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基于柑橘绿霉菌CYP51同源模建的定点突变与抗性机制分析

, PP. 81-86

Keywords: 同源模建,分子对接,定点突变,光谱分析

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Abstract:

为阐明杀菌剂与PdCYP51的相互作用及其抗性机制,基于最新解析的真核生物人类的CYP51晶体结构,同源模建了PdCYP51的三维结构,并选取商品化的杀菌剂烯唑醇进行分子对接,预测影响PdCYP51与烯唑醇相互作用的关键氨基酸。采用定点突变技术获得了PdCYP51-Y112H、F120L、F120D和S309A4种突变体(PdCYP51m)。结果表明,突变体蛋白PdCYP51-Y112H、F120L、F120D和S309A表达量均有不同程度的变化,PdCYP51-Y112H和F120L与未突变蛋白表达量相当,而PdCYP51-F120D和S309A表达量增加。4种突变体与杀菌剂的结合能力降低,其结合常数分别为1.28、0.18、1.03和1.31μmol/L,均大于未突变PdCYP51的0.12μmol/L。表明这些氨基酸是杀真菌剂烯唑醇与PdCYP51结合的关键氨基酸,PdCYP51与烯唑醇形成的疏水性空腔和稳定配体杀菌剂分子的作用力是酶蛋白与杀菌剂结合的重要因素。

References

[1]  闵晓芳,邓伯勋,陈丽锋,等. 柑橘采后致病青霉的鉴定. 果树学报,2007, 24(5): 653-656
[2]  Zhang Z F, Zhu Z R, Ma Z H, et al. A molecular mechanism of azoxystrobin resistance in Penicillium digitatum UV mutants and a PCR-based assay for detection of azoxystrobin-resistant strains in packing- or store-house isolates. International Journal of Food Microbiology, 2009,131(2/3): 157-161
[3]  Ma Z H, Michailides T J. Advances in understanding molecular mechanisms of fungicide resistance and molecular detection of resistant genotypes in phytopathogenic fungi. Crop Protection, 2005, 24 (10): 853-863
[4]  Feng L J, Wan Z, Wang X H, et al. Relationship between antifungal resistance of fluconazole resistant Candida albicans and mutations in ERG11 gene. Chinese Medical Journal, 2010, 123(5): 544-548
[5]  Morris G M, Goodsell D S, Halliday R S, et al. Automated docking using a Lamarckian genetic algorithm and an empirical binding free energy function. Journal of Computational Chemistry, 1998, 19(14): 1639-1662
[6]  Lepesheva G I, Waterman M R. Structural basis for conservation in the CYP51 family. Biochimica et Biophysica Acta, 2011, 1814(1): 88-89
[7]  Thompson J D, Higgins D G, Gibson T J. Clustal W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research, 1994, 22(22): 4673-4680
[8]  Eswar N, Webb B, Marti-Renom M A, et al. Comparative protein structure modeling using MODELLER. Current Protocols in Protein Science, 2007, 2(12): 15-32
[9]  Zhao L, Liu D L, Zhang Q Y, et al. Expression and homology modeling of sterol 14α-demethylase from Penicillium digitatum. FEMS Microbiology Letters, 2007, 277(1): 37-43
[10]  Bradford M M. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 1976, 72(1): 248-254
[11]  Omura T, Sato R. The carbon monoxide-binding pigment of liver microsomes. Ⅰ. Evidence for its hemoprotein nature. The Journal of Biological Chemistry, 1964, 239(7): 2370-2378
[12]  张?,黎晨,赵莉,等. 新型含氮化合物对柑桔绿霉菌的离体抑菌活性及其构效关系. 植物保护学报,2008,35(6):513-518
[13]  Gollapudy R, Ajmani S, Kulkarni S A. Modeling and interactions of Aspergillus fumigatus lanosterol 14-alpha demethylase \'A’ with azole antifungals. Bioorganic & Medicinal Chemistry, 2004, 12(11): 2937-2950
[14]  Rupp B, Raub S, Marian C, et al. Molecular design of two sterol 14 alpha-demethylase homology models and their interactions with the azole antifungals ketoconazole and bifonazole. Journal of Computer-Aided Molecular Design, 2005, 19(3): 149-163
[15]  Sheng C Q, Chen S H, Ji H T, et al. Evolutionary trace analysis of CYP51 family: implication for site-directed mutagenesis and novel antifungal drug design. Journal of Molecular Modeling, 2010, 16(2): 279-284
[16]  Lepesheva G I, Virus C, Waterman M R. Conservation in the CYP51 family. Role of the B’helix/BC loop and helices F and G in enzymatic function. Biochemistry, 2003, 42(30): 9091-9101
[17]  肖敏,杨娇艳,肖文精,等. 结合光谱法在DMIs类杀真菌剂筛选中的应用. 生物工程学报,2007,23(6):1129-1134
[18]  Zhang J H, Zhao L, Zhang J. Optimised expression and spectral analysis of the target enzyme CYP51 from Penicillium digitatum with possible new DMI fungicides. Pest Management Science, 2010, 66(12): 1344-1350
[19]  Delye C, Laigret F, Corio-Costet M F. A mutation in the 14α-demethylase gene of Uncinula necator that correlates with resistance to a sterol biosynthesis inhibitor. Applied and Environmental Microbiology, 1997, 63(8): 2966-2970
[20]  Delye C, Bousset L, Corio-Costet M F. PCR cloning and detection of point mutations in the eburicol 14α-demethylase (CYP51) gene from Erysiphe graminis f.sp. hordei, a "recalcitrant" fungus. Current Genetics, 1998, 34(5): 399-403
[21]  更多...
[22]  Joseph-Horne T, Hollomon D W. Molecular mechanisms of azole resistance in fungi. FEMS Microbiology Letters, 1997, 149(2): 141-149
[23]  Butters J A, Zhou M C, Hollomon D W. The mechanism of resistance to sterol 14α-demethylation inhibitors in a mutant (Erg 40) of Ustilago maydis. Pest Management Science, 2000, 56(3): 257-263

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