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The C-terminal domain of the Arabinosyltransferase Mycobacterium tuberculosis EmbC is a lectin-like carbohydrate binding module.

Alderwick LJ, Lloyd GS, Ghadbane H, May JW, Bhatt A, Eggeling L, Fütterer K, Besra GS - PLoS Pathog. (2011)

Bottom Line: Co-crystallisation with a cell wall-derived di-arabinoside acceptor analogue and structural comparison with ligand-bound CBMs suggest that EmbC(CT) 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 EmbC(CT), 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.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom.

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 (EmbC(CT)) 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 EmbC(CT) 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 EmbC(CT), 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.

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Related in: MedlinePlus

Schematic diagram of LAM synthesis and architecture of M. tuberculosis EmbC.A) Schematic representation of the stepwise assembly of LAM at the membrane of mycobacteria. The precursors of LAM are phosphatidylinositol mannosides (PIM), which contain a phosphatidyl-myo-inositol core unit. Initially, intracellular α-mannosyltransferases catalyse attachment of mannosyl units to inositol, followed by flipping of the glycolipid to the extracellular face of the membrane and further chain extension by membrane-embedded mannosyl- and arabinofuranosyl transferases to generate lipomannan (LM), lipoarabinomannan (LAM) and mannan-capped LAM (ManLAM). Relevant saccharide donor substrates are as follows: GDP-Man (guanosine-5′-diphosphate-α-D-mannose), PPM (C35/C50-polyprenyl-monophospho-mannose), DPA (β-D-arabinofuranosyl-1-monophosphoryl-decaprenol). ManT and AraT designate mannosyl- and arbinosyltransferases that are as yet uncharacterised. B) Topology diagram of EmbC based on the hydropathy analysis with TMHMM (www.cbs.dtu.dk/services/TMHMM/). Extracellular loops are labelled E1-E6 and CT, intracellular loops I1–I7. Functionally important sequence motifs, previously identified in references [10], [15], are indicated. The C-terminal domain (residues 719–1094) is shown as a ribbon diagram.
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ppat-1001299-g001: Schematic diagram of LAM synthesis and architecture of M. tuberculosis EmbC.A) Schematic representation of the stepwise assembly of LAM at the membrane of mycobacteria. The precursors of LAM are phosphatidylinositol mannosides (PIM), which contain a phosphatidyl-myo-inositol core unit. Initially, intracellular α-mannosyltransferases catalyse attachment of mannosyl units to inositol, followed by flipping of the glycolipid to the extracellular face of the membrane and further chain extension by membrane-embedded mannosyl- and arabinofuranosyl transferases to generate lipomannan (LM), lipoarabinomannan (LAM) and mannan-capped LAM (ManLAM). Relevant saccharide donor substrates are as follows: GDP-Man (guanosine-5′-diphosphate-α-D-mannose), PPM (C35/C50-polyprenyl-monophospho-mannose), DPA (β-D-arabinofuranosyl-1-monophosphoryl-decaprenol). ManT and AraT designate mannosyl- and arbinosyltransferases that are as yet uncharacterised. B) Topology diagram of EmbC based on the hydropathy analysis with TMHMM (www.cbs.dtu.dk/services/TMHMM/). Extracellular loops are labelled E1-E6 and CT, intracellular loops I1–I7. Functionally important sequence motifs, previously identified in references [10], [15], are indicated. The C-terminal domain (residues 719–1094) is shown as a ribbon diagram.

Mentions: Tuberculosis (TB) affects large parts of the world's population, particularly in developing countries [1]. The antibiotics isoniazid (INH) and ethambutol (EMB) [2] have been used for decades as frontline drugs to treat Mycobacterium tuberculosis infections, the causative agent of TB, but the rise of multi-drug resistant (MDR) and extensively drug resistant (XDR) strains poses a serious threat to present treatment options [3]. Both, 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]. Perturbations to the mAGP core tend to undermine viability of M. tuberculosis, a major reason why mAGP biosynthesis constitutes an attractive target for drug design efforts. The mycobacterial cell wall also 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 α(1→5), α(1→3) and β(1→2)-linked arabinofuranosyl (Araf) residues that are assembled in distinct structural motifs (Fig. 1A) [4], [5].


The C-terminal domain of the Arabinosyltransferase Mycobacterium tuberculosis EmbC is a lectin-like carbohydrate binding module.

Alderwick LJ, Lloyd GS, Ghadbane H, May JW, Bhatt A, Eggeling L, Fütterer K, Besra GS - PLoS Pathog. (2011)

Schematic diagram of LAM synthesis and architecture of M. tuberculosis EmbC.A) Schematic representation of the stepwise assembly of LAM at the membrane of mycobacteria. The precursors of LAM are phosphatidylinositol mannosides (PIM), which contain a phosphatidyl-myo-inositol core unit. Initially, intracellular α-mannosyltransferases catalyse attachment of mannosyl units to inositol, followed by flipping of the glycolipid to the extracellular face of the membrane and further chain extension by membrane-embedded mannosyl- and arabinofuranosyl transferases to generate lipomannan (LM), lipoarabinomannan (LAM) and mannan-capped LAM (ManLAM). Relevant saccharide donor substrates are as follows: GDP-Man (guanosine-5′-diphosphate-α-D-mannose), PPM (C35/C50-polyprenyl-monophospho-mannose), DPA (β-D-arabinofuranosyl-1-monophosphoryl-decaprenol). ManT and AraT designate mannosyl- and arbinosyltransferases that are as yet uncharacterised. B) Topology diagram of EmbC based on the hydropathy analysis with TMHMM (www.cbs.dtu.dk/services/TMHMM/). Extracellular loops are labelled E1-E6 and CT, intracellular loops I1–I7. Functionally important sequence motifs, previously identified in references [10], [15], are indicated. The C-terminal domain (residues 719–1094) is shown as a ribbon diagram.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3044687&req=5

ppat-1001299-g001: Schematic diagram of LAM synthesis and architecture of M. tuberculosis EmbC.A) Schematic representation of the stepwise assembly of LAM at the membrane of mycobacteria. The precursors of LAM are phosphatidylinositol mannosides (PIM), which contain a phosphatidyl-myo-inositol core unit. Initially, intracellular α-mannosyltransferases catalyse attachment of mannosyl units to inositol, followed by flipping of the glycolipid to the extracellular face of the membrane and further chain extension by membrane-embedded mannosyl- and arabinofuranosyl transferases to generate lipomannan (LM), lipoarabinomannan (LAM) and mannan-capped LAM (ManLAM). Relevant saccharide donor substrates are as follows: GDP-Man (guanosine-5′-diphosphate-α-D-mannose), PPM (C35/C50-polyprenyl-monophospho-mannose), DPA (β-D-arabinofuranosyl-1-monophosphoryl-decaprenol). ManT and AraT designate mannosyl- and arbinosyltransferases that are as yet uncharacterised. B) Topology diagram of EmbC based on the hydropathy analysis with TMHMM (www.cbs.dtu.dk/services/TMHMM/). Extracellular loops are labelled E1-E6 and CT, intracellular loops I1–I7. Functionally important sequence motifs, previously identified in references [10], [15], are indicated. The C-terminal domain (residues 719–1094) is shown as a ribbon diagram.
Mentions: Tuberculosis (TB) affects large parts of the world's population, particularly in developing countries [1]. The antibiotics isoniazid (INH) and ethambutol (EMB) [2] have been used for decades as frontline drugs to treat Mycobacterium tuberculosis infections, the causative agent of TB, but the rise of multi-drug resistant (MDR) and extensively drug resistant (XDR) strains poses a serious threat to present treatment options [3]. Both, 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]. Perturbations to the mAGP core tend to undermine viability of M. tuberculosis, a major reason why mAGP biosynthesis constitutes an attractive target for drug design efforts. The mycobacterial cell wall also 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 α(1→5), α(1→3) and β(1→2)-linked arabinofuranosyl (Araf) residues that are assembled in distinct structural motifs (Fig. 1A) [4], [5].

Bottom Line: Co-crystallisation with a cell wall-derived di-arabinoside acceptor analogue and structural comparison with ligand-bound CBMs suggest that EmbC(CT) 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 EmbC(CT), 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.

View Article: PubMed Central - PubMed

Affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom.

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 (EmbC(CT)) 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 EmbC(CT) 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 EmbC(CT), 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.

Show MeSH
Related in: MedlinePlus