Liang Yanqiu, Wang Qiong, Lu Jiapeng, Wang Yi, Liang Quanfeng, Luo Wei
The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, PR China.
Department of Biological and Agricultural Engineering, University of California, Davis, 1 Shields Ave, Davis, CA 95616, USA.
Int J Biol Macromol. 2025 Feb;288:138469. doi: 10.1016/j.ijbiomac.2024.138469. Epub 2024 Dec 5.
Leucine dehydrogenase (LeuDH) is a promising enzyme for the industrial production of L-tert-leucine (L-Tle), but its catalytic activity toward trimethylpyruvate (TMP) requires enhancement. In this study, we employed a semi-rational design approach involving homology modeling of LeuDH from Exiguobacterium sibiricum (EsiLeuDH) and molecular docking with TMP to predict potential mutation sites. These sites were tested using an alanine scanning strategy to assess their impact on enzymatic activity, followed by site-saturation mutagenesis and iterative saturation mutagenesis. The resulting mutant, EsiLeuDH-M3, exhibited a remarkable 306 % increase in specific enzymatic activity (104.69 U·mg), compared to the wild-type EsiLeuDH (WT). Molecular docking indicated that EsiLeuDH-M3 had an increased number of hydrogen bonds, improved stability, and an enlarged substrate-binding pocket. Moreover, molecular dynamics simulations suggested that EsiLeuDH-M3 possessed a more stable conformation but a more flexible pocket, allowing TMP to access the catalytic center more easily. Experiments examining the effects of different substrate concentrations on TMP bioconversion catalyzed by WT and EsiLeuDH-M3 indicated that EsiLeuDH-M3 tolerated higher TMP concentrations than the WT enzyme. Finally, L-Tle was produced using EsiLeuDH-M3 coupled with an NADH regeneration system, achieving a high conversion rate (91 %) of TMP at a substrate concentration of 0.7 M, which is expected to reduce production costs in the industrial application of L-Tle.
亮氨酸脱氢酶（LeuDH）是工业生产L-叔亮氨酸（L-Tle）的一种很有前景的酶，但它对三甲基丙酮酸（TMP）的催化活性需要提高。在本研究中，我们采用了一种半理性设计方法，包括对西伯利亚栖热菌（EsiLeuDH）的LeuDH进行同源建模以及与TMP进行分子对接，以预测潜在的突变位点。使用丙氨酸扫描策略测试这些位点对酶活性的影响，随后进行位点饱和诱变和迭代饱和诱变。所得突变体EsiLeuDH-M3与野生型EsiLeuDH（WT）相比，比酶活显著提高了306%（104.69 U·mg）。分子对接表明，EsiLeuDH-M3的氢键数量增加，稳定性提高，底物结合口袋扩大。此外，分子动力学模拟表明，EsiLeuDH-M3具有更稳定的构象但口袋更灵活，使TMP更容易进入催化中心。研究不同底物浓度对WT和EsiLeuDH-M3催化的TMP生物转化影响的实验表明，EsiLeuDH-M3比WT酶能耐受更高的TMP浓度。最后，使用EsiLeuDH-M3与NADH再生系统耦合生产L-Tle，在底物浓度为0.7 M时，TMP的转化率达到了91%，有望降低L-Tle工业应用中的生产成本。
