6ycn
From Proteopedia
Crystal structure of GcoA F169A bound to o-vanillin
Structural highlights
FunctionGCOA_AMYS7 Part of a two-component P450 system that efficiently O-demethylates diverse aromatic substrates such as guaiacol and a wide variety of lignin-derived monomers. Is likely involved in lignin degradation, allowing Amycolatopsis sp. ATCC 39116 to catabolize plant biomass. GcoA binds and processes the substrate with electrons supplied by the GcoB subunit.[1] Publication Abstract from PubMedBiological 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. Engineering a Cytochrome P450 for Demethylation of Lignin-Derived Aromatic Aldehydes.,Ellis ES, Hinchen DJ, Bleem A, Bu L, Mallinson SJB, Allen MD, Streit BR, Machovina MM, Doolin QV, Michener WE, Johnson CW, Knott BC, Beckham GT, McGeehan JE, DuBois JL JACS Au. 2021 Feb 4;1(3):252-261. doi: 10.1021/jacsau.0c00103. eCollection 2021, Mar 22. PMID:34467290[2] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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