Conformational switch upon substrate binding informs the rational design of CCoAOMT enzymes

Area of Science:

• Biochemistry
• Molecular Biology
• Computational Chemistry

Background:

• Poplar wood is a primary source for the paper industry, but lignin production generates significant industrial pollution.
• Reducing lignin content in papermaking improves pulp yield and decreases chemical reagent use and pollutant generation.
• Caffeoyl coenzyme A O-methyltransferase (CCoAOMT) regulates lignin biosynthesis, but its substrate recognition mechanism remains unclear.

Purpose of the Study:

• To elucidate the molecular recognition mechanism and conformational changes of CCoAOMT upon binding its substrate, caffeoyl coenzyme A (CCoA).
• To identify key residues and regions involved in substrate binding and allosteric regulation.
• To provide insights for designing enzymes with altered activity for industrial applications.

Main Methods:

• Comparative molecular dynamics (MD) simulations to analyze overall conformational changes.
• Free energy landscape (FEL) and conformation cluster analyses to investigate functional motion modes.
• Adaptive steered molecular dynamics (ASMD) simulations to observe substrate recognition and allostery.
• Binding free energy prediction and energy decomposition to identify key residues.
• Single mutation and folding entropy calculations to assess enzyme activity.

Main Results:

• CCoAOMT exhibits increased flexibility in regions outside the binding pocket (α1, α2, α6, α8), facilitating substrate binding.
• Functional motions involve regions β5, α2, and α8, with the enzyme's transport channel transitioning from open to closed upon substrate binding.
• Key residues (e.g., Y188, D218) form crucial hydrogen bonds anchoring the CCoA substrate.
• Six potential low-activity CCoAOMT mutants (K1L, N39G, I40P, T42N, N174D, D218V) were identified.

Conclusions:

• The study clarifies the dynamic conformational changes and substrate recognition process of CCoAOMT.
• Identified key residues and regions provide a basis for enzyme engineering to control lignin biosynthesis.
• Findings contribute to developing more sustainable papermaking processes by optimizing lignin content.