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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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Isothermal titration of FMN domains with FMN.Binding isotherms for the titration of FMN into AgCPR. The points are fit                            to a one-set-of-sites model. Reaction were carried out in BES buffer                            (100 mM, pH 7.0 and 25°C) as described in Materials and Methods. The thermodynamic parameters                            of ΔS, ΔG, ΔH, and Kd are listed                            in Table 2.
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pone-0020574-g004: Isothermal titration of FMN domains with FMN.Binding isotherms for the titration of FMN into AgCPR. The points are fit to a one-set-of-sites model. Reaction were carried out in BES buffer (100 mM, pH 7.0 and 25°C) as described in Materials and Methods. The thermodynamic parameters of ΔS, ΔG, ΔH, and Kd are listed in Table 2.

Mentions: The binding affinity of FMN to the isolated FMN-binding domains of both A. gambiae and hCPR were also examined by titration of the apo-FMN binding domains (Figure 4). Both isotherms show reasonable binding and saturation of the apo-proteins is achieved with excess flavin. The Kd for FMN binding to the isolated apo-human FMN-binding domain was calculated to be 23±0.9 nM which is almost 4-fold stronger than for the isolated apo-A. gambiae FMN-binding domain which was calculated to be 83±2.0 nM.


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)

Isothermal titration of FMN domains with FMN.Binding isotherms for the titration of FMN into AgCPR. The points are fit                            to a one-set-of-sites model. Reaction were carried out in BES buffer                            (100 mM, pH 7.0 and 25°C) as described in Materials and Methods. The thermodynamic parameters                            of ΔS, ΔG, ΔH, and Kd are listed                            in Table 2.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105092&req=5

pone-0020574-g004: Isothermal titration of FMN domains with FMN.Binding isotherms for the titration of FMN into AgCPR. The points are fit to a one-set-of-sites model. Reaction were carried out in BES buffer (100 mM, pH 7.0 and 25°C) as described in Materials and Methods. The thermodynamic parameters of ΔS, ΔG, ΔH, and Kd are listed in Table 2.
Mentions: The binding affinity of FMN to the isolated FMN-binding domains of both A. gambiae and hCPR were also examined by titration of the apo-FMN binding domains (Figure 4). Both isotherms show reasonable binding and saturation of the apo-proteins is achieved with excess flavin. The Kd for FMN binding to the isolated apo-human FMN-binding domain was calculated to be 23±0.9 nM which is almost 4-fold stronger than for the isolated apo-A. gambiae FMN-binding domain which was calculated to be 83±2.0 nM.

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