THE eutD GENE OF Escherichia coli ENCODES A PROTEIN WITH PHOSPHOTRANSACETYLASE ENZYME ACTIVITY

BOLOGNA, FEDERICO P.; CAMPOS BERMUDEZ, VALERIA A.; ANDREO, CARLOS S.; DRINCOVICH, MARÍA F.

San Miguel deTucumán, Tucumán, Argentina

Congreso; XLV Reunión anual de SAIB; 2009

<!-- /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin:0cm; margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman";} @page Section1 {size:595.3pt 841.9pt; margin:72.0pt 90.0pt 72.0pt 90.0pt; mso-header-margin:35.4pt; mso-footer-margin:35.4pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> Phosphotransacetylases (PTA) reversibly converts acetyl-CoA and inorganic phosphate to acetyl-P and CoASH. Distinct isoforms of PTA are expressed in prokaryotic organisms, where they play diverse metabolic roles. Surveys of sequenced microbial genomes reveal two classes of Pta enzymes. Class I enzymes (PtaI) are ~350 residues in length, whereas class II enzymes (PtaII) are twice as long as PtaI (~700 residues). PtaI enzymes share end-to-end homology with the C-terminal domain of PtaII enzymes; hence, it is inferred that the active site of PtaII enzymes is located within their C-terminal domain. Two PTA genes have been identified by sequence homology in E. coli: pta and eutD. eutD belongs to the PtaI class and it is codified within the 17-gene eut operon. The aim of the present work was the cloning and expression of EutD. The protein was purified and kinetically characterized. The product of eutD displays PTA activity, although the properties of this enzyme were different from that of the Pta protein. The reverse activity was tested and the Km for acetyl-CoA was obtained. Finally, the metabolic regulation and oligomeric state of the recombinant EutD were analyzed. All in all, these results indicates that EutD and Pta fulfil different roles in vivo.