RESUMO
Wild-type Streptomyces coelicolor A3(2) produces aminobacteriohopanetriol as the only elongated C35 hopanoid. The hopanoid phenotype of two mutants bearing a deletion of genes from a previously identified hopanoid biosynthesis gene cluster provides clues to the formation of C35 bacteriohopanepolyols. orf14 encodes a putative nucleosidase; its deletion induces the accumulation of adenosylhopane as it cannot be converted into ribosylhopane. orf18 encodes a putative transaminase; its deletion results in the accumulation of adenosylhopane, ribosylhopane, and bacteriohopanetetrol. Ribosylhopane was postulated twenty years ago as a precursor for bacterial hopanoids but was never identified in a bacterium. Absence of the transaminase encoded by orf18 prevents the reductive amination of ribosylhopane into aminobacteriohopanetriol and induces its accumulation. Its reduction by an aldose-reductase-like enzyme produces bacteriohopanetetrol, which is normally not present in S. coelicolor.
Assuntos
Streptomyces coelicolor/metabolismo , Triterpenos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Vias Biossintéticas , Deleção de Genes , Genes Bacterianos , Família Multigênica , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/metabolismo , Streptomyces coelicolor/química , Streptomyces coelicolor/genética , Transaminases/genética , Transaminases/metabolismo , Triterpenos/químicaRESUMO
Rapid and efficient light-induced fluorescence enhancement is demonstrated on a DMNPB-"caged" coumarin derivative carrying a His-tag recognition motif.
Assuntos
Corantes Fluorescentes/química , Histidina/química , Luz , Fotoquímica , Proteínas/química , Células HeLa , HumanosAssuntos
Compostos Bicíclicos Heterocíclicos com Pontes/agonistas , Canais Iônicos/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Antagonistas Nicotínicos/farmacologia , Piridinas/agonistas , Receptores Nicotínicos/efeitos dos fármacos , Animais , Linhagem Celular , Fluorescência , Humanos , Canais Iônicos/metabolismo , Microscopia Eletrônica de Transmissão , Neurônios/metabolismo , Ratos , Receptores Nicotínicos/metabolismo , Fatores de TempoRESUMO
We developed an engineered site-directed labeling method (Foucaud et al., 2001) to investigate ligand receptor interactions on the acetylcholine (ACh)- binding site of nicotinic acetylcholine receptors (nAChRs). The method uses cysteine receptor mutants, together with cysteine-reactive ligand analogs, to generate a site-directed covalent reaction within the binding site. We selected epibatidine (EPB) as a prototypical ligand, acting at all types of nAChRs with sufficient affinity to allow this study. Accordingly, we synthesized three cysteine-reactive derivatives, all modified at the C-3 of the pyridine ring of the alkaloid with NCS; -NHCOCH2Cl, and -CH2Cl groups, respectively (Fig. 1). The binding properties have been established on rat brain, alpha7-5HT3 chimera, and Torpedo membranes, respectively, whereas the functional properties were tested on alpha4beta2 and alpha7 receptor expressed in oocytes and Cys-less muscular receptor expressed in HEK cells (Sakr et al., 2005).