利用定點飽和突變的方法針對酵母菌中氧化鯊烯環化酵素內高度保留之芳香族胺基酸進行功能性的分析

Abstract

在固醇類生合成途徑中，酵素催化直鏈狀的氧化鯊烯形成羊毛硬脂醇的步驟，在近半世紀以來引起科學家極大的興趣。而在真菌與動物體中，由氧化鯊烯-羊毛硬脂醇環化酵素負責催化這個複雜的環化與骨架重排的反應。為了了解酵素中一些具有高度保留性的芳香族胺基酸所具有的功能，我們利用定點飽和突變的方式對酵母菌內其氧化鯊烯環化酵素中Trp390、Phe528與Trp587等三個胺基酸進行功能性的全面分析。在Trp390的功能性分析中，我們發現當Trp換成其他三個帶正電荷基團的胺基酸與Gly時，因為無法互補原本酵素的功能而使酵素失去活性，而在其他七個突變株ERG7W390X (X= Asn, Glu, Thr, Cys, Phe, Tyr, Pro）中，連帶產生了Achilleol A與CamelliolC。這個結果顯示了Trp390不僅會影響開環步驟中的Asp456，還對A環的形成有極大的影響。

對照Trp587的產物分析結果可以發現，除了ERG7W587F與 ERG7Trp587Tyr外其他突變株都會使酵素失去活性，這表示這個胺基酸在酵素中扮演著極為重要的角色。而在ERG7W587F與 ERG7W587Y突變株中得到了三環與四環的產物，也顯示了Trp587在整個環化與骨架重排反應上具有重要的影響。

在Phe528飽和定點突變中，有六個突變株（ERG7F528G、ERG7F528A、ERG7F528I、ERG7F528Q、ERG7F528M 與ERG7F528Y）會連帶生成單環的產物的Achilleol A與CamelliolC，不過其生成量都很少。另外，有三個胺基酸（Asp、Arg與Thr）在功能性補充中，無法互補原本酵素的正常功能。這個實驗結果顯示了Phe528可能與酵素受質通道具有一定的關聯。

The enzymatic cyclization of oxidosqualene is one of the most remarkable step in the biosynthesis of steroids and triterpenoids. Oxidosqualene-lanosterol cyclase (ERG7) catalyzes the complex cyclization/rearrangement of (3S)-2,3-oxidosqualene to lanosterol in fungi and mammals. In order to clarify the functional role of these highly conserved aromatic amino acids, site-saturated mutagenesis experiments on Trp390, Trp587 and Phe528 residues of Saccharomyces cerevisiae oxidosqualene-lanosterol cyclase (ERG7) were carried out. Genetic complementation results of ERG7Tyr390 showed that three positive-charged amino acids (His, Lys, Arg) and Gly substitutions cannot complement the yeast viability. Two truncated monocyclic intermediates, achilleol A and camelliol C, were concomitantly produced from ERG7W390X (X= Asn, Glu, Thr, Cys, Phe, Tyr, Pro) . These results suggested that the functional role of Trp390 in affecting both Asp456 to protonate the epoxide ring and the A ring formation.

Functional analysis of Trp587 residue showed that most of the substituted mutantions fail to complement the cyclase activity in a yeast ERG7 deficient strain, TKW14C2, except for the ERG7W587F and ERG7W587Y mutants, indicating the importance of this position for the catalytic function of ERG7. Moreover, diverse tricyclic and tetracyclic products were isolated from the ERG7W587F and ERG7W587Y mutants. The result suggested the possible stabilizing role of Trp587 in the ERG7-catalyzed cyclization/rearrangement cascade.

The site-saturated mutations on the ERG7F528 revealed that trace amount of two truncated monocyclic intermediates, achilleol A and camelliol C, were concomitantly produced from six ERG7F528X (X=Gly, Ala, Ile, Gln, Met, Tyr). In the functional analysis of ERG7F528X mutants, only three mutations (X=Asp, Arg, Thr) cannot complement the cyclase activity. The homology modeling studies suggested that Phe528 is located in the substrate entrance channel and possibly affect enzymatic activity through substrate binding.