5TL4

Crystal structure of Sphingomonas paucimobilis aryl O-demethylase LigM

Summary for 5TL4

• Entry DOI: 10.2210/pdb5tl4/pdb
• Descriptor: Vanillate/3-O-methylgallate O-demethylase, CHLORIDE ION, SODIUM ION, ... (6 entities in total)
• Functional Keywords: demethylase, tetrahydrofolate-dependent, aryl, transferase
• Biological source: Sphingomonas paucimobilis
• Total number of polymer chains: 4
• Total formula weight: 211773.03

Authors

Kohler, A.C.,Adams, P.D.,Simmons, B.A.,Sale, K.L. (deposition date: 2016-10-10, release date: 2017-04-12, Last modification date: 2024-03-06)

Primary citation

Kohler, A.C.,Mills, M.J.L.,Adams, P.D.,Simmons, B.A.,Sale, K.L.
Structure of aryl O-demethylase offers molecular insight into a catalytic tyrosine-dependent mechanism.
Proc. Natl. Acad. Sci. U.S.A., 114:E3205-E3214, 2017

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--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 , 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--methylgallate, which are common byproducts of biofuel production. Here, we characterize aryl -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 -demethylation and as a component of synthetic biology efforts to valorize previously underused aromatic compounds.

PubMed: 28373573
DOI: 10.1073/pnas.1619263114

Experimental method

X-RAY DIFFRACTION (1.751 Å)