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Enabling microbial syringol conversion through structure-guided protein engineering

DOI: 10.1073/pnas.1820001116 DOI Help

Authors: Melodie M. Machovina (Montana State University) , Sam J. B. Mallinson (University of Portsmouth) , Brandon C. Knott (National Renewable Energy Laboratory) , Alexander W. Meyers (National Renewable Energy Laboratory) , Marc Garcia-borrĂ s (University of California) , Lintao Bu (National Renewable Energy Laboratory) , Japheth E. Gado (National Renewable Energy Laboratory; University of Kentucky) , April Oliver (Montana State University) , Graham P. Schmidt (National Renewable Energy Laboratory) , Daniel J. Hinchen (University of Portsmouth) , Michael F. Crowley (National Renewable Energy Laboratory) , Christopher W. Johnson (National Renewable Energy Laboratory) , Ellen L. Neidle (University of Georgia) , Christina M. Payne (University of Kentucky) , Kendall N. Houk (University of California) , Gregg T. Beckham (National Renewable Energy Laboratory; Oak Ridge National Laboratory) , John E. Mcgeehan (University of Portsmouth) , Jennifer L. Dubois (Montana State University)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Proceedings Of The National Academy Of Sciences

State: Published (Approved)
Published: June 2019
Diamond Proposal Number(s): 17212

Open Access Open Access

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 O-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 O-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 Pseudomonas putida KT2440 with the GcoA-F169A variant. Taken together, our findings highlight the significant potential and plasticity of cytochrome P450 aromatic O-demethylases in the biological conversion of lignin-derived aromatic compounds.

Journal Keywords: demethylase; P450; lignin; biorefinery

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: I04-Macromolecular Crystallography

Documents:
1820001116.full.pdf