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Johannes Zschocke - One of the best experts on this subject based on the ideXlab platform.
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Riboflavin-responsive glutaryl CoA dehydrogenase deficiency
Molecular Genetics and Metabolism, 2006Co-Authors: Ronald A. Chalmers, Murray D. Bain, Johannes ZschockeAbstract:We report here riboflavin responsiveness in a patient with glutaryl CoA dehydrogenase (GCDH) deficiency, compound heterozygous for the S139L and P248L mutations and with 20% residual GCDH enzyme activity in vitro. Our results suggest the mitochondrial GCDH homotetramer remains intact with one of these mutations associated with the binding site of the single FAD cofactor and that pharmacological doses of the cofactor precursor may be sufficient to induce an increase in activity in the mutant GCDH enzyme, although not sufficient to normalise urinary organic acid excretion. Serine139 is one of nine conserved amino acid residues that line the binding site of the protein and is in close proximity to both substrate and FAD cofactor. It is possible that steric alterations caused by substitution of serine with leucine at this position may be overcome with high cofactor concentrations. P248L is also associated with some residual GCDH activity in other patients and the unique combination of S139L with P248L may also explain the results in our patient. Responsiveness to riboflavin in our patient has been compared with two other patients with glutaric aciduria type 1 and minimal residual GCDH activity, one with homozygosity for the R257Q mutation and one with heterozygosity for the G354S mutation and a novel G156V mutation. A low lysine diet reduced glutaric acid excretion in our riboflavin-responsive GCDH-deficient patient almost to control values. She is now 21 years of age and clinically and neurologically normal.
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Late-onset neurologic disease in Glutaryl-CoA dehydrogenase deficiency.
Neurology, 2005Co-Authors: S. Külkens, Johannes Zschocke, G. F. Hoffmann, Sven W. Sauer, Inga Harting, Stephan Gruber, Olaf Bodamer, Stefan KölkerAbstract:Neurologic disease in Glutaryl-CoA dehydrogenase (GCDH) deficiency usually presents with acute encephalopathic crises before 2 years of age. The authors report two previously asymptomatic patients with macrocephaly presenting with progressive neurologic deterioration and a severe leukoencephalopathy during adolescence or adulthood.
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Management of movement disorders in Glutaryl-CoA dehydrogenase deficiency: anticholinergic drugs and botulinum toxin as additional therapeutic options.
Journal of Inherited Metabolic Disease, 2004Co-Authors: Johannes Zschocke, Georg F Hoffmann, G. Zara, Alessandro P. BurlinaAbstract:Glutaric aciduria type I is an inborn error of metabolism due to the deficiency of Glutaryl-CoA dehydrogenase, an enzyme responsible for the catabolism of lysine, hydroxylysine and tryptophan. The most important neurological symptoms include dyskinesia and dystonia, which can be focal, segmental or generalized. Treatment of the extrapyramidal syndrome is often unsatisfactory. We report our experience in the treatment of generalized and focal dystonia with anticholinergic drugs and botulinum toxin type A, respectively. Both therapies proved beneficial.
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Correlation of genotype and phenotype in Glutaryl-CoA dehydrogenase deficiency.
Journal of Inherited Metabolic Disease, 2004Co-Authors: Ernst Christensen, Antonia Ribes, Begoña Merinero, Johannes ZschockeAbstract:We have investigated the correlation between genotype and phenotype in a large number of patients with glutaric aciduria type I (GA I). The deficiency of Glutaryl-CoA dehydrogenase has been confirmed in the Rigshospitalet's laboratory in 215 patients since 1975. Most of the patients were of European ancestry. Complete absence of enzyme activity was found in more than half of the patients, while 34% of patients had a residual activity up to 5% and a few patients had a residual activity of 5-15%. In four exceptional cases, a very high residual activity of up to 30% was found. Enzyme studies are thus a reliable method for confirming the diagnosis of GA I, although it may be difficult to distinguish exceptional 'mild' cases from heterozygous carriers for GA I. Three of the patients with very high residual activity are compound heterozygous for the missense mutations R227P and V400M, both of which are associated with residual enzyme activity of 8-10% in homozygous patients. Patients with a mild mutation on at least one chromosome frequently show unusual biochemical findings such as low or normal urinary excretion of glutaric acid and mild or only slightly increased excretion of 3-hydroxyglutaric acid. In contrast, patients with severe mutations such as R402W or A293T on both alleles have no residual activity and show the typical urinary metabolite pattern. Clinical data were available for a subgroup of 79 patients. No correlation with the biochemical phenotype or the genotype could be established.
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mutation analysis in glutaric aciduria type i
Journal of Medical Genetics, 2000Co-Authors: Johannes Zschocke, Elfriede Quak, Per Guldberg, G. F. HoffmannAbstract:Glutaric aciduria type 1 (GA1), resulting from the genetic deficiency of Glutaryl-CoA dehydrogenase (GDH), is a relatively common cause of acute metabolic brain damage in infants. Encephalopathic crises may be prevented by carnitine supplementation and diet, but diagnosis can be difficult as some patients do not show the typical excretion of large amounts of glutaric and 3-hydroxyglutaric acids in the urine. We present a rapid and efficient denaturing gradient gel electrophoresis (DGGE) method for the identification of mutations in the Glutaryl-CoA dehydrogenase ( GCDH ) gene that may be used for the molecular diagnosis of GA1 in a routine setting. Using this technique, we identified mutations on both alleles in 48 patients with confirmed GDH deficiency, while no mutations were detected in other patients with clinical suspicion of GA1 but normal enzyme studies. There was a total of 38 different mutations; 27 mutations were found in single patients only, and 21 mutations have not been previously reported. Fourteen mutations involved hypermutable CpG sites. The commonest GA1 mutation in Europeans is R402W, which accounts for almost 40% of alleles in patients of German origin. GCDH gene haplotypes were determined through the analysis of polymorphic markers in all families, and three CpG mutations were associated with different haplotypes, possibly reflecting independent recurrence. The high sensitivity of the DGGE method allows the rapid and cost efficient diagnosis of GA1 in instances where enzyme analyses are not available or feasible, despite the marked heterogeneity of the disease.
Georg F Hoffmann - One of the best experts on this subject based on the ideXlab platform.
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complementary dietary treatment using lysine free arginine fortified amino acid supplements in glutaric aciduria type i a decade of experience
Molecular Genetics and Metabolism, 2012Co-Authors: Stefan Kölker, Chris Mühlhausen, Cheryl R. Greenberg, David M. Koeller, Jana Heringer, Edith Muller, Esther M Maier, Regina Ensenauer, Andrea Schlune, Georg F HoffmannAbstract:Abstract The cerebral formation and entrapment of neurotoxic dicarboxylic metabolites (Glutaryl-CoA, glutaric and 3-hydroxyglutaric acid) are considered to be important pathomechanisms of striatal injury in glutaric aciduria type I (GA-I). The quantitatively most important precursor of these metabolites is lysine. Recommended therapeutic interventions aim to reduce lysine oxidation (low lysine diet, emergency treatment to minimize catabolism) and to enhance physiologic detoxification of Glutaryl-CoA via formation of glutarylcarnitine (carnitine supplementation). It has been recently shown in Gcdh −/− mice that cerebral lysine influx and oxidation can be modulated by arginine which competes with lysine for transport at the blood–brain barrier and the inner mitochondrial membrane [Sauer et al., Brain 134 (2011) 157–170]. Furthermore, short-term outcome of 12 children receiving arginine-fortified diet showed very promising results [Strauss et al., Mol. Genet. Metab. 104 (2011) 93–106]. Since lysine-free, arginine-fortified amino acid supplements (AAS) are commercially available and used in Germany for more than a decade, we evaluated the effect of arginine supplementation in a cohort of 34 neonatally diagnosed GA-I patients (median age, 7.43years; cumulative follow-up period, 221.6patientyears) who received metabolic treatment according to a published guideline [Kolker et al., J. Inherit. Metab. Dis. 30 (2007) 5–22]. Patients used one of two AAS product lines during the first year of life, resulting in differences in arginine consumption [group 1 (Milupa Metabolics): mean=111mg arginine/kg; group 2 (Nutricia): mean=145mg arginine/kg; p
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Maintenance treatment of Glutaryl-CoA dehydrogenase deficiency.
Journal of Inherited Metabolic Disease, 2004Co-Authors: Chris Mühlhausen, Georg F Hoffmann, Cheryl R. Greenberg, K. A. Strauss, Stefan Kölker, Jürgen G. Okun, E. R. Naughten, Kurt UllrichAbstract:This paper summarizes the published experience as well as results of the 3rd International Workshop on Glutaryl-CoA Dehydrogenase Deficiency held in October 2003 in Heidelberg, Germany, on the topic treatment of patients with Glutaryl-CoA dehydrogenase (GCDH) deficiency. So far no international recommendation for treatment of GCDH deficiency exists. Such an approach is hampered by several facts, namely the lack of an in-depth understanding of the pathophysiology of the disease, the lack of prospective studies, including the evaluation of drug monotherapy, and lack of objective documentation of clinical changes (e.g. video documentation) during pharmacotherapy.
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Emergency treatment in Glutaryl-CoA dehydrogenase deficiency.
Journal of Inherited Metabolic Disease, 2004Co-Authors: Stefan Kölker, Cheryl R. Greenberg, E. R. Naughten, Martin Lindner, E. Müller, Georg F HoffmannAbstract:The history of Glutaryl-CoA dehydrogenase deficiency is determined by acute encephalopathic crises that are precipitated by common febrile diseases, vaccinations or surgical interventions during infancy and early childhood. Such crises result in an irreversible destruction of the basal ganglia (in particular of the putamina), and consequently dystonia, dyskinesia and choreoathetosis. Secondary complications include feeding and speech problems, failure to thrive, recurrent aspiration, immobilization, severe motor deficits and early death. It is generally accepted that maintenance treatment based on dietary lysine or protein restriction and supplementation with carnitine (and riboflavin) is insufficient to prevent acute crises during intercurrent illnesses or conditions that enhance catabolic state. Consequently, outpatient and inpatient emergency therapies have been implemented. The present review describes a recommended approach to emergency therapy for this disease.
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Management of movement disorders in Glutaryl-CoA dehydrogenase deficiency: anticholinergic drugs and botulinum toxin as additional therapeutic options.
Journal of Inherited Metabolic Disease, 2004Co-Authors: Johannes Zschocke, Georg F Hoffmann, G. Zara, Alessandro P. BurlinaAbstract:Glutaric aciduria type I is an inborn error of metabolism due to the deficiency of Glutaryl-CoA dehydrogenase, an enzyme responsible for the catabolism of lysine, hydroxylysine and tryptophan. The most important neurological symptoms include dyskinesia and dystonia, which can be focal, segmental or generalized. Treatment of the extrapyramidal syndrome is often unsatisfactory. We report our experience in the treatment of generalized and focal dystonia with anticholinergic drugs and botulinum toxin type A, respectively. Both therapies proved beneficial.
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Challenges for basic research in Glutaryl-CoA dehydrogenase deficiency
Journal of Inherited Metabolic Disease, 2004Co-Authors: Stefan Kölker, Stephen I. Goodman, Georg F Hoffmann, K. A. Strauss, Jürgen G. Okun, David M. KoellerAbstract:During the last decades, efforts have been made to elucidate the complex mechanisms underlying neuronal damage in Glutaryl-CoA dehydrogenase deficiency. A combination of in vitro and in vivo investigations have facilitated the development of several hypotheses, including the probable pathogenic role of accumulating glutaric acid and 3-hydroxyglutaric acid. However, there are still many shortcomings that limit an evidence-based approach to treating this inborn error of metabolism. Major future goals should include generation of a suitable animal model for acute striatal necrosis, investigation of the formation, distribution and exact intra- and extracellular concentrations of accumulating metabolites, a deeper understanding of striatal vulnerability, and systematic investigation of effects on cerebral gene expression during development and of the modulatory role of inflammatory cytokines.
G. F. Hoffmann - One of the best experts on this subject based on the ideXlab platform.
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Late-onset neurologic disease in Glutaryl-CoA dehydrogenase deficiency.
Neurology, 2005Co-Authors: S. Külkens, Johannes Zschocke, G. F. Hoffmann, Sven W. Sauer, Inga Harting, Stephan Gruber, Olaf Bodamer, Stefan KölkerAbstract:Neurologic disease in Glutaryl-CoA dehydrogenase (GCDH) deficiency usually presents with acute encephalopathic crises before 2 years of age. The authors report two previously asymptomatic patients with macrocephaly presenting with progressive neurologic deterioration and a severe leukoencephalopathy during adolescence or adulthood.
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Neonatal screening for Glutaryl-CoA dehydrogenase deficiency
Journal of Inherited Metabolic Disease, 2004Co-Authors: Martin Lindner, Ernst Christensen, Cheryl R. Greenberg, Stefan Kölker, Andreas Schulze, G. F. HoffmannAbstract:Acute encephalopathic crisis in Glutaryl-CoA dehydrogenase deficiency results in an unfavourable disease course and poor outcome, dominated by dystonia, feeding problems, seizures and secondary complications, and quite often leading to early death. The prerequisite for the prevention of irreversible brain damage in this disease is the detection of affected patients and initiation of treatment before the manifestation of such crisis. Apart from macrocephaly there are no signs or symptoms characteristic for this disease in presymptomatic children and, thus, they are usually missed. In some countries, implementation of extended neonatal screening programmes using electrospray ionization tandem mass spectrometry (ESI-MS/MS) allows detection of affected newborns and start of therapy before onset of neurological complications. This article summarizes recent strategies, pitfalls and shortcomings of a mass screening for Glutaryl-CoA dehydrogenase deficiency using ESI-MS/MS. Furthermore, an alternative strategy, namely DNA-based neonatal screening for the Oji-Cree variant of this disease, is demonstrated. An optimization of diagnostic as well as therapeutic procedures must be achieved before GCDH deficiency unequivocally fulfills the criteria of a reliable and successful newborn screening programme.
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Excitotoxicity and bioenergetics in Glutaryl-CoA dehydrogenase deficiency.
Journal of Inherited Metabolic Disease, 2004Co-Authors: Stefan Kölker, G. F. Hoffmann, Kurt Ullrich, Sven W. Sauer, David M. Koeller, Friederike Hörster, Marina A. Schwab, Jürgen G. OkunAbstract:Summary: Glutaryl-CoA dehydrogenase deficiency is an inherited organic acid disorder with predominantly neurological presentation. The biochemical hallmark of this disease is an accumulation and enhanced urinary excretion of two key organic acids, glutaric acid and 3-hydroxyglutaric acid. If untreated, acute striatal damage is often precipitated by febrile illnesses during a vulnerable period of brain development in infancy or early childhood, resulting in a dystonic dyskinetic movement disorder. 3-Hydroxyglutaric and glutaric acids are structurally similar to glutamate, the main excitatory amino acid of the human brain, and are considered to play an important role in the pathophysiology of this disease. 3-Hydroxyglutaric acid induces excitotoxic cell damage specifically via activation of N-methyl-D-aspartate receptors. It has also been suggested that secondary amplification loops potentiate the neurotoxic properties of these organic acids. Probable mechanisms for this effect include cytokine-stimulated NO production, a decrease in energy metabolism, and reduction of cellular creatine phosphate levels. Finally, maturation-dependent changes in the expression of neuronal glutamate receptors may affect the vulnerability of the immature brain to excitotoxic cell damage in this disease.
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mutation analysis in glutaric aciduria type i
Journal of Medical Genetics, 2000Co-Authors: Johannes Zschocke, Elfriede Quak, Per Guldberg, G. F. HoffmannAbstract:Glutaric aciduria type 1 (GA1), resulting from the genetic deficiency of Glutaryl-CoA dehydrogenase (GDH), is a relatively common cause of acute metabolic brain damage in infants. Encephalopathic crises may be prevented by carnitine supplementation and diet, but diagnosis can be difficult as some patients do not show the typical excretion of large amounts of glutaric and 3-hydroxyglutaric acids in the urine. We present a rapid and efficient denaturing gradient gel electrophoresis (DGGE) method for the identification of mutations in the Glutaryl-CoA dehydrogenase ( GCDH ) gene that may be used for the molecular diagnosis of GA1 in a routine setting. Using this technique, we identified mutations on both alleles in 48 patients with confirmed GDH deficiency, while no mutations were detected in other patients with clinical suspicion of GA1 but normal enzyme studies. There was a total of 38 different mutations; 27 mutations were found in single patients only, and 21 mutations have not been previously reported. Fourteen mutations involved hypermutable CpG sites. The commonest GA1 mutation in Europeans is R402W, which accounts for almost 40% of alleles in patients of German origin. GCDH gene haplotypes were determined through the analysis of polymorphic markers in all families, and three CpG mutations were associated with different haplotypes, possibly reflecting independent recurrence. The high sensitivity of the DGGE method allows the rapid and cost efficient diagnosis of GA1 in instances where enzyme analyses are not available or feasible, despite the marked heterogeneity of the disease.
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glutaric aciduria type i from clinical biochemical and molecular diversity to successful therapy
Journal of Inherited Metabolic Disease, 1999Co-Authors: G. F. Hoffmann, Johannes ZschockeAbstract:The biochemical hallmark of glutaric aciduria type I (GA I) due to Glutaryl-CoA dehydrogenase deficiency is the accumulation of glutaric acid, and to a lesser degree of 3-hydroxyglutaric and glutaconic acids. Abnormal metabolites vary from gross organic aciduria to only slightly or intermittently elevated or even normal excretion of glutaric acid, making the diagnosis sometimes difficult. Close to 100 pathogenic mutations have been identified in the gene encoding Glutaryl-CoA dehydrogenase. Specific mutations correlate with low or no excretion of glutaric acid, but there appears to be no correlation between genotype and clinical phenotype.
Stefan Kölker - One of the best experts on this subject based on the ideXlab platform.
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complementary dietary treatment using lysine free arginine fortified amino acid supplements in glutaric aciduria type i a decade of experience
Molecular Genetics and Metabolism, 2012Co-Authors: Stefan Kölker, Chris Mühlhausen, Cheryl R. Greenberg, David M. Koeller, Jana Heringer, Edith Muller, Esther M Maier, Regina Ensenauer, Andrea Schlune, Georg F HoffmannAbstract:Abstract The cerebral formation and entrapment of neurotoxic dicarboxylic metabolites (Glutaryl-CoA, glutaric and 3-hydroxyglutaric acid) are considered to be important pathomechanisms of striatal injury in glutaric aciduria type I (GA-I). The quantitatively most important precursor of these metabolites is lysine. Recommended therapeutic interventions aim to reduce lysine oxidation (low lysine diet, emergency treatment to minimize catabolism) and to enhance physiologic detoxification of Glutaryl-CoA via formation of glutarylcarnitine (carnitine supplementation). It has been recently shown in Gcdh −/− mice that cerebral lysine influx and oxidation can be modulated by arginine which competes with lysine for transport at the blood–brain barrier and the inner mitochondrial membrane [Sauer et al., Brain 134 (2011) 157–170]. Furthermore, short-term outcome of 12 children receiving arginine-fortified diet showed very promising results [Strauss et al., Mol. Genet. Metab. 104 (2011) 93–106]. Since lysine-free, arginine-fortified amino acid supplements (AAS) are commercially available and used in Germany for more than a decade, we evaluated the effect of arginine supplementation in a cohort of 34 neonatally diagnosed GA-I patients (median age, 7.43years; cumulative follow-up period, 221.6patientyears) who received metabolic treatment according to a published guideline [Kolker et al., J. Inherit. Metab. Dis. 30 (2007) 5–22]. Patients used one of two AAS product lines during the first year of life, resulting in differences in arginine consumption [group 1 (Milupa Metabolics): mean=111mg arginine/kg; group 2 (Nutricia): mean=145mg arginine/kg; p
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Late-onset neurologic disease in Glutaryl-CoA dehydrogenase deficiency.
Neurology, 2005Co-Authors: S. Külkens, Johannes Zschocke, G. F. Hoffmann, Sven W. Sauer, Inga Harting, Stephan Gruber, Olaf Bodamer, Stefan KölkerAbstract:Neurologic disease in Glutaryl-CoA dehydrogenase (GCDH) deficiency usually presents with acute encephalopathic crises before 2 years of age. The authors report two previously asymptomatic patients with macrocephaly presenting with progressive neurologic deterioration and a severe leukoencephalopathy during adolescence or adulthood.
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Maintenance treatment of Glutaryl-CoA dehydrogenase deficiency.
Journal of Inherited Metabolic Disease, 2004Co-Authors: Chris Mühlhausen, Georg F Hoffmann, Cheryl R. Greenberg, K. A. Strauss, Stefan Kölker, Jürgen G. Okun, E. R. Naughten, Kurt UllrichAbstract:This paper summarizes the published experience as well as results of the 3rd International Workshop on Glutaryl-CoA Dehydrogenase Deficiency held in October 2003 in Heidelberg, Germany, on the topic treatment of patients with Glutaryl-CoA dehydrogenase (GCDH) deficiency. So far no international recommendation for treatment of GCDH deficiency exists. Such an approach is hampered by several facts, namely the lack of an in-depth understanding of the pathophysiology of the disease, the lack of prospective studies, including the evaluation of drug monotherapy, and lack of objective documentation of clinical changes (e.g. video documentation) during pharmacotherapy.
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Neonatal screening for Glutaryl-CoA dehydrogenase deficiency
Journal of Inherited Metabolic Disease, 2004Co-Authors: Martin Lindner, Ernst Christensen, Cheryl R. Greenberg, Stefan Kölker, Andreas Schulze, G. F. HoffmannAbstract:Acute encephalopathic crisis in Glutaryl-CoA dehydrogenase deficiency results in an unfavourable disease course and poor outcome, dominated by dystonia, feeding problems, seizures and secondary complications, and quite often leading to early death. The prerequisite for the prevention of irreversible brain damage in this disease is the detection of affected patients and initiation of treatment before the manifestation of such crisis. Apart from macrocephaly there are no signs or symptoms characteristic for this disease in presymptomatic children and, thus, they are usually missed. In some countries, implementation of extended neonatal screening programmes using electrospray ionization tandem mass spectrometry (ESI-MS/MS) allows detection of affected newborns and start of therapy before onset of neurological complications. This article summarizes recent strategies, pitfalls and shortcomings of a mass screening for Glutaryl-CoA dehydrogenase deficiency using ESI-MS/MS. Furthermore, an alternative strategy, namely DNA-based neonatal screening for the Oji-Cree variant of this disease, is demonstrated. An optimization of diagnostic as well as therapeutic procedures must be achieved before GCDH deficiency unequivocally fulfills the criteria of a reliable and successful newborn screening programme.
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Emergency treatment in Glutaryl-CoA dehydrogenase deficiency.
Journal of Inherited Metabolic Disease, 2004Co-Authors: Stefan Kölker, Cheryl R. Greenberg, E. R. Naughten, Martin Lindner, E. Müller, Georg F HoffmannAbstract:The history of Glutaryl-CoA dehydrogenase deficiency is determined by acute encephalopathic crises that are precipitated by common febrile diseases, vaccinations or surgical interventions during infancy and early childhood. Such crises result in an irreversible destruction of the basal ganglia (in particular of the putamina), and consequently dystonia, dyskinesia and choreoathetosis. Secondary complications include feeding and speech problems, failure to thrive, recurrent aspiration, immobilization, severe motor deficits and early death. It is generally accepted that maintenance treatment based on dietary lysine or protein restriction and supplementation with carnitine (and riboflavin) is insufficient to prevent acute crises during intercurrent illnesses or conditions that enhance catabolic state. Consequently, outpatient and inpatient emergency therapies have been implemented. The present review describes a recommended approach to emergency therapy for this disease.
Wolfgang Buckel - One of the best experts on this subject based on the ideXlab platform.
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Oxygen Exchange between Acetate and the Catalytic Glutamate Residue in Glutaconate CoA-transferase from Acidaminococcus fermentans IMPLICATIONS FOR THE MECHANISM OF CoA-ESTER HYDROLYSIS
Journal of Biological Chemistry, 1999Co-Authors: Thorsten Selmer, Wolfgang BuckelAbstract:Abstract The exchange of oxygen atoms between acetate, Glutaryl-CoA, and the catalytic glutamate residue in glutaconate CoA-transferase from Acidaminococcus fermentans was analyzed using [18O2]acetate together with matrix-assisted laser desorption/ionization time of flight mass spectrometry of an appropriate undecapeptide. The exchange reaction was shown to be site-specific, reversible, and required both Glutaryl-CoA and [18O2]acetate. The observed exchange is in agreement with the formation of a mixed anhydride intermediate between the enzyme and acetate. In contrast, with a mutant enzyme, which was converted to a thiol ester hydrolyase by replacement of the catalytic glutamate residue by aspartate, no 18O uptake from H2 18O into the carboxylate was detectable. This result is in accord with a mechanism in which the carboxylate of aspartate acts as a general base in activating a water molecule for hydrolysis of the thiol ester intermediate. This mechanism is further supported by the finding of a significant hydrolyase activity of the wild-type enzyme using acetyl-CoA as substrate, whereas Glutaryl-CoA is not hydrolyzed. The small acetate molecule in the substrate binding pocket may activate a water molecule for hydrolysis of the nearby enzyme-CoA thiol ester.
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biochemistry of glutaric aciduria type i activities of in vitro expressed wild type and mutant cdna encoding human glutaryl coa dehydrogenase
Journal of Inherited Metabolic Disease, 1999Co-Authors: Michael Liesert, Johannes Zschocke, Georg F Hoffmann, N Muhlhauser, Wolfgang BuckelAbstract:Glutaric aciduria type I (GA I; McKusick 231670) is caused by deficiency of Glutaryl-CoA dehydrogenase activity (EC 1.3.99.7), a key enzyme in the catabolism of the amino acids lysine, hydroxylysine and tryptophan (Besrat et al 1969; Goodman et al 1975). The failure to metabolize Glutaryl-CoA may give rise to elevated levels of glutarate, 3-hydroxyglutarate and glutaconate in the body fluids of the patient. Recent studies showed the leading role of 3-hydroxyglutarate in the pathogenesis of GA I (Baric et al, unpublished). Yet the source of this dicarboxylic acid is not well understood. Glutaryl-CoA dehydrogenase catalyses the reaction in two steps: dehydrogenation of Glutaryl-CoA to glutaconyl-CoA and decarboxylation of glutaconyl-CoA to crotonyl-CoA, which can be measured separately (Hartel et al 1993). Whereas the hydration of glutaconyl-CoA to 3-hydroxyglutarylCoA is catalysed by methylglutaconase (EC 4.2.1.18; Liesert and Buckel, unpublished), the formation of glutaconyl-CoA is less clear. It could arise from Glutaryl-CoA dehydrogenase mutants in which the decarboxylase activity is more affected than the dehydrogenase activity. Another possibility would be a nonspecific oxidation of Glutaryl-CoA by medium-chain acyl-CoA dehydrogenase (MCAD, EC 1.3.99.3) or similar enzymes. In order to address this question, mechanistically and clinically interesting mutants were introduced into a modified human Glutaryl-CoA dehydrogenase cDNA that can be expressed in Escherichia coli.
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CONVERSION OF GLUTACONATE COA-TRANSFERASE FROM ACIDAMINOCOCCUS FERMENTANS INTO AN ACYL-COA HYDROLASE BY SITE-DIRECTED MUTAGENESIS
FEBS Letters, 1997Co-Authors: Matthias Mack, Wolfgang BuckelAbstract:Abstract The heterooctameric (αβ)4 glutaconate CoA-transferase (EC 2.8.3.12) from the anaerobic bacterium Acidaminococcus fermentans catalyses the transfer of CoASH from acetyl-CoA to the 1-carboxylate of glutaconate. During this reaction the glutamate residue 54 of the β-subunit (βE54) forms a CoA-ester. The single amino acid replacement βE54D resulted in a drastic change of enzymatic function. The CoA-transferase activity decreased from 140 to less than 0.01 s−1, whereas the acyl-CoA hydrolase activity increased from less than 0.01 to 16 s−1. The new enzyme was able to catalyse the hydrolysis of Glutaryl-CoA, acetyl-CoA and 3-butenoyl-CoA. Since the mutants βE54A and βE54N showed neither acyl-CoA hydrolase nor CoA-transferase activity, it was concluded that the aspartate carboxylate of the mutant βE54D acted as a general base which facilitated the attack of water at the thiolester carbonyl. Surprisingly, Km for Glutaryl-CoA hydrolysis by the mutant (0.7 μM) as compared to CoA-transfer by the wild-type (28 μM) was 40 times lower. A 65 kDa protein, obtained by fusing the genes, gctA–gctB, coding for glutaconate CoA-transferase, retained 30% of the wild-type activity. Comparison of the amino acid sequences of 13 related enzymes demonstrated that Nature already has applied gene fusion in the case of pig heart CoA-transferase and has been using the E→D mutation for catalysis by a yeast acetyl-CoA hydrolase. © 1997 Federation of European Biochemical Societies.
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Purification of Glutaryl-CoA dehydrogenase from Pseudomonas sp., an enzyme involved in the anaerobic degradation of benzoate
Archives of Microbiology, 1993Co-Authors: Ulrich Härtel, Georg Fuchs, Jürgen Koch, Elke Eckel, Dietmar Linder, Wolfgang BuckelAbstract:Cell-free extracts of Pseudomonas sp. strains KB 740 and K 172 both contained high levels of Glutaryl-CoA dehydrogenase when grown anaerobically on benzoate or other aromatic compounds and with nitrate as electron acceptor. These aromatic compounds have in common benzoyl-CoA as the central aromatic intermediate of anerobic metabolism. The enzymatic activity was almost absent in cells grown aerobically on benzoate regardless whether nitrate was present. Glutaryl-CoA dehydrogenase activity was also detected in cell-free extracts of Rhodopseudomonas, Rhodomicrobium and Rhodocyclus after phototrophic growth on benzoate. Parallel to the induction of Glutaryl-CoA dehydrogenase as measured with ferricenium ion as electron acceptor, an about equally high glutaconyl-CoA decarboxylase activity was detected in cell-free extracts. The latter activity was measured with the NAD-dependent assay, as described for the biotin-containing sodium ion pump glutaconyl-CoA decarboxylase from glutamate fermenting bacteria. Glutaryl-CoA dehydrogenase was purified to homogeneity from both Pseudomonas strains. The enzymes catalyse the decarboxylation of glutaconyl-CoA at about the same rate as the oxidative decarboxylation of Glutaryl-CoA. The green enzymes are homotetramers (m=170 kDa) and contain 1 mol FAD per subunit. No inhibition was observed with avidin indicating the absence of biotin. The N-terminal sequences of the enzymes from both strains are similar (65%).