title: Expression and homology modeling of sterol 14alpha-demethylase from Penicillium digitatum.
abstract: Green mold of citrus, caused by Penicillium digitatum, is the most serious postharvest disease of citrus. Sterol 14alpha-demethylase (CYP51) is one of the key enzymes of sterol biosynthesis in biological kingdoms and is a prime target of antifungal drugs. To exploit novel 14alpha-demethylase inhibitor (DMI) fungicides, DNA and total RNA were isolated from P. digitatum. The CYP51 of P. digitatum was cloned and expressed in Escherichia coli, yielding recombinant protein with a molecular weight of c. 59 kDa. The P. digitatum CYP51 protein (PdCYP51) was purified and polyclonal antibodies were prepared. Compared with the sequence of P. digitatum PD5 in GenBank, there were four mutated nucleotides which resulted in four mutated amino acids. The three-dimensional (3D) model of P. digitatum CYP51 was established based on structure template of 1e9x.pdb and diniconazole was docked into the active site by FlexX. According to spectral data, it is suggested that the purified soluble protein had high affinity with diniconazole, a potent inhibitor of CYP51 reaction in fungi. At the same time, these spectral data suggested that the 3D model and the docking model were reasonable, which we hope can be used to provide a virtual screening of novel DMI drugs.
authors.affiliation: College of Life Sciences, Central China Normal University, Wuhan, China, and Key Laboratory of Pesticide & Chemical Biology (CCNU), Ministry of Education, Wuhan, China.
authors.forename: Li
authors.initials: L
authors.lastname: Zhao
authors.forename: Deli
authors.initials: D
authors.lastname: Liu
authors.forename: Qingye
authors.initials: Q
authors.lastname: Zhang
authors.forename: Shan
authors.initials: S
authors.lastname: Zhang
authors.forename: Jian
authors.initials: J
authors.lastname: Wan
authors.forename: Wenjing
authors.initials: W
authors.lastname: Xiao
mesh_terms: D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000378:metabolism / Q000494:pharmacology; D001483:Base Sequence; D001665:Binding Sites; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010407:Penicillium / Q000201:enzymology* / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D058886:Sterol 14-Demethylase; D040681:Structural Homology, Protein; D014230:Triazoles / Q000378:metabolism / Q000494:pharmacology
chemical_list: D000935:Antifungal Agents; D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D014230:Triazoles; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D058886:Sterol 14-Demethylase; C049346:diniconazole