# Production of a Human Metabolite of Atorvastatinin by Bacterial CYP102A1 Peroxygenase
## 4 Conclusions  
In this study, we found a simple enzymatic strategy of CYP102A1 enzymes for a high synthesis efficiency for the 4-OH atorvastatin by using bacterial CYP102A1 peroxygenase activity with hydrogen peroxide. A set of CYP102A1 mutants with high catalytic activity toward atorvastatin were obtained using enzyme library generation, high-throughput screening of highly active mutants, and enzymatic characterization of the mutants. In H\({}_{2}\)O\({}_{2}\)-supported reactions, the mutant M371 among the tested mutants showed the highest catalytic activity of atorvastatin 4-hydroxylation. Improved enzymatic properties of CYP102A1 peroxygenase activity over the NADPH-supported activity were found. This result shows that CYP102A1 peroxygenase activity can catalyze atorvastatin 4-hydroxylation by peroxide-dependent oxidation with high catalytic activity. These results suggest that the peroxygenase activity of CYP102A1 mutants can be developed to produce drugs' metabolites to further study their efficacy and safety.  
**Supplementary Materials:** The following are available online at [[https://www.mdpi.com/2076-341](https://www.mdpi.com/2076-341)]([https://www.mdpi.com/2076-341](https://www.mdpi.com/2076-341)) 7/11/2/603/s1, Table S1: The amino acid sequences of M16V2 and CYP102A1 mutants, Table S2: Kinetic parameters of atorvastatin 4-hydroxylation by CYP102A1 supported by NADPH and dissociation constants for atorvastatin, Figure S1: Proposed pathway for the atorvastatin metabolism in human, Figure S2: Standard curves of internal standard, quercetin, and atorvastatin, Figure S3: Dependence of atorvastatin 4-hydroxylation by CYP102A1 mutant on the H\({}_{2}\)O\({}_{2}\) concentration, Figure S4. HPLC chromatograms of atorvastatin and its metabolite produced by CYP102A1 mutant with and without NADPH, Figure S5: LC/MS spectra of atorvastatin and its major metabolite produced by CYP102A1 mutant (M371), Figure S6: Measurement of H\({}_{2}\)O\({}_{2}\) concentration during atorvastatin 4-hydroxylation by whole M371 via in situ H\({}_{2}\)O\({}_{2}\) generation, Figure S7: Binding titration of 4-OH atorvastatin to the CYP102A1.  
Figure 8: Binding titration of atorvastatin to the CYP102A1. The samples for binding titration contained 0–100 \(\mu\)M atorvastatinatin in 100 mM of a potassium phosphate buffer (pH 7.4) and 1 \(\mu\)M of M179 (**A**), M221 (**B**), M371 (**C**), and M387 (**D**). Spectrally determined dissociation constants (\(K_{\rm d}\)) were also shown.  
**Author Contributions:** Conceptualization, S.-J.Y. and C.-H.Y.; writing--original draft preparation, T.H.H.N. and C.-H.Y.; supervision, S.-J.Y. and C.-H.Y.; funding acquisition, C.-H.Y. All authors have read and agreed to the published version of the manuscript.  
This research was funded by the National Research Foundation of Korea (NRF-2018R1A4A1023882), Republic of Korea.  
**Institutional Review Board Statement:** Not applicable.  
**Informed Consent Statement:** Not applicable.  
**Data Availability Statement:** The data presented in this study are available on request from the corresponding author.  
**Conflicts of Interest:** The authors declare that they have no conflict of interest.