Crystal structure of human branched-chain alpha-ketoacid dehydrogenase and the molecular basis of multienzyme complex deficiency in maple syrup urine disease

BACKGROUND: Mutations in components of the extraordinarily large alpha-ketoacid dehydrogenase multienzyme complexes can lead to serious and often fatal disorders in humans, including maple syrup urine disease (MSUD). In order to obtain insight into the effect of mutations observed in MSUD patients, we determined the crystal structure of branched-chain alpha-ketoacid dehydrogenase (E1), the 170 kDa alpha(2)beta(2) heterotetrameric E1b component of the branched-chain alpha-ketoacid dehydrogenase multienzyme complex. RESULTS: The 2.7 A resolution crystal structure of human E1b revealed essentially the full alpha and beta polypeptide chains of the tightly packed heterotetramer. The position of two important potassium (K(+)) ions was determined. One of these ions assists a loop that is close to the cofactor to adopt the proper conformation. The second is located in the beta subunit near the interface with the small C-terminal domain of the alpha subunit. The known MSUD mutations affect the functioning of E1b by interfering with the cofactor and K(+) sites, the packing of hydrophobic cores, and the precise arrangement of residues at or near several subunit interfaces. The Tyr-->Asn mutation at position 393-alpha occurs very frequently in the US population of Mennonites and is located in a unique extension of the human E1b alpha subunit, contacting the beta' subunit. CONCLUSIONS: Essentially all MSUD mutations in human E1b can be explained on the basis of the structure, with the severity of the mutations for the stability and function of the protein correlating well with the severity of the disease for the patients. The suggestion is made that small molecules with high affinity for human E1b might alleviate effects of some of the milder forms of MSUD.     

The natural osmolyte trimethylamine N-oxide (TMAO) restores the ability of mutant tau to promote microtubule assembly

Coding region and intronic mutations in the gene for microtubule-associated protein tau cause frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17). Most coding region mutations effect a reduced ability of tau protein to interact with microtubules and lead to the formation of a filamentous pathology made of hyperphosphorylated tau. Here we show that trimethylamine N-oxide (TMAO) restores the ability of tau with FTDP-17 mutations to promote microtubule assembly. To mimic phosphorylation, serine and threonine residues in tau were singly or multiply mutated to glutamic acid, resulting in a reduced ability of tau to promote microtubule assembly. With the exception of the most heavily substituted protein (27 glutamic acid residues), TMAO increased the ability of mutant tau to promote microtubule assembly. However, it had no significant effect on heparin-induced assembly of tau into filaments.     

Gene preference in maple syrup urine disease

Untreated maple syrup urine disease (MSUD) results in mental and physical disabilities and often leads to neonatal death. Newborn-screening programs, coupled with the use of protein-modified diets, have minimized the severity of this phenotype and allowed affected individuals to develop into productive adults. Although inheritance of MSUD adheres to rules for single-gene traits, mutations in the genes for E1alpha, E1beta, or E2 of the mitochondrial branched-chain alpha-ketoacid dehydrogenase complex can cause the disease. Randomly selected cell lines from 63 individuals with clinically diagnosed MSUD were tested by retroviral complementation of branched-chain alpha-ketoacid dehydrogenase activity to identify the gene locus for mutant alleles. The frequencies of the mutations were 33% for the E1alpha gene, 38% for the E1beta gene, and 19% for the E2 gene. Ten percent of the tested cell lines gave ambiguous results by showing no complementation or restoration of activity with two gene products. These results provide a means to establish a genotype/phenotype relationship in MSUD, with the ultimate goal of unraveling the complexity of this single-gene trait. This represents the largest study to date providing information on the genotype for MSUD.     

A T-to-A substitution in the E1 alpha subunit gene of the branched-chain alpha-ketoacid dehydrogenase complex in two cell lines derived from Menonite maple syrup urine disease patients

We cloned and sequenced cDNAs of the E1 alpha and E1 beta subunits of the branched chain alpha-ketoacid dehydrogenase complex (BCKDH) in two cell lines derived from two different Menonite MSUD patients (GM 1655, GM 1099). A T-to-A substitution which generates an asparagine in place of a tyrosine at amino acid 394 of the mature E1 alpha subunit was present in both alleles in these two cell lines, whereas cDNAs of the E1 beta subunit in these cell lines were identical to that of normal human lymphoid cell line and that of the clone from a human placenta cDNA library. It is suggested that the Menonite MSUD is caused by the missense mutation of the E1 alpha subunit of the BCKDH complex.     

Functional differences in the catabolism of branched-chain L-amino acids in cultured normal and maple syrup urine disease fibroblasts

Possible functional differences in the catabolism of the four branched-chain L-amino acids in maple syrup urine disease were assessed using cultured human skin fibroblast stains. Transamination and oxidative decarboxylation were comparatively studied in 90-min incubations with 1 mmole/liter of 1-14C-labeled substrates. In normal cell strains (n = 5), apparent transamination rates (sum of branched-chain 2-oxo[14C]acid and 14CO2 release; means expressed in nmole/90 min/mg of cell protein) were in the order L-leucine (32) greater than L-valine (17) greater than or equal to L-isoleucine (14) greater than L-allo-isoleucine (8); 14CO2 production was in the order L-valine (9) greater than L-isoleucine (6) greater than or equal to L-leucine (5) greater than L-allo-isoleucine (2). In variant (n = 5) as well as classical (n = 2) MSUD cell lines, branched-chain 2-oxo-[14C]acid release rates were generally comparable to the control values. As compared to the 14CO2 release in controls (= 100%), branched-chain 2-oxo acid dehydrogenase activity in MSUD fibroblasts was individually reduced and varied considerably between strains (residual activity 2-38%). Within individual strains, only small differences in the residual decarboxylation activity were observed in incubations with L-valine, L-leucine, and L-isoleucine. It was remarkably high, however, when L-allo-isoleucine was applied as a substrate. With the exception of L-allo-isoleucine, apparent total transamination rates of branched-chain L-amino acids were therefore distinctly lower in MSUD cells than in normal cells.     

Defective decarboxylase in branched chain ketoacid oxidase multienzyme complex in classic type of maple syrup urine disease

Summary Enzyme kinetic studies on tissue extract from a newborn child who had died from the classic type of maple syrup urine disease (MSUD) revealed an altered decarboxylase moiety of the branched chain ketoacid oxidase multienzyme complex. Of two kinetically different decarboxylase components present in normal human controls the one with higher affinity for the substrate -ketoisovalerate was absent in our patient.

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Zusammenfassung Es wurden enzymkinetische Untersuchungen des verzweigtkettigen -Ketosäureoxidase-Komplexes in angereicherten Gewebeextrakten eines neugeborenen Kindes beschrieben, das an sogenannter klassischer Ahornsirupkrankheit gestorben ist. Während in normalen Kontrollen zwei kinetisch unterscheidbare Komponenten mit unterschiedlicher Substrataffinität nachweisbar sind, wurde im Gewebe des Patienten nur die Komponente mit geringerer Affinität zur -Ketoisovaleriansäure gefunden. Dieses Ergebnis deutet darauf hin, daß bei der klassischen Ahornsirupkrankheit das Teilenzym Decarboxylase im Multienzymkomplex der verzweigtkettigen Ketosäureoxidase defekt ist.

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This work was supported in part by grants of the Deutsche Forschungsgemeinschaft and the Stiftung Volkswagenwerk.     

Hepatocyte transplantation (HTx) corrects selected neurometabolic abnormalities in murine intermediate maple syrup urine disease (iMSUD)

Skvorak et al. [1] demonstrated the therapeutic efficacy of HTx in a murine model of iMSUD, confirming significant metabolic improvement and survival. To determine the effect of HTx on extrahepatic organs, we examined the metabolic effects of HTx in brain from iMSUD animals. Amino acid analysis revealed that HTx corrected increased ornithine, partially corrected depleted glutamine, and revealed a trend toward alloisoleucine correction. For amino acid and monoamine neurotransmitters, decreased GABA was partially corrected with HTx, while the l-histidine dipeptide of GABA, homocarnosine, was decreased in iMSUD mice and hypercorrected following HTx. Elevated branched-chain amino acids (BCAA; leucine, isoleucine, and valine) in MSUD can deplete brain tyrosine and tryptophan (the precursors of monoamine neurotransmitters, dopamine (DA) and serotonin (5-hydroxytryptamine; 5-HT)) through competition via the large neutral amino acid transporter. HTx corrected decreased DA levels and the DA metabolite, 3-methoxytyramine, and partially corrected the DA intermediate 3,4-dihydroxyphenylacetate (DOPAC) and 5-HT levels, despite normal tyrosine and tryptophan levels in iMSUD mouse brain. We further observed enhanced intracellular turnover of both DA and 5-HT in iMSUD mouse brain, both of which partially corrected with HTx. Our results suggest new pathomechanisms of neurotransmitter metabolism in this disorder and support the therapeutic relevance of HTx in iMSUD mice, while providing proof-of-principle that HTx has corrective potential in extrahepatic organs.     

Reversion of the maple syrup urine disease phenotype of impaired branched chain alpha-ketoacid dehydrogenase complex activity in fibroblasts from an affected child

Branched chain alpha-ketoacid dehydrogenase is a heteroprotein complex of mitochondria and commits the branched chain alpha-ketoacids to their catabolic fate. Inherited nuclear mutations in humans decrease the activity of this complex and result in maple syrup urine disease. Here we demonstrate the restoration of branched chain alpha-ketoacid dehydrogenase activity to fibroblasts from a child with this disorder by transfection with a cDNA for the prebranched chain acyltransferase. Prior to transfection these fibroblasts contained the prebranched chain acyltransferase gene but failed to transcribe the gene and thus lacked the protein. Regulation of the restored complex by phosphorylation mechanisms resembles that of wild-type cells. These results describe a human cell modeling system for testing engineered proteins and support the possibility of gene replacement therapy for this human disorder.     

Molecular characterization of maple syrup urine disease patients from Tunisia

Maple syrup urine disease (MSUD) is a rare disorder of branched-chain amino acids (BCAA) metabolism caused by the defective function of branched-chain α-ketoacid dehydrogenase complex (BCKD). The disease causal mutations can occur either in BCKDHA, BCKDHB or DBT genes encoding respectively the E1α, E1β and E2 subunits of the complex. In this study we report the molecular characterization of 3 Tunisian patients with the classic form of MSUD. Two novel putative mutations have been identified: the alteration c.716A>G (p.Glu239Gly) in BCKDHB and a small deletion (c.1333_1336delAATG; p.Asn445X) detected in DBT gene.     

Morphological alterations and induction of oxidative stress in glial cells caused by the branched-chain alpha-keto acids accumulating in maple syrup urine disease

Maple syrup urine disease (MSUD) is an inherited neurometabolic disorder biochemically characterized by the accumulation of the branched-chain alpha-keto acids (BCKA) alpha-ketoisocaproic (KIC), alpha-keto-beta-methylvaleric (KMV) and alpha-ketoisovaleric (KIV) and their respective branched-chain alpha-amino acids in body fluids and tissues. Affected MSUD patients have predominantly neurological features, including cerebral edema and atrophy whose pathophysiology is not well established. In the present study we investigated the effects of KIC, KMV and KIV on cell morphology, cytoskeleton reorganization, actin immunocontent and on various parameters of oxidative stress, namely total antioxidant reactivity (TAR), glutathione (GSH) and nitric oxide concentrations, and on the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) in C6 glioma cells. We initially observed that C6 cultivated cells exposed for 3 h to the BCKA (1 and 10 mM) changed their usual rounded morphology to a fusiform or process-bearing cell appearance, while 24 h exposure to these organic acids elicited massive cell death. Rhodamine-labelled phalloidin analysis revealed that these organic acids induced reorganization of the actin cytoskeleton with no modifications on total actin content. It was also observed that 3h cell exposure to low doses of all BCKA (1 mM) resulted in a marked reduction of the non-enzymatic antioxidant defenses, as determined by TAR and GSH measurements. In addition, KIC provoked a reduced activity of SOD and GPx, whereas KMV caused a diminution of SOD activity. In contrast, CAT activity was not modified by the metabolites. Furthermore, nitric oxide production was significantly increased by all BCKA. Finally, we observed that the morphological features caused by BCKA on C6 cells were prevented by the use of the antioxidants GSH (1.0 mM), alpha-tocopherol (trolox; 10 microM) and Nomega-nitro-L-arginine methyl ester (L-NAME; 500 microM). These results strongly indicate that oxidative stress might be involved in the cell morphological alterations and death, as well as in the cytoskeletal reorganization elicited by the BCKA. It is presumed that these findings are possibly implicated in the neuropathological features observed in patients affected by MSUD.     

Counteracting osmolyte trimethylamine N-oxide destabilizes proteins at pH below its pKa. Measurements of thermodynamic parameters of proteins in the presence and absence of trimethylamine N-oxide

Earlier studies have reported that trimethylamine N-oxide (TMAO), a naturally occurring osmolyte, is a universal stabilizer of proteins because it folds unstructured proteins and counteracts the deleterious effects of urea and salts on the structure and function of proteins. This conclusion has been reached from the studies of the effect of TMAO on proteins in the pH range 6.0-8.0. In this pH range TMAO is almost neutral (zwitterionic form), for it has a pK(a) of 4.66 +/- 0.10. We have asked the question of whether the effect of TMAO on protein stability is pH-dependent. To answer this question we have carried out thermal denaturation studies of lysozyme, ribonuclease-A, and apo-alpha-lactalbumin in the presence of various TMAO concentrations at different pH values above and below the pK(a) of TMAO. The main conclusion of this study is that near room temperature TMAO destabilizes proteins at pH values below its pK(a), whereas it stabilizes proteins at pH values above its pK(a). This conclusion was reached by determining the T(m) (midpoint of denaturation), delta H(m) (denaturational enthalpy change at T(m)), delta C(p) (constant pressure heat capacity change), and delta G(D) degrees (denaturational Gibbs energy change at 25 degrees C) of proteins in the presence of different TMAO concentrations. Other conclusions of this study are that T(m) and delta G(D) degrees depend on TMAO concentration at each pH value and that delta H(m) and the delta C(p) are not significantly changed in presence of TMAO.     

Effects of abnormal metabolites of maple syrup urine disease on neurotransmitter receptor binding

The deficiency of keto acid decarboxylase in maple syrup urine disease results in the accumulation of branched chain amino acids and their corresponding keto acids in tissues and body fluids. The effects of abnormal metabolites were investigated on neurotransmitter receptor binding in rat brain. alpha-Keto acids caused selective in vitro decrease in alpha-adrenergic, beta-adrenergic receptor binding in synaptosomal preparations from rat brain. No significant changes were observed in binding of cholinergic, GABA, and dopamine receptors binding in appropriate rat brain preparations. These results indicate that selective inhibition of adrenergic receptor binding by branched chain keto acids may presumably account for neural abnormality in maple syrup urine disease.     

Measurement of trimethylamine and trimethylamine N-oxide independently in urine by fast atom bombardment mass spectrometry

We report a method based upon fast atom bombardment mass spectrometry (FAB-MS) and stable isotope dilution techniques for the measurement of urinary trimethylamine (TMA) and trimethylamine N-oxide (TMAOx). TMA is extracted from urine that was spiked with (15)N-labeled TMA. The extracted TMA isotopomers are quaternized with trideuteromethyl iodide and analyzed in FAB-MS with hexaethylene glycol as matrix. TMAOx is measured by evaporation of another sample of the urine spiked with (15)N-labeled TMAOx on the FAB probe and analyzed as for the TMA. The method allows the ready and simple distinguishing of controls and patients with TMAuria, and is useful in monitoring patients with the disorder. We give examples of its use in determining normal control ranges for these metabolites and in evaluating patients.