Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 4 de 4
Filtrar
Más filtros

Banco de datos
Tipo del documento
Intervalo de año de publicación
1.
Cell ; 183(4): 1086-1102.e23, 2020 11 12.
Artículo en Inglés | MEDLINE | ID: mdl-33186521

RESUMEN

Strategies for installing authentic ADP-ribosylation (ADPr) at desired positions are fundamental for creating the tools needed to explore this elusive post-translational modification (PTM) in essential cellular processes. Here, we describe a phospho-guided chemoenzymatic approach based on the Ser-ADPr writer complex for rapid, scalable preparation of a panel of pure, precisely modified peptides. Integrating this methodology with phage display technology, we have developed site-specific as well as broad-specificity antibodies to mono-ADPr. These recombinant antibodies have been selected and characterized using multiple ADP-ribosylated peptides and tested by immunoblotting and immunofluorescence for their ability to detect physiological ADPr events. Mono-ADPr proteomics and poly-to-mono comparisons at the modification site level have revealed the prevalence of mono-ADPr upon DNA damage and illustrated its dependence on PARG and ARH3. These and future tools created on our versatile chemical biology-recombinant antibody platform have broad potential to elucidate ADPr signaling pathways in health and disease.


Asunto(s)
ADP-Ribosilación , Proteínas Portadoras/metabolismo , Proteínas Nucleares/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , ADP-Ribosilación/efectos de los fármacos , Secuencia de Aminoácidos , Anticuerpos/metabolismo , Bencimidazoles/farmacología , Línea Celular Tumoral , Técnicas de Visualización de Superficie Celular , Daño del ADN , Glicósido Hidrolasas/metabolismo , Histonas/metabolismo , Humanos , Fosfatos/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Ftalazinas/farmacología , Piperazinas/farmacología , Poli(ADP-Ribosa) Polimerasa-1/química , Proteínas Recombinantes/metabolismo , Serina/metabolismo , Tirosina/metabolismo
2.
Angew Chem Int Ed Engl ; 60(11): 5704-5707, 2021 03 08.
Artículo en Inglés | MEDLINE | ID: mdl-33320413

RESUMEN

As an approach towards unraveling the nitrogenase mechanism, we have studied the binding of CO to the active-site FeMo-cofactor. CO is not only an inhibitor of nitrogenase, but it is also a substrate, undergoing reduction to hydrocarbons (Fischer-Tropsch-type chemistry). The C-C bond forming capabilities of nitrogenase suggest that multiple CO or CO-derived ligands bind to the active site. Herein, we report a crystal structure with two CO ligands coordinated to the FeMo-cofactor of the molybdenum nitrogenase at 1.33 Šresolution. In addition to the previously observed bridging CO ligand between Fe2 and Fe6 of the FeMo-cofactor, a new ligand binding mode is revealed through a second CO ligand coordinated terminally to Fe6. While the relevance of this state to nitrogenase-catalyzed reactions remains to be established, it highlights the privileged roles for Fe2 and Fe6 in ligand binding, with multiple coordination modes available depending on the ligand and reaction conditions.


Asunto(s)
Monóxido de Carbono/metabolismo , Nitrogenasa/metabolismo , Sitios de Unión , Monóxido de Carbono/química , Ligandos , Molibdoferredoxina/química , Molibdoferredoxina/metabolismo , Nitrogenasa/química
3.
Elife ; 4: e11620, 2015 Dec 16.
Artículo en Inglés | MEDLINE | ID: mdl-26673079

RESUMEN

Dinitrogen reduction in the biological nitrogen cycle is catalyzed by nitrogenase, a two-component metalloenzyme. Understanding of the transformation of the inert resting state of the active site FeMo-cofactor into an activated state capable of reducing dinitrogen remains elusive. Here we report the catalysis dependent, site-selective incorporation of selenium into the FeMo-cofactor from selenocyanate as a newly identified substrate and inhibitor. The 1.60 Å resolution structure reveals selenium occupying the S2B site of FeMo-cofactor in the Azotobacter vinelandii MoFe-protein, a position that was recently identified as the CO-binding site. The Se2B-labeled enzyme retains substrate reduction activity and marks the starting point for a crystallographic pulse-chase experiment of the active site during turnover. Through a series of crystal structures obtained at resolutions of 1.32-1.66 Å, including the CO-inhibited form of Av1-Se2B, the exchangeability of all three belt-sulfur sites is demonstrated, providing direct insights into unforeseen rearrangements of the metal center during catalysis.


Asunto(s)
Azotobacter vinelandii/enzimología , Molibdoferredoxina/metabolismo , Nitrogenasa/metabolismo , Selenio/metabolismo , Azotobacter vinelandii/química , Dominio Catalítico , Cristalografía por Rayos X , Cianatos/metabolismo , Modelos Moleculares , Molibdoferredoxina/química , Conformación Proteica , Compuestos de Selenio/metabolismo
4.
Science ; 345(6204): 1620-3, 2014 Sep 26.
Artículo en Inglés | MEDLINE | ID: mdl-25258081

RESUMEN

The mechanism of nitrogenase remains enigmatic, with a major unresolved issue concerning how inhibitors and substrates bind to the active site. We report a crystal structure of carbon monoxide (CO)-inhibited nitrogenase molybdenum-iron (MoFe)-protein at 1.50 angstrom resolution, which reveals a CO molecule bridging Fe2 and Fe6 of the FeMo-cofactor. The µ2 binding geometry is achieved by replacing a belt-sulfur atom (S2B) and highlights the generation of a reactive iron species uncovered by the displacement of sulfur. The CO inhibition is fully reversible as established by regain of enzyme activity and reappearance of S2B in the 1.43 angstrom resolution structure of the reactivated enzyme. The substantial and reversible reorganization of the FeMo-cofactor accompanying CO binding was unanticipated and provides insights into a catalytically competent state of nitrogenase.


Asunto(s)
Monóxido de Carbono/química , Molibdoferredoxina/química , Fijación del Nitrógeno , Dominio Catalítico , Cristalografía por Rayos X , Activación Enzimática , Ligandos , Molibdoferredoxina/antagonistas & inhibidores , Unión Proteica , Azufre/química
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA