# Production of a Human Metabolite of Atorvastatinin by Bacterial CYP102A1 Peroxygenase
## 3 Results and Discussion
### Spectral Binding Titration  
To examine the differences in the substrate atorvastatinin's binding affinity to mutants M179, M221, M371, and M387, spectral binding titration was performed (Figure 8). Binding of atorvastatinin to all of the mutants tested here produced a pronounced Type II spectral shift, with a decrease at 390 nm and an increase at 420 nm, showing an increase in the heme-domain's low-spin fraction. The mutant's \(K_{\text{d}}\) values ranged between 2.5 and 3.7 uM, although M221 showed a higher \(K_{\text{d}}\) value (17 uM) than others. When 4-OH atorvastatinin, the product, was added to M371, a modified Type I spectral shift was produced with a decrease at 420 nm and an increase at 390 nm (Figure S7). The \(K_{\text{d}}\) value of 4-OH atorvastatinin to whole M371 was 6.2 uM.  
Although deuterated analogs were not used as internal standards to the analysis of atorvastatinin and its metabolite by LC-MS, the kinetic findings in this study should be reliable because commercially obtained 4-OH atorvastatinin was also used to confirm the results of subsequent kinetic studies. We found the calibration methods used to quantify the metabolite using quercetin as an internal standard were similar to the direct comparison of external 4-OH atorvastatinin standard. The extraction efficiency of 4-OH atorvastatinin was 75% under the experimental conditions used in this study.  
Drug metabolites in humans produced by P450s are critical for evaluating drug efficacy and safety in the drug discovery and development process. Lower activities of P450 enzymes are suggested for the H\({}_{2}\)O\({}_{2}\)-supported reaction rather than the NADPH-supported reaction, which can be explained by the lack of general acid-base residues in the P450 active sites [17]. This study showed that several CYP102A1 mutants can catalyze human drug metabolites with high peroxygenase activity to produce the human metabolite 4-OH atorvastatinin without requiring NADPH, an expensive cofactor [29]. Our results suggest that the catalytic activities of CYP102A1 peroxygenase toward atorvastatinin can be improved by enzyme engineering via random mutagenesis and site-directed mutation. The CYP102A1 mutants selected by high-throughput screening [12; 20; 21] showed high catalytic activity toward atorvastatinin. Atorvastatinin inhibits HMG-CoA reductase and is a very popular clinical drug used for hypertension and hyperlipidemia. The 4-OH atorvastatinin is known as an active metabolite, which is an expensive commercial product. By further improving CYP102A1 peroxygenase activity and stability, the per  
Figure 7: The cosolvent’s effect on atorvastatinin hydroxylation supported by hydrogen peroxide. The reaction mixtures included atorvastatinin substrate at 200 uM, 0.20 uM whole M371, and indicated cosolvent in 100 mM of a potassium phosphate buffer (pH 7.4). **(A)** Cosolvent (methanol, ethanol, isopropanol, and acetonitrile) with 1%, 3%, 5%, and 10% (\(\
icefrac{{v}}{{v}}\)).  
used to prepare drug metabolites of atorvastatin and possibly other drugs for industrial purposes.