Janke Ronja, Görner Christian, Hirte Max, Brück Thomas, Loll Bernhard
Institut für Chemie und Biochemie, Abteilung Strukturbiochemie, Freie Universität Berlin, Takustrasse 6, 14195 Berlin, Germany.
Fachgebiet Industrielle Biokatalyse, Technische Universität München, Lichtenbergstrasse 4, 85748 Garching, Germany.
Acta Crystallogr D Biol Crystallogr. 2014 Jun;70(Pt 6):1528-37. doi: 10.1107/S1399004714005513. Epub 2014 May 23.
Sesquiterpenes and diterpenes are a diverse class of secondary metabolites that are predominantly derived from plants and some prokaryotes. The properties of these natural products encompass antitumor, antibiotic and even insecticidal activities. Therefore, they are interesting commercial targets for the chemical and pharmaceutical industries. Owing to their structural complexity, these compounds are more efficiently accessed by metabolic engineering of microbial systems than by chemical synthesis. This work presents the first crystal structure of a bacterial diterpene cyclase, CotB2 from the soil bacterium Streptomyces melanosporofaciens, at 1.64 Å resolution. CotB2 is a diterpene cyclase that catalyzes the cyclization of the linear geranylgeranyl diphosphate to the tricyclic cyclooctat-9-en-7-ol. The subsequent oxidation of cyclooctat-9-en-7-ol by two cytochrome P450 monooxygenases leads to bioactive cyclooctatin. Plasticity residues that decorate the active site of CotB2 have been mutated, resulting in alternative monocyclic, dicyclic and tricyclic compounds that show bioactivity. These new compounds shed new light on diterpene cyclase reaction mechanisms. Furthermore, the product of mutant CotB2(W288G) produced the new antibiotic compound (1R,3E,7E,11S,12S)-3,7,18-dolabellatriene, which acts specifically against multidrug-resistant Staphylococcus aureus. This opens a sustainable route for the industrial-scale production of this bioactive compound.
倍半萜和二萜是一类多样的次生代谢产物，主要来源于植物和一些原核生物。这些天然产物的特性包括抗肿瘤、抗菌甚至杀虫活性。因此，它们是化学和制药行业感兴趣的商业目标。由于其结构复杂性，通过微生物系统的代谢工程比化学合成更有效地获得这些化合物。这项工作展示了来自土壤细菌黑色产色链霉菌的细菌二萜环化酶CotB2的首个晶体结构，分辨率为1.64 Å。CotB2是一种二萜环化酶，催化线性香叶基香叶基二磷酸环化生成三环环辛-9-烯-7-醇。随后，两种细胞色素P450单加氧酶将环辛-9-烯-7-醇氧化生成生物活性环辛菌素。修饰CotB2活性位点的可塑性残基已发生突变，产生了具有生物活性的单环、二环和三环化合物。这些新化合物为二萜环化酶反应机制提供了新的线索。此外，突变体CotB2(W288G)的产物产生了新的抗生素化合物(1R,3E,7E,11S,12S)-3,7,18-多拉贝拉三烯，它对耐多药金黄色葡萄球菌具有特异性作用。这为这种生物活性化合物的工业规模生产开辟了一条可持续的途径。
