Heterogeneity of mutations in Maple Syrup Urine Disease (MSUD): screening and identification of affected E1α and E1β subunits of the branched-chain α-keto-acid dehydrogenase multienzyme complex

Maple syrup urine disease (MSUD) is an autosomal recessive disease caused by a deficiency in subunits of the branched-chain α-keto-acid dehydrogenase complex (BCKDH). To characterize the mutations present in five patients with MSUD (four classic and one intermediate), three-step analyses were established: (1) identification of the involved subunit by complementation analysis using three different cell lines derived from homozygotes having E1α, E2β or the E2 mutant gene; (2), screening for a mutation site in cDNA of the corresponding subunit by RT-PCR-SSCP and (3), mutant analysis by sequencing the amplified cDNA fragment. Four single-base missense mutations, R115W, Q1556K, A209T and I282T, were detected in the E1α subunit. A single-base missense mutation H156R and three frame-shift mutations to generate stop codons downstream, including an 11-bp deletion of the tandem repeat in exon 1, a single-base (T) deletion and a single-base (G) insertion, were identified in the E1β subunit gene. All except one (11-bp deletion in E1β (Nobukini, Y., Mitsubuchi, H., Akaboshi, I., Indo, Y., Endo, F., Yoshioka, A. and Matsuda, I. (1991) J. Clin. Invest. 87, 1862–1866)) were novel mutations. The sites of amino-acid substitution were all conserved in other species. Thus, mutations causing MSUD are heterogeneous.     

Biochemical basis of type IB (E1beta ) mutations in maple syrup urine disease. A prevalent allele in patients from the Druze kindred in Israel.

Maple syrup urine disease (MSUD) is a metabolic disorder associated with often-fatal ketoacidosis, neurological derangement, and mental retardation. In this study, we identify and characterize two novel type IB MSUD mutations in Israeli patients, which affect the E1beta subunit in the decarboxylase (E1) component of the branched-chain alpha-ketoacid dehydrogenase complex. The recombinant mutant E1 carrying the prevalent S289L-beta (TCG --> TTG) mutation in the Druze kindred exists as a stable inactive alphabeta heterodimer. Based on the human E1 structure, the S289L-beta mutation disrupts the interactions between Ser-289-beta and Glu-290-beta', and between Arg-309-beta and Glu-290-beta', which are essential for native alpha(2)beta(2) heterotetrameric assembly. The R133P-beta (CGG --> CCG) mutation, on the other hand, is inefficiently expressed in Escherichia coli as heterotetramers in a temperature-dependent manner. The R133P-beta mutant E1 exhibits significant residual activity but is markedly less stable than the wild-type, as measured by thermal inactivation and free energy change of denaturation. The R133P-beta substitution abrogates the coordination of Arg-133-beta to Ala-95-beta, Glu-96-beta, and Ile-97-beta, which is important for strand-strand interactions and K(+) ion binding in the beta subunit. These findings provide new insights into folding and assembly of human E1 and will facilitate DNA-based diagnosis for MSUD in the Israeli population.     

Roles of active site and novel K+ ion-binding site residues in human mitochondrial branched-chain alpha-ketoacid decarboxylase/dehydrogenase

The human mitochondrial branched-chain alpha-ketoacid decarboxylase/dehydrogenase (BCKD) is a heterotetrameric (alpha(2)beta(2)) thiamine diphosphate (TDP)-dependent enzyme. The recently solved human BCKD structure at 2.7 A showed that the two TDP-binding pockets are located at the interfaces between alpha and beta' subunits and between alpha' and beta subunits. In the present study, we show that the E76A-beta' mutation results in complete inactivation of BCKD. The result supports the catalytic role of the invariant Glu-76-beta' residue in increasing basicity of the N-4' amino group during the proton abstraction from the C-2 atom on the thiazolium ring. A substitution of His-146-beta' with Ala also renders the enzyme completely inactive. The data are consistent with binding of the alpha-ketoacid substrate by this residue based on the Pseudomonas BCKD structure. Alterations in Asn-222-alpha, Tyr-224-alpha, or Glu-193-alpha, which coordinates to the Mg(2+) ion, result in an inactive enzyme (E193A-alpha) or a mutant BCKD with markedly higher K(m) for TDP and a reduced level of the bound cofactor (Y224A-alpha and N222S-alpha). Arg-114-alpha, Arg-220-alpha, and His-291-alpha interact with TDP by directly binding to phosphate oxygens of the cofactor. We show that natural mutations of these residues in maple syrup urine disease (MSUD) patients (R114W-alpha and R220W-alpha) or site-directed mutagenesis (H291A-alpha) also result in an inactive or partially active enzyme, respectively. Another MSUD mutation (T166M-alpha), which affects one of the residues that coordinate to the K(+) ion on the alpha subunit, also causes inactivation of the enzyme and an attenuated ability to bind TDP. In addition, fluorescence measurements establish that Trp-136-beta in human BCKD is the residue quenched by TDP binding. Thus, our results define the functional roles of key amino acid residues in human BCKD and provide a structural basis for MSUD.     

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.     

A 17-bp insertion and a Phe215----Cys missense mutation in the dihydrolipoyl transacylase (E2) mRNA from a thiamine-responsive maple syrup urine disease patient WG-34.

We have amplified the cDNA for the transacylase (E2) subunit of the branched-chain alpha-ketoacid dehydrogenase (BCKAD) complex from a thiamine-responsive MSUD cell line (WG-34) by the polymerase chain reaction. Sequencing of the amplified WG-34 cDNA showed a 17-bp insertion (AAATACCTTGTTACCAG) apparently resulting from an aberrant splicing of the E2 gene, and a missense (T----G) mutation that changes Phe215 to Cys in the E2 subunit. The existence of these two mutations was confirmed by probing the amplified E2 cDNA or genomic DNA with allele-specific oligonucleotides. The above results support the thesis that the thiamine-responsive MSUD patient (WG-34) is a compound heterozygote at the E2 locus. The implication of the E2 mutations for the thiamine-responsiveness observed in this patient is discussed.     

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.     

Maple syrup urine disease: identification and carrier-frequency determination of a novel founder mutation in the Ashkenazi Jewish population

Maple syrup urine disease (MSUD) is a rare, autosomal recessive disorder of branched-chain amino acid metabolism. We noted that a large proportion (10 of 34) of families with MSUD that were followed in our clinic were of Ashkenazi Jewish (AJ) descent, leading us to search for a common mutation within this group. On the basis of genotyping data suggestive of a conserved haplotype at tightly linked markers on chromosome 6q14, the BCKDHB gene encoding the E1beta subunit was sequenced. Three novel mutations were identified in seven unrelated AJ patients with MSUD. The locations of the affected residues in the crystal structure of the E1beta subunit suggested possible mechanisms for the deleterious effects of these mutations. Large-scale population screening of AJ individuals for R183P, the mutation present in six of seven patients, revealed that the carrier frequency of the mutant allele was approximately 1/113; the patient not carrying R183P had a previously described homozygous mutation in the gene encoding the E2 subunit. These findings suggested that a limited number of mutations might underlie MSUD in the AJ population, potentially facilitating prenatal diagnosis and carrier detection of MSUD in this group.     

Occurrence of a 2-bp (AT) deletion allele and a nonsense (G-to-T) mutant allele at the E2 (DBT) locus of six patients with maple syrup urine disease: multiple-exon skipping as a secondary effect of the mutations.

We have identified two novel mutant alleles in the transacylase (E2) gene of the human branched-chain alpha-keto acid dehydrogenase (BCKAD) complex in 6 of 38 patients with maple syrup urine disease (MSUD). One mutation, a 2-bp (AT) deletion in exon 2 of the E2 gene, causes a frameshift downstream of residue (-26) in the mitochondrial targeting presequence. The second mutation, a G-to-T transversion in exon 6 of the E2 gene, produces a premature stop codon at Glu-163 (E163*). Transfection of constructs harboring the E163* mutation into an E2-deficient MSUD cell line produced a truncated E2 subunit. However, this mutant E2 chain is unable to assemble into a 24-mer cubic structure and is degraded in the cell. The 2-bp (AT) deletion and the E163* mutant alleles occur in either the homozygous or compound-heterozygous state in the 6 of 38 unrelated MSUD patients studied. Moreover, an array of precise single- and multiple-exon deletions were observed in many amplified E2 mutant cDNAs. The latter results appear to represent secondary effects on RNA processing that are associated with the MSUD mutations at the E2 locus.     

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.     

Occurrence of a Tyr393----Asn (Y393N) mutation in the E1 alpha gene of the branched-chain alpha-keto acid dehydrogenase complex in maple syrup urine disease patients from a Mennonite population

Maple syrup urine disease (MSUD) is caused by a deficiency in the mitochondrial branched-chain alpha-keto acid dehydrogenase complex. The incidence of MSUD in the Philadelphia Mennonites is 1/176 births resulting from consanguinity. In this study, we amplified cDNAs for the decarboxylase E1 alpha subunit of the branched-chain alpha-keto acid dehydrogenase complex from a classical MSUD patient and from an obligatory heterozygote of a Mennonite family by the PCR. Sequencing of the amplified cDNAs disclosed at codon 393 of the mature E1 alpha polypeptide a base substitution changing a tyrosine (encoded by TAC) to an asparagine residue (encoded by AAC), which is designated Y393N. A segment of the E1 alpha gene containing the 5' portion of exon 9 was amplified. Probing of the amplified genomic DNA with allele-specific oligonucleotide probes showed that the mutation in the E1 alpha gene was homozygous in six Mennonites affected with classical MSUD and was present in heterozygous carriers. The identification of the MSUD mutation in the Philadelphia Mennonites will facilitate diagnosis and carrier detection for this population.     

Two novel mutations in the BCKDHB gene (R170H, Q346R) cause the classic form of maple syrup urine disease (MSUD)

Maple syrup urine disease (MSUD) is an autosomal recessive metabolic disorder that is caused by mutations in the subunits of the branched-chain α-ketoacid dehydrogenase (BCKD) complex. BCKD is a mitochondrial complex encoded by four nuclear genes (BCKDHA, BCKDHB, DBT, and DLD) and is involved in the metabolism of branched-chain amino acids (BCAAs). In this study, we investigated the DNA sequences of BCKDHA, BCKDHB and DBT genes for mutations in a Chinese newborn with the classic form of MSUD and predicted the associated conformational changes using molecular modeling. We identified two previously unreported mutations in the BCKDHB gene, R170H (c.509G>A) in exon 5 and Q346R (c.1037 A>G) in exon 9. In silico analysis of the two novel missense mutations revealed that the mutation R170H-β alters the spatial orientation with both Y195-β' and S206-α, which results in unstable β-β' assembly and an unstable K(+) ion binding loop of the α subunit, respectively; The Q346R mutation is predicted to disrupt the spatial conformation between Q346-β and I357-β', which reduces the affinity of the β-β' subunits. These results indicate that R170-β and Q346-β are crucial for the activity of the E1 component. These two novel mutations, R170H and Q346R result in the patient's clinical manifestation of the classic form of MSUD.     

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.     

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.     

Conjugative mapping of pyruvate, 2-ketoglutarate, and branched-chain keto acid dehydrogenase genes in Pseudomonas putida mutants.

Branched-chain keto acid dehydrogenase, an enzyme in the common pathway of branched-chain amino acid catabolism of Pseudomonas putida, is a multienzyme complex which catalyzes the oxidative decarboxylation of branched-chain keto acids. The objective of the present study was to isolate strains with mutations of this and other keto acid dehydrogenases and to map the location of the mutations on the chromosome of P. putida. Several strains with mutations of branched-chain keto acid dehydrogenase, two pyruvate and two 2-ketoglutarate dehydrogenase, were isolated, and the defective subunits were identified by biochemical analysis. By using a recombinant XYL-K plasmid to mediate conjugation, these mutations were mapped in relation to a series of auxotrophic and other catabolic mutations. The last time of entry recorded was at approximately 35 min, and the data were consistent with a single point of entry. Branched-chain keto acid dehydrogenase mutations affecting E1, E1 plus E2, and E3 subunits mapped at approximately 35 min. One other strain affected in the common pathway was deficient in branched-chain amino acid transaminase, and the mutation was mapped at 16 min. The mutations in the two pyruvate dehydrogenase mutants, one deficient in E1 and the other deficient in E1 plus E2, mapped at 22 minutes. The 2-ketoglutarate dehydrogenase mutation affecting the E1 subunit mapped at 12 minutes. A 2-ketoglutarate dehydrogenase mutant deficient in E3 was isolated, but the mutation proved too leaky to map.     

Activities of branched-chain 2-oxo acid dehydrogenase and its components in skin fibroblasts from normal and classical-maple-syrup-urine-disease subjects.

1. Comparisons of the activity and kinetics of the branched-chain 2-oxo acid dehydrogenase in cultured skin fibroblasts from normal and classical maple-syrup-urine-disease (MSUD) subjects provide a kinetic explanation for the enzyme defect. 2. In the intact cell assays, normal fibroblasts demonstrated hyperbolic kinetics with 3-methyl-2-oxo[1-14C]butyrate as a substrate. Intact fibroblasts from four classical MSUD patients showed no decarboxylation over a substrate concentration range of 0.25 to 5.0 mM, and thiamin (4 mM) was without effect. 3. The overall reaction of the multienzyme complex was efficiently reconstituted by using a disrupted-cell system. Normals again showed typical hyperbolic kinetics at the 2-oxo acid concentrations of 0.1 to 5 mM. The Vmax. and apparent Km values were 0.10 +/- 0.02 m-unit/mg of protein and 0.05-0.1 mM respectively, with 3-methyl-2-oxobutyrate. In contrast, classical MSUD patients exhibited sigmoidal kinetics (Hill coefficient, 2.5) with activity approaching 40-60% of the normal value at 5 mM substrate. The K0.5 values from the Hill plots for MSUD patients were 4-7 mM. 4. The E1 (branched-chain 2-oxo acid decarboxylase) component of the multienzyme complex was measured in disrupted-particulate preparations. Normals again showed hyperbolic kinetics with the 2-oxo acid, whereas MSUD preparations exhibited sigmoidal kinetics with the activity of E1 strictly dependent on substrate concentration. Apparent Km or K0.5 were 0.1 and 1.0 mM for normal and MSUD subjects respectively. 5. Measurements of E2 (dihydrolipoyl transacylase) and E3 (dihydrolipoyl dehydrogenase) in MSUD preparations showed them to be in the normal range. 6. The above data suggest a defect in the E1 step of branched-chain 2-oxo acid dehydrogenase in classical MSUD patients.     

Molecular mechanism for regulation of the human mitochondrial branched-chain alpha-ketoacid dehydrogenase complex by phosphorylation

The human mitochondrial branched-chain alpha-ketoacid dehydrogenase complex (BCKDC) is a 4 MDa macromolecular machine comprising three catalytic components (E1b, E2b, and E3), a kinase, and a phosphatase. The BCKDC overall activity is tightly regulated by phosphorylation in response to hormonal and dietary stimuli. We report that phosphorylation of Ser292-alpha in the E1b active site channel results in an order-to-disorder transition of the conserved phosphorylation loop carrying the phosphoryl serine. The conformational change is triggered by steric clashes of the phosphoryl group with invariant His291-alpha that serves as an indispensable anchor for the phosphorylation loop through bound thiamin diphosphate. Phosphorylation of Ser292-alpha does not severely impede the E1b-dependent decarboxylation of alpha-ketoacids. However, the disordered loop conformation prevents phosphorylated E1b from binding the E2b lipoyl-bearing domain, which effectively shuts off the E1b-catalyzed reductive acylation reaction and therefore completely inactivates BCKDC. This mechanism provides a paradigm for regulation of mitochondrial alpha-ketoacid dehydrogenase complexes by phosphorylation.     

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.     

Roles of His291-alpha and His146-beta' in the reductive acylation reaction catalyzed by human branched-chain alpha-ketoacid dehydrogenase: refined phosphorylation loop structure in the active site

We report here that alterations of either His291-alpha or His146-beta' in the active site of human branched-chain alpha-ketoacid dehydrogenase (E1b) impede both the decarboxylation and the reductive acylation reactions catalyzed by E1b as well as the binding of cofactor thiamin diphosphate (ThDP). In a refined human E1b active-site structure, His291-alpha, which aligns with His407 in Escherichia coli pyruvate dehydrogenase and His263 in yeast transketolase, is on a largely ordered phosphorylation loop. The imidazole ring of His291-alpha in E1b coordinates to the terminal phosphate oxygen atoms of bound ThDP. The N3 atom of wild-type His146-beta', which can be protonated, binds a water molecule and points toward the aminopyrimidine ring of ThDP. Remarkably, the H291A-alpha mutation results in a complete order-to-disorder transition of the loop region, which precludes the binding of the substrate lipoyl-bearing domain to E1b. The H146A-beta' mutation, on the other hand, does not alter the loop structure, but nullifies the reductive acylation activity of E1b. Our results suggest that: 1) His291-alpha plays a structural rather than a catalytic role in the binding of cofactor ThDP and the lipoyl-bearing domain to E1b, and 2) His146-beta' is an essential catalytic residue, probably functioning as a proton donor in the reductive acylation of lipoamide on the lipoyl-bearing domain.