Heterologous expression and characterization of the purified oxygenase component of Rhodococcus globerulus P6 biphenyl dioxygenase and of chimeras derived from it
Heterologous expression and characterization of the purified oxygenase component of Rhodococcus globerulus P6 biphenyl dioxygenase and of chimeras derived from it
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DOI：
10.1128/jb.181.16.4805-4811.1999
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发表时间：
1999-08-01
影响因子：
3.2
通讯作者：
Sylvestre, M
中科院分区：
文献类型：
--
作者：
Chebrou, H;Hurtubise, Y;Sylvestre, M
在这项工作中，我们已经纯化了His标记的加氧酶（ht-加氧酶）的组成部分球红球菌P6联苯双加氧酶。将P6加氧酶的α或β亚基与假单胞菌属菌株LB 400或睾丸酮丛毛单胞菌B-356的相应亚基交换，以产生新的嵌合体，所述嵌合体是纯化的ht-蛋白，并命名为ht-α（P6）β（P6）ht-α（P6）β（LB 400）、ht-α（P6）β（B-356）、ht-α（B-356）β（P6）、和ht-α（B-356）β（P6）ht-α（P6）β（P6）ht-α（P6）β（LB 400）ht-α（P6）β（B-356）在携带P6 bphA 1和bphA 2，P6 bphA 1和LB 400 bphE的重组大肠杆菌细胞中没有表达活性，或P6 bphA 1和B-356 bphE，因为P6加氧酶α亚基的[2Fe-2S] Rieske簇在这些克隆中没有正确组装。另一方面，ht-α（LB 400）β（P6）和ht-α（B-356）β（P6）在大肠杆菌中产生活性。此外，活性纯化的ht-α（P6）β（P6）、ht-α（P6）β（LB 400）ht-α（P6）β（B-356）显示出Rieske型蛋白的典型光谱，这些光谱是从携带衍生自新穿梭E.大肠杆菌-假单胞菌载体pEP 31，设计用于在假单胞菌中产生ht-蛋白。对这些纯化的嵌合体对选定的氯联苯的底物选择性模式的分析表明，由α！从不同的联苯双加氧酶募集的β亚基不是由两个亚基中的任一个特异性决定的。
In this work, we have purified the His-tagged oxygenase (ht-oxygenase) component of Rhodococcus globerulus P6 biphenyl dioxygenase. The a or beta subunit of P6 oxygenase was exchanged with the corresponding subunit of Pseudomonas sp, strain LB400 or of Comamonas testosteroni B-356 to create new chimeras that were purified ht-proteins and designated ht-alpha(P6)beta(P6) ht-alpha(P6)beta(LB400), ht-alpha(P6)beta(B-356), ht-alpha(B-356)beta(P6), and ht-alpha(B-356)beta(P6) ht-alpha(P6)beta(P6) ht-alpha(P6)beta(LB400) ht-alpha(P6)beta(B-356) were not expressed active in recombinant Escherichia coli cells carrying P6 bphA1 and bphA2, P6 bphA1 and LB400 bphE, or P6 bphA1 and B-356 bphE because the [2Fe-2S] Rieske cluster of P6 oxygenase a subunit was not assembled correctly in these clones. On the other hand ht-alpha(LB400)beta(P6) and ht-alpha(B-356)beta(P6) were produced active in E, coli. Furthermore, active purified ht-alpha(P6)beta(P6), ht-alpha(P6)beta(LB400) ht-alpha(P6)beta(B-356) showing typical spectra for Rieske-type proteins, were obtained from Pseudomonas putida KT2440 carrying constructions derived from the new shuttle E. coli-Pseudomonas vector pEP31, designed to produce ht-proteins in Pseudomonas. Analysis of the substrate selectivity pattern of these purified chimeras toward selected chlorobiphenyls indicate that the catalytic capacity of hybrid enzymes comprised of an a! and a beta subunit recruited from distinct biphenyl dioxygenases is not determined specifically by either one of the two subunits.