Ellis, Emerald S.Hinchen, Daniel J.Bleem, AlissaBu, LintaoMallinson, Sam J. B.Allen, Mark D.Streit, Bennett R.Machovina, Melodie M.Doolin, Quinlan V.Michener, William E.Johnson, Christopher W.Knott, Brandon C.Beckham, Gregg T.McGeehan, John E.DuBois, Jennifer L.2022-08-312022-08-312021-03Ellis, E. S., Hinchen, D. J., Bleem, A., Bu, L., Mallinson, S. J., Allen, M. D., ... & DuBois, J. L. (2021). Engineering a cytochrome P450 for demethylation of lignin-derived aromatic aldehydes. JACS Au, 1(3), 252-261.2691-3704https://scholarworks.montana.edu/handle/1/17036Biological funneling of lignin-derived aromatic compounds is a promising approach for valorizing its catalytic depolymerization products. Industrial processes for aromatic bioconversion will require efficient enzymes for key reactions, including demethylation of O-methoxy-aryl groups, an essential and often rate-limiting step. The recently characterized GcoAB cytochrome P450 system comprises a coupled monoxygenase (GcoA) and reductase (GcoB) that catalyzes oxidative demethylation of the O-methoxy-aryl group in guaiacol. Here, we evaluate a series of engineered GcoA variants for their ability to demethylate o-and p-vanillin, which are abundant lignin depolymerization products. Two rationally designed, single amino acid substitutions, F169S and T296S, are required to convert GcoA into an efficient catalyst toward the o- and p-isomers of vanillin, respectively. Gain-of-function in each case is explained in light of an extensive series of enzyme-ligand structures, kinetic data, and molecular dynamics simulations. Using strains of Pseudomonas putida KT2440 already optimized for p-vanillin production from ferulate, we demonstrate demethylation by the T296S variant in vivo. This work expands the known aromatic O-demethylation capacity of cytochrome P450 enzymes toward important lignin-derived aromatic monomers.en-UScc-byhttps://creativecommons.org/licenses/by/4.0/cytochrome p450Article