Metabolic role of cytoplasmic isozymes of 5,10-methylenetetrahydrofolate dehydrogenase in Saccharomyces cerevisiae.
Metabolic role of cytoplasmic isozymes of 5,10-methylenetetrahydrofolate dehydrogenase in Saccharomyces cerevisiae.
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酿酒酵母中5,10-亚甲基四氢叶酸脱氢酶细胞质同工酶的代谢作用。
DOI：
10.1021/bi952713d
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发表时间：
1996
期刊：
影响因子：
--
通讯作者：
Appling,DR
中科院分区：
文献类型：
--
作者：
West,MG;Horne,DW;Appling,DR
Saccharomycescerevisiaepossesses two cytosolic 5,10-methylenetetrahydrofolate (CH2-THF) dehydrogenases that differ in their redox cofactor specificity: an NAD-dependent dehydrogenase encoded by theMTD1gene and an NADP-dependent activity as part of the trifunctional C1-THF synthase encoded by theADE3gene. The experiments described here were designed to define the metabolic roles of the NAD- and NADP-dependent CH2-THF dehydrogenases in one-carbon interconversions andde novopurine biosynthesis. Growth studies showed that the NAD-dependent CH2-THF dehydrogenase is interchangeable with the NADP-dependent CH2-THF dehydrogenase when flow of one-carbon units is in the oxidative direction but that it does not participate significantly when flux is in the reductive direction.13C NMR experiments with [2-13C]glycine and unlabeled formate confirmed the latter conclusion. Direct measurements of cellular folate coenzyme levels revealed substantial levels of 10-formyl-THF (CHO-THF), the one-carbon donor used in purine synthesis, in the purine-requiringade3deletion strain. Thus, CHO-THF is necessary but not sufficient forde novopurine synthesis in yeast. Disruption of theMTD1gene in this strain resulted in undetectable CHO-THF, indicating that the NAD-dependent CH2-THF dehydrogenase was responsible for CHO-THF production in theade3deletion strain. Finally, we examined the ability of wild-type and catalytically-inactive domains of the cytoplasmic C1-THF synthase to complement the adenine auxotrophy of theade3deletion strain. Both the dehydrogenase/cyclohydrolase (D/C) domain and the synthetase domain could functionally replace the full-length protein, but, at least for the D/C domain, complementation was not dependent on catalytic activity. These results reveal a catalytic role for the NAD-dependent CH2-THF dehydrogenase in the oxidation of cytoplasmic one-carbon units and indicate that the cytoplasmic C1-THF synthase plays both catalytic and noncatalytic roles inde novopurine biosynthesis in yeast.
