Eddine Ali Nasser, von Kries Jens P, Podust Mikhail V, Warrier Thulasi, Kaufmann Stefan H E, Podust Larissa M
Max-Planck-Institute for Infection Biology, Berlin, Germany.
J Biol Chem. 2008 May 30;283(22):15152-9. doi: 10.1074/jbc.M801145200. Epub 2008 Mar 26.
A universal step in the biosynthesis of membrane sterols and steroid hormones is the oxidative removal of the 14alpha-methyl group from sterol precursors by sterol 14alpha-demethylase (CYP51). This enzyme is a primary target in treatment of fungal infections in organisms ranging from humans to plants, and development of more potent and selective CYP51 inhibitors is an important biological objective. Our continuing interest in structural aspects of substrate and inhibitor recognition in CYP51 led us to determine (to a resolution of 1.95A) the structure of CYP51 from Mycobacterium tuberculosis (CYP51(Mt)) co-crystallized with 4,4'-dihydroxybenzophenone (DHBP), a small organic molecule previously identified among top type I binding hits in a library screened against CYP51(Mt). The newly determined CYP51(Mt)-DHBP structure is the most complete to date and is an improved template for three-dimensional modeling of CYP51 enzymes from fungal and prokaryotic pathogens. The structure demonstrates the induction of conformational fit of the flexible protein regions and the interactions of conserved Phe-89 essential for both fungal drug resistance and catalytic function, which were obscure in the previously characterized CYP51(Mt)-estriol complex. DHBP represents a benzophenone scaffold binding in the CYP51 active site via a type I mechanism, suggesting (i) a possible new class of CYP51 inhibitors targeting flexible regions, (ii) an alternative catalytic function for bacterial CYP51 enzymes, and (iii) a potential for hydroxybenzophenones, widely distributed in the environment, to interfere with sterol biosynthesis. Finally, we show the inhibition of M. tuberculosis growth by DHBP in a mouse macrophage model.
膜甾醇和甾体激素生物合成中的一个普遍步骤是，甾醇14α-脱甲基酶（CYP51）将甾醇前体中的14α-甲基氧化去除。这种酶是治疗从人类到植物等生物体真菌感染的主要靶点，开发更有效和更具选择性的CYP51抑制剂是一个重要的生物学目标。我们一直对CYP51中底物和抑制剂识别的结构方面感兴趣，这促使我们确定了结核分枝杆菌CYP51（CYP51(Mt)）与4,4'-二羟基二苯甲酮（DHBP）共结晶的结构（分辨率为1.95Å），DHBP是一种小分子有机化合物，先前在针对CYP51(Mt)筛选的文库中被鉴定为I型结合最佳命中物之一。新确定的CYP51(Mt)-DHBP结构是迄今为止最完整的，是对来自真菌和原核病原体的CYP51酶进行三维建模的改进模板。该结构展示了柔性蛋白区域构象契合的诱导以及保守的Phe-89的相互作用，Phe-89对真菌耐药性和催化功能都至关重要，而这在先前表征的CYP51(Mt)-雌三醇复合物中并不明确。DHBP代表一种通过I型机制结合在CYP51活性位点的二苯甲酮支架，这表明（i）可能存在一类针对柔性区域的新型CYP51抑制剂，（ii）细菌CYP51酶具有替代催化功能，（iii）广泛分布于环境中的羟基二苯甲酮有可能干扰甾醇生物合成。最后，我们在小鼠巨噬细胞模型中展示了DHBP对结核分枝杆菌生长的抑制作用。
