Covalent modification of citrate lyase ligase from Clostridium sphenoides by phosphorylation/dephosphorylation

Abstract

Citrate lyase ligase was shown to be present in Clostridium sphenoides actively degrading citrate. In contrast to citrate lyase ligase from C. sporosphaeroides and Streptococcus lactis, the enzyme from C. sphenoides was under stringent regulatory control. The alteration of the kinetic properties of the enzyme after depletion of citrate suggested the presence of two different enzyme species in different phases of growth: active and partially active citrate lyase ligase. These enzymes were purified from in vivo 32P-labeled C. sphenoides cells, which were grown on low-phosphate medium containing 40 mM citrate and 1 mCi [32P]orthophosphate. During enzyme purification only the active form of citrate lyase ligase was shown to be radioactively labeled. Growth experiments with 14C-labeled precursors of purines and pyrimidines and subsequent purification of active citrate lyase ligase indicated that the 32P labeling of the enzyme was not due to the incorporation of a nucleotide. Inactivation of the ligase after its treatment with acid phosphatase also suggested that the active form of the enzyme is phosphorylated. Citrate lyase ligase, therefore, is the first known enzyme in an anaerobic bacterium whose activity is modulated by phosphorylation/dephosphorylation. both organisms that the intracellular pool size of L-glutamate is dependent on the level of its precursor, citrate, in the medium [12, 131. It is, therefore, reasonable that L-glutamate plays a key role in the regulation of citrate metabolism in these organisms. A high concentration of L-glutamate signals that citrate is available and that it can be cleaved via the fermentation pathway, while at low concentrations citrate is utilized for the biosynthesis of L-glutamate. In R. gelatinosus a futile cycle is avoided by the deacetylation (inactivation) of citrate lyase after the exhaustion of citrate in the medium. The enzyme catalyzing this reaction is citrate lyase deacetylase, whose activity is strongly inhibited by L-glutamate [13].After citrate exhaustion another futile cycle has to be avoided, the one between citrate lyase deacetylase and citrate lyase ligase. The latter enzyme has been shown to be rapidly inactivated in R . gelatinosus [9]. A similar inactivation has been observed in C. sphenoides and it has now been studied in detail in this organism. In this communication we present conclusive evidence that a covalent modification of citrate lyase ligase occurs and that this modification consists of a phosphorylation/dephosphorylation of this enzyme. MATERIALS AND METHODS Organism and growth conditionsClostridium sphenoides strain C 2 (DSM 614) was used for this investigation. The growth medium contained 40 mM trisodium citrate and was prepared as described in [12].Before the total consumption of citrate (17 h) growth was stopped and the harvested cells were stored at -20°C (yield 2.5-3.5 g wet weight/l). In this phase of growth, citrate lyase ligase was present in its active form. For the purification of partially active citrate lyase ligase cells were harv...

Covalent modification of proteins in Escherichia coli growing anaerobically with nitrate as electron acceptor

“…sphenoides [14]. All these results prompted us to conduct further radioactive experiments with E. coli in order to detect new protein species which are modified by covalent modification.…”

Citrate metabolism in anaerobic bacteria

“…Since these microbes utilize citrate also for the biosynthesis of L-glutamate via the first steps of the reactions of the tricarboxylic acid cycle [1][2][3], this amino acid plays a very important role in the regulation of citrate metabolism. In C. sphenoides, L-glutamate directly interacts with citrate lyase and it also favors the activation of citrate lyase activating enzyme (citrate lyase ligase) [19]. On the other hand, in Rc.…”

“…Recent investigations in vitro also show that the purified citrate lyase ligase is inactivated by incubation with alkaline phosphatase, which suggests that enzyme regulation by phosphorylation and dephosphorylation is the possible mechanism of this process [38]. Such a mechanism could be demonstrated for the ligase from C. sphenoides [19]. Although the physiological conditions for citrate lyase and ligase activation and inactivation are well defined, the activity of citrate lyase ligase, however, could not be reconstituted in vitro.…”

“…gelatinosus [50]. In contrast to the corresponding ligases from E. aerogenes [36], C. sphenoides [19] and S. lactis [51], citrate lyase ligase from Rc. gelatinosus is extremely unstable and is rapidly inactivated in cell extracts [35].…”

Protein Phosphorylation in Bacillus thuringiensis during Growth and -Endotoxin Production

“…In some species the phosphorylation of specific proteins has been characterized and related to the control of their activities. Thus the acetate-dependent attenuation of isocitrate dehydrogenase activity in Escherichia coli is mediated by phosphorylation (Nimmo, 1984) and in Clostridium sphenoides the active form of citrate lyase ligase has been shown to be phosphorylated (Antranikian et al, 1985). Furthermore, in Streptococcus pyogenes the HPr component of the phosphotransferase system is phosphorylated at both a serine and a histidine residue (Reizer et al, 1985).…”