进化后的联苯双加氧酶的稳态动力学表征，该酶获得了对苯和甲苯的新降解能力。

Steady-state kinetic characterization of evolved biphenyl dioxygenase, which acquired novel degradation ability for benzene and toluene.

作者信息

Suenaga Hikaru, Sato Mika, Goto Masatoshi, Takeshita Mariko, Furukawa Kensuke

机构信息

Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.

出版信息

Biosci Biotechnol Biochem. 2006 Apr;70(4):1021-5. doi: 10.1271/bbb.70.1021.

DOI:10.1271/bbb.70.1021

PMID:16636475

Abstract

Biphenyl dioxygenase (Bph Dox) catalyzes initial oxygenation in the bacterial biphenyl degradation pathway. Bph Dox in Pseudomonas pseudoalcaligenes KF707 is a Rieske type three-component enzyme in which a large subunit (encoded by the bphA1 gene) plays an important role in the substrate specificity of Bph Dox. Steady-state kinetic assays using purified enzyme components demonstrated that KF707 Bph Dox had a kcat/Km of 33.1 x 10(3) (M(-1) s(-1)) for biphenyl. Evolved 1072 Bph Dox generated by the process of DNA shuffling (Suenaga, H. et al., J. Bacteriol., 184, 3682-3688 (2002)) exhibited enhanced degradation activity not only for biphenyl (kcat/Km of 62.2 x 10(3) [M(-1) s(-1)]) but also for benzene and toluene, compounds that are rarely attacked by KF707 Bph Dox. These results suggest that evolved 1072 Bph Dox acquires higher affinities and catalytic efficiencies for various substrates than the original KF707 enzyme.

摘要

联苯双加氧酶（Bph Dox）催化细菌联苯降解途径中的初始氧化反应。假产碱假单胞菌KF707中的Bph Dox是一种 Rieske 型三组分酶，其中大亚基（由bphA1基因编码）在Bph Dox的底物特异性中起重要作用。使用纯化的酶组分进行的稳态动力学分析表明，KF707 Bph Dox对联苯的kcat/Km为33.1×10³（M⁻¹ s⁻¹）。通过DNA改组过程产生的进化型1072 Bph Dox（Suenaga, H.等人，《细菌学杂志》，184, 3682 - 3688 (2002)）不仅对联苯表现出增强的降解活性（kcat/Km为62.2×10³ [M⁻¹ s⁻¹]），而且对苯和甲苯也有增强的降解活性，而这些化合物很少被KF707 Bph Dox攻击。这些结果表明，进化型1072 Bph Dox对各种底物的亲和力和催化效率比原始的KF707酶更高。