釀酒酵母中表現並鑑定植物順式異戊二烯轉移酶LLA66

Abstract

異戊二烯轉移酶 (prenyltransferase)中有一種類的功能是催化將特定數量的異戊烯焦磷酸 (isopentenyl diphosphate, IPP)與法尼基焦磷酸 (farnesyl diphosphate, FPP)進行連續縮合反應，將碳鏈延長到指定的長度。根據受質異戊烯焦磷酸酵素反應時所形成的雙鍵立體化學，而將異戊二烯轉移酶分類為順式和反式。順式異戊二烯轉移酶主要合成較長的產物作為脂質載體，主導真核生物中糖蛋白或細菌中肽聚醣的生物合成。然而因為植物中的順式異戊烯基轉移酶通常存在幾種同源亞型，所以功能較少被仔細了解。來自麝香百合花的順式異戊烯基轉移酶基因LLA66是在絨氈層和小孢子中，所鑑定的第一個異戊烯基轉移酶。本次研究中，我們利用在釀酒酵母 (Saccharomyces cerevisiae)表現重組蛋白LLA66，發現它是一種獨特的中鏈萜類合成酶。藉由高效液相色譜法 (HPLC)及薄層色譜 （TLC）分析中，顯示產物為45個碳鏈的萜類。此外在使用Ni-NTA親和層析純化有6個His標記的LLA66蛋白時，我們還發現純化後的蛋白質溶液中具有將IPP轉化為法尼醇(farnesol)的催化活性。通過進一步的數據，我們認為是IPP：DMAPP異構酶、FPP合成酶和鹼性磷酸酶。但它們是否會形成蛋白質複合物是需要實驗的進一步驗證。

A group of prenyltransferases catalyze chain elongation of farnesyl diphosphate (FPP) to designated lengths by consecutive condensation reactions with specific numbers of isopentenyl diphosphate (IPP). According to the stereochemistry of the double bonds formed by IPP condensation, these prenyltransferase are classified as cis- and trans-types. Cis-prenyltransferases synthesize longer products as lipid carriers to mediate biosynthesis of peptidoglycan in bacteria or glycoproteins in eukaryotes. However, the functions of cis-prenyltransferases in plants are less understood because there are several homologous isoforms in different compartments. The cis-prenyltransferase gene LLA66 from Lilium longiflorum anther is the first prenyltransferase identified in the tapetum and microspores. As reported here, we have produced the recombinant LLA66 in Saccharomyces cerevisiae, performed in-vitro characterization of the enzyme and found it is a unique medium-chain synthase, and was resolved C45 isoprenoids in thin layer chromatography (TLC) and HPLC. After purification of the hexa-His-tagged LLA66 using Ni-NTA affinity chromatography, we also identified enzyme activities to convert IPP into farnesol in the purified protein mixture. By further analysis, we thought that a protein mixture of IPP:DMAPP isomerase, FPP synthase and alkaline phosphatase was co-purified with LLA66. We will further characterize the protein complex.