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

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.