Title:Regioselective Oxygenation of Polyunsaturated Fatty Acids by the Thermostable P450 from Thermus thermophilus HB27
Volume: 4
Issue: 3
Author(s): Shibdas Banerjee, Dwaipayan Dutta Gupta and Shyamalava Mazumdar
Affiliation:
Keywords:
CYP175A1, epoxidation, hydroxylation, oxygenation, P450, polyunsaturated fatty acids, regioselectivity,
thermostability.
Abstract: 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.
