Characterization of then-Alkane and Fatty Acid Hydroxylating Cytochrome P450 Forms 52A3 and 52A4

Abstract

Two enzymes, P450 52A3 (P450Cm1) and 52A4 (P450Cm2), the genes of which belong to theCYP52multigene family occuring in the alkane-assimilating yeastCandida maltosa,have been characterized biochemically and compared in terms of their substrate specificities. For this purpose, both the P450 proteins and the correspondingC. maltosaNADPH-cytochrome P450 reductase were separately produced by expressing their cDNAs inSaccharomyces cerevisiae,purified, and reconstituted to active monooxygenase systems. Starting from microsomal fractions with a specific content of 0.75 nmol P450Cm1, 0.34 nmol P450Cm2, and 10.5 units reductase per milligram of protein, respectively, each individual recombinant protein was purified to homogeneity. P450 substrate difference spectra indicated strong type I spectral changes and high-affinity binding ofn-hexadecane (Ks= 26 μM) andn-octadecane (Ks= 27 μM) to P450Cm1, whereas preferential binding to P450Cm2 was observed using lauric acid (Ks= 127 μM) and myristic acid (Ks= 134 μM) as substrates. These substrate selectivities were further substantiated by kinetic parameters, determined forn-alkane and fatty acid hydroxylation in a reconstituted system, which was composed of the purified components and phospholipid, as well as in microsomes obtained after coexpressing each of the P450 proteins with the reductase. The highest catalytic activities within the reconstituted system were measured forn-hexadecane hydroxylation to 1-hexadecanol by P450Cm1 (Vmax= 27 μM× min−1,Km= 54 μM) and oxidation of lauric acid to 16-hydroxylauric acid by P450Cm2 (Vmax= 30 μM× min−1,Km= 61 μM). We conclude that P450Cm1 and P450Cm2 exhibit overlapping but distinct substrate specificities due to different chain-length dependencies and preferences for eithern-alkanes or fatty acids.