Oxygenation cascade in conversion of n-alkanes to α,ω-dioic acids catalyzed by cytochrome p450 52A3

Purified recombinant cytochrome P450 52A3 and the corresponding NADPH-cytochrome P450 reductase from the alkane-assimilating yeast Candida maltosa were reconstituted into an active alkane monooxygenase system. Besides the primary product, 1-hexadecanol, the conversion of hexadecane yielded up to five additional metabolites, which were identified by gas chromatography-electron impact mass spectrometry as hexadecanal, hexadecanoic acid, 1,16-hexadecanediol, 16-hydroxyhexadecanoic acid, and 1,16-hexadecanedioic acid. As shown by substrate binding studies, the final product 1,16-hexadecanedioic acid acts as a competitive inhibitor of n-alkane binding and may be important for the metabolic regulation of the P450 activity. Kinetic studies of the individual sequential reactions revealed high V-max values for the conversion of hexadecane, 1-hexadecanol, and hexadecanal (27, 23, and 69 min(-1), respectively), whereas the oxidation of hexadecanoic acid, 1,16-hexadecanediol, and 16-hydroxyhexadecanoic acid occurred at significantly lower rates (9, 9, and 5 min(-1), respectively). 1-Hexadecanol was found to be the main branch point between mono- and diterminal oxidation. Taken together with data on the incorporation of O-18(2)-derived oxygen into the hexadecane oxidation products, the present study demonstrates that a single P450 form is able to efficiently catalyze a cascade of sequential mono- and diterminal monooxygenation reactions from n-alkanes to alpha,omega-dioic acids with high regioselectivity.