Publications

• Biosynthesis of the purines. XIV. Conversion of (alpha-N-formyl) glycinamide ribotide to (alpha-N-formyl) glycinamidine ribotide; purification and requirements of the enzyme system.

  MELNICK I., BUCHANAN J.M.

  J Biol Chem 225:157-162(1957) [PubMed] [EuropePMC]

• Formylglycinamide ribonucleotide synthetase from Escherichia coli: cloning, sequencing, overproduction, isolation, and characterization.

  Schendel F.J., Mueller E., Stubbe J., Shiau A., Smith J.M.

  The purL gene of Escherichia coli encoding the enzyme formylglycinamidine ribonucleotide (FGAM) synthetase which catalyzes the conversion of formylglycinamide ribonucleotide (FGAR), glutamine, and MgATP to FGAM, glutamate, ADP, and Pi has been cloned and sequenced. The mature protein, as deduced b ... >> More

  The purL gene of Escherichia coli encoding the enzyme formylglycinamidine ribonucleotide (FGAM) synthetase which catalyzes the conversion of formylglycinamide ribonucleotide (FGAR), glutamine, and MgATP to FGAM, glutamate, ADP, and Pi has been cloned and sequenced. The mature protein, as deduced by the structural gene sequence, contains 1628 amino acids and has a calculated Mr of 141,418. Comparison of the purL control region to other pur loci control regions reveals a common region of dyad symmetry which may be the binding site for the "putative" repressor protein. Construction of an overproducing strain permitted purification of the protein to homogeneity. N-Terminal sequence analysis and comparison of glutamine binding domain sequences (Ebbole & Zalkin, 1987) confirm the amino acid sequence deduced from the gene sequence. The purified protein exhibits glutaminase activity of 0.02% the normal turnover, and NH3 can replace glutamine as a nitrogen donor with a Km = 1 M and a turnover of 3 min-1 (2% glutamine turnover). The enzyme forms an isolable (1:1) complex with glutamine: t1/2 is 22 min at 4 degrees C. This isolated complex is not chemically competent to complete turnover when FGAR and ATP are added, demonstrating that ammonia and glutamine are not covalently bound as a thiohemiaminal available to complete the chemical conversion to FGAM. hydroxylamine trapping experiments indicate that glutamine is bound covalently to the enzyme as a thiol ester. Initial velocity and dead-end inhibition kinetic studies on FGAM synthetase are most consistent with a sequential mechanism in which glutamine binds followed by rapid equilibrium binding of MgATP and then FGAR. Incubation of [18O]FGAR with enzyme, ATP, and glutamine results in quantitative transfer of the 18O to Pi. << Less

  Biochemistry 28:2459-2471(1989) [PubMed] [EuropePMC]

• The formylglycinamide ribonucleotide amidotransferase complex from Bacillus subtilis: metabolite-mediated complex formation.

  Hoskins A.A., Anand R., Ealick S.E., Stubbe J.

  Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) catalyzes the ATP- and glutamine-dependent formation of formylglycinamidine ribonucleotide, ADP, P(i), and glutamate in the fourth step of de novo purine biosynthesis. Like all amidotransferases (ATs), FGAR-AT is proposed to channel ammon ... >> More

  Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) catalyzes the ATP- and glutamine-dependent formation of formylglycinamidine ribonucleotide, ADP, P(i), and glutamate in the fourth step of de novo purine biosynthesis. Like all amidotransferases (ATs), FGAR-AT is proposed to channel ammonia between a glutaminase and AT domain. In Gram-negative bacteria and eukaryotes, FGAR-AT is a single approximately 140 kDa protein. In archae and Gram-positive bacteria, the FGAR-AT is formed from three proteins: PurS (10 kDa), PurQ (25 kDa, a glutaminase), and smPurL (80 kDa, an AT). This is the only known AT to require a third structural component (PurS) for activity. Here we report the first purification and biochemical characterization of a three-component AT from Bacillus subtilis. Efforts to isolate an intact FGAR-AT focused initially on coexpression of PurS, smPurL, and PurQ. However, all attempts to purify the complex resulted in separation of the constituent proteins. PurS, smPurL, and PurQ were therefore separately expressed and purified to homogeneity. PurQ had a glutaminase activity of 0.002 s(-1), and smPurL had an ammonia-dependent AT activity of 0.044 s(-1). Reconstitution of PurS, smPurL, and PurQ at a ratio of 2:1:1 gave an activity of 2.49 s(-1), similar to that previously reported for the Escherichia coli 140 kDa FGAR-AT (5.00 s(-1)). PurS was essential for the glutamine-dependent FGAR-AT activity. Surprisingly, activity was found to be absolutely dependent on the presence of Mg2+ and ADP, and a stable FGAR-AT complex of 2PurS/1smPurL/1PurQ was detected only in the presence of Mg2+, ADP, and glutamine. The implications of these observations are discussed with respect to ammonia channeling. << Less

  Biochemistry 43:10314-10327(2004) [PubMed] [EuropePMC]

• Purification of, generation of monoclonal antibodies to, and mapping of phosphoribosyl N-formylglycinamide amidotransferase.

  Barnes T.S., Bleskan J.H., Hart I.M., Walton K.A., Barton J.W., Patterson D.

  5'-Phosphoribosyl N-formylglycinamide (FGAR) amidotransferase (EC 6.3.5.3) catalyzes the fourth reaction in the de novo synthesis of purines, that is, the conversion of FGAR to 5'-phosphoribosyl N-formylglycinamidine (FGAM). This is the only step of the pathway for which a vertebrate gene has not ... >> More

  5'-Phosphoribosyl N-formylglycinamide (FGAR) amidotransferase (EC 6.3.5.3) catalyzes the fourth reaction in the de novo synthesis of purines, that is, the conversion of FGAR to 5'-phosphoribosyl N-formylglycinamidine (FGAM). This is the only step of the pathway for which a vertebrate gene has not been cloned. FGAR amidotransferase has been highly purified from Chinese hamster ovary (CHO) cells, and this preparation has been used to generate monoclonal antibodies in mice. Two of these antibodies, designated BD4 and DD2, have been shown to recognize a 150-kDa protein in CHO-K1 cells that is of very low abundance in Ade-B cells, a CHO line in which FGAR amidotransferase activity is undetectable. Furthermore, the protein recognized by these antibodies is 5-10-fold more abundant in Azr cells. The CHO Azr cell line was made resistant to azaserine, a potent inhibitor of FGAR amidotransferase, and displays a 5-10-fold increase in FGAR amidotransferase activity over the parental K1 line. FGAR amidotransferase activity and the 150-kDa protein recognized by both monoclonal antibodies were found to immunoprecipitate concomitantly using antibody BD4. Monoclonal antibody DD2 cross-reacted with a human protein of identical molecular mass. A number of Ade-B/human hybrid cells were generated by somatic cell fusion and subsequent 5-bromo-2-deoxyuridine segregation. Analysis of these lines, together with two independently generated human/mouse hybrid cell lines, by both cytogenetics and immunoblotting with antibody DD2 revealed that the human FGAR amidotransferase gene is located on chromosome 17p. << Less

  Biochemistry 33:1850-1860(1994) [PubMed] [EuropePMC]

• Complexed structures of formylglycinamide ribonucleotide amidotransferase from Thermotoga maritima describe a novel ATP binding protein superfamily.

  Morar M., Anand R., Hoskins A.A., Stubbe J., Ealick S.E.

  Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) catalyzes the ATP-dependent synthesis of formylglycinamidine ribonucleotide (FGAM) from formylglycinamide ribonucleotide (FGAR) and glutamine in the fourth step of the purine biosynthetic pathway. FGAR-AT is encoded by the purL gene. Two ... >> More

  Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) catalyzes the ATP-dependent synthesis of formylglycinamidine ribonucleotide (FGAM) from formylglycinamide ribonucleotide (FGAR) and glutamine in the fourth step of the purine biosynthetic pathway. FGAR-AT is encoded by the purL gene. Two types of PurL have been detected. The first type, found in eukaryotes and Gram-negative bacteria, consists of a single 140 kDa polypeptide chain and is designated large PurL (lgPurL). The second type, small PurL (smPurL), is found in archaea and Gram-positive bacteria and consists of an 80 kDa polypeptide chain. SmPurL requires two additional gene products, PurQ and PurS, for activity. PurL is a member of a protein superfamily that contains a novel ATP-binding domain. Structures of several members of this superfamily are available in the unliganded form. We determined five different structures of FGAR-AT from Thermotoga maritima in the presence of substrates, a substrate analogue, and a product. These complexes have allowed a detailed description of the novel ATP-binding motif. The availability of a ternary complex enabled mapping of the active site, thus identifying potential residues involved in catalysis. The complexes show a conformational change in the active site compared to the unliganded structure. Surprising discoveries, an ATP molecule in an auxiliary site of the protein and the conformational changes associated with its binding, provoke speculation about the regulatory role of the auxiliary site in formation of the PurLSQ complex as well as the evolutionary relationship of PurLs from different organisms. << Less

  Biochemistry 45:14880-14895(2006) [PubMed] [EuropePMC]