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PMID:15301530

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Citation

Hoskins, AA, Anand, R, Ealick, SE and Stubbe, J (2004) The formylglycinamide ribonucleotide amidotransferase complex from Bacillus subtilis: metabolite-mediated complex formation. Biochemistry 43:10314-27

Abstract

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.

Links

PubMed Online version:10.1021/bi049127h

Keywords

Adenosine Diphosphate/metabolism; Ammonia/metabolism; Bacillus subtilis/enzymology; Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor/genetics; Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor/isolation & purification; Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor/metabolism; Cloning, Molecular; Glutamine/metabolism; Macromolecular Substances; Magnesium/metabolism; Substrate Specificity

Significance

Annotations

Gene product Qualifier GO Term Evidence Code with/from Aspect Extension Notes Status

BACSU:PURL

enables

GO:0004642: phosphoribosylformylglycinamidine synthase activity

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

BACSU:PURL

enables

GO:0005524: ATP binding

ECO:0000314: direct assay evidence used in manual assertion

F

Seeded From UniProt

complete

BACSU:PURL

involved_in

GO:0006164: purine nucleotide biosynthetic process

ECO:0000314: direct assay evidence used in manual assertion

P

Seeded From UniProt

complete

Notes

See also

References

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