6HQM

Crystal structure of GcoA F169I bound to guaiacol

Summary for 6HQM

• Entry DOI: 10.2210/pdb6hqm/pdb
• Descriptor: Cytochrome P450, PROTOPORPHYRIN IX CONTAINING FE, Guaiacol, ... (4 entities in total)
• Functional Keywords: cytochrome, p450, lignin., oxidoreductase
• Biological source: Amycolatopsis sp. ATCC 39116
• Total number of polymer chains: 1
• Total formula weight: 46159.39

Authors

Mallinson, S.J.B.,Hinchen, D.J.,Allen, M.D.,Johnson, C.W.,Beckham, G.T.,McGeehan, J.E. (deposition date: 2018-09-25, release date: 2019-07-03, Last modification date: 2024-01-24)

Primary citation

Machovina, M.M.,Mallinson, S.J.B.,Knott, B.C.,Meyers, A.W.,Garcia-Borras, M.,Bu, L.,Gado, J.E.,Oliver, A.,Schmidt, G.P.,Hinchen, D.J.,Crowley, M.F.,Johnson, C.W.,Neidle, E.L.,Payne, C.M.,Houk, K.N.,Beckham, G.T.,McGeehan, J.E.,DuBois, J.L.
Enabling microbial syringol conversion through structure-guided protein engineering.
Proc.Natl.Acad.Sci.USA, 116:13970-13976, 2019

PubMed Abstract: Microbial conversion of aromatic compounds is an emerging and promising strategy for valorization of the plant biopolymer lignin. A critical and often rate-limiting reaction in aromatic catabolism is -aryl-demethylation of the abundant aromatic methoxy groups in lignin to form diols, which enables subsequent oxidative aromatic ring-opening. Recently, a cytochrome P450 system, GcoAB, was discovered to demethylate guaiacol (2-methoxyphenol), which can be produced from coniferyl alcohol-derived lignin, to form catechol. However, native GcoAB has minimal ability to demethylate syringol (2,6-dimethoxyphenol), the analogous compound that can be produced from sinapyl alcohol-derived lignin. Despite the abundance of sinapyl alcohol-based lignin in plants, no pathway for syringol catabolism has been reported to date. Here we used structure-guided protein engineering to enable microbial syringol utilization with GcoAB. Specifically, a phenylalanine residue (GcoA-F169) interferes with the binding of syringol in the active site, and on mutation to smaller amino acids, efficient syringol -demethylation is achieved. Crystallography indicates that syringol adopts a productive binding pose in the variant, which molecular dynamics simulations trace to the elimination of steric clash between the highly flexible side chain of GcoA-F169 and the additional methoxy group of syringol. Finally, we demonstrate in vivo syringol turnover in KT2440 with the GcoA-F169A variant. Taken together, our findings highlight the significant potential and plasticity of cytochrome P450 aromatic -demethylases in the biological conversion of lignin-derived aromatic compounds.

PubMed: 31235604
DOI: 10.1073/pnas.1820001116

Experimental method

X-RAY DIFFRACTION (1.85 Å)