We engineered the CYP175A1 domain of 175RF for the hydroxylation of unnatural substrates. The active site of CYP175A1 is better suited to a linear aliphatic chain of the natural substrate, β-carotene, resulting in a narrow active site. In particular, Q67, Y68, W269, and I270 have large side chains (Fig. S4), which may prevent larger molecules such as testosterone from binding to the active site. In order to allow the entry of large molecules into the active site, we constructed three mutants: 175RFm1, W269P/I270A; 175RFm2, Q67G/Y68I; 175RFm3, Q67G/Y68I/W269P/I270A. The mutants were expressed in E. coli and purified. The reduced CO-difference spectrum of 175RFm1 showed an
Nilotinib peak at 450 nm and did not show a peak at 420 nm, whereas the reduced CO-difference spectra of 175RFm2 and 175RFm3 had a minor peak at 420 nm in addition to a major peak at 450 nm (data not shown). Although 175RF did not show any hydroxylation of testosterone, all mutants could hydroxylate
testosterone ( Table 1). In particular, 175RFm2 had efficient catalytic activity toward testosterone and produced 2β-hydroxytestosterone as a major metabolite ( Fig. 3). Furthermore, we evaluated the effect of these mutations of the active site on the hydroxylation of β-carotene ( Table 1). The β-carotene hydroxylation activity of 175RFm1 and 175RFm2 was significantly lower (450- and 2000-fold, respectively) than that of 175RF. Furthermore, 175RFm3 completely abolished the β-carotene hydroxylation activity.
