RESUMEN
An analogue 2 of coenzyme A (CoA) has been prepared in which the geminal methyl groups are replaced with hydrogens. An NMR titration study was conducted and shifts in frequency of protons in the pantetheine portion of the molecule upon titration of the adenine base were observed as has been previously reported with CoA. These studies indicate that the geminal dimethyl groups are not essential for adoption of a partially folded conformation in solution. Based on 1H-1H coupling constants, the distribution of conformations about the carbon-carbon bonds in the region of the methyl deletion were estimated. The results suggest that the conformer distribution is similar to that of CoA, but with small increases in population of the anti conformers. A simple model compound containing the didemethyl pantoamide moiety was prepared and subjected to similar conformational analysis. The coupling constants and predicted conformer distribution were almost identical to that of the CoA analogue, indicating that the conformer distribution is controlled by local interactions and not influenced by interactions between distant parts of the CoA molecule. The acetyl derivative of 2 was a fairly good substrate for the acetyl-CoA utilizing enzymes carnitine acetyltransferase, chloramphenicol acetyltransferase, and citrate synthase, with 1.3- to 10-fold increased Km values and 2.5- to 11-fold decreases in Vmax. The combined results indicate that the geminal dimethyl groups of CoA have modest effects on function and minimal effects on conformation.
Asunto(s)
Coenzima A/química , Carnitina O-Acetiltransferasa/química , Carnitina O-Acetiltransferasa/metabolismo , Cloranfenicol O-Acetiltransferasa/química , Cloranfenicol O-Acetiltransferasa/metabolismo , Espectroscopía de Resonancia Magnética , Estructura Molecular , Conformación ProteicaRESUMEN
INTRODUCTION: The polyketide natural products are assembled by a series of decarboxylation/condensation reactions of simple carboxylic acids catalyzed by polyketide synthase (PKS) complexes. The growing chain is assembled on acyl carrier protein (ACP), an essential component of the PKS. ACP requires posttranslational modification on a conserved serine residue by covalent attachment of a 4'-phosphopantetheine (P-pant) cofactor to yield active holo-ACP. When ACPs of Streptomyces type II aromatic PKS are overproduced in E. coli, however, typically little or no active holo-ACP is produced, and the ACP remains in the inactive apo-form. RESULTS: We demonstrate that E. coli holo-ACP synthase (ACPS), a fatty acid biosynthesis enzyme, can catalyze P-pant transfer in vitro to the Streptomyces PKS ACPs required for the biosynthesis of the polyketide antibiotics granaticin, frenolicin, oxytetracycline and tetracenomycin. The catalytic efficiency of this P-pant transfer reaction correlates with the overall negative charge of the ACP substrate. Several coenzyme A analogs, modified in the P-pant portion of the molecule, are likewise able to serve as substrates in vitro for ACPS. CONCLUSIONS: E coli ACPS can serve as a useful reagent for the preparation of holo-forms of Streptomyces ACPs as well as holo-ACPs with altered phosphopantetheine moieties. Such modified ACPs should prove useful for studying the role of particular ACPs and the phosphopantetheine cofactor in the subsequent reactions of polyketide and fatty acid biosynthesis.
Asunto(s)
Proteína Transportadora de Acilo/metabolismo , Coenzima A/metabolismo , Escherichia coli/enzimología , Streptomyces/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Antibacterianos/metabolismo , Clonación Molecular , Cartilla de ADN , Estructura Molecular , Complejos Multienzimáticos/metabolismo , Panteteína/análogos & derivados , Panteteína/metabolismo , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Transferasas (Grupos de Otros Fosfatos Sustitutos)/biosíntesis , Transferasas (Grupos de Otros Fosfatos Sustitutos)/aislamiento & purificaciónRESUMEN
A device is described which is relatively easy to construct and which can be used to protect brain implants affixed to the skull of large primates. The device is essentially a stainless steel ring cemented around the brain implant. A plexiglass dome can then be attached to the ring to protect the implant. Further, a second device can be constructed which attaches to the ring and which provides gentle leverage to the brain implant to remove adhered obturators.