Elucidation of the structural and mechanistic basis of pollutants-and signal-transmitter-degrading dioxygenases and their directed evolution

1.测定了嗜热嗜热菌HB8高原儿茶酸2，3-双加氧酶的晶体结构。对HPCD进行了功能表征。该酶对过氧化氢表现出异常的耐受性，对O&lt；2-&Gt；2具有较高的K_m值。用改进的测定方法测定了邻苯二酚2，3-双加氧酶(MPC)和2，3-二羟基联苯1，2-双加氧酶(BPHC)的动力学参数和底物结合常数。基于晶体结构的功能性质比较表明，在BPHC的情况下，C3-取代基与活性中心残基的相互作用促进了底物的结合，在MPC的情况下，C3取代基的作用促进了底物的解离。我们证明了底物结合率与取代基的吸电子性质、取代基对底物络合物(EA络合物)稳定性的空间效应呈负相关，与…呈正相关更重要的是MPC-苯酚络合物(EI络合物)的稳定性和取代基的吸电子性质。这些结果支持一种假设，即儿茶酚的结合是通过类似于EI络合物的中间体进行的，在该中间体中，儿茶酚以单齿方式与活性中心亚铁离子结合。我们发现MPC与EA络合物的氧结合速率与取代基的吸电子性质呈负相关。结果表明，邻苯二酚通过铁中心向O_2的单电子转移激活了O_2的后续反应。MPC的氧结合部位是由底物Hisl99、A202、Leu155和Phe191的苯环形成的。在BPHC中，Leu155分别被Val取代，在HPCD中，分别被Asn取代。我们发现C4取代的儿茶酚对BPHC的灭活作用类似于自杀。BPHC-4-甲基儿茶酚络合物的晶体结构显示了与3-苯基儿茶酚不同的异常双齿结合模式。综上所述，我们揭示了底物识别和O_2活化的结构和机理基础。基于合理的设计，外源性双加氧酶的定向进化现在是可能的。较少

1. Crystal structure of homoprotocatechuate 2,3-dioxygenase from Thermus thermophilus HB8 (HPCD) has been determined. Functional characterization of HPCD has been carried out. The enzyme shows unusual tolerance for hydrogen peroxide and a large K_m value for O_<2->2. The kinetic parameters and substrate binding constants of catechol 2,3-dioxygenase (Mpc) and 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC) have been determined using improved assay methods. Comparison of the functional properties on the basis of their crystal structures has revealed that the interaction of C3-substituent group with the active site residues facilitates substrate binding in the case of BphC and substrate dissociation in the case of Mpc, respectively.3. We have demonstrated negative correlation between the substrate binding rate of Mpc and electron-withdrawing nature of the substituent, steric effect of the substituent on the stability of the Mpc-substrate complex (EA complex), and positive correlation between … More the stability of Mpc-phenol complex (EI complex) and electron-withdrawing nature of the substituent. These results support a hypothesis that the binding of catechol proceeds through an intermediate resembling to EI complex in which catechol binds monodentately to the active site ferrous ion.4. We have found negative correlation between the rate of oxygen binding to EA complex of Mpc and electron-withdrawing nature of the substituent. The result indicates that one-electron transfer from catechol to O_2 via the iron center activatesO_2 for the subsequent reactions. The oxygen binding site of Mpc is formed by the benzene ring of substrate, Hisl99, A202, Leu155, and Phe191. Leu155 is replaced by Val in that of BphC, and by Asn in that of HPCD, respectively.5. We have found suicide-like inactivation of BphC by C4-substituted catechols. Crystal structure of the BphC-4-methylcatechol complex has revealed an aberrant bidentate binding mode different from that of 3-phenylcatechol.In conclusion, in the present study, we have revealed structural and mechanistic basis of substrate recognition and O_2 activation. Directed evolution of extradiol dioxygenases is now possible based on rational design. Less