3-Methylglutaconic aciduria type I is caused by mutations in AUH

3-Methylglutaconic aciduria type I is an autosomal recessive disorder clinically characterized by various symptoms ranging from delayed speech development to severe neurological handicap. This disorder is caused by a deficiency of 3-methylglutaconyl-CoA hydratase, one of the key enzymes of leucine degradation. This results in elevated urinary levels of 3-methylglutaconic acid, 3-methylglutaric acid, and 3-hydroxyisovaleric acid. By heterologous expression in Escherichia coli, we show that 3-methylglutaconyl-CoA hydratase is encoded by the AUH gene, whose product had been reported elsewhere as an AU-specific RNA-binding protein. Mutation analysis of AUH in two patients revealed a nonsense mutation (R197X) and a splice-site mutation (IVS8-1G-->A), demonstrating that mutations in AUH cause 3-methylglutaconic aciduria type I.     

Biochemical characterization of human 3-methylglutaconyl-CoA hydratase and its role in leucine metabolism

The metabolic disease 3-methylglutaconic aciduria type I (MGA1) is characterized by an abnormal organic acid profile in which there is excessive urinary excretion of 3-methylglutaconic acid, 3-methylglutaric acid and 3-hydroxyisovaleric acid. Affected individuals display variable clinical manifestations ranging from mildly delayed speech development to severe psychomotor retardation with neurological handicap. MGA1 is caused by reduced or absent 3-methylglutaconyl-coenzyme A (3-MG-CoA) hydratase activity within the leucine degradation pathway. The human AUH gene has been reported to encode for a bifunctional enzyme with both RNA-binding and enoyl-CoA-hydratase activity. In addition, it was shown that mutations in the AUH gene are linked to MGA1. Here we present kinetic data of the purified gene product of AUH using different CoA-substrates. The best substrates were (E)-3-MG-CoA (V(max) = 3.9 U.mg(-1), K(m) = 8.3 microM, k(cat) = 5.1 s(-1)) and (E)-glutaconyl-CoA (V(max) = 1.1 U.mg(-1), K(m) = 2.4 microM, k(cat) = 1.4 s(-1)) giving strong evidence that the AUH gene encodes for the major human 3-MG-CoA hydratase in leucine degradation. Based on these results, a new assay for AUH activity in fibroblast homogenates was developed. The only missense mutation found in MGA1 phenotypes, c.719C>T, leading to the amino acid exchange A240V, produces an enzyme with only 9% of the wild-type 3-MG-CoA hydratase activity.     

Identification of enzymes involved in oxidation of phenylbutyrate

In recent years the short-chain fatty acid, 4-phenylbutyrate (PB), has emerged as a promising drug for various clinical conditions. In fact, PB has been Food and Drug Administration-approved for urea cycle disorders since 1996. PB is more potent and less toxic than its metabolite, phenylacetate (PA), and is not just a pro-drug for PA, as was initially assumed. The metabolic pathway of PB, however, has remained unclear. Therefore, we set out to identify the enzymes involved in the β-oxidation of PB. We used cells deficient in specific steps of fatty acid β-oxidation and ultra-HPLC to measure which enzymes were able to convert PB or its downstream products. We show that the first step in PB oxidation is catalyzed solely by the enzyme, medium-chain acyl-CoA dehydrogenase. The second (hydration) step can be catalyzed by all three mitochondrial enoyl-CoA hydratase enzymes, i.e., short-chain enoyl-CoA hydratase, long-chain enoyl-CoA hydratase, and 3-methylglutaconyl-CoA hydratase. Enzymes involved in the third step include both short- and long-chain 3-hydroxyacyl-CoA dehydrogenase. The oxidation of PB is completed by only one enzyme, i.e., long-chain 3-ketoacyl-CoA thiolase. Taken together, the enzymatic characteristics of the PB degradative pathway may lead to better dose finding and limiting the toxicity of this drug.     

Induction of oxidative stress by the metabolites accumulating in 3-methylglutaconic aciduria in cerebral cortex of young rats

3-methylglutaconic (MGT), 3-methylglutaric (MGA) and occasionally 3-hydroxyisovaleric (OHIVA) acids accumulate in a group of diseases known as 3-methylglutaconic aciduria (MGTA). Although the clinical presentation of MGTA is mainly characterized by neurological symptoms, the mechanisms of brain damage in this disease are poorly known. In the present study we investigated the in vitro effect of MGT, MGA and OHIVA on various parameters of oxidative stress in cerebral cortex from young rats. Thiobarbituric acid-reactive substances (TBA-RS) and chemiluminescence were significantly increased by MGT, MGA and OHIVA, indicating that these metabolites induce lipid oxidative damage. Furthermore, the addition of melatonin, alpha-tocopherol and superoxide dismutase plus catalase fully prevented MGT-induced increase on TBA-RS, suggesting that free radicals were involved in this effect. These metabolites also provoked protein oxidative damage determined by increased carbonyl formation and sulfhydryl oxidation, but did not induce superoxide generation in submitochondrial particles. It was also verified that MGA and MGT significantly decreased the non-enzymatic antioxidant defenses in cerebral cortex supernatants and that melatonin and alpha-tocopherol totally blocked MGA-induced GSH reduction. The data indicate that the metabolites accumulating in MGTA elicit oxidative stress in vitro in the cerebral cortex. It is therefore presumed that this pathomechanism may be involved in the brain damage observed in patients affected by MGTA.     

3-Methylglutaconyl-CoA hydratase from Acinetobacter sp

Acinetobacter strain IVS-B aerobically grows on isovalerate as sole carbon and energy source. Isovalerate is metabolised via isovaleryl-CoA, an intermediate of the oxidative (S)-leucine degradation pathway. A 3-methylglutaconyl-CoA hydratase (EC 4.2.1.18) was purified 65-fold to apparent homogeneity from cell-free extracts of isovalerate-grown cells of Acinetobacter strain IVS-B. The enzyme was found to be a homotetramer (115.2 kDa) composed of four identical subunits of 28.8 kDa not containing any cofactors. The enzyme was shown to catalyse the hydration of (E)-glutaconyl-CoA (k _cat=18 s⁻¹, K _m=40 μM) and the dehydration of (S)-3-hydroxyglutaryl-CoA (k _cat=13 s⁻¹, K _m=52 μM), albeit with somewhat lower catalytic efficiencies as compared to the 3-methyl derivatives, 3-methylglutaconyl-CoA (k _cat=138 s⁻¹, K _m=14 μM) and (S)-3-hydroxy-3-methylglutaryl-CoA (k _cat=60 s⁻¹, K _m=36 μM). Thus, the mechanistically simple syn-addition of water to the (E)-isomer of 3-methylglutaconyl-CoA of the leucine degradative pathway leading to the common intermediate (S)-3-hydroxy-3-methylglutaryl-CoA was assigned as the major physiological role to this enzyme. The amino acid sequence of 3-methylglutaconyl-CoA hydratase from Acinetobacter sp. was found to be related to over 100 prokaryotic enoyl-CoA hydratases (up to 50% identity), possibly all being 3-methylglutaconyl-CoA hydratases.An erratum to this article can be found at     

POLG mutation in a patient with cataracts, early-onset distal muscle weakness and atrophy, ovarian dysgenesis and 3-methylglutaconic aciduria

Mutations in POLG account for one of the most frequent nuclear encoded causes of mitochondrial disorders to date. Individuals harboring POLG mutations exhibit fairly heterogeneous clinical presentations leading to increasing difficulties in classifying these patients into defined clinical phenotypes. This study aims to investigate the molecular basis of a mitochondrial cytopathy in a patient with 3-methylglutaconic aciduria and to expand the clinical phenotype associated with POLG mutations. Clinical, molecular and genetic analyses as well as neurophysiological examinations were carried out for a 23-year-old woman of mixed Caucasian and Latin American ancestry with a history of cataracts diagnosed at age 1 year, she had onset of distal muscle weakness at age 2 years progressing to atrophy and ovarian dysgenesis at puberty. The patient was found to have 3-methylglutaconic acid with normal 3 hydroxyisovaleric acid on urine organic acid analysis. POLG sequencing was done and a heterozygous variant, c.2851T>A (p.Y951N) was found which is predicted to be deleterious. There are limited reports of POLG mutations in individuals with 3-methylglutaconic aciduria. This case report of a young woman with a heterozygous mutation in POLG, presenting with muscle weakness and atrophy at a young age aims to aid clinicians in similar challenging diagnostic situations as well as enhances our understanding of POLG-related disease phenotypes.     

The coenzyme A-dependent, non-β-oxidation pathway and not direct deacetylation is the major route for ferulic acid degradation in Delftia acidovorans

The gene loci fcs and ech, encoding feruloyl-CoA synthetase and enoyl-CoA hydratase/aldolase, respectively, are involved in the ferulic acid catabolism in Delftia acidovorans. The amino acid sequence deduced from ech exhibited 51% identity to the enoyl-CoA hydratase/aldolase from Pseudomonas sp. strain HR199, indicating that the enzyme from D. acidovorans represents a new lineage of this protein. The genes fcs and ech were expressed in Escherichia coli enabling the recombinant strain to transform ferulic acid to vanillin as revealed by photometric and HPLC analysis. An fcs deficient mutant of D. acidovorans was unable to grow on ferulic acid. The obtained data suggest that in contrast to a previous publication the biotechnologically interesting direct non-oxidative deacetylation mechanism of ferulic acid cleavage is not realized in D. acidovorans. Instead, ferulic acid degradation in D. acidovorans proceeds via a coenzyme A-dependent non-beta-oxidative pathway.     

Analysis of the beta-oxidation of trans-unsaturated fatty acid in recombinant Saccharomyces cerevisiae expressing a peroxisomal PHA synthase reveals the involvement of a reductase-dependent pathway

The degradation of fatty acids having cis- or trans-unsaturated bond at an even carbon was analyzed in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate production in the peroxisome. Polyhydroxyalkanaote is synthesized by the polymerization of the beta-oxidation intermediates 3-hydroxy-acyl-CoAs via a bacterial polyhydroxyalkanoate synthase targeted to the peroxisome. The synthesis of polyhydroxyalkanoate in cells grown in media containing 10-cis-heptadecenoic acid was dependent on the presence of 2,4-dienoyl-CoA reductase activity as well as on Delta3,Delta2-enoyl-CoA isomerase activity. The synthesis of polyhydroxyalkanoate from 10-trans-heptadecenoic acid in mutants devoid of 2,4-dienoyl-CoA reductase revealed degradation of the trans fatty acid directly via the enoyl-CoA hydratase II activity of the multifunctional enzyme (MFE), although the level of polyhydroxyalkanoate was 10-25% to that of wild type cells. Polyhydroxyalkanoate produced from 10-trans-heptadecenoic acid in wild type cells showed substantial carbon flux through both a reductase-dependent and a direct MFE-dependent pathway. Flux through beta-oxidation was more severely reduced in mutants devoid of Delta3,Delta2-enoyl-CoA isomerase compared to mutants devoid of 2,4-dienoyl-CoA reductase. It is concluded that the intermediate 2-trans,4-trans-dienoyl-CoA is metabolized in vivo in yeast by both the enoyl-CoA hydratase II activity of the multifunctional protein and the 2,4-dienoyl-CoA reductase, and that the synthesis of the intermediate 3-trans-enoyl-CoA in the absence of the Delta3,Delta2-enoyl-CoA isomerase leads to the blockage of the direct MFE-dependent pathway in vivo.     

Evolution of function in the crotonase superfamily: (3S)-methylglutaconyl-CoA hydratase from Pseudomonas putida

Members of the enoyl-CoA hydratase (crotonase) superfamily catalyze different overall reactions that utilize a common catalytic strategy delivered by a shared structural scaffold; the substrates are usually acyl esters of coenzyme A, and the intermediates are usually thioester enolate anions stabilized by a conserved oxyanion hole. In many bacterial genomes, orthologous members that contain homologues of acid/base catalyst Glu164 but not of Glu144 in rat mitochondrial crotonase are encoded by operons of which the functions have not been assigned. Focusing on the orthologues from Pseudomonas aeruginosa and P. putida, we have determined that these operons encode enzymes in leucine catabolism with the unknown enzyme assigned as (3S)-methylglutaconyl-CoA hydratase (MGCH), which catalyzes the syn-hydration of (E)-3-methylglutaconyl-CoA to (3S)-hydroxymethylglutaryl-CoA. The discovery that bacterial MGCHs catalyze hydration of enoyl-CoAs utilizing a single active-site residue contrasts with the paradigm crotonases as well as with the recently identified mammalian MGCHs that use homologues of both Glu144 and Glu164 in crotonase. Substrate analogues lacking a gamma-carboxylate have been shown to be competitive inhibitors of the enzyme, and installation of a glutamate for the "missing" homologue of Glu144 fails to introduce hydratase activity with the substrate analogues. Thus, bacterial MGCHs may provide an example of opportunistic evolution in which a carboxylate group of the substrate functionally replaces one of the active site glutamate residues in the reactions catalyzed by crotonases and the eukaryotic MGCHs.     

Futile cycling of intermediates of fatty acid biosynthesis toward peroxisomal beta-oxidation in Saccharomyces cerevisiae

The flux of fatty acids toward beta-oxidation was analyzed in Saccharomyces cerevisiae by monitoring polyhydroxyalkanoate synthesis in the peroxisome from the polymerization, by a bacterial polyhydroxyalkanoate synthase, of the beta-oxidation intermediates 3-hydroxyacyl-CoAs. Synthesis of polyhydroxyalkanoate was dependent on the beta-oxidation enzymes acyl-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase multifunctional protein, which are involved in generating 3-hydroxyacyl-CoAs, and on the peroxin PEX5, which is involved in the import of proteins into the peroxisome. In wild type cells grown in media containing fatty acids, the polyhydroxyalkanoate monomer composition was largely influenced by the nature of the external fatty acid, such that even-chain monomers are generated from oleic acid and odd-chain monomers are generated from heptadecenoic acid. In contrast, polyhydroxyalkanoate containing predominantly 3-hydroxyoctanoate, 3-hydroxydecanoate, and 3-hydroxydodecanoate was synthesized in a mutant deficient in the peroxisomal 3-ketothiolase (fox3 Delta 0) growing either on oleic acid or heptadecenoic acid as well as in wild type and fox3 Delta 0 mutants grown on glucose or raffinose, indicating that 3-hydroxyacyl-CoAs used for polyhydroxyalkanoate synthesis were generated from the degradation of intracellular short- and medium-chain fatty acids by the beta-oxidation cycle. Inhibition of fatty acid biosynthesis with cerulenin blocked the synthesis of polyhydroxyalkanoate from intracellular fatty acids but still enabled the use of extracellular fatty acids for polymer production. Mutants affected in the synthesis of lipoic acid showed normal polyhydroxyalkanoate synthesis capacity. Together, these results uncovered the existence of a substantial futile cycle whereby short- and medium-chain intermediates of the cytoplasmic fatty acid biosynthetic pathway are directed toward the peroxisomal beta-oxidation pathway.     

Conservation of central nervous system glutaryl-coenzyme A dehydrogenase in fruit-eating bats with glutaric aciduria and deficient hepatic glutaryl-coenzyme A dehydrogenase

The adult fruit-eating bat, Rousettus aegypticus, excretes massive amounts of glutaric acid in the urine (20-70 mumol/mg creatinine) comparable to those of humans affected with the inherited metabolic disorder, glutaric aciduria type I. Glutaric acid was quantified by sequential liquid partition chromatography and gas chromatography. Oral loading with the amino acid precursors of glutaric acid, L-lysine and L-tryptophan, resulted in significant increases in glutaric acid excretion above the base-line values. Glutaryl-CoA dehydrogenase activity was assayed in adult bat tissues and compared with the same tissues in the rat using methods of 14CO2 evolution from 1,5-[14C]glutaryl-CoA. A severe deficiency of glutaryl-CoA dehydrogenase activity was found in the bat liver and kidney, whereas brain and spinal cord levels were similar to those in the rat. Reverse phase high performance liquid chromatography analysis of the metabolites in the assay mixture showed negligible hydrolysis of [14C]glutaryl-CoA to free [14C]glutaric acid and complete conversion of the product [14C]crotonyl-CoA to 3-hydroxy[14C]butyryl-CoA. The adult bat, with its huge glutaric acid excretion and deficient liver glutaryl-CoA dehydrogenase, metabolically mimics patients affected with glutaric aciduria type I. The bat does not, however, display the neurologic manifestations seen in patients. This may be explained by conservation of glutaryl-CoA dehydrogenase activity in the central nervous system of the bat.     

Degradation of anthracene by Mycobacterium sp. strain LB501T proceeds via a novel pathway,through o-phthalic acid

Mycobacterium sp. strain LB501T utilizes anthracene as a sole carbon and energy source. We analyzed cultures of the wild-type strain and of UV-generated mutants impaired in anthracene utilization for metabolites to determine the anthracene degradation pathway. Identification of metabolites by comparison with authentic standards and transient accumulation of o-phthalic acid by the wild-type strain during growth on anthracene suggest a pathway through o-phthalic acid and protocatechuic acid. As the only productive degradation pathway known so far for anthracene proceeds through 2,3-dihydroxynaphthalene and the naphthalene degradation pathway to form salicylate, this indicates the existence of a novel anthracene catabolic pathway in Mycobacterium sp. LB501T.     

Conversion of mevalonic acid to gamma, gamma-dimethylallyl pyrophosphate by Mycoplasma

Henrikson, Carl V. (University of South Dakota, Vermillion), and Paul F. Smith. Conversion of mevalonic acid to gamma,gamma-dimethylallyl pyrophosphate by Mycoplasma. J. Bacteriol. 92:701-706. 1966.-Three representative strains of Mycoplasma, M. laidlawii strain B, Mycoplasma sp. avian strain J, and M. hominis type 2 strain O7, were examined for the presence or absence of enzymes associated with the biosynthetic pathway from mevalonic acid to gamma,gamma-dimethylallyl pyrophosphate. M. laidlawii served as a control organism, since it is capable of de novo biosynthesis of carotenoids. All four enzymes, namely, adenosine triphosphate (ATP)-mevalonate 5-phosphotransferase (EC 2.7.1.36), ATP-5-phosphomevalonate phosphotransferase (EC 2.7.4.2), ATP-5-pyrophosphomevalonate carboxy-lyase (EC 4.1.1.33), and isopentenylpyrophosphate Delta(3),Delta(2)-isomerase (EC 5.3.3.2), were demonstrated in this organism. Mycoplasma sp. avian strain J, which contains all enzymes necessary for the biosynthesis of mevalonic acid, lacks the first three of the above enzymes but contains isopentenyl pyrophosphate Delta(3),Delta(2)-isomerase. M. hominis, which lacks the enzymes necessary for the biosynthesis of mevalonic acid, also is deficient in the enzymes involved in its conversion to gamma,gamma-dimethylallyl pyrophosphate.     

3-Methylcrotonyl-Coenzyme A Carboxylase Is a Component of the Mitochondrial Leucine Catabolic Pathway in Plants

3-Methylcrotonyl-coenzyme A carboxylase (MCCase) is a mitochondrial biotin-containing enzyme whose metabolic function is not well understood in plants. In soybean (Glycine max) seedlings the organ-specific and developmentally induced changes in MCCase expression are regulated by mechanisms that control the accumulation of MCCase mRNA and the activity of the enzyme. During soybean cotyledon development, when seed-storage proteins are degraded, leucine (Leu) accumulation peaks transiently at 8 d after planting. The coincidence between peak MCCase expression and the decline in Leu content provides correlative evidence that MCCase is involved in the mitochondrial catabolism of Leu. Direct evidence for this conclusion was obtained from radiotracer metabolic studies using extracts from isolated mitochondria. These experiments traced the metabolic fate of [U-¹⁴C]Leu and NaH¹⁴CO₃, the latter of which was incorporated into methylglutaconyl-coenzyme A (CoA) via MCCase. These studies directly demonstrate that plant mitochondria can catabolize Leu via the following scheme: Leu → α-ketoisocaproate → isovaleryl-CoA → 3-methylcrotonyl-CoA → 3-methylglutaconyl-CoA → 3-hydroxy-3-methylglutaryl-CoA → acetoacetate + acetyl-CoA. These findings demonstrate for the first time, to our knowledge, that the enzymes responsible for Leu catabolism are present in plant mitochondria. We conclude that a primary metabolic role of MCCase in plants is the catabolism of Leu.     

Abnormal urinary excretion of unsaturated dicarboxylic acids in patients with medium-chain acyl-CoA dehydrogenase deficiency

Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most frequently described metabolic disorder of fatty acid oxidation in humans. Acute episodes are usually characterized biochemically by the appearance of nonketotic dicarboxylic aciduria. In addition, other abnormal metabolites, such as suberylglycine, n-hexanoylglycine, 3-phenylpropionylglycine, and octanoylcarnitine, are excreted in the urine. Urinary organic acids were determined using dual capillary column gas-liquid chromatography and gas-liquid chromatography/mass spectrometry. In three cases of MCAD deficiency we observed a disproportionate increase in the excretion of unsaturated dicarboxylic acids compared to either fasting control children with expected ketotic dicarboxylic aciduria or patients with nonketotic dicarboxylic aciduria not associated with MCAD deficiency. The most significant increase was in the urinary excretion of cis-4-decendioic acid. Additionally, the urinary excretions of cis-3-octenedioic and cis-5-decenedioic acids were slightly decreased whereas the excretion of cis-5-dodecenedioic acid was increased. These data are consistent with the notion that as a result of MCAD deficiency the metabolic oxidation of unsaturated fatty acids such as linoleate and oleate is inhibited more than saturated fatty acids.     

Degradation of Anthracene by Mycobacterium sp. Strain LB501T Proceeds via a Novel Pathway, through o-Phthalic Acid

Mycobacterium sp. strain LB501T utilizes anthracene as a sole carbon and energy source. We analyzed cultures of the wild-type strain and of UV-generated mutants impaired in anthracene utilization for metabolites to determine the anthracene degradation pathway. Identification of metabolites by comparison with authentic standards and transient accumulation of o-phthalic acid by the wild-type strain during growth on anthracene suggest a pathway through o-phthalic acid and protocatechuic acid. As the only productive degradation pathway known so far for anthracene proceeds through 2,3-dihydroxynaphthalene and the naphthalene degradation pathway to form salicylate, this indicates the existence of a novel anthracene catabolic pathway in Mycobacterium sp. LB501T.     

Manganese deficiency leads to elevated amino acid pools in citric acid accumulating Aspergillus niger

Free amino acid pools have been investigated in a citric acid accumulating strain of Aspergillus niger during batch growth under manganese sufficient and deficient conditions by means of an improved chromatographic method. Studies on the mycelial content of several nitrogenous compounds under manganese sufficient and deficient conditions showed that manganese deficiency resulted in lower amino acid pool sizes during trophophase and considerable accumulation during idiophase, and in a reduction of the protein and nucleic acid contents. Addition of cycloheximide to mycelia grown with sufficient manganese also caused an elevation of free amino acid pool sizes, thus indicating that impairment of protein synthesis by manganese deficiency is responsible for the observed rise in amino acid concentration. Furthermore it was observed that the manganese deficient mycelia excreted high amounts of all amino acids suggesting that manganese deficiency may also affect membrane permeability.