Characterization of a superior biocatalyst for pravastatin production

Lead Research Organisation: University of Manchester
Department Name: Life Sciences

Abstract

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.

Technical Summary

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.

Publications

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Guengerich FP (2013) Unusual cytochrome p450 enzymes and reactions. in The Journal of biological chemistry

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McLean K (2015) Cytochrome P450

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McLean KJ (2015) Biological diversity of cytochrome P450 redox partner systems. in Advances in experimental medicine and biology

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McLean KJ (2015) Single-step fermentative production of the cholesterol-lowering drug pravastatin via reprogramming of Penicillium chrysogenum. in Proceedings of the National Academy of Sciences of the United States of America

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McLean KJ (2015) Single-step fermentative production of the cholesterol-lowering drug pravastatin via reprogramming of Penicillium chrysogenum. in Proceedings of the National Academy of Sciences of the United States of America

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Munro AW (2013) What makes a P450 tick? in Trends in biochemical sciences

 
Description The project was an industrial partnership between the Munro group at the University of Manchester and DSM in Delft, The Netherlands. The aim of the project was to characterize and to engineer a microbial cytochrome P450 enzyme for its use in the synthesis of the cholesterol-lowering drug pravastatin, with the ultimate aim that this could be used in an industrial process at DSM for the production of the drug. The project covered a lot of ground and a large amount of work was done to engineer the enzyme toward this industrial application. Structural, kinetic, spectroscopic and biochemical characterizations of the wild-type form of the P450 (formally named CYP105AS1 from Amycolatopsis orientalis) were done, and various mutants forms were similarly characterized, leading to the final enzyme (containing 5 mutations, named P450prava). In this way, assessment of the influence of mutations introduced could be quantified at each stage in the process in order to observe how beneficial mutations led to production of the final P450prava enzyme. These studies demonstrated a progressive improvement in compactin affinity achieved by targeted mutagenesis. The wild-type P450 (where P450s are types of enzymes that catalyze the insertion of oxygen atoms into their substrates) was shown to catalyze efficient hydroxylation of the substrate compactin, but produced the wrong form of the product - specifically the inactive epi-pravastatin rather that pravastatin. This issue was solved by progressive enzyme engineering, leading to the P450prava enzyme form producing pravastatin in substantial excess over epi-pravastatin. In order for the P450prava enzyme to function efficiently in compactin hydroxylation, a range of heterologous electron transfer (redox) partners were tested for their efficiency in passing electrons from the natural electron donor molecules NADH and NADPH to P450prava (these included proteins such as flavodoxins, ferredoxins and their associated reductases) and for their productivity in pravastatin formation. An "optimized" system with P450prava was ultimately developed in which the P450 was genetically fused to a different sort of redox partner enzyme - a phthalate dioxygenase reductase-like enzyme. This provided a catalytically self-sufficient system with higher electron transfer rates than achieved by reconstituting P450prava with separate redox partners, and was substantially more efficient than for the enzyme reconstituted with redox partners from the host bacterium (Amycolatopsis orientalis). The structure of the wild-type CYP105AS1 enzyme was determined using the method of X-ray crystallography, which enabled modeling of the compactin binding mode and facilitated the design of mutations to further improve compactin binding and to enable its stereo-selective oxidation, ultimately leading to the effective formation of pravastatin (over its inactive epimer epi-pravastatin) in the final P450prava form. The final evolved P450prava variant contained 5 active site mutations and led to near-complete production of pravastatin over epi-pravastatin. The structure of this "optimized" P450prava mutant in complex with compactin was solved, highlighting future rational mutagenesis strategies that might further enhance pravastatin production. Efficient compactin conversion to pravastatin was demonstrated at DSM using Penicillium chrysogenum fermentation with the optimized P450prava mutant. Levels of pravastatin produced exceeded those obtained from a previous industrial method (that required two fermentation steps), and was achieved in a single fermentation - thus improving both yield and cost efficiency substantially. The majority of data collected were published in the leading journal PNAS, and the researched received international coverage and was featured in a number of online publications, including in Nature Chemical Biology. In more recent work we have done further protein engineering of the P450prava enzyme to improve enantioselectivity of the enzyme, including use of different softwares to guide active site mutagenesis and to enhance stereoselectivity for the desired reaction.
Exploitation Route The work done has obvious applications for exploitation in the industrial production of pravastatin. The structural studies also illustrate how binding and oxidation of compactin might be further improved, as well as providing routes to oxidative modifications of other statin-related molecules to diversify the structures of these types of molecule for further drug characterization. In ongoing work we are using P450Prava and other mutants of the enzyme in studies examining transformations of compactin, pravastatin and other statin drugs.
Sectors Chemicals,Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

URL http://www.nature.com/nchembio/journal/v11/n4/full/nchembio.1783.html
 
Description BBSRC Industrial CASE studentship
Amount £80,000 (GBP)
Funding ID BB/K012282/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2013 
End 09/2017
 
Description Manchester Institute of Biotechnology Open Day - annual event from 2012 onwards 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Scientific demonstrations to senior secondary school students to enthuse them about a scientific career and to provide advice on career development and the courses on offer at the University of Manchester.

Annual event - such that lessons are learned from one year's activity and are carried forward to the following year's presentations.
Year(s) Of Engagement Activity 2012,2013,2014,2015
 
Description Schools visit (Wilmslow) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Presentation to primary school children in final year on general science/genetics - talk sparked questions and general discussion

Students registered interests in scientific career. Invite for further talk in following year obtained.
Year(s) Of Engagement Activity 2007,2008,2009,2010,2011