# Production of a Human Metabolite of Atorvastatinin by Bacterial CYP102A1 Peroxygenase
## 3 Results and Discussion  
### Hydroxylation of Atorvastatin  
At first, we screened the atorvastatintion hydroxylation activities of a set of CYP102A1 mutants, which showed high activities toward several drugs supported by NADPH [20, 21]. We found some mutants showed higher catalytic activity of atorvastatinatin 4-hydroxylation when compared to those of our previous work [12]. The mutants M179 (0.89 min\({}^{-1}\)),M221 (1.25 min\({}^{-1}\)), M371 (2.0 min\({}^{-1}\)), and M387 (0.83 min\({}^{-1}\)) showed 3.2-7.8-fold higher formation rate of 4-OH atorvastatin thin than the template M16V2 to make an enzyme library by error-prone PCR (0.26 min\({}^{-1}\)) (Figure 2). Based on these results, M16V2, M179, M221, M371, and M387 were selected and used for studying atorvastatin hydroxylation activities supported by H\({}_{2}\)O\({}_{2}\). Each mutant bore amino acid changes compared to WT CYP102A1, as described in Table S1. Another major metabolite of atorvastatin, 2-OH product, in the human liver, was not found in the CYP102A1-catalyzed reaction. The mutants M179, M221, M371, and M387 showed 7.5-fold, 7.1-fold, 76.6-fold, and 19.5-fold higher rates of 4-OH atorvastatinin formation than M16V2, respectively, when the reaction was performed with the externally added H\({}_{2}\)O\({}_{2}\) (10 mM). We found that 10 mM was the most optimal H\({}_{2}\)O\({}_{2}\) concentration to support the peroxygenase activity of CYP102A1 (Figure S3). The mutant M371 (1.8 min\({}^{-1}\)) showed the highest catalytic activity toward atorvastatinin among the tested mutants (Figure 2). M371\({}^{\prime}\)s catalytic activities toward atorvastatinin did not show large differences between NADPH- and H\({}_{2}\)O\({}_{2}\) supported reactions (Figure S4). Based on these results, M371 showed the highest product formation rates in both the NADPH and H\({}_{2}\)O\({}_{2}\) dependent reactions. Thus, we selected M371 for constructing the heme domain in an expression vector. The major metabolites and substrates were characterized by the HPLC (Figure 3) and LC-MS (Figure S5) results. Figure 2: Activity of atorvastatinin 4-hydroxylation catalyzed by CYP102A1 mutants. The reaction mixtures included atorvastatinin substrate (200 uM) and 0.20 uM of CYP102A1 in 100 mM of a potassium phosphate buffer (pH 7.4). For NADPH-supported reactions, an NADPH-generating system was used to initiate the reaction, which was incubated for 30 min at 37 \({}^{\circ}\)C. For H\({}_{2}\)O\({}_{2}\) supported reactions, 10 mM H\({}_{2}\)O\({}_{2}\) was used to initiate the reaction, which was incubated for 10 min at 37 \({}^{\circ}\)C.  
Figure 4: Kinetic parameters of atorvastatin 4-hydroxylation by whole M371 and M371-heme in the presence of H\({}_{2}\)O\({}_{2}\). The reactions include 5–200 uM atorvastatin substrate and 0.20 uM of CYP102A1 M371 whole enzyme (\(\Circuit\)) or heme domain in 100 mM of a potassium phosphate buffer (pH 7.4) (\(\bullet\)). We added 10 mM H\({}_{2}\)O\({}_{2}\) to initiate the reaction, which was incubated for 5 min at 37 \({}^{\circ}\)C.  
Figure 5: Total turnover numbers of atorvastatin 4-hydroxylation by CYP102A1 supported by H\({}_{2}\)O\({}_{2}\). The reaction mixtures included atorvastatin substrate at concentration of 200 uM and 0.20 uM of whole M371 (\(\Circuit\)) or heme-domain in 100 mM of a potassium phosphate buffer (pH 7.4) (\(\bullet\)). We added 10 mM H\({}_{2}\)O\({}_{2}\) to initiate the reaction and the reaction mixtures were incubated for 30 s, 1, 2, 3, 4, 5, 10, 20, and 30 min at 37 \({}^{\circ}\)C.  
K\({}_{\rm m}\) value of the H\({}_{2}\)O\({}_{2}\)-supported reaction was 2.8-fold lower than that of the NADPH-supported reaction. This result means that the H\({}_{2}\)O\({}_{2}\)-supported reaction showed a higher catalytic efficiency (3.3-fold) than that of the NADPH-supported reaction.