分析經雙點突變後之阿拉伯芥中氧化鲨烯-環阿屯醇環化酵素的功能與機制

Abstract

氧化鲨烯-羊毛硬脂醇環化酵素 Oxidosqualene-Lanosterol Cyclase ( OSC ) 在哺乳動物及真菌中分別將2,3-氧化鲨烯 (3S)2,3-oxidosqualene 環化重組生成羊毛硬脂醇 ( Lanosterol )；而在高等植物中，氧化鲨烯-環阿屯醇環化酵素Oxidosqualene-Cycloartenol Synthase ( CAS ) 可將2,3-氧化鲨烯環化重組生成環阿屯醇 ( Cycloartenol )。為了解 CAS 與 OSC 間分子演化的相關性，並且進一步探討CAS環化/重組的機制，以及產物的專一性與多樣性，我們將含有單一突變且具OSC活性之突變株CASY410C，依據 Squalene-Hopene Cyclase ( SHC ) 之X-射線晶體結構為模板，推測活性區域上29個胺基酸進行改變成丙胺酸之定點突變，利用質體交換 ( Plasmid Shuffle ) 的方式進行篩選。發現29個胺基酸中Y118、L124、G127、L179、T215、G366、P367、V368、L372、C484、E548、Y616與C730，對於CAS 環化重組的機制沒有顯著的影響；F123、W217、W221、M254、H257、V261、Y262、W416、F472、F550、I553、W610、F726、I732、Y734與Y737在CAS催化的過程中，可能協助受質環化與重組，擔任決定酵素活性有無的關鍵角色。 在分析CAS含雙點突變之產物的過程中，利用打破具雙點突變之酵母菌，以萃取的方式從酵母菌細胞中擷取出非可皂化的脂質。利用薄層色層分析片 ( TLC ) 了解產物的分佈、以矽膠的管柱 ( Silica column ) 將產物纯化分離、氣相色層分析 ( GC-MS ) 推其分子量，再利用核磁共振光譜學 ( NMR ) 分析產物的結構。由產物分析結果發現，包含活性區域靠近受質接受區下方之 M254、H257、V261的雙點突變效應下，有相同的新產物生成。新產物分別是24-Methlene 24,25 -Dihydrolanosterol、4,4-Dimethyl fecosterol與4-Methyl Fecosterol，分子量分別為 440、 426 與 412。我們推測CASY410C M254A、CASY410C H257A 與 CASY410C V261A 突變效應，失去環化的活性而轉變成固醇類甲基轉移酶的功能性 ( Sterol Methyl Transferase, SMT )，使得羊毛硬脂醇改變代謝途徑產生 24-Methlene 24, 25-Dihydrolanosterol，隨即經細胞中C-14α的去甲基酶作用後，進而形成4,4-Dimethyl Fecosterol、4-Methyl Fecosterol 與 Fecosterol，進入野生型麥角脂醇生合成途徑的下游，最後形成終產物麥角脂醇 ( Ergosterol )。

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 algae and photosynthetic plants. To study evolutionary relationships between OSC and CAS, cyclization / rearrangement mechanism, as well as product specificity and/or diversity, twenty-nine non-alanine residues located on the putative active site cavity surface of CASY410C mutant, a CAS mutant which changes its enzymatic activity from CAS to OSC, were mutated to alanine and assayed for their ability to complement the OSC deficiency and to study the effect of double mutations. All of the mutations were verified by restriction mapping and DNA sequencing, followed by genetic counter-selection. Among them, 13 out of 29 mutations failed to complement the ERG7 disruption indicating that these residues are crucial for the catalytic function of the enzyme. The nonsaponifiable lipids of inactive mutants were isolated and characterized by TLC, GC-MS and NMR. The CASV261AY410C mutant、CASM254AY410C mutant and CASH257AY410C mutant, which alone failed to complement the viability of cyclase-deficient yeast strain but accumulated 24-methylene-24,25-dihydrolanosterol, 4,4-dimethyl fecosterol and 4-methyl fecosterol, when both wild-type OSC and CAS double mutations were present, were isolated. The results indicated that a dramatic enzymatic activity change from oxidosqualene cyclase to sterol methyl transferase and concomitantly generated an alternative ergosterol biosynthetic pathway, were derived from the above-mentioned double mutations. In addition, the discovery here will provide important information with which to understand the active site topology for both enzymes and have significant consequences in relation to rational drug design and to the molecular engineering of the ergosterol biosynthetic pathway.