Abstract
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.
Highlights
Tuberculosis (TB) affects large parts of the world’s population, in developing countries [1]
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
Summary
Tuberculosis (TB) affects large parts of the world’s population, in developing countries [1]. INH and EMB inhibit the synthesis of essential components of the mycobacterial cell wall. This unique and highly impermeable barrier surrounds a single phospholipid bilayer membrane and is composed of an outer segment of solvent-extractable lipids, glycans and proteins, and a covalently linked inner segment, known as the mycolyl-arabinogalactan-peptidoglycan (mAGP) core [4]. The mycobacterial cell wall encompasses various membrane-anchored lipoglycans, a group that includes lipoarabinomannan (LAM), which plays a key role in modulating the host immune response [5]. The arabinogalactan (AG) segment of the mAGP core and LAM both contain D-arabinan polymer, composed of a(1R5), a(1R3) and b(1R2)-linked arabinofuranosyl (Araf) residues that are assembled in distinct structural motifs (Fig. 1A) [4,5]
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