Instituto de Química, Universidade Estadual Paulista-UNESP, CP 355, Araraquara 14800-900, SP, Brazil.
Centro de Pesquisa e Inovação em Biodiversidade e Fármacos, Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos 13563-120, SP, Brazil.
Molecules. 2021 Nov 11;26(22):6806. doi: 10.3390/molecules26226806.
Friedelin, a pentacyclic triterpene found in the leaves of the Celastraceae species, demonstrates numerous biological activities and is a precursor of quinonemethide triterpenes, which are promising antitumoral agents. Friedelin is biosynthesized from the cyclization of 2,3-oxidosqualene, involving a series of rearrangements to form a ketone by deprotonation of the hydroxylated intermediate, without the aid of an oxidoreductase enzyme. Mutagenesis studies among oxidosqualene cyclases (OSCs) have demonstrated the influence of amino acid residues on rearrangements during substrate cyclization: loss of catalytic activity, stabilization, rearrangement control or specificity changing. In the present study, friedelin synthase from (Celastraceae) was expressed heterologously in . Site-directed mutagenesis studies were performed by replacing phenylalanine with tryptophan at position 473 (Phe473Trp), methionine with serine at position 549 (Met549Ser) and leucine with phenylalanine at position 552 (Leu552Phe). Mutation Phe473Trp led to a total loss of function; mutants Met549Ser and Leu552Phe interfered with the enzyme specificity leading to enhanced friedelin production, in addition to α-amyrin and β-amyrin. Hence, these data showed that methionine 549 and leucine 552 are important residues for the function of this synthase.
从卫矛科植物叶子中分离得到的五环三萜化合物friedelin，具有多种生物活性，同时也是醌甲基三萜类化合物的前体，后者具有抗肿瘤的潜力。Friedelin 是由角鲨烯环化形成的，涉及一系列重排反应，通过羟基中间物的去质子化形成酮，该过程无需氧化还原酶的参与。对角鲨烯环化酶（OSC）的诱变研究表明，氨基酸残基会影响底物环化过程中的重排：丧失催化活性、稳定性、重排控制或特异性改变。本研究在大肠杆菌中异源表达了 （Celastraceae）的 friedelin 合酶。通过将第 473 位的苯丙氨酸突变为色氨酸（Phe473Trp）、第 549 位的蛋氨酸突变为丝氨酸（Met549Ser）以及第 552 位的亮氨酸突变为苯丙氨酸（Leu552Phe），对其进行定点突变。突变体 Phe473Trp 完全丧失了功能；突变体 Met549Ser 和 Leu552Phe 则会影响酶的特异性，导致 friedelin 产量增加，同时还会生成 α-香树脂醇和 β-香树脂醇。因此，这些数据表明，甲硫氨酸 549 和亮氨酸 552 是该合酶功能的重要残基。
