Eltis L D, Hofmann B, Hecht H J, Lünsdorf H, Timmis K N
GBF, National Research Center for Biotechnology, Braunschweig, Federal Republic of Germany.
J Biol Chem. 1993 Feb 5;268(4):2727-32.
2,3-Dihydroxybiphenyl 1,2-dioxygenase, an enzyme of the biphenyl biodegradation pathway that cleaves the first of the aromatic rings, was purified to apparent homogeneity from Pseudomonas sp. strain LB400 that had been engineered to hyperexpress the bphC gene. The enzyme had a subunit molecular mass of 33.2 kDa as determined by SDS-polyacrylamide electrophoresis. Kinetic studies indicate a KM of 7 +/- 1 microM for 2,3-dihydroxybiphenyl. The enzyme is strongly inhibited by substrate (Kss = 300 +/- 10 microM). Catechol, 3-methylcatechol, and 4-methylcatechol were cleaved less efficiently and showed weaker substrate inhibition. 3,4-Dihydroxybiphenyl was not a substrate for the enzyme. Ammonium sulfate and polyethylene glycol 6000 were used as precipitants to obtain two different crystal forms. Crystals grown from ammonium sulfate and polyethylene glycol 6000 had space groups of P4(2)2(1)2 and I222, respectively. Electron microscopy indicates that the enzyme is an octamer (265 kDa) consisting of subunits arranged in two planar tetramers in a staggered conformation.
2,3-二羟基联苯1,2-双加氧酶是联苯生物降解途径中的一种酶，可裂解第一个芳香环，它从经过基因工程改造以超表达bphC基因的假单胞菌属LB400菌株中纯化至表观均一。通过SDS-聚丙烯酰胺凝胶电泳测定，该酶的亚基分子量为33.2 kDa。动力学研究表明，2,3-二羟基联苯的KM为7±1 μM。该酶受到底物的强烈抑制（Kss = 300±10 μM）。儿茶酚、3-甲基儿茶酚和4-甲基儿茶酚的裂解效率较低，底物抑制作用较弱。3,4-二羟基联苯不是该酶的底物。硫酸铵和聚乙二醇6000用作沉淀剂，获得了两种不同的晶体形式。从硫酸铵和聚乙二醇6000中生长的晶体的空间群分别为P4(2)2(1)2和I222。电子显微镜显示该酶是一个八聚体（265 kDa），由亚基以交错构象排列成两个平面四聚体组成。
