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

Schrimsher, Jeffrey L.; Schendel, Frederick J.; Stubbe, JoAnne

doi:10.1021/bi00363a027

Cited by 33 publications

(35 citation statements)

References 28 publications

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“…AIRS was assayed spectrophotometrically as described by Schrimsher et al (1986), and ␤-HBD and Glu dehydrogenase as described by Shelp et al (1983) and Atkins et al (1975), respectively. Intactness of membranes in bacteroid preparations was assessed by measuring the activity of ␤-HBD before and after treatment of bacteroids with Triton X-100.…”

Section: Enzyme Assaysmentioning

confidence: 99%

Dual Intracellular Localization and Targeting of Aminoimidazole Ribonucleotide Synthetase in Cowpea

Goggin¹,

Lipscombe²,

Fedorova³

et al. 2003

Plant Physiology

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De novo purine biosynthesis is localized to both mitochondria and plastids isolated from Bradyrhizobium sp.-infected cells of cowpea (Vigna unguiculata L. Walp) nodules, but several of the pathway enzymes, including aminoimidazole ribonucleotide synthetase (AIRS [EC 6.3.3.1], encoded by Vupur5), are encoded by single genes. Immunolocalization confirmed the presence of AIRS protein in both organelles. Enzymatically active AIRS was purified separately from nodule mitochondria and plastids. N-terminal sequencing showed that these two isoforms matched the Vupur5 cDNA sequence but were processed at different sites following import; the mitochondrial isoform was five amino acids longer than the plastid isoform. Electrospray tandem mass spectrometry of a trypsin digest of mitochondrial AIRS identified two internal peptides identical with the amino acid sequence deduced from Vupur5 cDNA. Western blots of proteins from mitochondria and plastids isolated from root tips showed a single AIRS protein present at low levels in both organelles. 35 S-AIRS protein translated from a Vupur5 cDNA was imported into isolated pea (Pisum sativum) leaf chloroplasts in vitro by an ATPdependent process but not into import-competent mitochondria from several plant and non-plant sources. Components of the mature protein are likely to be important for import because the N-terminal targeting sequence was unable to target green fluorescent protein to either chloroplasts or mitochondria in Arabidopsis leaves. The data confirm localization of the protein translated from the AIRS gene in cowpea to both plastids and mitochondria and that it is cotargeted to both organelles, but the mechanism underlying import into mitochondria has features that are yet to be identified.In root nodules of legumes of the tribe Phaseoleae, symbiotically fixed N 2 is assimilated by de novo purine biosynthesis, after which the purines are oxidized to allantoin and allantoic acid (Atkins and Smith, 2000). It is in this form that fixed N is exported in xylem from the nodule to the shoot. The 10 enzymes of the purine biosynthetic pathway have been well studied in relation to their role in cancer metabolism, and the genes encoding them cloned from a wide variety of organisms. However, in plants, far less is known about the enzymes of the pathway, although genes encoding a number have now been isolated (Atkins and Smith, 2000).The cDNA sequences of all the plant purine biosynthetic (pur) genes studied have 5Ј extensions relative to the coding regions of the corresponding Escherichia coli genes (Senecoff and Meagher, 1993;Chapman et al., 1994;Schnorr et al., 1994Schnorr et al., , 1996Kim et al., 1995;Senecoff et al., 1996;Smith et al., 1998). These extensions potentially encode N-terminal, organelle-targeting sequences and indicate that in contrast to other eukaryotes (Gooljarsingh et al., 2001), the pathway is likely to be organelle-localized in plants. This was confirmed in a recent study that showed that in cowpea (Vigna unguiculata L. Walp) nodules, both the mitochondria ...

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Section: Enzyme Assaysmentioning

confidence: 99%

Dual Intracellular Localization and Targeting of Aminoimidazole Ribonucleotide Synthetase in Cowpea

Goggin¹,

Lipscombe²,

Fedorova³

et al. 2003

Plant Physiology

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“…The identification of mutations in the biosynthetic gene purI (encodes AIR synthetase in S. enterica, homologous to purM in Escherichia coli) that caused a conditional requirement for thiamine was unexpected. One of these mutants is the subject of this paper.AIR synthetase catalyzes the conversion of formylglycinamidine ribonucleotide (FGAM) to AIR, ADP, and P i (39) and has been purified and characterized from both chicken liver and E. coli (34,35). Kinetic studies with the E. coli enzyme suggested a sequential mechanism in which ATP bound first and ADP was released last (35).…”

mentioning

confidence: 99%

“…AIR synthetase catalyzes the conversion of formylglycinamidine ribonucleotide (FGAM) to AIR, ADP, and P i (39) and has been purified and characterized from both chicken liver and E. coli (34,35). Kinetic studies with the E. coli enzyme suggested a sequential mechanism in which ATP bound first and ADP was released last (35).…”

mentioning

confidence: 99%

Altered Pathway Routing in a Class of Salmonella enterica Serovar Typhimurium Mutants Defective in Aminoimidazole Ribonucleotide Synthetase

et al. 2001

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In Salmonella enterica serovar Typhimurium, purine nucleotides and thiamine are synthesized by a branched pathway. The last known common intermediate, aminoimidazole ribonucleotide (AIR), is formed from formylglycinamidine ribonucleotide (FGAM) and ATP by AIR synthetase, encoded by the purI gene in S. enterica. Reduced flux through the first five steps of de novo purine synthesis results in a requirement for purines but not necessarily thiamine. To examine the relationship between the purine and thiamine biosynthetic pathways, purI mutants were made (J. L. Zilles and D. M. Downs, Genetics 143:37-44, 1996). Unexpectedly, some mutant purI alleles (R35C/E57G and K31N/A50G/L218R) allowed growth on minimal medium but resulted in thiamine auxotrophy when exogenous purines were supplied. To explain the biochemical basis for this phenotype, the R35C/E57G mutant PurI protein was purified and characterized kinetically. The K m of the mutant enzyme for FGAM was unchanged relative to the wild-type enzyme, but the V max was decreased 2.5-fold. The K m for ATP of the mutant enzyme was 13-fold increased. Genetic analysis determined that reduced flux through the purine pathway prevented PurI activity in the mutant strain, and purR null mutations suppressed this defect. The data are consistent with the hypothesis that an increased FGAM concentration has the ability to compensate for the lower affinity of the mutant PurI protein for ATP.In Salmonella enterica serovar Typhimurium LT2, purines and thiamine are synthesized via a divergent pathway where 5-aminoimidazole ribonucleotide (AIR) is the intermediate at the branch point (Fig. 1). Since the cellular purine requirement is approximately 10 3 -fold higher than the thiamine requirement (based on auxotrophic requirements), this pathway provides a model to address control of an important metabolic branch point. Previous genetic and molecular analyses demonstrated that even 1% of the wild-type level of AIR synthetase was sufficient to supply the cellular requirement for thiamine but not purines (J. L. Zilles and D. M. Downs, submitted for publication), indicating that thiamine synthesis can be maintained even when flux through the common pathway is severely reduced. Under this condition, thiamine synthesis could continue if levels of the substrate (presumed to be AIR) remained above the K m for the first committed thiamine enzyme or if there were metabolite channeling between PurI and the thiamine enzyme (thought to be ThiC). Mutational analysis of purI, encoding the final common enzyme AIR synthetase, was pursued to clarify the parameters controlling thiamine synthesis with changing flux through the purine pathway.The synthesis of AIR, via the pathway common to purine and thiamine synthesis, appears to be regulated only in response to exogenous purines. There are three known levels of regulation on this pathway: (i) transcription of pur genes is repressed by PurR (with its corepressors hypoxanthine and guanine) (17,18,22,28,33,39), (ii) allosteric inhibition of the first committed...

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“…Otherwise, the assay mixture was the same as for Gln-GAR. In a second method, PRA was generated chemically (19) in a reaction mixture containing 50 mM Tris-HCl (pH 9.2), 500 mM NH 4 Cl, and 250 mM ribose-5-phosphate incubated at 37°C for 15 min. An aliquot of the PRA mixture was diluted fivefold into a 50-l GARS reaction mixture that also contained 50 mM Tris-HCl, 50 g of bovine serum albumin, 10 mM ATP, 5 mM […”

mentioning

confidence: 99%

Temperature-Dependent Function of the Glutamine Phosphoribosylpyrophosphate Amidotransferase Ammonia Channel and Coupling with Glycinamide Ribonucleotide Synthetase in a Hyperthermophile

Bera¹,

Chen²,

Smith

et al. 2000

J Bacteriol

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Genes encoding glutamine phosphoribosylpyrophosphate amidotransferase (GPAT) and glycinamide ribonucleotide synthetase (GARS) from Aquifex aeolicus were expressed in Escherichia coli, and the enzymes were purified to near homogeneity. Both enzymes were maximally active at a temperature of at least 90°C, with half-lives of 65 min for GPAT and 60 h for GARS at 80°C. GPAT activity is known to depend upon channeling of NH 3 from a site in an N-terminal glutaminase domain to a distal phosphoribosylpyrophosphate site in a C-terminal domain where synthesis of phosphoribosylamine (PRA) takes place. The efficiency of channeling of NH 3 for synthesis of PRA was found to increase from 34% at 37°C to a maximum of 84% at 80°C. The mechanism for transfer of PRA to GARS is not established, but diffusion between enzymes as a free intermediate appears unlikely based on a calculated PRA half-life of approximately 0.6 s at 90°C. Evidence was obtained for coupling between GPAT and GARS for PRA transfer. The coupling was temperature dependent, exhibiting a transition between 37 and 50°C, and remained relatively constant up to 90°C. The calculated PRA chemical half-life, however, decreased by a factor of 20 over this temperature range. These results provide evidence that coupling involves direct PRA transfer through GPAT-GARS interaction rather than free diffusion.The first two steps in de novo purine nucleotide synthesis involve reactions in which labile enzyme intermediates are transferred between distal, noncontiguous sites. In the first reaction, catalyzed by glutamine phosphoribosylpyrophosphate (PRPP) amidotransferase (GPAT), NH 3 , generated by hydrolysis of glutamine at an N-terminal glutaminase domain, is sequestered from H 2 O by transfer through a 20-Å tunnel to a C-terminal PRPP site where synthesis of phosphoribosylamine (PRA) takes place (11). Channeling of NH 3 through a hydrophobic tunnel prevents its release from the enzyme and also avoids the likelihood of protonation of NH 3 to NH 4 ϩ , which is not a substrate (13). These steps are shown by equations 1 and 2, and the synthesis of glycinamide ribonucleotide (GAR) by the second enzyme, GAR synthetase (GARS), is given in equation 3.Glutamine ϩ H 2 O 3 glutamate ϩ NH 3(1)The GPAT half reactions are coupled, and NH 3 is channeled by transient forms of the enzyme, in which closing of a flexible loop upon PRPP binding activates the glutaminase domain and forms an NH 3 channel between the active sites (1, 2, 4, 11). PRA, the product of the second GPAT half reaction, is susceptible to hydrolysis to ribose 5-phosphate, with a half-life of 5 s under physiological conditions at 37°C (17). The kinetics for the synthesis of GAR by Escherichia coli GPAT and GARS were found to be inconsistent with free diffusion of PRA between enzymes and, as a result, a direct-transfer mechanism was proposed (17). Since evidence for a GPAT-GARS physical interaction was not obtained, direct PRA transfer was assumed to involve a transient channeling interaction between GPAT and GARS. The transient...

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

Cited by 33 publications

References 28 publications

Dual Intracellular Localization and Targeting of Aminoimidazole Ribonucleotide Synthetase in Cowpea

Dual Intracellular Localization and Targeting of Aminoimidazole Ribonucleotide Synthetase in Cowpea

Altered Pathway Routing in a Class of Salmonella enterica Serovar Typhimurium Mutants Defective in Aminoimidazole Ribonucleotide Synthetase

Temperature-Dependent Function of the Glutamine Phosphoribosylpyrophosphate Amidotransferase Ammonia Channel and Coupling with Glycinamide Ribonucleotide Synthetase in a Hyperthermophile

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