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Structural-functional studies of Burkholderia cenocepacia D-glycero-β-D-manno-heptose 7-phosphate kinase (HldA) and characterization of inhibitors with antibiotic adjuvant and antivirulence properties.

Lee TW, Verhey TB, Antiperovitch PA, Atamanyuk D, Desroy N, Oliveira C, Denis A, Gerusz V, Drocourt E, Loutet SA, Hamad MA, Stanetty C, Andres SN, Sugiman-Marangos S, Kosma P, Valvano MA, Moreau F, Junop MS - J. Med. Chem. (2013)

Bottom Line: HldA is structurally similar to members of the PfkB carbohydrate kinase family and appears to catalyze heptose phosphorylation via an in-line mechanism mediated mainly by a conserved aspartate, Asp270.Moreover, we report the structures of HldA in complex with two potent inhibitors in which both inhibitors adopt a folded conformation and occupy the nucleotide-binding sites.Together, these results provide important insight into the mechanism of HldA-catalyzed heptose phosphorylation and necessary information for further development of HldA inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biomedical Sciences and Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, 1280 Main Street West, Hamilton, Ontario, L8S 4K1, Canada.

ABSTRACT
As an essential constituent of the outer membrane of Gram-negative bacteria, lipopolysaccharide contributes significantly to virulence and antibiotic resistance. The lipopolysaccharide biosynthetic pathway therefore serves as a promising therapeutic target for antivirulence drugs and antibiotic adjuvants. Here we report the structural-functional studies of D-glycero-β-D-manno-heptose 7-phosphate kinase (HldA), an absolutely conserved enzyme in this pathway, from Burkholderia cenocepacia. HldA is structurally similar to members of the PfkB carbohydrate kinase family and appears to catalyze heptose phosphorylation via an in-line mechanism mediated mainly by a conserved aspartate, Asp270. Moreover, we report the structures of HldA in complex with two potent inhibitors in which both inhibitors adopt a folded conformation and occupy the nucleotide-binding sites. Together, these results provide important insight into the mechanism of HldA-catalyzed heptose phosphorylation and necessary information for further development of HldA inhibitors.

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Lipopolysaccharide (LPS). (A) Schematic diagram of thestructure of LPS. (B) Biosynthesis of ADP-l-glycero-β-d-manno-heptose, a precursor for the biosynthesis of the innercore region of LPS, from d-sedoheptulose 7-phosphate. Thephosphorylation reaction converting d-glycero-β-d-manno-heptose-7-phosphate (M7P) to d-glycero-β-d-manno-heptose-1,7-bisphosphate (GMB) is catalyzed by HldA.(C) Two potent inhibitors of HldA. IC50 values againstHldA are the following: 0.81 μM for 1; 0.23 μMfor 2.
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fig1: Lipopolysaccharide (LPS). (A) Schematic diagram of thestructure of LPS. (B) Biosynthesis of ADP-l-glycero-β-d-manno-heptose, a precursor for the biosynthesis of the innercore region of LPS, from d-sedoheptulose 7-phosphate. Thephosphorylation reaction converting d-glycero-β-d-manno-heptose-7-phosphate (M7P) to d-glycero-β-d-manno-heptose-1,7-bisphosphate (GMB) is catalyzed by HldA.(C) Two potent inhibitors of HldA. IC50 values againstHldA are the following: 0.81 μM for 1; 0.23 μMfor 2.

Mentions: The defining characteristic of Gram-negativebacteria is the presence of an outer membrane, an asymmetric lipidbilayer whose outer leaflet is mainly composed of lipopolysaccharide(LPS). LPS forms a dense mesh and helps to anchor proteins in theouter membrane. On one hand, this maintains the structural integrityof the outer membrane, which is essential for adhesion to host cellsand interaction with host immune effectors including serum components.This implicates LPS as a key virulence factor.7 On the other hand, the dense hydrophilic mesh of LPS renders theouter membrane impermeable to hydrophobic molecules, which includemany currently available antibiotics such as macrolides.8 LPS comprises lipid A, a core oligosaccharide(OS) and, in some bacterial species, an O-antigen polysaccharide chain.The core OS can be further divided into an inner core region consistingof 2-keto-3-deoxy-d-manno-octosonic acid (KDO) and heptose(prevalently l-α-d-manno-heptose) residuesand an outer core region consisting mostly of hexose residues (Figure 1A).9,10 Lipid A and KDO are indispensablefor cell viability and conserved in virtually all Gram-negative bacteria.11 Their biosynthetic pathways have become targetsfor the development of traditional antibiotics.12,13 Interestingly, mutant cells with minimal LPS (i.e., LPS comprisinglipid A and KDO only) are viable but display the so-called deep-roughphenotype. In particular, they exhibit increased sensitivity to hydrophobicmolecules owing to the lack of negatively charged groups in the truncatedLPS for cross-linking by divalent cations, which disfavors the formationof a dense mesh in the outer membrane. In most species, these negativelycharged groups are contributed by phosphorylated heptose residues.11


Structural-functional studies of Burkholderia cenocepacia D-glycero-β-D-manno-heptose 7-phosphate kinase (HldA) and characterization of inhibitors with antibiotic adjuvant and antivirulence properties.

Lee TW, Verhey TB, Antiperovitch PA, Atamanyuk D, Desroy N, Oliveira C, Denis A, Gerusz V, Drocourt E, Loutet SA, Hamad MA, Stanetty C, Andres SN, Sugiman-Marangos S, Kosma P, Valvano MA, Moreau F, Junop MS - J. Med. Chem. (2013)

Lipopolysaccharide (LPS). (A) Schematic diagram of thestructure of LPS. (B) Biosynthesis of ADP-l-glycero-β-d-manno-heptose, a precursor for the biosynthesis of the innercore region of LPS, from d-sedoheptulose 7-phosphate. Thephosphorylation reaction converting d-glycero-β-d-manno-heptose-7-phosphate (M7P) to d-glycero-β-d-manno-heptose-1,7-bisphosphate (GMB) is catalyzed by HldA.(C) Two potent inhibitors of HldA. IC50 values againstHldA are the following: 0.81 μM for 1; 0.23 μMfor 2.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3585733&req=5

fig1: Lipopolysaccharide (LPS). (A) Schematic diagram of thestructure of LPS. (B) Biosynthesis of ADP-l-glycero-β-d-manno-heptose, a precursor for the biosynthesis of the innercore region of LPS, from d-sedoheptulose 7-phosphate. Thephosphorylation reaction converting d-glycero-β-d-manno-heptose-7-phosphate (M7P) to d-glycero-β-d-manno-heptose-1,7-bisphosphate (GMB) is catalyzed by HldA.(C) Two potent inhibitors of HldA. IC50 values againstHldA are the following: 0.81 μM for 1; 0.23 μMfor 2.
Mentions: The defining characteristic of Gram-negativebacteria is the presence of an outer membrane, an asymmetric lipidbilayer whose outer leaflet is mainly composed of lipopolysaccharide(LPS). LPS forms a dense mesh and helps to anchor proteins in theouter membrane. On one hand, this maintains the structural integrityof the outer membrane, which is essential for adhesion to host cellsand interaction with host immune effectors including serum components.This implicates LPS as a key virulence factor.7 On the other hand, the dense hydrophilic mesh of LPS renders theouter membrane impermeable to hydrophobic molecules, which includemany currently available antibiotics such as macrolides.8 LPS comprises lipid A, a core oligosaccharide(OS) and, in some bacterial species, an O-antigen polysaccharide chain.The core OS can be further divided into an inner core region consistingof 2-keto-3-deoxy-d-manno-octosonic acid (KDO) and heptose(prevalently l-α-d-manno-heptose) residuesand an outer core region consisting mostly of hexose residues (Figure 1A).9,10 Lipid A and KDO are indispensablefor cell viability and conserved in virtually all Gram-negative bacteria.11 Their biosynthetic pathways have become targetsfor the development of traditional antibiotics.12,13 Interestingly, mutant cells with minimal LPS (i.e., LPS comprisinglipid A and KDO only) are viable but display the so-called deep-roughphenotype. In particular, they exhibit increased sensitivity to hydrophobicmolecules owing to the lack of negatively charged groups in the truncatedLPS for cross-linking by divalent cations, which disfavors the formationof a dense mesh in the outer membrane. In most species, these negativelycharged groups are contributed by phosphorylated heptose residues.11

Bottom Line: HldA is structurally similar to members of the PfkB carbohydrate kinase family and appears to catalyze heptose phosphorylation via an in-line mechanism mediated mainly by a conserved aspartate, Asp270.Moreover, we report the structures of HldA in complex with two potent inhibitors in which both inhibitors adopt a folded conformation and occupy the nucleotide-binding sites.Together, these results provide important insight into the mechanism of HldA-catalyzed heptose phosphorylation and necessary information for further development of HldA inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biomedical Sciences and Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, 1280 Main Street West, Hamilton, Ontario, L8S 4K1, Canada.

ABSTRACT
As an essential constituent of the outer membrane of Gram-negative bacteria, lipopolysaccharide contributes significantly to virulence and antibiotic resistance. The lipopolysaccharide biosynthetic pathway therefore serves as a promising therapeutic target for antivirulence drugs and antibiotic adjuvants. Here we report the structural-functional studies of D-glycero-β-D-manno-heptose 7-phosphate kinase (HldA), an absolutely conserved enzyme in this pathway, from Burkholderia cenocepacia. HldA is structurally similar to members of the PfkB carbohydrate kinase family and appears to catalyze heptose phosphorylation via an in-line mechanism mediated mainly by a conserved aspartate, Asp270. Moreover, we report the structures of HldA in complex with two potent inhibitors in which both inhibitors adopt a folded conformation and occupy the nucleotide-binding sites. Together, these results provide important insight into the mechanism of HldA-catalyzed heptose phosphorylation and necessary information for further development of HldA inhibitors.

Show MeSH