Prokaryotic functional expression and activity comparison of three CYP9A genes from the polyphagous pestHelicoverpa armigera
AbstractCytochrome P450s (CYPs or P450s) have been long recognized as very important enzymes in the metabolism of xenobiotic and endogenous compounds, but only a few CYPs have been functionally characterized in insects. The effort in functional characterization of insect P450s is heavily hindered by technical difficulties in preparing active, individual P450 enzymes directly from the target insect. In this paper, we describe the functional expression of two additional pyrethroid resistance-associated CYP9A genes (CYP9A12andCYP9A17) from the polyphagous pestHelicoverpa armigerain the facileEscherichia coli. The functionality ofE. coliproduced CYP9A12, CYP9A14, and CYP9A17 was investigated and activities of these CYP9As were compared against three probe substrates after reconstitution with NADPH-dependent cytochrome P450 reductase. The results showed that active forms of CYP9A12 and CYP9A17 were expressed inE. coliwith a content of about 1.0–1.5 nmol mg−1protein in membrane preparations.In vitroassays showed that CYP9A14 was capable of catalyzing O-dealkylation of methoxyresorufin (MROD), ethoxyresorufin (EROD), and benzyloxyresorufin (BROD), while CYP9A12 and CYP9A17 exhibited only MROD and EROD activities. Kinetic studies demonstrated that CYP9A14 had the greatestkcat/Kmvalue for MROD, and CYP9A17 for EROD, while the lowestkcat/Kmvalues for both MROD and EROD were observed for CYP9A12. The distinct biochemical traits suggest that the three paralogous CYP9As may play different roles in xenobiotic metabolism in this important pest.