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Biochemical comparison of Anopheles gambiae and human NADPH P450 reductases reveals different 2'-5'-ADP and FMN binding traits.

Lian LY, Widdowson P, McLaughlin LA, Paine MJ - PLoS ONE (2011)

Bottom Line: Close functional similarities were found with respect to the steady state kinetics of cytochrome c reduction, with rates (k(cat)) of 105 s(-1) and 88 s(-1), respectively, for mosquito and human CPR.However, the inhibitory effects of 2',5'-ADP on activity were different; the IC(50) value of AgCPR for 2',5'-ADP was significantly higher (6-10 fold) than human CPR (hCPR) in both phosphate and phosphate-free buffer, indicative of a decrease in affinity for 2',5'-ADP.Furthermore, AgCPR was an order of magnitude more sensitive than hCPR to the reductase inhibitor diphenyliodonium chloride (IC(50) = 28 µM±2 and 361±31 µM respectively).

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Liverpool, Liverpool, United Kingdom. Lu-Yun.Lian@liverpool.ac.uk

ABSTRACT
NADPH-cytochrome P450 oxidoreductase (CPR) plays a central role in chemical detoxification and insecticide resistance in Anopheles gambiae, the major vector for malaria. Anopheles gambiae CPR (AgCPR) was initially expressed in Eschericia coli but failed to bind 2',5'-ADP Sepharose. To investigate this unusual trait, we expressed and purified a truncated histidine-tagged version for side-by-side comparisons with human CPR. Close functional similarities were found with respect to the steady state kinetics of cytochrome c reduction, with rates (k(cat)) of 105 s(-1) and 88 s(-1), respectively, for mosquito and human CPR. However, the inhibitory effects of 2',5'-ADP on activity were different; the IC(50) value of AgCPR for 2',5'-ADP was significantly higher (6-10 fold) than human CPR (hCPR) in both phosphate and phosphate-free buffer, indicative of a decrease in affinity for 2',5'-ADP. This was confirmed by isothermal titration calorimetry where binding of 2',5'-ADP to AgCPR (K(d) = 410±18 nM) was ∼10 fold weaker than human CPR (K(d) = 38 nM). Characterisation of the individual AgFMN binding domain revealed much weaker binding of FMN (K(d) = 83±2.0 nM) than the equivalent human domain (K(d) = 23±0.9 nM). Furthermore, AgCPR was an order of magnitude more sensitive than hCPR to the reductase inhibitor diphenyliodonium chloride (IC(50) = 28 µM±2 and 361±31 µM respectively). Taken together, these results reveal unusual biochemical differences between mosquito CPR and the human form in the binding of small molecules that may aid the development of 'smart' insecticides and synergists that selectively target mosquito CPR.

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Sequence alignment of human and mosquito CPR.Cofactor binding sites are as defined by Wang et al                        [11].                        Conserved residues associated with 2′-phosphate binding (S596, R597,                        K602 and R634 [17], [32]) are shown with an asterisk. The FMN binding                        domain spanning amino acids E69 to G254 that was expressed in E.                            coli is underlined.
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pone-0020574-g001: Sequence alignment of human and mosquito CPR.Cofactor binding sites are as defined by Wang et al [11]. Conserved residues associated with 2′-phosphate binding (S596, R597, K602 and R634 [17], [32]) are shown with an asterisk. The FMN binding domain spanning amino acids E69 to G254 that was expressed in E. coli is underlined.

Mentions: Despite its great medical importance, there is relatively little known about the biochemistry and function of the individual components of the P450 complex of A. gambiae. Most recently, CPR from the mosquito A. minimus has been characterised [12], [13]. The mosquito enzyme shared close biochemical similarities with other CPR family members (Figure 1), although weak binding FMN and FAD cofactors [12] points to potential species differences. We have mapped the tissue distribution of CPR in A. gambiae and discovered high levels of expression in specialised mosquito cells (oenocytes) that suggests key physiological roles for CPR in metabolic processes and pheromone production/metabolism [14]. As expected, CPR gene knockdown by RNAi greatly increases the susceptibility of A. gambiae to permethrin, a widely used pyrethroid insecticide, emphasising the important chemoprotective role of the P450 monooxygenase complex in this organism [14], and validating its potential as a target for the development of chemical inhibitors that might enhance insecticidal activity.


Biochemical comparison of Anopheles gambiae and human NADPH P450 reductases reveals different 2'-5'-ADP and FMN binding traits.

Lian LY, Widdowson P, McLaughlin LA, Paine MJ - PLoS ONE (2011)

Sequence alignment of human and mosquito CPR.Cofactor binding sites are as defined by Wang et al                        [11].                        Conserved residues associated with 2′-phosphate binding (S596, R597,                        K602 and R634 [17], [32]) are shown with an asterisk. The FMN binding                        domain spanning amino acids E69 to G254 that was expressed in E.                            coli is underlined.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105092&req=5

pone-0020574-g001: Sequence alignment of human and mosquito CPR.Cofactor binding sites are as defined by Wang et al [11]. Conserved residues associated with 2′-phosphate binding (S596, R597, K602 and R634 [17], [32]) are shown with an asterisk. The FMN binding domain spanning amino acids E69 to G254 that was expressed in E. coli is underlined.
Mentions: Despite its great medical importance, there is relatively little known about the biochemistry and function of the individual components of the P450 complex of A. gambiae. Most recently, CPR from the mosquito A. minimus has been characterised [12], [13]. The mosquito enzyme shared close biochemical similarities with other CPR family members (Figure 1), although weak binding FMN and FAD cofactors [12] points to potential species differences. We have mapped the tissue distribution of CPR in A. gambiae and discovered high levels of expression in specialised mosquito cells (oenocytes) that suggests key physiological roles for CPR in metabolic processes and pheromone production/metabolism [14]. As expected, CPR gene knockdown by RNAi greatly increases the susceptibility of A. gambiae to permethrin, a widely used pyrethroid insecticide, emphasising the important chemoprotective role of the P450 monooxygenase complex in this organism [14], and validating its potential as a target for the development of chemical inhibitors that might enhance insecticidal activity.

Bottom Line: Close functional similarities were found with respect to the steady state kinetics of cytochrome c reduction, with rates (k(cat)) of 105 s(-1) and 88 s(-1), respectively, for mosquito and human CPR.However, the inhibitory effects of 2',5'-ADP on activity were different; the IC(50) value of AgCPR for 2',5'-ADP was significantly higher (6-10 fold) than human CPR (hCPR) in both phosphate and phosphate-free buffer, indicative of a decrease in affinity for 2',5'-ADP.Furthermore, AgCPR was an order of magnitude more sensitive than hCPR to the reductase inhibitor diphenyliodonium chloride (IC(50) = 28 µM±2 and 361±31 µM respectively).

View Article: PubMed Central - PubMed

Affiliation: School of Biological Sciences, University of Liverpool, Liverpool, United Kingdom. Lu-Yun.Lian@liverpool.ac.uk

ABSTRACT
NADPH-cytochrome P450 oxidoreductase (CPR) plays a central role in chemical detoxification and insecticide resistance in Anopheles gambiae, the major vector for malaria. Anopheles gambiae CPR (AgCPR) was initially expressed in Eschericia coli but failed to bind 2',5'-ADP Sepharose. To investigate this unusual trait, we expressed and purified a truncated histidine-tagged version for side-by-side comparisons with human CPR. Close functional similarities were found with respect to the steady state kinetics of cytochrome c reduction, with rates (k(cat)) of 105 s(-1) and 88 s(-1), respectively, for mosquito and human CPR. However, the inhibitory effects of 2',5'-ADP on activity were different; the IC(50) value of AgCPR for 2',5'-ADP was significantly higher (6-10 fold) than human CPR (hCPR) in both phosphate and phosphate-free buffer, indicative of a decrease in affinity for 2',5'-ADP. This was confirmed by isothermal titration calorimetry where binding of 2',5'-ADP to AgCPR (K(d) = 410±18 nM) was ∼10 fold weaker than human CPR (K(d) = 38 nM). Characterisation of the individual AgFMN binding domain revealed much weaker binding of FMN (K(d) = 83±2.0 nM) than the equivalent human domain (K(d) = 23±0.9 nM). Furthermore, AgCPR was an order of magnitude more sensitive than hCPR to the reductase inhibitor diphenyliodonium chloride (IC(50) = 28 µM±2 and 361±31 µM respectively). Taken together, these results reveal unusual biochemical differences between mosquito CPR and the human form in the binding of small molecules that may aid the development of 'smart' insecticides and synergists that selectively target mosquito CPR.

Show MeSH
Related in: MedlinePlus