Characterization of the physiological substrate for lipopolysaccharide heptosyltransferases I and II

Gronow, Sabine; Oertelt, Clemens; Ervelä, Elise; Zamyatina, Alla; Kosma, Paul; Skurnik, Mikael; Holst, Otto

doi:10.1177/09680519010070040701

Cited by 27 publications

(4 citation statements)

References 24 publications

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“…17 The use of ADP-Lglycero-β-D-manno-heptose by heptosyltransferases involved in the LPS biosynthesis has been clearly established by studies showing that the α pyranose is not a substrate and that ADP-D-glycero-β-D-mannoheptose can serve as a substrate but with a tenfold reduced activity. 18,19 The WaaC protein, which is presented here, belongs to the neonatal meningitidis E. coli (NMEC) strain RS218. This E. coli strain possesses the K1 capsular polysaccharide and is associated with septicemia and urinary tract infections in children.…”

Section: Introductionmentioning

confidence: 99%

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

Grizot¹,

Salem²,

Vongsouthi³

et al. 2006

Journal of Molecular Biology

154

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Section: Introductionmentioning

confidence: 99%

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

Grizot¹,

Salem²,

Vongsouthi³

et al. 2006

Journal of Molecular Biology

154

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“…The recognition of side chain conformation as a contributing factor to transition-state stabilization by GTs opens up a new avenue for the development of a next generation of inhibitors designed to take advantage of the inherent binding preference of the glycosyltransferases. A clue as to how this might be achieved is provided by the E. coli heptosyltransferase WaaC, which processes the natural substrate 78 ADP-L-glycero-β-Dmanno-heptose (6) 10 times more effectively than the sidechain isomer ADP-D-glycero-β-D-manno-heptose (7). 79 Crystallographic studies by Ducruix and co-workers revealed that the substrate analogue ADP-2-deoxy-2-fluoro-L-glycero-β-Dgluco-heptose, whose side chain is predicted to take up the gg conformation in free solution, 80 is bound in the active site with its side chain held in the gg conformation by hydrogen bonding to lysine 192 (Figure 8).…”

Section: ■ Results and Discussionmentioning

confidence: 99%

“…A clue as to how this might be achieved is provided by the E. coli heptosyltransferase WaaC, which processes the natural substrate ADP- l - glycero -β- d - manno -heptose ( 6 ) 10 times more effectively than the side-chain isomer ADP- d - glycero -β- d - manno -heptose ( 7 ) . Crystallographic studies by Ducruix and co-workers revealed that the substrate analogue ADP-2-deoxy-2-fluoro- l - glycero -β- d - gluco -heptose, whose side chain is predicted to take up the gg conformation in free solution, is bound in the active site with its side chain held in the gg conformation by hydrogen bonding to lysine 192 (Figure ).…”

Section: Resultsmentioning

confidence: 99%

Side Chain Conformation Restriction in the Catalysis of Glycosidic Bond Formation by Leloir Glycosyltransferases, Glycoside Phosphorylases, and Transglycosidases

Quirke

Crich

2021

ACS Catal.

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Carbohydrate side chain conformation is an important factor in the control of reactivity at the anomeric center, i.e. in the making and breaking of glycosidic bonds, whether by chemical means or, for hydrolysis, by glycoside hydrolases. In nature glycosidic bond formation is catalyzed out by glycosyltransferases (GTs), glycoside phosphorylases, and transglycosidases. By an analysis of 118 crystal structures of sugar nucleotide dependent (Leloir) GTs, 136 crystal structures of glycoside phosphorylases, and 54 crystal structures of transglycosidases bound to hexopyranosides or their analogues at the donor site (−1 site), we determined that most enzymes that catalyze glycoside synthesis, be they GTs, glycoside phosphorylases, or transglycosidases, restrict their substrate side chains to the most reactive gauche,gauche (gg) conformation to achieve maximum stabilization of the oxocarbenium ion-like transition state for glycosyl transfer. The galactose series deviates from this trend, with α-galactosyltransferases preferentially restricting their substrates to the secondmost reactive gauche,trans (gt) conformation and β-galactosyltransferases favoring the least reactive trans,gauche (tg) conformation. This insight will help promote the design and development of improved, conformationally restricted GT inhibitors that take advantage of these inherent side chain preferences.

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“…ADPH isolation has been previously reported [25,26], and optimizations of that protocol are described below. Luria-Bertani (LB) media was prepared freshly or within 24 h of use; 2 L portions were made in 4 L flasks.…”

Section: Adph Isolation and Purificationmentioning

confidence: 99%

Extraction of ADP-Heptose and Kdo2-Lipid A from E. coli Deficient in the Heptosyltransferase I Gene

et al. 2021

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The enzymes involved in lipopolysaccharide (LPS) biosynthesis, including Heptosyltransferase I (HepI), are critical for maintaining the integrity of the bacterial cell wall, and therefore these LPS biosynthetic enzymes are validated targets for drug discovery to treat Gram-negative bacterial infections. Enzymes involved in the biosynthesis of lipopolysaccharides (LPSs) utilize substrates that are synthetically complex, with numerous stereocenters and site-specific glycosylation patterns. Due to the relatively complex substrate structures, characterization of these enzymes has necessitated strategies to generate bacterial cells with gene disruptions to enable the extraction of these substrates from large scale bacterial growths. Like many LPS biosynthetic enzymes, Heptosyltransferase I binds two substrates: the sugar acceptor substrate, Kdo2-Lipid A, and the sugar donor substrate, ADP-l-glycero-d-manno-heptose (ADPH). HepI characterization experiments require copious amounts of Kdo2-Lipid A and ADPH, and unsuccessful extractions of these two substrates can lead to serious delays in collection of data. While there are papers and theses with protocols for extraction of these substrates, they are often missing small details essential to the success of the extraction. Herein detailed protocols are given for extraction of ADPH and Kdo2-Lipid A (KLA) from E. coli, which have had proven success in the Taylor lab. Key steps in the extraction of ADPH are clearing the extract through ultracentrifugation and keeping all water that touches anything in the extraction, including filters, at a pH of 8.0. Key steps in the extraction of KLA are properly lysing the dried down cells before starting the extraction, maximizing yield by allowing precipitate to form overnight, appropriately washing the pellet with phenol and dissolving the KLA in 1% TEA using visual cues, rather than a specific volume. These protocols led to increased yield and a higher success rate of extractions thereby enabling the characterization of HepI.

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Characterization of the physiological substrate for lipopolysaccharide heptosyltransferases I and II

Cited by 27 publications

References 24 publications

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

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

Side Chain Conformation Restriction in the Catalysis of Glycosidic Bond Formation by Leloir Glycosyltransferases, Glycoside Phosphorylases, and Transglycosidases

Extraction of ADP-Heptose and Kdo2-Lipid A from E. coli Deficient in the Heptosyltransferase I Gene

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