Hurtubise Y, Barriault D, Sylvestre M
Institut National de la Recherche Scientifique-Santé, Pointe-Claire, Québec, H9R 1G6 Canada.
J Bacteriol. 1998 Nov;180(22):5828-35. doi: 10.1128/JB.180.22.5828-5835.1998.
Biphenyl dioxygenase (BPH dox) oxidizes biphenyl on adjacent carbons to generate 2,3-dihydro-2,3-dihydroxybiphenyl in Comamonas testosteroni B-356 and in Pseudomonas sp. strain LB400. The enzyme comprises a two-subunit (alpha and beta) iron sulfur protein (ISPBPH), a ferredoxin (FERBPH), and a ferredoxin reductase (REDBPH). B-356 BPH dox preferentially catalyzes the oxidation of the double-meta-substituted congener 3,3'-dichlorobiphenyl over the double-para-substituted congener 4,4'-dichlorobiphenyl or the double-ortho-substituted congener 2,2'-dichlorobiphenyl. LB400 BPH dox shows a preference for 2,2'-dichlorobiphenyl, and in addition, unlike B-356 BPH dox, it can catalyze the oxidation of selected chlorobiphenyls such as 2,2',5,5'-tetrachlorobiphenyl on adjacent meta-para carbons. In this work, we examine the reactivity pattern of BPH dox toward various chlorobiphenyls and its capacity to catalyze the meta-para dioxygenation of chimeric enzymes obtained by exchanging the ISPBPH alpha or beta subunit of strain B-356 for the corresponding subunit of strain LB400. These hybrid enzymes were purified by an affinity chromatography system as His-tagged proteins. Both types, the chimera with the alpha subunit of ISPBPH of strain LB400 and the beta subunit of ISPBPH of strain B-356 (the alphaLB400 betaB-356 chimera) and the alphaB-356betaLB400 chimera, were functional. Results with purified enzyme preparations showed for the first time that the ISPBPH beta subunit influences BPH dox's reactivity pattern toward chlorobiphenyls. Thus, if the alpha subunit were the sole determinant of the enzyme reactivity pattern, the alphaB-356betaLB400 chimera should have behaved like B-356 ISPBPH; instead, its reactivity pattern toward the substrates tested was similar to that of LB400 ISPBPH. On the other hand, the alphaLB400 betaB-356 chimera showed features of both B-356 and LB400 ISPBPH where the enzyme was able to metabolize 2,2'- and 3, 3'-dichlorobiphenyl and where it was able to catalyze the meta-para oxygenation of 2,2',5,5'-tetrachlorobiphenyl.
在睾丸酮丛毛单胞菌B - 356和假单胞菌属菌株LB400中，联苯双加氧酶（BPH dox）将联苯相邻碳上的原子氧化，生成2,3 - 二氢 - 2,3 - 二羟基联苯。该酶由一个双亚基（α和β）铁硫蛋白（ISPBPH）、一个铁氧化还原蛋白（FERBPH）和一个铁氧化还原蛋白还原酶（REDBPH）组成。与双对位取代的同系物4,4'-二氯联苯或双邻位取代的同系物2,2'-二氯联苯相比，B - 356 BPH dox优先催化双间位取代的同系物3,3'-二氯联苯的氧化反应。LB400 BPH dox表现出对2,2'-二氯联苯的偏好，此外，与B - 356 BPH dox不同的是，它可以催化选定的氯代联苯（如2,2',5,5'-四氯联苯）在相邻间位 - 对位碳上的氧化反应。在这项研究中，我们研究了BPH dox对各种氯代联苯的反应模式，以及它催化通过将菌株B - 356的ISPBPHα或β亚基与菌株LB400的相应亚基交换而获得的嵌合酶进行间位 - 对位双加氧反应的能力。这些杂合酶通过亲和色谱系统作为带有组氨酸标签的蛋白质进行纯化。两种类型，即具有菌株LB400的ISPBPHα亚基和菌株B - 356的ISPBPHβ亚基的嵌合体（αLB400βB - 356嵌合体）和αB - 356βLB400嵌合体，都是有功能的。纯化酶制剂的结果首次表明，ISPBPHβ亚基影响BPH dox对氯代联苯的反应模式。因此，如果α亚基是酶反应模式的唯一决定因素，αB - 356βLB400嵌合体应该表现得像B - 356 ISPBPH；相反，它对所测试底物的反应模式与LB400 ISPBPH相似。另一方面，αLB400βB - 356嵌合体表现出B - 356和LB400 ISPBPH的特征，该酶能够代谢2,2'-和3,3'-二氯联苯，并且能够催化2,2',5,5'-四氯联苯的间位 - 对位加氧反应。
