Download MendeleyStructure of aryl O-demethylase offers molecular insight into a catalytic tyrosine-dependent mechanism.
Kohler, A.C., Mills, M.J.L., Adams, P.D., Simmons, B.A., Sale, K.L.(2017) Proc Natl Acad Sci U S A 114: E3205-E3214
- PubMed: 28373573
- DOI: https://doi.org/10.1073/pnas.1619263114
- Primary Citation of Related Structures:
5TL4 - PubMed 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 from Sphingomonas 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 aryl O -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 aryl O -demethylation and as a component of synthetic biology efforts to valorize previously underused aromatic compounds.
Organizational Affiliation:
Joint BioEnergy Institute, Emeryville, CA 94608.
