Molecular reconstruction of flavocytochrome P450 BM3

黄细胞色素 P450 BM3 的分子重建

• 批准号: BB/K001884/1
• 负责人: Andrew Munro
• 金额: $ 58.8万
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2012
• 资助国家: 英国
• 起止时间: 2012 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FK001884%2F1
• 关键词: Molecular; reconstruction; flavocytochrome; P450; BM3

Determining the structure of biological molecules is critical for understanding their physiological function. Excellent examples include Crick and Watson's DNA structure, the structure of hemoglobin solved by Max Perutz and (more recently) the structures of the complex molecular machines (ATP synthase and the respiratory complexes) that facilitate energy generation from oxidation of foodstuffs (lipids, carbohydrates etc) to enable life. The major techniques used to determine the three dimensional organization of atoms in biological (and other) molecules are X-ray crystallography (diffraction of X-rays from crystals of the molecule, e.g. a protein) and nuclear magnetic resonance (NMR) that is used on molecules in solution. However, there are limitations to application of these methods for structural determination of large biomolecules (such as complex proteins and enzymes). Typically, determining structures of molecules larger in size than 30 kDa becomes challenging and expensive for NMR. While X-ray diffraction methods are more "tolerant" to the size of the molecule in question, it is absolutely dependent on ability to obtain crystals of the target molecule. This can be difficult (or impossible), and even if crystals are obtained they may not diffract X-rays sufficiently well to enable a structure to be solved. Crystals of large, complex proteins containing several subunits or individual structural segments (domains) are often difficult to crystallize, particularly if regions of the protein are highly mobile in solution.The subject of this proposal is an important member of the cytochrome P450 (P450) class of enzymes named flavocytochrome P450 BM3 (BM3). P450s are critical in human drug/xenobiotic detoxification, and play important roles in steroid metabolism. They do this by introducing oxygen atoms into their substrates. P450s are widely studied due to their importance in drug metabolism, as well as being important drug targets in their own right, but to date there are no true structural data to describe how P450s interact with their partner enzymes -notably cytochrome P450 reductase (CPR). In this work, we will apply a new combination of tools (spectroscopic, modelling, protein engineering and analytical methods) to define the structural organization of the BM3 enzyme - which is a "model" system in the P450 enzyme family, and which is also a natural "fusion enzyme" formed by direct linkage of a fatty acid metabolizing P450 to a CPR enzyme. While mammalian P450s and CPR are membrane associated, insoluble proteins, BM3 is fully soluble and can be prepared in large amounts for characterization. The BM3 protein has several domains and we have shown recently that it is functional as a dimer. BM3 can be broken down (by genetic engineering) into its individual domains (e.g. the CPR and P450 modules) and structures of its individual domains have been solved by X-ray crystallography. However, intact BM3 has been refractory to crystallography, for reasons described above. The crux of this proposal is to provide a structural model for the BM3 dimer through synergistic application of diverse spectroscopic, modelling and other methods to enable aspects of shape, domain mobility and inter-monomer distances to be defined, allowing the structure of the BM3 dimer to be reconstructed. The model will then be validated, and tools developed for BM3 will then be applied to another important enzyme - nitric oxide synthase (NOS) which has a similar dimeric architecture to BM3, but a more complicated mode of regulation due to interactions with the calcium binding protein calmodulin. NOS has also been refractory to crystallization, but is critical for human neurotransmission, immune function and blood pressure control. Collectively, these studies on BM3/NOS will provide a new route to determining the structural layout of complex multidomain proteins, as well as insights into how these heme-binding proteins interact with their linked CPR partner.

确定生物分子的结构对于理解其生理功能至关重要。很好的例子包括克里克和沃森的 DNA 结构、马克斯·佩鲁茨解决的血红蛋白结构以及（最近）复杂分子机器（ATP 合成酶和呼吸复合物）的结构，这些分子机器促进食品（脂质、碳水化合物等）氧化产生能量以维持生命。用于确定生物（和其他）分子中原子三维组织的主要技术是 X 射线晶体学（来自分子晶体（例如蛋白质）的 X 射线衍射）和用于溶液中分子的核磁共振 (NMR)。然而，这些方法在大生物分子（例如复杂蛋白质和酶）的结构测定中的应用存在局限性。通常，对于 NMR 来说，确定尺寸大于 30 kDa 的分子的结构变得具有挑战性且昂贵。虽然 X 射线衍射方法对所讨论的分子的大小更“宽容”，但它绝对取决于获得目标分子晶体的能力。这可能很困难（或不可能），而且即使获得了晶体，它们也可能无法很好地衍射 X 射线以解析结构。含有多个亚基或单个结构片段（结构域）的大而复杂的蛋白质晶体通常很难结晶，特别是当蛋白质区域在溶液中具有高度流动性时。本提案的主题是细胞色素 P450 (P450) 类酶的重要成员，名为黄素细胞色素 P450 BM3 (BM3)。 P450 在人类药物/异生物质解毒中至关重要，并在类固醇代谢中发挥重要作用。他们通过将氧原子引入其基底来实现这一点。 P450 由于其在药物代谢中的重要性以及其本身就是重要的药物靶点而受到广泛研究，但迄今为止，还没有真正的结构数据来描述 P450 如何与其伴侣酶（尤其是细胞色素 P450 还原酶 (CPR)）相互作用。在这项工作中，我们将应用新的工具组合（光谱、建模、蛋白质工程和分析方法）来定义BM3酶的结构组织——BM3酶是P450酶家族中的“模型”系统，也是由脂肪酸代谢P450与CPR酶直接连接形成的天然“融合酶”。哺乳动物 P450 和 CPR 是膜相关的不溶性蛋白质，而 BM3 是完全可溶的，可以大量制备用于表征。 BM3 蛋白有多个结构域，我们最近证明它具有二聚体的功能。 BM3 可以（通过基因工程）分解为其各个结构域（例如 CPR 和 P450 模块），并且其各个结构域的结构已通过 X 射线晶体学解析。然而，由于上述原因，完整的 BM3 难以进行晶体学分析。该提案的关键是通过多种光谱、建模和其他方法的协同应用，为BM3二聚体提供结构模型，以定义形状、域迁移率和单体间距离等方面，从而重建BM3二聚体的结构。然后，该模型将得到验证，为 BM3 开发的工具将应用于另一种重要的酶——一氧化氮合酶 (NOS)，该酶具有与 BM3 相似的二聚体结构，但由于与钙结合蛋白钙调蛋白的相互作用，其调节模式更为复杂。 NOS 也难以结晶，但对人类神经传递、免疫功能和血压控制至关重要。总的来说，这些关于 BM3/NOS 的研究将为确定复杂多域蛋白的结构布局提供新途径，并深入了解这些血红素结合蛋白如何与其连接的 CPR 伙伴相互作用。

项目成果

Overview on theoretical studies discriminating the two-oxidant versus two-state-reactivity models for substrate monoxygenation by cytochrome P450 enzymes.

区分细胞色素 P450 酶底物单氧合的双氧化剂与双态反应模型的理论研究概述。

• DOI: 10.2174/15680266113136660155
• 发表时间: 2013
• 期刊: Current topics in medicinal chemistry
• 影响因子: 3.4
• 作者: De Visser SP

P450-Catalyzed Regio- and Diastereoselective Steroid Hydroxylation: Efficient Directed Evolution Enabled by Mutability Landscaping

• DOI: 10.1021/acscatal.8b00389
• 发表时间: 2018-04-01
• 期刊: ACS CATALYSIS
• 影响因子: 12.9
• 作者: Acevedo-Rocha, Carlos G.;Gamble, Charles G.;Reetz, Manfred T.
• 通讯作者: Reetz, Manfred T.

Expression, Purification, and Biochemical Characterization of the Flavocytochrome P450 CYP505A30 from Myceliophthora thermophila.

• DOI: 10.1021/acsomega.7b00450
• 发表时间: 2017-08-31
• 期刊: ACS omega
• 影响因子: 4.1
• 作者: Baker GJ;Girvan HM;Matthews S;McLean KJ;Golovanova M;Waltham TN;Rigby SEJ;Nelson DR;Blankley RT;Munro AW
• 通讯作者: Munro AW