Enabling microbial syringol conversion through structure-guided protein engineering.
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.(2019) Proc Natl Acad Sci U S A 116: 13970-13976
- PubMed: 31235604 
- DOI: https://doi.org/10.1073/pnas.1820001116
- Primary Citation of Related Structures:  
6HQK, 6HQL, 6HQM, 6HQN, 6HQO, 6HQP, 6HQQ, 6HQR, 6HQS, 6HQT - 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 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.
Organizational Affiliation: 
Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT 59717.