Green J M, Nichols B P
Department of Biological Sciences, University of Illinois, Chicago 60680.
J Biol Chem. 1991 Jul 15;266(20):12971-5.
p-Aminobenzoate, a component of the vitamin folate, is one of seven compounds derived from the aromatic precursor chorismate in Escherichia coli. Historically the gene products of pabA and pabB were assumed to be sufficient for de novo p-aminobenzoate biosynthesis. Recent studies, however, have shown that these proteins, as nonidentical subunits of a single enzyme, act on chorismate to form a diffusible intermediate, most likely 4-amino-4-deoxychorismate. This intermediate is then converted to p-aminodeoxychorismate lyase (Nichols, B. P., Seibold, A. S., and Doktor, S. Z. (1989) J. Biol. Chem. 264, 8597-8601). Here we describe partial characterization of the intermediate and the purification of aminodeoxychorismate lyase 4100-fold to near homogeneity. Further purification of this enzyme by high pressure liquid chromatography permitted isolation of a pure sample that yielded N-terminal sequence. A 64-fold redundant oligonucleotide probe was used to identify a lambda clone containing the gene encoding aminodeoxychorismate lyase. The aminodeoxychorismate lyase gene, designated pabC, was mapped to 25 min on the E. coli chromosome and lies on a 7.5-kilobase pair EcoRI fragment. A strain harboring a pACYC184 recombinant containing pabC overproduced aminodeoxychorismate lyase activity 77-fold.
对氨基苯甲酸是维生素叶酸的组成成分之一，是大肠杆菌中由芳香族前体分支酸衍生而来的七种化合物之一。历史上，人们认为pabA和pabB的基因产物足以进行对氨基苯甲酸的从头生物合成。然而，最近的研究表明，这些蛋白质作为单一酶的不同亚基，作用于分支酸形成一种可扩散的中间体，很可能是4-氨基-4-脱氧分支酸。然后这种中间体被转化为对氨基脱氧分支酸裂解酶（尼科尔斯，B.P.，西博尔德，A.S.，和多克特，S.Z.（1989年）《生物化学杂志》264卷，8597 - 8601页）。在这里，我们描述了该中间体的部分特性以及氨基脱氧分支酸裂解酶4100倍纯化至接近均一性的过程。通过高压液相色谱对该酶进一步纯化，得到了一个产生N端序列的纯样品。一个64倍冗余的寡核苷酸探针被用于鉴定一个包含编码氨基脱氧分支酸裂解酶基因的λ克隆。氨基脱氧分支酸裂解酶基因，命名为pabC，被定位在大肠杆菌染色体上25分钟处，位于一个7.5千碱基对的EcoRI片段上。一个携带含有pabC的pACYC184重组体的菌株，其氨基脱氧分支酸裂解酶活性过量产生了77倍。
