title: Structure of arylO-demethylase offers molecular insight into a catalytic tyrosine-dependent mechanism.
abstract: Some strains of soil and marine bacteria have evolved intricate metabolic pathways for using environmentally derived aromatics as a carbon source. Many of these metabolic pathways go through intermediates such as vanillate, 3-O-methylgallate, and syringate. Demethylation of these compounds is essential for downstream aryl modification, ring opening, and subsequent assimilation of these compounds into the tricarboxylic acid (TCA) cycle, and, correspondingly, there are a variety of associated aryl demethylase systems that vary in complexity. Intriguingly, only a basic understanding of the least complex system, the tetrahydrofolate-dependent aryl demethylase LigM fromSphingomonas paucimobilis, a bacterial strain that metabolizes lignin-derived aromatics, was previously available. LigM-catalyzed demethylation enables further modification and ring opening of the single-ring aromatics vanillate and 3-O-methylgallate, which are common byproducts of biofuel production. Here, we characterize arylO-demethylation by LigM and report its 1.81-Å crystal structure, revealing a unique demethylase fold and a canonical folate-binding domain. Structural homology and geometry optimization calculations enabled the identification of LigM's tetrahydrofolate-binding site and protein-folate interactions. Computationally guided mutagenesis and kinetic analyses allowed the identification of the enzyme's aryl-binding site location and determination of its unique, catalytic tyrosine-dependent reaction mechanism. This work defines LigM as a distinct demethylase, both structurally and functionally, and provides insight into demethylation and its reaction requirements. These results afford the mechanistic details required for efficient utilization of LigM as a tool for arylO-demethylation and as a component of synthetic biology efforts to valorize previously underused aromatic compounds.
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA 94608.|Biomass Science and Conversion Technology Department, Sandia National Laboratories, Livermore, CA 94550.
authors.forename: Amanda C
authors.initials: AC
authors.lastname: Kohler
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA 94608.|Biomass Science and Conversion Technology Department, Sandia National Laboratories, Livermore, CA 94550.
authors.forename: Matthew J L
authors.initials: MJL
authors.lastname: Mills
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA 94608.|Biosciences Area, Lawrence Berkeley National Laboratory, Berkeley, CA 94720.|Department of Bioengineering, University of California, Berkeley, CA 94720.
authors.forename: Paul D
authors.initials: PD
authors.lastname: Adams
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA 94608.|Biosciences Area, Lawrence Berkeley National Laboratory, Berkeley, CA 94720.
authors.forename: Blake A
authors.initials: BA
authors.lastname: Simmons
authors.affiliation: Joint BioEnergy Institute, Emeryville, CA 94608; klsale@lbl.gov.|Biomass Science and Conversion Technology Department, Sandia National Laboratories, Livermore, CA 94550.
authors.forename: Kenneth L
authors.initials: KL
authors.lastname: Sale
mesh_terms: D002384:Catalysis; D018360:Crystallography, X-Ray; D007700:Kinetics; D053858:Metabolic Networks and Pathways; D010090:Oxidoreductases, O-Demethylating / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D020578:Sphingomonas / Q000201:enzymology*; D014443:Tyrosine / Q000378:metabolism*
chemical_list: D014443:Tyrosine; D010090:Oxidoreductases, O-Demethylating
keywords: aryl metabolism; biocatalysis; demethylase; lignin; tetrahydrofolate