The substrate specificity in the O-demethylation of 4-alkylguaiacols by cytochrome P450 AgcAP450

Alkylguaiacols are lignin-derived products obtained by reductive catalytic fractionation (RCF) of lignocellulosic biomass. The recently discovered AgcA(P450) enzyme from the CYP255A1 family oxidizes a range of 4-alkylguaiacols, having a preference for bulkier substrates such as 4-propylguaiacol over small substrates such as guaiacol. The substrate specificity of AgcA(P450) for 4-propylguaiacol, 4-ethylguaiacol, 4-methylguaiacol, and guaiacol was studied using molecular dynamics (MD) simulations. Our results showed that AgcA(P450) adopts an open conformation of the substrate access channel, allowing a water flux into the active site, and therefore resulting in a bigger pocket to accommodate bulkier substrates. MD simulations disclosed that the smaller substrates could not stably bind in the catalytic site, and thus it is unlikely for them to be transformed. Further, our quantum mechanics molecular mechanics (QM/MM) study shows that the activation energy barrier for the rate limiting step of the O-demethylation, i.e. hydrogen atom abstraction, is the lowest for AgcA(P450) in complex with 4-propylguaicol, compared to that of the enzyme in complex with the other smaller substrates. This unravels the structural feature of the lignin degradation enzyme responsible for the preference toward bulky substrates, and hence sets a basis for engineering enzymes to be exploited for the sustainable conversion of lignocellulosic biomass.