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Wolfgang Buckel - One of the best experts on this subject based on the ideXlab platform.
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cloning and expression of the two genes coding for l Serine Dehydratase from peptostreptococcus asaccharolyticus relationship of the iron sulfur protein to both l Serine Dehydratases from escherichia coli
Journal of Bacteriology, 1997Co-Authors: Antje E M Hofmeister, Susanne Textor, Wolfgang BuckelAbstract:The structural genes sdhA and sdhB, coding for the alpha- and beta-subunits of the [4Fe-4S] cluster containing L-Serine Dehydratase from Peptostreptococcus asaccharolyticus, have been cloned and sequenced. Expression of modified sdhB together with sdhA in Escherichia coli led to overproduction of active His6-tagged L-Serine Dehydratase. E. coli MEW22, deficient in the L-Serine Dehydratase L-SD1, was complemented by this sdhBA construct. The derived amino acid sequence of SdhBA shares similarities with both monomeric L-Serine Dehydratases, L-SD1 and L-SD2, from E. coli and with a putative L-Serine Dehydratase from Haemophilus influenzae, which suggests that these three enzymes are also iron-sulfur proteins.
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Dehydratase from Peptostreptococcus asaccharolyticus: Relationship of the Iron-Sulfur Protein to Both L-Serine Dehydratases from Escherichia coli
1996Co-Authors: Antje E M Hofmeister, Susanne Textor, Wolfgang Buckel, J. BacteriolAbstract:and expression of the two genes coding for L-Serine Dehydratase from Peptostreptococcus asaccharolyticus: relationship of the iron-sulfur protein to both L-Serine Dehydratases from Escherichia coli
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iron sulfur cluster containing l Serine Dehydratase from peptostreptococcus asaccharolyticus correlation of the cluster type with enzymatic activity
FEBS Letters, 1994Co-Authors: Antje E M Hofmeister, S P J Albracht, Wolfgang BuckelAbstract:Abstract Investigations were performed with regard to the function of the iron—sulfur cluster of l -Serine Dehydratase from Peptostreptococcus asaccharolyticus , an enzyme which is novel in the class of deaminating hydro-lyases in that it lacks pyridoxal-5′-phosphate. Anaerobically purified l -Serine Dehydratase from P. asaccharolyticus revealed EPR spectra characteristic of a [3Fe4S] + cluster constituting 1% of the total enzyme concentration. Upon incubation of the enzyme under air the intensity of the [3Fe4S] + signal increased correlating with the loss of enzymatic activity. Addition of l -Serine prevented this. Hence, active l -Serine Dehydratase probably contains a diamagnetic [4Fe4S] 2+ cluster which is converted by oxidation and loss of one iron ion to a paramagnetic [3Fe4S] + cluster, resulting in inactivation of the enzyme. In analogy to the mechanism elucidated for aconitase, it is proposed that l -Serine is coordinated via its hydroxyl and carboxyl groups to the labile iron atom of the [4Fe4S] 2+ cluster.
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the iron sulfur cluster containing l Serine Dehydratase from peptostreptococcus asaccharolyticus stereochemistry of the deamination of l threonine
FEBS Journal, 1992Co-Authors: Antje E M Hofmeister, Stefan Berger, Wolfgang BuckelAbstract:The stereochemistry of the deamination of l-threonine to 2-oxobutyrate, catalyzed by purified l-Serine Dehydratase of Peptostreptococcus asaccharolyticus, was elucidated. For this purpose the enzyme reaction was carried out with unlabelled l-threonine in 2H2O and in 3HOH, as well as with l-[3-3H]threonine in unlabelled water. Isotopically labelled 2-oxobutyrate thus formed was directly reduced in a coupled reaction with l- or d-lactate dehydrogenase and NADH. The (2R)- or (2S)-2-hydroxybutyrate species obtained were then subjected to configurational analyses of their labelled methylene group. The results from 1H-NMR spectroscopy and, after degradation of 2-hydroxy-butyrate to propionate, the transcarboxylase assay consistently indicated that the deamination of l-threonine catalyzed by l-Serine Dehydratase of P. asaccharolyticus proceeds with inversion and retention in a 2:1 ratio. This partial racemization is the first ever to be observed for a reaction catalyzed by Serine Dehydratase, therefore confirming the distinction of the l-Serine Dehydratase of P. asaccharolyticus as an iron-sulfur protein from those Dehydratases dependent on pyridoxal phosphate. For the latter enzymes exclusively, retention has been reported.
David J. Kelly - One of the best experts on this subject based on the ideXlab platform.
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metabolomic analysis of the food borne pathogen campylobacter jejuni application of direct injection mass spectrometry for mutant characterisation
Metabolomics, 2014Co-Authors: Matthew P Davey, Robert M Howlett, Paul W Quick, David J. KellyAbstract:Campylobacter jejuni is the most frequent cause of human food-borne bacterial gastroenteritis but its physiology and biochemistry are poorly understood. Only a few amino-acids can be catabolised and these are known to be important for host colonization. Here we have established methods for rapid high throughput analyses of global metabolism in C. jejuni using direct injection mass spectrometry (DIMS) to compare metabolite fingerprints of wild-type and mutant strains. Principal component analyses show that the metabolic fingerprint of mutants that have a genomic deletion in genes for key amino-acid catabolic enzymes (either sdaA, Serine Dehydratase; aspA, aspartase or aspB, aspartate:glutamate transaminase) can easily be distinguished from the isogenic parental strain. Assignment of putative metabolites showed predictable changes directly associated with the particular metabolic lesion in these mutants as well as more extensive changes in the aspA mutant compared to the sdaA or aspB strains. Further analyses of a cj0150c mutant strain, which has no obvious phenotype, suggested a role for Cj0150 in the conversion of cystathionine to homocysteine. Our results show that DIMS is a useful technique for probing the metabolism of this important pathogen and may help in assigning function to genes encoding novel enzymes with currently unknown metabolic roles.
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amino acid dependent growth of campylobacter jejuni key roles for aspartase aspa under microaerobic and oxygen limited conditions and identification of aspb cj0762 essential for growth on glutamate
Molecular Microbiology, 2008Co-Authors: Edward Guccione, Maria Del Rocio Leonkempis, Bruce M Pearson, Edward Hitchin, Francis Mulholland, Pauline M Van Diemen, Mark P Stevens, David J. KellyAbstract:Amino acids are key carbon and energy sources for the asaccharolytic food-borne human pathogen Campylobacter jejuni. During microaerobic growth in amino acid rich complex media, aspartate, glutamate, proline and Serine are the only amino acids significantly utilized by strain NCTC 11168. The catabolism of aspartate and glutamate was investigated. An aspartase (aspA) mutant (unable to utilize any amino acid except Serine) and a Cj0762c (aspB) mutant lacking aspartate:glutamate aminotransferase (unable to utilize glutamate), were severely growth impaired in complex media, and an aspA sdaA mutant (also lacking Serine Dehydratase) failed to grow in complex media unless supplemented with pyruvate and fumarate. Aspartase was shown by activity and proteomic analyses to be upregulated by oxygen limitation, and aspartate enhanced oxygen-limited growth of C. jejuni in an aspA-dependent manner. Stoichiometric aspartate uptake and succinate excretion involving the redundant DcuA and DcuB transporters indicated that in addition to a catabolic role, AspA can provide fumarate for respiration. Significantly, an aspA mutant of C. jejuni 81-176 was impaired in its ability to persist in the intestines of outbred chickens relative to the parent strain. Together, our data highlight the dual function of aspartase in C. jejuni and suggest a role during growth in the avian gut.
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L-Serine catabolism via an oxygen-labile L-Serine Dehydratase is essential for colonization of the avian gut by Campylobacter jejuni.
Infection and immunity, 2004Co-Authors: Jyoti Velayudhan, Michael A. Jones, Paul A. Barrow, David J. KellyAbstract:Campylobacter jejuni is a microaerophilic, asaccharolytic bacterium. The identity of the carbon and energy sources used by C. jejuni in vivo is unknown, but the genome sequence of strain NCTC11168 indicates the presence of genes for catabolism of a limited range of amino acids, including Serine. Specific omission of L-Serine from a defined medium containing a mixture of amino acids led to a dramatic decrease in cell yields. As C. jejuni does not have a biosynthetic Serine requirement, this supports earlier suggestions that L-Serine is a preferentially catabolized amino acid. Serine transport was found to be mediated by at least two systems in strain 11168; a high-capacity, low-affinity L-Serine-specific system encoded by Cj1625c (sdaC) and a higher-affinity L-Serine/L-threonine system responsible for residual L-Serine transport in an sdaC mutant. Catabolism of L-Serine to pyruvate and ammonia is carried out by SdaA (encoded by Cj1624c), which was overexpressed, purified, and shown to be an oxygen-labile iron-sulfur enzyme. L-Serine Dehydratase activity in an sdaA mutant was reduced 10-fold compared to that in the wild type, but the residual activity (due to the anabolic L-threonine Dehydratase) could not support either growth on or utilization of L-Serine in defined media. However, although sdaA mutants showed no obvious growth defect in complex media, they completely failed to colonize 3-week-old chickens as assayed both by cloacal swabs taken over a 6-week period and by cecal colony counts postmortem. In contrast, the isogenic parent strain colonized chickens to high levels within 1 week of inoculation. The results show that an active SdaA is essential for colonization of the avian gut by C. jejuni and imply that catabolism of L-Serine is crucially important for the growth of this bacterium in vivo.
Antje E M Hofmeister - One of the best experts on this subject based on the ideXlab platform.
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cloning and expression of the two genes coding for l Serine Dehydratase from peptostreptococcus asaccharolyticus relationship of the iron sulfur protein to both l Serine Dehydratases from escherichia coli
Journal of Bacteriology, 1997Co-Authors: Antje E M Hofmeister, Susanne Textor, Wolfgang BuckelAbstract:The structural genes sdhA and sdhB, coding for the alpha- and beta-subunits of the [4Fe-4S] cluster containing L-Serine Dehydratase from Peptostreptococcus asaccharolyticus, have been cloned and sequenced. Expression of modified sdhB together with sdhA in Escherichia coli led to overproduction of active His6-tagged L-Serine Dehydratase. E. coli MEW22, deficient in the L-Serine Dehydratase L-SD1, was complemented by this sdhBA construct. The derived amino acid sequence of SdhBA shares similarities with both monomeric L-Serine Dehydratases, L-SD1 and L-SD2, from E. coli and with a putative L-Serine Dehydratase from Haemophilus influenzae, which suggests that these three enzymes are also iron-sulfur proteins.
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Dehydratase from Peptostreptococcus asaccharolyticus: Relationship of the Iron-Sulfur Protein to Both L-Serine Dehydratases from Escherichia coli
1996Co-Authors: Antje E M Hofmeister, Susanne Textor, Wolfgang Buckel, J. BacteriolAbstract:and expression of the two genes coding for L-Serine Dehydratase from Peptostreptococcus asaccharolyticus: relationship of the iron-sulfur protein to both L-Serine Dehydratases from Escherichia coli
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iron sulfur cluster containing l Serine Dehydratase from peptostreptococcus asaccharolyticus correlation of the cluster type with enzymatic activity
FEBS Letters, 1994Co-Authors: Antje E M Hofmeister, S P J Albracht, Wolfgang BuckelAbstract:Abstract Investigations were performed with regard to the function of the iron—sulfur cluster of l -Serine Dehydratase from Peptostreptococcus asaccharolyticus , an enzyme which is novel in the class of deaminating hydro-lyases in that it lacks pyridoxal-5′-phosphate. Anaerobically purified l -Serine Dehydratase from P. asaccharolyticus revealed EPR spectra characteristic of a [3Fe4S] + cluster constituting 1% of the total enzyme concentration. Upon incubation of the enzyme under air the intensity of the [3Fe4S] + signal increased correlating with the loss of enzymatic activity. Addition of l -Serine prevented this. Hence, active l -Serine Dehydratase probably contains a diamagnetic [4Fe4S] 2+ cluster which is converted by oxidation and loss of one iron ion to a paramagnetic [3Fe4S] + cluster, resulting in inactivation of the enzyme. In analogy to the mechanism elucidated for aconitase, it is proposed that l -Serine is coordinated via its hydroxyl and carboxyl groups to the labile iron atom of the [4Fe4S] 2+ cluster.
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the iron sulfur cluster containing l Serine Dehydratase from peptostreptococcus asaccharolyticus stereochemistry of the deamination of l threonine
FEBS Journal, 1992Co-Authors: Antje E M Hofmeister, Stefan Berger, Wolfgang BuckelAbstract:The stereochemistry of the deamination of l-threonine to 2-oxobutyrate, catalyzed by purified l-Serine Dehydratase of Peptostreptococcus asaccharolyticus, was elucidated. For this purpose the enzyme reaction was carried out with unlabelled l-threonine in 2H2O and in 3HOH, as well as with l-[3-3H]threonine in unlabelled water. Isotopically labelled 2-oxobutyrate thus formed was directly reduced in a coupled reaction with l- or d-lactate dehydrogenase and NADH. The (2R)- or (2S)-2-hydroxybutyrate species obtained were then subjected to configurational analyses of their labelled methylene group. The results from 1H-NMR spectroscopy and, after degradation of 2-hydroxy-butyrate to propionate, the transcarboxylase assay consistently indicated that the deamination of l-threonine catalyzed by l-Serine Dehydratase of P. asaccharolyticus proceeds with inversion and retention in a 2:1 ratio. This partial racemization is the first ever to be observed for a reaction catalyzed by Serine Dehydratase, therefore confirming the distinction of the l-Serine Dehydratase of P. asaccharolyticus as an iron-sulfur protein from those Dehydratases dependent on pyridoxal phosphate. For the latter enzymes exclusively, retention has been reported.
Michael D. Toney - One of the best experts on this subject based on the ideXlab platform.
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ionization state of pyridoxal 5 phosphate in d Serine Dehydratase dialkylglycine decarboxylase and tyrosine phenol lyase and the influence of monovalent cations as inferred by 31p nmr spectroscopy
Biochimica et Biophysica Acta, 2006Co-Authors: K D Schnackerz, John W Keller, Robert S Phillips, Michael D. ToneyAbstract:Abstract The 31 P NMR spectroscopy of three pyridoxal 5′-phosphate-dependent enzymes, monomeric d -Serine Dehydratase, tetrameric dialkylglycine decarboxylase and tetrameric tyrosine phenol-lyase, whose enzymatic activities are dependent on alkali metal ions, was studied. 31 P NMR spectra of the latter two enzymes have never been reported, their 3D-structures, however, are available. The cofactor phosphate chemical shift of all three enzymes changes by ∼3 ppm as a function of pH, indicating that the phosphate group changes from being monoanionic at low pH to dianionic at high pH. The 31 P NMR signal of the phosphate group of pyridoxal 5′-phosphate provides a measure of the active site changes that occur when various alkali metal ions are bound. Structural information is used to assist in the interpretation of the chemical shift changes observed. For d -Serine Dehydratase, no structural data are available but nevertheless the metal ion arrangement in the PLP binding site can be predicted from 31 P NMR data.
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ionization state of pyridoxal 5 phosphate in d Serine Dehydratase dialkylglycine decarboxylase and tyrosine phenol lyase and the influence of monovalent cations as inferred by 31p nmr spectroscopy
Biochimica et Biophysica Acta, 2006Co-Authors: K D Schnackerz, John W Keller, Robert S Phillips, Michael D. ToneyAbstract:The 31P NMR spectroscopy of three pyridoxal 5'-phosphate-dependent enzymes, monomeric D-Serine Dehydratase, tetrameric dialkylglycine decarboxylase and tetrameric tyrosine phenol-lyase, whose enzymatic activities are dependent on alkali metal ions, was studied. 31P NMR spectra of the latter two enzymes have never been reported, their 3D-structures, however, are available. The cofactor phosphate chemical shift of all three enzymes changes by approximately 3 ppm as a function of pH, indicating that the phosphate group changes from being monoanionic at low pH to dianionic at high pH. The 31P NMR signal of the phosphate group of pyridoxal 5'-phosphate provides a measure of the active site changes that occur when various alkali metal ions are bound. Structural information is used to assist in the interpretation of the chemical shift changes observed. For D-Serine Dehydratase, no structural data are available but nevertheless the metal ion arrangement in the PLP binding site can be predicted from 31P NMR data.
Robert M Howlett - One of the best experts on this subject based on the ideXlab platform.
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metabolomic analysis of the food borne pathogen campylobacter jejuni application of direct injection mass spectrometry for mutant characterisation
Metabolomics, 2014Co-Authors: Matthew P Davey, Robert M Howlett, Paul W Quick, David J. KellyAbstract:Campylobacter jejuni is the most frequent cause of human food-borne bacterial gastroenteritis but its physiology and biochemistry are poorly understood. Only a few amino-acids can be catabolised and these are known to be important for host colonization. Here we have established methods for rapid high throughput analyses of global metabolism in C. jejuni using direct injection mass spectrometry (DIMS) to compare metabolite fingerprints of wild-type and mutant strains. Principal component analyses show that the metabolic fingerprint of mutants that have a genomic deletion in genes for key amino-acid catabolic enzymes (either sdaA, Serine Dehydratase; aspA, aspartase or aspB, aspartate:glutamate transaminase) can easily be distinguished from the isogenic parental strain. Assignment of putative metabolites showed predictable changes directly associated with the particular metabolic lesion in these mutants as well as more extensive changes in the aspA mutant compared to the sdaA or aspB strains. Further analyses of a cj0150c mutant strain, which has no obvious phenotype, suggested a role for Cj0150 in the conversion of cystathionine to homocysteine. Our results show that DIMS is a useful technique for probing the metabolism of this important pathogen and may help in assigning function to genes encoding novel enzymes with currently unknown metabolic roles.
