Báo cáo khoa học: Functional dissection of Escherichia coli phosphotransacetylase structural domains

Functional dissection of Escherichia coli

phosphotransacetylase structural domains and analysis

of key compounds involved in activity regulation

Valeria Alina Campos-Bermudez, Federico Pablo Bologna, Carlos Santiago Andreo and

Marı´a Fabiana Drincovich

Centro de Estudios Fotosinte´ticos y Bioquı´micos (CEFOBI), Universidad Nacional de Rosario, Argentina

Introduction

The successful adaptation of Escherichia coli to nutri￾tional changes depends primarily on metabolic

switches from programs that allow rapid growth on

abundant nutrients to others that permit survival in

their absence. One important switch, called ‘the acetate

switch’, involves the transition from the production to

the utilization of acetate from the medium [1]. During

exponential growth on rich medium, E. coli cells

excrete acetate into the environment as a way, among

other reasons, to recycle CoA and regenerate NAD+,

Keywords

acetyl-phosphate; activity regulation;

Escherichia coli; phosphotransacetylase;

protein domain

Correspondence

M. F. Drincovich, Suipacha 531, 2000

Rosario, Argentina

Fax: +54 341 4370044

Tel: +54 341 4371955

E-mail: [email protected]

(Received 15 January 2010, revised

11 February 2010, accepted 12 February

2010)

doi:10.1111/j.1742-4658.2010.07617.x

Escherichia coli phosphotransacetylase (Pta) catalyzes the reversible inter￾conversion 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 dif￾ferent 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 expres￾sion of the maximal catalytic activity, stabilization of the hexameric

native state, and Pta activity regulation by NADH, ATP, phosphoenol￾pyruvate, and pyruvate. The truncated protein Pta-F3 was able to com￾plement 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.

Abbreviations

AckA, acetate kinase; Acs, acetyl-CoA synthetase; CDD, Conserved Domain Database; IPTG, isopropyl thio-b-D-galactoside;

PEP, phosphoenolpyruvate; Pta, phosphotransacetylase.

FEBS Journal 277 (2010) 1957–1966 ª 2010 The Authors Journal compilation ª 2010 FEBS 1957