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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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Catalytic mechanism ofHldA. (A) On the basis of the positions of the two water molecules(shown in red and labeled W) located between AMP·PN and M7P (bothshown with stick models) in protomer A, an additional phosphate group(circled in red) was modeled onto AMP·PN. The head-on alignmentof the γ-phosphate group of AMP·PNP with the 1-hydroxylgroup of M7P is indicated by a dotted line. Residues of the GXGD signaturemotif are shown with stick models as well, while the magnesium ionis shown in cyan. (B) In protomer B, the 1-phosphate group of GMBforms hydrogen bonds with the main-chain NH groups of residues ofthe GXGD signature motif and an ionic interaction with the magnesiumion (all indicated by dashed lines). (C) Proposed catalytic mechanismof HldA.
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fig6: Catalytic mechanism ofHldA. (A) On the basis of the positions of the two water molecules(shown in red and labeled W) located between AMP·PN and M7P (bothshown with stick models) in protomer A, an additional phosphate group(circled in red) was modeled onto AMP·PN. The head-on alignmentof the γ-phosphate group of AMP·PNP with the 1-hydroxylgroup of M7P is indicated by a dotted line. Residues of the GXGD signaturemotif are shown with stick models as well, while the magnesium ionis shown in cyan. (B) In protomer B, the 1-phosphate group of GMBforms hydrogen bonds with the main-chain NH groups of residues ofthe GXGD signature motif and an ionic interaction with the magnesiumion (all indicated by dashed lines). (C) Proposed catalytic mechanismof HldA.

Mentions: Interestingly, in protomer A, two water moleculeswere located between the β-phosphate group of AMP·PN andthe 1-hydroxyl group of M7P. It is possible that the electron densitiesof these two water molecules actually belong to two of the oxygenatoms of the dynamically disordered γ-phosphate group of AMP·PNP.On the basis of the positions of these two water molecules, the γ-phosphategroup was modeled. The phosphorus atom of the γ-phosphate groupof AMP·PNP and the oxygen atom of the 1-hydroxyl group of M7Pare aligned head-on at a distance of 3.1 Å (Figure 6A). In protomer B, the 1-phosphate group of GMB forms hydrogenbonds through OP1 with the main-chain NH groups of Gly269B and Asp270Band through OP2 with the main-chain NH group of Gly267. OP3 formsa dative bond with the magnesium ion. The distance between N3B ofthe β-phosphate group of AMP·PN and the phosphorus atomof the 1-phosphate group of GMB is 4.8 Å (Figure 6B). These observations together strongly indicate the feasibilityof an in-line mechanism, which has been proposed for other membersof the PfkB carbohydrate kinase family, being adopted by the HldA-catalyzedphosphorylation. Asp270 of the GXGD signature motif acts as a catalyticbase, with its γ-carboxylate group deprotonating the 1-hydroxylgroup of M7P. The deprotonatation of the 1-hydroxyl group of M7P favorsits oxygen atom performing a nucleophilic attack at the phosphorusatom of the γ-phosphate group of ATP. The GXGD signature motifforms an anion hole at the N-terminus of α9, in which the positiveend of the helix dipole of α9 and the hydrogen bonds donatedby the main-chain NH groups of the GXGD signature motif, togetherwith the magnesium ion, help to accommodate the additional negativecharge in the γ-phosphate group of ATP during the nucleophilicattack, thereby lowering the energy of the transition state. The nucleophilicattack results in the formation of a phosphoester bond between the1-hydroxyl group of M7P and the γ-phosphate group of ATP andin the dissociation of the phosphodiester bond between the β-and the γ-phosphate groups of ATP, thereby producing GMB andADP (Figure 6C).


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)

Catalytic mechanism ofHldA. (A) On the basis of the positions of the two water molecules(shown in red and labeled W) located between AMP·PN and M7P (bothshown with stick models) in protomer A, an additional phosphate group(circled in red) was modeled onto AMP·PN. The head-on alignmentof the γ-phosphate group of AMP·PNP with the 1-hydroxylgroup of M7P is indicated by a dotted line. Residues of the GXGD signaturemotif are shown with stick models as well, while the magnesium ionis shown in cyan. (B) In protomer B, the 1-phosphate group of GMBforms hydrogen bonds with the main-chain NH groups of residues ofthe GXGD signature motif and an ionic interaction with the magnesiumion (all indicated by dashed lines). (C) Proposed catalytic mechanismof HldA.
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Related In: Results  -  Collection

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Show All Figures
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fig6: Catalytic mechanism ofHldA. (A) On the basis of the positions of the two water molecules(shown in red and labeled W) located between AMP·PN and M7P (bothshown with stick models) in protomer A, an additional phosphate group(circled in red) was modeled onto AMP·PN. The head-on alignmentof the γ-phosphate group of AMP·PNP with the 1-hydroxylgroup of M7P is indicated by a dotted line. Residues of the GXGD signaturemotif are shown with stick models as well, while the magnesium ionis shown in cyan. (B) In protomer B, the 1-phosphate group of GMBforms hydrogen bonds with the main-chain NH groups of residues ofthe GXGD signature motif and an ionic interaction with the magnesiumion (all indicated by dashed lines). (C) Proposed catalytic mechanismof HldA.
Mentions: Interestingly, in protomer A, two water moleculeswere located between the β-phosphate group of AMP·PN andthe 1-hydroxyl group of M7P. It is possible that the electron densitiesof these two water molecules actually belong to two of the oxygenatoms of the dynamically disordered γ-phosphate group of AMP·PNP.On the basis of the positions of these two water molecules, the γ-phosphategroup was modeled. The phosphorus atom of the γ-phosphate groupof AMP·PNP and the oxygen atom of the 1-hydroxyl group of M7Pare aligned head-on at a distance of 3.1 Å (Figure 6A). In protomer B, the 1-phosphate group of GMB forms hydrogenbonds through OP1 with the main-chain NH groups of Gly269B and Asp270Band through OP2 with the main-chain NH group of Gly267. OP3 formsa dative bond with the magnesium ion. The distance between N3B ofthe β-phosphate group of AMP·PN and the phosphorus atomof the 1-phosphate group of GMB is 4.8 Å (Figure 6B). These observations together strongly indicate the feasibilityof an in-line mechanism, which has been proposed for other membersof the PfkB carbohydrate kinase family, being adopted by the HldA-catalyzedphosphorylation. Asp270 of the GXGD signature motif acts as a catalyticbase, with its γ-carboxylate group deprotonating the 1-hydroxylgroup of M7P. The deprotonatation of the 1-hydroxyl group of M7P favorsits oxygen atom performing a nucleophilic attack at the phosphorusatom of the γ-phosphate group of ATP. The GXGD signature motifforms an anion hole at the N-terminus of α9, in which the positiveend of the helix dipole of α9 and the hydrogen bonds donatedby the main-chain NH groups of the GXGD signature motif, togetherwith the magnesium ion, help to accommodate the additional negativecharge in the γ-phosphate group of ATP during the nucleophilicattack, thereby lowering the energy of the transition state. The nucleophilicattack results in the formation of a phosphoester bond between the1-hydroxyl group of M7P and the γ-phosphate group of ATP andin the dissociation of the phosphodiester bond between the β-and the γ-phosphate groups of ATP, thereby producing GMB andADP (Figure 6C).

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