Emergence of Multifunctional Oxygenase Activities by Random Priming Recombination*
Emergence of Multifunctional Oxygenase Activities by Random Priming Recombination*
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DOI：
10.1074/jbc.m101323200
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发表时间：
2001-06
期刊：
影响因子：
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通讯作者：
H. Suenaga;M. Goto;K. Furukawa
中科院分区：
文献类型：
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作者：
H. Suenaga;M. Goto;K. Furukawa
联苯双加氧酶(BPH Dox)负责联苯的初始加氧反应。BPH Dox的大亚基(BphA1)在确定包括多氯联苯(PCbs)在内的与联苯相关的化合物的底物特异性方面起着至关重要的作用。假单胞菌产碱假单胞菌KF707的BPH Dox功能进化是通过bphA1基因的随机启动重组完成的，包括两轮体外重组和突变，然后进行体内活性增强的筛选。进化的BPH Dox获得了新颖的多功能降解能力，不仅可以降解多氯联苯，还可以降解二苯并呋喃、二苯并-对二恶英、二苯并噻吩和荧烯，这些化合物几乎不会被原始的KF707 BPH Dox攻击。二苯并呋喃和二苯并对二恶英的氧化方式为角式和侧式双氧化，二苯并噻吩亚磺酸的氧化方式为二苯并噻吩亚磺化，荧烯的氧化方式为单氧式。这些酶对多氯联苯同系物的降解能力也有所增强，保留了2，3-双加氧酶活性，并获得了3，4-双加氧酶活性，这取决于多氯联苯同系物的氯取代度。进一步的突变分析表明，BphA1第376位氨基酸与多功能加氧酶活性和氧合方式的获得密切相关。
Biphenyl dioxygenase (Bph Dox) is responsible for the initial dioxygenation of biphenyl. The large subunit (BphA1) of Bph Dox plays a crucial role in determination of substrate specificity of biphenyl-related compounds including polychlorinated biphenyls (PCBs). Functional evolution of Bph Dox ofPseudomonas pseudoalcaligenes KF707 was accomplished by random priming recombination of the bphA1 gene, involving two rounds of in vitro recombination and mutation followed by selection for increased activity in vivo. Evolved Bph Dox acquired novel and multifunctional degradation capabilities not only for PCBs but also for dibenzofuran, dibenzo-p-dioxin, dibenzothiophene, and fluorene, the compounds scarcely attacked by the original KF707 Bph Dox. The modes of oxygenation were angular and lateral dioxygenation for dibenzofuran and dibenzo-p-dioxin, sulfoxidation for dibenzothiophene, and mono-oxygenation for fluorene. These enzymes also exhibited enhanced degradation abilities for PCB congeners, retaining 2,3-dioxygenase activity and gaining 3,4-dioxygenase activity, depending on the chlorine substitution of PCB congeners. Further mutation analysis revealed that the amino acid at position 376 in BphA1 is significantly involved in the acquisition of multifunctional oxygenase activities and mode of oxygenation.