9IA1

Crystal Structure of AgcA bound to 4-ethylguaiacol

This is a non-PDB format compatible entry.

Summary for 9IA1

• Entry DOI: 10.2210/pdb9ia1/pdb
• Descriptor: Cytochrome P450, PROTOPORPHYRIN IX CONTAINING FE, 4-ethyl-2-methoxy-phenol, ... (4 entities in total)
• Functional Keywords: lignin, heme, o-demethylation, p450, cytochrome, oxidoreductase
• Biological source: Rhodococcus rhodochrous
• Total number of polymer chains: 2
• Total formula weight: 90512.86

Authors

Hinchen, D.J.,Zahn, M.,Wolf, M.E.,Eltis, L.D.,McGeehan, J.E. (deposition date: 2025-02-07, release date: 2026-07-15)

Primary citation

Wolf, M.E.,Hinchen, D.J.,Zahn, M.,McGeehan, J.E.,Eltis, L.D.
Engineering a Cytochrome P450 O -Demethylase for the Bioconversion of Hardwood Lignin.
Acs Synth Biol, 2026

PubMed Abstract: Lignin is a sustainable alternative to petroleum as a feedstock for the chemical industry. Emergent strategies for lignin valorization involve tandem processes in which biomass is chemo-catalytically fractionated, followed by bioconversion of the depolymerized lignin by microbial cell factories. A rate-limiting step in this bioconversion is -demethylation of the lignin-derived monomers. The reductive catalytic fractionation of hardwood biomass generates high yields of two classes of monomers: 4-alkylguaiacols and 4-alkylsyringols. The former are demethylated by AgcA, a cytochrome P450, and AgcB, the cognate reductase, but there are no known enzymes that convert the latter. To develop a biocatalyst that can efficiently transform these monomers, we studied and rationally engineered AgcAB. A 1.82 Å resolution crystal structure of AgcA from EP4 in complex with 4-ethylguaiacol identified residues Leu78, Ala293, and Phe166 as potential specificity determinants. Substitution of Ala293 and Leu78 decreased the specificity of AgcA for alkylguaiacols. Substitution of Phe166 yielded a variant that bound 4-propylsyringol but did not transform it. In contrast, the corresponding variant in the RHA1 homologue, AgcA Y166A, catalyzed the -demethylation of both methoxy groups of 4-propylsyringol with a of 8500 M s for the first -demethylation, nearly 7-fold higher than WT AgcA. Engineering RHA1 to express the variant yielded a strain that transformed 4-propylsyringol and 4-propylguaiacol simultaneously. Moreover, the engineered strain converted some of the 4-propylsyringol to pentanoyl-CoA, consistent with catabolism via the -cleavage pathway that catabolizes 4-alkylguaiacols. Exometabolomics validated the conversion of 4-propylsyringol via this pathway and identified -demethylation and extradiol ring cleavage as bottlenecks for its transformation. These studies improve our understanding of a critical lignin-degrading enzyme system and significantly advance the development of a biocatalyst to convert these monomers.

PubMed: 42406683
DOI: 10.1021/acssynbio.6c00337

Experimental method

X-RAY DIFFRACTION (1.82 Å)