From the School of Biomolecular Science and Engineering, Vidyasirimedhi Institute of Science and Technology (VISTEC), Wangchan Valley, Rayong 21210 and.
the Department of Biochemistry and Center for Excellence in Protein and Enzyme Technology, Faculty of Science, Mahidol University, Bangkok 14000, Thailand.
J Biol Chem. 2018 Nov 30;293(48):18525-18539. doi: 10.1074/jbc.RA118.005538. Epub 2018 Oct 3.
Halogenated phenol and nitrophenols are toxic compounds that are widely accumulated in the environment. Enzymes in the operon from the bacterium DTP0602 have the potential for application as biocatalysts in the degradation of many of these toxic chemicals. HadA monooxygenase previously was identified as a two-component reduced FAD (FADH)-utilizing monooxygenase with dual activities of dehalogenation and denitration. However, the partner enzymes of HadA, that is, the flavin reductase and quinone reductase that provide the FADH for HadA and reduce quinone to hydroquinone, remain to be identified. In this report, we overexpressed and purified the flavin reductases, HadB and HadX, to investigate their functional and catalytic properties. Our results indicated that HadB is an FMN-dependent quinone reductase that converts the quinone products from HadA to hydroquinone compounds that are more stable and can be assimilated by downstream enzymes in the pathway. Transient kinetics indicated that HadB prefers NADH and menadione as the electron donor and acceptor, respectively. We found that HadX is an FAD-bound flavin reductase, which can generate FADH for HadA to catalyze dehalogenation or denitration reactions. Thermodynamic and transient kinetic experiments revealed that HadX prefers to bind FAD over FADH and that HadX can transfer FADH from HadX to HadA via free diffusion. Moreover, HadX rapidly catalyzed NADH-mediated reduction of flavin and provided the FADH for a monooxygenase of a different system. Combination of all three flavin-dependent enzymes, HadA/HadB/HadX, reconstituted an effective dehalogenation and denitration cascade, which may be useful for future bioremediation applications.
卤代酚和硝基酚是广泛积累在环境中的有毒化合物。来自细菌 DTP0602 的操纵子中的酶具有作为许多这些有毒化学物质降解的生物催化剂的应用潜力。HadA 单加氧酶先前被鉴定为一种具有两种活性的二组分还原 FAD（FADH）利用单加氧酶，具有脱卤和脱硝双重活性。然而，HadA 的伴侣酶，即提供 FADH 的黄素还原酶和醌还原酶，以及将醌还原为氢醌，仍有待鉴定。在本报告中，我们过表达和纯化了黄素还原酶 HadB 和 HadX，以研究它们的功能和催化特性。我们的结果表明，HadB 是一种 FMN 依赖性醌还原酶，它将 HadA 的醌产物转化为更稳定的氢醌化合物，这些化合物可以被途径中的下游酶同化。瞬变动力学表明，HadB 优先选择 NADH 和甲萘醌分别作为电子供体和受体。我们发现，HadX 是一种 FAD 结合的黄素还原酶，它可以产生 FADH 供 HadA 催化脱卤或脱硝反应。热力学和瞬变动力学实验表明，HadX 优先与 FAD 结合而不是 FADH，并且 HadX 可以通过自由扩散将 FADH 从 HadX 转移到 HadA。此外，HadX 可快速催化黄素介导的 NADH 还原，并为不同系统的单加氧酶提供 FADH。三种黄素依赖性酶（HadA/HadB/HadX）的组合，重建了一个有效的脱卤和脱硝级联反应，这可能对未来的生物修复应用有用。
