Ji H, Zhang W, Zhou Y, Zhang M, Zhu J, Song Y, Lü J
School of Pharmacy, Second Military Medical University, Shanghai, People's Republic of China.
J Med Chem. 2000 Jun 29;43(13):2493-505. doi: 10.1021/jm990589g.
The three-dimensional structure of lanosterol 14alpha-demethylase (P450(14DM), CYP51) of Candida albicans was modeled on the basis of crystallographic coordinates of four prokaryotic P450s: P450BM3, P450cam, P450terp, and P450eryF. The P450(14DM) sequence was aligned to those of known proteins using a knowledge-based alignment method. The main chain coordinates of the core regions were transferred directly from the corresponding coordinates of P450BM3. The side chain conformations of the core regions were determined by the conformations of the equivalent residues with the highest homologous scores in four crystal structures. The model was then refined using molecular mechanics and molecular dynamics. The reliability of the resulting model was assessed by Ramachandran plots, Profile-3D, hydropathy plot analysis, and by analyzing the consistency of the model with the experimental data. The structurally and functionally important residues such as the heme binding residues, the residues interacting with redox-partner protein and/or involved in electron transfer, the residues lining substrate access channel, and the substrate binding residues were identified from the model. These residues are candidates for further site-directed mutagenesis and site-specific antipeptide antibody binding experiments. The active analogue approach was employed to search the pharmacophoric conformations for 14 azole antifungals. The resulting bioactive conformations were docked into the active site of lanosterol 14alpha-demethylase of Candida albicans. All 14 azole antifungals are shown to have a similar docking mode in the active site. The halogenated phenyl group of azole inhibitors is deep in the same hydrophobic binding cleft as the 17-alkyl chain of substrate. The pi-pi stacking interaction might exist between halogenated phenyl ring of inhibitors and the aromatic ring of residue Y132. The long side chains of some inhibitors such as itraconazole and ketoconazole surpass the active site and interact with the residues in the substrate access channel. To compare with mammalian enzymes, structurally selective residues of the active site of fungal lanosterol 14alpha-demethylase are distributed in the C terminus of F helix, beta6-1 sheet and beta6-2 sheet.
基于四种原核细胞色素P450（P450BM3、P450cam、P450terp和P450eryF）的晶体学坐标，对白色念珠菌羊毛甾醇14α-脱甲基酶（P450(14DM)，CYP51）的三维结构进行了模拟。使用基于知识的比对方法，将P450(14DM)序列与已知蛋白质的序列进行比对。核心区域的主链坐标直接从P450BM3的相应坐标转移而来。核心区域的侧链构象由四个晶体结构中同源性得分最高的等效残基的构象确定。然后使用分子力学和分子动力学对模型进行优化。通过拉氏图、Profile-3D、亲水性图谱分析以及分析模型与实验数据的一致性来评估所得模型的可靠性。从模型中鉴定出结构和功能上重要的残基，如血红素结合残基、与氧化还原伴侣蛋白相互作用和/或参与电子传递的残基、构成底物进入通道内壁的残基以及底物结合残基。这些残基是进一步进行定点诱变和位点特异性抗肽抗体结合实验的候选对象。采用活性类似物方法搜索14种唑类抗真菌药物的药效构象。将所得的生物活性构象对接至白色念珠菌羊毛甾醇14α-脱甲基酶的活性位点。结果显示，所有14种唑类抗真菌药物在活性位点具有相似的对接模式。唑类抑制剂的卤代苯基与底物的17-烷基链位于同一疏水结合裂隙深处。抑制剂的卤代苯环与残基Y132的芳香环之间可能存在π-π堆积相互作用。一些抑制剂（如伊曲康唑和酮康唑）的长侧链超出活性位点并与底物进入通道中的残基相互作用。为了与哺乳动物酶进行比较，真菌羊毛甾醇14α-脱甲基酶活性位点的结构选择性残基分布在F螺旋的C末端、β6-1片层和β6-2片层中。
