Impact of Missense Mutations on AFB1 Metabolism in Bovine Cytochrome P4503A Isoforms: A Computational Mutagenesis and Molecular Docking Analysis

Abstract

Cytochrome P450 3A (CYP3A) enzymes catalyze the metabolism of a wide range of endogenous and exogenous compounds. Genetic variations in the 3 CYP3A isoforms (CYP3A28, CYP3A74, and CYP3A76) may influence their expression and activity, leading to inter-individual differences in xenobiotic metabolism. In domestic cattle, understanding how genetic variations modulate CYP3A activity is crucial for both its therapeutic implications (clinical efficacy and adverse drug effects) and food safety (residues in foodstuff). Here, we updated the variant calling of CYP3As in 300 previously sequenced Piedmontese beef cattle, using the most recent reference genome, which contains an updated, longer sequence for CYP3A28. All but one previously identified missense variants were confirmed and a new variant, R105W in CYP3A28, was discovered. Through computational mutagenesis and molecular docking, we computationally predicted the impact of all identified CYP3A variant enzymes on protein stability and their affinity for aflatoxin B1 (AFB1), a potent carcinogen and food contaminant. For CYP3A28, we also computationally predicted its affinity for the probe substrate nifedipine (NIF). We found that CYP3A28 with R105W variant cannot accommodate NIF nor AFB1 in the binding pocket, thus affecting their metabolism. Our work provides computational foundation and prioritized ranking of CYP3A variants for future experimental validations.

Keywords: CYP3A; Piedmontese cattle breed; aflatoxin B1; cattle genetic variants; computational mutagenesis; cytochrome P450; molecular docking; nifedipine; single nucleotide variants.

Figure 1

NIF accommodation in the CYP3A28 substrate pocket. Black dashes indicated hydrogen bonds, while red dashes indicated π–cation interaction. The CYP backbone is shown in grey ribbon, the heme group in orange, the residues in violet and NIF in green.

Figure 2

AFB1 (light blue) and variants (blue) location in the CYP3A28 (a), CYP3A74 (b), and CYP3A76 (c) isoforms.

Figure 3

Docking poses of AFB1 against CYP3A28 (a), CYP3A74 (b,c), and CYP3A76 (d,e) models. CYP backbone is shown in grey ribbon, the heme group in orange, the residues in violet, and AFB1 in blue. Black dashes indicated hydrogen bonds, while yellow and red dashes indicated π–π stacking and π–cation interactions, respectively.