Quick-soaking of crystals reveals unprecedented insights into the catalytic mechanism of glycosyltransferases

Albesa‐Jové, David; Cifuente, Javier O.; Trastoy, Beatriz; Guerin, Marcelo E.

doi:10.1016/bs.mie.2019.02.034

Cited by 7 publications

(4 citation statements)

References 50 publications

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“…Two catalytic mechanisms have been proposed for retaining GTs, (i) a double-displacement mechanism that involves a nucleophilic residue that forms a covalent sugar–enzyme intermediate and in the absence of a nucleophilic residue (ii) a substrate-assisted front-face reaction mechanism that involves the formation of an oxocarbenium ion-like transition state. ,− In support of the substrate-assisted front-face reaction mechanism, recent structural work on GT-A GTs using a quick-soaking method has provided unprecedented insights by trapping snapshots along the reaction cycle. − That is the case for the retaining GTs glucosyl-3-phosphoglycerate synthase (GpgS) and α-1,3-galactosyltransferase (α3GalT). Although the latter contains a putative nucleophile in the active site (Glu317), the native ternary complexes determined in a productive mode for catalysis in the presence of their sugar donor, acceptor substrates, and metal cofactors support the substrate-assisted front-face reaction mechanism, suggesting a common reaction mechanism for all retaining GTs. ,, …”

Section: Discussionmentioning

confidence: 99%

Dissecting the Structural and Chemical Determinants of the “Open-to-Closed” Motion in the Mannosyltransferase PimA from Mycobacteria

et al. 2020

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The phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential peripheral membrane glycosyltransferase that initiates the biosynthetic pathway of phosphatidyl-myoinositol mannosides (PIMs), key structural elements and virulence factors of Mycobacterium tuberculosis. PimA undergoes functionally important conformational changes, including (i) -helix-to--strand and -strand-to--helix transitions, and (ii) an 'open-to-closed' motion between the two Rossmann-fold domains, a conformational change necessary to generate a catalytically competent active site. In previous work, we established that GDP-Man and GDP stabilize the enzyme and facilitate the switch to a more compact active state. To determine the structural contribution of the mannose ring in such activation mechanism we analyzed a series of chemical derivatives, including mannose-phosphate (Man-P) and mannose-pyrophosphateribose (Man-PP-RIB), and additional GDP derivatives, as pyrophosphate-ribose (PP-RIB) and GMP, by the combined used of X-ray crystallography, limited proteolysis, circular dichroism, isothermal titration calorimetry and Small Angle X-ray Scattering methods. Although the phosphate is present, we found that the mannose ring, neither covalently attached to phosphate (Man-P) nor to PP-RIB (Man-PP-RIB), does promote the switch to the active compact form of the enzyme. Therefore, the nucleotide moiety of GDP-Man, and not the sugar ring, facilitates the 'open-to-closed' motion, with the -phosphate group providing the high affinity binding to PimA. Altogether, the experimental data, contribute to a better understanding of the structural determinants involved in the 'open-to-closed' motion observed not only in PimA, but also visualized/predicted in other glycosyltransfeases. In addition, the experimental data might prove useful for the discovery/development of PimA and/or glycosyltransferase inhibitors.

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Section: Discussionmentioning

confidence: 99%

Dissecting the Structural and Chemical Determinants of the “Open-to-Closed” Motion in the Mannosyltransferase PimA from Mycobacteria

et al. 2020

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“…Besides Hs UGGT1 and Hs UGGT2, the human genome encodes 10 more genes containing a GlycosylTransferase-A (GT-A) or a GlycosylTransferase-B (GT-B) domain. From sequence alignment, it appears that the YW clamp providing the 5M-8OH-Q binding platform is specific to UGGTs (GT24 family 61 ; Figure S2 ). It is therefore unlikely that 5M-8OH-Q binds other GT-A and GT-B domains in human proteins in the same way it binds UGGTs.…”

Section: Discussionmentioning

confidence: 99%

A quinolin-8-ol sub-millimolar inhibitor of UGGT, the ER glycoprotein folding quality control checkpoint

Guay,

Ibba,

Kiappes

et al. 2023

iScience

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“…Both UGGT isoforms are inhibited (Figure 3), a result that agrees with the conservation of the 5M-8OH-Q binding site in the catalytic domain of the two proteins. Besides Hs UGGT1 and Hs UGGT2, the human genome encodes 10 more genes containing GT-A and GT-B glycosyltransferase domains, but from sequence alignment, it appears that the YW clamp providing the 5M-8OH-Q binding platform is specific to UGGTs (GT24 family(54)), Figure S3). Our model of the Man 9 GlcNAc 2 glycan bound to the catalytic domain of Ct UGGT shows the 5M-8OH-Q binding site partially overlapping with the Man 9 GlcNAc 2 glycan binding site, which is also shared between UGGT1 and UGGT2(42).…”

Section: Discussionmentioning

confidence: 99%

A quinolin-8-ol sub-millimolar inhibitor of UGGT, the ER glycoprotein folding quality control checkpoint

Guay¹,

Ibba

Kiappes

et al. 2022

Preprint

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The Endoplasmic Reticulum (ER) glycoprotein folding Quality Control (ERQC) machinery aids folding of glycoproteins in the ER. Misfolded glycoprotein recognition and ER-retention is mediated by the ERQC checkpoint enzyme, the 170 kDa UDP-Glucose glycoprotein glucosyltransferase (UGGT). UGGT modulation is a promising strategy for broad-spectrum antivirals, rescue-of-secretion therapy in rare disease caused by responsive mutations in glycoprotein genes, and many cancers, but to date no selective UGGT inhibitors are known. Towards the generation of selective UGGT inhibitors, we determined the crystal structures of the catalytic domain of Chaetomium thermophilum UGGT (CtUGGTGT24), alone and in complex with the inhibitor UDP-2-deoxy-2-fluoro-D-glucose (U2F). Using the CtUGGTGT24 crystals, we carried out a fragment-based lead discovery screen via X-ray crystallography and discovered that the small molecule 5-[(morpholin-4-yl)methyl]quinolin-8-ol (5M-8OH-Q) binds a CtUGGTGT24 'WY' conserved surface motif that is not present in other GT24 family glycosyltransferases. The 5M-8OH-Q molecule has a 613 μM binding affinity for human UGGT1in vitro as measured by saturation transfer difference NMR spectroscopy. The 5M-8OH-Q molecule inhibits both human UGGT1 and UGGT2 activity at concentrations higher than 750 μM in modified HEK293-6E cells. The compound is toxic in cellula and in planta at concentrations higher than 1 mM. A few off-target effects are also observed upon 5M-8OH-Q treatment. Based on an in silico model of the interaction between UGGT and its substrate N-glycan, the 5M-8OH-Q molecule likely works as a competitive inhibitor, binding to the site of recognition of the first GlcNAc residue of the substrate N-glycan.

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Quick-soaking of crystals reveals unprecedented insights into the catalytic mechanism of glycosyltransferases

Cited by 7 publications

References 50 publications

Dissecting the Structural and Chemical Determinants of the “Open-to-Closed” Motion in the Mannosyltransferase PimA from Mycobacteria

Dissecting the Structural and Chemical Determinants of the “Open-to-Closed” Motion in the Mannosyltransferase PimA from Mycobacteria

A quinolin-8-ol sub-millimolar inhibitor of UGGT, the ER glycoprotein folding quality control checkpoint

A quinolin-8-ol sub-millimolar inhibitor of UGGT, the ER glycoprotein folding quality control checkpoint

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