A close inspection of the substrate binding pocket from the crystal structure of CYP175A1 and CYP102A1 shows that the pockets/active sites of these two enzymes are similar and mostly surrounded by hydrophobic residues. Hence, both of them would prefer hydrophobic substrates to show catalytic activity. Long chain saturated fatty acids (Lauric acid, myristic acid, palmitic acid and stearic acid) are substrates of P450BM-3. However, no substrate other than β-carotene was earlier reported for the native CYP175A1. It is known that Thermus thermophilus HB27 produces lipolytic enzymes. In the Biochemistry and Systems Biology and the Chemical Biology and Bioinorganic Chemistry groups, we systematically screened several fatty acids (saturated and monounsaturated) for their potential as substrates for CYP175A1.
The results showed that the wild type enzyme could catalyze the reaction of mono-unsaturated fatty acids but not of saturated fatty acids. The product analyses using ESI-MS and GC-MS revealed an important regioselectivity in the CYP175A1 catalyzed monooxygenation of the monoenoic fatty acids depending on the ethylenic double bond (C=C) configuration. When the double bond was in cis-configuration, an epoxy fatty acid was found to be the major product and two allyl-hydroxy fatty acids were found to be the minor products. But, when the double bond was in trans-configuration the product distribution was reversed. The oxygenation efficiency was found to be the highest for Palmitoleic acid (chain length C16) but there was no direct correlation of the activity with the chain length or the position of unsaturation of the fatty acid. Molecular docking calculations showed that the ‘U’-type conformations of the monoenoic fatty acids are particularly responsible for their binding in the enzyme pocket and that is also consistent with the observed regioselectivity in the oxygenation reaction. The present results provide evidences that CYP175A1 can catalyze regioselective oxygenation reaction of several monoenoic fatty acids though it cannot catalyze the oxygenation of the corresponding saturated analogues. These studies provide critical information on the nature of the enzyme pocket and on the possible natural substrate of this orphan enzyme. For further information, visit our lab website