College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China.
College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, China.
Int J Biol Macromol. 2024 Apr;264(Pt 2):130484. doi: 10.1016/j.ijbiomac.2024.130484. Epub 2024 Feb 29.
The oxidative reaction of Fusarium mycotoxin deoxynivalenol (DON) using the dehydrogenase is a desirable strategy and environmentally friendly to mitigate its toxicity. However, a critical issue for these dehydrogenases shows widespread substrate promiscuity. In this study, we conducted pocket reshaping of Devosia strain A6-243 pyrroloquinoline quinone (PQQ)-dependent dehydrogenase (DADH) on the basis of protein structure and kinetic analysis of substrate libraries to improve preference for particular substrate DON (10a). The variant presented an increased preference for substrate 10a and enhanced catalytic efficiency. A 4.7-fold increase in preference for substrate 10a was observed. Kinetic profiling and molecular dynamics (MD) simulations provided insights into the enhanced substrate specificity and activity. Moreover, the variant exhibited stronger conversion of substrate 10a to 3-keto-DON compared to the wild DADH. Overall, this study provides a feasible protocol for the redesign of PQQ-dependent dehydrogenases with favourable substrate specificity and catalytic activity, which is desperately needed for DON antidote development.
利用脱氢酶使镰刀菌霉菌毒素脱氧雪腐镰刀菌烯醇（DON）发生氧化反应是一种可取的策略，并且对环境友好，可以减轻其毒性。然而，这些脱氢酶的一个关键问题是表现出广泛的底物混杂性。在这项研究中，我们根据蛋白质结构和底物文库的动力学分析，对德沃斯氏菌 A6-243 吡咯喹啉醌（PQQ）依赖性脱氢酶（DADH）进行口袋重塑，以提高对特定底物 DON（10a）的偏好性。该变体对底物 10a 的偏好性增加，催化效率提高。观察到对底物 10a 的偏好性增加了 4.7 倍。动力学分析和分子动力学（MD）模拟提供了对增强的底物特异性和活性的深入了解。此外，与野生型 DADH 相比，该变体显示出更强的将底物 10a 转化为 3-酮-DON 的能力。总的来说，这项研究为具有有利的底物特异性和催化活性的 PQQ 依赖性脱氢酶的重新设计提供了可行的方案，这是开发 DON 解毒剂所急需的。
