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

Purified recombinant cytochrome P450 52A3 and the corresponding NADPH-cytoc hrome P450 reductase from the alkane-assimilating yeast Candida maltosa wer e reconstituted into an active alkane monooxygenase system. Besides the pri mary product, 1-hexadecanol, the conversion of hexadecane yielded up to fiv e additional metabolites, which were identified by gas chromatography-elect ron impact mass spectrometry as hexadecanal, hexadecanoic acid, 1,16-hexade canediol, 16-hydroxyhexadecanoic acid, and 1,16-hexadecanedioic acid. As sh own by substrate binding studies, the final product 1,16-hexadecanedioic ac id 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 conversi on of hexadecane, 1-hexadecanol, and hexadecanal (27, 23, and 69 min(-1), r espectively), whereas the oxidation of hexadecanoic acid, 1,16-hexadecanedi ol, 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 da ta on the incorporation of O-18(2)-derived oxygen into the hexadecane oxida tion products, the present study demonstrates that a single P450 form is ab le to efficiently catalyze a cascade of sequential mono- and diterminal mon ooxygenation reactions from n-alkanes to alpha,omega-dioic acids with high regioselectivity.