Central Intermediary Metabolism

From whole genome analysis and from early biochemical and radiotracer studies we know that L. pneumophila contains a complete glycolytic pathway as well as key components of the pentose phosphate pathway and Entner Doudoroff pathway (see Figure 7.2). L. pneumophila lacks sugar transporters and must rely on gluconeogenesis for synthesis of sugars for biosynthesis of peptidoglycan, LPS, ribose, and deoxyribose and other cellular components. The bacteria can be grown in chemically defined medium and the addition of glucose to this medium does not change the growth rate (Ristrof et al., 1981; Warren and Miller, 1979). However, no studies have examined the nutritional requirements of L. pneu-mophila during intracellular growth or whether phosphorylated or nucleotide sugars might be utilized under these conditions. Generally bacteria that must synthesize sugars from Krebs cycle intermediates express several major gluco-neogenic enzymes. There are several major enzymes of gluconeogenesis that connects the Krebs cycle with the gluconeogenic pathway. PEP carboxylase, which converts oxaloacetate (OAA) to phosphoenol pyruvate (PEP), has been

Phosphoenolpyruvate Carboxylase

Figure 7.2. Metabolic pathways of L. pneumophila. The metabolic pathways depicted include the Krebs cycle, Entner Doudoroff (ED), Pentose Phosphate and Embden Myerhof pathways. The enzyme abbreviations used are common and key metabolic reactions are indicated by thickened arrows. Key enzymes of these pathways are discussed in the text. Legionella lacks transporters for sugars and therefore must rely on gluconeogenesis for biosynthesis of sugars.

Figure 7.2. Metabolic pathways of L. pneumophila. The metabolic pathways depicted include the Krebs cycle, Entner Doudoroff (ED), Pentose Phosphate and Embden Myerhof pathways. The enzyme abbreviations used are common and key metabolic reactions are indicated by thickened arrows. Key enzymes of these pathways are discussed in the text. Legionella lacks transporters for sugars and therefore must rely on gluconeogenesis for biosynthesis of sugars.

measured in cell free extracts (specific activity for this enzyme is 8 nmoles per min per mg of protein), and a second enzyme of this pathway, PEP carboxy kinase, was 8.4 nmoles per min per mg protein. Another major enzyme is PEP synthase which produces PEP by ATP-dependent phosphorylation of pyruvate. Pyruvate carboxylase produces OAA from pyruvate with ATP and CO2 and exhibits a high specific activity (140 nmoles per min per mg protein). The abundance of these classes of enzymes strongly supports a gluconeogenic metabolism and suggests that much of the carbon entering the Krebs cycle is directed to synthesis of glucose, fructose, and ribose sugars. L. pneumophila contains a complete catabolic Embden Myerhof pathway, but the direction of the pathway (catabolic versus gluconeogenic) is determined by the specific activities of key enzymes at several points in this pathway. In this regard, the key metabolic enzyme of the gluconeogenic pathway is fructose 1,6 biphosphatase whose activity in cell extracts from L. pneumophila was tenfold higher than that measured for phospho-fructokinase, the key catabolic enzyme that forms fructose 1,6 biphosphate. Interestingly, the fructose 1,6 biphosphatase gene is not present in the genomes of all three sequenced strains of L. pneumophila and in close relative Coxiella burnetii. The lack of an annotated gene for an enzyme activity is not uncommon as in silico pathway reconstructions have demonstrated for other bacteria. There are generally three possible explanations assuming the enzyme data are robust: (i) the functional gene might have diverged sufficiently that it is not picked up on BLAST searches; (ii) a paralog enzyme of related function might catalyze the reaction; or (iii) an enzyme that is unidirectional in some bacteria may be bidirectional or bifunctional in other bacteria. In this regard, a further examination of the 6-phosphofructokinase enzyme of L. pneumophila and C. burnetii, which typically catalyzes the forward reaction, indicates that the enzyme is of the PfkA class that uses PPi (pyrophosphate) instead of ATP. These enzymes are bifunc-tional (catalyzing the phosphorylation as well as the phosphatase activity). In contrast, enzymes of the PfkB class are not reversible and in enteric bacteria the phosphofructokinase enzyme catalyzes the formation of fructose 1,6 biphosphate and the fructose 1,6-biphosphatase catalyzes formation of F6P. Since this enzyme step is critical in synthesis of fructose-6-P (see Figure. 7.2), it is likely that essentiality testing would demonstrate the necessity of pfkA in gluconeogen-esis. Further studies with purified PfkA would be required to formally test substrate specificities and enzyme direction.

A second key gene that is absent in L. pneumophila is 6-phosphogluconate dehydrogenase which decarboxylates 6-phosphogluconate to produce ribulose 5 phosphate, which is subsequently converted to ribose-5-P and used for synthesis of ribose and deoxyribose (DNA and RNA synthesis). In other bacteria, such as Helicobacter pylori, ribose sugars are produced from the products of the Entner Doudoroff pathway together with fructose 6-P that is generated via gluconeogene-sis (Hoffman, 2001; Chalker et al., 2001). In H. pylori, fructose-1,6-biphosphatase activity is essential as mutations in the gene render H. pylori non-viable (Chalker et al., 2001). In this example, H. pylori lacks phosphofructokinase activity and is thereby blocked in the forward direction of glycolysis, despite its ability to utilize glucose. The major enzymes associated with the Pentose Phosphate Pathway of L. pneumophila (transketolase and transaldolase) catalyze many of the interconversions between fructose-6-P, glyceraldehyde-3-P (generated by the ED pathway) to produce all the intermediates required for synthesis of ribose and deoxyribose sugars, as well as 4-carbon compounds required for synthesis of vitamins and aromatic amino acids. Little attention has been paid to metabolic systems in L. pneumophila and metabolism is quite relevant to pathogenesis and persistence.

This is especially true for the planktonic cyst form that exhibits no respiratory activity and is near dormant metabolically (Garduño et al., 2002).

The Krebs cycle is complete and begins with the oxidative decarboxylation of pyruvate to produce acetyl CoA which condenses with oxaloacetate to form citrate (citrate synthase). However, the measured activity of pyruvate dehydrogenase was low (1.64 nmoles per min per mg protein) in cellular extracts suggesting that little pyruvate is converted to acetyl CoA (Keen and Hoffman, 1984). Generally pyruvate oxidation is required for generation of acetyl CoA to drive the Krebs cycle. However, it is quite likely that the required acetyl CoA is generated from fatty acid catabolism in concert with the many phospholipases noted for this organism. The deamination of L-aspartate, a major carbon source, produces sufficient oxaloacetate to condense with acetyl CoA to produce citrate. Such reactions would spare consumption of pyruvate by the Krebs cycle. Other organic acid intermediates of the Krebs cycle are derived from glutamate, serine, and related amino acids. In natural amoebic hosts, all of the amino acids would be obtained from the host. As mentioned earlier, much of the carbon required for biosynthesis of peptidoglycan, LPS, nucleic acids, and some of the amino acids associated with protein synthesis must be directed into the gluconeogenic route and this is supported by the high specific activities of gluconeogenic enzymes and Krebs cycle enzymes relative to those activities measured for the Embden Myerhof pathway.

The complete Krebs cycle is depicted in Figure. 7.2. The aconitase enzyme that converts citrate to isocitrate has received some attention as it requires iron for biological activity. In iron-starved bacteria, the activity of this enzyme decreases and is part of the phenotype associated with iron-restricted growth. In addition to the oxoglutarate dehydrogenase system (dihydrolipoamide dehydrogenase and dihy-drolipoamide succinyltransferase), L. pneumophila also contains the KorA and KorB subunits of oxoglutarate ferredoxin oxidoreductase that is commonly found in anaerobic bacteria. While the genes are present, direct enzymatic activity for these proteins has not been examined. It is possible that this alternate pathway functions under oxygen-limiting conditions that might exist during cyst morphogenesis late in the growth cycle in vivo. The POR and KOR enzymes are usually coupled with ferredoxin or flavodoxin that can function under anaerobic conditions. The specific activity for a-ketoglutarate dehydrogenase (NAD) was robust (~20 nmoles per min per mg protein) indicating that this is the major enzyme complex in bacteria grown in vitro. Most of the Krebs cycle enzymes are of high specific activity relative to glycolytic enzymes. Neither the genome sequence nor direct enzyme assay found any evidence for the glyoxylate bypass in L. pneu-mophila (Keen and Hoffman, 1984; Cazalet et al., 2004). While the isocitrate dehydrogenase exhibited specificity for NADP, the malate dehydrogenase exhibited specificity for NAD. One of the most active enzymes detected in cell free extracts of L. pneumophila was glutamate-aspartate transaminase which transfers the amino group from glutamate to oxaloacetate to produce aspartate. In order for there to be available oxaloacetate for this reaction (assuming an abundance of glutamate), conversion of serine to pyruvate and subsequent CO2 fixation to oxaloacetate would be required. It is not clear how the carbon and nitrogen balance is maintained or when in the growth cycle synthesis of storage polymers of poly-p-hydroxybutyrate is initiated. Generally synthesis of PHBA occurs under conditions of high carbon and low nitrogen.

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  • basso
    WHAT THE the Krebs Cycle, intermediates?
    8 years ago
  • ALEXIS
    How is gluconeogenisis connected to krebs cycle?
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