利用定點突變研究酵母菌中氧化鯊烯-羊毛硬脂醇環化酵素與反應機制間的關係

Abstract

對2,3-氧化鯊烯進行環化反應以產生脂醇和三□類天然物，為自然界中所發現之最神奇複雜與吸引人的生物轉變之一。在哺乳類、蕈類與植物中氧化鯊烯環化□(OSC)催化(3S)-2,3-氧化鯊烯進行複雜的環化/重組反應，以分別生成羊毛硬脂醇和環阿屯醇。為了進一步了解酵母菌中氧化鯊烯環化□之結構與產物專一性間的關係，我們針對公認活化區域Asp-456上游的12個非氨基丙酸殘基(氨基酸序列453-441)進行氨基丙酸掃描法的定點突變，以研究其突變效應。這些氨基丙酸之突變株(Thr-453-Ala、Tyr-452-Ala、Gly-451-Ala、Gln-450-Ala、Thr-449-Ala、Lys-448-Ala、Thr-447-Ala、Ser-446-Ala、Phe-445-Ala、Gly-444-Ala、Trp-443-Ala、Gly-441-Ala)利用互補於去除酵母菌環化□基因ERG7之菌株進行篩選。結果顯示突變株Lys-448-Ala (pFHCOSCRS-6)、Phe-445-Ala (pFHCOSCRS-10)和Trp-443-Ala (pFHCOSCRS-12)經轉殖入染色體上氧化鯊烯環化□已缺陷的酵母菌之存活與否具有決定性的影響。以上結果暗示Lys-448、Phe-445和Trp-443對於OSC之反應活性本身(和/或)決定其產物專一性方面，可能扮演著相當重要的角色。 另外，經由萃取酵母菌中麥角脂醇生合成途徑中之成分，進行管柱與薄層分析法分離後確認其成分，結果發現上述置換無法催化反應的進行而停留在受質，氧化鯊烯。再者，經由分子模擬之結果亦暗示Lys-448可能與鄰近的兩個α-helixes具有交互作用力，來穩定酵素整體之結構以進行環化作用。而在(氧化)鯊烯環化□中被完全保留之Phe-445，具推測其帶有之陽離子-π作用來穩定受質B環形成時帶正電的C-8。比對中高度相似性的Trp-443，在三維立體空間中與Asp-456則分別位於受質環氧基之兩側，因此可能在反應過程中負責穩定碳-2上所形成第一個帶正電的高能中間過渡產物以幫助A環的合成。由以上的研究結果，預期對增進氧化鯊烯環化□之環化反應機構，以及其酵素之特定氨基酸所扮演的結構-功能關係的了解，將有極大之助益。

The conversion of (3S)-2,3-oxidosqualene (OS) into sterols and tritepenes represents one of the most remarkable and fascinating biotransformations found in nature. The reaction is catalyzed by oxidosqualene cyclases (OSCs) which transform OS to lanosterol in mammals and fungi versus cycloartenol in algae and photosynthetic plants. To study the relationship between structural modifications and product-specificity alterations, twelve non-alanine residues (sequences 441-453) located upstream the putative active site domain of oxidosqualene-lanosterol cyclase of yeast Saccharomyces cerevisiae have been mutated to alanine. The alanine-scanning mutants (Thr-453-Ala、Tyr-452-Ala、Gly-451-Ala、Gln-450-Ala、Thr-449-Ala、Lys-448-Ala、Thr-447-Ala、Ser-446-Ala、Phe-445-Ala、Gly-444-Ala、Trp-443-Ala、Gly-441-Ala) were assayed for their ability to complement the cyclase-deficient, ERG7 knockout, S. cerevisiae strain. Among them, Lys-448-Ala (pFHCOSCRS-6)、Phe-445Ala (pFHCOSCRS-10) and Trp-443-Ala (pFHCOSCRS-12) failed to complement the ERG7 disruption, indicating that Lys-448、Phe-445 and Trp-443 are essential for catalytic function of OSC. In addition, analysis of nonsoponifiable lipid by TLC revealed that these cyclization reactions halted at oxidosqualene. Furthermore, homology modeling of the cyclase also revealed that the amino side chain of Lys-448 might interact with two proximal α-helixes to hold the whole enzyme active structure. Combination of the previous studies and the results of homology modeling suggested the highly conserved Phe-445 might stabilize the C-8 cation intermediate during B-ring formation via cation-π interaction. In parallel, homology modeling of the cyclase also revealed that the Trp-443, located in opposite position with Asp-456 around the epoxide of the substrate in 3-D structure, might be involved in stabilizing the high-energy C-2 cation intermediate during the A-ring formation. In conclusion, in combination of alanine-scanning mutations and analysis of their cyclization products as well as the molecular modeling results, would facilitate better understanding regarding the cyclization/rearrangement mechanism and structure-function relationships of amino acid residues of oxidosqualene cyclases.