Chemoenzymatic Synthesis, Inhibition Studies, and X-ray Crystallographic Analysis of the Phosphono Analog of UDP-Galp as an Inhibitor and Mechanistic Probe for UDP-Galactopyranose Mutase

Partha, Sarathy Karunan; Sadeghi-Khomami, Ali; Slowski, Kathryn; Kotake, Toshihisa; Thomas, Neil R.; Jakeman, David L.; Sanders, D.A.R.

doi:10.1016/j.jmb.2010.08.053

Cited by 39 publications

(58 citation statements)

References 51 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…(DI) were synthesized as reported previously (29,39,41). Donor substrates UDP-Gal and UDP-Glc were purchased from Sigma-Aldrich.…”

Section: Methodsmentioning

confidence: 99%

“…1A) generates UDP-C-Gal, a non-hydrolyzable substrate designed to be isosteric to UDP-Gal, which could be used as a probe of the GTA/GTB glycosyl transfer reaction. The substrate analog was previously used in structural studies to probe the UDP galactopyranose mutase reaction but was found to bind the enzyme in an alternate conformation; the ␤-phosphate and galactosyl moiety of the analog were shifted relative to the "U-shaped" conformation of natural donor (39).…”

Section: Homologous Glycosyltransferases ␣-(133)-n-acetylgalactosaminmentioning

confidence: 99%

See 1 more Smart Citation

High Resolution Structures of the Human ABO(H) Blood Group Enzymes in Complex with Donor Analogs Reveal That the Enzymes Utilize Multiple Donor Conformations to Bind Substrates in a Stepwise Manner

Gagnon

Meloncelli

Zheng

et al. 2015

Journal of Biological Chemistry

View full text Add to dashboard Cite

Background: Substrate hydrolysis has impeded structural investigation of human ABO(H) glycosyltransferase specificity. Results: Complexes with natural and isosteric non-hydrolyzable donor analogs show multiple stable intermediate donor binding conformations. Conclusion: Subtle stereochemical differences from natural donor prevent isosteric donor analog from displaying full mimicry. Significance: High resolution structural analysis provides insight into inhibitor development and the multistage process of substrate binding.

show abstract

“…(DI) were synthesized as reported previously (29,39,41). Donor substrates UDP-Gal and UDP-Glc were purchased from Sigma-Aldrich.…”

Section: Methodsmentioning

confidence: 99%

Section: Homologous Glycosyltransferases ␣-(133)-n-acetylgalactosaminmentioning

confidence: 99%

High Resolution Structures of the Human ABO(H) Blood Group Enzymes in Complex with Donor Analogs Reveal That the Enzymes Utilize Multiple Donor Conformations to Bind Substrates in a Stepwise Manner

Gagnon

Meloncelli

Zheng

et al. 2015

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…16,18–20,22,24–30 Though inhibitors have emerged, 18–20,31 few have been shown to function against microbial pathogens. We previously developed a fluorescence polarization (FP) assay to identify competitive UGM inhibitors.…”

mentioning

confidence: 99%

Virtual Screening for UDP-Galactopyranose Mutase Ligands Identifies a New Class of Antimycobacterial Agents

et al. 2015

View full text Add to dashboard Cite

Galactofuranose (Galf) is present in glycans critical for the virulence and viability of several pathogenic microbes, including Mycobacterium tuberculosis, yet the monosaccharide is absent from mammalian glycans. Uridine 5′-diphosphate-galactopyranose mutase (UGM) catalyzes the formation of UDP-Galf, which is required to produce Galf-containing glycoconjugates. Inhibitors of UGM have therefore been sought, both as antimicrobial leads and as tools to delineate the roles of Galf in cells. Obtaining cell permeable UGM probes by either design or high throughput screens has been difficult, as has elucidating how UGM binds small molecule, noncarbohydrate inhibitors. To address these issues, we employed structure-based virtual screening to uncover new inhibitor chemotypes, including a triazolothiadiazine series. These compounds are among the most potent antimycobacterial UGM inhibitors described. They also facilitated determination of a UGM–small molecule inhibitor structure, which can guide optimization. A comparison of results from the computational screen and a high-throughput fluorescence polarization (FP) screen indicated that the scaffold hits from the former had been evaluated in the FP screen but missed. By focusing on promising compounds, the virtual screen rescued false negatives, providing a blueprint for generating new UGM probes and therapeutic leads.

show abstract

“…Several crystal structures of bacterial UGMs have been determined (18–23). These structures revealed the essential UGM fold and provided insight into several aspects of UGM biochemistry, including the structural basis of substrate recognition and the catalytic mechanism.…”

mentioning

confidence: 99%

Crystal Structures of Trypanosoma cruzi UDP-Galactopyranose Mutase Implicate Flexibility of the Histidine Loop in Enzyme Activation

et al. 2012

View full text Add to dashboard Cite

Chagas disease is a neglected tropical disease caused by the protozoan parasite Trypanosoma cruzi. Here we report crystal structures of the galactofuranose biosynthetic enzyme UDP-galactopyranose mutase (UGM) from T. cruzi, which are the first structures of this enzyme from a protozoan parasite. UGM is an attractive target for drug design because galactofuranose is absent in humans but is an essential component of key glycoproteins and glycolipids in trypanosomatids. Analysis of the enzyme-UDP noncovalent interactions and sequence alignments suggests that substrate recognition is exquisitely conserved among eukaryotic UGMs and distinct from that of bacterial UGMs. This observation has implications for inhibitor design. Activation of the enzyme via reduction of the FAD induces profound conformational changes, including a 2.3-Å movement of the histidine loop (Gly60-Gly61-His62), rotation and protonation of the imidazole of His62, and cooperative movement of residues located on the si face of the FAD. Interestingly, these changes are substantially different from those described for Aspergillus fumigatus UGM, which is 45 % identical to T. cruzi UGM. The importance of Gly61 and His62 for enzymatic activity was studied with the site-directed mutant enzymes G61A, G61P, and H62A. These mutations lower the catalytic efficiency by factors of 10–50, primarily by decreasing kcat. Considered together, the structural, kinetic, and sequence data suggest that the middle Gly of the histidine loop imparts flexibility that is essential for activation of eukaryotic UGMs. Our results provide new information about UGM biochemistry and suggest a unified strategy for designing inhibitors of UGMs from the eukaryotic pathogens.

show abstract

Chemoenzymatic Synthesis, Inhibition Studies, and X-ray Crystallographic Analysis of the Phosphono Analog of UDP-Galp as an Inhibitor and Mechanistic Probe for UDP-Galactopyranose Mutase

Cited by 39 publications

References 51 publications

High Resolution Structures of the Human ABO(H) Blood Group Enzymes in Complex with Donor Analogs Reveal That the Enzymes Utilize Multiple Donor Conformations to Bind Substrates in a Stepwise Manner

High Resolution Structures of the Human ABO(H) Blood Group Enzymes in Complex with Donor Analogs Reveal That the Enzymes Utilize Multiple Donor Conformations to Bind Substrates in a Stepwise Manner

Virtual Screening for UDP-Galactopyranose Mutase Ligands Identifies a New Class of Antimycobacterial Agents

Crystal Structures of Trypanosoma cruzi UDP-Galactopyranose Mutase Implicate Flexibility of the Histidine Loop in Enzyme Activation

Contact Info

Product

Resources

About