Strategies for the construction of insect P450 fusion enzymes

Abstract

Cytochrome P450 monooxygenases (P450s) are ubiquitous enzymes with a broad substrate spectrum. Insect P450s are known to catalyze reactions such as the detoxification of insecticides and the synthesis of hydrocarbons, which makes them useful for many industrial processes. Unfortunately, it is difficult to utilize P450s effectively because they must be paired with cytochrome P450 reductases (CPRs) to facilitate electron transfer from reduced nicotinamide adenine dinucleotide phosphate (NADPH). Furthermore, eukaryotic P450s and CPRs are membrane-anchored proteins, which means they are insoluble and therefore difficult to purify when expressed in their native state. Both challenges can be addressed by creating fusion proteins that combine the P450 and CPR functions while eliminating membrane anchors, allowing the production and purification of soluble multifunctional polypeptides suitable for industrial applications. Here we discuss several strategies for the construction of fusion enzymes combining insect P450 with CPRs.