删除Loop区域表面不稳定氨基酸提高 (R)-ω-转氨酶热稳定性

Deletion of a dynamic surface loop improves thermostability of (R)-selective amine transaminase from Aspergillus terreus

摘要:

手性胺是一类具有重要价值的医药及精细化工中间体，如何实现手性胺类化合物的不对称合成是目前人们普遍关注的一个焦点问题。ω-转氨酶 (ω-Transaminase，ω-TA) 是一类能直接合成对映体手性胺的天然生物催化剂。相比于 (S)-ω-TA，(R)-ω-TA的研究较少，但其需求量随着手性胺类药物的发展日趋增大。提高具有潜在应用价值的 (R)-ω-TA的热稳定性，将有利于手性胺的制备。本文利用PyMOL软件和YASARA软件预测来源于土曲霉Aspergillus terreus的 (R)-ω-TA中具有高温度因子 (B-factor) 的Loop区域，通过定点突变对Loop区域表面不稳定氨基酸逐步进行删除获得突变酶。结果表明，突变酶R131del和突变酶P132-E133del半失活温度分别为41.1 ℃和39.4 ℃，比野生酶提高了2.6 ℃和0.9 ℃；在40 ℃下的半衰期分别为15.0 min和10.0 min，为野生酶的2.2倍和1.5倍。此外，在400 K和10 ns的分子模拟条件下，突变酶R131del在Loop区域的均方根涨落 (Root mean square fluctuation，RMSF) 比野生型低，突变酶P132-E133del在Loop区域增加了4个氢键。本研究通过删除 (R)-ω-转氨酶Loop区域表面不稳定氨基酸提高了该蛋白的热稳定性，同时也为其他酶热稳定性的理性设计提供了方法学指导。

Abstract:

Chiral amines are important building blocks for the synthesis of pharmaceutical products and fine chemicals. Highly stereoselective synthesis of chiral amines compounds through asymmetric amination has attracted more and more attention. ω-transaminases (ω-TAs) are a promising class of natural biocatalysts which provide an efficient and environment-friendly access to production of chiral amines with stringent enantioselectivity and excellent catalytic efficiency. Compared with (S)-ω-TA, the research focused on (R)-ω-TA was relatively less. However, increasing demand for chiral (R)-amines as pharmaceutical intermediates has rendered industrial applications of (R)-ω-TA more attractive. Improving the thermostability of (R)-ω-TA with potential biotechnological application will facilitate the preparation of chiral amines. In this study, the dynamic surface loop with higher B-factor from Aspergillus terreus (R)-ω-TA was predicted by two computer softwares (PyMOL and YASARA). Then mutant enzymes were obtained by deleting amino acid residues of a dynamic surface loop using site-directed mutagenesis. The results showed that the best two mutants R131del and P132-E133del improved thermostability by 2.6 ℃ and 0.9 ℃ in T5010 (41.1 ℃ and 39.4 ℃, respectively), and 2.2-fold and 1.5-fold in half-life (t1/2) at 40 ℃ (15.0 min and 10.0 min, respectively), compared to that of wild type. Furtherly, the thermostability mechanism of the mutant enzymes was investigated by molecular dynamics (MD) simulation and intermolecular interaction analysis. R131del in the loop region has lower root mean square fluctuation (RMSF) than the wild type at 400 K for 10 ns, and mutant enzyme P132-E133del increases four hydrogen bonds in the loop region. In this study, we obtain two stability-increased mutants of (R)-ω-TA from A. terreus by deleting its dynamic surface loop and also provide methodological guidance for the use of rational design to enhance the thermal stability of other enzymes.