RESUMO
Mammalian cyclic GMP-AMP synthase (cGAS) and its homologue dinucleotide cyclase in Vibrio cholerae (VcDncV) produce cyclic dinucleotides (CDNs) that participate in the defense against viral infection. Recently, scores of new cGAS/DncV-like nucleotidyltransferases (CD-NTases) were discovered, which produce various CDNs and cyclic trinucleotides (CTNs) as second messengers. Here, we present the crystal structures of EcCdnD, a CD-NTase from Enterobacter cloacae that produces cyclic AMP-AMP-GMP, in its apo-form and in complex with ATP, ADP and AMPcPP, an ATP analogue. Despite the similar overall architecture, the protein shows significant structural variations from other CD-NTases. Adjacent to the donor substrate, another nucleotide is bound to the acceptor binding site by a non-productive mode. Isothermal titration calorimetry results also suggest the presence of two ATP binding sites. GTP alone does not bind to EcCdnD, which however binds to pppApG, a possible intermediate. The enzyme is active on ATP or a mixture of ATP and GTP, and the best metal cofactor is Mg2+. The conserved residues Asp69 and Asp71 are essential for catalysis, as indicated by the loss of activity in the mutants. Based on structural analysis and comparison with VcDncV and RNA polymerase, a tentative catalytic pathway for the CTN-producing EcCdnD is proposed.
Assuntos
Trifosfato de Adenosina/química , Enterobacter cloacae/química , Magnésio/química , Nucleotídeos Cíclicos/química , Nucleotidiltransferases/química , Sítios de Ligação , Varredura Diferencial de Calorimetria , Catálise , Cristalografia por Raios X , Enterobacter cloacae/enzimologia , Guanosina Trifosfato/química , Ligantes , Mutação , Nucleotidiltransferases/síntese químicaAssuntos
Nucleotidiltransferases/análise , Staphylococcus/enzimologia , Sequência de Aminoácidos , Ácidos Carboxílicos/análise , Cromatografia em Gel , Ativação Enzimática , Fluorescência , Nucleotidiltransferases/síntese química , Peptídeos/síntese química , Fenilalanina/análise , Relação Estrutura-AtividadeRESUMO
The polypeptide corresponding to the amino acid sequence from residue 6 through 47 in staphylococcal nuclease has been synthesized by the solid-phase method. The synthetic product closely resembles the corresponding native polypeptide in both physical and chemical properties. The synthetic peptide may be recombined with the complimentary native peptide comprising residues 49 through 149 to form an active, semisynthetic enzyme. The "functional purification" of the crude, synthetic polypeptide by affinity chromatography was found to yield a synthetic fraction of greatly enhanced specific activity. This purification was accomplished on a column of Sepharose to which the complimentary native peptide had been covalently bound.