Characterization of a superior biocatalyst for pravastatin production

用于普伐他汀生产的优质生物催化剂的表征

• 批准号: BB/G014329/1
• 负责人: Andrew Munro
• 金额: $ 42.42万
• 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: 2009
• 资助国家: 英国
• 起止时间: 2009 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FG014329%2F1
• 关键词: Characterization; superior; biocatalyst; pravastatin; production

Recent years have seen a revolution in the public's understanding of the relationship between diet and human health. In particular, there has been widespread realization of the dangers associated with a cholesterol rich diet. Although cholesterol is essential for such functions as regulation of cell membrane fluidity and in the synthesis of steroid hormones, there is also clearly a dark side to the presence of excess cholesterol in the bloodstream. This is since excess cholesterol (hypercholesterolemia) is associated with excessive amounts of cholesterol carried by low density lipoproteins (LDL cholesterol). It is clear that high levels of LDL cholesterol (compared to levels of cholesterol carried by high density lipoproteins, HDL cholesterol) is a major risk factor for coronary heart disease. High LDL cholesterol is associated with atheroma development in arteries and the condition known as atherosclerosis. This has led to health advice focused on encouraging individuals to reduce the fat content of their diets and to take regular exercise to avoid excess body fat and to reduce heart disease. However, the intervention of the pharmaceutical industries has provided new ways in which the plasma cholesterol levels of patients can be decreased. The development of the class of drugs known as statins has been one of the major breakthroughs in human healthcare over the last two decades. These drugs act to lower plasma cholesterol levels, and their primary mechanism of action is the inhibition of a key human enzyme called 3-hydroxy-3-methylglutaryl-CoA reductase (HMG CoA reductase), which is the rate-limiting enzyme step in the pathway leading to cholesterol synthesis. The statins are a group of drugs that have their origins in the discovery of a natural compound (compactin) found in a fungus, and which was shown to have good cholesterol lowering properties. Since compactin itself was not stable enough for clinical use, derivatives were created and other molecules with a similar mode of action were prepared to provide useful drugs. One of the most effective of these is pravastatin, which is derived from compactin by the action of a heme-containing protein known as a cytochrome P450 (or P450), which introduces an oxygen atom onto the substrate. The first P450 (from a bacterium) shown to catalyse this reaction (P450sca-2) has been used commercially, but studies on this enzyme by the partner (DSM) on this application revealed the enzyme to be substantially inferior to another P450 enzyme (informally named P450prava) isolated from an alternative bacterium. The major objectives of this proposal are the production, purification and characterization of the P450prava enzyme, including the determination of its 3-dimensional structure by the method of x-ray crystallography. The work proposed also includes examination of partner proteins for the P450, which are required for delivery of electrons to P450prava to enable it to perform its catalytic function in pravastatin formation. These partner proteins will be purified and their interactions with P450prava quantified to identify the best system and conditions that lead to optimal production of pravastatin. Since P450prava has a tendency to introduce oxygen on the wrong side of a ring structure on the substrate compactin, the technique of rational mutagenesis will be used to alter the structure of the P450 in important regions that control compactin binding. The aim is to alter the position of oxygen insertion (to the correct side of the ring) in order to form the most active form of pravastatin as the major product. Preliminary studies have shown that this strategy will be successful. The overall outcome of the proposal will be the generation of a new catalyst (P450prava) for production of a leading statin drug (pravastatin), the determination of its structure and function, and its improvement as a catalyst by mutagenesis to facilitate improved commercial pravastatin production.

近年来，公众对饮食与人体健康之间关系的理解发生了革命性的变化。特别是，人们已经广泛认识到与富含胆固醇的饮食相关的危险。虽然胆固醇对于调节细胞膜流动性和合成类固醇激素等功能是必不可少的，但血液中过量胆固醇的存在也有明显的阴暗面。这是因为过量的胆固醇（高胆固醇血症）与低密度脂蛋白（LDL胆固醇）携带的过量胆固醇有关。很明显，高水平的低密度脂蛋白胆固醇（与高密度脂蛋白胆固醇携带的胆固醇水平相比）是冠心病的主要危险因素。高LDL胆固醇与动脉粥样硬化的发展和称为动脉粥样硬化的状况有关。这导致了健康建议的重点是鼓励个人减少饮食中的脂肪含量，并定期锻炼，以避免过多的身体脂肪和减少心脏病。然而，制药业的干预提供了降低患者血浆胆固醇水平的新方法。他汀类药物的开发是过去二十年来人类医疗保健的重大突破之一。这些药物可降低血浆胆固醇水平，其主要作用机制是抑制一种名为3-羟基-3-甲基戊二酰辅酶A还原酶（HMG CoA还原酶）的关键人体酶，该酶是导致胆固醇合成的途径中的限速酶步骤。他汀类药物是一组药物，其起源于在真菌中发现的天然化合物（compactin）的发现，并且被证明具有良好的降胆固醇特性。由于康伐他汀本身对于临床应用来说不够稳定，因此产生了衍生物，并制备了具有类似作用模式的其他分子以提供有用的药物。其中最有效的一种是普伐他汀，它是通过一种被称为细胞色素P450（或P450）的含血红素蛋白质的作用从康伐他汀衍生而来的，该蛋白质将氧原子引入底物。第一个显示催化该反应的P450（来自细菌）（P450 sca-2）已经在商业上使用，但是该申请的合作伙伴（DSM）对该酶的研究显示该酶基本上劣于从替代细菌分离的另一种P450酶（非正式地称为P450 prava）。该提案的主要目标是P450 prava酶的生产，纯化和表征，包括通过X射线晶体学方法确定其三维结构。这项工作还包括检查P450的伴侣蛋白，这是向P450 prava传递电子所必需的，使其能够在普伐他汀形成中发挥催化作用。这些伴侣蛋白将被纯化，并与P450 prava定量的相互作用，以确定最佳的系统和条件，导致普伐他汀的最佳生产。由于P450 prava有一种倾向，以引入氧的环结构上的底物compactin的错误的一面，合理的诱变技术将被用来改变P450的结构在重要的区域，控制compactin的结合。目的是改变氧插入的位置（到环的正确侧），以形成作为主要产物的最活性形式的普伐他汀。初步研究表明，这一战略将取得成功。该提案的总体结果将是产生一种用于生产领先的他汀类药物（普伐他汀）的新催化剂（P450 prava），确定其结构和功能，并通过诱变将其作为催化剂进行改进，以促进普伐他汀的商业化生产。

项目成果

Cytochrome P450 - Structure, Mechanism, and Biochemistry

细胞色素 P450 - 结构、机制和生物化学

• DOI: 10.1007/978-3-319-12108-6_6
• 发表时间: 2015
• 作者: McLean K

Computation-Aided Engineering of Cytochrome P450 for the Production of Pravastatin.

• DOI: 10.1021/acscatal.2c03974
• 发表时间: 2022-12-16
• 期刊: ACS CATALYSIS
• 影响因子: 12.9
• 作者: Ashworth, Mark A.;Bombino, Elvira;de Jong, Rene M.;Wijma, Hein J.;Janssen, Dick B.;McLean, Kirsty J.;Munro, Andrew W.
• 通讯作者: Munro, Andrew W.

Encyclopedia of Biophysics

生物物理学百科全书

• DOI: 10.1007/978-3-642-16712-6_41
• 发表时间: 2013
• 作者: Munro A