Purification and Preliminary Characterization of a Serine Hydrolase Involved in the Microbial Degradation of Polychlorinated Biphenyls*
Purification and Preliminary Characterization of a Serine Hydrolase Involved in the Microbial Degradation of Polychlorinated Biphenyls*
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DOI：
10.1074/jbc.273.36.22943
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发表时间：
1998-09
期刊：
影响因子：
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通讯作者：
S. Seah;G. Terracina;J. Bolin;P. Riebel;V. Snieckus;L. Eltis
中科院分区：
文献类型：
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作者：
S. Seah;G. Terracina;J. Bolin;P. Riebel;V. Snieckus;L. Eltis
2-羟基-6-氧代-6-苯基六-2,4-二烯酸（6-苯基-HODA）水解酶（BphD）是一种由洋葱伯克霍尔德菌 LB400 的 bphD 基因编码的联苯生物降解途径的酶，经过超表达和纯化，达到明显的同质性。 SDS-聚丙烯酰胺凝胶电泳证实BphD的亚基分子量为32 kDa，凝胶过滤证实它是分子量为122,000的同源四聚体。该酶水解 6-苯基-HODA，ak cat 为 5.0 (± 0.07) s−1，ak cat/K m 为 2.0 (± 0.08) × 107 m −1 s−1（100 mm 磷酸盐，pH 7.5，25 °C）。 BphD 对其他 2-羟基-6-氧代六-2,4-二烯酸酯 (HODA) 的特异性随着 C6 取代基的大小而显着降低； 6-甲基-HODA 是 3-甲基儿茶酚的主题裂解产物，其水解特异性比 6-苯基-HODA 低约 2300 倍。相比之下，来自甲苯降解途径的同源水解酶 TodF 对 6-甲基-和 6-乙基-HODA 显示出最高的特异性（k cat/K m 分别为 2.0 (± 0.05) × 106 m −1s−1 和 9.0 (± 0.5) × 106 m −1 s−1 ）。 TodF 对 6-苯基-HODA 和 6-叔丁基-HODA 没有显示出可检测的活性。 BphD 和 TodF 均不能有效水解 5-甲基-HODA。通过监测产物形成确定的 BphD 的 k cat 约为通过监测底物消失确定的一半，表明在酶催化反应过程中底物利用和产物形成发生了一些解偶联。使用硫酸铵与聚乙二醇400作为沉淀剂，获得BphD晶体。观察到的衍射分辨率至少为 1.9 Å，自旋转函数的评估证实了 222 (D2) 分子对称性。
2-Hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (6-phenyl-HODA) hydrolase (BphD), an enzyme of the biphenyl biodegradation pathway encoded by the bphD gene ofBurkholderia cepacia LB400, was hyperexpressed and purified to apparent homogeneity. SDS-polyacrylamide gel electrophoresis confirmed that BphD has a subunit molecular mass of 32 kDa, while gel filtration demonstrated that it is a homotetramer of molecular weight 122,000. The enzyme hydrolyzed 6-phenyl-HODA with ak cat of 5.0 (± 0.07) s−1 and ak cat/K m of 2.0 (± 0.08) × 107 m −1 s−1 (100 mm phosphate, pH 7.5, 25 °C). The specificity of BphD for other 2-hydroxy-6-oxohexa-2,4-dienoates (HODAs) decreased markedly with the size of the C6 substituent; 6-methyl-HODA, themeta cleavage product of 3-methylcatechol, was hydrolyzed approximately 2300 times less specifically than 6-phenyl-HODA. By comparison, the homologous hydrolase from the toluene degradation pathway, TodF, showed highest specificity for 6-methyl- and 6-ethyl-HODA (k cat/K m of 2.0 (± 0.05) × 106 m −1s−1 and 9.0 (± 0.5) × 106 m −1 s−1, respectively). TodF showed no detectable activity toward 6-phenyl-HODA and 6-tert-butyl-HODA. Neither BphD nor TodF hydrolyzed 5-methyl-HODA efficiently. The k cat of BphD determined by monitoring product formation was about half that determined by monitoring substrate disappearance, suggesting that some uncoupling of substrate utilization and product formation occurs during the enzyme catalyzed reaction. Crystals of BphD were obtained using ammonium sulfate combined with polyethylene glycol 400 as the precipitant. Diffraction was observed to a resolution of at least 1.9 Å, and the evaluation of self-rotation functions confirmed 222 (D2) molecular symmetry.