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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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Nucleotide-bindingsite. Only the site in protomer A is shown, as the nucleotide-bindinginteractions in protomer B are essentially the same. AMP·PN,M7P, and all of the residues involved are shown with stick models,while the magnesium ion is shown in cyan. For clarity, only some ofthe residues are labeled. All of the hydrogen bonds involved are indicatedby dashed lines.
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fig4: Nucleotide-bindingsite. Only the site in protomer A is shown, as the nucleotide-bindinginteractions in protomer B are essentially the same. AMP·PN,M7P, and all of the residues involved are shown with stick models,while the magnesium ion is shown in cyan. For clarity, only some ofthe residues are labeled. All of the hydrogen bonds involved are indicatedby dashed lines.

Mentions: In both protomers, the nucleotide-binding siteis constituted solely by residues from the α/β core. Theshapes of the outstanding electron densities clearly indicate thatthe adenine ring of the nucleotide is in the syn orientation,making van der Waals contacts with the main-chain atoms of Ser240and Glu241 in the loop connecting β11 to β12 and withthe side chains of Ala257, Ala259, Val262, and Val265 in the loopconnecting β13 to α9 and Val301 at the C-terminus of α10.The ribose ring of the nucleotide is in the C3′-endo conformation, with its 2′-hydroxyl group hydrogen-bondedto the γ-amide group of Asn294 in α10 and with its 3′-hydroxylgroup hydrogen-bonded to the main-chain carbonyl group of Gly243 atthe N-terminus of β12. The negative charges of the phosphategroups of the nucleotide are accommodated by the positive ends ofthe helix dipoles of α7 and α9, with the N-terminus ofα7 in a distance of approximately 6 Å from both the α-and the β-phosphate groups and with the N-terminus of α9in a distance of approximately 3.5 Å from the β-phosphategroup. The α-phosphate group forms hydrogen bonds through itsoxygen atom O1A with the main-chain NH group of Ser240, through O1Aand O2A with the γ-hydroxyl group of Ser240, and through O1Aand O3A with the γ-hydroxyl group of Thr238 at the C-terminusof β11. The β-phosphate group forms hydrogen bonds throughO1B with the γ-amide group of Asn202 at the N-terminus of α7and through N3B with the main-chain NH group of Gly269. Residues Asn202to Glu205 constitute the NXXE motif conserved in many members of thePfkB carbohydrate kinase family. The observed interaction suggeststhat the asparagine of this motif helps to align the phosphate groupsof the nucleotide for the phosphorylation reaction. Interestingly,O2B of the β-phosphate group forms a dative bond with a magnesiumion (Figure 4). The assignment of a magnesiumion instead of a water molecule at this position is strongly supportedby the quasi-octahedral coordination geometry exhibited by the sidechain carboxylate groups of Asp184 at the C-terminus of β9 andGlu205 at the N-terminus of α7, AMP·PN, GMB (in protomerB), and several water molecules in this region. Studies on ribokinasesand adenosine kinases from different species, which belong to thePfkB carbohydrate kinase family as well, have shown that divalentcations (presumably a magnesium ion in vivo) are required for catalysis. Moreover, mutagenesis studies on someother members of this family have shown that the glutamate of theNXXE motif, which corresponds to Glu205 in HldA, is important forthe binding of a magnesium ion in the active site.28 The γ-phosphate group of AMP·PNP could not belocated in either protomer because of the lack of electron densities.


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)

Nucleotide-bindingsite. Only the site in protomer A is shown, as the nucleotide-bindinginteractions in protomer B are essentially the same. AMP·PN,M7P, and all of the residues involved are shown with stick models,while the magnesium ion is shown in cyan. For clarity, only some ofthe residues are labeled. All of the hydrogen bonds involved are indicatedby dashed lines.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Nucleotide-bindingsite. Only the site in protomer A is shown, as the nucleotide-bindinginteractions in protomer B are essentially the same. AMP·PN,M7P, and all of the residues involved are shown with stick models,while the magnesium ion is shown in cyan. For clarity, only some ofthe residues are labeled. All of the hydrogen bonds involved are indicatedby dashed lines.
Mentions: In both protomers, the nucleotide-binding siteis constituted solely by residues from the α/β core. Theshapes of the outstanding electron densities clearly indicate thatthe adenine ring of the nucleotide is in the syn orientation,making van der Waals contacts with the main-chain atoms of Ser240and Glu241 in the loop connecting β11 to β12 and withthe side chains of Ala257, Ala259, Val262, and Val265 in the loopconnecting β13 to α9 and Val301 at the C-terminus of α10.The ribose ring of the nucleotide is in the C3′-endo conformation, with its 2′-hydroxyl group hydrogen-bondedto the γ-amide group of Asn294 in α10 and with its 3′-hydroxylgroup hydrogen-bonded to the main-chain carbonyl group of Gly243 atthe N-terminus of β12. The negative charges of the phosphategroups of the nucleotide are accommodated by the positive ends ofthe helix dipoles of α7 and α9, with the N-terminus ofα7 in a distance of approximately 6 Å from both the α-and the β-phosphate groups and with the N-terminus of α9in a distance of approximately 3.5 Å from the β-phosphategroup. The α-phosphate group forms hydrogen bonds through itsoxygen atom O1A with the main-chain NH group of Ser240, through O1Aand O2A with the γ-hydroxyl group of Ser240, and through O1Aand O3A with the γ-hydroxyl group of Thr238 at the C-terminusof β11. The β-phosphate group forms hydrogen bonds throughO1B with the γ-amide group of Asn202 at the N-terminus of α7and through N3B with the main-chain NH group of Gly269. Residues Asn202to Glu205 constitute the NXXE motif conserved in many members of thePfkB carbohydrate kinase family. The observed interaction suggeststhat the asparagine of this motif helps to align the phosphate groupsof the nucleotide for the phosphorylation reaction. Interestingly,O2B of the β-phosphate group forms a dative bond with a magnesiumion (Figure 4). The assignment of a magnesiumion instead of a water molecule at this position is strongly supportedby the quasi-octahedral coordination geometry exhibited by the sidechain carboxylate groups of Asp184 at the C-terminus of β9 andGlu205 at the N-terminus of α7, AMP·PN, GMB (in protomerB), and several water molecules in this region. Studies on ribokinasesand adenosine kinases from different species, which belong to thePfkB carbohydrate kinase family as well, have shown that divalentcations (presumably a magnesium ion in vivo) are required for catalysis. Moreover, mutagenesis studies on someother members of this family have shown that the glutamate of theNXXE motif, which corresponds to Glu205 in HldA, is important forthe binding of a magnesium ion in the active site.28 The γ-phosphate group of AMP·PNP could not belocated in either protomer because of the lack of electron densities.

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