Wahls W P, Song J M, Smith G R
Fred Hutchinson Cancer Research Center, Seattle, Washington 98104.
J Biol Chem. 1993 Nov 15;268(32):23792-8.
In eukaryotes C1-5,6,7,8-tetrahydrofolate (THF) synthase is a trifunctional enzyme that catalyzes the interconversion of reduced forms of folate to supply activated one-carbon units required for a variety of metabolic pathways. The enzymatic activities include 10-formyl-THF synthetase (EC 6.3.4.3), 5,10-methenyl-THF cyclohydrolase (EC 3.5.4.9), and 5,10-methylene-THF dehydrogenase (EC 1.5.1.5). In bacteria separate, monofunctional or bifunctional polypeptides catalyze the same reactions. We have purified C1-THF synthase from the fission yeast Schizosaccharomyces pombe and found its physical and enzymatic properties similar to those of other eukaryotic C1-THF synthase enzymes. Unexpectedly, the S. pombe enzyme bound strongly (Keq = 100 pM) to single-stranded DNA, but not to double-stranded DNA or to RNA. The binding was sequence-independent, apparently not cooperative, and not detectably inhibited by C1-THF synthase substrates or cofactors. Trifunctional cytoplasmic enzyme from Saccharomyces cerevisiae and monofunctional (synthetase) enzyme from Clostridium acidiurici also bound tightly to single-stranded DNA, while bifunctional (dehydrogenase and cyclohydrolase) enzyme from Escherichia coli did not, suggesting that single-stranded DNA binding is a conserved function of the synthetase domain of C1-THF synthase enzymes.
在真核生物中，C1-5,6,7,8-四氢叶酸（THF）合酶是一种三功能酶，催化叶酸还原形式的相互转化，以提供各种代谢途径所需的活化一碳单位。其酶活性包括10-甲酰-THF合成酶（EC 6.3.4.3）、5,10-亚甲基四氢叶酸环水解酶（EC 3.5.4.9）和5,10-亚甲基-THF脱氢酶（EC 1.5.1.5）。在细菌中，单独的单功能或双功能多肽催化相同的反应。我们从裂殖酵母粟酒裂殖酵母中纯化了C1-THF合酶，发现其物理和酶学性质与其他真核C1-THF合酶相似。出乎意料的是，粟酒裂殖酵母酶与单链DNA紧密结合（平衡常数Keq = 100 pM），但不与双链DNA或RNA结合。这种结合不依赖于序列，显然不是协同的，并且不受C1-THF合酶底物或辅因子的可检测抑制。来自酿酒酵母的三功能细胞质酶和来自尿酸梭菌的单功能（合成酶）酶也与单链DNA紧密结合，而来自大肠杆菌的双功能（脱氢酶和环水解酶）酶则不结合，这表明单链DNA结合是C1-THF合酶酶合成酶结构域的保守功能。
