Expression of CYP2L1 in the yeast Pichia pastoris, and determination of catalytic activity with progesterone and testosterone

Abstract

Cytochrome P450 (P450) enzymes are widely distributed throughout the plant and animal kingdoms. In previous work, two forms of P450 partially purified from spiny lobster, Panulirus argus, hepatopancreas microsomes were shown to metabolize several substrates, including testosterone and progesterone at the 16α, 6β and 21 positions f James (1990) Archives of Biochemistry and Biophysics 282, 8–17]. We have recently cloned and sequenced a major form of P450, CYP2L1, from hepatopancreas cDNA [James et al. (1996) Archives of Biochemistry and Biophysics 329, 31–38]. CYP2L1 is one of at least four P450s detected in a cDNA library constructed from spiny lobster hepatopancreas. The spiny lobster CYP2L1 cDNA was introduced into the methylotrophic yeast Pichia pastoris and the crustacean CYP2L1 was expressed in microsomes of the methanol-induced yeast. On the basis of immunoblot analysis and ability to catalyse the 16α-hydroxylation of testosterone and progesterone, the catalytically active, heterologously expressed spiny lobster CYP2L1 appears to be very similar to the major spiny lobster hepatopancreas P450 previously purified in this laboratory. This is the first report of heterologous expression of a crustacean P450.