Characterization of Chlorophenol 4-Monooxygenase (TftD) and NADH:FAD Oxidoreductase (TftC) of Burkholderia cepacia AC1100
Characterization of Chlorophenol 4-Monooxygenase (TftD) and NADH:FAD Oxidoreductase (TftC) of Burkholderia cepacia AC1100
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DOI：
10.1074/jbc.m109.056135
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发表时间：
2010-01-15
影响因子：
4.8
通讯作者：
Kang, ChulHee
中科院分区：
文献类型：
--
作者：
Webb, Brian N.;Ballinger, Jordan W.;Kang, ChulHee
洋葱伯克霍尔德氏菌AC1100可完全降解2,4,5 - 三氯苯酚，在此过程中，一种依赖FADH₂的单加氧酶（TftD）和一种NADH：FAD氧化还原酶（TftC）催化起始步骤。TftD将2,4,5 - 三氯苯酚（2,4,5 - TCP）氧化为2,5 - 二氯 - 对 - 苯醌，后者经化学还原为2,5 - 二氯 - 对 - 氢醌（2,5 - DiCHQ）。然后，TftD将后者氧化为5 - 氯 - 2 - 羟基 - 对 - 苯醌。在这些过程中，TftC提供所有所需的FADH₂。我们分别测定了二聚体TftC和四聚体TftD的晶体结构，分辨率分别为2.0埃和2.5埃。TftC的结构与相关的黄素还原酶相似。TftC中堆叠的烟酰胺：异咯嗪环以及连续反应动力学表明，还原态的FAD在NADH氧化后离开TftC。TftD的结构也与已知的依赖FADH₂的单加氧酶结构相似。其在异咯嗪环re侧的His - 289残基与2,5 - DiCHQ的一个羟基处于氢键距离内。H289A突变导致对2,5 - DiCHQ的活性完全丧失，对2,4,5 - TCP的催化效率显著降低。因此，His - 289在2,4,5 - TCP和2,5 - DiCHQ的催化中起不同作用。这些结果支持TftC产生游离的FADH₂，并且TftD利用FADH₂分别转化2,4,5 - TCP和2,5 - DiCHQ。其他实验数据也支持FADH₂在TftC和TftD之间扩散，而这两种酶之间没有直接的物理相互作用。
Burkholderia cepacia AC1100 completely degrades 2,4,5-trichlorophenol, in which an FADH(2)-dependent monooxygenase (TftD) and an NADH: FAD oxidoreductase (TftC) catalyze the initial steps. TftD oxidizes 2,4,5-trichlorophenol (2,4,5-TCP) to 2,5-dichloro-p-benzoquinone, which is chemically reduced to 2,5-dichloro-p-hydroquinone (2,5-DiCHQ). Then, TftD oxidizes the latter to 5-chloro-2-hydroxy-p-benzoquinone. In those processes, TftC provides all the required FADH(2). We have determined the crystal structures of dimeric TftC and tetrameric TftD at 2.0 and 2.5 angstrom resolution, respectively. The structure of TftC was similar to those of related flavin reductases. The stacked nicotinamide: isoalloxazine rings in TftC and sequential reaction kinetics suggest that the reduced FAD leaves TftC after NADH oxidation. The structure of TftD was also similar to the known structures of FADH(2)-dependent monooxygenases. Its His-289 residue in the re-side of the isoalloxazine ring is within hydrogen bonding distance with a hydroxyl group of 2,5-DiCHQ. An H289 A mutation resulted in the complete loss of activity toward 2,5-DiCHQ and a significant decrease in catalytic efficiency toward 2,4,5-TCP. Thus, His-289 plays different roles in the catalysis of 2,4,5-TCP and 2,5-DiCHQ. The results support that free FADH(2) is generated by TftC, and TftD uses FADH(2) to separately transform 2,4,5-TCP and 2,5-DiCHQ. Additional experimental data also support the diffusion of FADH(2) between TftC and TftD without direct physical interaction between the two enzymes.