利用突變策略、抑制作用與SELEX技術針對氧化鯊烯環化酵素進行功能性之探討

Abstract

氧化鯊烯環化酵素(EC 5.4.99-) 為一群具有催化受質(3S)-2,3-氧化鯊烯((3S)-2,3-oxidosqulaene)進行一系列的環化與重組反應之同源酵素。在絕大多數的物種中，氧化鯊烯環化酵素會負責專一性的生物轉化作用，進而生成多樣的固醇類或三萜類產物。受質所進行的環化機制與催化酵素本身結構間所產生的協同關係更是極度複雜與引人注目的。為了闡明受質-氧化鯊烯在機制上所進行的環化與重組反應，我們設計了許多實驗，包括分子生物學上的突變策略以及利用生物有機化學的方式來加以探討。 在一開始，我們利用“丙氨酸掃描式突變” 以及“定點突變” 的方式，並配合酵母菌體內的“質體交換篩選法” ，來加以確定在酵母菌中氧化鯊烯環化酵素 (S.c.ERG7) 其內部對於催化反應或結構穩定上重要的氨基酸位置。由實驗的結果發現，在我們所感興趣的酥氨酸-509到異白氨酸-513的這段區域上，酪氨酸-510在突變效應的影響下產生非常迥異的催化活性。當酪氨酸-510置換成色氨酸或離氨酸時，酵母菌的轉殖株便無法補充其體內本身的氧化鯊烯環化酵素上的缺失而導致菌株的死亡。但在換成丙氨酸時，酵母菌的突變株卻是可以存活。因此，我們進行了各轉殖株其體內非皂化酯醇的分離與鑑定，結果發現酵母菌氧化鯊烯環化酵素酪氨酸-510的丙氨酸突變株 (S. cerevisiae ERG7Tyr510Ala) 可以產生單環的蓍醇A (achilleol A)、四環的羊毛硬脂醇 (lanosterol) 以及parkeol。而在致死的色氨酸或離氨酸的突變株中，酵母菌僅能產生兩個單環的產物分別為蓍醇A (achilleol A)或camelliol C。為了更進一步的瞭解在不同的氨基酸改變下對於氧化鯊烯環化酵素酪氨酸-510所造成突變效應，我們繼而利用“定點飽和突變” 的方式來進行探討。其結果發現，不同的酪氨酸-510突變株會產生單環、三環以及多種四環的生合成產物。此外，在原先所推測對於產物專一性非常重要的“催化氨基酸鹼性殘基對” (catalytic base-dyad)，即酪氨酸-510與組氨酸-234所形成的共伴效應，可以藉由不同的酪氨酸-510與組氨酸-234的雙點突變來加以闡明。另一方面，我們亦利用類似的定點飽和突變結合生物有機化學的特性，發現更多對於在不同階段的環化與重組反應中相當重要的一些氨基酸位置，並分別鑑定出多個中間態產物的結構。為了仔細地瞭解這些氨基酸在酵素活性區域中的重要性，我們也建構了一系列酵母菌氧化鯊烯環化酵素的蛋白質同源模擬結構，進而合理地解釋這些關鍵性的氨基酸位置對於其單離產物間的關係。之後，我們亦利用量子力學的高斯軟體Gaussian 03來預測在不同的酵素突變株中，不同的產物其在理論能階上的相對應位置與其產生的趨勢。另一方面，我們將酵母菌中氧化鯊烯-羊毛硬脂醇環化酵素與阿拉伯芥中氧化鯊烯-環阿屯醇環化酵素進行一系列嵌合體酵素資料庫 (chimeric enzyme library) 的建構，期望能找出在這兩個酵素中專門與產物專一性有關的特定區域。因此，我們一共設計了十種不同的嵌合體 (chimeras)，並藉由“質體交換篩選法” 來分析它們在酵母菌中的活性表現。但在所有的嵌合體轉殖菌株中，其體內的非皂化酯醇並沒有明顯的差異，這也許意味著過於粗糙的切割可能造成整體酵素在結構上的瓦解。之後，我們進一步地比較之前在三萜類合成酵素中，其嵌合體酵素資料庫的實驗數據後，我們認為在固醇類生合成酵素中，產物的專一性也可能僅來自於酵素活性區域上少數幾個關鍵的功能性氨基酸。 除了分子生物學外，我們也同步進行了不同的生物化學實驗，並且將重點放在哺乳類動物中氧化鯊烯-羊毛硬脂醇環化酵素其結構與功能間的關係。我們主要是利用管柱層析、生物有機化學及不同的抑制劑修飾等實驗，對於牛肝中的氧化鯊烯環化酵素進行一系列的探討。在一開始，我們成功地由牛肝中將氧化鯊烯-羊毛硬脂醇環化酵素純化出來，並藉由串聯式質譜儀加以鑑定。之後，我們也順利地將整段序列解讀出來並將其表現在酵母菌中。而由氨基酸的比較上，我們發現牛肝中的氧化鯊烯環化酵素與其他三種哺乳類動物的氧化鯊烯環化酵素在序列上有百分之八十以上的相同度。然而，為了更進一步的瞭解其中一個非常有效的抗氧化鯊烯環化酵素之特性抑制劑-Ro48-8071其作用機制與其明確的鍵結位置，我們進行了光親和性的分析與設計了五個以Ro48-8071為骨架的螢光性衍生物。然而，由抑制酵素活性的實驗與螢光光譜特性上，我們發現化學螢光修飾會大幅減弱Ro48-8071的抑制活性。另外，我們利用分子入塢實驗(molecular docking) 也合理的解釋經修飾後的螢光化合物在氧化鯊烯環化酵素的活性區域中，對於酵素立體空間所產生的障礙關係以及經此修飾對於抑制能力的影響。希望未來能更進一步的研發新型式的Ro48-8071衍生型螢光抑制劑，除了能幫助氧化鯊烯環化酵素在結構與功能性質上進行探討，也能進一步提供在蛋白質體學上的應用，或對於在降膽固醇藥物的篩選上有所幫助。另一方面，我們也利用了一個隨機選擇性的組合式核苷酸資料庫篩選法-SELEX對於氧化鯊烯環化酵素進行篩選，希望選擇出對其具有一定結合能力的最適體 (aptamer)。經過九次的篩選循環，我們成功的得到了多個最適體分子。針對這些最適體分子，我們除了證實了它們與牛肝中氧化鯊烯環化酵素的結合能力外並計算出其大約在nM範圍的親和力。然而最適體與氧化鯊烯環化酵素間的作用方式仍需更進一步的試驗來加以瞭解。期望這些與氧化鯊烯環化酵素有結合能力的分子，可以在未來不論是針對羊毛硬脂醇環化酵素或是針對膽固醇的生合成途徑皆能提供在醫療或診斷上的應用。 綜合以上所述，我們利用了不同的分子生物學實驗，包括丙氨酸掃描式突變、定點直接/飽和突變、區域置換實驗、同源模擬與量子力學等生物資訊的研究得以針對氧化鯊烯環化酵素其環化與重組反應在機制上有更深入的瞭解。另外一方面，藉由生物化學與生物有機化學的相互運用，我們更獲得許多寶貴的資訊。特別是在哺乳動物其氧化鯊烯環化酵素的取得，以及利用化學性修飾而得以針對抑制劑Ro48-8071其抑制作用有更進一步的瞭解。同時利用體外SELEX的實驗，更幫助我們對於未來針對合理地設計抗真菌類或降低膽固醇藥物的領域上開創一條展新的大道。

Abstract

Oxidosqualene cyclases (EC 5.4.99-) constitute a family of enzymes that catalyze diverse cyclization/rearrangement reactions of (3S)-2,3-oxidosqualene (OS) into a distinct array of sterols and triterpenes. Notably, the product specificity among most of cyclase enzymes is species-dependent. The relationship between cyclization mechanism and enzymatic structure is extremely complex and attractive. In order to further elucidate the cyclization/rearrangement reaction of oxidosqualene cyclases, experiments including different molecular biological mutagenesis approaches as well as the bioorganic studies were accordingly carried out. First, the alanine-scanning mutagenesis and site-directed mutagenesis coupled with in vivo plasmid shuffling selection were employed to identify the catalytic or structural important residues in oxidosqualene-lanosterol cyclase from Saccharomyces cerevisiae (S. cerevisiae ERG7). Among the investigated sequence segment from Thr-509 to Ile-513, Tyr-510 showed the catalytic discrepancy in the cyclase activity upon mutagenic effect. The yeast transformant failed to complement the cyclase-deficiency when this position was mutated to tryptophan or lysine residues, but still maintained the yeast viability in the S. cerevisiae ERG7Y510A mutant. After analysis of the nonsaponifiable lipid from S. cerevisiae ERG7Y510A, the monocyclic achilleol A, tetracyclic lanosterol and parkeol were identified. Moreover, two monocyclic compounds, achilleol A and camelliol C, were isolated from the lethal S. cerevisiae ERG7Y510K and S. cerevisiae ERG7Y510W mutants. In order to further investigate the mutated effects on this residue, the site-saturated mutagenesis was subsequently performed. Diverse products including monocyclic, tricyclic, and different tetracyclic products were isolated from the S. cerevisiae ERG7Y510X mutants. Moreover, the inherent influence on product specificity via an altered coordinative interaction between the hypothesized catalytic dyad, Tyr-510 and His-234, were further examined in more detail via construction and analysis of a different set of S. cerevisiae ERG7H234X/Y510X double mutations. Moreover, other catalytically important residues and their respective premature cyclization products, involved in different cyclization/rearrangement stages, were also discovered by using similarly executed site-saturated mutagenesis, coupled with bioorganic characterization. In order to carefully explore the importance of these crucial residues within the enzymatic active site, plausible homology modeling structures were subsequently created. The diverse array of product profiles which were isolated from various mutated S. cerevisiae ERG7 cyclases was broadly representative. Moreover, the products’ tendency in different mutated enzymes was consequently understood by using the quantum mechanics calculation of Gaussian 03. In addition, the combination of chimeric enzyme library between Saccharomyces cerevisiae lanosterol synthase and Arabidopsis thaliana cycloartenol synthase was also constructed to determine the critical functional domain responsible for the product specificity. Ten diverse domain swapping chimeras were successfully created, and their activities were subsequently confirmed via plasmid shuffling selection. No divergence of the nonsaponifiable lipid patterns was observed among these inactive chimeras, suggesting that the rough partition might disrupt the enzyme structure. After comparison with the previous experimental results from the triterpene synthases, the product specificity-determining residues among these sterol-biosynthetic cyclases might be determined by just several functional crucial residues within the enzymatic active site. In parallel to the ongoing molecular biology approaches, we also performed a number of biochemical studies, including chromatographic purification, bioorganic characterization, and inhibition studies to examine the structure-function relationships for mammalian lanosterol cyclase. After successful purification and tandem mass characterization of bovine liver OSC, the gene encoding bovine liver OSC was subsequently determined. The deduced amino acid sequence showed >80% identity to that of the other three mammalian lanosterol synthases. The bovine liver OSC gene was also successfully cloned and functionally expressed in a yeast erg7 disruption strain. Moreover, in order to better understand the inhibiting mechanism of one potent OSC inhibitor, Ro48-8071, as well as to solve the exact inhibitor binding site, the photoaffinity labeling and chemical fluorescent modification of Ro48-8071 was also carried out. Several Ro48-8071-based fluorescent probes were developed and their inhibitory activity or fluorescence characteristics were analyzed. The results of chemical modification of Ro48-8071 suggested that the fluorescent Ro48-8071 derivatives dramatically reduced its inhibitory activity for purified bovine liver OSC. Moreover, the interactions between fluorescent Ro48-8071 derivative and the active site of bovine liver OSC, as well as the orientation of these probes have obviously changed, based on the molecular docking experimental data. In the future, improved site-specific fluorescent probes should be developed and applied to the chemical proteomic field for effectively screening the OSC drugs. By another approach, a randomly selected combinatorial approach, SELEX, was utilized for screening the potential OSC-binding aptamer molecules of the bovine liver OSC. After nine rounds of SELEX screening, a diverse array of aptamer candidates was isolated from the single-strand DNA library. These aptamer molecules exhibited the definitive interaction with the targeted protein and also revealed the approximate nM range affinity for bovine liver OSC. However, the binding interaction between individual aptamers and the cyclase protein should be explored in the future. These obtained OSC-binding aptamers will be applied in the pharmaceutical or diagnostic applications for lanosterol synthase as well as for studies in the cholesterol pathway in the future. Thus, the combination of results obtained from the molecular biological approaches, including alanine-scanning, site-directed/saturated mutagenesis, domain swapping experiments, homology modeling structure, and quantum mechanics calculation, a better understanding of the cyclization/rearrangement mechanism of oxidosqualene cyclase has been achieved. In addition, the biochemical characterization coupled with the bioorganic studies toward mammalian lanosterol synthase provided valuable information, especially in obtaining the purified cyclase protein, and illustrating the inhibition mechanism of Ro48-8071. Moreover, the in vitro SELEX procedure opens new avenues in rational designing of antifungal agents and hypocholesteremic agents.