Structure of the Escherichia coli Heptosyltransferase WaaC: Binary Complexes with ADP AND ADP-2-deoxy-2-fluoro Heptose

Grizot, Sylvestre; Salem, M.; Vongsouthi, Vanida; Durand, Lionel; Moreau, F.; Dohi, Hirofumi; Vincent, Stéphane; Escaich, Sonia; Ducruix, A.

doi:10.1016/j.jmb.2006.07.057

Cited by 91 publications

(164 citation statements)

References 41 publications

(43 reference statements)

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“…An extended C-terminal ␣-helix of MurG (Fig. 1B, drawn in blue) has been proposed to function as a connector of the two domains, and in the formation of the two Rossmann folds architecture (21). The Alg13/14 complex shares much of this conserved architecture but lacks the linker peptide to connect the subunits (Fig.…”

Section: Discussionmentioning

confidence: 99%

Interaction between the C Termini of Alg13 and Alg14 Mediates Formation of the Active UDP-N-acetylglucosamine Transferase Complex

Gao

Moriyama

Miura

et al. 2008

Journal of Biological Chemistry

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The second step of eukaryotic N-linked glycosylation in endoplasmic reticulum is catalyzed by an UDP-N-acetylglucosamine transferase that is comprised of two subunits, Alg13 and Alg14. The interaction between Alg13 and 14 is crucial for UDP-GlcNAc transferase activity, so formation of the Alg13/14 complex is likely to play a key role in the regulation of N-glycosylation. Using a combination of bioinformatics and molecular biological methods, we have undertaken a functional analysis of yeast Alg13 and Alg14 proteins to elucidate the mechanism of their interaction. Our mutational studies demonstrated that a short C-terminal ␣-helix of Alg13 is required for interaction with Alg14 and for enzyme activity. Electrostatic surface views of the modeled Alg13/14 complex suggest the presence of a hydrophobic cleft in Alg14 that provides a pocket for the Alg13 C-terminal ␣-helix. Co-immunoprecipitation assays confirmed the C-terminal three amino acids of Alg14 are required for maintaining the integrity of Alg13/Alg14 complex, and this depends on their hydrophobicity. Modeling studies place these three Alg14 residues at the entrance of the hydrophobic-binding pocket, suggesting their role in the stabilization of the interaction between the C termini of Alg13 and Alg14. Together, these results demonstrate that formation of this hetero-oligomeric complex is mediated by a short C-terminal ␣-helix of Alg13 in cooperation with the last three amino acids of Alg14. In addition, deletion of the N-terminal ␤-strand of Alg13 caused the destruction of protein, indicating the structural importance of this region in protein stability.

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Section: Discussionmentioning

confidence: 99%

Interaction between the C Termini of Alg13 and Alg14 Mediates Formation of the Active UDP-N-acetylglucosamine Transferase Complex

Gao

Moriyama

Miura

et al. 2008

Journal of Biological Chemistry

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“…8B). Amino acid Thr-187 is located within the sugar-nucleotide binding site of WaaC (46). In the crystal structure of the WaaC-ADP complex, the ␤-phosphate interacts with Thr-187, Thr-188, and Lys-192.…”

Section: Lapb Is Essential For Bacterial Growth Inmentioning

confidence: 99%

Assembly of Lipopolysaccharide in Escherichia coli Requires the Essential LapB Heat Shock Protein

Klein

Kobylak

Lindner

et al. 2014

Journal of Biological Chemistry

255

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Background:The mechanism of coordination between LPS synthesis and translocation is unknown. Results: Two new proteins, LapA and LapB, co-purify with LPS transport proteins. lapB mutants display defects in lipid A and core assembly. Conclusion: lapB mutants accumulate precursor LPS core species and exhibit elevated levels of LpxC. Significance: Coordinated assembly of LPS is a critical step for targeting to the outer membrane.

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“…To date, the structures of six GTs in complex with NDP-2F-sugars have been resolved. [11,12] It is worth noting that the co-crystallization of glycosyltransferases with NDP-sugar analogues revealed substrate-induced conformational changes of the protein. These results are extremely important, since they provide the binding mode and the conformation of the donor within the catalytic pocket, which can facilitate the design of inhibitors and the understanding of the mechanism.…”

Section: Introductionmentioning

confidence: 99%

“…Moreover, we have recently reported the crystallographic structure of heptosyltransferase WaaC in complex with 2. [12] Synthetically, the key step for the preparation of 2 is the simultaneous and stereoselective installation of both fluorine atom at C-2 and the phosphoryl group at C-1 through a selectfluor-mediated…”

Section: Introductionmentioning

confidence: 99%

Stereoselective Glycal Fluorophosphorylation: Synthesis of ADP‐2‐fluoroheptose, an Inhibitor of the LPS Biosynthesis

Dohi

Périon

Durka

et al. 2008

Chemistry A European J

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Heptosides are found in important bacterial glycolipids such as lipopolysaccharide (LPS), the biosynthesis of which is targeted for the development of novel antibacterial agents. This work describes the synthesis of a fluorinated analogue of ADP‐L‐glycero‐β‐D‐manno‐heptopyranose, the donor substrate of the heptosyl transferase WaaC, which catalyzes the incorporation of this carbohydrate into LPS. Synthetically, the key step for the preparation of ADP‐2F‐heptose is the simultaneous and stereoselective installation of both the fluorine atom at C‐2 and the phosphoryl group at C‐1 through a selectfluor‐mediated (selectfluor=1‐chloromethyl‐4‐fluorodiazoniabicyclo[2.2.2]octane bis(triflate)) electrophilic addition/nucleophilic substitution involving a heptosylglycal. Therefore, we detail in this article 1) the stereoselective preparation of the key intermediates heptosylglycals, 2) the development of a new fluorophosphorylation procedure allowing an excellent β‐gluco stereoselectivity with “all‐equatorial” glycals, 3) the synthesis of the target ADP‐2F‐heptose, and 4) some comments on the contacts observed between the fluorine atom of the final molecule and the protein in the crystallographic structure of heptosyltransferase WaaC.

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Structure of the Escherichia coli Heptosyltransferase WaaC: Binary Complexes with ADP AND ADP-2-deoxy-2-fluoro Heptose

Cited by 91 publications

References 41 publications

Interaction between the C Termini of Alg13 and Alg14 Mediates Formation of the Active UDP-N-acetylglucosamine Transferase Complex

Interaction between the C Termini of Alg13 and Alg14 Mediates Formation of the Active UDP-N-acetylglucosamine Transferase Complex

Assembly of Lipopolysaccharide in Escherichia coli Requires the Essential LapB Heat Shock Protein

Stereoselective Glycal Fluorophosphorylation: Synthesis of ADP‐2‐fluoroheptose, an Inhibitor of the LPS Biosynthesis

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