A General Strategy to Synthesize ADP-7-Azido-heptose and ADP-Azido-mannoses and Their Heptosyltransferase Binding Properties

Li, Tianlei; Tikad, Abdellatif; Fu, Huixiao; Milicaj, Jozafina; Castro, Colleen D.; Lacritick, Marine; Pan, Weidong; Taylor, Erika A.; Vincent, Stéphane

doi:10.1021/acs.orglett.1c00048

Cited by 11 publications

(11 citation statements)

References 29 publications

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“…The K M value for hexose is approximately 1.4-fold lower than that of native heptose, again being less impacted than HepI, which exhibits a nearly 10-fold decrease in the Michaelis constant for the same reduction in substrate size. 69 The binding affinities determined by tryptophan fluorescence quenching yield similar affinities for O-deacylated acceptor and native donor. Overall, the substrate utilization profiles for the two enzymes are quite similar and the ability of HepII to also utilize a sugar acceptor substrate with fewer acyl chains has the potential to allow future work to obtain liganded crystal structures of HepII to validate the predicted ligand-binding interactions described here.…”

Section: Multiplementioning

confidence: 93%

Kinetic Characterization and Computational Modeling of Escherichia coli Heptosyltransferase II: Exploring the Role of Protein Dynamics in Catalysis for GT-B Glycosyltransferase

et al. 2022

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Glycosyltransferase (GT) enzymes promote the formation of glycosidic bonds between a sugar molecule and a diversity of substrates. Heptosyltransferase II (HepII) is a GT involved in the lipopolysaccharide (LPS) biosynthetic pathway that transfers the seven-carbon sugar (l-glycero-d-manno-heptose, Hep) onto a lipid-anchored glycopolymer (heptosylated Kdo2-Lipid A, Hep-Kdo2-Lipid A, or HLA). LPS plays a key role in Gram-negative bacterial sepsis, biofilm formation, and host colonization, and as such, LPS biosynthetic enzymes are targets for novel antimicrobial therapeutics. Three heptosyltransferases are involved in the inner-core LPS biosynthesis, with Escherichia coli HepII being the last to be quantitatively characterized in vivo. HepII shares modest sequence similarity with heptosyltransferase I (HepI) while maintaining a high degree of structural homology. Here, we report the first kinetic and biophysical characterization of HepII and demonstrate the properties of HepII that are shared with HepI, including sugar donor promiscuity and sugar acceptor-induced secondary structural changes, which results in significant thermal stabilization. HepII also has an increased catalytic efficiency and a significantly tighter binding affinity for both of its substrates compared to HepI. A structural model of the HepII ternary complex, refined by molecular dynamics simulations, was developed to probe the potentially important substrate–protein contacts. Ligand binding-induced changes in Trp fluorescence in HepII enabled the determination of substrate dissociation constants. Combined, these efforts meaningfully enhance our understanding of the heptosyltransferase family of enzymes and will aid in future efforts to design novel, potent, and specific inhibitors for this family of enzymes.

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Section: Multiplementioning

confidence: 93%

Kinetic Characterization and Computational Modeling of Escherichia coli Heptosyltransferase II: Exploring the Role of Protein Dynamics in Catalysis for GT-B Glycosyltransferase

et al. 2022

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“…The entropic contribution was estimated with the interaction entropy approximation. “Experimental” binding free energies were calculated from the Michaelis equilibrium constant ( K M ) for each of the ligands from previously determined kinetics constants. , Errors are reported as standard error and are assumed to propagate.…”

Section: Methodsmentioning

confidence: 99%

Ligand-Induced Conformational and Dynamical Changes in a GT-B Glycosyltransferase: Molecular Dynamics Simulations of Heptosyltransferase I Complexes

Hassan

Milicaj

Ramirez-Mondragon

et al. 2021

J. Chem. Inf. Model.

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Understanding the dynamical motions and ligand recognition motifs of heptosyltransferase I (HepI) can be critical to discerning the behavior of other glycosyltransferase (GT) enzymes. Prior studies in our lab have demonstrated that GTs in the GT-B structural class, which are characterized by their connection of two Rossman-like domains by a linker region, have conserved structural fold and dynamical motions, despite low sequence homology, therefore making discoveries found in HepI transferable to other GT-B enzymes. Through molecular dynamics simulations and ligand binding free energy analysis of HepI in the apo and bound complexes (for all kinetically relevant combinations of the native substrates/products), we have determined the energetically favored enzymatic pathway for ligand binding and release. Our principal component, dynamic cross correlation, and network analyses of the simulations have revealed correlated motions involving residues within the N-terminal domain communicating with C-terminal domain residues via both proximal amino acid residues and also functional groups of the bound substrates. Analyses of the structural changes, energetics of substrate/product binding, and changes in pK a have elucidated a variety of inter and intradomain interactions that are critical for enzyme catalysis. These data corroborate our experimental observations of protein conformational changes observed in both presteady state kinetic and circular dichroism analyses of HepI. These simulations provided invaluable structural insights into the regions involved in HepI conformational rearrangement upon ligand binding. Understanding the specific interactions governing conformational changes is likely to enhance our efforts to develop novel dynamics disrupting inhibitors against GT-B structural enzymes in the future.

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“…The K M for the hexose is approximately 1.4 fold lower than the native heptose, again being less impacted than HepI which exhibits a nearly 10 fold decrease in the Michaelis constant for the same reduction in susbtrate size. 69 The binding affinities determined by tryptophan fluorescence quenching yield similar affinities for O-deacylated acceptor and native donor. Overall, the substrate utilization profiles for the two enzymes are quite similar and the ability of HepII to also utilize a sugar acceptor substrate with fewer acyl chains has the potential to allow for future work to obtain liganded crystal structures of HepII to validate the predicted ligand-binding interactions described here.…”

Section: Discussionmentioning

confidence: 93%

Kinetic Characterization and Computational Modeling of the Escherichia coli Heptosyltransferase II: Exploring the Role of Protein Dynamics in Catalysis for a GT-B Glycosyltransferase

Hassan

Liu

Milicaj

et al. 2022

Preprint

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Glycosyltransferases (GTs) are enzymes that are uniquely adapted to promote the formation of a glycosidic bond between a sugar molecule and a wide variety of substrates. Heptosyltransferase II (HepII) is a GT involved in the lipopolysaccharide (LPS) biosynthetic pathway that transfers the seven-carbon sugar (L-glycero-D-manno-heptose; Hep) onto a lipid anchored glycopolymer (heptosylated Kdo2-Lipid A, Hep-Kdo2-Lipid A or HLA). LPS plays a key role in Gram-negative bacterial sepsis as a stimulator of the human immune response and has been used as an adjuvant in vaccines. As such, ongoing efforts towards inhibition of LPS biosynthetic enzymes to aid development of novel antimicrobial therapeutics has driven significant effort towards the characterization of these enzymes. Three heptosyltransferases are involved in the inner-core biosynthesis, with E. coli HepII being the last to be quantitatively characterized in vivo, as described herein. HepII shares modest sequence similarity with heptosyltransferase I (HepI) while maintaining a high degree of structural homology. Here we report the first kinetic and biophysical characterization of HepII and demonstrate the properties of HepII that are shared by HepI to include sugar donor promiscuity, and sugar acceptor induced secondary structural changes which results in significant thermal stabilization. HepII also has an increased catalytic efficiency and a significantly tighter binding affinity for both of its substrates, with an insensitivity to the number of acyl chains on the sugar acceptor. Additionally, a structural model of the HepII ternary complex, refined by molecular dynamics simulations, was developed to probe potentially important substrate-protein contacts and revealed the potential of Tryptophan (Trp) residues responsible for reporting on ligand binding. As was previously described for HepI, Tryptophan fluorescence in HepII allowed observation of substrate induced changes in Trp fluorescence intensity which enabled determination of substrate dissociation constants. Combined, these efforts meaningfully enhance our understanding of the Heptosyltransferase family of enzymes and will aid in future efforts to design novel, potent and specific inhibitors for this family of enzymes.

show abstract

A General Strategy to Synthesize ADP-7-Azido-heptose and ADP-Azido-mannoses and Their Heptosyltransferase Binding Properties

Cited by 11 publications

References 29 publications

Kinetic Characterization and Computational Modeling of Escherichia coli Heptosyltransferase II: Exploring the Role of Protein Dynamics in Catalysis for GT-B Glycosyltransferase

Kinetic Characterization and Computational Modeling of Escherichia coli Heptosyltransferase II: Exploring the Role of Protein Dynamics in Catalysis for GT-B Glycosyltransferase

Ligand-Induced Conformational and Dynamical Changes in a GT-B Glycosyltransferase: Molecular Dynamics Simulations of Heptosyltransferase I Complexes

Kinetic Characterization and Computational Modeling of the Escherichia coli Heptosyltransferase II: Exploring the Role of Protein Dynamics in Catalysis for a GT-B Glycosyltransferase

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