Characterization of a superior biocatalyst for 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.

Pravastatin is a leading statin drug used to inhibit the human enzyme HMG CoA reductase and to facilitate the reduction of plasma cholesterol levels. In turn, this decreases levels of LDL cholesterol in plasma and hence risk of atherosclerosis and coronary heart disease. Pravastatin can be generated from a natural product (compactin) produced by Penicillium citrinum, and previous studies identified a P450 oxidase enzyme (P450sca-2) from the bacterium Streptomyces carbophilus that performed the relevant hydroxylation reaction. This process is now off patent, and in preliminary work DSM and the applicants have demonstrated that a much superior compactin hydroxylase P450 catalyst exists in the bacterium Amycolatopsis orientalis. The enzyme, informally termed P450prava, is more readily expressed, more stable and has superior kinetic properties to P450sca-2. Major initial objectives of this proposal are the optimisation of expression, the purification and biochemical/biophysical characterization of P450prava (including substrate- and ligand-binding analysis), followed by crystallization of the P450 and the determination of its crystal structure in ligand-free and compactin-bound forms. Distinct redox partner systems support P450prava catalysis, including an A. orientalis ferredoxin and a phthalate dioxygenase reductase/P450prava fusion. These systems will be characterised by detailed kinetic methods and an optimal system identified. P450prava forms large amounts of a less-active epimer of pravastatin, and rational mutagenesis (targeted on areas known to control substrate binding) will be used to create variants that optimise pravastatin formation (at expense of epi-pravastatin). Preliminary studies show that the approach is sound and the objective feasible. The outcome of the project will be the generation of an optimised new catalyst and an efficient redox system for pravastatin production, that can be used commercially for fermentation of an important pharmaceutical.