Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 114, Issue 16, Pages E3205-E3214Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1619263114
Keywords
demethylase; biocatalysis; aryl metabolism; tetrahydrofolate; lignin
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Funding
- National Institutes of Health
- National Institute of General Medical Sciences
- Howard Hughes Medical Institute
- Office of Science, Office of Basic Energy Sciences, of the US Department of Energy [DE-AC02-05CH11231]
- US Department of Energy, Office of Science and Office of Biological and Environmental Research [DE-AC02-05CH11231]
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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-angstrom 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.
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