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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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Purification a spectral characterisation of AgCPR.A, SDS-polyacrylamide gel electrophoresis of AgCPR purified by                            nickel-agarose affinity chromatography. Lane 1, molecular weight                            standards (kDa); Lane 2, partially purified CPR fraction eluted with 250                            mM imidazole; Lane 3, thrombin cleaved AgCPR. B. absorption spectra of                            purified AgCPR(1.5 µM). Trace a is the oxidised spectrum; Trace b                            is the reduced spectrum with 1.5 µM NADPH and measured after 10                            seconds; trace c is the air-stable semiquinone measured after 30                            min.
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pone-0020574-g002: Purification a spectral characterisation of AgCPR.A, SDS-polyacrylamide gel electrophoresis of AgCPR purified by nickel-agarose affinity chromatography. Lane 1, molecular weight standards (kDa); Lane 2, partially purified CPR fraction eluted with 250 mM imidazole; Lane 3, thrombin cleaved AgCPR. B. absorption spectra of purified AgCPR(1.5 µM). Trace a is the oxidised spectrum; Trace b is the reduced spectrum with 1.5 µM NADPH and measured after 10 seconds; trace c is the air-stable semiquinone measured after 30 min.

Mentions: Initial attempts to purify full-length AgCPR expressed in E. coli using standard ion exchange and 2, 5′-ADP-Sepharose affinity chromatography [19], [20] failed, which suggested that AgCPR might have different nucleotide binding properties. Therefore, to examine this further N-terminally histidine tagged soluble forms, lacking the N-terminal membrane anchor, of AgCPR was expressed in E. coli and affinity purified over nickel agarose (Fig. 2A). Although CPR lacking the amino-terminal membrane anchor loses its ability to interact with P450 it is otherwise fully functional and capable of reducing a range of electron acceptors [11]. Equivalent preparations of human anchorless CPR were also produced [17], and comparative enzyme activities measured via the reduction of cytochrome c, the surrogate electron acceptor most commonly used for measuring diflavin reductase activity [21].


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)

Purification a spectral characterisation of AgCPR.A, SDS-polyacrylamide gel electrophoresis of AgCPR purified by                            nickel-agarose affinity chromatography. Lane 1, molecular weight                            standards (kDa); Lane 2, partially purified CPR fraction eluted with 250                            mM imidazole; Lane 3, thrombin cleaved AgCPR. B. absorption spectra of                            purified AgCPR(1.5 µM). Trace a is the oxidised spectrum; Trace b                            is the reduced spectrum with 1.5 µM NADPH and measured after 10                            seconds; trace c is the air-stable semiquinone measured after 30                            min.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105092&req=5

pone-0020574-g002: Purification a spectral characterisation of AgCPR.A, SDS-polyacrylamide gel electrophoresis of AgCPR purified by nickel-agarose affinity chromatography. Lane 1, molecular weight standards (kDa); Lane 2, partially purified CPR fraction eluted with 250 mM imidazole; Lane 3, thrombin cleaved AgCPR. B. absorption spectra of purified AgCPR(1.5 µM). Trace a is the oxidised spectrum; Trace b is the reduced spectrum with 1.5 µM NADPH and measured after 10 seconds; trace c is the air-stable semiquinone measured after 30 min.
Mentions: Initial attempts to purify full-length AgCPR expressed in E. coli using standard ion exchange and 2, 5′-ADP-Sepharose affinity chromatography [19], [20] failed, which suggested that AgCPR might have different nucleotide binding properties. Therefore, to examine this further N-terminally histidine tagged soluble forms, lacking the N-terminal membrane anchor, of AgCPR was expressed in E. coli and affinity purified over nickel agarose (Fig. 2A). Although CPR lacking the amino-terminal membrane anchor loses its ability to interact with P450 it is otherwise fully functional and capable of reducing a range of electron acceptors [11]. Equivalent preparations of human anchorless CPR were also produced [17], and comparative enzyme activities measured via the reduction of cytochrome c, the surrogate electron acceptor most commonly used for measuring diflavin reductase activity [21].

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