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

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-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:

Kohler, Amanda C. ^[1]; Mills, Matthew J. L. ^[1]; Adams, Paul D. ^[2]; Simmons, Blake A. ^[3]; Sale, Kenneth L. ^[1]

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+ Show Author Affiliations

1. Joint BioEnergy Institute, Emeryville, CA 94608,, Biomass Science and Conversion Technology Department, Sandia National Laboratories, Livermore, CA 94550,
2. Joint BioEnergy Institute, Emeryville, CA 94608,, Biosciences Area, Lawrence Berkeley National Laboratory, Berkeley, CA 94720,, Department of Bioengineering, University of California, Berkeley, CA 94720
3. Joint BioEnergy Institute, Emeryville, CA 94608,, Biosciences Area, Lawrence Berkeley National Laboratory, Berkeley, CA 94720,

Publication Date:

Mon Apr 03 00:00:00 EDT 2017

Research Org.:

Lawrence Berkeley National Laboratory (LBNL), Berkeley, CA (United States)

Sponsoring Org.:

USDOE Office of Science (SC), Biological and Environmental Research (BER)

OSTI Identifier:

1349696

Alternate Identifier(s):

OSTI ID: 1393223

Grant/Contract Number:  

AC02-05CH11231

Resource Type:

Published Article

Journal Name:

Proceedings of the National Academy of Sciences of the United States of America

Additional Journal Information:

Journal Name: Proceedings of the National Academy of Sciences of the United States of America Journal Volume: 114 Journal Issue: 16; Journal ID: ISSN 0027-8424

Publisher:

Proceedings of the National Academy of Sciences

Country of Publication:

United States

Language:

English

Subject:

59 BASIC BIOLOGICAL SCIENCES; dimethylase; biocatalysis; aryl metabolism; tetrahydrofolate; lignin