Barlowe C K, Appling D R
Department of Chemistry and Biochemistry, Clayton Foundation Biochemical Institute, University of Texas, Austin 78712.
Mol Cell Biol. 1990 Nov;10(11):5679-87. doi: 10.1128/mcb.10.11.5679-5687.1990.
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.
在真核生物中，10-甲酰四氢叶酸（甲酰-THF）合成酶、5,10-亚甲基四氢叶酸环水解酶和NADP(+)-依赖性5,10-亚甲基四氢叶酸脱氢酶活性存在于一种称为C1-THF合酶的单一多肽上。这种三功能酶由酿酒酵母中的ADE3基因编码，被认为负责为嘌呤从头合成提供一碳供体10-甲酰-THF。ADE3基因的缺失导致腺嘌呤营养缺陷型，推测是由于缺乏细胞质中的10-甲酰-THF。在本报告中，体外产生了影响酵母C1-THF合酶一种或多种催化活性的特定点突变，并通过基因替换将其转移到染色体ADE3位点。与ADE3缺失不同，使C1-THF合酶的所有三种活性失活的点突变并未导致对腺嘌呤的需求。在ade3缺失菌株中异源表达编码单功能10-甲酰-THF合成酶的尿酸梭菌基因，即使该单功能合成酶在体内具有催化活性，也不能恢复在无腺嘌呤情况下的生长。这些结果表明，除了C1-THF合酶之外的一种或多种酶可以产生足够的细胞质10-甲酰-THF，但该10-甲酰-THF用于嘌呤合成的有效利用需要C1-THF合酶的非酶功能。已从酵母细胞中鉴定并纯化了一种依赖NAD+进行催化的单功能5,10-亚甲基四氢叶酸脱氢酶（C.K.巴洛和D.R.阿普林，《生物化学》29:7089-7094，1990）。我们提出，表达全长但无催化活性的C1-THF合酶的菌株的特征可能是由于形成了一种涉及结构未改变的C1-THF合酶的嘌呤合成多酶复合物，并且这些菌株中必需一碳单位的产生是由一种依赖NAD+的5,10-亚甲基四氢叶酸脱氢酶完成的。
