Barlowe C K, Appling D R
Clayton Foundation Biochemical Institute, University of Texas, Austin 78712.
Biofactors. 1989 Mar;2(1):57-63.
In eukaryotes, 10-formyltetrahydrofolate (THF) synthetase, 5,10-methenyl-THF cyclohydrolase and 5,10-methylene-THF dehydrogenase activities are present on a single polypeptide termed C1-THF synthase. These reactions are generally catalyzed by three separate monofunctional enzymes in prokaryotic cells. In this report a general method for the generation, detection and analysis of specific mutations affecting the catalytic activity of any of the reactions catalyzed by C1-THF synthase or its monofunctional counterparts is described. The method relies on plasmid-borne expression of genes in strains of the yeast Saccharomyces cerevisiae that are missing one or more of the activities of C1-THF synthase. Specific segments of the gene are subjected in vitro to random mutagenesis, the mutant genes expressed in yeast and screened by phenotype for inactivating mutations. Plasmids encoding mutant enzymes are recovered for sequence analysis. One-step purification of C1-THF synthase from the yeast expression system is demonstrated. The feasibility and versatility of the method is shown with the yeast ADE3 gene encoding the cytoplasmic C1-THF synthase and the gene encoding the monofunctional 10-formyl-THF synthetase from Clostridium acidiurici.
在真核生物中,10-甲酰四氢叶酸(THF)合成酶、5,10-亚甲基四氢叶酸环水解酶和5,10-亚甲基四氢叶酸脱氢酶的活性存在于一种名为C1-THF合酶的单一多肽上。而在原核细胞中,这些反应通常由三种独立的单功能酶催化。本报告描述了一种用于产生、检测和分析影响C1-THF合酶或其单功能对应物所催化的任何反应催化活性的特定突变的通用方法。该方法依赖于在酿酒酵母菌株中通过质粒介导的基因表达,这些菌株缺乏C1-THF合酶的一种或多种活性。基因的特定片段在体外进行随机诱变,突变基因在酵母中表达,并通过表型筛选失活突变。回收编码突变酶的质粒进行序列分析。展示了从酵母表达系统中一步纯化C1-THF合酶的方法。用编码细胞质C1-THF合酶的酵母ADE3基因和编码来自尿酸梭菌的单功能10-甲酰四氢叶酸合成酶的基因展示了该方法的可行性和通用性。