Mammalian Pyrimidine Biosynthesis: Fresh Insights into an Ancient Pathway (2004)

Pyrimidine nucleotides play a critical role in cellular metabolism serving as activated precursors of RNA and DNA, CDP-diacylglycerol phosphoglyceride for the assembly of cell membranes and UDP-sugars for protein glycosylation and glycogen synthesis (1Jones M.E. Annu. Rev. Biochem. 1980; 49: 253-279Crossref PubMed Scopus (535) Google Scholar, 2Evans D.R. Hardie D.G. Coggins J.R., J.R. Multidomain Proteins-Structure and Function. Elsevier Science Publishers, New York1986: 283-331Google Scholar, 3Traut T.W. Mol. Cell. Biochem. 1994; 140: 1-22Crossref PubMed Scopus (1271) Google Scholar). In addition, uridine nucleotides act via extra-cellular receptors to regulate a variety of physiological processes (4Connolly G.P. Duley J.A. Trends Pharmacol. Sci. 1999; 20: 218-225Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar). There are two routes to the synthesis of pyrimidines; nucleotides can be recycled by the salvage pathways or synthesized de novo from small metabolites (Fig. 1). Most cells have several specialized passive and active transporters (5Cass C.E. Young J.D. Baldwin S.A. Biochem. Cell Biol. 1998; 76: 761-770Crossref PubMed Scopus (168) Google Scholar, 6Mascia L. Turchi G. Bemi V. Ipata P.L. Biochim. Biophys. Acta. 2001; 1524: 45-50Crossref PubMed Scopus (21) Google Scholar) that allow the reutilization of preformed pyrimidine nucleosides and bases. The relative contribution of the de novo and salvage pathways depends on cell type and developmental stage. In general, the activity of the de novo pathway is low in resting or fully differentiated cells where the need for pyrimidines is largely satisfied by the salvage pathways (7Fairbanks L.D. Bofill M. Ruckemann K. Simmonds H.A. J. Biol. Chem. 1995; 270: 29682-29689Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). In contrast, de novo pyrimidine biosynthesis (Fig. 1) is indispensable in proliferating cells in order to meet the increased demand for nucleic acid precursors and other cellular components. Consequently, the activity of the de novo pathway is subject to elaborate growth state-dependent control mechanisms. Pyrimidine biosynthesis is invariably up-regulated in tumors and neoplastic cells (8Weber G. Biochemistry (Mosc.). 2001; 66: 1164-1173Crossref PubMed Scopus (20) Google Scholar), and the pathway has been linked to the etiology or treatment of several other disorders including AIDS (9Bofill M. Fairbanks L. Ruckemann K. Lipman M. Simmonds H. J. Biol. Chem. 1995; 270: 29690-29697Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar), diabetes (10Kunjara S. Sochor M. Ali M. Drake A. Greenbaum A. McLean P. Biochem. Med. Metab. Biol. 1991; 46: 215-225Crossref PubMed Scopus (9) Google Scholar), and various autoimmune diseases (11Sanders S. Harisdangkul V. Am. J. Med. Sci. 2002; 323: 190-193Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) such as rheumatoid arthritis. This review focuses on the structure and regulation of the pyrimidine biosynthetic complexes and the interplay of the diverse control mechanisms operative in mammalian cells. Since the pioneering discoveries of Jones, Hoogenraad and others in 1971 (1Jones M.E. Annu. Rev. Biochem. 1980; 49: 253-279Crossref PubMed Scopus (535) Google Scholar), the physical association of the first three enzymes of the de novo pyrimidine pathway, carbamoyl-phosphate synthetase (CPSase), aspartate transcarbamylase (ATCase), and dihydroorotase (DHOase) has been documented in many animal cells. In 1977, Stark and his associates (12Coleman P. Suttle D. Stark G. J. Biol. Chem. 1977; 252: 6379-6385Abstract Full Text PDF PubMed Google Scholar) made the remarkable discovery that all three activities are carried on a single polypeptide. The 243-kDa CAD polypeptide associates to form hexamers and higher oligomers (13Lee L. Kelly R.E. Pastra-Landis S.C. Evans D.R. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 6802-6806Crossref PubMed Scopus (46) Google Scholar) so that the mass of the complex exceeds 1.4 MDa or about one-half the size of the ribosome. The domain structure (Fig. 2) has been mapped, and the function of each domain has been assigned (14Kim H. Kelly R.E. Evans D.R. J. Biol. Chem. 1992; 267: 7177-7184Abstract Full Text PDF PubMed Google Scholar). Carbamoyl-phosphate Synthetase—CPSase catalyzes the synthesis of carbamoyl phosphate from glutamine, bicarbonate, and two ATP molecules. Carbamoyl phosphate biosynthesis (Fig. 1, circled 1) is a complex process involving four partial reactions (15Anderson P.M. Walsh P.J. Wright P.A. Nitrogen Metabolism and Excretion. CRC Press Inc., Boca Raton, FL1995: 33-55Google Scholar, 16Evans D. Wiley Encyclopedia of Molecular Medicine. 5. John Wiley and Sons, Inc., New York2002: 449-453Google Scholar) catalyzed by the GLN, CPS.A, and CPS.B domains of the molecule (Fig. 2). The 40-kDa GLN domain is an amidotransferase that generates ammonia by glutamine hydrolysis. The synthetase domain consists of two homologous 60-kDa halves, CPS.A and CPS.B, thought to have arisen by an ancestral gene duplication and fusion (17Nyunoya H. Broglie K.E. Lusty C.J. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 2244-2246Crossref PubMed Scopus (36) Google Scholar). These subdomains are functionally equivalent (18Guy H.I. Evans D.R. J. Biol. Chem. 1996; 272: 13762-13769Abstract Full Text Full Text PDF Scopus (34) Google Scholar) but assume specialized functions when fused together in the intact molecule (19Britton H.G. Rubio V. Grisolia S. Eur. J. Biochem. 1979; 102: 521-530Crossref PubMed Scopus (35) Google Scholar, 20Post L.E. Post D.J. Raushel F.M. J. Biol. Chem. 1990; 265: 7742-7747Abstract Full Text PDF PubMed Google Scholar). CPS.A catalyzes the ATP-dependent activation of bicarbonate forming carboxyphosphate, which then reacts with ammonia to form carbamate. Carbamoyl phosphate is formed in a second ATP-dependent phosphorylation on CPS.B. All of these intermediates are labile but are effectively sequestered within the complex, passing between the active sites via narrow tunnels that snake through the interior of the molecule (21Thoden J.B. Holden H.M. Wesenberg G. Raushel F.M. Rayment I. Biochemistry. 1997; 36: 6305-6316Crossref PubMed Scopus (305) Google Scholar). CPSase catalyzes the rate-limiting step in de novo pyrimidine biosynthesis and, as discussed below, controls the flux through the pathway (1Jones M.E. Annu. Rev. Biochem. 1980; 49: 253-279Crossref PubMed Scopus (535) Google Scholar). Aspartate Transcarbamylase—The reaction of carbamoyl phosphate and aspartate to form carbamoyl aspartate is catalyzed by ATCase (Fig. 1, circled 2). A single CPSase in Escherichia coli supplies carbamoyl phosphate for both pyrimidine and arginine biosynthesis. Thus, the bacterial ATCase catalyzes the first committed step in the pyrimidine pathway and is allosterically regulated (22Lipscomb W.N. Adv. Enzymol. Relat. Areas Mol. Biol. 1994; 68: 67-151PubMed Google Scholar). In contrast, there are two CPSases in mammalian cells, CPSII, the CAD activity committed to pyrimidine biosynthesis, and CPSI (23Lusty C.J. Eur. J. Biochem. 1978; 85: 373-383Crossref PubMed Scopus (89) Google Scholar), an ammonia-dependent enzyme that initiates urea biosynthesis in the mitochondria. Because CAD CPSase catalyzes the first step in the pathway, ATCase is unregulated and there is no counterpart of the regulatory chain found in E. coli ATCase. The isolated CAD ATCase domain, from proteolytic digests (24Grayson D.R. Evans D.R. J. Biol. Chem. 1983; 258: 4123-4129Abstract Full Text PDF PubMed Google Scholar) or expressed in E. coli (25Maley J.A. Davidson J.N. Mol. Gen. Genet. 1988; 213: 278-284Crossref PubMed Scopus (27) Google Scholar), is a homotrimer of 34-kDa subunits. Kinetic and modeling studies showed that the mammalian domain shares a common catalytic mechanism, oligomeric structure, and tertiary-fold with the E. coli ATCase catalytic subunit (24Grayson D.R. Evans D.R. J. Biol. Chem. 1983; 258: 4123-4129Abstract Full Text PDF PubMed Google Scholar, 25Maley J.A. Davidson J.N. Mol. Gen. Genet. 1988; 213: 278-284Crossref PubMed Scopus (27) Google Scholar, 26Scully J.L. Evans D.R. Proteins. 1991; 9: 191-206Crossref PubMed Scopus (23) Google Scholar, 27Qiu Y. Davidson J.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 97-102Crossref PubMed Scopus (11) Google Scholar), including a composite active site comprised of residues from adjacent subunits. Qiu and Davidson (27Qiu Y. Davidson J.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 97-102Crossref PubMed Scopus (11) Google Scholar) showed that substitutions at the trimer interface disrupt the CAD oligomeric structure suggesting that the trimeric ATCase interactions are a crucial organizing element in the hexamer. Dihydroorotase—The 46-kDa CAD DHOase domain, which catalyzes the reversible condensation of carbamoyl aspartate to dihydroorotate (Fig. 1, circled 3), is a zinc metalloenzyme. Whereas the active site of E. coli DHOase has two zinc ions and a carboxylysine that bridges the metal centers (28Thoden J.B. Phillips Jr., G.N. Neal T.M. Raushel F.M. Holden H.M. Biochemistry. 2001; 40: 6989-6997Crossref PubMed Scopus (174) Google Scholar), the mammalian DHOase domain probably belongs to a different subgroup of the amidohydrolase superfamily (29Holm L. Sander C. Proteins. 1997; 28: 72-82Crossref PubMed Scopus (420) Google Scholar). An extensive phylogenetic analysis (30Fields C. Brichta D. Shephardson M. Farinha M. O'Donovan G. Paths Pyrimidines. 1999; 7: 49-63Google Scholar) classified DHOases into two major classes thought to have arisen by an ancestral gene duplication. Type I DHOases are the most ancient and include domains of multifunctional proteins, such as CAD, subunits of multienzyme complexes, and monofunctional enzymes. The type II enzymes, such as E. coli DHOase, are a more recent evolutionary development, are smaller (38 kDa), and have undergone appreciable changes in sequence. The isolated CAD DHOase domain (31Kelly R.E. Mally M.I. Evans D.R. J. Biol. Chem. 1986; 261: 6073-6083Abstract Full Text PDF PubMed Google Scholar, 32Musmanno L.A. Maley J.A. Davidson J.N. Gene (Amst.). 1991; 99: 211-216Crossref PubMed Scopus (13) Google Scholar, 33Williams N.K. Peide Y. Seymour K.K. Ralston G.B. Christopherson R.I. Protein Eng. 1993; 6: 333-340Crossref PubMed Scopus (19) Google Scholar, 34Zimmermann B.H. Evans D.R. Biochemistry. 1993; 32: 1519-1527Crossref PubMed Scopus (19) Google Scholar) has only one zinc atom and is larger than its bacterial counterpart consistent with its assignment as a type I DHOase. Mammalian DHOdhase is a 43-kDa flavoprotein (FMN) localized in the mitochondria that oxidizes dihydroorotate to orotate (Fig. 1, circled 4). The electrons are transferred directly to the respiratory chain via ubiquinone (1Jones M.E. Annu. Rev. Biochem. 1980; 49: 253-279Crossref PubMed Scopus (535) Google Scholar). Biochemical and microscopic studies (1Jones M.E. Annu. Rev. Biochem. 1980; 49: 253-279Crossref PubMed Scopus (535) Google Scholar, 35Carrey E.A. Dietz C. Glubb D.M. Loffler M. Lucocq J.M. Watson P.F. Reproduction. 2002; 123: 757-768Crossref PubMed Scopus (31) Google Scholar) showed that the enzyme is an integral membrane protein localized in the inner mitochondrial membrane with the active site facing the inner membrane space (Fig. 3). Mitochondrial import (36Rawls J. Knecht W. Diekert K. Lill R. Loffler M. Eur. J. Biochem. 2000; 267: 2079-2087Crossref PubMed Scopus (90) Google Scholar) is governed by an uncleaved bipartite sequence at the amino end of the polypeptide (Figs. 2 and 3) that consists of a mitochondrial targeting sequence (MT) and a membrane stop-transfer sequence (MA) that anchors the protein to the inner membrane. Structural studies showed that truncated human DHOdhase (37Liu S. Neidhardt E.A. Grossman T.H. Ocain T. Clardy J. Structure Fold. Des. 2000; 8: 25-33Abstract Full Text Full Text PDF Scopus (264) Google Scholar), lacking the bipartite sequence, consists of two domains (Fig. 2). The active site is located within the large catalytic domain (CAT), whereas the small domain (QT) forms a tunnel that leads directly to the bound FMN and provides access to ubiquinone. Orotate is completely buried on the distal side of FMN so it is unlikely to enter via the same tunnel. Instead, a flexible loop moves out of position to accommodate substrate binding on the distal side of FMN. Antiproliferative agents such as leflunomide bind within the tunnel blocking access of ubiquinone to the active site. UMP synthase is a bifunctional protein that catalyzes the last two steps of de novo pyrimidine biosynthesis (Fig. 1) (1Jones M.E. Annu. Rev. Biochem. 1980; 49: 253-279Crossref PubMed Scopus (535) Google Scholar). The mammalian protein (Fig. 2) consists of a 24-kDa orotate phosphoribosyltransferase (OPRTase) that catalyzes the transfer of PRPP to orotate forming OMP (Fig. 1, circled 5) and a 28-kDa orotidine-5′-phosphate decarboxylase (ODCase) that decarboxylates OMP (Fig. 1, circled 6) forming UMP (38Suttle D.P. Bugg B.Y. Winkler J.K. Kanalas J.J. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 1754-1758Crossref PubMed Scopus (54) Google Scholar). Sedimentation analysis of mouse UMP synthase (39Traut T.W. Payne R.C. Biochemistry. 1980; 19: 6068-6074Crossref PubMed Scopus (21) Google Scholar) identified a monomeric and two dimeric species, 5.1 S and 5.6 S. The monomer lacks ODCase activity, and the 5.1 S dimer is only partially active. The formation of the fully active 5.6 S dimer is induced by the binding of OMP or nucleotide analogs. CTPSase (Fig. 1, circled 9) catalyzes the ATP-dependent transfer of the amide nitrogen of glutamine to the C-4 position of UTP to form CTP (40Zalkin H. Methods Enzymol. 1985; 113: 282-287Crossref PubMed Scopus (27) Google Scholar). Although there are no structural studies, sequence analysis revealed that CTPSase consists of two domains (Fig. 2): an amidotransferase domain (GLN) that hydrolyzes glutamine and an amidator domain (AMD) that catalyzes the ATP-dependent phosphorylation of UTP and its subsequent reaction with ammonia to form CTP. This reaction is the rate-limiting step in the formation of cytosine nucleotides and as such represents another important control locus in pyrimidine biosynthesis. Biochemical and microscopic studies showed that CAD is primarily cytosolic (35Carrey E.A. Dietz C. Glubb D.M. Loffler M. Lucocq J.M. Watson P.F. Reproduction. 2002; 123: 757-768Crossref PubMed Scopus (31) Google Scholar, 41Chaparian M.G. Evans D.R. FASEB J. 1988; 2: 2982-2989Crossref PubMed Scopus (15) Google Scholar) with a smaller fraction in the nucleus. In the cytosolic compartment, CAD and UMP synthase are localized around and outside the mitochondria, and CAD appears to be associated with the cytoskeleton. Mitochondria are known to be anchored to the cytoskeletal network, so an interesting possibility is that CAD binds to and translocates along the filament to the mitochondria where DHOdhase is located (Fig. 4, path A). The physical association of CAD with the mitochondria is an attractive idea because physiological the the formation of carbamoyl aspartate dihydroorotate R.I. M.E. J. Biol. Chem. 1980; Full Text PDF PubMed Google Scholar). CAD the mitochondria allow a more of dihydroorotate by DHOdhase and the of carbamoyl aspartate in the The role of CAD in the M.G. Evans D.R. FASEB J. 1988; 2: 2982-2989Crossref PubMed Scopus (15) Google Scholar). and J.A. J. 1998; PubMed Google Scholar) found that CAD is associated with the in cells, where it anchors the protein at sites of active the a CAD in of and an DHOase domain, found within the M. M. S. J.L. G. J. Biol. Chem. Full Text PDF PubMed Google Scholar). J. S. D. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), a a protein fusion protein has been into cells it to the of CAD in cells. These studies showed that CAD is localized in the in resting cells or the of the cell but that a is into the the S when the demand for pyrimidine nucleotides a The of CAD in the the site of RNA and synthesis to be an but the of DHOdhase within the mitochondria synthesized in the have to and into the mitochondria where it is to and orotate then the (Fig. 4, path This to the of CAD in the nucleus. Thus, the role of CAD it is that a role in the regulation of the pathway or that CAD has a in or cell that is to pyrimidine biosynthesis. The nucleotide are within narrow in resting cells but to in cells T.W. Mol. Cell. Biochem. 1994; 140: 1-22Crossref PubMed Scopus (1271) Google Scholar). in (7Fairbanks L.D. Bofill M. Ruckemann K. Simmonds H.A. J. Biol. Chem. 1995; 270: 29682-29689Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar). The increased demand for nucleotides is satisfied in large by of de novo pyrimidine biosynthesis as a of increased enzyme and control mechanisms. Gene gene is at both the and and is up-regulated when resting cells enter the G.N. Davidson J.N. 1988; 7: Google Scholar, G.N. Davidson J.N. 1988; PubMed Scopus Google Scholar, G.N. Cell 1988; PubMed Scopus Google Scholar). binding to an found to be for the in CAD gene that at the as cells the K.E. P.J. Mol. Cell. Biol. 1997; PubMed Scopus Google Scholar, S. M. I. S. PubMed Scopus Google Scholar). the protein CAD is at the of by M. Cell Mol. Sci. PubMed Scopus Google Scholar). is known about the regulation of the the other pyrimidine biosynthetic enzymes, but there are many studies that that the of these enzymes, and in the are higher in tumors and other cells (8Weber G. Biochemistry (Mosc.). 2001; 66: 1164-1173Crossref PubMed Scopus (20) Google Scholar). The of cell growth probably the of CAD and other enzymes and the of de novo pyrimidine biosynthesis, but the changes in flux through the pathway control by and the activity of CPSase activity of CAD is the major locus of control of de novo pyrimidine biosynthesis (1Jones M.E. Annu. Rev. Biochem. 1980; 49: 253-279Crossref PubMed Scopus (535) Google Scholar). The enzyme is subject to by the end UTP and is allosterically activated by a UMP synthase substrate (Fig. is a and pyrimidine and because it is a substrate in the first step of biosynthesis. The bind to a regulatory (Fig. at the end of CPS.B in CAD H.I. Evans D.R. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar) and other CPSases J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. V. A. Lusty C.J. Rubio V. Biochemistry. 1999; PubMed Scopus Google Scholar). studies (1Jones M.E. Annu. Rev. Biochem. 1980; 49: 253-279Crossref PubMed Scopus (535) Google Scholar) that regulation of CAD the of pyrimidine biosynthesis in and of the UTP and PRPP in cells, and on the activity of the pyrimidine biosynthetic pathway that with the of these on the CAD CPSase A is by a recent analysis J.M. J. Davidson J.N. J. Mol. Biol. 1999; PubMed Scopus Google Scholar) that the of in UTP of CAD the pyrimidine to the precursors for E.A. D.G. Hardie D.G. J. 1985; PubMed Scopus Google Scholar) made the important discovery that protein A CAD at two sites (Fig. 2). of in the regulatory (Fig. 2) UTP E.A. D.G. Hardie D.G. J. 1985; PubMed Scopus Google Scholar) and the of the enzyme for PRPP H.I. L. Evans D.R. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). The of be to pyrimidine biosynthesis and allow of the nucleotide for cell be partially by the of PRPP into the growth state-dependent regulation of the pathway by the discovery H.I. P. M. E. Evans D.R. 2000; PubMed Scopus Google Scholar) that growth the via the of a single in the CPS.A domain of CAD (Fig. 2). This has no on CAD activity but UTP from an to a and PRPP changes in the be to the flux through the pyrimidine a (Fig. in the found to be both in and in Evans D.R. H.I. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar), in a in CPSase activity and of the CTP synthase activity the relative size of the UTP and CTP and the of pyrimidine and The enzyme is allosterically activated by and is by the end CTP (Fig. 1). The enzyme is by both and protein D.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The enzyme is a but the binding of the UTP and the formation of the active In contrast, the enzyme is an dimer that is known about the mammalian CTPSase is regulated by and CTP L. Adv. Med. Biol. 1998; PubMed Scopus Google Scholar), but or it is by phosphorylation is of the de low of pyrimidine biosynthesis is to resting cells. The activity of the pathway when cells enter the growth and then to as the Evans D.R. H.I. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The to growth is associated with a large in activity and the phosphorylation of the CAD site a UTP of CAD is and PRPP activation changes in the that can for the of pyrimidine biosynthesis. the and growth is and there is a in phosphorylation of The to PRPP and the activity of the pyrimidine biosynthetic pathway is The changes in CAD phosphorylation with the of the pathway as the cells S and are at the as pyrimidine biosynthesis is J. S. D. H. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The of of pyrimidine biosynthesis in cells, has been to the activity that leads to phosphorylation of the CAD site and a of phosphorylation of CAD S. H. J. PubMed Scopus (35) Google Scholar). These are consistent with a (Fig. 5) in which the of pyrimidine biosynthesis is in resting cells by UTP the cells enter the the phosphorylation of CAD as a and the nucleotide to CAD activity is primarily by which S as a of the and The steps in pyrimidine biosynthesis in all with the monofunctional bacterial proteins, the enzymes have a more complex structural and a more of The pathway is subject to diverse regulatory mechanisms including and and changes in the and interplay of these controls in the cell a