Carbocyclic glycinamide ribonucleotide is a substrate for glycinamide ribonucleotide transformylase

The carbocyclic analog of glycinamide ribonucleotide has been synthesized from the racemic parent trihydroxy cyclopentyl amine (B.L. Kam and N.J. Oppenheimer (1981) J. Org. Chem. 46, 3268-3272). This analog was accepted as a substrate (Km = 18 microM, Vmax = 0.23 mM/min) by mammalian glycinamide ribonucleotide transformylase (EC 2.1.2.2) with an efficiency comparable to that of the natural substrate glycinamide ribonucleotide (Km = 10 microM, Vmax = 0.27 mM/min). For each molecule of 10-formyl-5,8-dideazafolate cosubstrate consumed, 0.92 molecule of N-formyl carbocyclic glycinamide ribonucleotide was produced in the enzymatic reaction, indicating a 1:1 stoichiometry. These studies afford the first alternate nucleotide substrate for glycinamide ribonucleotide transformylase and suggest that the ribose ring oxygen of glycinamide ribonucleotide is not critical for enzyme recognition and binding.     

Preliminary crystallographic investigations of glycinamide ribonucleotide transformylase

Crystals of glycinamide ribonucleotide transformylase have been grown from 0.4 to 1 M ammonium sulfate, 0.6 to 1 M sodium-potassium phosphate, or 0.65 to 1 M citrate in the pH range 4.5-7.0. The single crystals display variable morphology with varying pH. The crystals belong to the orthorhombic space group C222 with cell dimensions a = 141.4 A, b = 98.2 A, c = 103.5 A. Co-crystals have also been obtained in the presence of the inhibitor 5,8-dideazafolate (KI = 18 microM) under similar crystallization conditions. Crystals of a chemically modified enzyme, iodinated at Cys-21, were grown under similar conditions within the pH range 6.5-7.0. These crystals are isomorphous with the unmodified enzyme. Crystals suitable for high resolution (less than 2.5 A) x-ray diffraction studies have been obtained for each of the above.     

Molecular structure of Escherichia coli PurT-encoded glycinamide ribonucleotide transformylase

In Escherichia coli, the PurT-encoded glycinamide ribonucleotide transformylase, or PurT transformylase, catalyzes an alternative formylation of glycinamide ribonucleotide (GAR) in the de novo pathway for purine biosynthesis. On the basis of amino acid sequence analyses, it is known that the PurT transformylase belongs to the ATP-grasp superfamily of proteins. The common theme among members of this superfamily is a catalytic reaction mechanism that requires ATP and proceeds through an acyl phosphate intermediate. All of the enzymes belonging to the ATP-grasp superfamily are composed of three structural motifs, termed the A-, B-, and C-domains, and in each case, the ATP is wedged between the B- and C-domains. Here we describe two high-resolution X-ray crystallographic structures of PurT transformylase from E. coli: one form complexed with the nonhydrolyzable ATP analogue AMPPNP and the second with bound AMPPNP and GAR. The latter structure is of special significance because it represents the first ternary complex to be determined for a member of the ATP-grasp superfamily involved in purine biosynthesis and as such provides new information about the active site region involved in ribonucleotide binding. Specifically in PurT transformylase, the GAR substrate is anchored to the protein via Glu 82, Asp 286, Lys 355, Arg 362, and Arg 363. Key amino acid side chains involved in binding the AMPPNP to the enzyme include Arg 114, Lys 155, Glu 195, Glu 203, and Glu 267. Strikingly, the amino group of GAR that is formylated during the reaction lies at 2.8 A from one of the gamma-phosphoryl oxygens of the AMPPNP.     

Mammalian glycinamide ribonucleotide transformylase: purification and some properties

Glycinamide ribonucleotide transformylase, the first of the two formyl group transferases of de novo purine biosynthesis requiring 10-formyltetrahydrofolate, has been purified 1500-fold, nearly to homogeneity, from the murine lymphoma cell line L5178Y. Purification of the enzyme was facilitated by the use of a gelatin protease "affinity" resin. This mammalian enzyme is a monomer of approximate Mr 110 000. The kinetic studies are consistent with a sequential reaction mechanism and yield Michaelis constants of 0.4 mM for the substrate, glycinamide ribonucleotide, and 0.25 microM for the cofactor analogue 10-formyl-5,8-dideazafolate. A minimum Vmax of 2 mumol/(min . mg) was obtained for the purified enzyme, from which a turnover number of 4 s-1 was calculated.     

Isolation of a multifunctional protein with aminoimidazole ribonucleotide synthetase, glycinamide ribonucleotide synthetase, and glycinamide ribonucleotide transformylase activities: characterization of aminoimidazole ribonucleotide synthetase

5-Aminoimidazole ribonucleotide (AIR) synthetase, glycinamide ribonucleotide (GAR) synthetase, and GAR transformylase activities from chicken liver exist on a single polypeptide of Mr 110,000 [Daubner, C. S., Schrimsher, J. L., Schendel, F. J., Young, M., Henikoff, S., Patterson, D., Stubbe, J., & Benkovic, S. J. (1985) Biochemistry 24, 7059-7062]. Details of copurification of these three activities through four chromatographic steps are reported. The ratios of these activities remain constant throughout the purification. AIR synthetase has an absolute requirement for K+ for activity and under these conditions has apparent molecular weights of 330,000, determined by Sephadex G-200 chromatography, and 133,000, determined by sucrose density gradient ultracentrifugation. Incubation of 18O-labeled formylglycinamidine ribonucleotide (FGAM) with AIR synthetase results in stoichiometric production of AIR, ADP, and [18O]Pi. NMR spectra of beta-FGAM and beta-AIR are reported.     

The apo and ternary complex structures of a chemotherapeutic target: human glycinamide ribonucleotide transformylase

Glycinamide ribonucleotide transformylase (GART; 10-formyltetrahydrofolate:5'-phosphoribosylglycinamide formyltransferase, EC 2.1.2.2), an essential enzyme in de novo purine biosynthesis, has been a chemotherapeutic target for several decades. The three-dimensional structure of the GART domain from the human trifunctional enzyme has been solved by X-ray crystallography. Models of the apoenzyme, and a ternary complex with the 10-formyl-5,8-dideazafolate cosubstrate and a glycinamide ribonucleotide analogue, hydroxyacetamide ribonucleotide [alpha,beta-N-(hydroxyacetyl)-d-ribofuranosylamine], are reported to 2.2 and 2.07 A, respectively. The model of the apoenzyme represents the first structure of GART, from any source, with a completely unoccupied substrate and cosubstrate site, while the ternary complex is the first structure of the human GART domain that is bound at both the substrate and cosubstrate sites. A comparison of the two models therefore reveals subtle structural differences that reflect substrate and cosubstrate binding effects and implies roles for the invariant residues Gly 133, Gly 146, and His 137. Preactivation of the DDF formyl group appears to be key for catalysis, and structural flexibility of the active end of the substrate may facilitate nucleophilic attack. A change in pH, rather than folate binding, correlates with movement of the folate binding loop, whereas the phosphate binding loop position does not vary with pH. The electrostatic surface potentials of the human GART domain and Escherichia coli enzyme explain differences in the binding affinity of polyglutamylated folates, and these differences have implications to future chemotherapeutic agent design.     

A multifunctional protein possessing glycinamide ribonucleotide synthetase, glycinamide ribonucleotide transformylase, and aminoimidazole ribonucleotide synthetase activities in de novo purine biosynthesis

Three activities on the pathway of purine biosynthesis de novo in chicken liver, namely, glycinamide ribonucleotide synthetase, glycinamide ribonucleotide transformylase, and aminoimidazole ribonucleotide synthetase, have been found to reside on the same polypeptide chain. Three diverse purification schemes, utilizing three different affinity resins, give rise to the same protein since the final material has identical specific activities for all three enzymatic reactions and a molecular weight on sodium dodecyl sulfate gels of about 110 000. A single antibody preparation precipitates all three activities and binds to the multifunctional protein obtained by two methods in Western blots. Partial chymotryptic digestion of the purified protein gives rise to two fragments, one possessing glycinamide ribonucleotide synthetase activity and the other containing glycinamide ribonucleotide transformylase activity.     

Evidence for a novel glycinamide ribonucleotide transformylase in Escherichia coli.

We demonstrate here that Escherichia coli synthesizes two different glycinamide ribonucleotide (GAR) transformylases, both catalyzing the third step in the purine biosynthetic pathway. One is coded for by the previously described purN gene (GAR transformylase N), and a second, hitherto unknown, enzyme is encoded by the purT gene (GAR transformylase T). Mutants defective in the synthesis of the purN- and the purT-encoded enzymes were isolated. Only strains defective in both genes require an exogenous purine source for growth. Our results suggest that both enzymes may function to ensure normal purine biosynthesis. Determination of GAR transformylase T activity in vitro required formate as the C1 donor. Growth of purN mutants was inhibited by glycine. Under these conditions GAR accumulated. Addition of purine compounds or formate prevented growth inhibition. The regulation of the level of GAR transformylase T is controlled by the PurR protein and hypoxanthine.     

Human glycinamide ribonucleotide transformylase: active site mutants as mechanistic probes

Human glycinamide ribonucleotide transformylase (GART) (EC 2.1.2.2) is a validated target for cancer chemotherapy, but mechanistic studies of this therapeutically important enzyme are limited. Site-directed mutagenesis, initial velocity studies, pH-rate studies, and substrate binding studies have been employed to probe the role of the strictly conserved active site residues, N106, H108, and D144, and the semiconserved K170 in substrate binding and catalysis. Only two conservative substitutions, N106Q and K170R, resulted in catalytically active enzymes, and these active mutant enzymes gave pH-rate profiles and a steady-state kinetic mechanism essentially identical to those of the native enzyme. All inactive mutants were able to bind both substrates, ruling out disrupted formation of the ternary complex as the source of inactivity. Differences between human and Escherichia coli GART, previously used as a model for the human enzyme, were evident.     

Active-site mapping and site-specific mutagenesis of glycinamide ribonucleotide transformylase from Escherichia coli

The affinity reagent N10-(bromoacetyl)-5,8-dideazafolate has previously been shown to inactivate glycinamide ribonucleotide transformylase (EC 2.1.2.2) from Escherichia coli in an active-site-directed manner with a 1:1 stoichiometry [Inglese et al. (1990) Biochemistry 29, 1436-1443]. After a series of mild proteolytic digestions, the dideazafolate label was localized to an active-site peptide attached by an ester linkage to the highly conserved residue Asp 144. Subsequent site-specific mutagenesis of Asp 144 to Asn 144 resulted in a catalytically inactive enzyme that retained the ability to bind substrates and inhibitors. The Asn 144 mutant could be further labeled with the affinity reagent in an active-site-directed stoichiometric fashion; however, the site of modification in this case was His 119. These results imply that Asp 144 may function as a general base within the catalytic center of the transformylase and is in close proximity to His 119 in the folded protein.     

Subcloning, characterization, and affinity labeling of Escherichia coli glycinamide ribonucleotide transformylase

Glycinamide ribonucleotide transformylase (GAR TFase; EC 2.1.2.2) has been purified 70-fold to apparent homogeneity from Escherichia coli harboring an expression vector encoding the purN gene product, GAR TFase. The protein is a monomer of Mr 23,241 and catalyzes a single reaction. Steady-state kinetic parameters for the enzyme have been obtained. The structural requirements for cofactor utilization have been investigated and found to parallel those of the multifunctional avian enzyme. The enzyme was inactivated with the affinity label N10-(bromoacetyl)-5,8-dideazafolate in a stoichiometric and active-site-specific manner. The ionization state of the cofactor analogue in the enzyme-cofactor complex appears to require the dissociation of the proton at N3 of the pyrimidine within the complex.     

PurT-encoded glycinamide ribonucleotide transformylase. Accommodation of adenosine nucleotide analogs within the active site

PurT-encoded glycinamide ribonucleotide transformylase, or PurT transformylase, functions in purine biosynthesis by catalyzing the formylation of glycinamide ribonucleotide through a catalytic mechanism requiring Mg(2+)ATP and formate. From previous x-ray diffraction analyses, it has been demonstrated that PurT transformylase from Escherichia coli belongs to the ATP-grasp superfamily of enzymes, which are characterized by three structural motifs referred to as the A-, B-, and C-domains. In all of the ATP-grasp enzymes studied to date, the adenosine nucleotide ligands are invariably wedged between the B- and C-domains, and in some cases, such as biotin carboxylase and carbamoyl phosphate synthetase, the B-domains move significantly upon nucleotide binding. Here we present a systematic and high-resolution structural investigation of PurT transformylase complexed with various adenosine nucleotides or nucleotide analogs including Mg(2+)ATP, Mg(2+)-5'-adenylylimidodiphosphate, Mg(2+)-beta,gamma-methyleneadenosine 5'-triphosphate, Mg(2+)ATPgammaS, or Mg(2+)ADP. Taken together, these studies indicate that the conformation of the so-called "T-loop," delineated by Lys-155 to Gln-165, is highly sensitive to the chemical identity of the nucleotide situated in the binding pocket. This sensitivity to nucleotide identity is in sharp contrast to that observed for the "P-loop"-containing enzymes, in which the conformation of the binding motif is virtually unchanged in the presence or absence of nucleotides.     

Assignment of a third purine biosynthetic gene (glycinamide ribonucleotide transformylase) to human chromosome 21.

Using a series of human-hamster hybrid cell lines, a gene coding for glycinamide ribonucleotide transformylase was mapped to human chromosome 21. The availability of hybrids containing only portions of chromosome 21 allowed the gene to be assigned to the region between the q11.2 and the q22.2 bands, inclusive. Differentiation of human and hamster glycinamide ribonucleotide transformylase was accomplished via an immunoprecipitation assay that employed a polyclonal antibody raised against the human enzyme.     

Mammalian glycinamide ribonucleotide transformylase. Kinetic mechanism and associated de novo purine biosynthetic activities

Glycinamide ribonucleotide transformylase catalyzes the conversion of glycinamide ribonucleotide and 10-formyltetrahydrofolate to formylglycinamide ribonucleotide and tetrahydrofolate. The enzyme purified from the murine lymphoma cell line L5178Y also catalyzes two other de novo purine biosynthetic activities, glycinamide ribonucleotide synthetase and aminoimidazole ribonucleotide synthetase. The transformylase reaction shows a 1:1 stoichiometry for substrate utilization and an optimum rate between pH 7.9 and 8.3. Initial velocity and dead-end inhibition patterns indicate that the kinetic mechanism of the transformylation reaction is ordered-sequential, with 10-formyltetrahydrofolate binding first. alpha, beta-Hydroxyacetamide ribonucleotide (alpha, beta-N-(hydroxyacetyl)-D-ribofuranosylamine) is shown to be an inhibitor of the transformylase, competitive against glycinamide ribonucleotide.     

An antibody probe to determine the native species of glycinamide ribonucleotide transformylase in chicken liver

Antibody probes of Western blots [Renart, J., Reiser, J., & Stark, G. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 3116] of chicken liver homogenates under various conditions revealed that glycinamide ribonucleotide transformylase can be rapidly proteolyzed in such homogenates. These findings, along with molecular weight measurements by ultracentrifugation, identify the true form of glycinamide ribonucleotide transformylase as a monomeric protein of 117000 daltons. This protein has been purified 400-fold in 44% yield from chicken liver in one step on an affinity column of 10-formyl-5,8-dideazafolate-Sepharose. Native glycinamide ribonucleotide transformylase retains full activity after proteolytic cleavage to a form (Mr 55000) similar to fragments seen in the Western blot of the homogenates. This phenomenon may be responsible for the previous identification of glycinamide ribonucleotide (GAR) transformylase as a dimer of 55000-dalton subunits. Similar analyses using antibodies to 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase [Mueller, W. T., & Benkovic, S. J. (1981) Biochemistry 20, 337] and trifunctional enzyme [Smith, G. K., Mueller, W. T., Wasserman, G. F., Taylor, W. D., & Benkovic, S. J. (1980) Biochemistry 19, 4313] confirm that these two proteins were isolated in their native forms.     

Effect of nitrous oxide-induced inactivation of vitamin B12 on glycinamide ribonucleotide transformylase and 5-amino-4-imidazole carboxamide transformylase

Exposure to nitrous oxide (N2O) in vivo is accompanied by oxidation of cob[I]-alamin to the inactive cob[III]alamin [1]. There is loss of methionine synthetase activity [2] and evidence of depressed supply of single carbon units at the formate level of oxidation [3,4,5]. We measured the effect of inactivation of B12 on the folate-dependent transformylases concerned in purine synthesis. After 24 h exposure to N2O there was a significant fall in glycinamide ribonucleotide transformylase (EC 2.1.2.2) and a significant increase in 5-amino-4-imidazole carboxamide transformylase (EC 2.1.2.3).     

In silico analysis of glycinamide ribonucleotide transformylase inhibition by PY873, PY899 and DIA

In humans, purine de novo synthesis pathway consists of multi-functional enzymes. Nucleotide metabolism enzymes are potential drug targets for treating cancer and autoimmune diseases. Glycinamide ribonucleotide transformylase (GART) is one of the most important trifunctional enzymes involved in purine synthesis. Previous studies have demonstrated the role of folate inhibitors against tumor activity. In this present study, three components of GART enzyme were targeted as receptor dataset and in silico analysis was carried out with folate ligand dataset. To accomplish the task, Autodock 4.2 was used for determining the docking compatibilities of ligand and receptor dataset. Taken together, it has been suggested that folate ligands could be potentially used as inhibitors of GART.     

Catalytic mechanism of Escherichia coli glycinamide ribonucleotide transformylase probed by site-directed mutagenesis and pH-dependent studies.

Site-directed mutagenesis followed by studies of the pH dependence of the kinetic parameters of the mutants has been used to probe the role of the active site residues and loops in catalysis by glycinamide ribonucleotide transformylase (EC 2.1.2.2). The analysis of the mutants of the strictly conserved active site residues, His108 and Asp144, revealed that His108 acts in a salt bridge with Asp144 as a general acid catalyst with a pK(a) value of 9.7. Asp144 also plays a key role in the preparation of the active site geometry for catalysis. The rate-limiting step in the pH range of 6-10 appears to be the catalytic steps involving tetrahedral intermediates, supported by the observation of a pL (L being H or D)-independent solvent deuterium isotope effect of 2. The ionization of the amino group of glycinamide ribonucleotide both as a free and as a bound form dominates the kinetic behavior at low pH. The analysis of a mutation, H121Q, within the loop spanning amino acids 111-131 suggests the closure of the loop is involved in the binding of the substrate. The kinetic behavior parallels pH effects revealed by a series of X-ray crystallographic structures of the apoenzyme and inhibitor-bound enzyme [Su, Y., Yamashita, M. M., Greasley, S. E. , Mullen, C. A., Shim, J. H., Jennings, P. A., Benkovic, S. J., and Wilson, I. A. (1998) J. Mol. Biol. 281, 485-499], permitting a more exact formulation of the probable catalytic mechanism.     

De novo purine nucleotide biosynthesis: cloning of human and avian cDNAs encoding the trifunctional glycinamide ribonucleotide synthetase-aminoimidazole ribonucleotide synthetase-glycinamide ribonucleotide transformylase by functional complementation in E. coli.

The trifunctional enzyme encoding glycinamide ribonucleotide synthetase (GARS)-aminoimidazole ribonucleotide synthetase (AIRS)-glycinamide ribonucleotide transformylase (GART) was cloned by functional complementation of an E. coli mutant using an avian liver cDNA expression library. In E. coli, genes encoding these separate activities (purD, purM, and purN, respectively) produce three proteins. The avian cDNA, in contrast, encodes a single polypeptide with all three enzyme activities. Using the avian DNA as a probe, a cDNA encoding the complete coding sequence of the trifunctional human enzyme was also isolated and sequenced. The deduced amino acid sequence of the human and avian polyproteins show extensive sequence homologies to the bacterial purD, purM, and purN encoded proteins. Avian and human liver RNAs appear to encode both a trifunctional enzyme (G-ARS-AIRS-GART) as well as an RNA which encodes only GARS. The trifunctional protein has been implicated in the pathology of Downs Syndrome and molecular tools are now available to explore this hypothesis. Initial efforts to compare the expression of GARS-AIRS-GART between a normal fibroblast cell line and a Downs Syndrome cell line indicate that the levels of RNA are similar.     

A quantum chemical study of the water-assisted mechanism in one-carbon unit transfer reaction catalyzed by glycinamide ribonucleotide transformylase

A theoretical study for the water-assisted mechanism in one-carbon unit transfer reaction catalyzed by glycinamide ribonucleotide transformylase (GAR Tfase) is investigated in which the proton transfers in an indirect way and the energy barrier for each transition state has been lowered about 80–100 kJ/mol when compared with the corresponding one in a no-water-involved mechanism. There are two possible pathways in each mechanism: one is concerted and the other is stepwise. Our results have verified the presumption from experiments that one water molecule can assist to achieve the whole reaction. Because the addition of this water molecule in the transition states can relax the strong strain in the unstable system and greatly lowered the energy barrier. The water-assisted paths are preferable to the no-water-involved ones and the bulk solvent effect of water is also discussed.