Nucleotide and deduced amino acid sequence of the E1 alpha subunit of human liver branched-chain alpha-ketoacid dehydrogenase

A 1552-bp cDNA for the E1 alpha subunit of branched-chain alpha-ketoacid dehydrogenase (BCKDH) was isolated from a human liver cDNA library. The cDNA contained a 1134-bp open reading frame that encoded 378 amino acid (aa) residues of the enzyme and 418 bp of 3'-untranslated sequence. The deduced amino acid sequence of the human protein shows 96% identity with that of the rat enzyme subunit. Those 117-aa residues surrounding the phosphorylation sites are completely conserved between man and rat. BCKDH E1 alpha showed considerable amino acid sequence similarity with pyruvate dehydrogenase E1 alpha, particularly in the region of the two principal phosphorylation sites of these proteins. Northern blots of human liver and skin fibroblasts demonstrated a single 1.8-kb mRNA band, with a higher level of E1 alpha mRNA in liver than in normal fibroblasts. Fibroblasts from a patient with thiamine-responsive maple syrup urine disease (MSUD) contained an mRNA of the same size and abundance as that of normal fibroblasts. Genomic DNA from normal and MSUD fibroblasts gave the same restriction maps on Southern blots, and the gene was approximately 10-kb in size.     

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.     

Assignment of the gene for the E1 alpha subunit of branched chain alpha-ketoacid dehydrogenase to chromosome 19

The gene encoding the E1 alpha subunit of branched chain alpha-ketoacid dehydrogenase was mapped to human chromosome 19. 32P-labeled human E1 alpha cDNA was hybridized with DNA derived from flow-sorted human chromosomes; it hybridized exclusively with that from chromosome 19.     

Molecular cloning of the mature E1b-beta subunit of human branched-chain alpha-keto acid dehydrogenase complex

We have isolated a cDNA encoding the E1b-beta subunit of the human branched-chain alpha-keto acid dehydrogenase complex. The human E1b-beta cDNA is 1401 base pairs in length. It encodes the entire mature E1b-beta subunit consisting of 342 amino acid residues, and a mitochondrial targeting presequence of 31 residues. The calculated molecular mass of the mature human E1b-beta subunit is 37,851 Da, and the calculated isoelectric point is pH 5.18. A hydropathy plot shows that the human E1b-beta subunit is highly hydrophobic. Northern blot analysis shows that the human E1b-beta mRNA is approximately 1.4 kb in size. It is present at the normal level in fibroblasts from two unrelated maple syrup urine disease patients.     

Molecular phenotypes in cultured maple syrup urine disease cells. Complete E1 alpha cDNA sequence and mRNA and subunit contents of the human branched chain alpha-keto acid dehydrogenase complex

The activity of the branched-chain alpha-keto acid dehydrogenase complex is deficient in patients with the inherited maple syrup urine disease (MSUD). To elucidate the molecular basis of this metabolic disorder, we have isolated three overlapping cDNA clones encoding the E1 alpha subunit of the human enzyme complex. The composite human E1 alpha cDNA consists of 1783 base pairs encoding the entire human E1 alpha subunit of 400 amino acids with calculated Mr = 45,552. The human E1 alpha and the previously isolated human E2 cDNAs were used as probes in Northern blot analysis with cultured fibroblasts and lymphoblasts from seven unrelated MSUD patients. The results along with those of Western blotting have revealed five distinct molecular phenotypes according to mRNA and protein-subunit contents. These consist of type I, where the levels of E1 alpha mRNA and E1 alpha and E1 beta subunits are normal in cells, but E1 activity is deficient; Type II, where the E1 alpha mRNA is present in normal quantity, whereas the contents of E1 alpha and E1 beta subunits are reduced; Type III, where the level of E1 alpha mRNA is markedly reduced with a concomitant loss of E1 alpha and E1 beta subunits; Type IV, where the contents of both E2 mRNA and E2 subunits are markedly reduced; and Type V, where the E2 mRNA is normally expressed, but the E2 subunit is markedly reduced or completely absent. Type V includes thiamin-responsive (WG-34) and certain classical MSUD cells. These molecular phenotypes have demonstrated the complexity of MSUD and identified the affected gene in different patients for further characterization.     

Molecular cloning of a cDNA for the E1 alpha subunit of rat liver branched chain alpha-ketoacid dehydrogenase

We have isolated a cDNA encoding the branched chain alpha-ketoacid dehydrogenase E1 alpha subunit. A rat liver lambda gt11 expression library was screened with antibody reactive with the 2-oxoisovalerate dehydrogenase (lipoamide) component. A positive clone, lambda BZ304, contains a 1.7-kilobase pair cDNA insert with a 1323-base pair open reading frame. Translation of the open reading frame predicts the 24 residues of the previously reported phosphorylation sites 1 and 2 for the bovine kidney and rabbit heart enzymes. The N-terminal sequence of purified E1 alpha was determined, and this sequence was found 40 residues from the beginning of the deduced peptide sequence. Northern blots of rat liver and muscle RNA demonstrate a single mRNA species of approximately 1.8 kilobase pairs in each tissue, suggesting that this cDNA is nearly full length.     

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.     

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 human pyruvate dehydrogenase complex. Isolation of cDNA clones for the E1 alpha subunit, sequence analysis, and characterization of the mRNA

cDNA clones corresponding to the entire length of mRNA for the alpha subunit of human pyruvate dehydrogenase (EC 1.2.4.1), the E1 component of the pyruvate dehydrogenase complex, have been isolated from liver cDNA libraries. Two classes of cDNA clones were obtained and these correspond to two forms of pyruvate dehydrogenase E1 alpha mRNA. Both mRNA species have been demonstrated in a variety of human tissues and cultured fibroblasts. The cDNA sequence has been determined and, from it, the protein sequence of the human E1 alpha subunit was deduced. The protein is synthesized with a typical mitochondrial import leader sequence and the peptide bond at which this sequence is cleaved after transport into the mitochondrion has been determined by direct amino acid sequencing of the mature E1 alpha subunit. The human pyruvate dehydrogenase E1 alpha subunit contains identical phosphorylation sites to those found in the corresponding porcine protein. Preliminary studies of pyruvate dehydrogenase E1 alpha mRNA in cultured fibroblasts from patients with severe pyruvate dehydrogenase deficiency have revealed considerable heterogeneity as would be expected from protein studies.     

Characterization of two cDNA clones for pyruvate dehydrogenase E1 beta subunit and its regulation in tricarboxylic acid cycle-deficient fibroblast

Two distinct types of cDNA clones encoding for the pyruvate dehydrogenase (PDH) E1 beta subunit were isolated from a human liver lambda gt11 cDNA library and characterized. These cDNA clones have identical nucleotide sequences for PDH E1 beta protein coding region but differ in their lengths and in the sequences of their 3'-untranslated regions. The smaller cDNA had an unusual polyadenylation signal within its protein coding region. The cDNA-deduced protein of PDH E1 beta subunit revealed a precursor protein of 359 amino acid residues (Mr 39,223) and a mature protein of 329 residues (Mr 35,894), respectively. Both cDNAs shared high amino acid sequence similarity with that isolated from human foreskin (Koike, K.K., Ohta, S., Urata, Y., Kagawa, Y., and Koike, M. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 41-45) except for three regions of frameshift mutation. These changes led to dramatic alterations in the local net charges and predicted protein conformation. One of the different sequences in the protein coding region of liver cDNA (nucleotide position 452-752) reported here was confirmed by sequencing the region after amplification of cDNA prepared from human skin fibroblasts by the polymerase chain reaction. Southern blot analysis verified simple patterns of hybridization with E1 beta cDNA, indicating that the PDH E1 beta subunit gene is not a member of a multigene family. The mechanisms of differential expression of the PDH E1 alpha and E1 beta subunits were also studied in established fibroblast cell lines obtained from patients with Leigh's syndrome and other forms of congenital lactic acidosis. In Northern blot analyses for PDH E1 alpha and E1 beta subunits, no apparent differences were observed between two Leigh's syndrome and the control fibroblasts studied: one species of PDH E1 alpha mRNA and three species of E1 beta mRNA were observed in all the cell lines examined. However, in one tricarboxylic acid cycle deficient fibroblast cell line, which has one-tenth of the normal enzyme activity, the levels of immunoreactive PDH E1 alpha and E1 beta subunits were markedly decreased as assessed by immunoblot analyses. These data indicated a regulatory mutation caused by either inefficient translation of E1 alpha and E1 beta mRNAs into protein or rapid degradation of both subunits upon translation. In contrast, the PDH E1 alpha and E1 beta subunits in two fibroblast cell lines from Leigh's syndrome patients appeared to be normal as judged by 1) enzyme activity, 2) mRNA Northern blot, 3) genomic DNA Southern blot, and 4) immunoblot analyses indicating that the lactic acidosis seen in these patients did not result from a single defect in either of these E1 alpha and E1 beta subunits of the PDH complex.     

Molecular basis of maple syrup urine disease: novel mutations at the E1 alpha locus that impair E1(alpha 2 beta 2) assembly or decrease steady-state E1 alpha mRNA levels of branched-chain alpha-keto acid dehydrogenase complex.

We report the occurrence of three novel mutations in the E1 alpha (BCKDHA) locus of the branched-chain alpha-keto acid dehydrogenase (BCKAD) complex that cause maple syrup urine disease (MSUD). An 8-bp deletion in exon 7 is present in one allele of a compound-heterozygous patient (GM-649). A single C nucleotide insertion in exon 2 occurs in one allele of an intermediate-MSUD patient (Lo). The second allele of patient Lo carries an A-to-G transition in exon 9 of the E1 alpha gene. This missense mutation changes Tyr-368 to Cys (Y368C) in the E1 alpha subunit. Both the 8-bp deletion and the single C insertion generate a downstream nonsense codon. Both mutations appear to be associated with a low abundance of the mutant E1 alpha mRNA, as determined by allele-specific oligonucleotide probing. Transfection studies strongly suggest that the Y368C substitution in the E1 alpha subunit impairs its proper assembly with the normal E1 beta. Unassembled as well as misassembled E1 alpha and E1 beta subunits are degraded in the cell.     

Natural osmolyte trimethylamine N-oxide corrects assembly defects of mutant branched-chain alpha-ketoacid decarboxylase in maple syrup urine disease

Maple syrup urine disease is caused by deficiency in the mitochondrial branched-chain alpha-ketoacid dehydrogenase (BCKD) complex. The clinical phenotype includes often fatal ketoacidosis, neurological derangement, and mental retardation. The type IA mutations Y393N-alpha, Y368C-alpha, and F364C-alpha, which occur in the E1alpha subunit of the decarboxylase (E1) component of the BCKD complex, impede the conversion of an alphabeta heterodimeric intermediate to a native alpha(2)beta(2) heterotetramer in the E1 assembly pathway. In the present study, we show that a natural osmolyte trimethylamine N-oxide (TMAO) at the optimal 1 m concentration restores E1 activity, up to 50% of the wild type, in the mutant E1 carrying the above missense mutations. TMAO promotes the conversion of otherwise trapped mutant heterodimers to active heterotetramers. This slow step does not involve dissociation/reassociation of the mutant heterodimers, which are preformed in the presence of chaperonins GroEL/GroES and Mg-ATP. The TMAO-stimulated mutant E1 activity is remarkably stable upon removal of the osmolyte, when cofactor thiamine pyrophosphate and the transacylase component of the BCKD complex are present. The above in vitro results offer the use of chemical chaperones such as TMAO as an approach to mitigate assembly defects caused by maple syrup urine disease mutations.     

Expression and assembly of a functional E1 component (alpha 2 beta 2) of mammalian branched-chain alpha-ketoacid dehydrogenase complex in Escherichia coli.

We have expressed an active recombinant E1 decarboxylase component of the mammalian branched-chain alpha-ketoacid dehydrogenase complex in Escherichia coli by subcloning mature E1 alpha and E1 beta subunit cDNA sequences into a bacterial expression vector. To permit affinity purification under native conditions, the mature E1 alpha subunit was fused with the affinity ligand E. coli maltose-binding protein (MBP) through an endoprotease Factor Xa-specific linker peptide. When co-expressed, the MBP-E1 alpha fusion and E1 beta subunits were shown to co-purify as a MBP-E1 component that exhibited both E1 activity and binding competence for recombinant branched-chain E2 component. In contrast, in vitro mixing of individually expressed MBP-E1 alpha and E1 beta did not result in assembly or produce E1 activity. Following proteolytic removal of the affinity ligand and linker peptide with Factor Xa, a recombinant E1 species was eluted from a Sephacryl S-300HR sizing column as an enzymatically active 160-kDa species. The latter showed 1:1 subunit stoichiometry, which was consistent with an alpha 2 beta 2 structure. The recovery of this 160-kDa recombinant E1 species (estimated at 0.07% of total lysate protein) was low, with the majority of the recombinant protein lost as insoluble aggregates. Our findings suggest that the concurrent expression of both E1 alpha and E1 beta subunits in the same cellular compartment is important for assembly of both subunits into a functional E1 alpha 2 beta 2 heterotetramer. By using this co-expression system, we also find that the E1 alpha missense mutation (Tyr-393----Asn) characterized in Mennonites with maple syrup urine disease prevents the assembly of soluble E1 heterotetramers.     

Structural and biochemical basis for novel mutations in homozygous Israeli maple syrup urine disease patients: a proposed mechanism for the thiamin-responsive phenotype

Maple syrup urine disease (MSUD) results from mutations affecting different subunits of the mitochondrial branched-chain alpha-ketoacid dehydrogenase complex. In this study, we identified seven novel mutations in MSUD patients from Israel. These include C219W-alpha (TGC to TGG) in the E1alpha subunit; H156Y-beta (CAT to TAT), V69G-beta (GTT to GGT), IVS 9 del[-7:-4], and 1109 ins 8bp (exon 10) in the E1beta subunit; and H391R (CAC to CGC) and S133stop (TCA to TGA) affecting the E2 subunit of the branched-chain alpha-ketoacid dehydrogenase complex. Recombinant E1 proteins carrying the C219W-alpha or H156Y-beta mutation show no catalytic activity with defective subunit assembly and reduced binding affinity for cofactor thiamin diphosphate. The mutant E1 harboring the V69G-beta substitution cannot be expressed, suggesting aberrant folding caused by this mutation. These E1 mutations are ubiquitously associated with the classic phenotype in homozygous-affected patients. The H391R substitution in the E2 subunit abolishes the key catalytic residue that functions as a general base in the acyltransfer reaction, resulting in a completely inactive E2 component. However, wild-type E1 activity is enhanced by E1 binding to this full-length mutant E2 in vitro. We propose that the augmented E1 activity is responsible for robust thiamin responsiveness in homozygous patients carrying the H391R E2 mutation and that the presence of a full-length mutant E2 is diagnostic of this MSUD phenotype. The present results offer a structural and biochemical basis for these novel mutations and will facilitate DNA-based diagnosis for MSUD in the Israeli population.     

Molecular heterogeneity for bovine maple syrup urine disease

In Poll Herefords, it is known that maple syrup urine disease results from a nonsense mutation in codon -6 of the gene for the Elα subunit of branched-chain α-keto acid dehydrogenase. The disease also occurs in Poll Shorthorns, but its molecular basis in this breed has not yet been determined. Allele-specific hybridization and allele-specific amplification, both based on the Poll Hereford mutation, failed to detect the mutant allele in Poll Shorthorn heterozygotes, and detected the normal allele in affected Poll Hereford-cross-Poll Shorthorn calves. These results demonstrate between breed molecular heterogeneity for bovine maple syrup urine disease.     

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.