In order to establish a fast and accurate method for novel DMIs fungicide screening, lanosterol 14alpha-demethylase of Magnaporthe grisea expressed in E. coli was used as target enzyme and the DMI fungicides diniconazole, tebuconazole, triadimenol and triadimefon were used as representative fungicides, the effects of enzyme activity, enzyme purity and concentration on the binding spectra were investigated. The results showed that active enzyme, elimination of interference of other P450s and proper enzyme concentration were necessary for obtaining accurate binding spectra. The Kd values of diniconazole, tebuconazole, triadimenol and triadimefon were 0.143 micromol/L, 0.24 micromol/L, 0.257 micromol/L and 0.307 micromol/L respectively, which significantly correlated to their 120h-EC50 values on the growth of Magnaporthe grisea. The results indicated that the binding spectra of fungicide and lanosterol 14alpha-demethylase can serve as a reliable and fast method for novel fungicide screening.
Cytochrome P-450 Enzyme System ; metabolism ; Fungicides, Industrial ; pharmacology ; Spectrophotometry ; methods ; Sterol 14-Demethylase ; Triazoles ; pharmacology

Sterol 14alpha-demethylase (CYP51), the most widely distributed member of the P450 superfamily, is the key enzyme in sterol biosynthesis pathway. CYP51 is not only an important model for fundamental P450 structure/function studies, but also an important target protein of cholesterol-lowering agents, antifungal drugs and herbicides. This article reviewed the research advances in CYP51 at various aspects, including sequence characteristics, physiological roles, catalytic properties in vitro, protein structure, structure-function relationships and inhibition of CYP51. The problems remained in current research and designations of CYP51 inhibitors are also discussed.
Cytochrome P-450 Enzyme Inhibitors ; Cytochrome P-450 Enzyme System ; genetics ; physiology ; Mutagenesis, Site-Directed ; Sequence Analysis, Protein ; Sterol 14-Demethylase

The cyp51 primers and two pairs of mutant primers which removed different transmembrane region were designed based on Ustilago maydis cyp51 gene structure analysis. The full cyp51 DNA fragment as well as mutant cyp51 genes were amplified and cloned by using the total DNA from Ustilago maydis as template, then subcloned into different expression vectors. The recombinant expression plasmids were transformed into Escherichia coli BL21 (DE3), BL21 (DE3) pLysS and Rosetta (DE3) respectively. A series of experiments leads to the finding that only pET32-YH-35 could be highly expressed at the optimal condition of 30 degrees C induced with 0.5 mmol/L IPTG The expressed protein (CYP51) showed biological activity by spectra analysis of the protein binding to 4 standard fungicides and to 14 XF-synthetic fungicide compounds, and only one XF-synthetic fungicide compound (XF-113) was similar to standard fungicides in binding constant. This compound is promising to be a new effective antifungal drug. These results will facilitate the further study on the mechanism of pathogenic fungi CYP51 and pesticide molecules, and will provide a new idea for efficient design and development of new anti-fungal drugs.
Antifungal Agents ; isolation & purification ; Cloning, Molecular ; Cytochrome P-450 Enzyme System ; genetics ; metabolism ; Escherichia coli ; genetics ; metabolism ; Fungal Proteins ; genetics ; metabolism ; Plasmids ; genetics ; Recombinant Proteins ; genetics ; metabolism ; Sterol 14-Demethylase ; Ustilago ; enzymology ; genetics

This study is to investigate the effect of Euphorbia humifusa effective fraction (EHEF) on the CYP51 enzyme activity, the lanosterol content and the MEP, SUB gene expression of Trichophyton rubrum. Trichophyton rubrum was treated by EHEF for 7 days at 26 degrees C. The activity of CYP51 enzyme of Trichophyton rubrum in the cell membrane was determined by using ELISA kit, and the lanosterol content was investigated by using high performance liquid chromatography (HPLC), and the MEP, SUB gene expression of Trichophyton rubrum was detected with the reverse transcription polymerase chain reaction (RT-PCR) method. Results showed that EHEF can decrease the membrane CYP51 enzyme activity, and it also can accumulate the fungal lanosterol in a dose-dependent manner, and it also can decrease the gene expression of MEP and SUB. The antifungal mechanism of EHEF may be related to the inhibition on CYP51 enzyme activity, and to the effects on fungal cell membrane ergosterol biosynthesis. It may also play an antifungal effect by inhibiting the MEP, SUB gene expression of fungal proteases.
Antifungal Agents ; isolation & purification ; pharmacology ; Cell Membrane ; drug effects ; metabolism ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Enzyme Activation ; drug effects ; Euphorbia ; chemistry ; Gene Expression Regulation, Fungal ; Lanosterol ; metabolism ; Metalloproteases ; metabolism ; Plants, Medicinal ; chemistry ; Sterol 14-Demethylase ; metabolism ; Subtilisins ; metabolism ; Trichophyton ; drug effects ; genetics ; metabolism