Gómez-Gil Leticia, Kumar Pravindra, Barriault Diane, Bolin Jeffrey T, Sylvestre Michel, Eltis Lindsay D
Department of Microbiology, Life Sciences Institute, University of British Columbia, Vancouver, BC V6T 1Z3, Canada.
J Bacteriol. 2007 Aug;189(15):5705-15. doi: 10.1128/JB.01476-06. Epub 2007 May 25.
Biphenyl dioxygenase (BPDO) catalyzes the aerobic transformation of biphenyl and various polychlorinated biphenyls (PCBs). In three different assays, BPDO(B356) from Pandoraea pnomenusa B-356 was a more potent PCB-degrading enzyme than BPDO(LB400) from Burkholderia xenovorans LB400 (75% amino acid sequence identity), transforming nine congeners in the following order of preference: 2,3',4-trichloro approximately 2,3,4'-trichloro > 3,3'-dichloro > 2,4,4'-trichloro > 4,4'-dichloro approximately 2,2'-dichloro > 2,6-dichloro > 2,2',3,3'-tetrachloro approximately 2,2',5,5'-tetrachloro. Except for 2,2',5,5'-tetrachlorobiphenyl, BPDO(B356) transformed each congener at a higher rate than BPDO(LB400). The assays used either whole cells or purified enzymes and either individual congeners or mixtures of congeners. Product analyses established previously unrecognized BPDO(B356) activities, including the 3,4-dihydroxylation of 2,6-dichlorobiphenyl. BPDO(LB400) had a greater apparent specificity for biphenyl than BPDO(B356) (k(cat)/K(m) = 2.4 x 10(6) +/- 0.7 x 10(6) M(-1) s(-1) versus k(cat)/K(m) = 0.21 x 10(6) +/- 0.04 x 10(6) M(-1) s(-1)). However, the latter transformed biphenyl at a higher maximal rate (k(cat) = 4.1 +/- 0.2 s(-1) versus k(cat) = 0.4 +/- 0.1 s(-1)). A variant of BPDO(LB400) containing four active site residues of BPDO(B356) transformed para-substituted congeners better than BPDO(LB400). Interestingly, a substitution remote from the active site, A267S, increased the enzyme's preference for meta-substituted congeners. Moreover, this substitution had a greater effect on the kinetics of biphenyl utilization than substitutions in the substrate-binding pocket. In all variants, the degree of coupling between congener depletion and O(2) consumption was approximately proportional to congener depletion. At 2.4-A resolution, the crystal structure of the BPDO(B356)-2,6-dichlorobiphenyl complex, the first crystal structure of a BPDO-PCB complex, provided additional insight into the reactivity of this isozyme with this congener, as well as into the differences in congener preferences of the BPDOs.
联苯双加氧酶（BPDO）催化联苯及各种多氯联苯（PCBs）的需氧转化。在三种不同的测定中，来自洋葱伯克霍尔德氏菌B-356的BPDO（B356）比来自嗜麦芽窄食单胞菌LB400的BPDO（LB400）（氨基酸序列同一性为75%）是一种更强效的多氯联苯降解酶，它按照以下偏好顺序转化9种同系物：2,3',4-三氯联苯≈2,3,4'-三氯联苯＞3,3'-二氯联苯＞2,4,4'-三氯联苯＞4,4'-二氯联苯≈2,2'-二氯联苯＞2,6-二氯联苯＞2,2',3,3'-四氯联苯≈2,2',5,5'-四氯联苯。除2,2',5,5'-四氯联苯外，BPDO（B356）转化每种同系物的速率都高于BPDO（LB400）。测定使用了全细胞或纯化酶，以及单个同系物或同系物混合物。产物分析确定了以前未被认识的BPDO（B356）活性，包括2,6-二氯联苯的3,4-二羟基化。BPDO（LB400）对联苯的表观特异性高于BPDO（B356）（k(cat)/K(m)=2.4×10⁶±0.7×10⁶ M⁻¹ s⁻¹，而k(cat)/K(m)=0.21×10⁶±0.04×10⁶ M⁻¹ s⁻¹）。然而，后者转化联苯的最大速率更高（k(cat)=4.1±0.2 s⁻¹，而k(cat)=0.4±0.1 s⁻¹）。含有BPDO（B356）四个活性位点残基的BPDO（LB400）变体转化对位取代同系物的能力优于BPDO（LB400）。有趣的是，一个远离活性位点的取代A267S增加了该酶对间位取代同系物的偏好。此外，这种取代对联苯利用动力学的影响比对底物结合口袋中取代的影响更大。在所有变体中，同系物消耗与氧气消耗之间的偶联程度与同系物消耗大致成比例。在2.4埃分辨率下，BPDO（B356）-2,6-二氯联苯复合物的晶体结构，即第一个BPDO-多氯联苯复合物的晶体结构，为该同工酶与这种同系物的反应性以及BPDO对同系物偏好的差异提供了更多见解。
