利用定點突變進行氧化鯊烯環化酵素家族假設活性區中胺基酸之特性探討

Abstract

氧化鯊烯環化酵素存在於大多數生物體之中，他們會將直鏈狀的氧化鯊烯，反應成特定的多環結構。在不同的生物物種，例如植物、動物與真菌間，會經由不同的反應機制而形成不同的產物。催化過程包含氧化鯊烯上環氧基開環的起始反應、中途複雜的環化和重組的步驟、以及最後的去質子反應，形成高度專一性的產物。為了比較不同類型的氧化鯊烯環化酵素，我們選用了酵母菌中的氧化鯊烯-羊毛硬脂醇環化酵素、阿拉伯芥中的環阿屯醇環化酵素及豌豆中的β-麥胚固醇環化酵素這三種來利用定點突變的方式，分析存在於酵素活性區中的相對應胺基酸，希望能從實驗分析找出它們的功能以及重要性。 在酵母菌的氧化鯊烯-羊毛硬脂醇環化酵素實驗中，我們利用定點飽和突變的方法，針對Cys703來做分析。分析結果顯示經由不同的胺基酸突變，產生了八種不同的產物，除了原先就會產生的羊毛硬脂醇之外，產生六種已知的環化中間物和一個先前未曾被發現過的未知物。而其中之一的四環產物較為被關注，它在十七號碳的型態擁有向上的氫和向下的長碳側鏈，與正常的結構相反，所以可以得知Cys703在決定十七號碳上的側鏈為向上或向下型態佔有一定的重要性。此外，除了此產物以外，其他環化中間物皆和Phe699突變點的分析十分類似，加上與離受質較遠的Cys703相比，Phe699為活性區第一層胺基酸且影響力大，可以推測Cys703和第一層胺基酸的穩定性息息相關。 另一方面為阿拉伯芥中的環阿屯醇環化酵素及豌豆中的β-麥胚固醇環化酵素的分析。前者與氧化鯊烯-羊毛硬脂醇環化酵素相同，在受質的摺疊會經由椅形-船形-椅形形成原脂醇碳陽離子中間物；而後者則會經由椅形-椅形-椅形形成達瑪烯碳陽離子中間物。此兩種酵素經由不同的結構和機制，分別形成環阿屯醇及β-麥胚固醇。實驗分析方法是利用丙胺酸掃描法，選定十五個不同位置的胺基酸做突變，探討產物的差異性。在環阿屯醇環化酵素的定點突變中，產生了一些和氧化鯊烯-羊毛硬脂醇環化酵素突變中出現過的產物，推測是因為結構和機制非常的類似，因此儘管是不同物種，但還是可以經由突變而產生相同的產物。而在β-麥胚固醇環化酵素的定點突變中，只分析出兩種產物，推測可能是因為在β-麥胚固醇環化酵素這種椅形-椅形-椅形的機制中需要比較精確的結構才可以使反應穩定，而詳細的反應機制則需要更進一步地針對活性區周圍的胺基酸做深入的研究。

Oxidosqualene cyclases catalyze the biotransformation of the linear form substrate, (3S)-2,3-oxidosqualene, into tetracyclic or pentacyclic triterpene product. Different species of organisms including S. cerevisiae ERG7, A. thaliana CAS1 and P. sativum PSY operate this complex reaction through different conformation intermediates within the oxidosqualene cyclization process. According to previous reports, by utilizing the diverse structural and stereochemical control in various catalytically important amino acid residue mutants, oxidosqualene cyclases produced diverse product profiles ranging from monocyclic to polycyclic triterpene alcohols. These data implied that the plastic enzyme could thus be redesigned to obtain a novel reactivity from this complex enzyme, but with the characteristics of well-known high product specificity. Moreover, in order to further illustrate other critical amino acids involved in the catalytic significance and enzymatic plasticity of ERG7, CAS1 and PSY, we described herein a series of site-saturated mutations on the Cys703 residue of ERG7 and fifteen alanine-scanning mutations of CAS1 and PSY, respectively. In the mutations of Cys703, a diverse products profile, including four known truncated cyclization tricyclic structures, four known tetracyclic structures and a novel product were identified from various ERG7C703X mutants. The product characterization and homology modeling results suggested that the Cys703 may indirectly affect the C-17 cation stabilization and the final deprotonation step, via the intermediary of the Phe699 residue, resulting in the production of either 17α or 17β side chain product derivatives. Structure-function-mechanism relationships of Cys703 and Phe699 on the catalytic activity of OSC could thus be discussed in a series of ERG7Phe699X/Cys703I double mutants. In parallel, alanine-scanning mutagenesis was carried out on putative active site residues of CAS1 and PSY to further analyze the mutagenic effect on oxidosqualene cyclization in different sources. For mutation on CAS1, the product profiles of different mutants showed that there are highly relevant comparisons between CAS1 and ERG7. For example, the same bicyclic compound (9R,10S)-polypoda-8(26),13E,17E,21-tetraen-3β-ol was found in the CAS1Tyr734 mutant and its corresponding residue in the ERG7Tyr707 mutant. The results show that although they exist in totally different species, the relationships between them are still relevent. For the PSY mutation, there were only β-amyrin and lupeol synthesized from these mutants. This phenomenon allows us to predict that the producing of β-amyrin in PSY needs more precise structural control of substrate in the enzymatic active site. Thus, the detailed understanding of critical residues should be investigated after the functional roles of the neighboring amino acids in the active site are confirmed.