Glycosyltransferase Family 47 - Revision history

Harry Brumer: fixed broken cite tags

2026-01-25T18:11:52Z

fixed broken cite tags

Daniel Tehrani at 16:17, 18 July 2025

2025-07-18T16:17:04Z

Daniel Tehrani at 16:07, 18 July 2025

2025-07-18T16:07:21Z

Harry Brumer: moved figure to tops of sections

2024-07-22T19:19:58Z

moved figure to tops of sections

Breeanna Urbanowicz at 19:30, 19 July 2024

2024-07-19T19:30:26Z

Harry Brumer: fixed headings

2024-07-12T22:45:21Z

fixed headings

Harry Brumer: fixed headings

2024-07-12T22:43:15Z

fixed headings

Harry Brumer: increased image sizes

2024-07-12T22:42:17Z

increased image sizes

Harry Brumer at 22:39, 12 July 2024

2024-07-12T22:39:20Z

Daniel Tehrani at 20:51, 12 July 2024

2024-07-12T20:51:55Z

@@ Line 65: / Line 65: @@
 GT47 enzymes are characterized by GT-B fold architecture comprised of two linked Rossmann-fold domains with a cleft between the domains containing the active site. GT47 enzymes bind their nucleotide sugar donor through interactions with the C-terminal Rossmann fold domain, while the acceptor substrate generally binds either in the cleft between the two domains or exclusively with the N-terminal Rossmann fold domain. The binding sites for donor and acceptor residues are generally comprised of loop regions extending from the respective Rossmann fold domains facing toward the cleft between the two domains <cite>Zhang2023 Moremen2019 Rini2022</cite>.
 == Family Firsts ==
-The first structure of a CAZy family GT47 was the cryo-EM structure of the human EXTL3, two domain protein that contained an active GT64 domain involved in adding the initial priming GlcNAc of the heparan sulfate biosynthesis pathway and a catalytically inactive GT47 domain <cite>WilsonL2022<cite>. Following this discovery, two cryo-EM structures of the EXT1-2 heterocomplex were elucidated <cite>LiH2023 LeisicoF2022<cite>. EXT1 contains a GT47 β1,4-GlcA transferase domain and an inactive GT64 α1,4GlcNAc transferase-like domain of EXT1, while EXT2 contains an inactive GT47 β1,4-GlcA transferase-like domain along with an active GT64 α1,4GlcNAc transferase domain <cite>LiH2023</cite>. Structures of UDP and acceptor co-complexes were determined for each of the enzyme active sites to map substrate interactions. The structures provided insight into the overall enzyme fold (GT-B) and catalytic site structure and mechanism (inverting) as a framework for studies on the other CAZy GT47 enzymes, especially the GT47s in plants that lack empirical structures.
+The first structure of a CAZy family GT47 was the cryo-EM structure of the human EXTL3, two domain protein that contained an active GT64 domain involved in adding the initial priming GlcNAc of the heparan sulfate biosynthesis pathway and a catalytically inactive GT47 domain <cite>WilsonL2022</cite>. Following this discovery, two cryo-EM structures of the EXT1-2 heterocomplex were elucidated <cite>LiH2023 LeisicoF2022</cite>. EXT1 contains a GT47 β1,4-GlcA transferase domain and an inactive GT64 α1,4GlcNAc transferase-like domain of EXT1, while EXT2 contains an inactive GT47 β1,4-GlcA transferase-like domain along with an active GT64 α1,4GlcNAc transferase domain <cite>LiH2023</cite>. Structures of UDP and acceptor co-complexes were determined for each of the enzyme active sites to map substrate interactions. The structures provided insight into the overall enzyme fold (GT-B) and catalytic site structure and mechanism (inverting) as a framework for studies on the other CAZy GT47 enzymes, especially the GT47s in plants that lack empirical structures.
 == References ==

@@ Line 65: / Line 65: @@
 GT47 enzymes are characterized by GT-B fold architecture comprised of two linked Rossmann-fold domains with a cleft between the domains containing the active site. GT47 enzymes bind their nucleotide sugar donor through interactions with the C-terminal Rossmann fold domain, while the acceptor substrate generally binds either in the cleft between the two domains or exclusively with the N-terminal Rossmann fold domain. The binding sites for donor and acceptor residues are generally comprised of loop regions extending from the respective Rossmann fold domains facing toward the cleft between the two domains <cite>Zhang2023 Moremen2019 Rini2022</cite>.
 == Family Firsts ==
-The first structure of a CAZy family GT47 was the cryo-EM structure of the human EXTL3, two domain protein that contained an active GT47 domain involved in adding the initial priming GlcNAc of the heparan sulfate biosynthesis pathway and an inactive GT64 <cite>WilsonL2022<cite>. Following this discovery, two cryo-EM structures of the EXT1-2 heterocomplex were elucidated <cite>LiH2023 LeisicoF2022<cite>. EXT1 contains a GT47 β1,4-GlcA transferase domain and an inactive GT64 α1,4GlcNAc transferase-like domain of EXT1, while EXT2 contains an inactive GT47 β1,4-GlcA transferase-like domain along with an active GT64 α1,4GlcNAc transferase domain <cite>LiH2023</cite>. Structures of UDP and acceptor co-complexes were determined for each of the enzyme active sites to map substrate interactions. The structures provided insight into the overall enzyme fold (GT-B) and catalytic site structure and mechanism (inverting) as a framework for studies on the other CAZy GT47 enzymes, especially the GT47s in plants that lack empirical structures.
+The first structure of a CAZy family GT47 was the cryo-EM structure of the human EXTL3, two domain protein that contained an active GT64 domain involved in adding the initial priming GlcNAc of the heparan sulfate biosynthesis pathway and a catalytically inactive GT47 domain <cite>WilsonL2022<cite>. Following this discovery, two cryo-EM structures of the EXT1-2 heterocomplex were elucidated <cite>LiH2023 LeisicoF2022<cite>. EXT1 contains a GT47 β1,4-GlcA transferase domain and an inactive GT64 α1,4GlcNAc transferase-like domain of EXT1, while EXT2 contains an inactive GT47 β1,4-GlcA transferase-like domain along with an active GT64 α1,4GlcNAc transferase domain <cite>LiH2023</cite>. Structures of UDP and acceptor co-complexes were determined for each of the enzyme active sites to map substrate interactions. The structures provided insight into the overall enzyme fold (GT-B) and catalytic site structure and mechanism (inverting) as a framework for studies on the other CAZy GT47 enzymes, especially the GT47s in plants that lack empirical structures.
 == References ==

@@ Line 65: / Line 65: @@
 GT47 enzymes are characterized by GT-B fold architecture comprised of two linked Rossmann-fold domains with a cleft between the domains containing the active site. GT47 enzymes bind their nucleotide sugar donor through interactions with the C-terminal Rossmann fold domain, while the acceptor substrate generally binds either in the cleft between the two domains or exclusively with the N-terminal Rossmann fold domain. The binding sites for donor and acceptor residues are generally comprised of loop regions extending from the respective Rossmann fold domains facing toward the cleft between the two domains <cite>Zhang2023 Moremen2019 Rini2022</cite>.
 == Family Firsts ==
-The first structure of a CAZy family GT47 was the cryo-EM structure of the human EXT1-2 heterocomplex containing a GT47 β1,4-GlcA transferase domain and an inactive GT64 α1,4GlcNAc transferase-like domain of EXT1, while EXT2 contains an inactive GT47 β1,4-GlcA transferase-like domain along with an active GT64 α1,4GlcNAc transferase domain <cite>LiH2023</cite>. Structures of UDP and acceptor co-complexes were determined for each of the enzyme active sites to map substrate interactions. The structures provided insight into the overall enzyme fold (GT-B) and catalytic site structure and mechanism (inverting) as a framework for studies on the other CAZy GT47 enzymes, especially the GT47s in plants that lack empirical structures.
+The first structure of a CAZy family GT47 was the cryo-EM structure of the human EXTL3, two domain protein that contained an active GT47 domain involved in adding the initial priming GlcNAc of the heparan sulfate biosynthesis pathway and an inactive GT64 <cite>WilsonL2022<cite>. Following this discovery, two cryo-EM structures of the EXT1-2 heterocomplex were elucidated <cite>LiH2023 LeisicoF2022<cite>. EXT1 contains a GT47 β1,4-GlcA transferase domain and an inactive GT64 α1,4GlcNAc transferase-like domain of EXT1, while EXT2 contains an inactive GT47 β1,4-GlcA transferase-like domain along with an active GT64 α1,4GlcNAc transferase domain <cite>LiH2023</cite>. Structures of UDP and acceptor co-complexes were determined for each of the enzyme active sites to map substrate interactions. The structures provided insight into the overall enzyme fold (GT-B) and catalytic site structure and mechanism (inverting) as a framework for studies on the other CAZy GT47 enzymes, especially the GT47s in plants that lack empirical structures.
 == References ==
@@ Line 96: / Line 96: @@
 #Schultink2013 Schultink A, Cheng K, Park YB, Cosgrove DJ, Pauly M. (2013) The Identification of Two Arabinosyltransferases from Tomato Reveals Functional Equivalency of Xyloglucan Side Chain Substituents. Plant Physiology. 2013;163(1):86-94.[https://doi.org/10.1104/pp.113.221788 DOI: 10.1104/pp.113.221788]
 #ZhuL2018 pmid=31245712
 </biblio>

@@ Line 54: / Line 54: @@
 == Kinetics and Mechanism ==
 GT47 enzymes employ an inverting catalytic mechanism where the hydroxyl group of an acceptor substrate presumably acts as a nucleophile in a SN2 single displacement reaction. The result is an inversion of the anomeric configuration of the transferred sugar from an α-linked sugar nucleotide donor to form a β-linked extended glycan product. While an SN2 mechanism would predict the deprotonation of the acceptor nucleophile by an enzyme associated catalytic base, the structure of the EXT1 active site did not appear to contain an appropriately positioned ionizable group to act as catalytic base <cite>LiH2023</cite>. Similar structural studies on the inverting GT-B fold glycosyltransferases, POFUT1 <cite>LiZ2017 Lira2018 Lira2011</cite> and AtFUT1 <cite>Urbanowicz2017</cite>, also indicated the lack of an appropriately positioned catalytic base for deprotonation. In these latter cases a non-canonical SN1-like mechanism was proposed. A similar SN1-like mechanism may also occur for the GT47 enzymes <cite>Zhang2023 Moremen2019</cite>.
 == Catalytic Residues ==
 Unlike GT-A fold enzymes, GT-B fold enzymes like GT47s lack the predictable catalytic features, such as a DxD motif, G-loop, xED, and C-term His, that are involved in sugar nucleotide and divalent cation interactions <cite>#Taujale2020</cite>. In place of the bridging interactions between the nucleotide sugar donor diphosphate residues and an enzyme bound divalent cation as found in GT-A fold enzymes, GT-B fold glycosyltransferases employ basic Lys and Arg side chains for interaction with the diphosphate <cite>Zhang2023 Moremen2019 Rini2022</cite>. Mutation of the active site Lys and Arg residues in the GT47 domain of EXT1 completely eliminated β1,4-GlcA transferase activity as well as co-polymerase activity for extension of heparan sulfate backbone synthesis <cite>LiH2023</cite>. Additional residues involved in donor and acceptor interactions were identified in the EXT1:UDP:acceptor complex during structural studies, but further mutagenesis studies were not performed to test function <cite>LiH2023</cite>. Analogous Lys and Arg residues can be identified in the putative donor binding sites in AlphaFold models plant GT47 enzymes, but their roles in catalysis have not been tested.
 == Three-dimensional structures ==
-[[File:EXT1EXT2 GT47 structure rendering figure 2 V5.jpg|thumb|400px|right|'''Figure 2: GT47 Domain of EXT1 in EXT1-2 Heterocomplex.''' A) Cartoon representation of EXT1 (Salmon and Green) and EXT2 (Gray) in the EXT1-2 heparan sulfate co-polymerase heterocomplex. The GT47 domain of EXT1 is highlighted in green, while the remaining GT47 domain is highlighted in salmon. The nucleotide bound to the active site shown is shown in pink, and the 4-mer heparan sulfate oligosaccharide acceptor is shown in cyan. B) Enlargement of the GT47 domain in EXT1, highlighting the two Rossman folds of the GT-B glycosyltransferase domain (β-strands of N-Term and C-Term Rossman Folds shown in yellow)..]]
+[[File:EXT1EXT2 GT47 structure rendering figure 2 V5.jpg|thumb|400px|right|'''Figure 2: GT47 Domain of EXT1 in EXT1-2 Heterocomplex.''' A) Cartoon representation of EXT1 (Salmon and Green) and EXT2 (Gray) in the EXT1-2 heparan sulfate co-polymerase heterocomplex. The GT47 domain of EXT1 is highlighted in green, while the remaining GT47 domain is highlighted in salmon. The nucleotide bound to the active site shown is shown in pink, and the 4-mer heparan sulfate oligosaccharide acceptor is shown in cyan. B) Enlargement of the GT47 domain in EXT1, highlighting the two Rossman folds of the GT-B glycosyltransferase domain (β-strands of N-Term and C-Term Rossman Folds shown in yellow).]]
 == Family Firsts ==
 The first structure of a CAZy family GT47 was the cryo-EM structure of the human EXT1-2 heterocomplex containing a GT47 β1,4-GlcA transferase domain and an inactive GT64 α1,4GlcNAc transferase-like domain of EXT1, while EXT2 contains an inactive GT47 β1,4-GlcA transferase-like domain along with an active GT64 α1,4GlcNAc transferase domain <cite>LiH2023</cite>. Structures of UDP and acceptor co-complexes were determined for each of the enzyme active sites to map substrate interactions. The structures provided insight into the overall enzyme fold (GT-B) and catalytic site structure and mechanism (inverting) as a framework for studies on the other CAZy GT47 enzymes, especially the GT47s in plants that lack empirical structures.

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 <!-- RESPONSIBLE CURATORS: Please replace the {{UnderConstruction}} tag below with {{CuratorApproved}} when the page is ready for wider public consumption -->
-{{UnderConstruction}}
+{{CuratorApproved}}
 * [[Author]]: [[User:Daniel Tehrani|Daniel Tehrani]] and [[User:Charlie Corulli|Charlie Corulli]]
 * [[Responsible Curator]]:  [[User:Breeanna Urbanowicz|Breeanna Urbanowicz]]

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 Unlike the previously mentioned polysaccharides, extensins are rod-like hydroxyproline rich glycoproteins (HRGP) that form crosslinked networks in the plant cell wall. These networks are reported to play a crucial role in regulating cell wall growth and development <cite>Showalter2016</cite>. A unique member of the GT47 family, Extensin Arabinose Deficient transferase (ExAD), is reported to synthesize the addition of the fourth arabinofuranose (Araf) on Araf substituted C4-hydroxyprolines (Hyps) creating Hyp-Araf4, a unique feature found on extensins <cite>Moller2017 Showalter2016</cite>.
-=== Animals''' ===
+=== Animals ===
 The abundance of GT47 family enzymes in mammals is more restricted and includes only members of the Exostosin (EXT) and Exostoslin-Like (EXTL) family of enzymes involved in heparan sulfate biosynthesis. Heparan sulfate is comprised of a repeat disaccharide polymer of ( GlcAβ1,4GlcNAcα1,4-)n that is further elaborated with extensive sulfation along the polymer chain. The disaccharide backbone repeat is elongated by the co-polymerase activity of the heterodimeric EXT1-EXT2 complex <cite>LiH2023</cite>. EXT1 and EXT2 are homologous two domain enzymes, and each protein chain contains a GT47 β1,4-GlcA transferase-like and a GT64 α1,4GlcNAc transferase-like domain. Surprisingly, only the GT47 domain of EXT1 and GT64 domain of EXT2 exhibit catalytic activity, while the other domains in each subunit are nonfunctional <cite>LiH2023</cite>. Additional EXT homologs include the EXTL proteins, EXTL1-3. EXTL1 and EXTL3 are two domain proteins, each harboring a GT47 and GT64 domain like EXT1 and EXT2. However, only the GT64 domains exhibit α1,4GlcNAc transferase activity, while their corresponding GT47 domains are inactive. In contrast, EXTL2 is a single GT64 domain enzyme with a α1,4GlcNAc transferase activity, while the corresponding GT47 domain present in other EXTs is missing. Thus, among the five mammalian EXT or EXTL homologs, only EXT1 contains a functional GT47 domain exhibiting β1,4-GlcA transferase activity.

@@ Line 30: / Line 30: @@
 Glycosyltransferases in GT47 catalyze the transfer of a wide variety of monosaccharides from activated donor sugar nucleotides onto a diversity of acceptor substrates found in plants, animals, insects, and bacteria <cite>Zhang2023</cite>. Donor sugar nucleotides for discrete clades of GT47 enzymes include UDP-Arabinofuranose (UDP-Araf), UDP-Arabinopyranose (UDP-Arap), UDP-Xylose (UDP-Xyl), UDP-Galactose (UDP-Gal), UDP-Galacturonic acid (UDP-GalA), and UDP-Glucuronic acid (UDP-GlcA, i.e. EXT1) <cite>Zhang2023 LiX2004 Harholt2006 Wu2009 Madson2003 Pena2012 LiH2023</cite>. Genes encoding members of the GT47 family are found across all domains of life, and known biochemical pathways GT47 enzymes include diverse  plant cell wall polysaccharides and glycoproteins and the heparan sulfate backbone <cite>Zhang2023 LiH2023</cite>.
-<u>'''Plants'''</u>
+=== Plants ===
 The GT47 family is highly diversified in plants, having an association with the biosynthesis of almost every class of plant cell wall polysaccharide <cite>Zhang2023</cite>. Although the enzymatic functions for the vast majority of plant GT47s are currently unknown, analysis of plant mutants has identified members predicted to use UDP-GalA, UDP-Gal, UDP-Arap, UDP-Araf, and UDP-Xyl as activated sugar donors. Known acceptor polysaccharide substrates include xyloglucan, xylan, galacto-glucomannan, xylo-galacturonan, cell wall extensins, and rhamnogalacturonan I. Most members of GT47 from plants have been identified through analysis of mutants.
@@ Line 49: / Line 49: @@
 Unlike the previously mentioned polysaccharides, extensins are rod-like hydroxyproline rich glycoproteins (HRGP) that form crosslinked networks in the plant cell wall. These networks are reported to play a crucial role in regulating cell wall growth and development <cite>Showalter2016</cite>. A unique member of the GT47 family, Extensin Arabinose Deficient transferase (ExAD), is reported to synthesize the addition of the fourth arabinofuranose (Araf) on Araf substituted C4-hydroxyprolines (Hyps) creating Hyp-Araf4, a unique feature found on extensins <cite>Moller2017 Showalter2016</cite>.
-<u>'''Animals'''</u>
+=== Animals''' ===
 The abundance of GT47 family enzymes in mammals is more restricted and includes only members of the Exostosin (EXT) and Exostoslin-Like (EXTL) family of enzymes involved in heparan sulfate biosynthesis. Heparan sulfate is comprised of a repeat disaccharide polymer of ( GlcAβ1,4GlcNAcα1,4-)n that is further elaborated with extensive sulfation along the polymer chain. The disaccharide backbone repeat is elongated by the co-polymerase activity of the heterodimeric EXT1-EXT2 complex <cite>LiH2023</cite>. EXT1 and EXT2 are homologous two domain enzymes, and each protein chain contains a GT47 β1,4-GlcA transferase-like and a GT64 α1,4GlcNAc transferase-like domain. Surprisingly, only the GT47 domain of EXT1 and GT64 domain of EXT2 exhibit catalytic activity, while the other domains in each subunit are nonfunctional <cite>LiH2023</cite>. Additional EXT homologs include the EXTL proteins, EXTL1-3. EXTL1 and EXTL3 are two domain proteins, each harboring a GT47 and GT64 domain like EXT1 and EXT2. However, only the GT64 domains exhibit α1,4GlcNAc transferase activity, while their corresponding GT47 domains are inactive. In contrast, EXTL2 is a single GT64 domain enzyme with a α1,4GlcNAc transferase activity, while the corresponding GT47 domain present in other EXTs is missing. Thus, among the five mammalian EXT or EXTL homologs, only EXT1 contains a functional GT47 domain exhibiting β1,4-GlcA transferase activity.

@@ Line 56: / Line 56: @@
 GT47 enzymes employ an inverting catalytic mechanism where the hydroxyl group of an acceptor substrate presumably acts as a nucleophile in a SN2 single displacement reaction. The result is an inversion of the anomeric configuration of the transferred sugar from an α-linked sugar nucleotide donor to form a β-linked extended glycan product. While an SN2 mechanism would predict the deprotonation of the acceptor nucleophile by an enzyme associated catalytic base, the structure of the EXT1 active site did not appear to contain an appropriately positioned ionizable group to act as catalytic base <cite>LiH2023</cite>. Similar structural studies on the inverting GT-B fold glycosyltransferases, POFUT1 <cite>LiZ2017 Lira2018 Lira2011</cite> and AtFUT1 <cite>Urbanowicz2017</cite>, also indicated the lack of an appropriately positioned catalytic base for deprotonation. In these latter cases a non-canonical SN1-like mechanism was proposed. A similar SN1-like mechanism may also occur for the GT47 enzymes <cite>Zhang2023 Moremen2019</cite>.
-[[File:GT47_mechanism_V5.jpg|thumb|300px|right|'''Figure 1: Proposed Mechanism of GT47 Domain in EXT1.''' GT47 enzymes employ an inverting catalytic mechanism through two potential inverting mechanisms. A) In the SN2 inverting mechanism, the hydroxyl group of an acceptor substrate (shown as GlcNAc) acts as nucleophile in a single displacement reaction leading to inversion in anomeric configuration of the transferred sugar. B) The lack of an appropriately positioned ionizable group to act as catalytic base in the EXT1 structure suggested a non-canonical SN1-like mechanism for the GT47 domain of EXT1. A similar SN1-like mechanism may also occur for other GT47 enzymes.]]
+[[File:GT47_mechanism_V7.jpg|thumb|300px|right|'''Figure 1: Proposed Mechanism of GT47 Domain in EXT1.''' GT47 enzymes employ an inverting catalytic mechanism through two potential inverting mechanisms. A) In the SN2 inverting mechanism, the hydroxyl group of an acceptor substrate (shown as GlcNAc) acts as nucleophile in a single displacement reaction leading to inversion in anomeric configuration of the transferred sugar. B) The lack of an appropriately positioned ionizable group to act as catalytic base in the EXT1 structure suggested a non-canonical SN1-like mechanism for the GT47 domain of EXT1. A similar SN1-like mechanism may also occur for other GT47 enzymes.]]
 == Catalytic Residues ==