Cloning and sequence analysis of the Escherichia coli metH gene encoding cobalamin-dependent methionine synthase and isolation of a tryptic fragment containing the cobalamin-binding domain.
Cloning and sequence analysis of the Escherichia coli metH gene encoding cobalamin-dependent methionine synthase and isolation of a tryptic fragment containing the cobalamin-binding domain.
复制标题
DOI：
10.1016/s0021-9258(18)80083-7
复制
发表时间：
1989-08
期刊：
影响因子：
--
通讯作者：
Ruma Banerjee;N. L. Johnston;J. K. Sobeski;Prasanta Datta;R. Matthews
中科院分区：
文献类型：
--
作者：
Ruma Banerjee;N. L. Johnston;J. K. Sobeski;Prasanta Datta;R. Matthews
通过与大肠杆菌中的甲硫氨酸原养型互补，从大肠杆菌K-12 DNA 的质粒文库中分离出编码钴胺素依赖性甲硫氨酸合酶(EC 2.1.1.13)的基因。缺乏钴胺素依赖性和非依赖性甲硫氨酸合酶活性的粘菌素（RK4536：metE，metHH）。使用含有结构基因缺失的一系列质粒的Maxicell表达来绘制该基因在克隆的DNA片段上的位置和方向。将含有该基因的6.3-kilobaseEcoRI-SalI片段克隆到测序载体pGEM3B中进行双链DNA测序； MetH编码区由3372个核苷酸组成。该酶是从过量生产的大肠杆菌菌株中纯化出来的。含有重组质粒的大肠杆菌，其中蛋氨酸合酶的水平比野生型大肠杆菌提高了30至40倍。大肠杆菌。重组酶是一种分子量为 123,640 的蛋白质，在标准测定中的周转数为 1,450 min-1。这些值将与之前报道的从大肠杆菌野生型菌株纯化的均质酶的分子量值 133,000 和周转数 1,240–1,560 min-1 进行比较。大肠杆菌 B（Frasca, V.、Banerjee, R. V.、Dunham, W. R.、Sands, R. H. 和 Matthews, R. G. (1988)Biochemistry27, 8458–8465）。胰蛋白酶对天然酶的有限蛋白水解导致酶活性丧失，但结合的钴胺素保留在分子量为 28,000 的肽片段上。该片段已显示从 1124 个残基推导的氨基酸序列的残基 643 延伸到残基 900。
A gene encoding cobalamin-dependent methionine synthase (EC 2.1.1.13) has been isolated from a plasmid library ofEscherichia coliK-12 DNA by complementation to methionine prototrophy in anE. colistrain lacking both cobalamin-dependent and -independent methionine synthase activities (RK4536:metE,metHH). Maxicell expression of a series of plasmids containing deletions in themetHstructural gene was employed to map the position and orientation of the gene on the cloned DNA fragment. A 6.3-kilobaseEcoRI-SalI fragment containing the gene was cloned into the sequencing vector pGEM3B for double-stranded DNA sequencing; the MetH coding region consists of 3372 nucleotides. The enzyme was purified from an overproducing strain ofE. coliharboring the recombinant plasmid, in which the level of methionine synthase was elevated 30- to 40-fold over wild-typeE. coli. Recombinant enzyme is a protein of 123,640 molecular weight and has a turnover number of 1,450 min-1 in the standard assay. These values are to be compared with previously reported values of 133,000 for the molecular weight and 1,240–1,560 min-1 for the turnover number of the homogenous enzyme purified from a wild-type strain ofE. coliB (Frasca, V., Banerjee, R. V., Dunham, W. R., Sands, R. H., and Matthews, R. G. (1988)Biochemistry27, 8458–8465). Limited proteolysis of the native enzyme with trypsin resulted in loss of enzyme activity but retention of bound cobalamin on a peptide fragment of 28,000 molecular weight. This fragment has been shown to extend from residue 643 to residue 900 of the 1124-residue deduced amino acid sequence.