Genomic and bioinformatic analysis of NADPH-cytochrome P450 reductase in Anopheles stephensi (Diptera: Culicidae)

Abstract

The cytochrome P450 monooxygenase (P450) enzyme system is a major mechanism of xenobiotic biotransformation. The nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase (CPR) is required for transfer of electrons from NADPH to P450. One CPR gene was identified in the genome of the malaria-transmitting mosquito Anopheles stephensi Liston (Diptera: Culicidae). The gene encodes a polypeptide containing highly conserved flavin mononucleotide-, flavin adenine dinucleotide-, and NADPH-binding domains, a unique characteristic of the reductase. Phylogenetic analysis revealed that the A. stephensi and other known mosquito CPRs belong to a monophyletic group distinctly separated from other insects in the same order, Diptera. Amino acid residues of CPRs involved in binding of P450 and cytochrome c are conserved between A. stephensi and the Norway rat Rattus norvegicus Berkenhout (Rodentia: Muridae). However, gene structure particularly within the coding region is evidently different between the two organisms. Such difference might arise during the evolution process as also seen in the difference of P450 families and isoforms found in these organisms. CPR in the mosquito A. stephensi is expected to be active and serve as an essential component of the P450 system.

Keywords: binding domain; gene structure; phylogenetic tree; sequence analysis.

Fig. 1.

The predicted sequence of A. stephensi CPR transcript. The sequence extends from the initiation codon to the stop codon. The upper and lower lines represent nucleotide and deduced amino acid sequences, respectively. The one-letter code for each amino acid is aligned with the second nucleotide of each codon. The predicted transmembrane region is underlined.

Fig. 2.

A neighbor-joining phylogenetic tree of CPRs in A. stephensi and other selected organisms. Gaps were excluded ( n  = 1,000). The branch length represents the accumulation of amino acid changes. The scale indicates 0.05 amino acid change per site. The vertical bars indicate phylogenetic clusters.

Fig. 3.

Intron–exon organization of representative CPR genes. Only mosquitoes with their genomes completed and released to the public were analyzed for gene structure. Rat CPR was included for genetic comparison. The coding regions shown here start from the initiation codon to the stop codon. The square boxes represent exons, and the lines connecting the boxes represent introns. The sizes of both exons and introns are not drawn to scale. The nucleotide length of each exon is presented in each box. Sequential exon numbers (in red) are shown either under (in the case of rat CPR gene) or above (in the case of mosquito CPR genes) the corresponding exons. Binding domains for FMN, FAD, and NADPH are shown in green, red, and blue, respectively.

Fig. 4.

Nucleotide and amino acid sequence comparisons of the last exons of the CPR coding regions between A. stephensi and other organisms. The one-letter code for each amino acid is aligned with the second nucleotide of each codon. The predicted secondary structures of the A. stephensi CPR are shown under the amino acid sequences. The green bar represents the alpha helix, and the blue arrow represents the beta sheet. Identical residues in amino acid sequences are highlighted. Organisms included in the analysis are R. norvegicus (representing a vertebrate); Drosophila melanogaster and Musca domestica (representing Diptera along with A. stephensi ), Tribolium castaneum (representing Coleoptera), and Spodoptera exigua (representing Lepidoptera).

Fig. 5.

Conserved regions for ligand bindings in the A. stephensi CPR. The multiple sequence alignment illustrates FMN-, FAD-, and NADPH-binding domains of mosquito CPRs according to Pfam. The binding domains are boxed (green, FMN; blue, FAD; purple, NADPH). Triangles above amino acids indicate catalytic residues involved in hydride transfer. Essential residues involved in ligand bindings are highlighted.

Fig. 5.

Conserved regions for ligand bindings in the A. stephensi CPR. The multiple sequence alignment illustrates FMN-, FAD-, and NADPH-binding domains of mosquito CPRs according to Pfam. The binding domains are boxed (green, FMN; blue, FAD; purple, NADPH). Triangles above amino acids indicate catalytic residues involved in hydride transfer. Essential residues involved in ligand bindings are highlighted.

Fig. 6.

Conserved residues involved in cytochrome P450 or cytochrome c interactions. The multiple sequence alignment shows two clusters of acidic residues of mosquito CPRs in comparison with rat CPR. The clusters are boxed, and identical residues are highlighted.