Engineering protonation conformation of l-aspartate-α-decarboxylase to relieve mechanism-based inactivation
Engineering protonation conformation of l-aspartate-α-decarboxylase to relieve mechanism-based inactivation
复制标题
DOI：
10.1002/bit.27316
复制
发表时间：
2020-03-02
影响因子：
3.8
通讯作者：
Wu, Jing
中科院分区：
文献类型：
--
作者：
Qian, Yuanyuan;Lu, Cui;Wu, Jing
l-天冬氨酸脱羧酶（PanD）失活机制导致活性位点的不可逆修饰，是l-天冬氨酸高效生产-丙氨酸的主要挑战。本研究采用构象动力学和结构定位相结合的半理性策略提高枯草芽孢杆菌PanD （BsPanD）的催化稳定性。通过位点饱和和c端缺失，生成了Q5 （BsPanD(I46V/I88M/K104S/I126*)）。Q5的催化半衰期和总周转率（TTN）分别是母体Q0的3.48倍和2.52倍。造成差异的原因是Tyr58的酚基与Ser25的丙酮基之间的距离d1延长（Q0为4.9埃，Q5为5.5埃），错误质子化发生的难度增加，A、B、C区残基的柔韧性降低，从而提高了正确质子化的概率。变异Q5与L -天冬氨酸酶（AspA）在15l生物反应器中偶联，生成了以富马酸为底物的线性级联体系，产β -丙氨酸为118.6 g/L，产物/催化剂（P/C）比为5.9 g/g，转化率为99%。这些结果表明，重塑PanD的质子化构象可以有效地缓解机制失活，提高催化稳定性。
Mechanism-based inactivation of l-aspartate-alpha-decarboxylase (PanD), which leads to irreversible modification of active site, is a major challenge in the efficient production of beta-alanine from L-aspartic acid. In this study, a semi-rational strategy that combined conformational dynamics and structural alignment was applied to increase the catalytic stability of Bacillus subtilis PanD (BsPanD). Using site-saturation and C-terminal deletion, the variant Q5 (BsPanD(I46V/I88M/K104S/I126*)) was generated. The catalytic half-life and the total turnover number (TTN) of Q5 were 3.48-fold and 2.52-fold higher, respectively, compared with that of the parent Q0. The reasons for the differences were the prolonged distance d1 between the phenolic group of Tyr58 and pyruvoyl group of Ser25 (4.9 angstrom in Q0 vs. 5.5 angstrom in Q5), an increased difficulty for incorrect protonation to occur, and the decreased flexibility of residues in regions A, B, and C, thereby enhancing the probability of correct protonation. Variant Q5, coupled with l-aspartase (AspA) in a 15-L bioreactor, generated a linear cascade system using fumaric acid as a substrate, yielding 118.6 g/L beta-alanine with a product/catalyst (P/C) ratio of 5.9 g/g and a conversion > 99%. These results showed that reshaping the protonation conformation of PanD can efficiently relieve mechanism-based inactivation and boost catalytic stability.