摘要
红球菌(Rhodococcus sp.)R04基因组有15种细胞色素P450单加氧酶,其中CYP125A18与结核分枝杆菌(Mycobacterium tuberculosis)和马红球菌(Rhodococcus equi)的CYP125有较高同源性。利用NCBI蛋白质数据库搜索同源序列,对Rhodococcus sp.R04的15种CYP450一级结构序列进行比对和系统发育分析;对CYP125A18基因进行了克隆表达,并用紫外分光光度法对蛋白质的光谱学特性以及与唑类药物互作情况进行分析。实验结果表明,Rhodococcus sp.R04 15种CYP450均含有保守的氨基酸序列和铁血红素催化中心。SDS-PAGE分析表明,CYP125A18分子量约为50 k D,CYP125A18还原态和CO结合后与CYP125A18氧化态的差示光谱表现为典型的CYP450光谱特性。CYP125A18与底物4-胆甾烯-3-酮结合后,血红素铁全部转变为高自旋状态;与唑类药物滴定后发生了II型光谱转变。解离常数表明,7种唑类药物与CYP125A18的亲和力由强到弱依次为酮康唑、益康唑、4-苯基咪唑、氟康唑、4-甲基-2-苯基咪唑、克霉唑、甲硝唑。上述发现对研究CYP125代谢胆固醇具有重要意义,同时为疾病耐药性研究及药物选择提供数据和理论支持。
The Rhodococcus sp. R04 genome contains 15 cytochrome P450 monooxygenases,including CYP125A18 that shares homology with counterparts from Mycobacterium tuberculosis and Rhodococcus equi. In this study,the amino acid sequences of those 15 CYP450 s were aligned for constructing a phylogenetic tree. Cloned CYP125A18 was expressed in E. coli BL21（ DE3）,and then analyzed for protein spectroscopic properties. Its interaction with azoles was investigated by using UV-Vis spectrophotometry. The results showed that all examined CYP450 s contained conserved sequences and a heme iron catalytic center. SDS-PAGE analysis showed that the apparent molecular weight of CYP125A18 was approximately 50 k D. Typical CYP450 characteristics were shown in difference spectra when CYP125A18 bound to CO in the reductive or oxidative states. Binding 4-cholesten-3-one,all heme irons were transformed to high spin state. The type II spectral shift occurred at post-titration with azole drugs.According to the dissociation constants of CYP125A18 from strong to weak,the seven tested azole drugs were ketoconazole, econazole, 4-phenyl imidazole, fluconazole, 4-methyl-2-phenyl imidazole,clotrimazole and metronidazole. These findings may provide valuable data for the study of drug resistance and selection.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2016年第3期295-304,共10页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金项目(No.30800030)
山西省自然科学基金(2014011030-3)资助~~
