Serrano-Posada Hugo, Centeno-Leija Sara, Rojas-Trejo Sonia, Stojanoff Vivian, Rodríguez-Sanoja Romina, Rudiño-Piñera Enrique, Sánchez Sergio
Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad Universitaria, 04510 México, DF, Mexico.
Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Avenida Universidad 2001, 62210 Cuernavaca, MOR, Mexico.
Acta Crystallogr F Struct Biol Commun. 2015 Sep;71(Pt 9):1194-9. doi: 10.1107/S2053230X15014363. Epub 2015 Aug 25.
Labdane-related diterpenoids are natural products with potential pharmaceutical applications that are rarely found in bacteria. Here, a putative class I labdane-related diterpene synthase (LrdC) identified by genome mining in a streptomycete was successfully crystallized using the microbatch method. Crystals of the LrdC enzyme were obtained in a holo form with its natural cofactor Mg(2+) (LrdC-Mg(2+)) and in complex with inorganic pyrophosphate (PPi) (LrdC-Mg(2+)-PPi). Crystals of native LrdC-Mg(2+) diffracted to 2.50 Å resolution and belonged to the trigonal space group P3221, with unit-cell parameters a = b = 107.1, c = 89.2 Å. Crystals of the LrdC-Mg(2+)-PPi complex grown in the same conditions as the native enzyme with PEG 8000 diffracted to 2.36 Å resolution and also belonged to the trigonal space group P3221. Crystals of the LrdC-Mg(2+)-PPi complex grown in a second crystallization condition with PEG 3350 diffracted to 2.57 Å resolution and belonged to the monoclinic space group P21, with unit-cell parameters a = 49.9, b = 104.1, c = 66.5 Å, β = 111.4°. The structure was determined by the single-wavelength anomalous dispersion (SAD) technique using the osmium signal from a potassium hexachloroosmate (IV) derivative.
赖百当类二萜是具有潜在药物应用价值的天然产物，在细菌中很少见。在此，通过基因组挖掘在链霉菌中鉴定出的一种假定的I类赖百当类二萜合酶（LrdC），利用微量分批法成功结晶。LrdC酶的晶体以全酶形式与天然辅因子Mg(2+)（LrdC-Mg(2+)）以及与无机焦磷酸（PPi）形成复合物（LrdC-Mg(2+)-PPi）的形式获得。天然LrdC-Mg(2+)晶体的衍射分辨率为2.50 Å，属于三方晶系空间群P3221，晶胞参数a = b = 107.1，c = 89.2 Å。在与天然酶相同条件下用聚乙二醇8000生长的LrdC-Mg(2+)-PPi复合物晶体的衍射分辨率为2.36 Å，也属于三方晶系空间群P3221。在第二种结晶条件下用聚乙二醇3350生长的LrdC-Mg(2+)-PPi复合物晶体的衍射分辨率为2.57 Å，属于单斜晶系空间群P21，晶胞参数a = 49.9，b = 104.1，c = 66.5 Å，β = 111.4°。该结构通过单波长反常色散（SAD）技术，利用六氯锇酸钾（IV）衍生物的锇信号确定。
