarbamylase deficiency). The holoenzyme is trimeric, and as in other transcarbamoylases, each subunit contains an N-terminal domain that binds carbamoyl phosphate and a C-terminal domain that binds l-ornithine. The active site is located in the cleft between domains and contains additional residues from an adjacent subunit. Binding ofN-phosphonacetyl-l-ornithine promotes domain closure. The resolution of the structure enables the role of active site residues in the catalytic mechanism to be critically examined. The side chain of Cys-303 is positioned so as to be able to interact with the δ-amino group of l-ornithine which attacks the carbonyl carbon of carbamoyl phosphate in the enzyme-catalyzed reaction. This sulfhydryl group forms a charge relay system with Asp-263 and the α-amino group of l-ornithine, instead of with His-302 and Glu-310, as previously proposed. In common with other ureotelic ornithine transcarbamoylases, the human enzyme lacks a loop of ∼20 residues between helix H10 and β-strand B10 which is present in prokaryotic ornithine transcarbamoylases but has a C-terminal extension of 10 residues that interacts with the body of the protein but is exposed. The sequence of this C-terminal extension is homologous to an interhelical loop found in several membrane proteins, including mitochondrial transport proteins, suggesting a possible mode of interaction with the inner mitochondrial membrane. The crystal structure of human ornithine transcarbamoylase complexed with the bisubstrate analogN-phosphonacetyl-l-ornithine has been solved at 1.85-Å resolution by molecular replacement. Deleterious mutations produce clinical hyperammonia that, if untreated, results in neurological symptoms or death (ornithine transcarbamylase deficiency). The holoenzyme is trimeric, and as in other transcarbamoylases, each subunit contains an N-terminal domain that binds carbamoyl phosphate and a C-terminal domain that binds l-ornithine. The active site is located in the cleft between domains and contains additional residues from an adjacent subunit. Binding ofN-phosphonacetyl-l-ornithine promotes domain closure. The resolution of the structure enables the role of active site residues in the catalytic mechanism to be critically examined. The side chain of Cys-303 is positioned so as to be able to interact with the δ-amino group of l-ornithine which attacks the carbonyl carbon of carbamoyl phosphate in the enzyme-catalyzed reaction. This sulfhydryl group forms a charge relay system with Asp-263 and the α-amino group of l-ornithine, instead of with His-302 and Glu-310, as previously proposed. In common with other ureotelic ornithine transcarbamoylases, the human enzyme lacks a loop of ∼20 residues between helix H10 and β-strand B10 which is present in prokaryotic ornithine transcarbamoylases but has a C-terminal extension of 10 residues that interacts with the body of the protein but is exposed. The sequence of this C-terminal extension is homologous to an interhelical loop found in several membrane proteins, including mitochondrial transport proteins, suggesting a possible mode of interaction with the inner mitochondrial membrane. ornithine transcarbamoylase aspartate transcarbamoylase carbamoyl phosphate bound l-ornithine ornithine transcarbamoylase deficiency N-phosphonacetyl-l-ornithine root mean square deviation water. Since ornithine transcarbamoylase (OTCase)1 catalyzes the formation of citrulline from carbamoyl phosphate (CP) andl-ornithine (ORN) in the urea cycle (1Snodgrass P.J. Biochemistry. 1968; 7: 3047-3051Crossref PubMed Scopus (68) Google Scholar), deleterious mutations in the human OTCase gene produce clinical hyperammonemia, with subsequent neurological symptoms or even death (ornithine transcarbamylase deficiency; OTCD). Approximately 140 mutations that give rise to OTCD have been identified (2Tuchman M. Morizono H. Rajagopal B.S. Plante R.J. Allewell N.M. J. Inherited Metab. Dis. 1998; 21: 40-58Crossref PubMed Scopus (0) Google Scholar), and several mutant enzymes have been purified and characterized biochemically (3Morizono H. Tuchman M. Rajagopal B.S. McCann M.T. Listrom C.D. Yuan X. Venugopal D. Barany G. Allewell N.M. Biochem. J. 1997; 322: 625-631Crossref PubMed Scopus (36) Google Scholar, 4Morizono H. Listrom C.D. Rajagopal B.S. Aoyagi M. McCann M.T. Allewell N.M. Tuchman M. Hum. Mol. Genet. 1997; 6: 963-968Crossref PubMed Scopus (15) Google Scholar). Establishing the structural basis of their deleterious effects is of considerable clinical importance, as well as biochemical interest.OTCase is homologous in structure and function to the catalytic trimer of Escherichia coli aspartate transcarbamoylase (ATCase), a widely studied allosteric enzyme (5Lipscomb W.N. Adv. Enzymol. Relat. Areas Mol. Biol. 1994; 68: 67-151PubMed Google Scholar). We have previously developed a homology model of human OTCase (6Tuchman M. Morizono H. Reish O. Yuan X. Allewell N.M. J. Med. Genet. 1995; 32: 680-688Crossref PubMed Scopus (49) Google Scholar) based on the ATCase structure (7Kosman R.P. Gouaux J.E. Lipscomb W.N. Proteins Struct. Funct. Genet. 1993; 15: 147-176Crossref PubMed Scopus (65) Google Scholar) which allowed the functional effects of some naturally occurring deleterious mutations that produce OTCD to be predicted. The crystal structures of unliganded catabolic Pseudomonas aeruginosaOTCase (8Villeret V. Tricot C. Stalon V. Dideberg O. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10762-10766Crossref PubMed Scopus (59) Google Scholar), unliganded E. coli OTCase (9Jin L. Seaton B.A. Head J.F. Nat. Struct. Biol. 1997; 4: 622-625Crossref PubMed Scopus (40) Google Scholar), liganded E. coli OTCase (10Ha Y. McCann M.T. Tuchman M. Allewell N.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9550-9555Crossref PubMed Scopus (48) Google Scholar), and unliganded anabolic Pseudomonas furiosus OTCase (11Villeret V. Clantin B. Tricot C. Legrain C. Roovers M. Stalon V. Glansdorff N. van Beeumen J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2801-2806Crossref PubMed Scopus (132) Google Scholar) confirmed many of these predictions. However, all the OTCase structures available to date are of bacterial enzymes and at only moderate resolution. All have significant sequence differences from the human enzyme (Fig.1), and some function anabolically to synthesize arginine. To establish structure-function relationships for the human enzyme with certainty, its crystal structure is required.We report herein the structure of human OTCase complexed with the bisubstrate analog N-phosphonacetyl-l-ornithine (PALO) at 1.8-Å resolution, the highest resolution OTCase crystal structure reported to date. 215 bound solvent molecules, some of which form clusters at the active site, can be identified. The resolution of the structure enables the function of active site residues to be examined with precision and a model of the catalytic mechanism to be proposed. We compare this structure with those of other transcarbamoylases, discuss possible functions for its unique regions, and describe the structural basis of the clinical effects of some common and/or severe mutations that produce OTCD. Since ornithine transcarbamoylase (OTCase)1 catalyzes the formation of citrulline from carbamoyl phosphate (CP) andl-ornithine (ORN) in the urea cycle (1Snodgrass P.J. Biochemistry. 1968; 7: 3047-3051Crossref PubMed Scopus (68) Google Scholar), deleterious mutations in the human OTCase gene produce clinical hyperammonemia, with subsequent neurological symptoms or even death (ornithine transcarbamylase deficiency; OTCD). Approximately 140 mutations that give rise to OTCD have been identified (2Tuchman M. Morizono H. Rajagopal B.S. Plante R.J. Allewell N.M. J. Inherited Metab. Dis. 1998; 21: 40-58Crossref PubMed Scopus (0) Google Scholar), and several mutant enzymes have been purified and characterized biochemically (3Morizono H. Tuchman M. Rajagopal B.S. McCann M.T. Listrom C.D. Yuan X. Venugopal D. Barany G. Allewell N.M. Biochem. J. 1997; 322: 625-631Crossref PubMed Scopus (36) Google Scholar, 4Morizono H. Listrom C.D. Rajagopal B.S. Aoyagi M. McCann M.T. Allewell N.M. Tuchman M. Hum. Mol. Genet. 1997; 6: 963-968Crossref PubMed Scopus (15) Google Scholar). Establishing the structural basis of their deleterious effects is of considerable clinical importance, as well as biochemical interest. OTCase is homologous in structure and function to the catalytic trimer of Escherichia coli aspartate transcarbamoylase (ATCase), a widely studied allosteric enzyme (5Lipscomb W.N. Adv. Enzymol. Relat. Areas Mol. Biol. 1994; 68: 67-151PubMed Google Scholar). We have previously developed a homology model of human OTCase (6Tuchman M. Morizono H. Reish O. Yuan X. Allewell N.M. J. Med. Genet. 1995; 32: 680-688Crossref PubMed Scopus (49) Google Scholar) based on the ATCase structure (7Kosman R.P. Gouaux J.E. Lipscomb W.N. Proteins Struct. Funct. Genet. 1993; 15: 147-176Crossref PubMed Scopus (65) Google Scholar) which allowed the functional effects of some naturally occurring deleterious mutations that produce OTCD to be predicted. The crystal structures of unliganded catabolic Pseudomonas aeruginosaOTCase (8Villeret V. Tricot C. Stalon V. Dideberg O. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10762-10766Crossref PubMed Scopus (59) Google Scholar), unliganded E. coli OTCase (9Jin L. Seaton B.A. Head J.F. Nat. Struct. Biol. 1997; 4: 622-625Crossref PubMed Scopus (40) Google Scholar), liganded E. coli OTCase (10Ha Y. McCann M.T. Tuchman M. Allewell N.M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9550-9555Crossref PubMed Scopus (48) Google Scholar), and unliganded anabolic Pseudomonas furiosus OTCase (11Villeret V. Clantin B. Tricot C. Legrain C. Roovers M. Stalon V. Glansdorff N. van Beeumen J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 2801-2806Crossref PubMed Scopus (132) Google Scholar) confirmed many of these predictions. However, all the OTCase structures available to date are of bacterial enzymes and at only moderate resolution. All have significant sequence differences from the human enzyme (Fig.1), and some function anabolically to synthesize arginine. To establish structure-function relationships for the human enzyme with certainty, its crystal structure is required. We report herein the structure of human OTCase complexed with the bisubstrate analog N-phosphonacetyl-l-ornithine (PALO) at 1.8-Å resolution, the highest resolution OTCase crystal structure reported to date. 215 bound solvent molecules, some of which form clusters at the active site, can be identified. The resolution of the structure enables the function of active site residues to be examined with precision and a model of the catalytic mechanism to be proposed. We compare this structure with those of other transcarbamoylases, discuss possible functions for its unique regions, and describe the structural basis of the clinical effects of some common and/or severe mutations that produce OTCD. We thank Dr. R. Sweet for assistance during data collection at beamline X12C in the National Synchrotron Light Source at Brookhaven National Laboratory. This facility is supported by the U. S. Department of Energy Offices of Health and Environmental Research and of Basic Energy Sciences and by the National Science Foundation. We also thank Drs. L. Banaszak and D. Ohlendorf for facilitating our use of equipment in the Kahlert Center for Structure Biology at the University of Minnesota. Some calculations were carried out on the Basic Science Computing Laboratory at the Minnesota Supercomputer Center. We thank Drs. G. Barany and D. Venugopal in the Department of Chemistry at the University of Minnesota for synthesizing PALO.