NADPH-cytochrome P450 oxidoreductase from the mosquito Anopheles minimus: Kinetic studies and the influence of Leu86 and Leu219 on cofactor binding and protein stability

Abstract

NADPH-cytochrome c oxidoreductase from the mosquito Anopheles minimus lacking the first 55 amino acid residues was expressed in Escherichia coli. The purified enzyme loses FMN, leading to an unstable protein and subsequent aggregation. To understand the basis for the instability, we constructed single and triple mutants of L86F, L219F, and P456A, with the first two residues in the FMN domain and the third in the FAD domain. The triple mutant was purified in high yield with stoichiometries of 0.97 FMN and 0.55 FAD. Deficiency in FAD content was overcome by addition of exogenous FAD to the enzyme. Both wild-type and the triple mutant follow a two-site Ping-Pong mechanism with similar kinetic constants arguing against any global structural changes. Analysis of the single mutants indicates that the proline to alanine substitution has no impact, but that both leucine to phenylalanine substitutions are essential for FMN binding and maximum stability of the enzyme.

Metabolism of Pyrethroids by Mosquito Cytochrome P450 Enzymes: Impact on Vector Control

“…Due to loss of flavin cofactors and instability of An. minimus CYPOR, we have identified two key amino acids (Leu86 and Leu219 in FMN binding domain) by amino sequence alignment and shown that mutations of the two leucine residues into conserved phenylalanine residues that are found conserved among other CYPORs could rescue loss of FAD cofactor and increase protein stability of mosquito CYPOR (Sarapusit et al, 2008(Sarapusit et al, , 2010. These mutations do not affect kinetic mechanism and constants of enzyme.…”

“…However its expression has been of poor yield as a result of inclusion bodies formation. An attempt to obtain soluble protein by deletion of the first 55 amino acid residues comprising of membrane binding region (55AnCYPOR) has been successful (Sarapusit et al, 2008). However the protein could not be purified by 2'5'-ADP affinity column, indicating that NADPH binding capacity of mosquito CYPOR is low and this is different from CYPORs of other organisms such as rat and human (Sarapusit, 2009).…”

“…gambiae CYPOR (Lian et al, 2011). Only under specific condition was 55AnCYPOR successfully expressed and purified to homogeneity by a combination of Ni 2+ NTA-affinity chromatography and G200-gel filtration chromatography (Sarapusit et al, 2008). Moreover both purified full-length (flAnCYPOR) and membrane-deleted 55AnCYPOR proteins readily lose FAD and FMN cofactors, they undergo www.intechopen.com aggregation and are unstable compared to rat and human CYPORs (Sarapusit et al, 2008(Sarapusit et al, , 2010.…”

“…Moreover both purified full-length (flAnCYPOR) and membrane-deleted 55AnCYPOR proteins readily lose FAD and FMN cofactors, they undergo www.intechopen.com aggregation and are unstable compared to rat and human CYPORs (Sarapusit et al, 2008(Sarapusit et al, , 2010. While supplementation of FAD could increase activity of both full-length and membrane-deleted forms, FMN supplementation could increase activity of full-length form only (Sarapusit et al, 2008(Sarapusit et al, , 2010. This behavior is different from membrane-deleted soluble CYPORs of rat and human in which exogenous FMN is readily incorporated into its FMNbinding site (Döhr et al, 2001;Shen et al, 1989).…”

“…Only under specific condition was 55AnCYPOR successfully expressed and purified to homogeneity by a combination of Ni 2+ NTA-affinity chromatography and G200-gel filtration chromatography (Sarapusit et al, 2008). Moreover both purified full-length (flAnCYPOR) and membrane-deleted 55AnCYPOR proteins readily lose FAD and FMN cofactors, they undergo www.intechopen.com aggregation and are unstable compared to rat and human CYPORs (Sarapusit et al, 2008(Sarapusit et al, , 2010. While supplementation of FAD could increase activity of both full-length and membrane-deleted forms, FMN supplementation could increase activity of full-length form only (Sarapusit et al, 2008(Sarapusit et al, , 2010.…”