Untangling the Reaction Mechanism of the Polysaccharide Lyase PL42 Using QM/MM Metadynamics Simulations

Polysaccharide lyase family 42 (PL42) enzymes cleave terminal Rha-GlcA motifs from gum arabic through β-elimination. However, the neutron-based catalytic proposal for FoRham1 has not been validated on a free-energy surface. Here, we combine QM/MM well-tempered metadynamics with umbrella sampling to analyze the reaction mechanism of the PL42 lyase FoRham1 at the atomic level. The calculations support a concerted yet highly asynchronous syn β-elimination, where His85 functions as the catalytic base/acid, with assistance from Asp83 and doubly protonated His105. Arg166 stabilizes the glucuronate carboxylate electrostatically. The reactive substrate conformation is an inverse-chair ¹C₄ state, and the +1 sugar follows a ¹C₄ → ¹C₄ → ¹H₂ pathway along the minimum-free-energy route. The metadynamics barrier (∼19 kcal mol^-1) is refined to 17.7 kcal mol^-1 through umbrella sampling, aligning reasonably with experimental data. Beyond confirming the proposed experimental mechanism, the results demonstrate that PL42 catalyzes β-elimination via a histidine-based catalytic system distinct from those of PL7 and PL38. Additionally, they expand the emerging conformational framework linking polysaccharide lyase mechanisms through their product-ring geometries.