Molecular docking and in vitro fermentation reveal plant-derived rottlerin targeting methyl-coenzyme M reductase to reduce enteric methane emissions from dairy cows

This study aimed to investigate the inhibitory effects of rottlerin, a plant-derived polyphenol compound, on methane (CH₄) production in dairy cow rumen fermentation by targeting methyl-coenzyme M reductase (MCR). Molecular docking and virtual screening revealed that rottlerin exhibited a high binding affinity (-8.300 kcal/mol) with key residues in the catalytic site of MCR (GLY-142 and GLN-230). In vitro rumen fermentation showed that rottlerin significantly reduced CH₄ production in a dose-dependent manner (up to 32.4%), while maintaining stable pH, dry matter digestibility, and VFA concentration. Microbial community analysis revealed a reduction in the relative abundance of Methanobrevibacter, a dominant hydrogenotrophic methanogen, and a simultaneous increase in Candidatus_Methanomethylophilus (P < 0.05), a methylotrophic methanogen, indicating a metabolic shift favoring methylotrophic rather than hydrogenotrophic methanogenesis. The in vitro MCR inhibition assay showed a concentration dependent inhibition of MCR activity (half-maximal inhibitory concentration [IC₅₀] = 23.72 μM), suggesting a direct interaction between rottlerin and MCR, consistent with the docking results. These findings highlight the potential of rottlerin as a naturally occurring, MCR-targeted feed additive for sustainable methane mitigation in dairy cows.