Bugg T D
Department of Chemistry, University of Southampton, UK.
Biochim Biophys Acta. 1993 Oct 6;1202(2):258-64. doi: 10.1016/0167-4838(93)90013-h.
The mhpB gene encoding 2,3-dihydroxyphenylpropionate 1,2-dioxygenase in Escherichia coli was subcloned from Clarke-Carbon plasmid pLC20-30 by complementation with an mhpB- strain LW366. Dioxygenase MhpB was purified using a five-step procedure from an overexpressing construct containing the mhpB gene, giving enzyme of > 95% homogeneity. The purified enzyme appeared as a 36-kDa subunit by SDS-PAGE, and had a native molecular mass of 134 kDa as determined by gel filtration. The apoenzyme obtained after chromatography could be re-activated by addition of Fe(II) and ascorbate to give the holoenzyme with a specific activity of 48 U/mg, which could be readily inactivated by oxidation or complexation of the Fe(II) cofactor, or simply by dilution. The substrate specificity of MhpB was examined, and as well as 2,3-dihydroxyphenylpropionate the enzyme was found to catalyse meta-ring cleavage of 3-methylcatechol and catechol, with reduced catalytic efficiency. The N-terminal sequence obtained for the purified enzyme showed significant sequence similarity with catechol 2,3-dioxygenase from Alcaligenes eutrophus, but none with catechol 2,3-dioxygenases from Pseudomonas.
通过与mhpB缺陷型菌株LW366互补，从克拉克 - 卡本质粒pLC20 - 30中克隆出大肠杆菌中编码2,3 - 二羟基苯丙酸1,2 - 双加氧酶的mhpB基因。使用五步程序从含有mhpB基因的过表达构建体中纯化双加氧酶MhpB，得到纯度大于95%的酶。通过SDS - PAGE分析，纯化后的酶呈现为36 kDa的亚基，通过凝胶过滤测定其天然分子量为134 kDa。层析后得到的脱辅基酶可通过添加Fe(II)和抗坏血酸重新激活，得到具有48 U/mg比活性的全酶，该全酶可通过Fe(II)辅因子的氧化或络合，或简单地通过稀释轻易失活。研究了MhpB的底物特异性，发现该酶除了能催化2,3 - 二羟基苯丙酸外，还能催化3 - 甲基邻苯二酚和邻苯二酚的间位环裂解，但其催化效率较低。纯化酶的N端序列与嗜碱假单胞菌的邻苯二酚2,3 - 双加氧酶具有显著的序列相似性，但与假单胞菌属的邻苯二酚2,3 - 双加氧酶没有序列相似性。
