臺灣杉單萜合成酶基因選殖與單萜二次環化機制

Abstract

萜類合成酶(terpene synthases)的蛋白質結構與其功能性間之關聯性，長期以來一直是相關酵素改質工程所面臨的最大挑戰。本研究首先藉由設計退化性引子(degenerate primer)、聚合酶連鎖反應(polymerase chain reaction, PCR)與cDNA末端快速增幅(rapid amplification of cDNA ends, RACE)等方式，順利地自臺灣杉(Taiwania cryptomerioides)葉部選殖到單萜合成酶基因Tc-αpin/teo與Tc-teo；再透過比較兩者胺基酸序列與產物上的差異性，輔以同源模擬(homology modeling)及定點突變(site-directed mutagenesis)等技術，成功地在Tc-αPIN/TEO蛋白質結構中，界定出三個對萜類(terpenes)二次環化機制(secondary cyclization mechanism)有決定性影響力的塑性殘基(plasticity residues)，分別為Y327、Y429與Y575。將這些tyrosine突變成phenylalanine的試驗結果顯示，其胺基酸支鏈(side chain)上之hydroxyl groups，可能藉由協同作用(synergistic effect)共同幫助碳陽離子(carbocation)進行二次環化。這些發現，或許能使我們對於複雜的萜類生合成步驟有更深入的瞭解，並作為未來蛋白質工程(protein engineering)試驗設計之參考。

The ambiguous protein structure-function correlation of terpene synthases has been a challenge for protein engineers. Thus, we intended to decipher the determinants associated with the product specificity of terpene synthases. Using polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) methods, we successfully cloned two monoterpene synthase genes, Tc-αpin/teo and Tc-teo, from Taiwania cryptomerioides. The enzymes encoded by these genes shared 97% similarity of amino acid sequences, but had different terpene product profiles. We adopted homology modeling to compare the structural differences between Tc-αPIN/TEO and Tc-TEO, and successfully identified three plasticity residues involved in secondary cyclization around the active site of Tc-αPIN/TEO, namely Y327, Y429 and Y575. In the site-directed mutagenesis experiments, converting these phenolic residues into phenylalanines resulted in a dramatic decrease in the synthesis of α-pinene, indicating that the hydroxyl group of tyrosine may play an important role in the synthesis of bicyclic terpene products. This finding may assist our understanding of the complex cyclization processes and protein engineering design in the future.