Zhang Dong, Zhu Xiuxiu, Hu Die, Wen Zheng, Zhang Chen, Wu Minchen
Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122 China.
School of Pharmaceutical Science, Jiangnan University, Wuxi, 214122 China.
3 Biotech. 2021 Feb;11(2):69. doi: 10.1007/s13205-020-02633-3. Epub 2021 Jan 13.
To enhance the specific activity and catalytic efficiency ( / ) of an NADH-dependent PPR, its directed modification was performed based on the computer-aided design using molecular docking simulation and multiple sequence alignment. Firstly, five single-site variants of an PPR-encoding gene () were amplified and expressed in BL21 (DE3). The asymmetric reduction of 20 mM phenylpyruvic acid (PPA) was carried out using 50 mg/mL / or / whole wet cells at 37 °C for 20 min, giving d-phenyllactic acid (PLA) with 41.1 or 44.3% yield, being 1.17- or 1.26-fold that by /. Secondly, double-site variants were obtained by saturation mutagenesis of Ala79 in PPR. Among all tested transformants, / exhibited the highest d-PLA yield of 85.3%. The specific activity and / of the purified PPR increased to 67.5 U/mg and 169.8 mM s, which were 3.0- and 13.2-fold those of PPR, respectively. Finally, the catalytic mechanism analysis of PPR by molecular docking simulation indicated that the replacement of Arg53 in PPR with Gln expanded its substrate-binding pocket, while that Ala79 with Val formed an additional π-sigma interaction with phenyl group of PPA.
The online version of this article (10.1007/s13205-020-02633-3) contains supplementary material, which is available to authorized users.
为提高依赖烟酰胺腺嘌呤二核苷酸（NADH）的丙酮酸还原酶（PPR）的比活性和催化效率（/），基于计算机辅助设计，利用分子对接模拟和多序列比对进行了定向改造。首先，扩增了PPR编码基因（）的5个单点突变体，并在大肠杆菌BL21（DE3）中表达。在37℃下，使用50mg/mL的/或/全湿细胞对20mM苯丙酮酸（PPA）进行不对称还原20分钟，得到d-苯乳酸（PLA），产率分别为41.1%或44.3%，是/产率的1.17倍或1.26倍。其次，通过对PPR中Ala79进行饱和诱变获得了双点突变体。在所有测试的转化体中，/的d-PLA产率最高，为85.3%。纯化后的PPR的比活性和/分别提高到67.5U/mg和169.8mM·s，分别是PPR的3.0倍和13.2倍。最后，通过分子对接模拟对PPR的催化机制分析表明，用谷氨酰胺取代PPR中的精氨酸53扩大了其底物结合口袋，而用缬氨酸取代丙氨酸79与PPA的苯基形成了额外的π-σ相互作用。
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