Characterization of a superior biocatalyst for 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，人们已经普遍认识到高胆固醇饮食的危害。尽管胆固醇在调节细胞膜流动性和合成类固醇激素等功能中起着至关重要的作用，但血液中过量胆固醇的存在显然也有不好的一面。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（与高密度脂蛋白所携带的胆固醇水平相比）是冠心病的主要危险因素。高低密度脂蛋白胆固醇与动脉粥样硬化的发展和动脉粥样硬化有关。因此，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，以避免体内脂肪过多，减少心脏病。然而，制药行业的干预为降低患者血浆胆固醇水平提供了新的途径。他汀类药物的开发是过去二十年来人类医疗保健领域的重大突破之一。这些药物的作用是降低血浆胆固醇水平，它们的主要作用机制是抑制一种关键的人体酶，即3-羟基-3-甲基戊二酰辅酶a还原酶（HMG CoA还原酶），这是导致胆固醇合成途径中的限速酶步骤。他汀类药物是一类药物，其起源是在真菌中发现的一种天然化合物（紧实蛋白），该化合物被证明具有良好的降胆固醇特性。由于压实蛋白本身不够稳定，不能用于临床，因此衍生品被创造出来，其他具有类似作用模式的分子被制备成有用的药物。其中最有效的一种是普伐他汀，它是通过一种称为细胞色素P450（或P450）的含血红素蛋白的作用从压实蛋白中衍生出来的，该蛋白将氧原子引入底物。第一个P450（来自细菌）被证明可以催化该反应（P450sca-2）已经在商业上使用，但是合作伙伴（DSM）对该酶的研究表明，该酶实质上不如从另一种细菌中分离出来的另一种P450酶（非正式命名为P450prava）。本提案的主要目标是P450prava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。提出的工作还包括检查P450的伴侣蛋白，这是将电子传递给P450prava以使其在普伐他汀形成中发挥催化作用所必需的。这些伙伴蛋白将被纯化，它们与P450prava的相互作用将被量化，以确定最佳的系统和条件，导致最佳的普伐他汀生产。由于P450prava倾向于将氧引入底物紧实蛋白环结构的错误一侧，因此将使用合理诱变技术来改变P450控制紧实蛋白结合的重要区域的结构。目的是改变氧插入的位置（在环的正确一侧），以形成最活跃的普伐他汀作为主要产物。初步研究表明，这一策略将会成功。该提案的总体结果将是生成一种新的催化剂（P450prava），用于生产一种领先的他汀类药物（普伐他汀），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。

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.