Raymond R K, Kastanos E K, Appling D R
The Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, 78712, USA.
Arch Biochem Biophys. 1999 Dec 15;372(2):300-8. doi: 10.1006/abbi.1999.1498.
The identification, expression, and assay of two Saccharomyces cerevisiae genes encoding methylenetetrahydrofolate reductases (MTHFR) is described. MTHFR catalyzes the reduction of 5, 10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, used to methylate homocysteine in methionine synthesis. The MET12 gene is located on chromosome XVI and encodes a protein of 657 amino acids. The MET13 gene is located on chromosome VII and encodes a protein of 599 amino acids. The deduced amino acid sequences of these two genes are 34% identical to each other and 32-37% identical to the human MTHFR. A phenotype for the single disruption of MET12 was not observed, however, single disruption of MET13 resulted in methionine auxotrophy. Double disruption of both MET12 and MET13 also resulted in methionine auxotrophy. Growth of the methionine auxotrophs was supported by both methionine and S-adenosylmethionine. Transcripts of both MET12 and MET13 were detected in total RNA from wild type cells grown in the presence or absence of methionine. The methionine requirement of the met12 met13 double disruptant was complemented by plasmid-borne MET13, but not MET12 even when a multicopy plasmid was used. Furthermore, overexpression of the human MTHFR in the met12 met13 double disruptant complemented the methionine auxotrophy of this strain. In contrast, overexpression of the Escherichia coli metF gene did not complement the methionine requirement of met12 met13 cells. Assays for MTHFR in crude extracts and expression of the yeast proteins in Escherichia coli verified that both MET12 and MET13 encode functional MTHFR isozymes.
本文描述了酿酒酵母中两个编码亚甲基四氢叶酸还原酶（MTHFR）的基因的鉴定、表达及检测。MTHFR催化5,10-亚甲基四氢叶酸还原为5-甲基四氢叶酸，后者用于蛋氨酸合成中同型半胱氨酸的甲基化。MET12基因位于第十六号染色体上，编码一个含657个氨基酸的蛋白质。MET13基因位于第七号染色体上，编码一个含599个氨基酸的蛋白质。这两个基因推导的氨基酸序列彼此间有34%的同源性，与人类MTHFR有32 - 37%的同源性。未观察到MET12单基因缺失的表型，然而MET13单基因缺失导致蛋氨酸营养缺陷型。MET12和MET13双基因缺失也导致蛋氨酸营养缺陷型。蛋氨酸和S-腺苷甲硫氨酸均可支持蛋氨酸营养缺陷型菌株的生长。在有或没有蛋氨酸存在的情况下生长的野生型细胞的总RNA中均检测到MET12和MET13的转录本。met12 met13双基因缺失菌株对蛋氨酸的需求可被携带MET13的质粒互补，但即使使用多拷贝质粒，MET12也不能互补。此外，人类MTHFR在met12 met13双基因缺失菌株中的过表达可互补该菌株的蛋氨酸营养缺陷型。相反，大肠杆菌metF基因的过表达不能互补met12 met13细胞对蛋氨酸的需求。粗提物中MTHFR的检测以及酵母蛋白在大肠杆菌中的表达证实MET12和MET13均编码功能性MTHFR同工酶。
