© 2008 The Authors Journal compilation © 2008 The Royal Entomological Society 125 Insect Molecular Biology (2008) 17(2), 125–135 doi: 10.1111/j.1365-2583.2007.00788.x Blackwell Publishing Ltd Characterization of inhibitors and substrates of Anopheles gambiae CYP6Z2 L. A. Mclaughlin*, U. Niazi†, J. Bibby†, J.-P. David‡, J. Vontas§, J. Hemingway¶, H. Ranson¶, M. J. Sutcliffe† and M. J. I. Paine¶ *Biomedical Research Centre, University of Dundee, Ninewells Hospital and Medical School, Dundee, United Kingdom; †Manchester Interdisciplinary Biocentre & School of Chemical Engineering and Analytical Science, University of Manchester, Manchester, United Kingdom; ‡Laboratoire d’Ecologie Alpine, UMR 5553 Université Joseph Fourier – CNRS, Grenoble, France; §Laboratory of Pesticide Science, Agricultural University of Athens, Greece; and ¶Liverpool School of Tropical Medicine, Liverpool, United Kingdom Abstract Three CYP6Z genes are linked to a major pyrethroid resistance locus in the mosquito Anopheles gambiae. We have expressed CYP6Z2 in Escherichia coli and produced a structural model in order to examine its role in detoxification. E. coli membranes co-express- ing CYP6Z2 and An. gambiae P450 reductase (AgCPR) catalysed the dealkylation of benzyloxyresorufin with kinetic parameters K m = 0.13 μM; K cat = 1.5 min -1 . The IC 50 values of a wide range of compounds were meas- ured. Pyrethroids cypermethrin and permethrin pro- duced low IC 50 values, but were not metabolized. Plant flavanoids were the most potent inhibitors. Several compounds were shown to be substrates, suggesting that CYP6Z2 has broad substrate specificity and plays an important chemo-protective role during the her- bivorous phase of the life-cycle. Keywords: P450, insecticide metabolism, mosquito, malaria, P450 reductase. Introduction The mosquito Anopheles gambiae is the major vector for Plasmodium falciparum, the parasite responsible for malaria which affects several million people worldwide. The use of insecticides, in particular those belonging to the pyrethroid class, is an important component of malaria control pro- grammes. However, resistance to pyrethroids has started to emerge, which poses a major threat to the control of mosquito-borne disease (N’guessan et al., 2007). The cytochrome P450 gene family, which catalyses the chemical transformation of both xenobiotic and endogenous com- pounds, is associated with insecticide resistance through increased metabolism of insecticides (Feyereisen, 1999; Ranson et al., 2002). Although the An. gambiae genome contains over one hundred P450 genes, only a handful of these have thus far been linked to insecticide resistance. CYP6Z1 and CYP325A3 are overexpressed in an estab- lished pyrethroid-resistant laboratory strain of An. gambiae (David et al., 2005), whereas CYP6Z2 and CYP6M2 are overexpressed in a pyrethroid-resistant field strain from Odumasy, Ghana (Muller et al., 2007). CYP6Z genes have also been associated with constitutive pyrethroid resistance by genetic mapping and quantitative PCR (Nikou et al ., 2003). As yet none of these enzymes have been functionally characterized. We have focused here on the CYP6Z gene family, which is comprised of four genes, three of which are tightly clus- tered on chromosome 3R (Nikou et al., 2003). Interestingly, all three have distinctive differences in life stage expression; CYP6Z1 is predominately expressed in adults, whereas CYP6Z2 is expressed at high levels in the larvae and to a lesser extent in adults, and finally CYP6Z3 is expressed predominantly in larvae and pupae (Nikou et al., 2003). The molecular basis for this variation in developmental expres- sion remains unclear but presumably reflects differences in genetic control mechanisms and endogenous function (Nikou et al., 2003). Here we have sought to define the function of these P450s by expressing CYP6Z2 in Escherichia coli with its cognate redox partner An. gambiae NADPH P450 reductase (AgCPR) and examining its inhibition and substrate preferences. Alongside, we have used homology modelling and docking techniques to gain insight at the Received 10 September 2007; accepted after revision 6 December 2007; first published online 15 February 2008. Correspondence: Mark J. I. Paine, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool, L3 5QA, United Kingdom. Tel.: +44 (0)151 705 3310; fax: +44 (0)151 705 3370; e-mail: m.j.paine@liverpool.ac.uk