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-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.
Laboratory of Bacterial Genetics, Fundación Instituto Leloir, IIBBA-CONICET, Av. Patricias Argentinas 435, C1405BWE Buenos Aires, Argentina.
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Salinas
Departamento de Química Inorgánica, Analítica, y Química Física/INQUIMAE CONICET, Córdoba, Argentina.|Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, C1428EHA Buenos Aires, Argentina.
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Petruk
Laboratory of Bacterial Genetics, Fundación Instituto Leloir, IIBBA-CONICET, Av. Patricias Argentinas 435, C1405BWE Buenos Aires, Argentina.
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Brukman
Laboratory of Bacterial Genetics, Fundación Instituto Leloir, IIBBA-CONICET, Av. Patricias Argentinas 435, C1405BWE Buenos Aires, Argentina.
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Bianco
Laboratory of Bacterial Genetics, Fundación Instituto Leloir, IIBBA-CONICET, Av. Patricias Argentinas 435, C1405BWE Buenos Aires, Argentina.
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Jacobs
Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Universitaria, Pabellón 2, C1428EHA Buenos Aires, Argentina.
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Marti
Laboratory of Bacterial Genetics, Fundación Instituto Leloir, IIBBA-CONICET, Av. Patricias Argentinas 435, C1405BWE Buenos Aires, Argentina.
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Ielpi
D001665:Binding Sites; D014453:Glucuronosyltransferase / Q000737:chemistry / Q000378:metabolism*; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D011135:Polysaccharides, Bacterial / Q000378:metabolism*; D066329:Protein Aggregates; D011485:Protein Binding; D011487:Protein Conformation; D014535:Uridine Diphosphate Glucuronic Acid / Q000378:metabolism*; D016959:Xanthomonas campestris / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism
D011135:Polysaccharides, Bacterial; D066329:Protein Aggregates; D014535:Uridine Diphosphate Glucuronic Acid; D014453:Glucuronosyltransferase; C002563:xanthan gum
GumK; conformational change; glycosyltransferase; membrane monotopic protein; xanthan