Oxygenation Cascade in Conversion of n-Alkanes to α,ω-Dioic Acids Catalyzed by Cytochrome P450 52A3

• Published in: The Journal of biological chemistry 1998-12, Vol.273 (49), p.32528-32534
• Main Authors: Scheller, Ulrich, Zimmer, Thomas, Becher, Dörte, Schauer, Frieder, Schunck, Wolf-Hagen
• Format: Article
• Language: English
• Subjects: 1-HEXADECANOL; ACIDE ORGANIQUE; ACIDOS ORGANICOS; ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; ALCOHOLES; ALCOHOLES GRASOS; ALCOHOLS; ALCOOL; ALCOOL GRAS; ALKANE MONOOXYGENASE; Alkanes - metabolism; CANDIDA MALTOSA; CARBOXYLIC ACIDS; Catalysis; CHEMICAL STRUCTURE; CITOCROMO P450; Cytochrome P-450 Enzyme System - metabolism; CYTOCHROME P450; Dicarboxylic Acids - metabolism; ENZYMIC ACTIVITY; ESTRUCTURA QUIMICA; FATTY ALCOHOLS; Fungal Proteins; Gas Chromatography-Mass Spectrometry; HEXADECANE; HIDROCARBUROS; HYDROCARBONS; HYDROCARBURE; Kinetics; METABOLITE; METABOLITES; METABOLITOS; Mixed Function Oxygenases; ORGANIC ACIDS; OXIDACION; OXIDATION; OXIDOREDUCTASES; OXIDORREDUCTASAS; OXYDATION; OXYDOREDUCTASE; Oxygen - metabolism; PROTEINAS RECOMBINANTES; PROTEINE RECOMBINANTE; RECOMBINANT PROTEINS; STRUCTURE CHIMIQUE; UNSPECIFIC MONOOXYGENASE
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.273.49.32528

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 highVmax 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 fromn-alkanes to α,ω-dioic acids with high regioselectivity.