RESUMEN
The adenylate-forming enzymes, including acyl-CoA synthetases, the adenylation domains of non-ribosomal peptide synthetases (NRPS), and firefly luciferase, perform two half-reactions in a ping-pong mechanism. We have proposed a domain alternation mechanism for these enzymes whereby, upon completion of the initial adenylation reaction, the C-terminal domain of these enzymes undergoes a 140 degrees rotation to perform the second thioester-forming half-reaction. Structural and kinetic data of mutant enzymes support this hypothesis. We present here mutations to Salmonella enterica acetyl-CoA synthetase (Acs) and test the ability of the enzymes to catalyze the complete reaction and the adenylation half-reaction. Substitution of Lys609 with alanine results in an enzyme that is unable to catalyze the adenylate reaction, while the Gly524 to leucine substitution is unable to catalyze the complete reaction yet catalyzes the adenylation half-reaction with activity comparable to the wild-type enzyme. The positions of these two residues, which are located on the mobile C-terminal domain, strongly support the domain alternation hypothesis. We also present steady-state kinetic data of putative substrate-binding residues and demonstrate that no single residue plays a dominant role in dictating CoA binding. We have also created two mutations in the active site to alter the acyl substrate specificity. Finally, the crystallographic structures of wild-type Acs and mutants R194A, R584A, R584E, K609A, and V386A are presented to support the biochemical analysis.
Asunto(s)
Acetato CoA Ligasa/química , Acetato CoA Ligasa/metabolismo , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/metabolismo , Salmonella enterica/enzimología , Acetato CoA Ligasa/aislamiento & purificación , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Sitios de Unión , Catálisis , Cristalización/métodos , Cristalografía por Rayos X , Glicina/química , Glicina/genética , Cinética , Lisina/química , Lisina/genética , Conformación Molecular , Estructura Molecular , Proteínas Mutantes/aislamiento & purificación , Salmonella enterica/genética , Especificidad por Sustrato/genética , Valina/química , Valina/genéticaRESUMEN
The Escherichia coli enterobactin synthetic cluster is composed of six proteins, EntA-EntF, that form the enterobactin molecule from three serine molecules and three molecules of 2,3-dihydroxybenzoic acid (DHB). EntC, EntB and EntA catalyze the three-step synthesis of DHB from chorismate. EntA is a member of the short-chain oxidoreductase (SCOR) family of proteins and catalyzes the final step in DHB synthesis, the NAD+-dependent oxidation of 2,3-dihydro-2,3-dihydroxybenzoic acid to DHB. The structure of EntA has been determined by multi-wavelength anomalous dispersion methods. Here, the 2.0 A crystal structure of EntA in the unliganded form is presented. Analysis of the structure in light of recent structural and bioinformatic analysis of other members of the SCOR family provides insight into the residues involved in cofactor and substrate binding.