利用定點突變對氧化鯊烯環化酵素的假設活性區進行功能性分析

Abstract

氧化鯊烯環化酵素(Oxidosqualene cyclase，OSC)，催化直鏈狀的氧化鯊烯經單一步驟環化生成四環或五環的三萜類。不同物種(氧化)鯊烯環化酵素活性區上的差異，誘導酵素進行不同的摺疊，形成產物的多樣性及複雜性。 為了解氧化鯊烯環化酵素假設活性區之胺基酸與受質環化/重組催化機制的關係，以同屬之鯊烯環化酵素(Squalene-Hopene cyclase, SHC)的X-ray晶體結構為模板，經序列比對推估出OSC的29個可能活性區位置，以定點突變進行環化重組的機制研究。經質體交換(plasmid shuffle)初步篩選發現有9個突變點(OSCF104T、OSCH146A、OSCW232A、OSCW232R、OSCH234A、OSCM532A、OSCW587A、OSCF699A與OSCY707A)無法存活，可能代表這些突變位置是催化反應的重要影響點。培養此9個含OSC突變株的酵母菌，經由產物分離與結構鑑定，以推測這些胺基酸在環化機制上所扮演的催化角色。 在OSCW232A、OSCH234A 突變株有新產物發現，OSCW232A與實驗室另一個突變株OSCH234Y產物相同，OSCH234A另多了一個未知物，進一步純化分離，NMR鑑定未知物結構為Protosta-20,24-dien-3ß-ol。藉由建構OSCW232A、OSCH234A產物圖譜，推論OSCW232A、OSCH234A在催化反應中調節環化的重組步驟，可能扮演穩定碳陽離子及脫氫的功能性角色。 為了更進一步探討OSCW232在環化機制的功用，進行飽和突變實驗(Saturated mutagenesis)，將此位置胺基酸突變成其餘19種氨基酸，觀察產物變化探討催化的功能性。利用氣相層析/質譜比對，發現有5個突變產物，除了OSCW232A原本的3個突變產物，另有2個未知物滯留時間26 min及26.5 min，目前正進一步分離此2個未知物，同時利用質譜及銀染TLC片推估此二者結構。並建構這些突變點的電腦模擬圖，進一步佐證W232在催化的功能角色。

Oxidosqualene cyclases (OSCs) constitute a family of enzymes which catalyze the conversion of linear oxidosqualene into polycyclic tetracyclic and pentacyclic triterpenoids in a one-step enzyme-catalyzed reaction. The OSC-catalyzed cyclization /rearrangement reaction represents one of the most remarkable and fascinating biotransformations found in nature. In order to clarify the function of the amino acid residues located on the putative active site cavity surface of oxidosqualene-lanosterol cyclase of yeast Saccharomyces cerevisiae, site-directed mutagenesis coupled with plasmid shuffle experiments were carried out. Following the assaying of twenty-nine alanine-scanning mutants for the complementation of yeast viability, nine inactive mutants(OSCF104T, OSCH146A, OSCW232A, OSCW232R, OSCH234A, OSCM532A, OSCW587A, OSCF699A and OSCY707A) were further analyzed for product profile. Further product isolation and characterization of each mutant showed that OSCW232A mutant produced Protosta-12,24-dien-3ß-ol, Lanosterol and Parkeol as end products, exhibiting the same product profile as that of OSCH234Y mutant. In parallel, the OSCH234A mutant produced Protosta-12,24-dien-3ß-ol, Lanosterol, Parkeol, and Protosta-20,24-dien -3ß-ol as end products. To completely characterize the functional role of W232 in OSC activity, the saturated mutagenesis experiments at W232 position were performed. Following the analysis of each mutant products, two additional new products with retention time (rt.) at 26.0 and 26.5 min were observed on GC-MS spectrometry. The EI low mass spectrum of rt. 26 min product showed a molecular ion at m/z 426 and fragment peaks at 357 ([M-C5H9]+) and 313 ([M-C8H15-H2]+), suggesting an intact tetracyclic ring. In summary, the results suggest a role for W232 in stabilizing a cationic intermediate, probably after protosteryl cation formation, and in determining the deprotonation position for diverse product profile.