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

Poplar is a deciduous tree belonging to the willow family, whose wood has the characteristics of light texture and toughness. It can be used to produce pulp, and is one of the main raw materials in the paper industry. A large amount of lignin is produced during pulping and papermaking process, where the generated waste liquid and pollutants are one of the main sources of industrial pollution. Regulating lignin content not only enables factories to process more pulp, but also greatly reduces the use of chemical reagents and the production of pollutants. Caffeoyl coenzyme A O-methyltransferase (CCoAOMT) is a class of S-adenosyl methionine (SAM) methyltransferases, which can effectively regulate the total amount of lignin biosynthesis. At present, molecular recognition mechanism of CCoAoMT with the substrate caffeoyl coenzyme A (CCoA) and corresponding conformational change both remain unclear, significantly restricting the design of weakly active enzymes. In this work, the overall conformational changes of CCoAOMT after binding CCoA were firstly explored by comparative molecular dynamics (MD) simulations. The results show that the four regions of α1, α2, α6 and α8 located outside binding pocket show greater flexibility, which is conducive to substrate binding. Through free energy landscape (FEL) and conformation cluster analyses, the representative functional motion modes were investigated, with the regions involved being β5, α2 and α8. According to adaptive steered molecular dynamics (ASMD) simulations, the complete recognition process of the receptor by SAM and CCoA, and allostery in the transport channel of substrates both were observed. With the binding of substrates, the structure of CCoAOMT gradually becomes more compact, accompanied by its transport channel from open to closed state. By binding free energy prediction and energy decomposition, a series of key residues favoring the receptor-substrate mutual recognition were proposed. Specifically, CCoA exhibits H-bonds with N170, D218, T42, Y188, N39, R186, N174 and R30, as well as hydrophobic interactions with K1 and I40. In particular, Y188 and D218 both anchor CCoA caffeoyl group to the Ca2+ active site by forming stable H-bonds. Based on exhaustive single mutation and folding entropy calculation, six mutants (K1L, N39G, I40P, T42N, N174D and D218V) may be candidates for low-activity CCoAOMT.