Crystallization and X-ray diffraction analysis of a putative bacterial class I labdane-related diterpene synthase.
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.
Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad Universitaria, 04510 México, DF, Mexico.
Hugo
H
Serrano-Posada
Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad Universitaria, 04510 México, DF, Mexico.
Sara
S
Centeno-Leija
Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Avenida Universidad 2001, 62210 Cuernavaca, MOR, Mexico.
Sonia
S
Rojas-Trejo
NSLS, Brookhaven National Laboratory, 75 Brookhaven Avenue, Building 725D, Upton, NY 11973-5000, USA.
Vivian
V
Stojanoff
Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad Universitaria, 04510 México, DF, Mexico.
Romina
R
Rodríguez-Sanoja
Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Avenida Universidad 2001, 62210 Cuernavaca, MOR, Mexico.
Enrique
E
Rudiño-Piñera
Departamento de Biología Molecular y Biotecnología, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Ciudad Universitaria, 04510 México, DF, Mexico.
Sergio
S
Sánchez
D019883:Alkyl and Aryl Transferases / Q000737:chemistry*; D000595:Amino Acid Sequence; D002850:Chromatography, Gel; D003460:Crystallization; D011756:Diphosphates / Q000737:chemistry; D004224:Diterpenes / Q000378:metabolism*; D008274:Magnesium / Q000737:chemistry; D008969:Molecular Sequence Data; D013302:Streptomyces / Q000201:enzymology*; D014961:X-Ray Diffraction
D011756:Diphosphates; D004224:Diterpenes; C433017:labdane; D019883:Alkyl and Aryl Transferases; C108877:terpene synthase; D008274:Magnesium
Streptomyces; diterpene synthase; genome mining; labdane-related diterpenoid