RESUMEN
The crystal structure of the ribosomal 50S subunit from Haloarcula marismortui in complex with the transition state analog CCdA-phosphate-puromycin (CCdApPmn) led to a mechanistic proposal wherein the universally conversed A2451 in the ribosomal active site acts as an "oxyanion hole" to promote the peptidyl transferase reaction [Nissen, P., Hansen, J., Ban, N., Moore, P.B., and Steitz, T.A. (2000) Science 289, 920-929]. In the model, close proximity (3 A) between the A2451 N3 and the nonbridging phosphoramidate oxygen of CCdApPmn suggested that the carbonyl oxyanion formed during the tetrahedral transition state is stabilized by hydrogen bonding to the protonated A2451 N3, the pKa of which must be perturbed substantially. We characterize the contribution of the putative hydrogen bond between the N3 of A2451 and the nonbridging phosphoramidate oxygen by using chemical protection and peptidyl transfer inhibition assays. If this putative hydrogen bond makes a significant thermodynamic contribution, then CCdApPmn-binding affinity to the 50S ribosomal subunit should be strongly pH-dependent, with affinity increasing as the pH is lowered. We report that CCdApPmn binds 50S ribosomes with essentially equal affinity at all pH values between 5.0 and 8.5. These data argue against a mechanism for peptidyl transfer in which a residue with near neutral pKa stabilizes the transition-state oxyanion, at least to the extent that CCdApPmn accurately mimics the transition state.
Asunto(s)
Peptidil Transferasas/metabolismo , ARN/metabolismo , AnionesRESUMEN
The ponA gene of Mycobacterium smegmatis encodes a 95-kDa penicillin binding protein, PBP1, that is similar to PBP1s of Mycobacterium tuberculosis and Mycobacterium leprae. Transposon disruption of ponA in M. smegmatis resulted in a PBP1-deficient mutant that was sensitive to beta-lactam antibiotics, was more permeable to glycine, and grew slowly in liquid culture.
Asunto(s)
Proteínas Bacterianas , Proteínas Portadoras/genética , Muramoilpentapéptido Carboxipeptidasa/genética , Mutación/genética , Mycobacterium smegmatis/genética , Permeabilidad de la Membrana Celular/genética , Medios de Cultivo , Glicina/metabolismo , Hexosiltransferasas/genética , Hexosiltransferasas/metabolismo , Cinética , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Mycobacterium smegmatis/enzimología , Mycobacterium smegmatis/crecimiento & desarrollo , Proteínas de Unión a las Penicilinas , Peptidil Transferasas/genética , Peptidil Transferasas/metabolismo , Reacción en Cadena de la Polimerasa , beta-Lactamasas/genética , beta-Lactamasas/metabolismoRESUMEN
The ponA gene of cosmid L222 of the Mycobacterium leprae genome library encodes a multimodular class A penicillin-binding protein (PBP), PBP1. The PBP, labelled with a polyhistidine sequence, has been produced in Escherichia coli, extracted from the membranes with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane-sulfonate (CHAPS) and purified by Ni2(+)-nitrilotriacetic acid-agarose chromatography. In contrast to the pon1-encoded class A PBP1, PBP1 undergoes denaturation at temperatures higher than 25 degrees C, it catalyzes acyl transfer reactions on properly structured thiolesters, and it binds penicillin with high affinity.
Asunto(s)
Proteínas Bacterianas , Proteínas Portadoras , Hexosiltransferasas/química , Hexosiltransferasas/metabolismo , Proteínas de la Membrana/química , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , Muramoilpentapéptido Carboxipeptidasa , Mycobacterium leprae/química , Penicilinas/metabolismo , Peptidil Transferasas/química , Peptidil Transferasas/metabolismo , Acilación , Cósmidos , Escherichia coli/genética , Hexosiltransferasas/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Complejos Multienzimáticos/genética , Mycobacterium leprae/genética , Mycobacterium leprae/metabolismo , Proteínas de Unión a las Penicilinas , Peptidil Transferasas/genética , Desnaturalización Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Solubilidad , Temperatura , Transformación BacterianaRESUMEN
Cosmid B577, a member of the collection of ordered clones corresponding to the genome of Mycobacterium leprae, contains a gene, provisionally called pon1, that encodes an 821-amino-acid-residue high-molecular-mass class A penicillin-binding protein, provisionally called PBP1. With similar amino acid sequences and modular designs, M. leprae PBP1 is related to Escherichia coli PBP1a and PBP1b, bienzymatic proteins with transglycosylase and transpeptidase activities. When produced in E. coli, His tag-labelled derivatives of M. leprae PBP1 adopt the correct membrane topology, with the bulk of the polypeptide chain on the surface of the plasma membrane. They defy attempts at solubilization with all the detergents tested except cetyltrimethylammonium bromide. The solubilized PBP1 derivatives can be purified by affinity chromatography on Ni2+-nitrilotriacetic acid agarose. They have low affinities for the usual penicillins and cephalosporins.