Biochemical Comparison of Anopheles gambiae and Human NADPH P450 Reductases Reveals Different 2′-5′-ADP and FMN Binding Traits

Lian, Lu-Yun, Widdowson, Philip, McLaughlin, Lesley A. and Paine, Mark ORCID: https://orcid.org/0000-0003-2061-7713 (2011) 'Biochemical Comparison of Anopheles gambiae and Human NADPH P450 Reductases Reveals Different 2′-5′-ADP and FMN Binding Traits'. PLoS ONE, Vol 6, Issue 5, e20574.

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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 29, 59-ADP Sepharose. To investigate this unusual trait, we expressed and purified a truncated histidinetagged 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 (kcat) of 105 s21 and 88 s21, respectively, for mosquito and human CPR. However, the inhibitory effects of 29,59-ADP on activity were different; the IC50 value of AgCPR for 29, 59 –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 29, 59- ADP. This was confirmed by isothermal titration calorimetry where binding of 29,59-ADP to AgCPR (Kd = 410618 nM) was ,10 fold weaker than human CPR (Kd = 38 nM). Characterisation of the individual AgFMN binding domain revealed much weaker binding of FMN (Kd=8362.0 nM) than the equivalent human domain (Kd=2360.9 nM). Furthermore, AgCPR was an order of magnitude more sensitive than hCPR to the reductase inhibitor diphenyliodonium chloride (IC50=28 mM62 and 361631 mM 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.

Item Type:	Article
Subjects:	QX Parasitology > Insects. Other Parasites > QX 515 Anopheles QX Parasitology > Insects. Other Parasites > QX 650 Insect vectors WC Communicable Diseases > Tropical and Parasitic Diseases > WC 750 Malaria
Faculty: Department:	Groups (2002 - 2012) > Vector Group
Digital Object Identifer (DOI):	https://doi.org/10.1371/journal.pone.0020574
Depositing User:	Users 183 not found.
Date Deposited:	02 Aug 2011 14:11
Last Modified:	06 Feb 2018 13:03
URI:	https://archive.lstmed.ac.uk/id/eprint/2269

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