Major factors affecting isocitrate lyase activity in Rhodobacter capsulatus B10 under phototrophic conditions (original) (raw)
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Isocitrate lyase of the facultative intracellular pathogen Rhodococcus equi
2002
Isocitrate lyase is the first enzyme of the glyoxylate shunt which is required for the assimilation of fatty acids and acetate. The intracellular pathogen Rhodococcus equi contains high activities of this enzyme following growth on acetate and lactate, indicating that it plays an important role in the metabolism of these substrates. The gene encoding isocitrate lyase (aceA) was cloned and
The control of the synthesis of isocitrate lyase in a thermophilic bacillus
Journal of general microbiology, 1979
From a strain of Bacillus stearothermophilus, devoid of active pyruvate carboxylase, a mutant (NG-15) was selected that grew on acetate in the presence of glucose. This mutant differed from its parent organism in possessing high activities of isocitrate lyase when grown on all carbon sources tested except nutrient broth, in possessing unusually low activities of NADP+-dependent isocitrate dehydrogenase and in containing increased amounts of isocitrate. Revertants of mutant NG-15 which regained the ability to synthesize active pyruvate carboxylase also synthesized isocitrate lyase and isocitrate dehydrogenase to the same extent as the wild-type strain. These results suggest that the regulatory mechanism for the synthesis of isocitrate lyase in the thermophile may be different from that in mesophilic bacilli.
Activation of isocitrate lyase and triosephosphate dehydrogenase in Azotobacter vinelandii extracts
Biochimica et Biophysica Acta, 1963
[s0citrate ]yase is 20treed in Azolobact, er vinelandii during growth on acetate. Reported requirements of isocitrate ]yase for Mg 2+ and cysteine have been confirmed, bu~ EDTA could replace cysteine for activation, and did so more efficiently. This obx'iat~ the serious interference by cysteipe in glyoxylic acid measurement. Isocitrate lyase has a half-maximum velocity at &6-zo-6 M threo-I,s-isocitrate and is not inhibited by high DL-isocitrate concentration (up to 8 raM). Triosephosphate dehydrogenase has been identified in ammonium sulphate fi'actions of crude extracts of Azotobacter. This enzyme is also activated by EDTA nlore efficiently than by cysteine. It is concluded that the~e activations by cysteine and EDTA are the result of binding of inhibitor}' heavy metals in the as,say medium, The possible nature of ."filch metals is discussed. Bi~im. Bio~kF~. Ac~. 67 (t~3) Z26-z39 I%OCI1-RATE I.YA~E AND TRI()SF.PIIOSPIlA'rE DEHYDROGENASE
Protein Expression and Purification, 1999
Monomeric isocitrate dehydrogenase was expressed in Corynebacterium glutamicum cells harboring pEK-icdES1, a plasmid carrying the gene for the enzyme. Two-to three-fold higher expression levels of the recombinant enzyme were observed in such cells when grown in fermentors, compared to those grown in shaker incubators. The enzyme was purified to homogeneity by ammonium sulfate fractionation, Sephadex G-150 gel filtration, FPLC Mono Q anion-exchange chromatography, and affinity gel chromatography. Approximately 4 mg of 98% pure recombinant enzyme was obtained per liter of bacterial culture. Our results also include optimum buffer conditions for purification and storage of the enzyme.
Isocitrate Lyase Activity Is Required for Virulence of the Intracellular Pathogen Rhodococcus equi
Infection and Immunity, 2005
Rhodococcus equi is an important pathogen of foals, causing severe pyogranulomatous pneumonia. Virulent R. equi strains grow within macrophages, a process which remains poorly characterized. A potential source of carbon for intramacrophage R. equi is membrane lipid-derived fatty acids, which following  oxidation are assimilated via the glyoxylate bypass. To assess the importance of isocitrate lyase, the first enzyme of the glyoxylate bypass, in virulence of a foal isolate of R. equi, a mutant was constructed by a strategy of single homologous recombination using a suicide plasmid containing an internal fragment of the R. equi aceA gene encoding isocitrate lyase. Complementation of the resulting mutant with aceA showed that the mutant was specific for this gene. Assessment of virulence in a mouse macrophage cell line showed that the mutant was killed, in contrast to the parent strain. Studies in the liver of intravenously infected mice showed enhanced clearance of the mutant. When four 3-week-old foals were infected intrabronchially, the aceA mutant was completely attenuated, in contrast to the parent strain. In conclusion, the aceA gene was shown to be essential for virulence of R. equi, suggesting that membrane lipids may be an important source of carbon for phagocytosed R. equi.
Serine319 and 321 Are Functional in Isocitrate Lyase from Escherichia coli
Current Microbiology, 1997
With site-directed mutagenesis, Ser319 and Ser321 in conserved stretch 3 of tetrameric isocitrate lyase from Escherichia coli were each substituted with alanine, cysteine, asparagine, or threonine in addition to simultaneous alanine/alanine substitutions. Besides their absolute conservation in all aligned isocitrate lyase sequences, the location of these serine residues, which flank a completely conserved proline, had been suggested in the active site in previous research by studies of photoinactivation of the enzyme by vanadate [Ko et al. (1992) J Biol Chem 267:91]. All substitutions for Ser321 and 319 except by threonine appreciably reduced the k cat of E. coli isocitrate lyase relative to that for wild-type (100) as follows:
Physiology of dark fermentative growth of Rhodopseudomonas capsulata
Journal of bacteriology, 1980
The photosynthetic bacterium Rhodopseudomonas capsulata can grow under anaerobic conditions with light as the energy source or, alternatively, in darkness with D-fructose or certain other sugars as the sole source of carbon and energy. Growth in the latter mode requires an "accessory oxidant" such as trimethylamine-N-oxide, and the resulting cells contain the photosynthetic pigments characteristic of R. capsulata (associated with intracytoplasmic membranes) and substantial deposits of poly-beta-hydroxybutyrate. In dark anaerobic batch cultures in fructose plus trimethylamine-N-oxide medium, trimethylamine formation parallels growth, and typical fermentation products accumulate, namely, CO2 and formic, acetic, and lactic acids. These products are also found in dark anaerobic continuous cultures of R. capsulata; acetic acid and CO2 predominate when fructose is limiting, whereas formic and lactic acids are observed at elevated concentrations when trimethylamine-N-oxide is the...
Microbial Physiology, 2005
During aerobic growth of Escherichia coli on acetate as sole source of carbon and energy, the organism requires the operation of the glyoxylate bypass enzymes, namely isocitrate lyase (ICL) and the anaplerotic enzyme malate synthase (MS). Under these conditions, the glyoxylate bypass enzyme ICL is in direct competition with the Krebs cycle enzyme isocitrate dehydrogenase (ICDH) for their common substrate and although ICDH has a much higher affinity for isocitrate, flux of carbon through ICL is assured by virtue of high intracellular level of isocitrate and the reversible phosphorylation/inactivation of a large fraction of ICDH. Reversible inactivation is due to reversible phosphorylation catalysed by ICDH kinase/phosphatase, which harbours both catalytic activities on the same polypeptide. The catalytic activities of ICDH kinase/phosphatase constitute a moiety conserved cycle, require ATP and exhibit ‘zero-order ultrasensitivity’. The structural gene encoding ICDH kinase/phosphatase...