TY - JOUR
T1 - Engineering a Cytochrome P450 for Demethylation of Lignin-Derived Aromatic Aldehydes
AU - Ellis, Emerald
AU - Hinchen, Daniel
AU - Bleem, Alissa
AU - Bu, Lintao
AU - Mallinson, Sam
AU - Allen, Mark
AU - Streit, Bennett
AU - Machovina, Melodie
AU - Doolin, Quinlan
AU - Michener, William
AU - Johnson, Christopher
AU - Knott, Brandon
AU - Beckham, Gregg
AU - McGeehan, John
AU - DuBois, Jennifer
N1 - Publisher Copyright:
© 2021 American Chemical Society. All rights reserved.
PY - 2021/3/22
Y1 - 2021/3/22
N2 - Biological 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.
AB - Biological 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.
KW - aromatic O-demethylation
KW - biological funneling
KW - cytochrome P450
KW - lignin
KW - protein engineering
UR - http://www.scopus.com/inward/record.url?scp=85111046911&partnerID=8YFLogxK
U2 - 10.1021/jacsau.0c00103
DO - 10.1021/jacsau.0c00103
M3 - Article
AN - SCOPUS:85111046911
SN - 2691-3704
VL - 1
SP - 252
EP - 261
JO - JACS Au
JF - JACS Au
IS - 3
ER -