Functional dissection of Escherichia coli phosphotransacetylase structural domains and analysis of key compounds involved in activity regulation.
Escherichia coli phosphotransacetylase (Pta) catalyzes the reversible interconversion of acetyl-CoA and acetyl phosphate. Both compounds are critical in E. coli metabolism, and acetyl phosphate is also involved in the regulation of certain signal transduction pathways. Along with acetate kinase, Pta plays an important role in acetate production when E. coli grows on rich medium; alternatively, it is involved in acetate utilization at high acetate concentrations. E. coli Pta is composed of three different domains, but only the C-terminal one, called PTA_PTB, is specific for all Ptas. In the present work, the characterization of E. coli Pta and deletions from the N-terminal region were performed. E. coli Pta acetyl phosphate-forming and acetyl phosphate-consuming reactions display different maximum activities, and are differentially regulated by pyruvate and phosphoenolpyruvate. These compounds activate acetyl phosphate production, but inhibit acetyl-CoA production, thus playing a critical role in defining the rates of the two Pta reactions. The characterization of three truncated Ptas, which all display Pta activity, indicates that the substrate-binding site is located at the C-terminal PTA_PTB domain. However, the N-terminal P-loop NTPase domain is involved in expression of the maximal catalytic activity, stabilization of the hexameric native state, and Pta activity regulation by NADH, ATP, phosphoenolpyruvate, and pyruvate. The truncated protein Pta-F3 was able to complement the growth on acetate of an E. coli mutant defective in acetyl-CoA synthetase and Pta, indicating that, although not regulated by metabolites, the Pta C-terminal domain is active in vivo.
Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI), Universidad Nacional de Rosario, Argentina.
Valeria Alina
VA
Campos-Bermudez
Federico Pablo
FP
Bologna
Carlos Santiago
CS
Andreo
María Fabiana
MF
Drincovich
D000084:Acetate Kinase / Q000378:metabolism*; D000105:Acetyl Coenzyme A / Q000378:metabolism*; D002384:Catalysis; D004926:Escherichia coli / Q000254:growth & development / Q000502:physiology*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D007700:Kinetics; D008954:Models, Biological; D010755:Organophosphates / Q000378:metabolism*; D010709:Phosphate Acetyltransferase / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000235:genetics
D029968:Escherichia coli Proteins; D010755:Organophosphates; D011994:Recombinant Proteins; C011632:acetyl phosphate; D000105:Acetyl Coenzyme A; D010709:Phosphate Acetyltransferase; D000084:Acetate Kinase