Purification and characterization of aminoimidazole ribonucleotide synthetase from Escherichia coli

Schrimsher, Jeffrey L.; Schendel, Frederick J.; Stubbe, Jo Anne; Smith, John L.

doi:10.1021/bi00363a028

Cited by 40 publications

(63 citation statements)

References 24 publications

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“…FGAM was produced from FGAR using purified E. coli FGAR amidotransferase and isolated as described (26,35). IPTG (isopropyl-␤-D-thiogalactopyranoside) was purchased from Fisher Biotech.…”

Section: Methodsmentioning

confidence: 99%

“…AIR synthetase activity was assayed by detecting formation of AIR using a previously described modification of the Bratton-Marshall assay for diazotizable amines (34,35). To conserve reagents, the assays were performed in small volumes.…”

Section: Methodsmentioning

confidence: 99%

“…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%

“…Kinetic studies with the E. coli enzyme suggested a sequential mechanism in which ATP bound first and ADP was released last (35). The structure of AIR syn-thetase from E. coli has recently been solved, and the enzyme is believed to represent a new class of ATP-binding proteins (21,27).…”

mentioning

confidence: 99%

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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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“…FGAM was produced from FGAR using purified E. coli FGAR amidotransferase and isolated as described (26,35). IPTG (isopropyl-␤-D-thiogalactopyranoside) was purchased from Fisher Biotech.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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