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Molecular genetic analysis of Saccharomyces cerevisiae C1-tetrahydrofolate synthase mutants reveals a noncatalytic function of the ADE3 gene product and an additional folate-dependent enzyme.
Mol Cell Biol. 1990 Nov; 10(11):5679-87.MC

Abstract

In eucaryotes, 10-formyltetrahydrofolate (formyl-THF) synthetase, 5,10-methenyl-THF cyclohydrolase, and NADP(+)-dependent 5,10-methylene-THF dehydrogenase activities are present on a single polypeptide termed C1-THF synthase. This trifunctional enzyme, encoded by the ADE3 gene in the yeast Saccharomyces cerevisiae, is thought to be responsible for the synthesis of the one-carbon donor 10-formyl-THF for de novo purine synthesis. Deletion of the ADE3 gene causes adenine auxotrophy, presumably as a result of the lack of cytoplasmic 10-formyl-THF. In this report, defined point mutations that affected one or more of the catalytic activities of yeast C1-THF synthase were generated in vitro and transferred to the chromosomal ADE3 locus by gene replacement. In contrast to ADE3 deletions, point mutations that inactivated all three activities of C1-THF synthase did not result in an adenine requirement. Heterologous expression of the Clostridium acidiurici gene encoding a monofunctional 10-formyl-THF synthetase in an ade3 deletion strain did not restore growth in the absence of adenine, even though the monofunctional synthetase was catalytically competent in vivo. These results indicate that adequate cytoplasmic 10-formyl-THF can be produced by an enzyme(s) other than C1-THF synthase, but efficient utilization of that 10-formyl-THF for purine synthesis requires a nonenzymatic function of C1-THF synthase. A monofunctional 5,10-methylene-THF dehydrogenase, dependent on NAD+ for catalysis, has been identified and purified from yeast cells (C. K. Barlowe and D. R. Appling, Biochemistry 29:7089-7094, 1990). We propose that the characteristics of strains expressing full-length but catalytically inactive C1-THF synthase could result from the formation of a purine-synthesizing multienzyme complex involving the structurally unchanged C1-THF synthase and that production of the necessary one-carbon units in these strains is accomplished by an NAD+ -dependent 5,10-methylene-THF dehydrogenase.

Authors+Show Affiliations

Department of Chemistry and Biochemistry, Clayton Foundation Biochemical Institute, University of Texas, Austin 78712.No affiliation info available

Pub Type(s)

Journal Article
Research Support, U.S. Gov't, P.H.S.

Language

eng

PubMed ID

2233711

Citation

Barlowe, C K., and D R. Appling. "Molecular Genetic Analysis of Saccharomyces Cerevisiae C1-tetrahydrofolate Synthase Mutants Reveals a Noncatalytic Function of the ADE3 Gene Product and an Additional Folate-dependent Enzyme." Molecular and Cellular Biology, vol. 10, no. 11, 1990, pp. 5679-87.
Barlowe CK, Appling DR. Molecular genetic analysis of Saccharomyces cerevisiae C1-tetrahydrofolate synthase mutants reveals a noncatalytic function of the ADE3 gene product and an additional folate-dependent enzyme. Mol Cell Biol. 1990;10(11):5679-87.
Barlowe, C. K., & Appling, D. R. (1990). Molecular genetic analysis of Saccharomyces cerevisiae C1-tetrahydrofolate synthase mutants reveals a noncatalytic function of the ADE3 gene product and an additional folate-dependent enzyme. Molecular and Cellular Biology, 10(11), 5679-87.
Barlowe CK, Appling DR. Molecular Genetic Analysis of Saccharomyces Cerevisiae C1-tetrahydrofolate Synthase Mutants Reveals a Noncatalytic Function of the ADE3 Gene Product and an Additional Folate-dependent Enzyme. Mol Cell Biol. 1990;10(11):5679-87. PubMed PMID: 2233711.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Molecular genetic analysis of Saccharomyces cerevisiae C1-tetrahydrofolate synthase mutants reveals a noncatalytic function of the ADE3 gene product and an additional folate-dependent enzyme. AU - Barlowe,C K, AU - Appling,D R, PY - 1990/11/1/pubmed PY - 1990/11/1/medline PY - 1990/11/1/entrez SP - 5679 EP - 87 JF - Molecular and cellular biology JO - Mol. Cell. Biol. VL - 10 IS - 11 N2 - In eucaryotes, 10-formyltetrahydrofolate (formyl-THF) synthetase, 5,10-methenyl-THF cyclohydrolase, and NADP(+)-dependent 5,10-methylene-THF dehydrogenase activities are present on a single polypeptide termed C1-THF synthase. This trifunctional enzyme, encoded by the ADE3 gene in the yeast Saccharomyces cerevisiae, is thought to be responsible for the synthesis of the one-carbon donor 10-formyl-THF for de novo purine synthesis. Deletion of the ADE3 gene causes adenine auxotrophy, presumably as a result of the lack of cytoplasmic 10-formyl-THF. In this report, defined point mutations that affected one or more of the catalytic activities of yeast C1-THF synthase were generated in vitro and transferred to the chromosomal ADE3 locus by gene replacement. In contrast to ADE3 deletions, point mutations that inactivated all three activities of C1-THF synthase did not result in an adenine requirement. Heterologous expression of the Clostridium acidiurici gene encoding a monofunctional 10-formyl-THF synthetase in an ade3 deletion strain did not restore growth in the absence of adenine, even though the monofunctional synthetase was catalytically competent in vivo. These results indicate that adequate cytoplasmic 10-formyl-THF can be produced by an enzyme(s) other than C1-THF synthase, but efficient utilization of that 10-formyl-THF for purine synthesis requires a nonenzymatic function of C1-THF synthase. A monofunctional 5,10-methylene-THF dehydrogenase, dependent on NAD+ for catalysis, has been identified and purified from yeast cells (C. K. Barlowe and D. R. Appling, Biochemistry 29:7089-7094, 1990). We propose that the characteristics of strains expressing full-length but catalytically inactive C1-THF synthase could result from the formation of a purine-synthesizing multienzyme complex involving the structurally unchanged C1-THF synthase and that production of the necessary one-carbon units in these strains is accomplished by an NAD+ -dependent 5,10-methylene-THF dehydrogenase. SN - 0270-7306 UR - https://www.unboundmedicine.com/medline/citation/2233711/Molecular_genetic_analysis_of_Saccharomyces_cerevisiae_C1_tetrahydrofolate_synthase_mutants_reveals_a_noncatalytic_function_of_the_ADE3_gene_product_and_an_additional_folate_dependent_enzyme_ L2 - http://mcb.asm.org/cgi/pmidlookup?view=long&pmid=2233711 DB - PRIME DP - Unbound Medicine ER -