# Production of a Human Metabolite of Atorvastatinin by Bacterial CYP102A1 Peroxygenase
## 1 Introduction  
Atorvastatinin is a cholesterol-lowering drug widely used in treating hypercholesterolemia and preventing cardiovascular disease [1, 2]. This statin drug inhibits 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase activity, which catalyzes the rate-limiting step in cholesterol biosynthesis [3]. The atorvastatinin can be hydroxylated to 2-OH and 4-OH atorvastatinin by cytochrome P450 (CYP or P450) enzyme, CYP3A4, in the human liver [4] (Figure 1, Figure S1). Along with this, atorvastatinin's hydroxylated metabolites have been reported as active metabolites. Among statin drugs, only the atorvastatinin has metabolites that can inhibit HMG-CoA reductase equiption hot that of the parent drug [5]. About 70% of the HMG-CoA reductase inhibition achieved with atorvastatinin has been observed with its 2-OH and 4-OH atorvastatinin [6]. In addition, these two active metabolites have higher efficacy in lowering LDL (low-density lipoprotein) cholesterol when compared with other statins [7]. However, systematic studies have not been performed on thesafety, efficacy, and toxicity of these metabolites, although the metabolites can contribute to atorvastatin's cholesterol-lowering effect.  
During the drug development process, which is guided by the U.S. Food and Drug Administration (FDA), the drug metabolites in safety testing (MIST) should be separated to evaluate the safety testing and drug toxicity [8; 9]. Although chemical synthesis methods can prepare some drug metabolites of concern, other metabolites such as chiral compounds may not be easily prepared by these methods. Those metabolites can be synthesized by P450 enzymes, including human P450s [10]. Particularly, the CYP102A1 (P450 BM3) from _Bacillus megaterium_ demonstrated as a possible bicostalyst for producing human metabolites, which could be useful in fine-chemical biosynthesis and the pharmaceutical fields [11; 12]. The CYP102A1 mutants, with new or enhanced catalytic activities toward drugs, could be created by site-directed mutagenesis, random mutagenesis, and rational design [13]. The CYP102A1 was also proposed for industrial application as a potential monooxygenase with high catalytic activities and stabilities [11; 14].  
The P450s' typical function is to catalyze the oxidation of a wide range of physiological substrates and foreign chemicals. P450s reductively activate molecular oxygen (O\({}_{2}\)) by using two electrons transferred to P450s by NAD(P)H via P450 reductase (CPR) [15]. Otherwise, hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)) can be used as an oxidant for oxygen activation of P450s via the peroxide shunt pathway [16]. Using peroxides is interesting for monooxygenation reactions catalyzed by P450s as the reaction does not require an electron transfer partner and cofactor NAD(P)H. The low-cost H\({}_{2}\)O\({}_{2}\) can lead to using the P450 peroxygenase in industrial-scale synthesis [17].  
Reports found that some of the CYP102A1 mutants obtained via random mutagenesis could catalyze regioselective hydroxylation of atorvastatin to produce 4-OH atorvastatin, which is one of two of the major metabolites in humans, by NADPH-dependent CYP102A1 catalyzed reaction [12] (Figure 1). In this work, we examined the peroxygenase activity of CYP102A1 mutants in having high atorvastatin 4-hydroxylation activity supported by H\({}_{2}\)O\({}_{2}\) and compared this to the mutants' activities supported by the NADPH regeneration system. 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.  
Figure 1: Chemical structures of atorvastatin and its metabolites, 2-OH and 4-OH atorvastatin. The metabolites are made in the human liver by CYP3A4. Bacterial CYP102A1 mutants can make only one metabolite, 4-OH atorvastatin.