Structure of aryl O -demethylase offers molecular insight into a catalytic tyrosine-dependent mechanism
- Joint BioEnergy Institute, Emeryville, CA 94608,, Biomass Science and Conversion Technology Department, Sandia National Laboratories, Livermore, CA 94550,
- Joint BioEnergy Institute, Emeryville, CA 94608,, Biosciences Area, Lawrence Berkeley National Laboratory, Berkeley, CA 94720,, Department of Bioengineering, University of California, Berkeley, CA 94720
- Joint BioEnergy Institute, Emeryville, CA 94608,, Biosciences Area, Lawrence Berkeley National Laboratory, Berkeley, CA 94720,
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 rin g opening of the single-ring aromatics vanillate and 3-Omethylgallate, which are common byproducts of biofuel production. 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. Our 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.
- Research Organization:
- Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC), Biological and Environmental Research (BER)
- Grant/Contract Number:
- AC02-05CH11231
- OSTI ID:
- 1349696
- Alternate ID(s):
- OSTI ID: 1393223
- Journal Information:
- Proceedings of the National Academy of Sciences of the United States of America, Journal Name: Proceedings of the National Academy of Sciences of the United States of America Vol. 114 Journal Issue: 16; ISSN 0027-8424
- Publisher:
- Proceedings of the National Academy of SciencesCopyright Statement
- Country of Publication:
- United States
- Language:
- English
Web of Science
Similar Records
A promiscuous cytochrome P450 aromatic O-demethylase for lignin bioconversion
Enabling microbial syringol conversion through structure-guided protein engineering