Formation of a covalent adduct in retaining β-Kdo glycosyl-transferase WbbB via substrate-mediated proton relay
The GT99 domain of the membrane-anchored WbbB glycosyltransferase (WbbBGT99) catalyzes the transfer of 3-deoxy-D-manno-oct-2-acid (β-Kdo) to an O-antigen saccharide acceptor with retention of stereochemistry. It has been proposed that the enzyme follows an unprecedented double-displacement mechanism...
| Autores: | , , , |
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| Tipo de recurso: | artículo |
| Estado: | Versión publicada |
| Fecha de publicación: | 2024 |
| País: | España |
| Institución: | Varias* (Consorci de Biblioteques Universitáries de Catalunya, Centre de Serveis Científics i Acadèmics de Catalunya) |
| Repositorio: | Recercat. Dipósit de la Recerca de Catalunya |
| OAI Identifier: | oai:dnet:recercat____::f2d74ff6cbc3134ec68994c1e9865d1f |
| Acceso en línea: | https://hdl.handle.net/2445/229478 |
| Access Level: | acceso abierto |
| Palabra clave: | Proteïnes portadores Espectrometria de masses Glicoproteïnes Carrier proteins Mass spectrometry Glycoproteins |
| Sumario: | The GT99 domain of the membrane-anchored WbbB glycosyltransferase (WbbBGT99) catalyzes the transfer of 3-deoxy-D-manno-oct-2-acid (β-Kdo) to an O-antigen saccharide acceptor with retention of stereochemistry. It has been proposed that the enzyme follows an unprecedented double-displacement mechanism involving the formation of covalent adduct between the Kdo sugar and an active site residue (Asp232) that is properly oriented for nucleophilic attack. Here we use QM/MM metadynamics simulations on recently reported crystal structures to provide theoretical evidence for the formation of such adduct and unveil the atomic details of the chemical reaction. Our results support the interpretation made on the basis of X-ray and mass spectrometry analyses. Moreover, we show that the formation of the β-Kdo-Asp232 adduct is assisted by the sugar Kdo-carboxylate group, which mediates the transfer of a proton from Asp232 towards the phosphate leaving group, alleviating electrostatic repulsion between the two negatively charged carboxylate groups. The computed mechanism also explains whyvHis265, previously proposed to act as a general acid, does not impairvcatalysis. This mechanism can be extended to other related enzymes, expanding the repertoire of GT mechanisms in Nature. |
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