利用飽和定點突變方法針對酵母菌氧化鯊烯環化酵素內 Ile705 和豌豆氧化鯊烯-β-麥胚固醇環化酵素假設活性區內的 Leu734 兩者的結構影響在環化及重組過程中的功能性分析

Abstract

在近半個世紀以來，讓許多有機生物化學家為之著迷的酵素-氧化鯊烯環化酵素，以氧化鯊烯作為反應起始物，在不同生物體中經由各式各樣的氧化鯊烯環化酵素會形成特定的環化產物。催化過程中包含氧化鯊烯上環氧基開環起始反應，經由複雜的環化/重組反應以及最後高度專一性的去質子化步驟形成環化產物。為了比較不同類型的氧化鯊烯環化酵素，我們選用了酵母菌氧化鯊烯-羊毛硬脂醇環化酵素和豌豆-□-麥胚固醇環化酵素兩種作分析，前者在受質的摺疊會經由椅形-船形-椅形 (chair-boat-chair) 形成原脂醇碳陽離子中間物 (Protosteryl cation intermediate) 而後者則會經由椅形-椅形-椅形 (chair-chair-chair) 形成達瑪烯碳陽離子中間物 (Dammarenyl cation intermediate)，兩種環化酵素分別會形成羊毛硬脂醇 (Lanosterol) 和 β-麥胚固醇 (β-Amyrin)。利用飽和定點突變的方式，分析存在於酵素假設活性區中的相對應胺基酸，Ile705及Leu734。由於在細菌SHC酵素活性區相對位置L607的研究中，L607的重要性不容被忽視，所以希望能從實驗分析找出它們的功能以及重要性。 實驗結果方面，在Ile705位置突變過後的酵母菌氧化鯊烯-羊毛硬脂醇環化酵素，產生了七種產物，除了原先就會產生的羊毛硬脂醇之外，產生五種已知的環化中間物和一個先前未曾被發現過的未知物。四環的新產物較為被關注的焦點是它在17號碳的型態擁有向上的氫，也就是長碳側鏈是向下的，此特殊構型我們將其定義為17α 構型，同時在ERG7I705F 突變株可發現兩種擁有17α構型的產物，所以可以得知ERG7I705在決定17號碳向上或向下型態佔有一定的重要性。此外，除了新產物以外，其他環化中間物皆和ERG7F699X的分析相同，加上與離受質較遠的I705相比，F699為活性區第一層胺基酸且影響力大，可以推測I705和第一層胺基酸的穩定性息息相關。 在豌豆-β-麥胚固醇環化酵素中Leu734的功能性分析結果方面，和Ile705的結果大相逕庭，L734的突變株沒有產生任何環化產物。而造成此結果的可能性是L734扮演著穩定受質的腳色，因此在L734突變過後，由於立障、酸鹼度、極性等環境的改變，從電腦模擬軟體的判斷推測鄰近胺基酸最有可能被影響是F728，穩定受質環境被破壞以致於沒有任何環化產物產生。另一方面，由於豌豆-β-麥胚固醇環化酵素活性區胺基酸的分析非常稀少，對於L734的功能性判斷也無法深入，所以對於其鄰近胺基酸的分析也是未來所必須面臨的。

Oxidosqualene-lanosterol cyclase (S. cerevisiae ERG7) catalyzes the biotransformation of the linear form substrate, oxidosqualene, into tetracyclic lanosterol in yeast and mammals. Different species of organisms including S. cerevisiae OSC (ERG7) and P. sativum βAS (PSY) operate through different conformational intermediates within the oxidosqualene cyclization process. According to previous reports, by utilizing the diverse structural and stereochemical control in various catalytically important amino acid residue mutants, oxidosqualene cyclase produced diverse product profiles ranging from mono- to polycyclic triterpene alcohols. These data implied that the direction for the plastic enzyme was redesigned to obtain a novel reactivity from this complex enzyme, but with the characteristic of well-known high product specificity. Moreover, in order to further illustrate other critical amino acids involved in the catalytic significance and/or enzymatic plasticity of OSC and PSY, we describe herein a series of site-saturated mutations of the Ile705 residue of ERG7 and Leu734 of PSY. In the mutations of I705, seven products including three known truncated cyclization tricyclic structures, three known tetracyclic structures, as well as one novel compound that contains a tetracyclic scaffold with a 17α side chain and a △20/22 double bond, were identified from various ERG7I705X mutants. From the product distribution of ERG7I705X mutants, we deduce that the Ile705 residue may affect the first-tired residues and the stereochemistry of exocyclic long side chain during the final step of cyclization, to produce either 17α or 17β□side chain derivatives in different mutants. The relationship of the structure-function-mechanisms of Ile705 on the catalysis activity of OSC will be discussed. However, mutation of L734 causing disruption of catalytic cyclization, β-amyrin synthase did not work in PSYL734X mutants. This result revealed that the L734 residue is crucial within the putative active site of cyclase and significantly different with the mutations of ERG7I705. PSYL734 may stabilize the substrate conformation with its neighbor residues, but the detailed function should be investigated after the functional roles of the neighboring amino acids in the active site are confirmed.