Properties of a mutant Escherichia coli phosphoenolpyruvate carboxylase deficient in coregulation by intermediary metabolites

Abstract

Phosphoenolpyruvate carboxylase of Escherichia coli is activated by three different mechanisms: contiguous by acetyl coenzyme A, precursor by fructose 1,6-bisphosphate, and compensatory feedback by cytidine 5'-diphosphate (CDP). Even though each activator can interact independently with the enzyme, synergistic effects are observed with some combinations, namely, fructose 1,6-bisphosphate or CDP (coregulators), with acetyl coenzyme A. A mutant was isolated that has a phosphoenolpyruvate carboxylase which is refractory to activation by fructose, 1,6-bisphosphate and CDP. The mutant enzyme was shown to be active primarily as the dimer and to lack cooperativity in substrate binding. The binding of acetyl coenzyme A and substrate, however, was essentially the same as that of the wild-type enzyme. The mutant cells grew extremely slowly on glucose alone as the sole carbon source. The only defect in the mutant appeared to be the inability of this enzyme to be activated by the coregulators. These data are consistent with the thesis that coregulation by fructose 1,6-bisphosphate or CDP is an essential requirement for the activation in vivo of this enzyme.

Properties of an Escherichia coli mutant deficient in phosphoenolpyruvate carboxylase catalytic activity

“…It has been stated earlier that no single mutation of phosphoenolpyruvate carboxylase has been found which alters only one characteristic of the enzyme (15,18,19). Therefore, it is important to describe each mutant as fully as possible and then compare structural differences which are characteristic of mutants expressing similar phenotypes.…”

“…We have described an altered phosphoenolpyruvate carboxylase, obtained from a mutant (PpcI), that is active primarily as the dimer (15). This mutant is unique in that it does not form kinetically productive complexes with fructose 1,6-bisphosphate, a site I activator (27), and cells containing this form of the enzyme grow very poorly on glucose as the sole carbon source. In an attempt to characterize more completely the regulatory and catalytic functions of this enzyme and to relate these functions to structure, other mutants have also been isolated.…”

Metabolic regulation of <i>Escherichia coli</i> cultivated under anaerobic and aerobic conditions in response to the specific pathway gene knockouts

“…From enzyme activity data, Ppc activity in the pykF mutant is up-regulated as compared to the parent strain. The reason for the high Ppc flux may be due to high PEP concentration and synergistic activation by FDP [35,36]. Since the product of the reaction through Ppc is OAA, which is the precursor for biosynthesis, the ppc gene knockout causes low biomass synthesis flux [18].…”

Deletingpckimproves growth and suppresses by-product formation during 1,3-propanediol fermentation byKlebsiella pneumoniae

“…PEP carboxylation can generate OAA, which can be used to replenish the C4 metabolites that are involved in the TCA cycle for cell synthesis. In E. coli, PEP carboxylation pathway-deficient strains do not grow on glucose-minimal medium because PEP carboxylation is the only reported anaplerotic reaction that converts C3 to C4 metabolites in central metabolism (McAlister et al 1981;Yang et al 2003Yang et al , 2014Kim et al 2004). Here, the severe growth defect that was found strongly indicates that PEP carboxylation could also be the only C3 anaplerotic reaction in Kl.…”

“…Rumen bacteria, such as Actinobacillus succinogenes, use PCK as the main enzyme for PEP carboxylation (Samuelov et al 1991;Van der Werf et al 1997;Lee et al 2006;McKinlay et al 2007), as bacteria can use the high-energy phosphate bonds of PEP to form ATP (Zhang et al 2009;Zelle et al 2010). However, Escherichia coli and some other bacteria use PPC as the main carboxylase (McAlister et al 1981;Chao and Liao 1993;Kim et al 2004). Based on genomic data, Kl.…”