阿拉伯芥中氧化鯊烯-環阿屯醇環化酵素的突變效應研究

Abstract

氧化鯊烯-羊毛硬脂醇環化酵素 (OSC) 和氧化鯊烯-環阿屯醇環化酵素 (CAS) 催化了複雜的環化/重組反應，在哺乳類動物和真菌中，可將 (3S)2,3-氧化鯊烯催化而成羊毛硬脂醇，而在光合植物中則生成環阿屯醇。為了了解胺基酸改變對反應之進行以及產物變化的影響，我們使用定點突變的方法，同時結合質體交換的策略。將H477之胺基酸和位於氧化鯊烯-環阿屯醇環化酵素的推測活性位置空腔之29個非胺基丙酸之胺基酸突變為胺基丙酸。再將突變株轉植進入已去除染色體ERG7基因的酵母菌菌株中，之後，分析突變株是否有將氧化鯊烯轉化而為羊毛硬脂醇的能力，並且利用薄層色層分析的方法分析新產物。在這些突變點中，H477C、H477F、H477W、H477S、H477A的突變株會催化受質氧化鯊烯，其產物會由環阿屯醇轉化為羊毛硬脂醇，造成環化產物之改變，但是在這些突變株當中，沒有突變株發現有新產物之生成。 再者，以鯊烯-蛇麻烯環化酵素為模板模擬之氧化鯊烯-環阿屯醇環化酵素之分子模擬為依據，其中 W217A、M254A、F550A、I553A、W416A、C484A、W610A、Y616A之胺基酸與抑制劑有較短之距離，因此將它們做進一步之討論。結果發現W416、C484、W610、Y616的改變可能使得活性喪失以致於TLC分析無法觀察到新產物。

Oxidosqualene-lanosterol cyclase (OSC) and oxidosqualene-cycloartenol synthase (CAS) catalyze the complex cyclization/rearrangement of (3S)2,3-oxidosqualene into lanosterol in mammal and fungi versus cycloartenol in photosynthetic plants. Site-directed mutagenesis coupled with plasmid shuffle strategies were applied to study the effect of amino acid alteration on product specificity. H477 and twenty nine non-alanine residues located on the putative active site cavity surface of oxidosqualene cycloartenol synthase have been mutated to alanine. Mutants were assayed for their ability to transform oxidosqualene to lanosterol, ERG7 knockout, Saccharomyces cerevisiae strain, and analyzed their truncated novel product by TLC method. Among them, H477C, H477F, H477W, H477S, H477A resulted in changing the cyclization product of oxidosqualene from cycloartenol to lanosterol, but novel product were undetected. Furthermore, according to CAS homology modeling, based on SHC template, W217A, M254A, F550A, I553A, W416A, C484A, W610A and Y616A were chosen for further investigation, due to their short distances from the inhibitor. It appeared that the alteration of W416, C484, W610 and Y616 might cause loss of enzyme activity such that no novel product was observed by TLC analysis.