3-溴異丁酸甲酯(methyl 3-bromo-2-methylpropionate, MBMP) 為製備抗高血壓藥物－Captopril、反轉錄病毒蛋白酶抑制劑、環氧合酶抑制劑、非天然胺基酸以及生物鹼的多重對掌性子，在製藥工業上扮演重要的角色。然而，酯水解酵素對MBMP 的鏡像選擇性差導致無法得到高光學純度之單一鏡像異構物是目前最大的瓶頸。
本研究建立在理性蛋白質工程的基礎上，藉由分子對接預測MBMP 與已解出蛋白質結構之偽單孢菌酯水解酵素之交互作用，呈現出酵素-受質之複合體。藉由檢視此複合體，本研究針對受質結合位附近的胺基酸進行定點突變，試圖提升酯水解酵素對MBMP 的鏡像選擇性，來獲得高光學純度之(S) 型產物。與預期之結果相同，偽單孢菌酯水解酵素對MBMP 的鏡像選擇性不高(E = 20 [S])。經由定點突變，本研究成功篩選到高鏡像選擇性的突變株Phe203Trp (E = 67 [S]) 以及Val161Met (E = 51 [S])。Phe203Trp 是藉由色胺酸上的多電子吲哚環與陰電性的甲基溴基團之間有排斥的電子效應的緣故來增加鏡像選擇性。另外，Val161Met 是藉由甲硫胺酸上的硫原子與碳結合之溴原子間有吸引的靜電作用力的關係來增加鏡像選擇性。出乎意料的，雙突變株V161M; F203W 的 E 值為 63 (S)，顯示出結合單突變株的優點沒有加乘效果。另一方面，充分運用電子效應的原理能夠改變酵素的鏡像選擇性，雙突變株V161W; F203L 反轉了酯水解酵素原有的鏡像選擇能力，其對相反的鏡像異構物具有低的鏡像選擇性(E = 2 [R])。
評估高鏡像選擇性之突變株，雙突變株V161M; F203W 具有高度的E 值且比單突變株Phe203Trp 的催化效率要快，實為有潛力運用在製藥工業上得到高光學純度之(S) 型產物，進一步參與藥物的合成。

Methyl 3-bromo-2-methylpropionate (MBMP) is a chiral synthon for the preparation of antihypertension drug－Captopril, retroviral protease inhibitors, cyclooxygenase inhibitors, unnatural amino acids, and alkaloids. MBMP is playing important role in pharmaceutical industry. However, the major bottleneck is the esterase catalyzing hydrolysis of MBMP with low-to-moderate enantioselectivity, which cannot obtain single enantiomer with high optical purity.
In this research, we based on the rational protein engineering and predicted interaction between MBMP and X-ray structure of esterase (EST) from Pseudomonas putida IFO12996 by molecular docking. By the docked EST-MBMP complexes, we focused mutations into the substrate binding site to increase enantioselectivity toward MBMP, getting the (S)-product with high optical purity. As we expected, wild-type EST showed moderate enantioselectivity toward MBMP (E = 20 [S]). By the site-directed mutagenesis, we identified the mutant, Phe203Trp, with high enantioselectivity (E = 67 [S]) and another mutant, Val161Met, also with high enantioselectivity (E = 51 [S]). The Phe203Trp increased enantioselectivity by introducing repulsive electronic effects between the electro-rich indole ring of Trp and the electronegative bromomethyl group. Furthermore, the Val161Met increased enantioselectivity through attractive electrostatic force between the sulfur and carbon-bonded Br. Surprisingly, the E value of double mutant, V161M; F203W, is 63 (S), showing that the individual advantages of single mutant are not additive. On the other hand, change in electronic effects potential controlled enantioselectivity, the double mutant, V161W; F203L, which invert the enantioselectivity of EST, has low enantioselectivity for the opposite enantiomer (E = 2 [R]).
Assessment of EST mutants, the double mutant, V161M; F203W, has relative high E value but better catalytic efficiency than Phe203Trp, so it is really the strongest competitive candidate for pharmaceutical industry to obtain (S)-product with high optical purity.

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