RESUMO
Dual-specificity protein phosphatases (DUSPs), which dephosphorylate both phosphoserine/threonine and phosphotyrosine, play vital roles in immune activation, brain function and cell-growth signalling. A family-wide structural library of human DUSPs was constructed based on experimental structure determination supplemented with homology modelling. The catalytic domain of each individual DUSP has characteristic features in the active site and in surface-charge distribution, indicating substrate-interaction specificity. The active-site loop-to-strand switch occurs in a subtype-specific manner, indicating that the switch process is necessary for characteristic substrate interactions in the corresponding DUSPs. A comprehensive analysis of the activity-inhibition profile and active-site geometry of DUSPs revealed a novel role of the active-pocket structure in the substrate specificity of DUSPs. A structure-based analysis of redox responses indicated that the additional cysteine residues are important for the protection of enzyme activity. The family-wide structures of DUSPs form a basis for the understanding of phosphorylation-mediated signal transduction and the development of therapeutics.
Assuntos
Fosfatases de Especificidade Dupla/química , Fosfatases de Especificidade Dupla/classificação , Inibidores Enzimáticos/química , Filogenia , Domínio Catalítico , Cristalografia por Raios X , Cisteína/química , Fosfatases de Especificidade Dupla/antagonistas & inibidores , Fosfatases de Especificidade Dupla/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Hidrólise , Modelos Moleculares , Oxirredução , Fosfosserina/química , Fosfotreonina/química , Fosfotirosina/química , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/classificação , Proteínas Recombinantes/genética , Transdução de Sinais , Homologia Estrutural de Proteína , Especificidade por SubstratoRESUMO
A series of l and dl forms of O-phosphorylated amino acids (serine, threonine, tyrosine) have been studied by using solid-state multinuclear NMR spectroscopy and ab initio calculations. Principal elements of the (13)C and (31)P chemical shielding tensors have been measured and discussed in relation to zwitterionic structures and intermolecular contacts. DFT calculations have been compared with experimental data showing their ability to reproduce experimentally obtained tensor values in this challenging class of compounds. The changes of orientation of (31)P chemical shielding tensor with respect to the molecular frame in the presence of hydrogen bonds have been revealed and discussed on the ground of theoretical calculations. The measurements of internuclear P...P distances, based on Zeeman magnetization exchange between (31)P spins with differing chemical shielding tensor orientations, were exploited for a clear distinction between enantiomers and racemates.
Assuntos
Aminoácidos/química , Ressonância Magnética Nuclear Biomolecular/métodos , Fosfosserina/química , Fosfotreonina/química , Fosfotirosina/química , Isótopos de Carbono , Fósforo/análise , Fosforilação , EstereoisomerismoRESUMO
Methyl-coenzyme M reductase (MCR) is a nickel enzyme catalyzing the formation of methane from methyl-coenzyme M and coenzyme B in all methanogenic archaea. The active purified enzyme exhibits the axial EPR signal MCR-red1 and in the presence of coenzyme M and coenzyme B the rhombic signal MCR-red2, both derived from Ni(I). Two other EPR-detectable states of the enzyme have been observed in vivo and in vitro designated MCR-ox1 and MCR-ox2 which have quite different nickel EPR signals and which are inactive. Until now the MCR-ox1 and MCR-ox2 states could only be induced in vivo. We report here that in vitro the MCR-red2 state is converted into the MCR-ox1 state by the addition of polysulfide and into a light-sensitive MCR-ox2 state by the addition of sulfite. In the presence of O(2) the MCR-red2 state was converted into a novel third state designated MCR-ox3 and exhibiting two EPR signals similar but not identical to MCR-ox1 and MCR-ox2. The formation of the MCR-ox states was dependent on the presence of coenzyme B. Investigations with the coenzyme B analogues S-methyl-coenzyme B and desulfa-methyl-coenzyme B indicate that for the induction of the MCR-ox states the thiol group of coenzyme B is probably not of importance. The results were obtained with purified active methyl-coenzyme M reductase isoenzyme I from Methanothermobacter marburgensis. They are discussed with respect to the nickel oxidation states in MCR-ox1, MCR-ox2 and MCR-ox3 and to a possible presence of a second redox active group in the active site. Electronic supplementary material to this paper can be obtained by using the Springer LINK server located at http://dx.doi.org/10.1007/s00775-001-0325-z.