Skip to content

Engineering a cytochrome P450 for demethylation of lignin-derived aromatic aldehydes

Research output: Contribution to journalArticlepeer-review

  • Emerald Ellis
  • Daniel James Hinchen
  • Alissa Bleem
  • Lintao Bu
  • Simon James Bradshaw Mallinson
  • Mark Devin Allen
  • Bennett Streit
  • Melodie M. Machovina
  • Quinlan Doolin
  • William E. Michener
  • Christopher W. Johnson
  • Brandon C. Knott
  • Gregg T. Beckham
  • Professor John McGeehan
  • Jennifer L. DuBois
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.
Original languageEnglish
Number of pages10
JournalJACS Au
Early online date4 Feb 2021
Publication statusEarly online - 4 Feb 2021


Related information

Relations Get citation (various referencing formats)

ID: 26102459