Structural basis for heparan sulfate co-polymerase action by the EXT1–2 complex

Li, Hua; Chapla, Digantkumar; Amos, Robert A.; Ramiah, Annapoorani; Moremen, Kelley W.; H, Li

doi:10.1038/s41589-022-01220-2

Cited by 18 publications

(23 citation statements)

References 57 publications

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“…2b–e). In each case, the aligned nucleotide was placed below the side chain of Asp407—a highly conserved residue that has since been shown to bind sugar nucleotides in other GT47 family members (Leisico et al ., 2022; Wilson et al ., 2022; Li et al ., 2023); hence, this is the likely binding pocket for UDP-Gal in At XLT2.…”

Section: Resultsmentioning

confidence: 99%

The biosynthesis, degradation, and function of cell wall β-xylosylated xyloglucan mirrors that of arabinoxyloglucan

Wilson

Neun

et al. 2023

Preprint

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SummaryXyloglucan is an abundant polysaccharide in many primary cell walls and in the human diet. Decoration of its α-xylosyl side chains with further sugars is critical for plant growth, even though the sugars themselves vary considerably between species. Plants in the Ericales order—prevalent in human diets—exhibit β1,2-linked xylosyl decorations. The biosynthetic enzymes responsible for adding these xylosyl decorations, as well as the hydrolases that remove them in the human gut, are unidentified.GT47 xyloglucan glycosyltransferase candidates were expressed in Arabidopsis andendo-xyloglucanase products from transgenic wall material were analysed by electrophoresis, mass spectrometry, and NMR. The activities of gut bacterial hydrolasesBoGH43A andBoGH43B on synthetic glycosides and xyloglucan oligosaccharides were measured by colorimetry and electrophoresis.CcXBT1 is a xyloglucan β-xylosyltransferase from coffee that can modify Arabidopsis xyloglucan and restore the growth of galactosyltransferase mutants. RelatedVmXST1 is a weakly active xyloglucan α-arabinofuranosyltransferase from cranberry.BoGH43A hydrolyses both α-arabinofuranosylated and β-xylosylated oligosaccharides.CcXBT1’s presence in coffee andBoGH43A’s promiscuity suggest that β-xylosylated xyloglucan is not only more widespread than thought, but might also nourish beneficial gut bacteria. The evolutionary instability of transferase specificity and lack of hydrolase specificity hint that, to enzymes, xylosides and arabinofuranosides are closely resemblant.

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Section: Resultsmentioning

confidence: 99%

The biosynthesis, degradation, and function of cell wall β-xylosylated xyloglucan mirrors that of arabinoxyloglucan

Wilson

Neun

et al. 2023

Preprint

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“…2b-e). In each case, the aligned nucleotide was placed below the sidechain of Asp407a highly conserved residue that has since been shown to bind sugar nucleotides in other GT47 family members (Leisico et al, 2022;Wilson et al, 2022;Li et al, 2023); hence, this is the likely binding pocket for UDP-Gal in AtXLT2.…”

Section: Resultsmentioning

confidence: 99%

The biosynthesis, degradation, and function of cell wall β‐xylosylated xyloglucan mirrors that of arabinoxyloglucan

Wilson,

Neun,

et al. 2023

New Phytologist

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Summary Xyloglucan is an abundant polysaccharide in many primary cell walls and in the human diet. Decoration of its α‐xylosyl sidechains with further sugars is critical for plant growth, even though the sugars themselves vary considerably between species. Plants in the Ericales order – prevalent in human diets – exhibit β1,2‐linked xylosyl decorations. The biosynthetic enzymes responsible for adding these xylosyl decorations, as well as the hydrolases that remove them in the human gut, are unidentified. GT47 xyloglucan glycosyltransferase candidates were expressed in Arabidopsis and endo‐xyloglucanase products from transgenic wall material were analysed by electrophoresis, mass spectrometry, and nuclear magnetic resonance (NMR) spectroscopy. The activities of gut bacterial hydrolases BoGH43A and BoGH43B on synthetic glycosides and xyloglucan oligosaccharides were measured by colorimetry and electrophoresis. CcXBT1 is a xyloglucan β‐xylosyltransferase from coffee that can modify Arabidopsis xyloglucan and restore the growth of galactosyltransferase mutants. Related VmXST1 is a weakly active xyloglucan α‐arabinofuranosyltransferase from cranberry. BoGH43A hydrolyses both α‐arabinofuranosylated and β‐xylosylated oligosaccharides. CcXBT1's presence in coffee and BoGH43A's promiscuity suggest that β‐xylosylated xyloglucan is not only more widespread than thought, but might also nourish beneficial gut bacteria. The evolutionary instability of transferase specificity and lack of hydrolase specificity hint that, to enzymes, xylosides and arabinofuranosides are closely resemblant.

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“…29,30 The general position of the NS 7-mer superimposes with the oligosaccharide bound to that of the GT64 GT-A domain of EXT2 (PDB ID 7sck; rmsd 2.75 Å over 184 Cαs) (Figure 3A). 29 The position of the acceptor nonreducing end GlcA (saccharide 7) is dictated through the potential CH-π interaction with Tyr604, a hydrogen bond interaction of the carboxylate with Arg524, and potential van der Waals interactions with side chains Phe529 and His508 (Figure 3A). Arg524 is also in the position to form a hydrogen bond with the O3 oxygen and NS moiety of the neighboring GlcNS unit (saccharide 6).…”

Section: ■ Resultsmentioning

confidence: 99%

“…While both EXT1 and EXT2 have N -terminal GlcA-T and C-terminal GlcNAc-T domains, the EXT1 enzyme performs the GlcA-T reaction while EXT2 performs the GlcNAc-T reaction . Like PmHS2, the active sites within the heterodimer are remote from one another and the polymerase mechanism is believed to be distributive in nature. , Interestingly, unlike PmHS2, this heterodimer forms a head-to-head arrangement. As EXT1 and EXT2 are type-II transmembrane proteins, the N -terminal helices anchor both proteins to the Golgi membrane, where the complex is located.…”

Section: Discussionmentioning

confidence: 99%

Structural and Functional Analysis of Heparosan Synthase 2 from Pasteurella multocida to Improve the Synthesis of Heparin

Stancanelli,

Krahn,

Viverette

et al. 2024

ACS Catal.

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Heparin is a widely used drug to treat thrombotic disorders in hospitals. Heparosan synthase 2 from Pasteurella multocida (PmHS2) is a key enzyme used for the chemoenzymatic synthesis of heparin oligosaccharides. It has both activities: glucosaminyl transferase activity and glucuronyl transferase activity; however, the mechanism to carry out the glycooligomerization is unknown. Here, we report crystal structures of PmHS2 constructs with bound uridine diphosphate (UDP) and a cryo-EM structure of PmHS2 in complex with UDP and a heptasaccharide (NS 7-mer) substrate. Using a liquid chromatography−mass spectrometry analytical method, we discovered that the enzyme displays both a two-step concerted oligomerization mode and a distributive oligomerization mode depending on the nonreducing end of the starting oligosaccharide primer. Removal of seven amino acid residues from the C-terminus results in an enzymatically active monomer instead of a dimer and loses the concerted oligomerization mode of activity. In addition, the monomer construct can transfer N-acetyl glucosamine at a substrate concentration that is ∼7-fold higher than a wildtype enzyme. It was also determined that an F529A mutant can transfer an N-sulfoglucosamine (GlcNS) saccharide from a previously inactive UDP-GlcNS donor. Performing the glyco-transfer reaction at a high substrate concentration and the capability of using unnatural donors are desirable to simplifying the chemoenzymatic synthesis to prepare heparin-based therapeutics.

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Structural basis for heparan sulfate co-polymerase action by the EXT1–2 complex

Cited by 18 publications

References 57 publications

The biosynthesis, degradation, and function of cell wall β-xylosylated xyloglucan mirrors that of arabinoxyloglucan

The biosynthesis, degradation, and function of cell wall β-xylosylated xyloglucan mirrors that of arabinoxyloglucan

The biosynthesis, degradation, and function of cell wall β‐xylosylated xyloglucan mirrors that of arabinoxyloglucan

Structural and Functional Analysis of Heparosan Synthase 2 from Pasteurella multocida to Improve the Synthesis of Heparin

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