利用飽和定點突變方法研究酵母菌氧化鯊烯環化酵素內的假設活性區胺基酸對於催化環化/重組反應的影響

Abstract

在酵母菌以及哺乳類動物中，氧化鯊烯環化酵素(OSC)催化(3S)-2,3-氧化鯊烯((3S)-2,3-oxidosqulaene)進行環化/重組反應而產生羊毛硬脂醇。這個複雜的環化/重組反應的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。我們利用飽和定點突變的方法來探討酵母菌中氧化鯊烯環化酵素的兩個胺基酸, Tyr99以及Trp443的功能以及在酵素催化反應的過程中所扮演的角色。在OSCTyr99的功能性分析中,當其殘基被突變成某些帶極性側鏈的胺基酸時可以有功能性補充的作用而支持其氧化鯊烯環化酵素(OSC)被突變的酵母菌存活下來，並且形成產物的多樣性。經由產物的分析我們分離出了之前未被發表過兩個新的三環碳陽離子中間產物，分別是(13αH)-isomalabarica-14Z,17E,21-trien-3β-ol以及 (13αH)-isomalabarica-14E,17E,21- trien-3β-ol。 經由實驗結果我們推測Tyr99在環化機制中可能是穩定椅形-船型(chair-boat)結構所形成的6-6-5三環馬可尼可夫碳-14陽離子(6-6-5 tricyclic Markovnikov C-14 cation)以及控制接下來碳-15脫氫反應的立體化學(stereochemical)。在分子模擬的分析中，Tyr99相對於碳-陽離子的空間位置不同於His234和Phe445，進而影響了酵素與其碳陽離子中間體之間的反應位向和靜電作用因此導致反應最後選擇不同位置進行去質子化的結果。而量子力學的計算結果顯示出酵素催化反應裡所產生的種種效應都對在三環馬可尼可夫碳陽離子的去質子化步驟中立體化學特性有重大影響。另ㄧ方面，關於OSCTrp443的飽和定點突變分析中，其中兩個突變，Trp443Ala以及Trp443Lys同時產生了兩個單環的產物achilleol A 和 camelliol C。而achilleol A 和 camelliol C這兩個單環產物証明了其生成是由於不完整的環化反應被迫終止在碳-10而形成碳陽離子中間體，再經由不同位置的去質子化反應所造成。綜合產物比例以及分子模擬的分析，推測OSCTrp443的功能可能是在於影響受質反反應前的摺疊構形以及穩定環氧基被質子化隨即A環形的中所產生的碳陽離子。最後，我們的實驗結果可以說明藉由飽和定點突變方法以及產物分離/分析的實驗可以更深入了解氧化鯊烯環化酵素的結構、功能以及機制之間的關係。

Oxidosqualene-lanosterol cyclase (ERG7) catalyzes the cyclization/rearrangement of (3S)-2,3-oxidosqualene to lanosterol in yeast and mammals. The complex cyclization/rearrangement reaction mechanism consists of initial epoxide protonation, four successive cationic cyclizations, four consecutive hydride or methyl group rearrangements, and a final highly specific deprotonation step. Site-saturated mutagenesis experiments were carried out on the Tyr99 and Trp443 positions of OSC in Saccharomyces cerevisiae to investigate their putative functional role in the catalytic cyclization. For functional analysis of OSCTyr99, some polar side-chain group substitutions genetically complemented yeast viability and produced spatially related product diversity. Product isolation and characterization revealed two novel tricyclic intermediates, (13αH)-isomalabarica-14Z,17E,21-trien-3β-ol and (13αH)-isomalabarica-14E,17E,21- trien-3β-ol, for the first time. These results suggested that the functional role of the Tyr99 in affecting both the chair-boat 6-6-5 tricyclic Markonikov C-14 cation stabilization and the stereochemistry of the protons at the C-15 position for subsequent deprotonation reaction. Homology modeling analysis, results revealed that Tyr99 residue is located spatially differently from that of His234 and Phe445 to the common C-14 cation which affects the orientation or electrostatic interaction between the enzyme and its cationic intermediate, and results in abstracting of a proton from a different position or orientation. The quantum mechanical calculation results suggested that the energetics of stereochemical control during the tricyclic Markovnikov cation deprotonation step could be affected by the inclusion of these enzymatic effects. The site-saturated mutagenesis applying on the OSCTrp443 revealed that two truncated monocyclic intermediates, achilleol A and camelliol C, were concomitantly produced from the Trp443Ala and Trp443Lys mutants. The formation of achilleol A and camelliol C were identified as evidence for premature truncation of C-10 cationic intermediates following the proton abstraction from different position. The product profile coupling with homology modeling analysis showed that the functional role of Trp443 of OSC might influence the substrate prefolding and stabilizing the epoxide protonation and A-ring formation. Finally, these results further exemplify the potential of site-saturated mutagenesis coupled with product isolation/characterization in elucidating the structure-function-mechanism relationships of the oxidosqualene cyclase.