ePrints Repository

The C-Terminal Domain of the Arabinosyltransferase Mycobacterium tuberculosis EmbC Is a Lectin-Like Carbohydrate Binding Module

Alderwick, Luke J. and Lloyd, Georgina S. and Ghadbane, Hemza and May, John W. and Bhatt, Apoorva and Eggeling, Lothar and Fütterer, Klaus and Besra, Gurdyal S. (2011) The C-Terminal Domain of the Arabinosyltransferase Mycobacterium tuberculosis EmbC Is a Lectin-Like Carbohydrate Binding Module. PLoS Pathogens, 7 (2). e1001299. ISSN 1553-7374

Loading
PDF (613Kb)

URL of Published Version: http://dx.doi.org/10.1371/journal.ppat.1001299

Identification Number/DOI: doi:10.1371/journal.ppat.1001299

The D-arabinan-containing polymers arabinogalactan (AG) and lipoarabinomannan (LAM) are essential components of the unique cell envelope of the pathogen Mycobacterium tuberculosis. Biosynthesis of AG and LAM involves a series of membrane-embedded arabinofuranosyl (Araf) transferases whose structures are largely uncharacterised, despite the fact that several of them are pharmacological targets of ethambutol, a frontline drug in tuberculosis therapy. Herein, we present the crystal structure of the C-terminal hydrophilic domain of the ethambutol-sensitive Araf transferase M. tuberculosis EmbC, which is essential for LAM synthesis. The structure of the C-terminal domain of EmbC (EmbCCT) encompasses two sub-domains of different folds, of which subdomain II shows distinct similarity to lectin-like carbohydrate-binding modules (CBM). Co-crystallisation with a cell wall-derived di-arabinoside acceptor analogue and structural comparison with ligand-bound CBMs suggest that EmbCCT contains two separate carbohydrate binding sites, associated with subdomains I and II, respectively. Single-residue substitution of conserved tryptophan residues (Trp868, Trp985) at these respective sites inhibited EmbC-catalysed extension of LAM. The same substitutions differentially abrogated binding of di- and penta-arabinofuranoside acceptor analogues to EmbCCT, linking the loss of activity to compromised acceptor substrate binding, indicating the presence of two separate carbohydrate binding sites, and demonstrating that subdomain II indeed functions as a carbohydrate-binding module. This work provides the first step towards unravelling the structure and function of a GT-C-type glycosyltransferase that is essential in M. tuberculosis. Author Summary Top Tuberculosis (TB), an infectious disease caused by the bacillus Mycobacterium tuberculosis, burdens large swaths of the world population. Treatment of active TB typically requires administration of an antibiotic cocktail over several months that includes the drug ethambutol. This front line compound inhibits a set of arabinosyltransferase enzymes, called EmbA, EmbB and EmbC, which are critical for the synthesis of arabinan, a vital polysaccharide in the pathogen's unique cell envelope. How precisely ethambutol inhibits arabinosyltransferase activity is not clear, in part because structural information of its pharmacological targets has been elusive. Here, we report the high-resolution structure of the C-terminal domain of the ethambutol-target EmbC, a 390-amino acid fragment responsible for acceptor substrate recognition. Combining the X-ray crystallographic analysis with structural comparisons, site-directed mutagenesis, activity and ligand binding assays, we identified two regions in the C-terminal domain of EmbC that are capable of binding acceptor substrate mimics and are critical for activity of the full-length enzyme. Our results begin to define structure-function relationships in a family of structurally uncharacterised membrane-embedded glycosyltransferases, which are an important target for tuberculosis therapy.

Type of Work:Article
Date:24 February 2011 (Publication)
School/Faculty:Colleges (2008 onwards) > College of Life & Environmental Sciences
Department:School of Biosciences
Subjects:QP Physiology
Institution:University of Birmingham
Copyright Holders:Public Library of Science
ID Code:832
Refereed:YES
Local Holdings:
Export Reference As : ASCII + BibTeX + Dublin Core + EndNote + HTML + METS + MODS + OpenURL Object + Reference Manager + Refer + RefWorks
Share this item :
QR Code for this page

Repository Staff Only: item control page