利用定點突變4,5多巴-雙加氧酶探討其受質選擇性及動力學之影響

Abstract

本研究主題就是在紫茉莉花中萃取的4,5-多巴雙加氧酶 (DOPA 4,5-dixoygenase, MjDOD)可催化左旋多巴 (Levodopa, L-DOPA)，但對多巴胺 (Dopamine, DA)也會產生相似的催化，然後運用不同位點突變胺基酸得其突變體，分別針對左旋多巴以及多巴胺進行酵素催化反應使其產生兩種不同的甜菜黃素 (Betaxanthin)，而這兩種不同的甜菜黃素在430 nm有最高吸收鋒，因此可以最終產物甜菜黃素在430 nm的吸收值與濃度作圖，再透過Michaelis–Menten equation得出最大反應速率 (Vmax) 和酵素親和力 (Km)。此外，4,5-多巴雙加氧酶中特定的氨基酸位置突變以改變對於基質的選擇性與活性，從中挑選出對於多巴胺或左旋多巴具有獨特專一性的4,5-多巴雙加氧酶突變體，分別有比起野生型對於L-DOPA更專一的F252Y突變體，還有對於DA有良好選擇性的雙點突變體N249D&F252Y。The 4,5-dopa dioxygenase purified from Mirabilis jalapa can catalyze levodopa (L-DOPA) and dopamine (DA) to produce two different betaxanthins (Betaxanthin). We thus performed site-directed mutagenesis to generate several mutants, and studied kinetics of mutated enzymes for L-DOPA and DA, respectively. In order to improve specificity, we changed the amino acids near the active sites to alter the hydrophilicity and steric barriers of the entire molecule, which results in changes in the binding ability to dopamine or L-DOPA. The four mutants that undergo site-directed mutagenesis were used for whole-cell bioassays. The detailed enzyme kinetics test was carried out. We totally build over 100 mutate strains, and using whole-cell biosensor for high-throughput screening. Finally, we use protein purification and Michaelis–Menten equation, successfully select the dopamine-specific mutation F252Y and the L-DOPA-specific double mutation N249D&F252Y.