On-line monitoring of intracellular ATP concentration in Escherichia coli fermentations

Lasko, Daniel R; Wang, Daniel I. C.

doi:10.1002/(sici)1097-0290(19961105)52:3<364::aid-bit2>3.0.co;2-i

Cited by 33 publications

(18 citation statements)

References 14 publications

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“…Although many of the critical process parameters cannot currently be measured online, intensive research efforts have been made to develop new sensors and sampling devices/ techniques (Schugerl et al, 1996). For example, online monitoring of green fluorescence protein (GFP) fluorescence to determine recombinant product concentration (Randers-Eichhorn et al, 1997), and online monitoring of firefly luciferase to track intracellular ATP concentration (Lasko and Wang, 1996) have been reported. In the case of GFP, green fluorescence could be monitored online and/or in vivo during fermentation to indicate product level (Albano et al, 1996(Albano et al, , 1998Cha et al, 1997Cha et al, , 1999aDeLisa et al, 1999c;Randers-Eichhorn et al, 1997).…”

Section: Introductionmentioning

confidence: 76%

Framework for online optimization of recombinant protein expression in high-cell-densityEscherichia coli cultures using GFP-fusion monitoring

Chae

DeLisa

Joon

et al. 2000

Biotechnol. Bioeng.

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Section: Introductionmentioning

confidence: 76%

Framework for online optimization of recombinant protein expression in high-cell-densityEscherichia coli cultures using GFP-fusion monitoring

Chae

DeLisa

Joon

et al. 2000

Biotechnol. Bioeng.

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“…Since the intracellular ATP concentration of enteric bacteria has been reported to be about 3 mM (4,26,29,58; for a review, see reference 37), we concluded that the concentration of acetyl-P is also about 3 mM. Implicit in this calculation is the assumption that all three of the phosphates within ATP are turned over at approximately the same rate.…”

Section: Discussionmentioning

confidence: 99%

The Intracellular Concentration of Acetyl Phosphate in Escherichia coli Is Sufficient for Direct Phosphorylation of Two-Component Response Regulators

et al. 2007

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Acetyl phosphate, the intermediate of the AckA-Pta pathway, acts as a global signal in Escherichia coli. Although acetyl phosphate clearly signals through two-component response regulators, it remains unclear whether acetyl phosphate acts as a direct phospho donor or functions through an indirect mechanism. We used two-dimensional thin-layer chromatography to measure the relative concentrations of acetyl phosphate, acetyl coenzyme A, ATP, and GTP over the course of the entire growth curve. We estimated that the intracellular concentration of acetyl phosphate in wild-type cells reaches at least 3 mM, a concentration sufficient to activate two-component response regulators via direct phosphoryl transfer.Bacterial cells must respond properly to diverse external and internal cues, a process that requires global signaling. The ideal global signal is metabolically inexpensive, short-lived, indicative only of significant changes, and capable of effecting the coordinated regulation of diverse cellular processes. Several lines of evidence suggest that the small molecule acetyl phosphate (acetyl-P) can serve as such a global signal.Acetyl-P is the high-energy, acid/base-labile intermediate of the reversible Pta-AckA pathway (Fig. 1). This pathway interconverts coenzyme A (HS-CoA), ATP, and acetate with acetyl coenzyme A (acetyl-CoA), ADP, and orthophosphate (P i ) (9, 43). The reversibility of this pathway permits both acetyl-CoA synthesis (acetate activation) and acetate evolution (acetogenesis). During acetogenesis, Pta synthesizes acetyl-P and HSCoA from acetyl-CoA and P i , while AckA generates ATP from acetyl-P and ADP. Simultaneously, AckA produces acetate, which cells excrete into the environment. Thus, the steadystate concentration of acetyl-P depends upon the rate of its formation catalyzed by Pta and the rate of its degradation catalyzed by AckA (for reviews, see references 45 and 59).Acetyl-P has been proposed to act as a global regulator by direct phosphorylation of response regulators (RRs) of the family of two-component signal transduction (2CST) pathways (34, 55). The simplest of these 2CST pathways consists of a histidine kinase (HK) and an RR. Using ATP as its phosphoryl donor, the HK autophosphorylates a conserved histidine residue. In turn, the RR autophosphorylates a conserved aspartate residue, using its phosphorylated cognate HK as its phosphoryl donor. Experiments performed in vitro have clearly demonstrated that acetyl-P can donate its phosphoryl group to purified RRs but not to HKs (for reviews, see references 52 and 57). This ability arises from its capacity to store energy. Acetyl-P possesses a larger ⌬G°of hydrolysis (Ϫ43.3 kJ/mol) than ATP possesses (Ϫ30.5 kJ/mol in complex with Mg 2ϩ ). This difference forms the basis for the role of acetyl-P in generating ATP (for a review, see reference 59).Although acetyl-P can function as a phosphoryl donor in vitro, its ability to function in vivo as a global signal has remained essentially unproven, despite a wealth of seemingly supportive data (for a r...

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“…Here, we report a comprehensive kinetic study of the forward reaction of AK, exploring concentrations of nucleotides and Mg 2ϩ that are comparable to those inside E. coli cells, [Mg 2ϩ ] ϳ 1-2 mM (38) and [ATP] up to 3 mM (39). We discovered a previously unreported phenomenon: an increase in the forward reaction rate of AK with increasing Mg 2ϩ concentrations, where the stoichiometry of Mg 2ϩ to the enzyme is greater than one.…”

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confidence: 99%

Direct Mg2+ Binding Activates Adenylate Kinase from Escherichia coli

Tan

Hanson

Yang

2009

Journal of Biological Chemistry

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We report evidence that adenylate kinase (AK) from Escherichia coli can be activated by the direct binding of a magnesium ion to the enzyme, in addition to ATP-complexed Mg 2؉ . By systematically varying the concentrations of AMP, ATP, and magnesium in kinetic experiments, we found that the apparent substrate inhibition of AK, formerly attributed to AMP, was suppressed at low magnesium concentrations and enhanced at high magnesium concentrations. This previously unreported magnesium dependence can be accounted for by a modified random bi-bi model in which Mg 2؉ can bind to AK directly prior to AMP binding. A new kinetic model is proposed to replace the conventional random bi-bi mechanism with substrate inhibition and is able to describe the kinetic data over a physiologically relevant range of magnesium concentrations. According to this model, the magnesium-activated AK exhibits a 23-؎ 3-fold increase in its forward reaction rate compared with the unactivated form. The findings imply that Mg 2؉ could be an important affecter in the energy signaling network in cells. Adenylate kinase (AK)2 is a ϳ24-kDa enzyme involved in cellular metabolism that catalyzes the reversible phosphoryl transfer reaction (1) as in Reaction 1.It is recognized to play an important role in cellular energetic signaling networks (2, 3). A deficiency in human AK function may lead to such illness as hemolytic anemia (4 -8) and coronary artery disease (9); the latter is thought to be caused by a disruption of the AMP signaling network of AK (10). The ubiquity of AK makes it an ideal candidate for investigating evolutionary divergence and natural adaptation at a molecular level (11,12). Indeed, extensive structure-function studies have been carried out for AK (reviewed in Ref. 13). Both structural and biophysical studies have suggested that large-amplitude conformational changes in AK are important for catalysis (14 -19). More recently, the functional roles of conformational dynamics have been investigated using NMR (20 -22), computer simulations (23-27), and single-molecule spectroscopy (28). Given the critical role of AK in regulating cellular energy networks and its use as a model system for understanding the functional roles of conformational changes in enzymes, it is imperative that the enzymatic mechanism of AK be thoroughly characterized and understood.The enzymatic reaction of adenylate kinase has been shown to follow a random bi-bi mechanism using isotope exchange experiments (29). Isoforms of adenylate kinases characterized from a wide range of species have a high degree of sequence, structure, and functional conservation. Although all AKs appear to follow the same random bi-bi mechanistic framework (15, 29 -33), a detailed kinetic analysis reveals interesting variations among different isoforms. For example, one of the most puzzling discrepancies is the change in turnover rates with increasing AMP concentration between rabbit muscle AK and Escherichia coli AK. Although the reactivity of rabbit muscle AK is slightly inhibited at high...

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On-line monitoring of intracellular ATP concentration in Escherichia coli fermentations

Cited by 33 publications

References 14 publications

Framework for online optimization of recombinant protein expression in high-cell-densityEscherichia coli cultures using GFP-fusion monitoring

Framework for online optimization of recombinant protein expression in high-cell-densityEscherichia coli cultures using GFP-fusion monitoring

The Intracellular Concentration of Acetyl Phosphate in Escherichia coli Is Sufficient for Direct Phosphorylation of Two-Component Response Regulators

Direct Mg2+ Binding Activates Adenylate Kinase from Escherichia coli

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