Cytochrome P450 hydroxylase (CYP) in actinomycetes plays an important role in the
biosynthesis and bioconversion of various secondary metabolites. Two unique CYPs named
CYP-sb21 and CYP-pa1, which were identified from Sebekia benihana and Pseudonocardia
autotrophica, respectively, were proven to transfer a hydroxyl group at the 4th or 9th N-methyl
leucine position of immunosuppressive agent cyclosporin A (CsA). Interestingly, these two
homologous CYPs showed different CsA regio-selectivities. CYP-sb21 exhibited preferential
hydroxylation activity at the 4th position over the 9th position, whereas CYP-pa1 showed the
opposite preference. To narrow down the CYP domain critical for CsA regio-selectivity, each
CYP was divided into four domains, and each domain was swapped with its counterpart from
the other CYP. A total of 18 hybrid CYPs were then individually tested for CsA regioselectivity.
Although most of the hybrid CYPs failed to exhibit a significant change in regioselectivity
in the context of CsA hydroxylation, hybrid CYP-pa1 swapped with the second
domain of CYP-sb21 showed a higher preference for the 9th position. Moreover, hybrid CYPsb21
containing seven amino acids from the 2nd domain of CYP-pa1 showed higher preference
for the 4th position. These results imply that the 2nd domain of CsA-specific CYP plays a
critical role in CsA regio-selectivity, thereby setting the stage for biotechnological application
of CsA regio-selective hydroxylation.

Keywords: Cytochrome P450, Cyclosporin A, Regio-selectivity, Actinomycetes