Abstract
Atorvastatin is a widely used statin drug that prevents cardiovascular disease and treats hyperlipidemia. The major metabolites in humans are 2-OH and 4-OH atorvastatin, which are active metabolites known to show highly inhibiting effects on 3-hydroxy-3-methylglutaryl-CoA reductase activity. Producing the hydroxylated metabolites by biocatalysts using enzymes and whole-cell biotransformation is more desirable than chemical synthesis. It is more eco-friendly and can increase the yield of desired products. In this study, we have found an enzymatic strategy of P450 enzymes for highly efficient synthesis of the 4-OH atorvastatin, which is an expensive commercial product, by using bacterial CYP102A1 peroxygenase activity with hydrogen peroxide without NADPH. We obtained a set of CYP102A1 mutants with high catalytic activity toward atorvastatin using enzyme library generation, high-throughput screening of highly active mutants, and enzymatic characterization of the mutants. In the hydrogen peroxide supported reactions, a mutant, with nine changed amino acid residues compared to a wild-type among tested mutants, showed the highest catalytic activity of atorvastatin 4-hydroxylation (1.8 min−1). This result shows that CYP102A1 can catalyze atorvastatin 4-hydroxylation by peroxide-dependent oxidation with high catalytic activity. The advantages of CYP102A1 peroxygenase activity over NADPH-supported monooxygenase activity are discussed. Taken together, we suggest that the P450 peroxygenase activity can be used to produce drugs’ metabolites for further studies of their efficacy and safety.
Highlights
Atorvastatin is a widely used statin drug that prevents cardiovascular disease and treats hyperlipidemia
We found that the peroxygenase activity of CYP102A1 can be used as a biocatalyst to catalyze the reaction of atorvastatin hydroxylation because CYP102A
We found that the peroxygenase activity of CYP102A1 can be used as a biocatalyst to catalyze the reaction of atorvastatin hydroxylation because CYP102A1 peroxygenase with a low cost has higher activity than that of the NADPH-supported activity
Summary
Atorvastatin is a widely used statin drug that prevents cardiovascular disease and treats hyperlipidemia. We have found an enzymatic strategy of P450 enzymes for highly efficient synthesis of the 4-OH atorvastatin, which is an expensive commercial product, by using bacterial CYP102A1 peroxygenase activity with hydrogen peroxide without NADPH. Atorvastatin is a cholesterol-lowering drug widely used in treating hypercholesterolemia and preventing cardiovascular disease [1,2] This statin drug inhibits 3-hydroxy3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase activity, which catalyzes the ratelimiting step in cholesterol biosynthesis [3]. About 70% of the HMG-CoA reductase inhibition achieved with atorvastatin has been observed with its 2-OH and 4-OH atorvastatin [6] These two active metabolites have higher efficacy in lowering LDL (low-density lipoprotein) cholesterol when compared with other statins [7]. Sis could catalyze regioselective hydroxylation of atorvastatin to produce 4-OH atorvas tatin, which is one of two of the major metabolites in humans, by NADPH-dependen
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