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-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 Vmax 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 18O2-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.
Scheller
U
U
Max-Delbrück-Center for Molecular Medicine, D-13122 Berlin-Buch, Germany.
Zimmer
T
T
Becher
D
D
Schauer
F
F
Schunck
W H
WH
D000473:Alkanes / Q000378:metabolism*; D002384:Catalysis; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D003998:Dicarboxylic Acids / Q000378:metabolism*; D005656:Fungal Proteins; D008401:Gas Chromatography-Mass Spectrometry; D007700:Kinetics; D006899:Mixed Function Oxygenases; D010100:Oxygen / Q000378:metabolism*
D000473:Alkanes; D003998:Dicarboxylic Acids; D005656:Fungal Proteins; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; C116330:CYP52A3 protein, Candida maltosa; D010100:Oxygen