Identification of CYP106A2 as a Regioselective Allylic Bacterial Diterpene Hydroxylase

The cytochrome P450 monooxygenase CYP106A2 from Bacillus megaterium ATCC 13368 catalyzes hydroxylations of a variety of 3-oxo-Delta(4)-steroids such as progesterone and deoxycorticosterone (DOC), mainly in the 15 beta-position. We combined a high-throughput screening and a rational approach for identifying new substrates of CYP106A2. The diterpene resin acid abietic acid was found to be a substrate and was docked into the active site of a CYP106A2 homology model to provide further inside into the structural basis of the regioselectivity of hydroxylation. The products of the hydroxylation reaction were analyzed by HPLC and the V-max and K-m values were calculated. The corresponding reaction products were analyzed by NMR spectroscopy and identified as 12 alpha- and 12 beta-hydroxyabietic acid. CYP106A2 was therefore identified as the first reported bacterial cytochrome P450 diterpene hydroxylase. Furthermore, an effective whole-cell catalyst for the selective allylic 12 alpha- and 12 beta-hydroxylation was applied to produce the hydroxylated products.