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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 oxidized AgCPR with 2′, 5′                            – ADP.Binding isotherms for the titration of 2′,5′-ADP into AgCPR.                            The points are fit to a one-set-of-sites model. Reactions 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 1.
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pone-0020574-g003: Isothermal titration of oxidized AgCPR with 2′, 5′ – ADP.Binding isotherms for the titration of 2′,5′-ADP into AgCPR. The points are fit to a one-set-of-sites model. Reactions 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 1.

Mentions: Since AgCPR appeared to bind weakly to 2′, 5′-ADP the binding properties of this molecule were examined in more detail. 2′, 5′-ADP is the adenosine-ribose moiety of NADPH, which binds through a bi-partite mode with the nicotinamide moiety, to separate binding pockets of CPR [17]. Thus to delineate the roles played by the nicotinamide and ribose moieties, we used isothermal titration calorimetry (ITC) to determine the binding affinities of the adenosine-ribose fragments (Table 1). As previously reported for hCPR [25], this was done in a phosphate-free buffer to prevent competitive interactions with free phosphate. Figure 3 represents the ITC binding isotherms resulting from the titration of AgCPR with 2′, 5′-ADP, NADP+ and 2′-AMP. The binding isotherms were exothermic and fit to a single-site model. The observed dissociation binding constant for the AgCPR - 2′5′-ADP interaction was 410±18 nM, with a binding stoichiometry n = 1.03. This is substantially weaker than hCPR under the same reaction conditions (Kd of 38±3.8 nM; this study and [25]).


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 oxidized AgCPR with 2′, 5′                            – ADP.Binding isotherms for the titration of 2′,5′-ADP into AgCPR.                            The points are fit to a one-set-of-sites model. Reactions 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 1.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105092&req=5

pone-0020574-g003: Isothermal titration of oxidized AgCPR with 2′, 5′ – ADP.Binding isotherms for the titration of 2′,5′-ADP into AgCPR. The points are fit to a one-set-of-sites model. Reactions 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 1.
Mentions: Since AgCPR appeared to bind weakly to 2′, 5′-ADP the binding properties of this molecule were examined in more detail. 2′, 5′-ADP is the adenosine-ribose moiety of NADPH, which binds through a bi-partite mode with the nicotinamide moiety, to separate binding pockets of CPR [17]. Thus to delineate the roles played by the nicotinamide and ribose moieties, we used isothermal titration calorimetry (ITC) to determine the binding affinities of the adenosine-ribose fragments (Table 1). As previously reported for hCPR [25], this was done in a phosphate-free buffer to prevent competitive interactions with free phosphate. Figure 3 represents the ITC binding isotherms resulting from the titration of AgCPR with 2′, 5′-ADP, NADP+ and 2′-AMP. The binding isotherms were exothermic and fit to a single-site model. The observed dissociation binding constant for the AgCPR - 2′5′-ADP interaction was 410±18 nM, with a binding stoichiometry n = 1.03. This is substantially weaker than hCPR under the same reaction conditions (Kd of 38±3.8 nM; this study and [25]).

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