利用定點突變研究不同物種之氧化鯊烯環化酵素假設活性區內氨基酸之角色

Abstract

氧化鯊烯環化酵素的催化機制在近半世紀以來，是生物化學及化學家公認最迷人且最具挑戰性的生化反應之一。直鏈狀的氧化鯊烯在不同物種的氧化鯊烯環化酵素內，經過單一步驟的酵素催化反應後，生成近二百種具有物種特異性的四至六環的三萜類化合物。為了探討同源的氧化鯊烯環化酵素如何在演化的過程中，藉由改變其活性位置的氨基酸結構以產生具有不同物種專一性之產物，我們利用丙氨酸掃描法，探討豌豆中香桂素合成酵素和阿拉伯芥中環阿屯醇酵素的假設活性區氨基酸其參與在酵素的結構與活性關係。並且，針對酵母菌氧化鯊烯環化酵素活性區域中 Cys457 進行飽和定點突變，進行功能性的全面分析。 在豌豆香桂素合成酵素中，我們發現PSYY259A 與 PSYG369A 單定點突變株會造成酵素失去其原本的活性；而在阿拉伯芥環阿屯醇酵素中，有五個定點突變株 CASY118A、CASL124A、CASW221A、CASM254A 和 CASP367A 會導致酵素失去原本活性，而 CASH257A 則是改變在環化最終步驟的脫氫反應，進而生成帕克醇。這些影響酵素活性的氨基酸突變，對於鄰近的氨基酸都具有一定程度的影響，而導致環化機制的失常。此外，利用不同載體表現PSYY259H並分析其突變株產物結果發現，若欲以酵母菌表現其他物種基因，需以具有高表現量特質之載體攜帶較佳。 在酵母菌 ERG7 飽和定點突變中，巨大的立體障礙（如Phe、Tyr與 Trp等氨基酸取代）與電荷影響（如His、Lys、Arg和Asp等取代）都可能會使酵素失去原本活性。此外，殘基過小的 Gly 取代也會影響酵素與受質的穩定性，改變產物分布，使單環的 Achilleol A 成為其主要產物。實驗結果顯示，Cys457 對於受質的開環反應有直接相關的影響，此外也會利用氨基酸殘基的立體障礙穩定環化機制的反應。

The enzymatic cyclization of oxidosqualene is one of the most remarkable steps in the biosynthesis of steroids and triterpenoids. Oxidosqualene cyclases catalyze the biotransformation of the linear form substrate, oxidosqualene, into nearly 200 skeletally diverse triterpene compounds. In order to investigate the evolutionary divergence for the production of species-dependent products from individual oxidosqualene cyclases-mediated cyclizations via changing their critical active site environments, the alanine-scanning mutagenesis on plant P. sativum βAS and A. thaliana CAS were carried out, respectively, to study the relationship between functional residues substitutions and the respective enzymatic activity. Moreover, site-saturated mutagenesis experiments on Cys457 of S. cerevisiae ERG7 were carried out to clarify functional role of Cys457. From the observation of mutagenic effect on either plant P. sativum βAS and A. thaliana CAS, PSYY259A and PSYG369A caused a dramatic disruption of catalytic function in βAS cyclization reaction. Moreover, CASY118A、CASL124A、CASW221A、CASM254A、CASH257A and CASP367A mutations also lost their original function. Among them, CASH257A mutation changed the deprotonation site and resulted in the production of parkeol. The homology model revealed that the position of aromatic amino acid in active site was affected by these mutations. Besides, the analysis result of NSL extraction of PSYY259H mutant with a galactose-inducible vector, pYES2, exhibited a distinct product profiles from that of original pRS314 vector, suggesting that pYES2 vector is a suitable plasmid for analysis of plant oxidosqualene cyclase. In parallel, the site-saturated mutations on Cys457 position of S. cerevisiae ERG7 showed that most of the substitutions successfully complemented the cyclase activity in a yeast ERG7 deficient strain, TKW14c2, except for the aromatic side chain substituted mutations（Phe、Tyr and Trp）and electronically charged side chain substitutions（His、Lys、Arg and Asp）. Conversely, the residue of Gly substitution is too small to stabilize the cyclization reaction and leads to a monocyclic achilleol A formation. The homology modeling study suggested that the functional role of Cys457 might affect Asp456 to initiate the epoxide ring opening as well as to stabilize the following A-ring cyclization.