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Why single snp analyses fail: epistatic structural effects in honey bee CYP336A1
Orientador(es)
Resumo(s)
Cytochrome P450 enzymes are central to pesticide metabolism and resistance, yet how these proteins diversify substrate specificity while maintaining catalytic function remains poorly understood. A genome-wide analysis of CYP336A1 (a nicotine-metabolizing P450) across 1467 Apis mellifera males from 25 countries spanning the Mediterranean, Middle East, Europe, and Cuba revealed an intricate haplotype architecture. Despite the detection of only 28 single-nucleotide variants (SNPs), 45 distinct haplotypes were detected for CYP336A1. Among these, 23 haplotypes carried at least four SNPs, and four harboured more than 10. A five-SNP haplotype (D202G; M207I; I222V; V226I; Q238K) dominated at 36% frequency, far exceeding the next most common single-SNP haplotype (D262N, 9%). Interestingly, this dominant haplotype was completely absent from the Iberian Peninsula, North Africa, and Oman and, consequently, from five A. mellifera subspecies: iberiensis, intermissa, jemenitica, mellifera and sahariensis. To investigate the functional impact of the identified variants, individually and in combination, we used in sillico protein structural approaches. Protein models were generated with trRosetta, validated with MolProbity, and evaluated using TM-score and RMSD via TM-Align. Structural modelling revealed remarkable fold congruency: the enzyme encoded by the five-SNP haplotype retained a near-identical fold as compared to the wild-type enzyme (TM-score = 0.998, RMSD = 0.34 Å), as did a rarer 13-SNP haplotype (2%) (TM-score = 0.998, RMSD = 0.38 Å). Individual SNPs also produced minimal backbone displacement (0.32–0.54 Å), suggesting that P450 diversification proceeds through subtle structural adjustments rather than major disruption. Moreover, most SNPs clustered within substrate-recognition regions, whereas catalytic residues remained invariant across haplotypes, demonstrating a partitioning between substrate-recognition/binding evolution and preservation of catalytic machinery.
Importantly, single-variant effects cannot predict multi-variant haplotype outcomes. As such, heavy reliance on individual SNPs for pesticide risk assessment may misestimate real metabolic capacity.
Descrição
Palavras-chave
Protein evolution Cytochrome p450 Protein modelling
Contexto Educativo
Citação
Li, Fernanda; Lima, Daniela; Bashir, Sana; Yadró Garcia, Carlos A.; Graaf, Dirk C. de; De Smet, Lina; Verbinnen, Gilles; Rosa-Fontana, Annelise; Rufino, José; Martín-Hernández, Raquel; Pinto, M. Alice; Henriques, Dora (2025). Why single snp analyses fail: epistatic structural effects in honey bee CYP336A1. In XXI International Meeting of the Portuguese Association for Evolutionary Biology. Bragança: Instituto Politécnico de Bragança, p. 73. ISBN 978-972-745-368-9