期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2017
卷号：114
期号：16
页码：E3205-E3214
DOI：10.1073/pnas.1619263114
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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.
