随机-半理性组合突变改造ω-转氨酶催化合成(R)-1-(1-萘基)乙胺

Engineering ω-transaminase by random mutagenesis and semi-rational design for the synthesis of (R)-(+)-1-(1-naphthyl)ethylamine

摘要:

(R)-1-(1-萘基)乙胺是合成拟钙剂药物盐酸西那卡塞的关键手性中间体，利用ω-转氨酶不对称还原1-萘乙酮合成(R)-1-(1-萘基)乙胺具有较好的应用前景。文中针对节杆菌属Arthrobacter sp.来源的ω-转氨酶，采用随机突变和半理性设计相结合的策略，获得了催化效率和热稳定性提高的突变酶F225M、C281I和F225M/C281I。与WT相比，双突变体F225M/C281I的kcat提高85%，Km下降56%，催化效率kcat/Km提高3.42倍。此外，F225M/C281I催化10 mmol/L 1-萘乙酮反应24 h的转化率提高了22%。分子对接和分子动力学模拟结果表明，F225M/C281I相比于WT增加了与底物1-萘乙酮之间的Pi-Pi相互作用力，导致其催化效率的提高；而且突变酶F225M/C281I的134–139位点残基的均方根波动 (RMSF) 相比WT明显降低，与半衰期的略微提高相关。

Abstract:

(R)-(+)-1-(1-naphthyl)ethylamine is a key chiral intermediate for the synthesis of calcimimetic drug cinacalcet hydrochloride. ω-Transaminase has been considered to be potential for producing (R)-(+)-1-(1-naphthyl)ethylamine by asymmetric reduction of 1-acetonaphthone. Here, ω-transaminase from Arthrobacter sp. was engineered by combinatorial strategies of random mutagenesis and semi-rational design. Variants F225M, C281I, F225M/C281I with improved catalytic efficiency and thermostability were obtained. Compared with WT, variant F225M/C281I showed 85% increased kcat, 56% decreased Km and 3.42-fold kcat/Km. Furthermore, 22% higher conversion rate was achieved by F225M/C281I at 10 mmol/L 1-acetonaphthone after 24 h. Based on molecular docking and molecular dynamics simulation, improved catalytic efficiency of F225M/C281I could be attributed to its increased Pi-Pi T-shaped interaction with substrate 1-acetonaphthone. Additionally, a slightly higher half-life of F225M/C281I was validated by its lower root-mean-square fluctuation (RMSF) value of loop 134–139 compared with WT.