Binding of the substrate UDP-glucuronic acid induces conformational changes in the xanthan gum glucuronosyltransferase

GumK is a membrane-associated glucuronosyltransferase of Xanthomonas campestris that is involved in xanthan gum biosynthesis. GumK belongs to the inverting GT-B superfamily and catalyzes the transfer of a glucuronic acid (GlcA) residue from uridine diphosphate (UDP)-GlcA (UDP-GlcA) to a lipid-PP-tri...

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Autores principales:	Salinas, Silvina R., Petruk, Ariel Alcides, Brukman, Nicolás G., Martí, Marcelo Adrián, Ielpi, Luis
Publicado:	2016
Materias:	Conformational change Glycosyltransferase GumK Membrane monotopic protein Xanthan Bins Biochemistry Circular dichroism spectroscopy Conformations Dichroism Glucose Molecular dynamics Proteins Conformational differences Glycosyl transferase Molecular dynamics simulations Substrate-binding sites Xanthomonas campestris Xanthan gum glucuronosyltransferase unclassified drug uridine diphosphate glucuronic acid xanthan xanthan gum glucuronosyltransferase bacterial polysaccharide protein aggregate protein binding amino acid substitution amino terminal sequence Article binding site carboxy terminal sequence circular dichroism complex formation conformational transition controlled study crystal structure enzyme conformation enzyme metabolism enzyme substrate complex hydrophobicity ligand binding membrane binding molecular docking molecular dynamics nonhuman priority journal protein aggregation protein degradation protein domain protein protein interaction turbidity chemistry enzymology metabolism protein conformation Binding Sites Glucuronosyltransferase Molecular Docking Simulation Molecular Dynamics Simulation Polysaccharides, Bacterial Protein Aggregates Protein Binding Protein Conformation Uridine Diphosphate Glucuronic Acid
Acceso en línea:	https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_17410126_v29_n6_p197_Salinas http://hdl.handle.net/20.500.12110/paper_17410126_v29_n6_p197_Salinas
Aporte de:	Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) de Universidad de Buenos Aires

id	paper:paper_17410126_v29_n6_p197_Salinas
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spelling	paper:paper_17410126_v29_n6_p197_Salinas2023-06-08T16:26:59Z Binding of the substrate UDP-glucuronic acid induces conformational changes in the xanthan gum glucuronosyltransferase Salinas, Silvina R. Petruk, Ariel Alcides Brukman, Nicolás G. Martí, Marcelo Adrián Ielpi, Luis Conformational change Glycosyltransferase GumK Membrane monotopic protein Xanthan Bins Biochemistry Circular dichroism spectroscopy Conformations Dichroism Glucose Molecular dynamics Proteins Conformational change Conformational differences Glycosyl transferase GumK Molecular dynamics simulations Substrate-binding sites Xanthan Xanthomonas campestris Xanthan gum glucuronosyltransferase unclassified drug uridine diphosphate glucuronic acid xanthan xanthan gum glucuronosyltransferase bacterial polysaccharide glucuronosyltransferase protein aggregate protein binding uridine diphosphate glucuronic acid xanthan amino acid substitution amino terminal sequence Article binding site carboxy terminal sequence circular dichroism complex formation conformational transition controlled study crystal structure enzyme conformation enzyme metabolism enzyme substrate complex hydrophobicity ligand binding membrane binding molecular docking molecular dynamics nonhuman priority journal protein aggregation protein degradation protein domain protein protein interaction turbidity chemistry enzymology metabolism molecular docking protein conformation Xanthomonas campestris Binding Sites Glucuronosyltransferase Molecular Docking Simulation Molecular Dynamics Simulation Polysaccharides, Bacterial Protein Aggregates Protein Binding Protein Conformation Uridine Diphosphate Glucuronic Acid Xanthomonas campestris GumK is a membrane-associated glucuronosyltransferase of Xanthomonas campestris that is involved in xanthan gum biosynthesis. GumK belongs to the inverting GT-B superfamily and catalyzes the transfer of a glucuronic acid (GlcA) residue from uridine diphosphate (UDP)-GlcA (UDP-GlcA) to a lipid-PP-trisaccharide embedded in the membrane of the bacteria. The structure of GumK was previously described in its apo- and UDP-bound forms, with no significant conformational differences being observed. Here, we study the behavior of GumK toward its donor substrate UDP-GlcA. Turbidity measurements revealed that the interaction of GumK with UDP-GlcA produces aggregation of protein molecules under specific conditions. Moreover, limited proteolysis assays demonstrated protection of enzymatic digestion when UDP-GlcA is present, and this protection is promoted by substrate binding. Circular dichroism spectroscopy also revealed changes in the GumK tertiary structure after UDP-GlcA addition. According to the obtained emission fluorescence results, we suggest the possibility of exposure of hydrophobic residues upon UDP-GlcA binding. We present in silico-built models of GumK complexed with UDP-GlcA as well as its analogs UDP-glucose and UDP-galacturonic acid. Through molecular dynamics simulations, we also show that a relative movement between the domains appears to be specific and to be triggered by UDP-GlcA. The results presented here strongly suggest that GumK undergoes a conformational change upon donor substrate binding, likely bringing the two Rossmann fold domains closer together and triggering a change in the N-terminal domain, with consequent generation of the acceptor substrate binding site. © 2016 The Author. Fil:Salinas, S.R. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Petruk, A.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Brukman, N.G. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Marti, M.A. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. Fil:Ielpi, L. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. 2016 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_17410126_v29_n6_p197_Salinas http://hdl.handle.net/20.500.12110/paper_17410126_v29_n6_p197_Salinas
institution	Universidad de Buenos Aires
institution_str	I-28
repository_str	R-134
collection	Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA)
topic	Conformational change Glycosyltransferase GumK Membrane monotopic protein Xanthan Bins Biochemistry Circular dichroism spectroscopy Conformations Dichroism Glucose Molecular dynamics Proteins Conformational change Conformational differences Glycosyl transferase GumK Molecular dynamics simulations Substrate-binding sites Xanthan Xanthomonas campestris Xanthan gum glucuronosyltransferase unclassified drug uridine diphosphate glucuronic acid xanthan xanthan gum glucuronosyltransferase bacterial polysaccharide glucuronosyltransferase protein aggregate protein binding uridine diphosphate glucuronic acid xanthan amino acid substitution amino terminal sequence Article binding site carboxy terminal sequence circular dichroism complex formation conformational transition controlled study crystal structure enzyme conformation enzyme metabolism enzyme substrate complex hydrophobicity ligand binding membrane binding molecular docking molecular dynamics nonhuman priority journal protein aggregation protein degradation protein domain protein protein interaction turbidity chemistry enzymology metabolism molecular docking protein conformation Xanthomonas campestris Binding Sites Glucuronosyltransferase Molecular Docking Simulation Molecular Dynamics Simulation Polysaccharides, Bacterial Protein Aggregates Protein Binding Protein Conformation Uridine Diphosphate Glucuronic Acid Xanthomonas campestris
spellingShingle	Conformational change Glycosyltransferase GumK Membrane monotopic protein Xanthan Bins Biochemistry Circular dichroism spectroscopy Conformations Dichroism Glucose Molecular dynamics Proteins Conformational change Conformational differences Glycosyl transferase GumK Molecular dynamics simulations Substrate-binding sites Xanthan Xanthomonas campestris Xanthan gum glucuronosyltransferase unclassified drug uridine diphosphate glucuronic acid xanthan xanthan gum glucuronosyltransferase bacterial polysaccharide glucuronosyltransferase protein aggregate protein binding uridine diphosphate glucuronic acid xanthan amino acid substitution amino terminal sequence Article binding site carboxy terminal sequence circular dichroism complex formation conformational transition controlled study crystal structure enzyme conformation enzyme metabolism enzyme substrate complex hydrophobicity ligand binding membrane binding molecular docking molecular dynamics nonhuman priority journal protein aggregation protein degradation protein domain protein protein interaction turbidity chemistry enzymology metabolism molecular docking protein conformation Xanthomonas campestris Binding Sites Glucuronosyltransferase Molecular Docking Simulation Molecular Dynamics Simulation Polysaccharides, Bacterial Protein Aggregates Protein Binding Protein Conformation Uridine Diphosphate Glucuronic Acid Xanthomonas campestris Salinas, Silvina R. Petruk, Ariel Alcides Brukman, Nicolás G. Martí, Marcelo Adrián Ielpi, Luis Binding of the substrate UDP-glucuronic acid induces conformational changes in the xanthan gum glucuronosyltransferase
topic_facet	Conformational change Glycosyltransferase GumK Membrane monotopic protein Xanthan Bins Biochemistry Circular dichroism spectroscopy Conformations Dichroism Glucose Molecular dynamics Proteins Conformational change Conformational differences Glycosyl transferase GumK Molecular dynamics simulations Substrate-binding sites Xanthan Xanthomonas campestris Xanthan gum glucuronosyltransferase unclassified drug uridine diphosphate glucuronic acid xanthan xanthan gum glucuronosyltransferase bacterial polysaccharide glucuronosyltransferase protein aggregate protein binding uridine diphosphate glucuronic acid xanthan amino acid substitution amino terminal sequence Article binding site carboxy terminal sequence circular dichroism complex formation conformational transition controlled study crystal structure enzyme conformation enzyme metabolism enzyme substrate complex hydrophobicity ligand binding membrane binding molecular docking molecular dynamics nonhuman priority journal protein aggregation protein degradation protein domain protein protein interaction turbidity chemistry enzymology metabolism molecular docking protein conformation Xanthomonas campestris Binding Sites Glucuronosyltransferase Molecular Docking Simulation Molecular Dynamics Simulation Polysaccharides, Bacterial Protein Aggregates Protein Binding Protein Conformation Uridine Diphosphate Glucuronic Acid Xanthomonas campestris
description	GumK is a membrane-associated glucuronosyltransferase of Xanthomonas campestris that is involved in xanthan gum biosynthesis. GumK belongs to the inverting GT-B superfamily and catalyzes the transfer of a glucuronic acid (GlcA) residue from uridine diphosphate (UDP)-GlcA (UDP-GlcA) to a lipid-PP-trisaccharide embedded in the membrane of the bacteria. The structure of GumK was previously described in its apo- and UDP-bound forms, with no significant conformational differences being observed. Here, we study the behavior of GumK toward its donor substrate UDP-GlcA. Turbidity measurements revealed that the interaction of GumK with UDP-GlcA produces aggregation of protein molecules under specific conditions. Moreover, limited proteolysis assays demonstrated protection of enzymatic digestion when UDP-GlcA is present, and this protection is promoted by substrate binding. Circular dichroism spectroscopy also revealed changes in the GumK tertiary structure after UDP-GlcA addition. According to the obtained emission fluorescence results, we suggest the possibility of exposure of hydrophobic residues upon UDP-GlcA binding. We present in silico-built models of GumK complexed with UDP-GlcA as well as its analogs UDP-glucose and UDP-galacturonic acid. Through molecular dynamics simulations, we also show that a relative movement between the domains appears to be specific and to be triggered by UDP-GlcA. The results presented here strongly suggest that GumK undergoes a conformational change upon donor substrate binding, likely bringing the two Rossmann fold domains closer together and triggering a change in the N-terminal domain, with consequent generation of the acceptor substrate binding site. © 2016 The Author.
author	Salinas, Silvina R. Petruk, Ariel Alcides Brukman, Nicolás G. Martí, Marcelo Adrián Ielpi, Luis
author_facet	Salinas, Silvina R. Petruk, Ariel Alcides Brukman, Nicolás G. Martí, Marcelo Adrián Ielpi, Luis
author_sort	Salinas, Silvina R.
title	Binding of the substrate UDP-glucuronic acid induces conformational changes in the xanthan gum glucuronosyltransferase
title_short	Binding of the substrate UDP-glucuronic acid induces conformational changes in the xanthan gum glucuronosyltransferase
title_full	Binding of the substrate UDP-glucuronic acid induces conformational changes in the xanthan gum glucuronosyltransferase
title_fullStr	Binding of the substrate UDP-glucuronic acid induces conformational changes in the xanthan gum glucuronosyltransferase
title_full_unstemmed	Binding of the substrate UDP-glucuronic acid induces conformational changes in the xanthan gum glucuronosyltransferase
title_sort	binding of the substrate udp-glucuronic acid induces conformational changes in the xanthan gum glucuronosyltransferase
publishDate	2016
url	https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_17410126_v29_n6_p197_Salinas http://hdl.handle.net/20.500.12110/paper_17410126_v29_n6_p197_Salinas
work_keys_str_mv	AT salinassilvinar bindingofthesubstrateudpglucuronicacidinducesconformationalchangesinthexanthangumglucuronosyltransferase AT petrukarielalcides bindingofthesubstrateudpglucuronicacidinducesconformationalchangesinthexanthangumglucuronosyltransferase AT brukmannicolasg bindingofthesubstrateudpglucuronicacidinducesconformationalchangesinthexanthangumglucuronosyltransferase AT martimarceloadrian bindingofthesubstrateudpglucuronicacidinducesconformationalchangesinthexanthangumglucuronosyltransferase AT ielpiluis bindingofthesubstrateudpglucuronicacidinducesconformationalchangesinthexanthangumglucuronosyltransferase
_version_	1768544384734724096

Binding of the substrate UDP-glucuronic acid induces conformational changes in the xanthan gum glucuronosyltransferase

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