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.

Engineering a highly sensitive biosensor for abscisic acid in mammalian cells

“…Moreover, this depletion could be reversed by treatment of cells with the CYP707A inhibitor, abscinazole‐E3M. The majority of CYP enzymes require the assistance of NADPH‐cytochrome P450 reductase for the electron transfer from NADPH [40]. However, the overexpressed AtCYP707A3 depleted ABA in HEK293T cells without expressing additional CYP reductase, indicating the involvement of endogenous CYP reductase.…”

Genome-wide identification, molecular evolution and gene expression of P450 gene family in Cyrtotrachelus buqueti

“…According to the branching situation, C. buqueti and Dendroctonus ponderosae are more closely related, but compared with the red-brown weevil, C. buqueti is more closely related to the latter. The red palm weevil is a pest that is seriously harmful to palm plants [ 34 ] ,whose larvae feed on the main stem cause irreversible damage to the host plant and severely cause plant death in the short term [ 5 ] Studies have shown that it is the most destructive pest of palm trees worldwide, and functional analysis has demonstrated that the up-regulation of P450 gene expression has evolved into adaptation to insecticide stress caused by exposure to the systemic neonicotinoid insecticide imidacloprid [ 35 ]. C. buqueti is most closely related to Rhynchophorus ferrugineus , and it is speculated that the large C. buqueti has the same relationship with insecticides.…”

“…However, the molecular basis for the transcriptional regulation of Cytochrome P450 remains largely unknown [ 4 ]. Studies have shown that it is difficult to utilize P450 effectively now because they must be paired with cytochrome P450 reductase (CPRs) to facilitate electron transfer from reduced nicotinamide adenine dinucleotide phosphoric acid (NADPH) [ 5 ]. Insects are the most prosperous biological population on the earth, the role of P450 involves insect growth, development, feeding and other processes, it is also involved in the synthesis and metabolism of many endogenous compounds in insects, and metabolic detoxification is one of the important functions of the enzyme system.…”

The role of P450 enzymes in malaria and other vector‐borne infectious diseases

“…To note, some evidences from non‐mosquito insects (e.g., most studied Drosophila melanogaster fruit fly) could be suggestive for the strategies of efficient in vitro P450 expression or fusion enzyme construction 62,63 for in vitro studies of vector CYPs. Other interesting studies regard mitochondrial CYP12A4 and CYP12D1 involvement in Drosophila insecticide resistance 64,65 or detoxification enzyme targeting in Spodoptera littoralis 66 …”