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Title: Structure of aryl O -demethylase offers molecular insight into a catalytic tyrosine-dependent mechanism

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, andmore » 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.« less
Authors:
 [1] ;  [1] ;  [2] ;  [3] ;  [1]
  1. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Sandia National Lab. (SNL-CA), Livermore, CA (United States). Biomass Science and Conversion Technology Dept.
  2. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biosciences Area; Univ. of California, Berkeley, CA (United States). Dept. of Bioengineering
  3. Joint BioEnergy Inst. (JBEI), Emeryville, CA (United States); Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States). Biosciences Area
Publication Date:
Grant/Contract Number:
AC02-05CH11231
Type:
Published Article
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 114; Journal Issue: 16; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences, Washington, DC (United States)
Research Org:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; dimethylase; biocatalysis; aryl metabolism; tetrahydrofolate; lignin
OSTI Identifier:
1349696
Alternate Identifier(s):
OSTI ID: 1393223

Kohler, Amanda C., Mills, Matthew J. L., Adams, Paul D., Simmons, Blake A., and Sale, Kenneth L.. Structure of aryl O -demethylase offers molecular insight into a catalytic tyrosine-dependent mechanism. United States: N. p., Web. doi:10.1073/pnas.1619263114.
Kohler, Amanda C., Mills, Matthew J. L., Adams, Paul D., Simmons, Blake A., & Sale, Kenneth L.. Structure of aryl O -demethylase offers molecular insight into a catalytic tyrosine-dependent mechanism. United States. doi:10.1073/pnas.1619263114.
Kohler, Amanda C., Mills, Matthew J. L., Adams, Paul D., Simmons, Blake A., and Sale, Kenneth L.. 2017. "Structure of aryl O -demethylase offers molecular insight into a catalytic tyrosine-dependent mechanism". United States. doi:10.1073/pnas.1619263114.
@article{osti_1349696,
title = {Structure of aryl O -demethylase offers molecular insight into a catalytic tyrosine-dependent mechanism},
author = {Kohler, Amanda C. and Mills, Matthew J. L. and Adams, Paul D. and Simmons, Blake A. and Sale, Kenneth L.},
abstractNote = {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.},
doi = {10.1073/pnas.1619263114},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
number = 16,
volume = 114,
place = {United States},
year = {2017},
month = {4}
}

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