Genetic and physiological characterization of Escherichia coli mutants deficient in phosphoenolpyruvate carboxykinase activity

Abstract

Mutants doubly deficient in phosphoenolpyruvate carboxykinase (pck) and phosphoenolpyruvate synthetase (pps) were unable to grow with succinate as the sole carbon source. A number of pck mutations isolated from pps strains by penicillin selection mapped at 74 min on the Escherichia coli chromosome, between glpD and aroB. Several of the strains containing these mutations had a protein antigenically related to phosphoenolpyruvate carboxykinase, and, therefore, the mutations probably represented mutations in the structural gene for this enzyme. Phosphoenolpyruvate carboxykinase was regulated at the level of transcription by catabolite repression. Enzyme levels also increased in stationaryphase cultures by a mechanism independent of cyclic adenosine monophosphate or the product of the relA gene.

The PEP—pyruvate—oxaloacetate node as the switch point for carbon flux distribution in bacteria: We dedicate this paper to Rudolf K. Thauer, Director of the Max-Planck-Institute for Terrestrial Microbiology in Marburg, Germany, on the occasion of his 65th birthday

“…In all these organisms, PEP carboxykinase gene expression is low when they grow on glycolytic substrates and higher when they grow on gluconeogenic substrates. In E. coli and S. meliloti, the respective pckA gene additionally is strongly induced in the stationary growth phase [5,6,96], whereas in R. palustris it is strongly induced in the exponential growth phase, irrespective of the carbon source and under both anaerobic light and aerobic dark conditions [97]. Such a log-phase induction has not been found for other pckA genes characterized to date.…”

Characterization of phosphoenolpyruvate carboxykinase from Corynebacterium glutamicum

“…In contrast to other prokaryotic PEP carboxykinases, the enzyme from C. glutamicum did not use ADP as cosubstrate, but was completely dependent on the presence of IDP, which is the common nucleotide observed for the enzyme from eukaryotes [8][9][10][11]. The g m for the various substrates is in the same range as observed for other PEP carboxykinases.…”

“…The g m for the various substrates is in the same range as observed for other PEP carboxykinases. Also the dependence on divalent manganese ions for full activity has been reported for several other enzymes [8][9][10][11].…”

“…PEP carboxykinase from various eukaryotic sources has been studied, but reports on PEP carboxykinase activity in bacteria have been sporadic [8,9]. PEP carboxykinase has been observed in Arthrobacter, Escherichia coli, Nocardia, Salmonella and very recently in Anaerobiospirillum, but has not yet been reported in C. glutamicum [8][9][10][11]. In this paper we describe, for the first time, the presence of PEP carboxykinase in C. glutamicum, together with some enzyme properties.…”

Involvement of cAMP-CRP in transcription activation and repression of thepckgene encoding PEP carboxykinase, the key enzyme of gluconeogenesis

“…Phosphoenolpyruvate carboxykinase (Pck) catalyzes the addition of bicarbonate to phosphoenolpyruvate to form four-carbon compound oxaloacetate, providing substrates for gluconeogenesis. Transcription of the pck gene increases in the stationary phase and is under the catabolite repression (Goldie & Sanwal, 1980;Goldie & Medina, 1990). Accordingly, the pck promoter was identified as a novel CRP target (Shimada et al, 2011b).…”