Quercetin-metabolizing CYP6AS enzymes of the pollinator Apis mellifera (Hymenoptera: Apidae) Wenfu Mao a , Sanjeewa G. Rupasinghe b , Reed M. Johnson a , Arthur R. Zangerl a , Mary A. Schuler b , May R. Berenbaum a, ⁎ a Department of Entomology, University of Illinois, Urbana, Illinois 61801-3795, USA b Department of Cell and Developmental Biology, University of Illinois, Urbana, Illinois 61801, USA abstract article info Article history: Received 4 June 2009 Received in revised form 23 August 2009 Accepted 25 August 2009 Available online 6 September 2009 Keywords: Apis mellifera Cytochrome P450 monooxygenase CYP6AS subfamily Detoxification Flavonoid metabolism Honey Pollinator Although the honey bee (Apis mellifera) genome contains far fewer cytochrome P450 genes associated with xenobiotic metabolism than other insect genomes sequenced to date, the CYP6AS subfamily, apparently unique to hymenopterans, has undergone an expansion relative to the genome of the jewel wasp (Nasonia vitripennis). The relative dominance of this family in the honey bee genome is suggestive of a role in processing phytochemicals encountered by honey bees in their relatively unusual diet of honey (comprising concentrated processed nectar of many plant species) and bee bread (a mixture of honey and pollen from many plant species). In this study, quercetin was initially suggested as a shared substrate for CYP6AS1, CYP6AS3, and CYP6AS4, by its presence in honey, extracts of which induce transcription of these three genes, and by in silico substrate predictions based on a molecular model of CYP6AS3. Biochemical assays with heterologously expressed CYP6AS1, CYP6AS3, CYP6AS4 and CYP6AS10 enzymes subsequently confirmed their activity toward this substrate. CYP6AS1, CYP6AS3, CYP6AS4 and CYP6AS10 metabolize quercetin at rates of 0.5 ± 0.1, 0.5 ± 0.1, 0.2 ± 0.1, and 0.2 ± 0.1 pmol quercetin/ pmol P450/min, respectively. Substrate dockings and sequence alignments revealed that the positively charged amino acids His107 and Lys217 and the carbonyl group of the backbone between Leu302 and Ala303 are essential for quercetin orientation in the CYP6AS3 catalytic site and its efficient metabolism. Multiple replacements in the catalytic site of CYP6AS4 and CYP6AS10 and repositioning of the quercetin molecule likely account for the lower metabolic activities of CYP6AS4 and CYP6AS10 compared to CYP6AS1 and CYP6AS3. © 2009 Elsevier Inc. All rights reserved. 1. Introduction Cytochrome P450 monooxygenases (P450s) constitute a large superfamily of heme-thiolate enzymes in a wide range of organisms that catalyze the NADPH-associated reductive cleavage of oxygen to produce a functionalized product and water (http://drnelson.utmem. edu/CytochromeP450.html). P450s catalyze a broad diversity of reactions (Guengerich, 2001) that in insects contribute to the biosynthesis of endogenous pheromones and hormones as well as the detoxification of natural and synthetic xenobiotics (Feyereisen, 2005, 2006; Li et al., 2007). Insect genomes typically contain large numbers of P450 genes, consistent with the hypothesis that this gene superfamily has expanded by multiple gene duplication events associated with “animal–plant warfare” as represented by reciprocal selective responses between herbivores and their host plants (Gonzalez and Nebert, 1990). The available collection of annotated P450s across all available genomes has been subdivided into four major clades. Genes in the CYP3 clade, such as the insect-specific CYP6 family, have been extensively implicated in xenobiotic metabolism (Feyereisen, 2006; Li et al., 2007). Not all interactions between plants and herbivores, however, constitute “warfare”—since the late Cretaceous many angiosperm plant species have produced sugar-rich nectars for the express purpose of attracting and rewarding mutualistic partner species that contribute to pollination (Brandenburg et al., 2009). For herbivores, the toxicological consequences of consuming nectar differ dramati- cally from the consequences of consuming chemically well-defended foliar tissues. The first genome sequence of a pollinator species became available in 2006 with the completion of the genome of the western honey bee Apis mellifera (Honey Bee Consortium, 2006). Annotation of the complete P450 inventory of this genome (http:// drnelson.utmem.edu/CytochromeP450.html) revealed 46 genes, a reduction on the order of 50% or greater in this gene superfamily relative to other insect genomes (Claudianos et al., 2006). Several unique aspects of A. mellifera biology are likely to have contributed to this reduction. One aspect is the relatively innocuous nature of this pollinator's diet, which differs considerably from that of typical herbivores. In contrast with foliage-feeding herbivores that consume Comparative Biochemistry and Physiology, Part B 154 (2009) 427–434 ⁎ Corresponding author. Dept. of Entomology, 320 Morrill Hall, University of Illinois, 505 S Goodwin, Urbana, IL 61801-3795, USA. Tel.: +1 217 333 7785. E-mail address: maybe@uiuc.edu (M.R. Berenbaum). 1096-4959/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpb.2009.08.008 Contents lists available at ScienceDirect Comparative Biochemistry and Physiology, Part B journal homepage: www.elsevier.com/locate/cbpb