Skip to main content
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1993 Jun;175(11):3591–3597. doi: 10.1128/jb.175.11.3591-3597.1993

Evidence for a novel glycinamide ribonucleotide transformylase in Escherichia coli.

P Nygaard 1, J M Smith 1
PMCID: PMC204760  PMID: 8501063

Abstract

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.

Full text

PDF
3591

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Almassy R. J., Janson C. A., Kan C. C., Hostomska Z. Structures of apo and complexed Escherichia coli glycinamide ribonucleotide transformylase. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):6114–6118. doi: 10.1073/pnas.89.13.6114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Axley M. J., Grahame D. A., Stadtman T. C. Escherichia coli formate-hydrogen lyase. Purification and properties of the selenium-dependent formate dehydrogenase component. J Biol Chem. 1990 Oct 25;265(30):18213–18218. [PubMed] [Google Scholar]
  3. Bochner B. R., Ames B. N. ZTP (5-amino 4-imidazole carboxamide riboside 5'-triphosphate): a proposed alarmone for 10-formyl-tetrahydrofolate deficiency. Cell. 1982 Jul;29(3):929–937. doi: 10.1016/0092-8674(82)90455-x. [DOI] [PubMed] [Google Scholar]
  4. Caperelli C. A. Mammalian glycinamide ribonucleotide transformylase: purification and some properties. Biochemistry. 1985 Mar 12;24(6):1316–1320. doi: 10.1021/bi00327a008. [DOI] [PubMed] [Google Scholar]
  5. Choi K. Y., Zalkin H. Structural characterization and corepressor binding of the Escherichia coli purine repressor. J Bacteriol. 1992 Oct;174(19):6207–6214. doi: 10.1128/jb.174.19.6207-6214.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Daubner S. C., Young M., Sammons R. D., Courtney L. F., Benkovic S. J. Structural and mechanistic studies on the HeLa and chicken liver proteins that catalyze glycinamide ribonucleotide synthesis and formylation and aminoimidazole ribonucleotide synthesis. Biochemistry. 1986 May 20;25(10):2951–2957. doi: 10.1021/bi00358a033. [DOI] [PubMed] [Google Scholar]
  7. Dev I. K., Harvey R. J. A complex of N5,N10-methylenetetrahydrofolate dehydrogenase and N5,N10-methenyltetrahydrofolate cyclohydrolase in Escherichia coli. Purification, subunit structure, and allosteric inhibition by N10-formyltetrahydrofolate. J Biol Chem. 1978 Jun 25;253(12):4245–4253. [PubMed] [Google Scholar]
  8. Dev I. K., Harvey R. J. N10-Formyltetrahydrofolate is the formyl donor for glycinamide ribotide transformylase in Escherichia coli. J Biol Chem. 1978 Jun 25;253(12):4242–4244. [PubMed] [Google Scholar]
  9. Dev I. K., Harvey R. J. Sources of one-carbon units in the folate pathway of Escherichia coli. J Biol Chem. 1982 Feb 25;257(4):1980–1986. [PubMed] [Google Scholar]
  10. Dimri G. P., Ames G. F., D'Ari L., Rabinowitz J. C. Physical map location of the Escherichia coli gene encoding the bifunctional enzyme 5,10-methylene-tetrahydrofolate dehydrogenase/5,10-methenyl-tetrahydrofolate cyclohydrolase. J Bacteriol. 1991 Sep;173(17):5251–5251. doi: 10.1128/jb.173.17.5251.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Ebbole D. J., Zalkin H. Cloning and characterization of a 12-gene cluster from Bacillus subtilis encoding nine enzymes for de novo purine nucleotide synthesis. J Biol Chem. 1987 Jun 15;262(17):8274–8287. [PubMed] [Google Scholar]
  12. Flannigan K. A., Hennigan S. H., Vogelbacker H. H., Gots J. S., Smith J. M. Purine biosynthesis in Escherichia coli K12: structure and DNA sequence studies of the purHD locus. Mol Microbiol. 1990 Mar;4(3):381–392. doi: 10.1111/j.1365-2958.1990.tb00605.x. [DOI] [PubMed] [Google Scholar]
  13. Gots J. S., Benson C. E., Jochimsen B., Koduri K. R. Microbial models and regulatory elements in the control of purine metabolism. Ciba Found Symp. 1977;(48):23–41. doi: 10.1002/9780470720301.ch3. [DOI] [PubMed] [Google Scholar]
  14. He B., Shiau A., Choi K. Y., Zalkin H., Smith J. M. Genes of the Escherichia coli pur regulon are negatively controlled by a repressor-operator interaction. J Bacteriol. 1990 Aug;172(8):4555–4562. doi: 10.1128/jb.172.8.4555-4562.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Henikoff S., Furlong C. E. Sequence of a Drosophila DNA segment that functions in Saccharomyces cerevisiae and its regulation by a yeast promoter. Nucleic Acids Res. 1983 Feb 11;11(3):789–800. doi: 10.1093/nar/11.3.789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Henikoff S., Keene M. A., Sloan J. S., Bleskan J., Hards R., Patterson D. Multiple purine pathway enzyme activities are encoded at a single genetic locus in Drosophila. Proc Natl Acad Sci U S A. 1986 Feb;83(3):720–724. doi: 10.1073/pnas.83.3.720. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Houlberg U., Hove-Jensen B., Jochimsen B., Nygaard P. Identification of the enzymatic reactions encoded by the purG and purI genes of Escherichia coli. J Bacteriol. 1983 Jun;154(3):1485–1488. doi: 10.1128/jb.154.3.1485-1488.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Houlberg U., Jensen K. F. Role of hypoxanthine and guanine in regulation of Salmonella typhimurium pur gene expression. J Bacteriol. 1983 Feb;153(2):837–845. doi: 10.1128/jb.153.2.837-845.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Inglese J., Johnson D. L., Shiau A., Smith J. M., Benkovic S. J. Subcloning, characterization, and affinity labeling of Escherichia coli glycinamide ribonucleotide transformylase. Biochemistry. 1990 Feb 13;29(6):1436–1443. doi: 10.1021/bi00458a014. [DOI] [PubMed] [Google Scholar]
  20. Jensen K. F., Houlberg U., Nygaard P. Thin-layer chromatographic methods to isolate 32P-labeled 5-phosphoribosyl-alpha-1-pyrophosphate (PRPP): determination of cellular PRPP pools and assay of PRPP synthetase activity. Anal Biochem. 1979 Oct 1;98(2):254–263. doi: 10.1016/0003-2697(79)90138-6. [DOI] [PubMed] [Google Scholar]
  21. LENNOX E. S. Transduction of linked genetic characters of the host by bacteriophage P1. Virology. 1955 Jul;1(2):190–206. doi: 10.1016/0042-6822(55)90016-7. [DOI] [PubMed] [Google Scholar]
  22. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  23. Meng L. M., Kilstrup M., Nygaard P. Autoregulation of PurR repressor synthesis and involvement of purR in the regulation of purB, purC, purL, purMN and guaBA expression in Escherichia coli. Eur J Biochem. 1990 Jan 26;187(2):373–379. doi: 10.1111/j.1432-1033.1990.tb15314.x. [DOI] [PubMed] [Google Scholar]
  24. Meng L. M., Nygaard P. Identification of hypoxanthine and guanine as the co-repressors for the purine regulon genes of Escherichia coli. Mol Microbiol. 1990 Dec;4(12):2187–2192. doi: 10.1111/j.1365-2958.1990.tb00580.x. [DOI] [PubMed] [Google Scholar]
  25. Messenger L. J., Zalkin H. Glutamine phosphoribosylpyrophosphate amidotransferase from Escherichia coli. Purification and properties. J Biol Chem. 1979 May 10;254(9):3382–3392. [PubMed] [Google Scholar]
  26. Meyer E., Leonard N. J., Bhat B., Stubbe J., Smith J. M. Purification and characterization of the purE, purK, and purC gene products: identification of a previously unrecognized energy requirement in the purine biosynthetic pathway. Biochemistry. 1992 Jun 2;31(21):5022–5032. doi: 10.1021/bi00136a016. [DOI] [PubMed] [Google Scholar]
  27. Plamann M. D., Stauffer G. V. Regulation of the Escherichia coli glyA gene by the metR gene product and homocysteine. J Bacteriol. 1989 Sep;171(9):4958–4962. doi: 10.1128/jb.171.9.4958-4962.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Randerath K., Randerath E. Ion-exchange thin-layer chromatography. XIV. Separation of nucleotide sugars and nucleoside monophosphates on PEI-cellulose. Anal Biochem. 1965 Dec;13(3):575–579. doi: 10.1016/0003-2697(65)90356-8. [DOI] [PubMed] [Google Scholar]
  29. Ravnikar P. D., Somerville R. L. Genetic characterization of a highly efficient alternate pathway of serine biosynthesis in Escherichia coli. J Bacteriol. 1987 Jun;169(6):2611–2617. doi: 10.1128/jb.169.6.2611-2617.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Rex J. H., Aronson B. D., Somerville R. L. The tdh and serA operons of Escherichia coli: mutational analysis of the regulatory elements of leucine-responsive genes. J Bacteriol. 1991 Oct;173(19):5944–5953. doi: 10.1128/jb.173.19.5944-5953.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Rohlman C. E., Matthews R. G. Role of purine biosynthetic intermediates in response to folate stress in Escherichia coli. J Bacteriol. 1990 Dec;172(12):7200–7210. doi: 10.1128/jb.172.12.7200-7210.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Schild D., Brake A. J., Kiefer M. C., Young D., Barr P. J. Cloning of three human multifunctional de novo purine biosynthetic genes by functional complementation of yeast mutations. Proc Natl Acad Sci U S A. 1990 Apr;87(8):2916–2920. doi: 10.1073/pnas.87.8.2916. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schrimsher J. L., Schendel F. J., Stubbe J. Isolation of a multifunctional protein with aminoimidazole ribonucleotide synthetase, glycinamide ribonucleotide synthetase, and glycinamide ribonucleotide transformylase activities: characterization of aminoimidazole ribonucleotide synthetase. Biochemistry. 1986 Jul 29;25(15):4356–4365. doi: 10.1021/bi00363a027. [DOI] [PubMed] [Google Scholar]
  34. Smith G. K., Mueller W. T., Benkovic P. A., Benkovic S. J. On the cofactor specificity of glycinamide ribonucleotide and 5-aminoimidazole-4-carboxamide ribonucleotide transformylase from chicken liver. Biochemistry. 1981 Mar 3;20(5):1241–1245. doi: 10.1021/bi00508a029. [DOI] [PubMed] [Google Scholar]
  35. Smith J. M., Daum H. A., 3rd Nucleotide sequence of the purM gene encoding 5'-phosphoribosyl-5-aminoimidazole synthetase of Escherichia coli K12. J Biol Chem. 1986 Aug 15;261(23):10632–10636. [PubMed] [Google Scholar]
  36. Staben C., Rabinowitz J. C. Nucleotide sequence of the Saccharomyces cerevisiae ADE3 gene encoding C1-tetrahydrofolate synthase. J Biol Chem. 1986 Apr 5;261(10):4629–4637. [PubMed] [Google Scholar]
  37. Steiert J. G., Rolfes R. J., Zalkin H., Stauffer G. V. Regulation of the Escherichia coli glyA gene by the purR gene product. J Bacteriol. 1990 Jul;172(7):3799–3803. doi: 10.1128/jb.172.7.3799-3803.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Tiedeman A. A., Smith J. M. lacZY gene fusion cassettes with KanR resistance. Nucleic Acids Res. 1988 Apr 25;16(8):3587–3587. doi: 10.1093/nar/16.8.3587. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Urbanowski M. L., Stauffer G. V. Role of homocysteine in metR-mediated activation of the metE and metH genes in Salmonella typhimurium and Escherichia coli. J Bacteriol. 1989 Jun;171(6):3277–3281. doi: 10.1128/jb.171.6.3277-3281.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Westby C. A., Gots J. S. Genetic blocks and unique features in the biosynthesis of 5'-phosphoribosyl-N-formylglycinamide in Salmonella typhimurium. J Biol Chem. 1969 Apr 25;244(8):2095–2102. [PubMed] [Google Scholar]
  41. White J. H., Lusnak K., Fogel S. Mismatch-specific post-meiotic segregation frequency in yeast suggests a heteroduplex recombination intermediate. Nature. 1985 May 23;315(6017):350–352. doi: 10.1038/315350a0. [DOI] [PubMed] [Google Scholar]
  42. Whitehead T. R., Park M., Rabinowitz J. C. Distribution of 10-formyltetrahydrofolate synthetase in eubacteria. J Bacteriol. 1988 Feb;170(2):995–997. doi: 10.1128/jb.170.2.995-997.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Winans S. C., Elledge S. J., Krueger J. H., Walker G. C. Site-directed insertion and deletion mutagenesis with cloned fragments in Escherichia coli. J Bacteriol. 1985 Mar;161(3):1219–1221. doi: 10.1128/jb.161.3.1219-1221.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Woods R. A., Jackson I. E. The accumulation of glycinamide ribotide by ade3 and ade8 mutants of Saccharomyces cerevisiae. Biochem Biophys Res Commun. 1973 Aug 6;53(3):787–793. doi: 10.1016/0006-291x(73)90161-7. [DOI] [PubMed] [Google Scholar]
  45. Zalkin H., Dixon J. E. De novo purine nucleotide biosynthesis. Prog Nucleic Acid Res Mol Biol. 1992;42:259–287. doi: 10.1016/s0079-6603(08)60578-4. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES