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Characterization and kinetics of phosphopantothenoylcysteine synthetase from Enterococcus faecalis.

ABSTRACT: The enzyme phosphopantothenoylcysteine synthetase (PPCS) catalyzes the nucleotide-dependent formation of phosphopantothenoylcysteine from (R)-phosphopantothenate and L-cysteine in the biosynthetic pathway leading to the formation of the essential biomolecule, coenzyme A. The Enterococcus faecalis gene coaB encodes a novel monofunctional PPCS which has been cloned into pET23a and expressed in Escherichia coli BL21 AI. The heterologous expression system yielded 30 mg of purified PPCS per liter of cell culture. The purified enzyme chromatographed as a homodimer of 28 kDa subunits on Superdex HR 200 gel filtration resin. The monofunctional protein displayed a nucleotide specificity for cytidine 5'-triphosphate (CTP) analogous to that seen for bifunctional PPCS expressed by most prokaryotes. Kinetic characterization, utilizing initial velocity and product inhibition studies, found the mechanism of PPCS to be Bi Uni Uni Bi Ping-Pong, with the nucleotide CTP binding first and CMP released last. Michaelis constants were 156, 17, and 86 microM for CTP, (R)-phosphopantothenate, and L-cysteine, respectively, and the k(cat) was 2.9 s(-1). [carboxyl-(18)O]Phosphopantothenate was prepared by hydrolysis of methyl pantothenate with Na(18)OH, followed by enzymatic phosphorylation with E. faecalis pantothenate kinase (PanK). The fate of the carboxylate oxygen of labeled phosphopantothenate, during the course of the PPCS-catalyzed reaction with CTP and L-cysteine, was monitored by (31)P NMR spectroscopy. The results show that the carboxylate oxygen of the phosphopantothenate is recovered with the CMP formed during the reaction, indicative of the formation of a phosphopantothenoyl cytidylate catalytic intermediate, which is consistent with the kinetic mechanism.

SUBMITTER: Yao J

PROVIDER: S-EPMC2892170 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Characterization and kinetics of phosphopantothenoylcysteine synthetase from Enterococcus faecalis.

Yao Jiangwei J Patrone James D JD Dotson Garry D GD

Biochemistry 20090301 12

The enzyme phosphopantothenoylcysteine synthetase (PPCS) catalyzes the nucleotide-dependent formation of phosphopantothenoylcysteine from (R)-phosphopantothenate and L-cysteine in the biosynthetic pathway leading to the formation of the essential biomolecule, coenzyme A. The Enterococcus faecalis gene coaB encodes a novel monofunctional PPCS which has been cloned into pET23a and expressed in Escherichia coli BL21 AI. The heterologous expression system yielded 30 mg of purified PPCS per liter of ...[more]

PMID: 19182993

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Project description:UnlabelledThe bacterial stringent response (SR) is a conserved stress tolerance mechanism that orchestrates physiological alterations to enhance cell survival. This response is mediated by the intracellular accumulation of the alarmones pppGpp and ppGpp, collectively called (p)ppGpp. In Enterococcus faecalis, (p)ppGpp metabolism is carried out by the bifunctional synthetase/hydrolase E. faecalis Rel (RelEf) and the small alarmone synthetase (SAS) RelQEf. Although Rel is the main enzyme responsible for SR activation in Firmicutes, there is emerging evidence that SASs can make important contributions to bacterial homeostasis. Here, we showed that RelQEf synthesizes ppGpp more efficiently than pppGpp without the need for ribosomes, tRNA, or mRNA. In addition to (p)ppGpp synthesis from GDP and GTP, RelQEf also efficiently utilized GMP to form GMP 3'-diphosphate (pGpp). Based on this observation, we sought to determine if pGpp exerts regulatory effects on cellular processes affected by (p)ppGpp. We found that pGpp, like (p)ppGpp, strongly inhibits the activity of E. faecalis enzymes involved in GTP biosynthesis and, to a lesser extent, transcription of rrnB by Escherichia coli RNA polymerase. Activation of E. coli RelA synthetase activity was observed in the presence of both pGpp and ppGpp, while RelQEf was activated only by ppGpp. Furthermore, enzymatic activity of RelQEf is insensitive to relacin, a (p)ppGpp analog developed as an inhibitor of "long" RelA/SpoT homolog (RSH) enzymes. We conclude that pGpp can likely function as a bacterial alarmone with target-specific regulatory effects that are similar to what has been observed for (p)ppGpp.ImportanceAccumulation of the nucleotide second messengers (p)ppGpp in bacteria is an important signal regulating genetic and physiological networks contributing to stress tolerance, antibiotic persistence, and virulence. Understanding the function and regulation of the enzymes involved in (p)ppGpp turnover is therefore critical for designing strategies to eliminate the protective effects of this molecule. While characterizing the (p)ppGpp synthetase RelQ of Enterococcus faecalis (RelQEf), we found that, in addition to (p)ppGpp, RelQEf is an efficient producer of pGpp (GMP 3'-diphosphate). In vitro analysis revealed that pGpp exerts complex, target-specific effects on processes known to be modulated by (p)ppGpp. These findings provide a new regulatory feature of RelQEf and suggest that pGpp may represent a new member of the (pp)pGpp family of alarmones.

Dataset Information

Characterization and kinetics of phosphopantothenoylcysteine synthetase from Enterococcus faecalis.

Publications

Characterization and kinetics of phosphopantothenoylcysteine synthetase from Enterococcus faecalis.

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