Pyruvate metabolism and the phosphorylation state of isocitrate dehydrogenase in Escherichia coli

During growth of Escherichia coli on acetate, isocitrate dehydrogenase (ICDH) is partially inactivated by phosphorylation and is thus rendered rate-limiting in the Krebs cycle so that the intracellular concentration of isocitrate rises which, in turn, permits an increased flux of carbon through the anaplerotic sequence of the glyoxylate bypass. A large number of metabolites stimulate ICDH phosphatase and inhibit ICDH kinase in the wild-type (E. coli ML308) and thus regulate the utilization of isocitrate by the two competing enzymes, ICDH and isocitrate lyase. Addition of pyruvate to acetate grown cultures triggers a rapid dephosphorylation and threefold activation of ICDH, both in the wild-type (ML308) and in mutants lacking pyruvate dehydrogenase (ML308/Pdh-), PEP synthase (ML308/Pps-) or both enzymes (ML308/Pdh-Pps-). Pyruvate stimulates the growth on acetate of those strains with an active PEP synthase but inhibits the growth of those strains that lack this enzyme. When pyruvate is exhausted, ICDH is again inactivated and the growth rate reverts to that characteristic of growth on acetate. Because pyruvate stimulates dephosphorylation of ICDH in strains with differing capabilities for pyruvate metabolism, it seems likely that pyruvate itself is a sufficient signal to activate the dephosphorylation mechanism, but this does not discount the importance of other signals under other circumstances.     

Compensatory phosphorylation of isocitrate dehydrogenase. A mechanism for adaptation to the intracellular environment

When Escherichia coli grows on acetate, the flow of isocitrate through the glyoxylate bypass is regulated, in part, through the phosphorylation of isocitrate dehydrogenase. In addition to its role in adaptation to alternative carbon sources, this phosphorylation system responds to variation in the intracellular level of isocitrate dehydrogenase. This system can compensate for changes in the cellular level of isocitrate dehydrogenase in excess of 10-fold, maintaining a nearly constant activity for isocitrate dehydrogenase during growth on acetate. The behavior of the phosphorylation system exhibited considerable strain-specific variation. This was most clearly demonstrated using mutants which lacked the ability to phosphorylate isocitrate dehydrogenase. In two strains, mutation of the gene for isocitrate dehydrogenase kinase/phosphatase rendered the cells unable to grow on acetate. In contrast, a third strain was relatively insensitive to a mutation in this gene. This lack of phenotypic expression appears to result from a lower cellular level of isocitrate dehydrogenase in this strain which renders the phosphorylation (and consequent inhibition) of isocitrate dehydrogenase less essential. The gene for isocitrate dehydrogenase kinase/phosphatase (aceK) was located in the glyoxylate bypass operon, downstream from the genes for isocitrate lyase and malate synthase.     

Isocitrate dehydrogenase kinase/phosphatase: aceK alleles that express kinase but not phosphatase activity.

For Escherichia coli, growth on acetate requires the induction of the enzymes of the glyoxylate bypass, isocitrate lyase and malate synthase. The branch point between the glyoxylate bypass and the Krebs cycle is controlled by phosphorylation of isocitrate dehydrogenase (IDH), inhibiting that enzyme's activity and thus forcing isocitrate through the bypass. This phosphorylation cycle is catalyzed by a bifunctional enzyme, IDH kinase/phosphatase, which is encoded by aceK. We have employed random mutagenesis to isolate novel alleles of aceK. These alleles were detected by the loss of ability to complement an aceK null mutation. The products of one class of these alleles retain IDH kinase activity but have suffered reductions in IDH phosphatase activity by factors of 200 to 400. Selective loss of the phosphatase activity also appears to have occurred in vivo, since cells expressing these alleles exhibit phenotypes which are reminiscent of strains lacking IDH; these strains are auxotrophic for glutamate. Assays of cell-free extracts confirmed that this phenotype resulted from nearly quantitative phosphorylation of IDH. The availability of these novel alleles of aceK allowed us to assess the significance of the precise control which is a characteristic of the IDH phosphorylation cycle in vivo. The fractional phosphorylation of IDH was varied by controlled expression of one of the mutant alleles, aceK3, in a wild-type strain. Reduction of IDH activity to 50% of the wild-type level did not adversely affect growth on acetate. However, further reductions inhibited growth, and growth arrest occurred when the IDH activity fell to 15% of the wild-type level. Thus, although wild-type cells maintain a precise effective IDH activity during growth on acetate, this precision is not critical.     

Control of metabolic interconversion of isocitrate dehydrogenase between the catalytically active and inactive forms in Escherichia coli

The enzymic interconversion of Escherichia coli isocitrate dehydrogenase (ICDH) between the catalytically active and inactive forms is mediated through the activities of ICDH-kinase/phosphatase in response to changes in the metabolic environment. In this study, the use of mutant strains devoid of isocitrate lyase ( aceA:: Tn10 ) and pyruvate dehydrogenase activities revealed that the signal which triggers the reversible inactivation of ICDH in vivo is not directly related to acetate itself, but rather to the need to maintain high intracellular levels of isocitrate and free co-enzyme A. The use of these mutants also revealed, rather unexpectedly, that acetate grown cells contain more ICDH protein than those grown with other carbon sources and that the catalytic activity of ICDH kinase/phosphatase is in excess of cellular demands. Furthermore, this study also revealed the presence of a 50-kDa (±2 kDa) acetate-specific polypeptide, the identity of which has yet to be established.     

The isocitrate dehydrogenase phosphorylation cycle. Identification of the primary rate-limiting step

In Escherichia coli, the branch point between the Krebs cycle and the glyoxylate bypass is regulated by the phosphorylation of isocitrate dehydrogenase (IDH). Phosphorylation inactivates IDH, forcing isocitrate through the bypass. This bypass is essential for growth on acetate but does not serve a useful function when alternative carbon sources, such as glucose or pyruvate, are also present. When pyruvate or glucose is added to a culture growing on acetate, the cells responded by dephosphorylating IDH and thus inhibiting the flow of isocitrate through the glyoxylate bypass. In an effort to identify the primary rate-limiting step in the response of IDH phosphorylation to alternative carbon sources, we have examined the response rates of congenic strains of E. coli which express different levels of IDH kinase/phosphatase, the bifunctional protein which catalyzes this phosphorylation cycle. The rate of the pyruvate-induced dephosphorylation of IDH was proportional to the level of IDH kinase/phosphatase, indicating that IDH kinase/phosphatase was primarily rate-limiting for dephosphorylation. However, the identity of the primary rate-limiting step appears to depend on the stimulus, since the rate of dephosphorylation of IDH in response to glucose was independent of the level of IDH kinase/phosphatase.     

Role of energetic coenzyme pools in the production of L-carnitine by Escherichia coli

The aim of this work was to understand the steps controlling the biotransformation of trimethylammonium compounds into L(-)-carnitine by Escherichia coli. The high-cell density reactor steady-state levels of carbon source (glycerol), biotransformation substrate (crotonobetaine), acetate (anaerobiosis product) and fumarate (as an electron acceptor) were pulsed by increasing them fivefold. Following the pulse, the evolution of the enzyme activities involved in the biotransformation process of crotonobetaine into L(-)-carnitine (crotonobetaine hydration), in the synthesis of acetyl-CoA (ACS: acetyl-CoA synthetase and PTA: ATP: acetate phosphotransferase) and in the distribution of metabolites for the tricarboxylic acid (ICDH: isocitrate dehydrogenase) and glyoxylate (ICL: isocitrate lyase) cycles was monitored. In addition, the levels of carnitine, the cell ATP content and the NADH/NAD(+) ratio were measured in order to assess the importance and participation of these energetic coenzymes in the catabolic system. The results provided an experimental demonstration of the important role of the glyoxylate shunt during biotransformation and the need for high levels of ATP to maintain metabolite transport and biotransformation. Moreover, the results obtained for the NADH/NAD(+) pool indicated that it is correlated with the biotransformation process at the NAD(+) regeneration and ATP production level in anaerobiosis. More importantly, a linear correlation between the NADH/NAD(+) ratio and the levels of the ICDH and ICL (carbon and electron flows) and the PTA and ACS (acetate and ATP production and acetyl-CoA synthesis) activity levels was assessed. The main metabolic pathway operating during cell metabolic perturbation with a pulse of glycerol and acetate in the high-cell density membrane reactor was that related to ICDH and ICL, both regulating the carbon metabolism, together with PTA and ACS enzymes (regulating ATP production).     

Purification and characterization of Acinetobacter calcoaceticus isocitrate lyase.

Acinetobacter calcoaceticus is capable of growing on acetate or compounds that are metabolized to acetate. During adaptation to growth on acetate, A. calcoaceticus B4 exhibits an increase in NADP(+)-isocitrate dehydrogenase and isocitrate lyase activities. In contrast, during adaptation to growth on acetate, Escherichia coli exhibits a decrease in NADP(+)-isocitrate dehydrogenase activity that is caused by reversible phosphorylation of specific serine residues on this enzyme. Also, in E. coli, isocitrate lyase is believed to be active only in the phosphorylated form. This phosphorylation of isocitrate lyase may regulate entry of isocitrate into the glyoxylate bypass. To understand the relationships between these two isocitrate-metabolizing enzymes and the metabolism of acetate in A. calcoaceticus B4 better, we have purified isocitrate lyase to homogeneity. Physical and kinetic characterization of the enzyme as well as the inhibitor specificity and divalent cation requirement have been examined.     

Isocitrate dehydrogenase kinase/phosphatase

In Escherichia coli, isocitrate dehydrogenase (IDH) is regulated by phosphorylation. This phosphorylation cycle is catalyzed by an unusual, bifunctional protein:IDH kinase/phosphatase. IDH kinase/phosphatase is expressed from a single gene, aceK, and both activities are catalyzed by the same polypeptide. The amino acid sequence of IDH kinase/phosphatase does not exhibit the characteristics which are typical of other protein kinases, although it does contain a consensus ATP binding site. The available evidence suggests that the IDH kinase and IDH phosphatase reactions occur at the same active site and that the IDH phosphatase reaction results from the back reaction of IDH kinase tightly coupled to ATP hydrolysis. The function of the IDH phosphorylation cycle is to control the flux of isocitrate through the glyoxylate bypass. This pathway is essential for growth on acetate because it prevents the quantitative loss of the acetate carbons as CO2 in the Krebs' cycle. IDH kinase/phosphatase monitors general metabolism by responding to the levels of a wide variety of metabolites, many of which activate IDH phosphatase and inhibit IDH kinase. The ability of IDH kinase/phosphatase to monitor general metabolism allows. the IDH phosphorylation cycle to compensate for substantial perturbations of the system, such as a 15-fold overproduction of IDH. The significance of the cellular level of IDH kinase/phosphatase has also been evaluated. The level of this protein is in great excess of that required for steady-state growth on acetate. In contrast, IDH kinase/phosphatase is, in some cases, rate-limiting for the dephosphorylation of IDH which results when preferred carbon sources are added to cultures growing on acetate.     

The reversible phosphorylation of isocitrate dehydrogenase of Salmonella typhimurium

The 46,000 dalton phosphoprotein in Salmonella typhimurium is isocitrate dehydrogenase, an enzyme at the branch point between the glyoxylate and Krebs cycle pathways. The enzyme is phosphorylated by a kinase which is controlled by growth conditions; and it is dephosphorylated by a phosphatase. Acetate, ethanol, α-methylglucoside, and deoxyglucose cause an activation of the phosphorylation reaction in intact cells. A number of other compounds are found to affect the kinase and phosphatase activities. The reversible phosphorylation of isocitrate dehydrogenase plays a major role in the control of the Krebs cycle and glyoxylate pathways.     

The phosphorylation of Escherichia coli isocitrate dehydrogenase in intact cells.

The isocitrate dehydrogenase of Escherichia coli ML308 can be reversibly activated by addition of pyruvate to cells growing on acetate [Bennett & Holms (1975) J. Gen. Microbiol. 87, 37-51]. By using cells pulse-labelled with [32P]Pi we showed that the activation and inactivation of the enzyme in these conditions correlate with its dephosphorylation and rephosphorylation respectively. Incubation of cell extracts prepared during an activation/inactivation cycle with purified isocitrate dehydrogenase phosphatase confirmed that the pyruvate-induced activation of the dehydrogenase goes essentially to completion. The results show that the reversible changes in the activity of the dehydrogenase in cells grown on acetate are solely due to phosphorylation/dephosphorylation. Inactive 32P-labelled isocitrate dehydrogenase was isolated from cells incubated with [32P]Pi in the presence of acetate. Both this material and purified enzyme phosphorylated in vitro were digested with chymotrypsin, and the phosphopeptides were isolated and analysed. Only one phosphopeptide was observed in each case; the results show that the residue phosphorylated in vivo is identical with that phosphorylated by purified isocitrate dehydrogenase kinase in vitro.     

Glyoxylate bypass operon of Escherichia coli: cloning and determination of the functional map.

In Escherichia coli, a single operon encodes the metabolic and regulatory enzymes of the glyoxylate bypass. The metabolic enzymes, isocitrate lyase and malate synthase, are expressed from aceA and aceB, and the regulatory enzyme, isocitrate dehydrogenase kinase/phosphatase, is expressed from aceK. We cloned this operon and determined its functional map by deletion analysis. The order of the genes in this operon is aceB-aceA-aceK, with aceB proximal to the promoter, consistent with the results of previous experiments using genetic techniques. The promoter was identified by S1 nuclease mapping, and its nucleotide sequence was determined. Isocitrate lyase and malate synthase were readily identified by autoradiography after the products of the operon clone were labeled by the maxicell procedure and then resolved by electrophoresis. In contrast, isocitrate dehydrogenase kinase/phosphatase, expressed from the same plasmid, was undetectable. This observation is consistent with a striking downshift in expression between aceA and aceK.     

Regulation of isocitrate lyase inRhodobacter capsulatus E1F1

InRhodobacter capsulatus E1F1, isocitrate lyase (ICL) (EC 4.5.3.1) is a regulatory enzyme whose levels are increased in the presence of acetate as the sole carbon source. Acetate activated isocitrate lyase in a process dependent on energy supply and de novo protein synthesis. In contrast to isocitrate lyase, isocitrate dehydrogenase (ICDH) activity was independent of the carbon source used for growth and significantly increased in darkened cells. Pyruvate or yeast extract prevented in vivo activation of isocitrate lyase in cells growing on acetate. The enzyme was reversibly inactivated to a great extent in vitro by pyruvate and other oxoacids presumably involved in acetate metabolism. These results suggest that, inR. capsulatus E1F1, isocitrate lyase is regulated by both enzyme synthesis and oxoacid inactivation.     

Structural basis of the substrate specificity of bifunctional isocitrate dehydrogenase kinase/phosphatase

Isocitrate dehydrogenase kinase/phosphatase (AceK) regulates entry into the glyoxylate bypass by reversibly phosphorylating isocitrate dehydrogenase (ICDH). On the basis of the recently determined structure of the AceK-ICDH complex from Escherichia coli, we have classified the structures of homodimeric NADP(+)-ICDHs to rationalize and predict which organisms likely contain substrates for AceK. One example is Burkholderia pseudomallei (Bp). Here we report a crystal structure of Bp-ICDH that exhibits the necessary structural elements required for AceK recognition. Kinetic analyses provided further confirmation that Bp-ICDH is a substrate for AceK. We conclude that the highly stringent AceK binding sites on ICDH are maintained only in Gram-negative bacteria.     

Molecular characterization of a bifunctional glyoxylate cycle enzyme, malate synthase/isocitrate lyase, in Euglena gracilis

Euglena gracilis induced glyoxylate cycle enzymes when ethanol was fed as a sole carbon source. We purified, cloned and characterized a bifunctional glyoxylate cycle enzyme from E. gracilis (EgGCE). This enzyme consists of an N-terminal malate synthase (MS) domain fused to a C-terminal isocitrate lyase (ICL) domain in a single polypeptide chain. This domain order is inverted compared to the bifunctional glyoxylate cycle enzyme in Caenorhabditis elegans, an N-terminal ICL domain fused to a C-terminal MS domain. Purified EgGCE catalyzed the sequential ICL and MS reactions. ICL activity of purified EgGCE increased in the existence of acetyl-CoA at a concentration of micro-molar order. We discussed the physiological roles of the bifunctional glyoxylate cycle enzyme in these organisms as well as its molecular evolution.     

Structural and mechanistic insights into the bifunctional enzyme isocitrate dehydrogenase kinase/phosphatase AceK

The switch between the Krebs cycle and the glyoxylate bypass is controlled by isocitrate dehydrogenase kinase/phosphatase (AceK). AceK, a bifunctional enzyme, phosphorylates and dephosphorylates isocitrate dehydrogenase (IDH) with its unique active site that harbours both the kinase and ATP/ADP-dependent phosphatase activities. AceK was the first example of prokaryotic phosphorylation identified, and the recent characterization of the structures of AceK and its complex with its protein substrate, IDH, now offers a new understanding of both previous and future endeavours. AceK is structurally similar to the eukaryotic protein kinase superfamily, sharing many of the familiar catalytic and regulatory motifs, demonstrating a close evolutionary relationship. Although the active site is shared by both the kinase and phosphatase functions, the catalytic residues needed for phosphatase function are readily seen when compared with the DXDX(T/V) family of phosphatases, despite the fact that the phosphatase function of AceK is strictly ATP/ADP-dependent. Structural analysis has also allowed a detailed look at regulation and its stringent requirements for interacting with IDH.