Selective π-1 oxidation of fatty acids by CYP147G1 from Mycobacterium marinum

Background: Cyp147G1 is one of 47 cytochrome P450 encoding genes in Mycobacterium marinum M, a pathogenic bacterium with a high degree of sequence similarity to Mycobacterium tuberculosis and Mycobacterium ulcerans. Cyp147G1 is one of only two of these cyp genes which are closely associated with a complete electron transfer system. Methods: The substrate range of the enzyme was tested in vitro and the activity of CYP147G1 was reconstituted in vivo by co-producing the P450 with the ferredoxin and ferredoxin reductase. Results: Substrates of CYP147G1 include fatty acids ranging from octanoic to hexadecanoic acid. CYP147G1 catalysed the selective hydroxylation of linear and co-2 methyl branched fatty acids at the omega-1 position (>= 98%). Oxidation of omega-1 methyl branched fatty acids generated the omega and omega-1 hydroxylation products in almost equal proportions, indicating altered position of hydrogen abstraction. Conclusions: This selectivity of fatty acid hydroxylation inferred that linear species must bind in the active site of the enzyme with the terminal methyl group sequestered so that abstraction at the C-H bonds of the omega-1 position is favoured. With branched substrates, one of the methyl groups must be close to the compound I oxygen atom and enable hydroxylation at the terminal methyl group to compete with the reaction at the omega-1C-H bond. General significance: Hydroxy fatty acids are widely used for industrial, food and medical purposes. CYP147G1 demonstrates high regioselectivity for hydroxylation at a sub-terminal position on a broad range of linear fatty acids, not seen in other CYP enzymes.