6YCJ

Crystal structure of GcoA T296S bound to guaiacol

6YCJ の概要

• エントリーDOI: 10.2210/pdb6ycj/pdb
• 分子名称: Aromatic O-demethylase, cytochrome P450 subunit, PROTOPORPHYRIN IX CONTAINING FE, Guaiacol, ... (4 entities in total)
• 機能のキーワード: aromatic catabolism, cytochrome p450, lignin valorization, protein engineering, oxidoreductase
• 由来する生物種: Amycolatopsis sp. (strain ATCC 39116 / 75iv2)
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 46179.37

構造登録者

Mallinson, S.J.B.,Hinchen, D.J.,Ellis, E.S.,Beckham, G.T.,DuBois, J.L.,McGeehan, J.E. (登録日: 2020-03-18, 公開日: 2021-02-17, 最終更新日: 2024-01-24)

主引用文献

Ellis, E.S.,Hinchen, D.J.,Bleem, A.,Bu, L.,Mallinson, S.J.B.,Allen, M.D.,Streit, B.R.,Machovina, M.M.,Doolin, Q.V.,Michener, W.E.,Johnson, C.W.,Knott, B.C.,Beckham, G.T.,McGeehan, J.E.,DuBois, J.L.
Engineering a Cytochrome P450 for Demethylation of Lignin-Derived Aromatic Aldehydes.
Jacs Au, 1:252-261, 2021

PubMed Abstract: 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 -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 methoxy-aryl group in guaiacol. Here, we evaluate a series of engineered GcoA variants for their ability to demethylate -and -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 - and -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 KT2440 already optimized for -vanillin production from ferulate, we demonstrate demethylation by the T296S variant . This work expands the known aromatic demethylation capacity of cytochrome P450 enzymes toward important lignin-derived aromatic monomers.

PubMed: 34467290
DOI: 10.1021/jacsau.0c00103

実験手法

X-RAY DIFFRACTION (1.64 Å)