Strachan P D, Freer A A, Fewson C A
Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, UK.
Biochem J. 1998 Aug 1;333 ( Pt 3)(Pt 3):741-7. doi: 10.1042/bj3330741.
A method was developed for the purification of catechol 1, 2-dioxygenase from Rhodococcus rhodochrous NCIMB 13259 that had been grown in the presence of benzyl alcohol. The enzyme has very similar apparent Km (1-2 microM) and Vmax (13-19 units/mg of protein) values for the intradiol cleavage of catechol, 3-methylcatechol and 4-methylcatechol and it is optimally active at pH9. Cross-linking studies indicate that the enzyme is a homodimer. It contains 0.6 atoms of Fe per subunit. The enzyme was crystallized with 15% (w/v) poly(ethylene glycol) 4000/0.33 M CaCl2/25 mM Tris (pH7.5) by using a microseeding technique. Preliminary X-ray characterization showed that the crystals are in space group C2 with unit-cell dimensions a=111.9 A, b=78.1 A, c=134.6 A, beta=100 degrees. An oligonucleotide probe, made by hemi-nested PCR, was used to clone the gene encoding catechol 1,2-dioxygenase (catA). The deduced 282-residue sequence corresponds to a protein of molecular mass 31539 Da, close to the molecular mass of 31558 Da obtained by electrospray MS of the purified enzyme. catA was subcloned into the expression vector pTB361, allowing the production of catechol 1,2-dioxygenase to approx. 40% of the total cellular protein. The deduced amino acid sequence of the enzyme has 56% and 75% identity with the catechol 1, 2-dioxygenases of Arthrobacter mA3 and Rhodococcus erythropolis AN-13 respectively, but less than 35% identity with intradiol catechol and chlorocatechol dioxygenases of Gram-negative bacteria.
已开发出一种从在苯甲醇存在下生长的红平红球菌NCIMB 13259中纯化儿茶酚1,2 - 双加氧酶的方法。该酶对儿茶酚、3 - 甲基儿茶酚和4 - 甲基儿茶酚的间位裂解具有非常相似的表观Km（1 - 2微摩尔）和Vmax（13 - 19单位/毫克蛋白质）值，并且在pH9时具有最佳活性。交联研究表明该酶是一种同型二聚体。每个亚基含有0.6个铁原子。通过微种子技术，该酶在15%（w/v）聚乙二醇4000/0.33 M氯化钙/25 mM Tris（pH7.5）中结晶。初步X射线表征表明晶体属于空间群C2，晶胞尺寸为a = 111.9 Å，b = 78.1 Å，c = 134.6 Å，β = 100°。通过半巢式PCR制备的寡核苷酸探针用于克隆编码儿茶酚1,2 - 双加氧酶（catA）的基因。推导的282个残基序列对应于分子量为31539 Da的蛋白质，接近通过纯化酶的电喷雾质谱获得的31558 Da的分子量。catA被亚克隆到表达载体pTB361中，使得儿茶酚1,2 - 双加氧酶的产量达到总细胞蛋白的约40%。该酶推导的氨基酸序列与节杆菌mA3和红平红球菌AN - 13的儿茶酚1,2 - 双加氧酶分别具有56%和75%的同一性，但与革兰氏阴性菌的间位儿茶酚和氯儿茶酚双加氧酶的同一性小于35%。
