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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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HldA–inhibitor interactions. Only the nucleotide-bindingsite in protomer A of HldA:M7P:1 is shown, as the inhibitor-bindinginteractions are essentially the same in protomer B of HldA:M7P:1 and both protomers of His6-HldA:1 and HldA:2. Inhibitor 1, M7P, and allof the residues involved are shown with stick models. For clarity,only some of the residues are labeled. All of the hydrogen bonds involvedare indicated by dotted lines.
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fig10: HldA–inhibitor interactions. Only the nucleotide-bindingsite in protomer A of HldA:M7P:1 is shown, as the inhibitor-bindinginteractions are essentially the same in protomer B of HldA:M7P:1 and both protomers of His6-HldA:1 and HldA:2. Inhibitor 1, M7P, and allof the residues involved are shown with stick models. For clarity,only some of the residues are labeled. All of the hydrogen bonds involvedare indicated by dotted lines.

Mentions: Both inhibitors 1 and 2 improve their shape complementarity with the nucleotide-bindingsites of HldA by adopting a folded conformation, with their phenyltriazine,linker, and benzothiazole moieties roughly corresponding to the adenine,the ribose, and the phosphate moieties of AMP·PN, respectively(Figure 9A). However, compared with AMP·PN,these inhibitors penetrate the nucleotide-binding sites more deeply,thereby enhancing their hydrophobic interactions with the protein,which constitute the majority of HldA–inhibitor interactions(Figure 9B). These inhibitors make van derWaals contacts with the main-chain atoms of Ser240, Glu241, Gly243,Gly269, and Gly298 and with the side chains of Asn202, Thr238, Met244,Ala257, Ala259, Val262, Val265, Ala268, Val272, Ala297, and Val301.Only two hydrogen bonds were observed between these inhibitors andHldA. N3 of the benzothiazole moiety forms a hydrogen bond with themain-chain NH group of Ser240, while the NH group of the linker moietyforms a hydrogen bond with the β-amide group of Asn294. Thecarboxylate group of the benzothiazole moiety forms a dipole–dipoleinteraction with the β-amide group of Asn202 (Figure 10). Inhibitor 1 has a methoxyl substituentat phenyl C2 and C6, whereas inhibitor 2 has a chloridesubstituent at the corresponding positions. Nevertheless, no significantdifferences were observed between these inhibitors in their interactionswith HldA. For both inhibitors, one of the substituents protrudesinto the solvent, whereas the other interacts with the benzothiazolemoiety in a lipophilic (for methoxyl) or a chloride−π(for chloride) manner (Figure 9A). Importantly,in protomer A of HldA:M7P:1, no interactions were observedbetween inhibitor 1 and M7P, indicating that the bindingof either ligand does not favor or disfavor that of the other (Figure 10). This strongly suggests that inhibitors 1 and 2 do not use the exclusion of M7P as astrategy for inhibiting HldA. Kinetic studies showed that these inhibitorsare purely ATP-competitive.24


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)

HldA–inhibitor interactions. Only the nucleotide-bindingsite in protomer A of HldA:M7P:1 is shown, as the inhibitor-bindinginteractions are essentially the same in protomer B of HldA:M7P:1 and both protomers of His6-HldA:1 and HldA:2. Inhibitor 1, M7P, and allof the residues involved are shown with stick models. For clarity,only some of the residues are labeled. All of the hydrogen bonds involvedare indicated by dotted lines.
© Copyright Policy
Related In: Results  -  Collection

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

fig10: HldA–inhibitor interactions. Only the nucleotide-bindingsite in protomer A of HldA:M7P:1 is shown, as the inhibitor-bindinginteractions are essentially the same in protomer B of HldA:M7P:1 and both protomers of His6-HldA:1 and HldA:2. Inhibitor 1, M7P, and allof the residues involved are shown with stick models. For clarity,only some of the residues are labeled. All of the hydrogen bonds involvedare indicated by dotted lines.
Mentions: Both inhibitors 1 and 2 improve their shape complementarity with the nucleotide-bindingsites of HldA by adopting a folded conformation, with their phenyltriazine,linker, and benzothiazole moieties roughly corresponding to the adenine,the ribose, and the phosphate moieties of AMP·PN, respectively(Figure 9A). However, compared with AMP·PN,these inhibitors penetrate the nucleotide-binding sites more deeply,thereby enhancing their hydrophobic interactions with the protein,which constitute the majority of HldA–inhibitor interactions(Figure 9B). These inhibitors make van derWaals contacts with the main-chain atoms of Ser240, Glu241, Gly243,Gly269, and Gly298 and with the side chains of Asn202, Thr238, Met244,Ala257, Ala259, Val262, Val265, Ala268, Val272, Ala297, and Val301.Only two hydrogen bonds were observed between these inhibitors andHldA. N3 of the benzothiazole moiety forms a hydrogen bond with themain-chain NH group of Ser240, while the NH group of the linker moietyforms a hydrogen bond with the β-amide group of Asn294. Thecarboxylate group of the benzothiazole moiety forms a dipole–dipoleinteraction with the β-amide group of Asn202 (Figure 10). Inhibitor 1 has a methoxyl substituentat phenyl C2 and C6, whereas inhibitor 2 has a chloridesubstituent at the corresponding positions. Nevertheless, no significantdifferences were observed between these inhibitors in their interactionswith HldA. For both inhibitors, one of the substituents protrudesinto the solvent, whereas the other interacts with the benzothiazolemoiety in a lipophilic (for methoxyl) or a chloride−π(for chloride) manner (Figure 9A). Importantly,in protomer A of HldA:M7P:1, no interactions were observedbetween inhibitor 1 and M7P, indicating that the bindingof either ligand does not favor or disfavor that of the other (Figure 10). This strongly suggests that inhibitors 1 and 2 do not use the exclusion of M7P as astrategy for inhibiting HldA. Kinetic studies showed that these inhibitorsare purely ATP-competitive.24

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