Background: CYP175A1 is a thermophilic P450 with high potential to invoke as an industrially viable biocatalyst though very little is known about the natural substrate of this enzyme. The crystal structure of CYP175A1 is similar to its mesophilic analogue CYP102A1, which is a fatty acid metabolizing enzyme (J Biol Chem 1990; 265: 4233-9). We have earlier shown that CYP175A1 catalyzes regioselective mono-oxygenation of different monounsaturated fatty acids (Biochemistry 2012; 51: 7880-90). The present work highlights screening of several important polyunsaturated fatty acids for their potentiality as substrate of this orphan P450 enzyme.
Methods: Both molecular modeling/docking as well as spectrophotometric screening of the substrates along with detailed activity assay and mass spectrometric identification of the products have been carried out to determine the reactivity and selectivity of oxygenation of polyunsaturated fatty acids by CYP175A1.
Results: Our investigations showed that polyunsaturated fatty acids (arachidonic acid, linoleic acid, α-linolenic acid and - linolenic acid) can be oxygenated by the enzymatic action of CYP175A1 although the enzyme did not show any detectable activity on the corresponding saturated analogues. Results further showed that unlike the monounsaturated fatty acids, the polyunsaturated fatty acids undergo mono- as well as di-oxygenation reactions. Analyses of products also suggested that the monooxygenation of these fatty acids is highly regioselective and is regulated by the number and position of the double bonds in the fatty acids.
Conclusion: CYP175A1 may have important roles in lipid metabolism and thus can be used for regioselective oxygenation of different fatty acids. The present study also improves our current understanding on the nature of the enzyme pocket and of the possible natural substrate of this orphan enzyme.