title: Optimised expression and spectral analysis of the target enzyme CYP51 from Penicillium digitatum with possible new DMI fungicides.
abstract: BACKGROUND
  Sterol 14α-demethylase (CYP51), a key target of azole (DMI) fungicides, can be expressed in both prokaryotes and eukaryotes. Green mould of citrus, caused by Penicillium digitatum (Pers.) Sacc., is a serious post-harvest disease. To develop specific and more effective fungicides against this disease, the characteristics of the interaction between sterol 14α-demethylase from P. digitatum (PdCYP51) and possible new fungicides were analysed. The cyp51 gene of P. digitatum was cloned and expressed under different conditions in Escherichia coli (Mig.) Cast. & Chalm., and the binding spectra of PdCYP51 were explored by the addition of two commercial azoles and four new nitrogen compounds.
  
  
  RESULTS
  The yield of soluble protein (PdCYP51) was largest when expressed in Rosetta (DE3) induced by 0.5 mM IPTG for 8 h at 30 °C. Compound B (7-methoxy-2H-benzo[b][1,4]thiazine-3-amine) showed the strongest binding activity of the four new nitrogen compounds, with a K(d) value of 0.268 µM. The K(d) values of the six compounds were significantly correlated with their EC(50) values.
  
  
  CONCLUSIONS
  The spectral analysis and bioassay results could be used to screen the new chemical entities effectively. Compound B, selected by virtual screening from a commercial chemical library, is a candidate for a new DMI fungicide. These results provide a theoretical basis and new ideas for efficient design and development of new antifungal agents.
authors.affiliation: Hubei Key Laboratory of Genetic Regulation and Integrative Biology, College of Life Sciences, Central China Normal University, Wuhan, China.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Zhang
authors.forename: Li
authors.initials: L
authors.lastname: Zhao
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
authors.forename: Rui
authors.initials: R
authors.lastname: Han
authors.forename: Shuxiang
authors.initials: S
authors.lastname: Li
authors.forename: Yongze
authors.initials: Y
authors.lastname: Yuan
authors.forename: Jian
authors.initials: J
authors.lastname: Wan
authors.forename: Wenjing
authors.initials: W
authors.lastname: Xiao
authors.forename: Deli
authors.initials: D
authors.lastname: Liu
mesh_terms: D001393:Azoles / Q000494:pharmacology*; D005656:Fungal Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D005659:Fungicides, Industrial / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects*; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D010407:Penicillium / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D058886:Sterol 14-Demethylase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D001393:Azoles; D005656:Fungal Proteins; D005659:Fungicides, Industrial; D058886:Sterol 14-Demethylase