CHARACTERIZATION OF THE N-ALKANE AND FATTY-ACID HYDROXYLATING CYTOCHROME-P450 FORMS 52A3 AND 52A4

Two enzymes, P450 52A3 (P450Cm1) and 52A4 (P450Cm2), the genes of whic h belong to the CYP52 multigene family occuring in the alkane-assimila ting yeast Candida maltosa, have been characterized biochemically and compared in terms of their substrate specificities. For this purpose, both the P450 proteins and the corresponding C. maltosa NADPH-cytochro me P450 reductase were separately produced by expressing their cDNAs i n Saccharomyces cerevisiae, purified, and reconstituted to active mono oxygenase systems. Starting from microsomal fractions with a specific content of 0.75 nmol P450Cm1, 0.34 nmol P450Cm2, and 10.5 units reduct ase per milligram of protein, respectively, each individual recombinan t protein was purified to homogeneity. P450 substrate difference spect ra indicated strong type I spectral changes and high-affinity binding of n-hexadecane (K-s = 26 mu M) and n-octadecane (K-s = 27 mu M) to P4 50Cm1, whereas preferential binding to P450Cm2 was observed using laur ic acid (K-s = 127 mu M) and myristic acid (K-s = 134 mu M) as substra tes. These substrate selectivities were further substantiated by kinet ic parameters, determined for n-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 coexpressin g each of the P450 proteins with the reductase. The highest catalytic activities within the reconstituted system were measured for n-hexadec ane hydroxylation to 1-hexadecanol by P450Cm1 (V-max = 27 mu M x min(- 1), K-m = 54 mu M) and oxidation of lauric acid to 16-hydroxylauric ac id by P450Cm2 (V-max = 30 mu M x min(-1), K-m = 61 mu M). We conclude that P450Cm1 and P450Cm2 exhibit overlapping but distinct substrate sp ecificities due to different chain-length dependencies and preferences for either n-alkanes or fatty acids. (C) 1996 Academic Press, Inc.