摘要
乙酰辅酶A羧化酶 (AcetylCoACarboxylaseEC 6.4.1 .2 ,ACC)催化依赖于ATP的乙酰辅酶A羧化形成丙二酸单酰辅酶A ,该反应是脂肪酸生物合成途径中的第一步 ,也是受到调控的关键一步。根据结核分枝杆菌 (M .tuberculosis)和天蓝色链霉菌 (S .coelicolor)中ACC -α亚基的氨基酸保守序列和地中海拟无枝菌酸菌U32对氨基酸密码子的使用偏好 ,设计简并引物以U32基因组DNA为模板扩增出一条约 2 5 0bp的片段 ,并以此片段作探针成功地从U32基因组cosmid文库中克隆到相应的ACC -α亚基的编码基因accA。该基因对应的ORF长1 797bp,编码一个 5 98个氨基酸的蛋白 ,推算出的分子量是 63,71 4Da ;基因G +Cmol%含量为70 .1 % ,符合U32基因结构特征 ,距起始密码子GTG上游 6个碱基处有链霉菌典型的RBS序列AGGAGG ,并有生物素羧化酶特征的ATP结合区。利用pET2 8(b)系统构建表达载体 ,在E .coliBL2 1 (DE3)中实现了accA的诱导表达 ,产物大部分以可溶形式存在 ,并通过WesternBlot证明该蛋白上确有共价结合的生物素。NorthernBlot分析了各种氮源对accA基因转录水平的不同影响。
Acetyl CoA carboxylase (EC 6.4.1.2, ACC) catalyzes the ATP-dependent carboxylation of acetyl CoA to yield malonyl CoA, which is the first committed step in fatty acid synthesis. A pair of degenerate PCR primers were designed according to the conserved amino acid sequence of AccA from M. tuberculosis and S. coelicolor. The product of the PCR amplification, a DNA fragment of 250bp was used as a probe for screening the U32 genomic cosmid library and its gene, accA, coding the biotinylated protein subunit of acetyl CoA carboxylase, was successfully cloned from U32. The accA ORF encodes a 598-amino-acid protein with the calculated molecular mass of 63.7kD, with 70.1% of G+C content. A typical Streptomyces RBS sequence, AGGAGG, was found at the -6 position upstream of the start codon GTG. Analysis of the deduced amino acid sequence showed the presence of biotin-binding site and putative ATP-bicarbonate interaction region, which suggested the U32 AccA may act as a biotin carboxylase as well as a biotin carrier protein. Gene accA was then cloned into the pET28(b) vector and expressed solubly in E. coli BL21(DE3) by 0.1mmol/L IPTG induction. Western blot confirmed the covalent binding of biotin with AccA. Northern blot analyzed transcriptional regulation of accA by 5 different nitrogen sources. =
出处
《微生物学报》
CAS
CSCD
北大核心
2003年第1期56-64,共9页
Acta Microbiologica Sinica
基金
国家自然科学基金重点项目资助 ( 1 0 0 39630 0 1 0 )
