Identification of a cysteine residue at the active site of <i>Escherichia coli</i> isocitrate lyase

Nimmo, Hugh G.; Douglas, Fiona; Kleanthous, Colin; Campbell, David G.; MacKintosh, Carol

doi:10.1042/bj2610431

Cited by 19 publications

(20 citation statements)

References 23 publications

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“…3. For the E. coli enzyme, lysine 193 and cysteine 195, both located within the first of these regions, as well as serine 319 and serine 321, situated in the third conserved domain, were identified as essential for catalysis (10,21,22,34). In accordance with these findings, the C. glutamicum enzyme possesses identical amino acids at the respective sites, indicating the importance of these amino acids in catalysis.…”

supporting

confidence: 58%

Characterization of the isocitrate lyase gene from Corynebacterium glutamicum and biochemical analysis of the enzyme

1994

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Isocitrate lyase is a key enzyme in the glyoxylate cycle and is essential as an anaplerotic enzyme for growth on acetate as a carbon source. It is assumed to be of major importance in carbon flux control in the amino acid-producing organism Corynebacterium ghltaml'cum. In crude extracts of C. glutamicum, the specific activities of isocitrate lyase were found to be 0.01 U/mg of protein after growth on glucose and 2.8 U/mg of protein after growth on acetate, indicating tight regulation. The isocitrate lyase gene, aceA, was isolated, subcloned, and characterized. The predicted gene product of aceA consists of 432 amino acids (M1, 47,228) (27, 28; for a review, see reference 46). Particularly in Escherichia coli, regulation of carbon flux between the two cycles has been subject to intensive investigation. It was found that in E. coli, ICL is formed only when acetate is the sole carbon source of the medium (24). ICL shows low affinity towards isocitrate, and ICD exhibits high affinity for its substrate. To allow significant amounts of carbon to enter the glyoxylate cycle, ICD in E. coli is substantially inactivated by reversible phosphorylation during growth on acetate (27,28,49). Both phosphorylation and dephosphorylation is catalyzed by one enzyme, ICD kinase/phosphatase (28). When acetate is the sole carbon source, the kinase activity of this enzyme leads to phosphorylation and thus to inactivation of ICD. When glucose is added, ICD is dephosphorylated, resulting in the restoration of ICD activity. Besides this regulation of ICD, the E. coli ICL is activated by phosphorylation (37) and is inhibited by a variety of metabolites, e.g., phosphoenolpyruvate (PEP), 3-phosphoglycerate, and succinate (31, 38), thereby allowing fine control of the carbon flux.Since ICL has a key position in the central metabolism, increased attention has been focused on the genetics of ICL in

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confidence: 58%

Characterization of the isocitrate lyase gene from Corynebacterium glutamicum and biochemical analysis of the enzyme

1994

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“…This cysteine is strictly conserved in all known ICL, consistent with its crucial role in catalysis (29,30,50,51). This cysteine was proposed to be required for deprotonation of a carboxylate of succinate during the catalytic reaction (52).…”

Section: Discussionmentioning

confidence: 58%

Regulation by Glutathionylation of Isocitrate Lyase from Chlamydomonas reinhardtii

Bedhomme

Zaffagnini

Marchand

et al. 2009

Journal of Biological Chemistry

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Post-translational modification of protein cysteine residues is emerging as an important regulatory and signaling mechanism. We have identified numerous putative targets of redox regulation in the unicellular green alga Chlamydomonas reinhardtii. One enzyme, isocitrate lyase (ICL), was identified both as a putative thioredoxin target and as an S-thiolated protein in vivo. ICL is a key enzyme of the glyoxylate cycle that allows growth on acetate as a sole source of carbon. The aim of the present study was to clarify the molecular mechanism of the redox regulation of Chlamydomonas ICL using a combination of biochemical and biophysical methods. The results clearly show that purified C. reinhardtii ICL can be inactivated by glutathionylation and reactivated by glutaredoxin, whereas thioredoxin does not appear to regulate ICL activity, and no inter-or intramolecular disulfide bond could be formed under any of the conditions tested. Glutathionylation of the protein was investigated by mass spectrometry analysis, Western blotting, and site-directed mutagenesis. The enzyme was found to be protected from irreversible oxidative inactivation by glutathionylation of its catalytic Cys 178 , whereas a second residue, Cys 247 , becomes artifactually glutathionylated after prolonged incubation with GSSG. The possible functional significance of this post-translational modification of ICL in Chlamydomonas and other organisms is discussed.

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“…Previous studies on ICL enzymes and elucidation of the crystal structure of the enzymes with bound inhibitors (5,6,21,26,34), and similar studies of PEP mutase from Mytilus edulis (19,20), identified residues that affect the catalytic activities of these enzymes. Mutations in equivalent residues in the prpB gene were constructed, including residues C123 (the proposed active site base of ICL), D58, H125, K121, and R122 (Fig.…”

Section: Overproduction and Purification Of H 6 Prpb Proteinmentioning

confidence: 95%

“…Mechanisms of catalysis have also been proposed for the ICL proteins based on three-dimensional structures and data from active-site mutant proteins (5,6,34). Based on ICL structures with bound inhibitors and previous inhibition experiments using 3-bromopyruvate and iodoacetate, it was concluded that a cysteinyl residue was most likely the active-site residue required for isocitrate cleavage (5,6,21,26,34). It is interesting that an equivalently positioned cysteinyl residue is not conserved in PEP mutase, yet is conserved in CPEP mutase and the lyases (Fig.…”

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confidence: 99%

Residues C123 and D58 of the 2-Methylisocitrate Lyase (PrpB) Enzyme ofSalmonella entericaAre Essential for Catalysis

Grimek¹,

Holden

Rayment

et al. 2003

J Bacteriol

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The prpB gene of Salmonella enterica serovar Typhimurium LT2 encodes a protein with 2-methylisocitrate (2-MIC) lyase activity, which cleaves 2-MIC into pyruvate and succinate during the conversion of propionate to pyruvate via the 2-methylcitric acid cycle. This paper reports the isolation and kinetic characterization of wild-type and five mutant PrpB proteins. Wild-type PrpB protein had a molecular mass of approximately 32 kDa per subunit, and the biologically active enzyme was comprised of four subunits. Optimal 2-MIC lyase activity was measured at pH 7.5 and 50°C, and the reaction required Mg 2؉ ions; equimolar concentrations of Mn 2؉ ions were a poor substitute for Mg 2؉ (28% specific activity). Dithiothreitol (DTT) or reduced glutathione (GSH) was required for optimal activity; the role of DTT or GSH was apparently not to reduce disulfide bonds, since the disulfide-specific reducing agent Tris(2-carboxyethyl)phosphine hydrochloride failed to substitute for DTT or GSH. The K m of PrpB for 2-MIC was measured at 19 M, with a k cat of 105 s ؊1 . Mutations in the prpB gene were introduced by site-directed mutagenesis based on the active-site residues deemed important for catalysis in the closely related phosphoenolpyruvate mutase and isocitrate lyase enzymes. Residues D58, K121, C123, and H125 of PrpB were changed to alanine, and residue R122 was changed to lysine. Nondenaturing polyacrylamide gel electrophoresis indicated that all mutant PrpB proteins retained the same oligomeric state of the wild-type enzyme, which is known to form tetramers. The PrpB K121A , PrpB H125A , and PrpB R122K mutant proteins formed enzymes that had 1,050-, 750-, and 2-fold decreases in k cat for 2-MIC lyase activity, respectively. The PrpB D58A and PrpB C123A proteins formed tetramers that displayed no detectable 2-MIC lyase activity indicating that both of these residues are essential for catalysis. Based on the proposed mechanism of the closely related isocitrate lyases, PrpB residue C123 is proposed to serve as the active site base, and residue D58 is critical for the coordination of a required Mg 2؉ ion.Propionate catabolism in Salmonella enterica serovar Typhimurium LT2 (herein referred to as serovar Typhimurium) occurs via the 2-methylcitric acid (2-MC) cycle (Fig. 1) (18). The 2-MC cycle of propionate metabolism was first identified in the yeast Yarrowia lipolytica and several years later was found to exist in prokaryotes (8,18,36,37). The prokaryotic enzymes of this pathway have been characterized. PrpE is the propionyl coenzyme A synthetase (14, 17), PrpC is the 2-MC synthase (18, 39), PrpD is the 2-MC dehydratase (8, 15), and PrpB is the 2-methylisocitrate (2-MIC) lyase (7,18). This work focuses on the serovar Typhimurium PrpB enzyme, which catalyzes the cleavage of 2-MIC into pyruvate and succinate (15).Comparisons of the amino acid sequence of the PrpB protein to other protein sequences identified it as a homolog of carboxyphosphonoenolpyruvate (CPEP) mutase, phosphoenolpyruvate (PEP) mutase, and isocitrate lyases (ICL) ...

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Identification of a cysteine residue at the active site of Escherichia coli isocitrate lyase

Cited by 19 publications

References 23 publications

Characterization of the isocitrate lyase gene from Corynebacterium glutamicum and biochemical analysis of the enzyme

Characterization of the isocitrate lyase gene from Corynebacterium glutamicum and biochemical analysis of the enzyme

Regulation by Glutathionylation of Isocitrate Lyase from Chlamydomonas reinhardtii

Residues C123 and D58 of the 2-Methylisocitrate Lyase (PrpB) Enzyme ofSalmonella entericaAre Essential for Catalysis

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