利用定點飽和突變技術對於酵母菌氧化鯊烯環化酵素中Gly383及Thr384進行功能性分析並藉由表現之蒺藜苜蓿轉醣酵素對三萜類化合物進行醣基衍生化之研究

Abstract

在酵母菌以及哺乳類動物中，氧化鯊烯環化酵素(ERG7或OSC)催化直鏈狀的氧化鯊烯((3S)-2,3-oxidosqulaene)進行環化/重組反應而產生羊毛硬脂醇。在不同的生物物種中，像是動物、真菌和植物會藉由不同的環化酵素和反應機制形成不同的產物。這個複雜的環化/重組反應機制，包含了氧化鯊烯上的環氧基(epoxide)被質子化而起始環化反應，以及一連串的氫化基、甲基的重組和最後高度專一性的去質子化步驟。根據先前的研究，在環化酵素中對於活性區域及立體化學結構具有重要影響的胺基酸進行突變，可以得到單環、三環和四環等多樣性固醇類產物。為了更進一步瞭解其他關鍵胺基酸對於OSC在環化過程及反應機制所扮演的角色，利用飽和定點突變的方式，分析存在於酵素假設活性區中的相對應胺基酸，Gly383 及Thr384。在ERG7T384X 和 ERG7G383X突變株中，分離出許多四環和三環產物，包括parkeol、9β-lanosta-7,24-dien-3β-ol、protosta-16,24-dien-3β-ol 和 (13αH)-isomalabarica-14(26),17,21-trien-3β-ol 等。而藉由酵素結構模擬圖我們也注意到Gly383 和Thr384位在受質C-17原脂醇碳陽離子旁的環狀區域（loop）上。Gly383 和Thr384 對於穩定C-14 和C-17原脂醇碳陽離子，以及最後脫氫反應的C8 / C-9 碳陽離子中間物似乎扮演重要的角色，對這兩個胺基酸進行突變可能會對酵素活性區域的結構及環化/重組反應造成影響，而得到這些非專一性的多樣性產物。 三萜皂苷是一群具有相似化學結構的多樣性產物，由三萜皂苷配基和醣基所組成，為分布於高等植物中的二級代謝產物且多方面的生物活性可被廣泛應用。大部分的三萜皂苷其C-3上的OH 基都接有醣基或醣鏈，而此結構也被認為是其具有生物活性的關鍵。在利用定點突變研究氧化鯊烯環化酵素重排和環化機制的實驗當中，分離出許多四環的中間物及脫氫位置不同的產物，為了使這些產物有更多的應用性。我們合成出由蒺藜苜蓿所分離出的轉醣酵素基因-UGT73K1，並得到大小為 52-kDa 的蛋白質。未來希望將氧化鯊烯環化酵素的突變產物作為此轉醣酵素的受質，在其C-3上的OH 基接上醣基並作相關的活性測試，使此多樣性產物能有廣泛的生物活性，也期盼在生物製藥方面有進一步的發展。

Oxidosqualene-lanosterol cyclase (OSC or ERG7) catalyzes the cyclization/rearrangement of the linear form substrate, oxidosqualene, into tetracyclic lanosterol in yeast and mammals. Different species of organisms including S. cerevisiae OSC, A. thaliana CAS and P. sativum PSY operate through different conformational intermediates within the oxidosqualene cyclization process. The postulated cyclization/rearrangement reaction encompasses an acid-catalyzed epoxide protonation, consecutive cationic/π interaction-directed tetracyclic ring cyclizations, hydrides or methyl groups rearrangement, and a final highly specific deprotonation step. According to previous reports, the mutated OSC produced diverse product profiles ranging from mono- to polycyclic triterpene alcohols by utilizing the diverse structural and stereochemical control in various catalytically important residues mutant. In order to further illustrate other critical amino acids involved in the catalytic significance and/or enzymatic plasticity of OSC, site-saturated mutagenesis experiments were carried out on the Gly383 and Thr384 of ERG7 to investigate their functional roles in the oxidosqualene cyclization/rearrangement reaction. Various tetracyclic or tricyclic truncated products, including parkeol, 9β-lanosta-7,24-dien-3β-ol, protosta-16,24-dien-3β-ol, and (13αH)-isomalabarica-14(26),17,21-trien-3β-ol were isolated from the ERG7T384X and ERG7G383X mutants. In parallel, we also observed that the Gly383 and Thr384 are located on the loop of ERG7 below the C-17 protosteryl cation from the examination of homology models. These results indicated that the Gly383 and Thr384 residues may play an important role in stabilizing C-17 and C-14 protosteryl cation as well as the final C-9 lanosteryl cationic intermediate. The mutated substitution in these two residues, Gly383 and Thr384, may affect the structure of protein active site that interfere the cyclization/rearrangement reaction cascade and thus resulted in the generation of the altered cyclized/deportonated product. Triterpene saponins are a group of diverse compounds with a similar chemical structure, consisting of a triterpene aglycone and sugars moiety. They are a class of plant natural products with a wide range of bioactivities. The presence of a sugar chain attached to the aglycone at its C-3 hydroxyl position is the common feature shared by all saponins and also a critical factor influencing the biological activity of many saponins. In order to further functionalize the isolated intermediate structures among the putative OS-cyclization/rearrangement cascades from various ERG7 mutants and subject them for the application of pharmaceutical field in future. We try to attach a sugar moiety to the C-3 hydroxyl position of tetracyclic products in ERG7 mutants. Herein, we first obtained one glucosyltransferase gene, UGT73K1, from Medicago truncatula and expressed it with predict molecular mass about 52-kDa, revealed by a SDS-PAGE. In the future, various OSC mutants’ products will be substrates of UGT73K1, and apply these structural derivatives for various bioactivity analyses.