The purine ribonucleotide inosine 5'-monophosphate (IMP) is assembled on 5-phospho-alpha-D-ribose 1-diphosphate (PRPP), with atoms derived from aspartate, glutamine, glycine, N10-formyl-tetrahydrofolate, and carbon dioxide. Although several of the individual reactions in this sequence are reversible, as indicated by the double-headed arrows in the diagram, other irreversible steps drive the pathway in the direction of IMP synthesis in the normal cell. All of these reactions are thus annotated here only in the direction of IMP synthesis. Guanosine 5'-monophosphate (GMP) and adenosine 5'-monophosphate (AMP) are synthesized from IMP (Zalkin & Dixon 1992).
The pyrimidine orotate (orotic acid) is synthesized in a sequence of four reactions, deriving its atoms from glutamine, bicarbonate, and aspartate. A single multifunctional cytosolic enzyme catalyzes the first three of these reactions, while the last one is catalyzed by an enzyme associated with the inner mitochondrial membrane. In two further reactions, catalyzed by a bifunctional cytosolic enzyme, orotate reacts with 1-phosphoribosyl 5-pyrophosphate (PRPP) to yield orotidine 5'-monophosphate, which is decarboxylated to yield uridine 5'-monophosphate (UMP). While several individual reactions in this pathway are reversible, other irreversible reactions drive the pathway in the direction of UMP biosynthesis in the normal cell. All reactions are thus annotated here only in the forward direction.<p>This pathway has been most extensively analyzed at the genetic and biochemical level in hamster cell lines. All three enzymes have also been purified from human sources, however, and the key features of these reactions have been confirmed from studies of this human material (Jones 1980).<p>All other pyrimidines are synthesized from UMP. The reactions annotated here, catalyzed by dCMP deaminase and dUTP diphosphatase yield dUMP, which in turn is converted to TMP by thymidylate synthase.
View original pathway at:Reactome.</div>
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Iwahana H, Fujimura M, Ii S, Kondo M, Moritani M, Takahashi Y, Yamaoka T, Yoshimoto K, Itakura M.; ''Molecular cloning of a human cDNA encoding a trifunctional enzyme of carbamoyl-phosphate synthetase-aspartate transcarbamoylase-dihydroorotase in de Novo pyrimidine synthesis.''; PubMedEurope PMCScholia
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Suchi M, Mizuno H, Kawai Y, Tsuboi T, Sumi S, Okajima K, Hodgson ME, Ogawa H, Wada Y.; ''Molecular cloning of the human UMP synthase gene and characterization of point mutations in two hereditary orotic aciduria families.''; PubMedEurope PMCScholia
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Barnes TS, Bleskan JH, Hart IM, Walton KA, Barton JW, Patterson D.; ''Purification of, generation of monoclonal antibodies to, and mapping of phosphoribosyl N-formylglycinamide amidotransferase.''; PubMedEurope PMCScholia
Bader B, Knecht W, Fries M, Löffler M.; ''Expression, purification, and characterization of histidine-tagged rat and human flavoenzyme dihydroorotate dehydrogenase.''; PubMedEurope PMCScholia
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Nakamura J, Lou L.; ''Biochemical characterization of human GMP synthetase.''; PubMedEurope PMCScholia
Ito K, Uchino H.; ''Control of pyrimidine biosynthesis in human lymphocytes. Simultaneous increase in activities of glutamine-utilizing carbamyl phosphate synthetase and aspartate transcarbamylase in phytohemagglutinin-stimulated human peripheral lymphocytes and their enzyme co-purification.''; PubMedEurope PMCScholia
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Vethe NT, Bremer S, Bergan S.; ''IMP dehydrogenase basal activity in MOLT-4 human leukaemia cells is altered by mycophenolic acid and 6-thioguanosine.''; PubMedEurope PMCScholia
Mori K, Ikeda M, Ariumi Y, Dansako H, Wakita T, Kato N.; ''Mechanism of action of ribavirin in a novel hepatitis C virus replication cell system.''; PubMedEurope PMCScholia
Gooljarsingh LT, Ramcharan J, Gilroy S, Benkovic SJ.; ''Localization of GAR transformylase in Escherichia coli and mammalian cells.''; PubMedEurope PMCScholia
Holmes EW, Wyngaarden JB, Kelley WN.; ''Human glutamine phosphoribosylpyrophosphate amidotransferase. Two molecular forms interconvertible by purine ribonucleotides and phosphoribosylpyrophosphate.''; PubMedEurope PMCScholia
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Yablonski MJ, Pasek DA, Han BD, Jones ME, Traut TW.; ''Intrinsic activity and stability of bifunctional human UMP synthase and its two separate catalytic domains, orotate phosphoribosyltransferase and orotidine-5'-phosphate decarboxylase.''; PubMedEurope PMCScholia
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Cytosolic AMP, GMP, and IMP stimulate the association of phosphoribosyl pyrophosphate amidotransferase (PPAT) dimers to form tetramers (Holmes et al. 1973a,b).
Two isoforms of adenylosuccinate synthetase, ADSS and ADSSL1, catalyze the reaction of IMP, aspartate, and GTP to form adenylosuccinate, GDP, and orthophosphate (Powell et al. 1992; Sun et al. 2005). ADSS is a homotetramer (unpublished crystallographic data - PDB 2V40) and ADSSL1 is inferred to be a tetramer likewise.
Inorganic pyrophosphate (PPi) is continuously produced as a result of ATP-utilising biosynthesis of protein, RNA, and DNA. Inorganic pyrophosphatase (PPase) catalyses the hydrolysis of PPi into two orthophosphates (Pi) thereby minimizing the cellular level of PPi and driving otherwise reversible reactions in the direction of PPi generation. Phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPP) is a dimeric protein, binding a Mg2+ in each subunit which can mediate the hydrolysis of PPi to 2xPi (Yokoi et al. 2003, Koike et al. 2006). LHPP is expressed in thyrocytes, located in the cytosol and nucleoplasm. In addition, LHPP is more prominently expressed in hyperfunctional states of the thyroid, such as in Graves disease and autonomously functional thyroid nodule (AFTN) (Koike et al. 2006).
The decarboxylation of orotidine 5'-monophosphate (OMP) to form uridine 5'-monophosphate (UMP) is catalyzed by the orotidine 5'-phosphate decarboxylase activity of the bifunctional "uridine monophosphate synthetase (orotate phosphoribosyl transferase and orotidine 5'-decarboxylase)" protein. While purified human protein has not been characterized in detail, the close similarity of the human gene to that encoding the well-studied hamster protein, and the demonstration that mutations in the human gene are associated with failure to convert orotate to UMP in vivo, provide convincing evidence that the human uridine monophosphate synthetase protein indeed catalyzes these two reactions (McClard et al. 1980; Suchi et al. 1997). The active form of the human protein is a dimer (Yablonski et al. 1996; Wittmann et al. 2008).
The synthesis of orotidine 5'-monophosphate (OMP) from orotate and 5-phospho-alpha-D-ribose 1-diphosphate (PRPP) is catalyzed by the orotate phosphoribosyltransferase activity of the bifunctional "uridine monophosphate synthetase (orotate phosphoribosyl transferase and orotidine 5'-decarboxylase)" protein. The reaction itself is freely reversible, but is pulled in the forward direction in vivo by the irreversible hydrolysis of pyrophosphate. While purified human protein has not been characterized in detail, the close similarity of the human gene to that encoding the well-studied hamster protein, and the demonstration that mutations in the human gene are associated with failure to convert orotate to UMP in vivo, provide convincing evidence that the human uridine monophosphate synthetase protein indeed catalyzes these two reactions (McClard et al. 1980; Suchi et al. 1997). The active form of the human protein is a dimer (Yablonski et al. 1996; Wittmann et al. 2008).
Dihydroorotate dehydrogenase catalyzes the oxidation of dihydroorotate to orotate (orotic acid). The enzyme is located in the inner mitochondrial membrane oriented so that cytosolic dihydroorotate molecules have access to it, and orotate is released into the cytosol. The reducing equivalents generated by the reaction are transferred by ubiquinone (Coenzyme Q) to the electron transport chain within the inner mitochondrial membrane. There is no evidence for the involvement of NAD+ as an acceptor of reducing equivalents in this reaction in mammalian cells (Jones 1980). In contrast, the reaction catalyzed by the enzyme isolated from the anaerobic bacterium Clostridium oroticum requires NAD+ as a cofactor but also proceeds strongly in the direction of dihydroorotate synthesis consistent with the greater electronegativity of NAD+ (Lieberman and Kornberg 1953).
Early studies of purified rat liver enzyme by Forman and Kennedy suggested the presence of flavin mononucleotide and iron-sulfur complexes as cofactors. More recent work by Bader, Beuneu, and their colleagues with recombinant human protein expressed in insect cells has confirmed the presence of flavin mononucleotide, at a stoichiometry of one molecule per molecule of apoenzyme but suggests that iron-sulfur complexes, if indeed they are involved in the oxidation and electron transport process, are not an integral part of the dihydroorotate dehydrogenase holoenzyme.
The synthesis of dihydroorotate from N-carbamoyl L-aspartate is catalyzed by the dihydroorotase activity of cytosolic trifunctional CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase) protein. This activity has not been directly demonstrated in experimental studies of the purified human protein, but has been inferred from the behavior of the purified hamster protein and the high degree of sequence similarity between the cloned hamster and human genes (Iwahana et al. 1996). Also on the basis of this similarity, the active human protein is annotated as a hexamer (Lee et al. 1985).
The synthesis of N-carbamoyl L-aspartate from carbamoyl phosphate and L-aspartate is catalyzed by the aspartate carbamoyltansferase activity of cytosolic trifunctional CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase) protein (Ito and Uchino 1973; Iwahana et al. 1996). The purified human protein is active in several different oligomerization states as is its Syrian hamster homologue. The most abundant form of the latter is a hexamer, and the active human protein is annotated as a hexamer by inference (Ito and Uchino 1973; Lee et al. 1985).
The synthesis of carbamoyl phosphate from glutamine, bicarbonate, and ATP is catalyzed by the carbamoyl-phosphate synthase (glutamine-hydrolyzing) activity of cytosolic trifunctional CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase) protein (Ito and Uchino 1973; Iwahana et al. 1996). The purified human protein is active in several different oligomerization states, as is its Syrian hamster homologue. The most abundant form of the latter is a hexamer, and the active human protein is annotated as a hexamer by inference (Ito and Uchino 1973; Lee et al. 1985).
Cytosolic GMP synthase (GMPS) catalyzes the reaction of xanthosine 5'-monophosphate (XMP), ATP, glutamine and water to form guanosine 5'-monophosphate (GMP), AMP, glutamate and pyrophosphate. GMPS is a monomer (Hirst et al. 1994; Nakamura and Lou 1995).
Two human isoenzymes, IMP dehydrogenase 1 and 2 (IMPDH1,2) catalyze the irreversible dehydrogenation of inosine 5'-monophosphate (IMP) to form xanthosine 5'-monophosphate (XMP). The active forms of both isoenzymes are homotetramers, and they are nearly identical in their catalytic efficiencies and their susceptibility to inhibition by XMP (Carr et al. 1993; Colby et al. 1999; Hager et al. 1995). Both enzymes occur as homotetramers (Colby et al. 1999 - IMPDH2; unpubliched data in PDB 1JCN IMPDH1). A variety of experiments suggest that IMPDH1 and 2 have distinct functions in vivo. While IMPDH1 is expressed at constant levels, IMPDH2 is expressed at elevated levels in tumor cells and in mitotic normal cells. In humans, heterozygosity for mutant forms of IMPDH1 is associated with a form of retinitis pigmentosa (Bowne et al. 2002). In laboratory mice, mutations that disrupt the homologue of IMPDH1 have no obvious effect at the level of the whole organism, while ones that disrupt IMPDH2 are lethal (Gu et al. 2003).
This reaction is the rate limiting step in the synthesis of guanosine 5'-monophosphate (GMP) from IMP, and GMP competitively inhibits the well-characterized bacterial IMP dehydrogenase enzyme. Evidence for an inhibitory effect of GMP on the human isoenzymes has not been reported. Rather, they appear to be inhibited by XMP; in addition, transcription of one or both IMP dehydrogenase mRNAs may be inhibited by high cellular GMP concentrations (Glesne et al. 1991).
The reversible synthesis of inosine 5'-monophosphate from 5'-phosphoribosyl-5-formaminoimidazole-4-carboxamide (FAICAR) is catalyzed by the IMP cyclohydrolase activity of the bifunctional 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase enzyme. This cytosolic protein occurs primarily as a dimer (Rayl et al. 1996; Vergis et al. 2001) and may further associate with other enzymes of de novo IMP biosynthesis under some metabolic conditions (An et al. 2008).
The irreversible transfer of a formyl group to 5'-phosphoribosyl-5-aminoimidazole-4-carboxamide (AICAR), to yield 5'-phosphoribosyl-5-formaminoimidazole-4-carboxamide (FAICAR), is catalyzed by the phosphoribosylaminoimidazolecarboxamide formyltransferase activity of the bifunctional 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase enzyme. This cytosolic protein occurs primarily as a dimer (Rayl et al. 1996; Vergis et al. 2001) and may further associate with other enzymes of de novo IMP biosynthesis under some metabolic conditions (An et al. 2008).
The reversible conversion of 5'-phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide (SAICAR) to 5'-phosphoribosyl-5-aminoimidazole-4-carboxamide (AICAR) and fumarate is catalyzed by adenylosuccinate lyase. The active form of this enzyme is a cytosolic tetramer (Stone et al. 1993), and fluoresence microscopy of cultured human cells suggests that it may associate with other enzymes of de novo IMP biosynthesis under some metabolic conditions (An et al. 2008). The enzyme also catalyzes the conversion of adenylosuccinate to adenosine 5'-monophosphate and fumarate. Humans lacking the enzyme accumulate dephosphorylated forms of both substrates, indicating that the enzyme mediates both reactions in vivo as well (Race et al. 2000).
The conversion of 5'-phosphoribosyl-5-aminoimidazole-4-carboxylate (CAIR) and aspartate to 5'-phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide (SAICAR), accompanied by the conversion of ATP to ADP and orthophosphate, is catalyzed by the phosphoribosylaminoimidazole succinocarboxamide synthetase domain of the bifunctional protein "phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase" (PAICS) (Schild et al. 1990). The enzyme is an octamer (Li et al. 2007); it may associate in the cytosol with other enzymes of de novo IMP biosynthesis under some metabolic conditions (An et al. 2008).
The reversible carboxylation of 5'-phosphoribosyl-5-aminoimidazole (AIR) to form 5'-phosphoribosyl-5-aminoimidazole-4-carboxylate (CAIR) is catalyzed by the phosphoribosylaminoimidazole carboxylase domain of the bifunctional protein "phosphoribosylaminoimidazole carboxylase, phosphoribosylaminoimidazole succinocarboxamide synthetase" (PAICS) (Schild et al. 1990). The enzyme is an octamer (Li et al. 2007); it may associate in the cytosol with other enzymes of de novo IMP biosynthesis under some metabolic conditions (An et al. 2008).
The irreversible synthesis of cytosolic 5'-phosphoribosyl-5-aminoimidazole (AIR) from 5'-phosphoribosylformylglycinamidine (FGAM), accompanied by the conversion of ATP to ADP and orthophosphate, is catalyzed by the phosphoribosylaminoimidazole synthetase domain of the trifunctional protein, "phosphoribosylglycinamide formyltransferase, phosphoribosylglycinamide synthetase, phosphoribosylaminoimidazole synthetase" (GART) (Aimi et al. 1990). The active form of the protein is cytosolic and may co-localize with other enzymes of de novo IMP biosynthesis under some metabolic conditions (Gooljarsingh et al. 2001; An et al. 2008).
The irreversible transfer of an amino group from L-glutamine to 5'-phosphoribosylformylglycinamide (FGAR), forming 5'-phosphoribosylformylglycinamidine (FGAM) and glutamate, accompanied by the conversion of ATP to ADP and orthophosphate, is catalyzed by phosphoribosylformylglycinamidine synthetase. The human enzyme has been purified and characterized biochemically (Barnes et al. 1994). Fluoresence microscopy studies of cultured human cells have shown that the enzyme is cytosolic and suggest that it may co-localize with other enzymes of de novo IMP biosynthesis under some metabolic conditions (An et al. 2008).
The irreversible transfer of a formyl group to cytosolic 5-phosphoribosylglycinamide (GAR) to form 5'-phosphoribosylformylglycinamide (FGAR) is catalyzed by the phosphoribosylglycinamide formyltransferase domain of the trifunctional protein, "phosphoribosylglycinamide formyltransferase, phosphoribosylglycinamide synthetase, phosphoribosylaminoimidazole synthetase" (GART) (Aimi et al. 1990; Zhang et al. 2002). Fluoresence microscopy studies of cultured human cells have shown that GART is cytosolic and suggest that it may co-localize with other enzymes of de novo IMP biosynthesis under some metabolic conditions (Gooljarsingh et al. 2001; An et al. 2008).
The synthesis of cytosolic 5-phosphoribosylglycinamide from 5-phosphoribosylamine and glycine, accompanied by the conversion of ATP to ADP and orthophosphate, is catalyzed by the phosphoribosylglycinamide synthetase domain of the trifunctional protein, "phosphoribosylglycinamide formyltransferase, phosphoribosylglycinamide synthetase, phosphoribosylaminoimidazole synthetase" (GART) (Aimi et al. 1990). The active form of the protein is cytosolic (Gooljarsingh et al. 2001; An et al. 2008).
Cytosolic PPAT (phosphoribosyl pyrophosphate amidotransferase) catalyzes the reaction of 5-phospho-alpha-D-ribose 1-diphosphate (PRPP), water, and L-glutamine to form 5-phosphoribosylamine, L-glutamate, and pyrophosphate. This event is the committed step in de novo purine synthesis. The reaction itself is reversible, but it is pulled strongly in the direction of 5'-phosphoribosylamine synthesis by the irreversible hydrolysis of the pyrophosphate that is also formed in the reaction. Fluoresence microscopy studies of cultured human cells have shown that PPAT is cytosolic and suggest that it may co-localize with other enzymes of de novo IMP biosynthesis under some metabolic conditions (An et al. 2008). The PPAT enzyme is inferred to be an iron-sulfur protein, like its well-characterized B. subtilis homologue, because incubation of purified enzyme with molecular oxygen or chelating agents inactivates it, and activity can be restored by incubation with ferrous iron and inorganic sulfide. The stoichiometry of the iron-sulfur moiety and its role in enzyme activity remain unknown (Itakura and Holmes 1979). The fully active form of the enzyme is a dimer, which can associate further to form a tetramer with sharply reduced activity (Holmes et al. 1973b; Iwahana et al. 1993). Interaction of the enzyme with inosine 5'-monophosphate (IMP), guanosine 5'-monophosphate (GMP), and adenosine 5'-monophosphate (AMP), end products of de novo purine biosynthesis, favors tetramer formation, while interaction with 5-phospho-alpha-D-ribose 1-diphosphate (PRPP), a required substrate, favors formation of the active dimer. Kinetic studies suggest that the enzyme's binding site for GMP and IMP is separate from its AMP binding site (Holmes et al. 1973a).
The irreversible conversion of adenylosuccinate to adenosine 5'-monophosphate and fumarate is catalyzed by adenylosuccinate lyase. The active form of this enzyme is a cytosolic tetramer (Stone et al. 1993). The enzyme also catalyzes the conversion of 5'-phosphoribosyl-5-aminoimidazole-4-N-succinocarboxamide (SAICAR) to 5'-phosphoribosyl-5-aminoimidazole-4-carboxamide (AICAR) and fumarate. Humans lacking the enzyme accumulate dephosphorylated forms of both substrates, indicating that the enzyme mediates both reactions in vivo as well (Race et al. 2000).
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dehydrogenase
holoenzymeAnnotated Interactions
Early studies of purified rat liver enzyme by Forman and Kennedy suggested the presence of flavin mononucleotide and iron-sulfur complexes as cofactors. More recent work by Bader, Beuneu, and their colleagues with recombinant human protein expressed in insect cells has confirmed the presence of flavin mononucleotide, at a stoichiometry of one molecule per molecule of apoenzyme but suggests that iron-sulfur complexes, if indeed they are involved in the oxidation and electron transport process, are not an integral part of the dihydroorotate dehydrogenase holoenzyme.
This reaction is the rate limiting step in the synthesis of guanosine 5'-monophosphate (GMP) from IMP, and GMP competitively inhibits the well-characterized bacterial IMP dehydrogenase enzyme. Evidence for an inhibitory effect of GMP on the human isoenzymes has not been reported. Rather, they appear to be inhibited by XMP; in addition, transcription of one or both IMP dehydrogenase mRNAs may be inhibited by high cellular GMP concentrations (Glesne et al. 1991).
dehydrogenase
holoenzyme