RESUMO
UMP kinase catalyses the phosphorylation of UMP by ATP to yield UDP and ADP. In prokaryotes, the reaction is carried out by a hexameric enzyme, activated by GTP and inhibited by UTP. In the present study, Streptococcus pneumoniae UMP kinase was studied as a target for antibacterial research and its interest was confirmed by the demonstration of the essentiality of the gene for cell growth. In the presence of MnCl2 or MgCl2, the saturation kinetics of recombinant purified UMP kinase was hyperbolic for UMP (K(m)=0.1 mM) and sigmoidal for ATP (the substrate concentration at half-saturation S0.5=9.4+/-0.7 mM and n=1.9+/-0.1 in the presence of MgCl2). GTP increased the affinity for ATP and decreased the Hill coefficient (n). UTP decreased the affinity for ATP and only slightly increased the Hill coefficient. The kcat (175+/-13 s(-1) in the presence of MgCl2) was not affected by the addition of GTP or UTP, whose binding site was shown to be different from the active site. The hydrodynamic radius of the protein similarly decreased in the presence of ATP or GTP. There was a shift in the pH dependence of the activity when the ATP concentration was switched from low to high. These results support the hypothesis of an allosteric transition from a conformation with low affinity for ATP to a form with high affinity, which would be induced by the presence of ATP or GTP.
Assuntos
Trifosfato de Adenosina/metabolismo , Núcleosídeo-Fosfato Quinase/metabolismo , Streptococcus pneumoniae/enzimologia , Difosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Regulação Alostérica/efeitos dos fármacos , Sítio Alostérico , Sequência de Aminoácidos , Varredura Diferencial de Calorimetria , Catálise/efeitos dos fármacos , Cátions/metabolismo , Cromatografia em Gel , Clonagem Molecular , Estabilidade Enzimática , Guanosina Trifosfato/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Ligantes , Cloreto de Magnésio/farmacologia , Dados de Sequência Molecular , Núcleosídeo-Fosfato Quinase/química , Núcleosídeo-Fosfato Quinase/genética , Fosforilação/efeitos dos fármacos , Desnaturação Proteica , Streptococcus pneumoniae/genética , Especificidade por Substrato , Temperatura , Uridina Monofosfato/metabolismoRESUMO
The formation of the Mtr2-Mex67 heterodimer is essential for yeast mRNA export as it constitutes a key nuclear component for shuttling mRNA between the nuclear and cytoplasm compartments through the nuclear pore complex. We report the crystal structures of apo-Mtr2 from the human pathogen Candida albicans and of its complex with the Mex67 NTF2-like domain. Compared with other members of the NTF2 fold family, Mtr2 displays novel structural features involved in the nuclear export of the large ribosomal subunit and consistent with a dual functional role of Mtr2 during yeast nuclear export events. The structure of the Mtr2-Mex67 NTF2-like domain complex, which overall is similar to those of the human and Saccharomyces cerevisiae homologs, unveils three putative Phe-Gly repeat binding sites, of which one contributes to the heterodimer interface. These structures exemplify an unrecognized adaptability of the NTF2 building block in evolution, identify novel structural determinants associated with key biological functions at the molecular surface of the yeast Mtr2-Mex67 complex, and suggest that the yeast and human mRNA export machineries may differ.
Assuntos
Núcleo Celular/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas Nucleares/química , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Ligação a RNA/química , Proteínas de Saccharomyces cerevisiae/química , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Sítios de Ligação , Candida albicans/metabolismo , Cromatografia em Gel , Cristalografia por Raios X , Citoplasma/metabolismo , Dimerização , Proteínas Fúngicas/química , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
We report on the production of hydrocortisone, the major adrenal glucocorticoid of mammals and an important intermediate of steroidal drug synthesis, from a simple carbon source by recombinant Saccharomyces cerevisiae strains. An artificial and fully self-sufficient biosynthetic pathway involving 13 engineered genes was assembled and expressed in a single yeast strain. Endogenous sterol biosynthesis was rerouted to produce compatible sterols to serve as substrates for the heterologous part of the pathway. Biosynthesis involves eight mammalian proteins (mature forms of CYP11A1, adrenodoxin (ADX), and adrenodoxin reductase (ADR); mitochondrial forms of ADX and CYP11B1; 3beta-HSD, CYP17A1, and CYP21A1). Optimization involved modulating the two mitochondrial systems and disrupting of unwanted side reactions associated with ATF2, GCY1, and YPR1 gene products. Hydrocortisone was the major steroid produced. This work demonstrates the feasibility of transfering a complex biosynthetic pathway from higher eukaryotes into microorganisms.
