On the mechanism of phosphoenolpyruvate synthetase (PEPs) and its inhibition by sodium fluoride: potential magnesium and aluminum fluoride complexes of phosphoryl transfer.

Phosphoenolpyruvate synthase (PEPs) catalyzes the conversion of pyruvate to phosphoenolpyruvate (PEP) using a two-step mechanism invoking a phosphorylated-His intermediate. Formation of PEP is an initial step in gluconeogenesis, and PEPs is essential for growth of Escherichia coli on 3-carbon sources such as pyruvate. The production of PEPs has also been linked to bacterial virulence and antibiotic resistance. As such, PEPs is of interest as a target for antibiotic development, and initial investigations of PEPs have indicated inhibition by sodium fluoride. Similar inhibition has been observed in a variety of phospho-transfer enzymes through the formation of metal fluoride complexes within the active site. Herein we quantify the inhibitory capacity of sodium fluoride through a coupled spectrophotometric assay. The observed inhibition provides indirect evidence for the formation of a MgF3(-) complex within the enzyme active site and insight into the phospho-transfer mechanism of PEPs. The effect of AlCl3 on PEPs enzyme activity was also assessed and found to decrease substrate binding and turnover.

The plastidial glucan, water dikinase (GWD) catalyses multiple phosphotransfer reactions.

The plant genome encodes at least two distinct and evolutionary conserved plastidial starch-related dikinases that phosphorylate a low percentage of glucosyl residues at the starch granule surface. Esterification of starch favours the transition of highly ordered α-glucans to a less ordered state and thereby facilitates the cleavage of interglucose bonds by hydrolases. Metabolically most important is the phosphorylation at position C6, which is catalysed by the glucan, water dikinase (GWD). The reactions mediated by recombinant wild-type GWD from Arabidopsis thaliana (AtGWD) and from Solanum tuberosum (StGWD) were studied. Two mutated proteins lacking the conserved histidine residue that is indispensible for glucan phosphorylation were also included. The wild-type GWDs consume approximately 20% more ATP than is required for glucan phosphorylation. Similarly, although incapable of phosphorylating α-glucans, the two mutated dikinase proteins are capable of degrading ATP. Thus, consumption of ATP and phosphorylation of α-glucans are not strictly coupled processes but, to some extent, occur as independent phosphotransfer reactions. As revealed by incubation of the GWDs with [γ-(33) P]ATP, the consumption of ATP includes the transfer of the γ-phosphate group to the GWD protein but this autophosphorylation does not require the conserved histidine residue. Thus, the GWD proteins possess two vicinal phosphorylation sites, both of which are transiently phosphorylated. Following autophosphorylation at both sites, native dikinases flexibly use various terminal phosphate acceptors, such as water, α-glucans, AMP and ADP. A model is presented describing the complex phosphotransfer reactions of GWDs as affected by the availability of the various acceptors.

Cloning and characterization of Escherichia coli DUF299: a bifunctional ADP-dependent kinase--Pi-dependent pyrophosphorylase from bacteria.

BACKGROUND: Phosphoenolpyruvate synthetase (PEPS; EC 2.7.9.2) catalyzes the synthesis of phosphoenolpyruvate from pyruvate in Escherichia coli when cells are grown on a three carbon source. It also catalyses the anabolic conversion of pyruvate to phosphoenolpyruvate in gluconeogenesis. A bioinformatics search conducted following the successful cloning and expression of maize leaf pyruvate, orthophosphate dikinase regulatory protein (PDRP) revealed the presence of PDRP homologs in more than 300 bacterial species; the PDRP homolog was identified as DUF299. RESULTS: This paper describes the cloning and expression of both PEPS and DUF299 from E. coli and establishes that E. coli DUF299 catalyzes both the ADP-dependent inactivation and the Pi-dependent activation of PEPS. CONCLUSION: This paper represents the first report of a bifunctional regulatory enzyme catalysing an ADP-dependent phosphorylation and a Pi-dependent pyrophosphorylation reaction in bacteria.

A CBM20 low-affinity starch-binding domain from glucan, water dikinase.

The family 20 carbohydrate-binding module (CBM20) of the Arabidopsis starch phosphorylator glucan, water dikinase 3 (GWD3) was heterologously produced and its properties were compared to the CBM20 from a fungal glucoamylase (GA). The GWD3 CBM20 has 50-fold lower affinity for cyclodextrins than that from GA. Homology modelling identified possible structural elements responsible for this weak binding of the intracellular CBM20. Differential binding of fluorescein-labelled GWD3 and GA modules to starch granules in vitro was demonstrated by confocal laser scanning microscopy and yellow fluorescent protein-tagged GWD3 CBM20 expressed in tobacco confirmed binding to starch granules in planta.

Phosphoenolpyruvate synthetase and pyruvate, phosphate dikinase of Thermoproteus tenax: key pieces in the puzzle of archaeal carbohydrate metabolism.

The interconversion of phosphoenolpyruvate and pyruvate represents an important control point of the Embden-Meyerhof-Parnas (EMP) pathway in Bacteria and Eucarya, but little is known about this site of regulation in Archaea. Here we report on the coexistence of phosphoenolpyruvate synthetase (PEPS) and the first described archaeal pyruvate, phosphate dikinase (PPDK), which, besides pyruvate kinase (PK), are involved in the catalysis of this reaction in the hyperthermophilic crenarchaeote Thermoproteus tenax. The genes encoding T. tenax PEPS and PPDK were cloned and expressed in Escherichia coli, and the enzymic and regulatory properties of the recombinant gene products were analysed. Whereas PEPS catalyses the unidirectional conversion of pyruvate to phosphoenolpyruvate, PPDK shows a bidirectional activity with a preference for the catabolic reaction. In contrast to PK of T. tenax, which is regulated on transcript level but exhibits only limited regulatory potential on protein level, PEPS and PPDK activities are modulated by adenosine phosphates and intermediates of the carbohydrate metabolism. Additionally, expression of PEPS is regulated on transcript level in response to the offered carbon source as revealed by Northern blot analyses. The combined action of the differently regulated enzymes PEPS, PPDK and PK represents a novel way of controlling the interconversion of phosphoenolpyruvate and pyruvate in the reversible EMP pathway, allowing short-term and long-term adaptation to different trophic conditions. Comparative genomic analyses indicate the coexistence of PEPS, PPDK and PK in other Archaea as well, suggesting a similar regulation of the carbohydrate metabolism in these organisms.

The starch-related R1 protein is an alpha -glucan, water dikinase.

To determine the enzymatic function of the starch-related R1 protein it was heterologously expressed in Escherichia coli and purified to apparent homogeneity. Incubation of the purified protein with various phosphate donor and acceptor molecules showed that R1 is capable of phosphorylating glucosyl residues of alpha-glucans at both the C-6 and the C-3 positions in a ratio similar to that occurring naturally in starch. Phosphorylation occurs in a dikinase-type reaction in which three substrates, an alpha-polyglucan, ATP, and H(2)O, are converted into three products, an alpha-polyglucan-P, AMP, and orthophosphate. The use of ATP radioactively labeled at either the gamma or beta positions showed that solely the beta phosphate is transferred to the alpha-glucan. The apparent K(m) of the R1 protein for ATP was calculated to be 0.23 microM and for amylopectin 1.7 mg x ml(-1). The velocity of in vitro phosphorylation strongly depends on the type of the glucan. Glycogen was an extremely poor substrate; however, the efficiency of phosphorylation strongly increased if the glucan chains of glycogen were elongated by phosphorylase. Mg(2+) ions proved to be essential for activity. Incubation of R1 with radioactively labeled ATP in the absence of an alpha-glucan showed that the protein phosphorylates itself with the beta, but not with the gamma phosphate. Autophosphorylation precedes the phosphate transfer to the glucan indicating a ping-pong reaction mechanism.

Improving lycopene production in Escherichia coli by engineering metabolic control.

Metabolic engineering has achieved encouraging success in producing foreign metabolites in a variety of hosts. However, common strategies for engineering metabolic pathways focus on amplifying the desired enzymes and deregulating cellular controls. As a result, uncontrolled or deregulated metabolic pathways lead to metabolic imbalance and suboptimal productivity. Here we have demonstrated the second stage of metabolic engineering effort by designing and engineering a regulatory circuit to control gene expression in response to intracellular metabolic states. Specifically, we recruited and altered one of the global regulatory systems in Escherichia coli, the Ntr regulon, to control the engineered lycopene biosynthesis pathway. The artificially engineered regulon, stimulated by excess glycolytic flux through sensing of an intracellular metabolite, acetyl phosphate, controls the expression of two key enzymes in lycopene synthesis in response to flux dynamics. This intracellular control loop significantly enhanced lycopene production while reducing the negative impact caused by metabolic imbalance. Although we demonstrated this strategy for metabolite production, it can be extended into other fields where gene expression must be closely controlled by intracellular physiology, such as gene therapy.

Protein splicing in the absence of an intein penultimate histidine.

Protein splicing is a self-catalytic process in which an intervening sequence, termed an intein, is excised from a protein precursor, and the flanking polypeptides are religated. The conserved intein penultimate His facilitates this reaction by assisting in Asn cyclization, which results in C-terminal splice junction cleavage. However, many inteins do not have a penultimate His. Previous splicing studies with 2 such inteins yielded contradictory results. To resolve this issue, the splicing capacity of 2 more inteins without penultimate His residues was examined. Both the Methanococcus jannaschii phosphoenolpyruvate synthase and RNA polymerase subunit A' inteins spliced. Splicing of the phosphoenolpyruvate synthase intein improved when its penultimate Phe was changed to His, but splicing of the RNA polymerase subunit A' intein was inhibited when its penultimate Gly was changed to His. We propose that inteins lacking a penultimate His (i) arose by mutation from ancestors in which a penultimate His facilitated splicing, (ii) that loss of this His inhibited, but may not have blocked, splicing, and (iii) that selective pressure for efficient expression of the RNA polymerase yielded an intein that utilizes another residue to assist Asn cyclization, changing the intein active site so that a penultimate His now inhibits splicing.