Evans C T, Owens D D, Sumegi B, Kispal G, Srere P A
Pre-Clinical Science Unit, Veterans Administration Medical Center, Dallas, Texas 75216.
Biochemistry. 1988 Jun 28;27(13):4680-6. doi: 10.1021/bi00413a015.
Citrate synthase is a key enzyme of the Krebs tricarboxylic acid cycle and catalyzes the stereospecific synthesis of citrate from acetyl coenzyme A and oxalacetate. The amino acid sequence and three-dimensional structure of pig citrate synthase dimers are known, and regions of the enzyme involved in substrate binding and catalysis have been identified. A cloned complementary DNA sequence encoding pig citrate synthase has been isolated from a pig kidney lambda gt11 cDNA library after screening with a synthetic oligonucleotide probe. The complete nucleotide sequence of the 1.5-kilobase cDNA was determined. The coding region consists of 1395 base pairs and confirms the amino acid sequence of purified pig citrate synthase. The derived amino acid sequence of pig citrate synthase predicts the presence of a 27 amino acid N-terminal leader peptide whose sequence is consistent with the sequences of other mitochondrial signal peptides. A conserved amino acid sequence in the mitochondrial leader peptides of pig citrate synthase and yeast mitochondrial citrate synthase was identified. To express the pig citrate synthase cDNA in Escherichia coli, we employed the inducible T7 RNA polymerase/promoter double plasmid expression vectors pGP1-2 and pT7-7 [Tabor, S., & Richardson, C. C. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1074-1078]. The pig citrate synthase cDNA was modified to delete the N-terminal leader sequence; then by use of a synthetic oligonucleotide linker, the modified cDNA was cloned into pT7-7 immediately following the initiator Met. A glutamate-requiring (citrate synthase deficient), recA- E. coli mutant, DEK15, was transformed with pGP1-2 and then pT7-7PCS. pT7-7PCS complemented the E. coli gltA mutation.(ABSTRACT TRUNCATED AT 250 WORDS)
柠檬酸合酶是三羧酸循环的关键酶，催化由乙酰辅酶A和草酰乙酸立体定向合成柠檬酸。猪柠檬酸合酶二聚体的氨基酸序列和三维结构已为人所知，并且已经鉴定出该酶中参与底物结合和催化的区域。在用合成寡核苷酸探针筛选后，从猪肾λgt11 cDNA文库中分离出了编码猪柠檬酸合酶的克隆互补DNA序列。测定了1.5千碱基cDNA的完整核苷酸序列。编码区由1395个碱基对组成，证实了纯化的猪柠檬酸合酶的氨基酸序列。猪柠檬酸合酶推导的氨基酸序列预测存在一个27个氨基酸的N端前导肽，其序列与其他线粒体信号肽的序列一致。鉴定出了猪柠檬酸合酶和酵母线粒体柠檬酸合酶的线粒体前导肽中的保守氨基酸序列。为了在大肠杆菌中表达猪柠檬酸合酶cDNA，我们使用了可诱导的T7 RNA聚合酶/启动子双质粒表达载体pGP1-2和pT7-7 [Tabor, S., & Richardson, C. C. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 1074-1078]。对猪柠檬酸合酶cDNA进行修饰以删除N端前导序列；然后使用合成寡核苷酸接头，将修饰后的cDNA紧接起始甲硫氨酸后克隆到pT7-7中。用pGP1-2然后用pT7-7PCS转化需要谷氨酸（柠檬酸合酶缺陷）、recA-的大肠杆菌突变体DEK15。pT7-7PCS互补了大肠杆菌的gltA突变。（摘要截短至250字）
