RESUMO
This work illustrates the highly selective fluorescence detection of ATP in the presence of other competing anions, such as AMP, ADP, PPi and other phosphates by using a set of hydroxide-bridged dizinc(ii) complexes offering a cavity lined with hydrogen bonds and other interactive forces. ATP, as a whole, was recognized by the synergic combination of Zn-phosphate bonding, ππ stacking between the adenine ring of ATP and the pyridine ring of the dizinc complex and hydrogen bonding interactions that modulate the cavity structure of the dizinc complexes.
Assuntos
Difosfato de Adenosina/química , Monofosfato de Adenosina/química , Trifosfato de Adenosina/química , Complexos de Coordenação/química , Difosfatos/química , Hidróxidos/química , Zinco/química , Difosfato de Adenosina/isolamento & purificação , Monofosfato de Adenosina/isolamento & purificação , Trifosfato de Adenosina/isolamento & purificação , Cristalografia por Raios X , Difosfatos/isolamento & purificação , Fluorescência , Ligação de Hidrogênio , Modelos Moleculares , Estrutura MolecularRESUMO
Calyculin C, a minor derivative of the calyculins, has an additional methyl group on C32 of calyculin A. A recent biosynthetic study of calyculins revealed that an end product of calyculin biosynthesis is the pyrophosphate form, phosphocalyculin A. However, the pyrophosphate counterpart derived from calyculin C had not been reported. We isolated phosphocalyculin C as a minor pyrophosphate derivative, by a detailed investigation of an extract from the sponge Discodermia calyx. The treatment of phosphocalyculin C with the D. calyx cell-free extract significantly enhanced its cytotoxicity, providing molecular evidence for its role as the protoxin of calyculin C.
Assuntos
Difosfatos/isolamento & purificação , Difosfatos/toxicidade , Oxazóis/isolamento & purificação , Oxazóis/toxicidade , Poríferos , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Citotoxinas/isolamento & purificação , Citotoxinas/farmacologia , Relação Dose-Resposta a Droga , Toxinas Marinhas , CamundongosRESUMO
Octaprenyl pyrophosphate synthase (OPPs) is involved in the synthesis of the side chains of ubiquinone and menaquinone and catalyzes consecutive condensation reactions of farnesyl pyrophosphate with isopentenyl pyrophosphate to generate polyprenyl pyrophosphate and pyrophosphate. In order to investigate the roles played by OPPs in the metabolism of ubiquinone and menaquinone and the enzymatic mechanisms of these enzymes, analysis of the structure-function relationship of OPPs from Helicobacter pylori was initiated. The gene for OPPs was cloned, the protein was expressed, purified and crystallized and a diffraction data set was collected to 2.00â Å resolution. The crystals belonged to space group P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 109.33, c = 103.41â Å.
Assuntos
Alquil e Aril Transferases/química , Alquil e Aril Transferases/metabolismo , Difosfatos/química , Difosfatos/metabolismo , Helicobacter pylori/enzimologia , Catálise , Cristalização , Cristalografia por Raios X/métodos , Difosfatos/isolamento & purificação , Helicobacter pylori/genética , Relação Estrutura-Atividade , Difração de Raios XRESUMO
Human Vgamma2Vdelta2 T cells are stimulated by prenyl pyrophosphates, such as isopentenyl pyrophosphate (IPP), and play important roles in mediating immunity against microbial pathogens and have potent anti-tumor activity. (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) has been identified as a metabolite in the 2-C-methyl-D-erythritol-4 phosphate (MEP) pathway for isoprenoid biosynthesis that is used by many bacteria and protozoan parasites. We find that HMBPP is the major Vgamma2Vdelta2 T-cell antigen for many bacteria, including Mycobacterium tuberculosis, Yersinia enterocolitica and Escherichia coli. HMBPP was a 30 000-fold more potent antigen than IPP. Using mutant bacteria, we show that bacterial antigen levels for Vgamma2Vdelta2 T cells are controlled by MEP pathway enzymes and find no evidence for the production of 3-formyl-1-butyl pyrophosphate. Moreover, HMBPP reactivity required only germ line-encoded Vgamma2Vdelta2 TCR elements and is present at birth. Importantly, we show that bacterial HMBPP levels correlated with their ability to expand Vgamma2Vdelta2 T cells in vivo upon engraftment into severe combined immunodeficiency-beige mice. Thus, the production of HMBPP by a microbial-specific isoprenoid pathway plays a major role in determining whether bacteria will stimulate Vgamma2Vdelta2 T cells in vivo. This preferential stimulation by a common microbial isoprenoid metabolite allows Vgamma2Vdelta2 T cells to respond to a broad array of pathogens using this pathway.