Functional dissection of Escherichia coli phosphotransacetylase structural domains and analysis of key compounds involved in activity regulation

CAMPOS-BERMUDEZ, V. A.; BOLOGNA, F.; ANDREO, CARLOS S; MARIA FABIANA DRINCOVICH

FEBS JOURNAL

WILEY-BLACKWELL PUBLISHING, INC

Año: 2010 vol. 277 p. 1957 - 1966

1742-464X

Escherichia coli phosphotransacetylase (Pta) catalyses the reversible interconvertion of acetyl-CoA and acetyl-P. Both compounds are critical in E. coli metabolism, whereas acetyl-P 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 concentration. E. coli Pta is composed by three different domains, but only the C-terminal one, called PTA_PTB, is specific for all Ptas and the unique domain in certain Ptas. In the present work, the characterization of E. coli Pta and deletions from the N-terminal region was performed. E. coli Pta acetyl-P forming and consuming reactions display different maximum activities and are differentially regulated by pyruvate and PEP. These compounds activate acetyl-P production, but inhibit acetyl-CoA production, thus playing a critical role in defining the rate of the two Pta reactions. The characterization of three Pta truncated proteins, 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 the expression of the maximal catalytic activity, in the stabilization of the hexameric native state and in Pta activity regulation by NADH, ATP, PEP 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.