G2Cdb::Gene report

Gene id
Gene symbol
Homo sapiens
dihydrolipoamide branched chain transacylase E2
G00000456 (Mus musculus)

Databases (8)

Curated Gene
OTTHUMG00000010921 (Vega human gene)
ENSG00000137992 (Ensembl human gene)
1629 (Entrez Gene)
811 (G2Cdb plasticity & disease)
DBT (GeneCards)
248610 (OMIM)
Marker Symbol
HGNC:2698 (HGNC)
Protein Sequence
P11182 (UniProt)

Literature (35)

Pubmed - other

  • Defining the human deubiquitinating enzyme interaction landscape.

    Sowa ME, Bennett EJ, Gygi SP and Harper JW

    Department of Pathology, Harvard Medical School, Boston, MA 02115, USA.

    Deubiquitinating enzymes (Dubs) function to remove covalently attached ubiquitin from proteins, thereby controlling substrate activity and/or abundance. For most Dubs, their functions, targets, and regulation are poorly understood. To systematically investigate Dub function, we initiated a global proteomic analysis of Dubs and their associated protein complexes. This was accomplished through the development of a software platform called CompPASS, which uses unbiased metrics to assign confidence measurements to interactions from parallel nonreciprocal proteomic data sets. We identified 774 candidate interacting proteins associated with 75 Dubs. Using Gene Ontology, interactome topology classification, subcellular localization, and functional studies, we link Dubs to diverse processes, including protein turnover, transcription, RNA processing, DNA damage, and endoplasmic reticulum-associated degradation. This work provides the first glimpse into the Dub interaction landscape, places previously unstudied Dubs within putative biological pathways, and identifies previously unknown interactions and protein complexes involved in this increasingly important arm of the ubiquitin-proteasome pathway.

    Funded by: NIA NIH HHS: AG085011, R01 AG011085, R01 AG011085-16; NIGMS NIH HHS: GM054137, GM67945, R01 GM054137, R01 GM054137-14, R01 GM067945

    Cell 2009;138;2;389-403

  • Molecular genetics of maple syrup urine disease in the Turkish population.

    Gorzelany K, Dursun A, Coşkun T, Kalkanoğlu-Sivri SH, Gökçay GF, Demirkol M, Feyen O and Wendel U

    Department of General Pediatrics, Heinrich-Heine University, Düsseldorf, Germany.

    In maple syrup urine disease (MSUD), disease-causing mutations can affect the BCKDHA, BCKDHB or DBT genes encoding for the E1alpha, E1beta and E2 subunits of the multienzyme branched-chain alpha-keto acid dehydrogenase (BCKDH) complex. Here we summarize the MSUD genotypes of a cohort of 32 unrelated Turkish patients in whom both alleles at a single gene locus harbored presumable disease-causing nucleotide changes. The patients had different forms of MSUD, ranging from the severe classical form (26 patients) to severe and mild variants (6 patients). In all except two patients (92%), the mutations occurred homozygously. The mutational spectrum included 27 different sequence variations--12 changes in the BCKDHA, 10 in the BCKDHB, and 5 in the DBT genes. In 37% (12 patients) of a total of 64 alleles, the supposed disease-causing mutations were located in the BCKDHA gene, in 44% (14 patients) in the BCKDHB gene and in 19% (6 patients) in the DBT gene. The mutational profile is heterogeneous, although two mutations occurred three times and five mutations occurred twice. There was no cluster for a single mutation except for c.773G>A (p.Cys258Tyr) in the BCKDHA gene, a hypothetical founder mutation in the Camlidere population.

    The Turkish journal of pediatrics 2009;51;2;97-102

  • Early diagnosis of maple syrup urine disease using polymerase chain reaction-based mutation detection.

    Silao CL, Padilla CD and Matsuo M

    Institute of Human Genetics, National Institutes of Health Philippines, Manila, Philippines.

    Background: Maple syrup urine disease (MSUD) is an autosomal recessive disorder caused by defective activity of the branched-chain alpha-ketoacid dehydrogenase enzyme complex. Early diagnosis and management of MSUD are imperative for preventing permanent neurological impairments. In the Philippines, a 4.7 kb deletion in the dihydrolipoamide branched-chain transacylase E2 (DBT) gene has been commonly identified in MSUD patients. Polymerase chain reaction (PCR) amplification of a junction fragment between intron 10 and exon 11 has been used to detect this deletion. The purpose of the present paper was to use PCR-based mutation detection of the deletion mutation to diagnose MSUD in neonates in order to provide proper diagnosis and effective treatment.

    Methods: A region encompassing exon 11 and the junction fragment of the E2 (DBT) gene was PCR amplified from genomic DNA prepared from two neonates at risk for MSUD.

    Results: PCR amplification of both exon 11 and the junction fragment from one of the neonates demonstrated that this case was a heterozygous carrier of the deletion. Thus, normal feeding was started. For the other neonate, PCR amplification of the junction fragment was successful, whereas the region encompassing exon 11 was not amplified. This neonate was genotyped as homozygous for the deletion, and treatment for MSUD was provided immediately.

    Conclusion: Examination of the deletion mutation in the E2 (DBT) gene facilitated early MSUD diagnosis and was beneficial for the determination of the proper course of treatment.

    Pediatrics international : official journal of the Japan Pediatric Society 2008;50;3;312-4

  • Molecular and structural analyses of maple syrup urine disease and identification of a founder mutation in a Portuguese Gypsy community.

    Quental S, Macedo-Ribeiro S, Matos R, Vilarinho L, Martins E, Teles EL, Rodrigues E, Diogo L, Garcia P, Eusébio F, Gaspar A, Sequeira S, Furtado F, Lança I, Amorim A and Prata MJ

    IPATIMUP-Institute of Pathology and Molecular Immunology of University of Porto, Rua Dr. Roberto Frias, s/n, 4200-465 Porto, Portugal. mquental@ipatimup.pt

    Maple syrup urine disease (MSUD) is an autosomal recessive disorder, caused by the defective function of the branched-chain alpha-ketoacid dehydrogenase complex (BCKD). BCKD is a mitochondrial complex, encoded by four nuclear genes (BCKDHA, BCKDHB, DBT and DLD), involved in the metabolism of branched-chain amino acids (BCAAs). Since the MSUD mutational spectrum has not been previously assessed in Portugal, in this study we present the molecular characterization of 30 MSUD Portuguese patients. Seventeen putative mutations have been identified (six in BCKDHA, five in BCKDHB and six in DBT); seven of them are here described for the first time. The most common mutation identified was a C deletion in BCKDHA gene (c.117delC; p.R40GfsX23), already reported in the Spanish population. Interestingly, it was found in all patients of a Gypsy community from South of the country, so a founder effect is probably responsible for the high incidence of the disease in this community. Structural models of MSUD missense mutations have been performed to understand their pathogenic effect, in order to elucidate and often to predict the severity of a mutation clinical consequence.

    Molecular genetics and metabolism 2008;94;2;148-56

  • The layered structure of human mitochondrial DNA nucleoids.

    Bogenhagen DF, Rousseau D and Burke S

    Department of Pharmacological Sciences, State University of New York at Stony Brook, Stony Brook, New York 11794-8651, USA. dan@pharm.sunysb.edu

    Mitochondrial DNA (mtDNA) occurs in cells in nucleoids containing several copies of the genome. Previous studies have identified proteins associated with these large DNA structures when they are biochemically purified by sedimentation and immunoaffinity chromatography. In this study, formaldehyde cross-linking was performed to determine which nucleoid proteins are in close contact with the mtDNA. A set of core nucleoid proteins is found in both native and cross-linked nucleoids, including 13 proteins with known roles in mtDNA transactions. Several other metabolic proteins and chaperones identified in native nucleoids, including ATAD3, were not observed to cross-link to mtDNA. Additional immunofluorescence and protease susceptibility studies showed that an N-terminal domain of ATAD3 previously proposed to bind to the mtDNA D-loop is directed away from the mitochondrial matrix, so it is unlikely to interact with mtDNA in vivo. These results are discussed in relation to a model for a layered structure of mtDNA nucleoids in which replication and transcription occur in the central core, whereas translation and complex assembly may occur in the peripheral region.

    Funded by: NIEHS NIH HHS: R01-ES12039

    The Journal of biological chemistry 2008;283;6;3665-75

  • Description of the mutations in 15 subjects with variant forms of maple syrup urine disease.

    Flaschker N, Feyen O, Fend S, Simon E, Schadewaldt P and Wendel U

    Department of General Paediatrics, University Children's Hospital, Heinrich-Heine-University, Düsseldorf, Germany.

    Background: In maple syrup urine disease (MSUD), disease-causing mutations can affect the BCKDHA, BCKDHB or DBT genes encoding for the E1 alpha, E1 beta and E2 subunits of the multienzyme branched-chain 2-keto acid dehydrogenase (BCKD) complex.

    Aim: The aim of this study was to screen DNA samples of 15 subjects with distinct well-characterized variant MSUD phenotypes for mutations in the three genes in order to demonstrate a potential correlation between specific nucleotide changes and particular variant phenotypes.

    Methods: The exonic coding sequences of all three genes were studied using genomic DNA and cellular RNA derived from peripheral blood leukocytes.

    Results: In 37% of the cases (total 30 alleles), disease-causing mutations were located in the BCKDHA, in 46% in the BCKDHB, and in 13% in the DBT gene. Novel mutations occurring homozygously were p.Ala328Thr in the BCKDHA gene and p.Gly249_Lys257del in the DBT gene. Both are associated with a mild MSUD variant. The same holds true for the novel mutations p.Pro200Ala in BCKDHB and p.Phe307Ser in DBT which were identified in heterozygous fashion. Among the known mutant alleles, p.Gly278Ser in the BCKDHB gene was relatively frequent and also associated with a mild MSUD variant.

    Conclusion: The results of this study indicate that genotyping may be predictive of clinical severity of variant MSUD phenotypes and might be of prognostic value particularly in subjects with variant MSUD identified in newborn screening in whom early treatment fortunately slows the natural course of the disease.

    Journal of inherited metabolic disease 2007;30;6;903-9

  • Large-scale mapping of human protein-protein interactions by mass spectrometry.

    Ewing RM, Chu P, Elisma F, Li H, Taylor P, Climie S, McBroom-Cerajewski L, Robinson MD, O'Connor L, Li M, Taylor R, Dharsee M, Ho Y, Heilbut A, Moore L, Zhang S, Ornatsky O, Bukhman YV, Ethier M, Sheng Y, Vasilescu J, Abu-Farha M, Lambert JP, Duewel HS, Stewart II, Kuehl B, Hogue K, Colwill K, Gladwish K, Muskat B, Kinach R, Adams SL, Moran MF, Morin GB, Topaloglou T and Figeys D

    Protana, Toronto, Ontario, Canada.

    Mapping protein-protein interactions is an invaluable tool for understanding protein function. Here, we report the first large-scale study of protein-protein interactions in human cells using a mass spectrometry-based approach. The study maps protein interactions for 338 bait proteins that were selected based on known or suspected disease and functional associations. Large-scale immunoprecipitation of Flag-tagged versions of these proteins followed by LC-ESI-MS/MS analysis resulted in the identification of 24,540 potential protein interactions. False positives and redundant hits were filtered out using empirical criteria and a calculated interaction confidence score, producing a data set of 6463 interactions between 2235 distinct proteins. This data set was further cross-validated using previously published and predicted human protein interactions. In-depth mining of the data set shows that it represents a valuable source of novel protein-protein interactions with relevance to human diseases. In addition, via our preliminary analysis, we report many novel protein interactions and pathway associations.

    Molecular systems biology 2007;3;89

  • Structure of the subunit binding domain and dynamics of the di-domain region from the core of human branched chain alpha-ketoacid dehydrogenase complex.

    Chang CF, Chou HT, Lin YJ, Lee SJ, Chuang JL, Chuang DT and Huang TH

    Genomics Research Center and Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 115, Republic of China.

    The homo-24-meric dihydrolipoyl transacylase (E2) scaffold of the human branched-chain alpha-ketoacid dehydrogenase complex (BCKDC) contains the lipoyl-bearing domain (hbLBD), the subunit-binding domain (hbSBD) and the inner core domain that are linked to carry out E2 functions in substrate channeling and recognition. In this study, we employed NMR techniques to determine the structure of hbSBD and dynamics of several truncated constructs from the E2 component of the human BCKDC, including hbLBD (residues 1-84), hbSBD (residues 111-149), and a di-domain (hbDD) (residues 1-166) comprising hbLBD, hbSBD and the interdomain linker. The solution structure of hbSBD consists of two nearly parallel helices separated by a long loop, similar to the structures of the SBD isolated from other species, but it lacks the short 3(10) helix. The NMR results show that the structures of hbLBD and hbSBD in isolated forms are not altered by the presence of the interdomain linker in hbDD. The linker region is not entirely exposed to solvent, where amide resonances associated with approximately 50% of the residues are observable. However, the tethering of these two domains in hbDD significantly retards the overall rotational correlation times of hbLBD and hbSBD, changing from 5.54 ns and 5.73 ns in isolated forms to 8.37 ns and 8.85 ns in the linked hbDD, respectively. We conclude that the presence of the interdomain linker restricts the motional freedom of the hbSBD more significantly than hbLBD, and that the linker region likely exists as a soft rod rather than a flexible string in solution.

    Funded by: NIDDK NIH HHS: DK-26578

    The Journal of biological chemistry 2006;281;38;28345-53

  • Diversification of transcriptional modulation: large-scale identification and characterization of putative alternative promoters of human genes.

    Kimura K, Wakamatsu A, Suzuki Y, Ota T, Nishikawa T, Yamashita R, Yamamoto J, Sekine M, Tsuritani K, Wakaguri H, Ishii S, Sugiyama T, Saito K, Isono Y, Irie R, Kushida N, Yoneyama T, Otsuka R, Kanda K, Yokoi T, Kondo H, Wagatsuma M, Murakawa K, Ishida S, Ishibashi T, Takahashi-Fujii A, Tanase T, Nagai K, Kikuchi H, Nakai K, Isogai T and Sugano S

    Life Science Research Laboratory, Central Research Laboratory, Hitachi, Ltd., Kokubunji, Tokyo, 185-8601, Japan.

    By analyzing 1,780,295 5'-end sequences of human full-length cDNAs derived from 164 kinds of oligo-cap cDNA libraries, we identified 269,774 independent positions of transcriptional start sites (TSSs) for 14,628 human RefSeq genes. These TSSs were clustered into 30,964 clusters that were separated from each other by more than 500 bp and thus are very likely to constitute mutually distinct alternative promoters. To our surprise, at least 7674 (52%) human RefSeq genes were subject to regulation by putative alternative promoters (PAPs). On average, there were 3.1 PAPs per gene, with the composition of one CpG-island-containing promoter per 2.6 CpG-less promoters. In 17% of the PAP-containing loci, tissue-specific use of the PAPs was observed. The richest tissue sources of the tissue-specific PAPs were testis and brain. It was also intriguing that the PAP-containing promoters were enriched in the genes encoding signal transduction-related proteins and were rarer in the genes encoding extracellular proteins, possibly reflecting the varied functional requirement for and the restricted expression of those categories of genes, respectively. The patterns of the first exons were highly diverse as well. On average, there were 7.7 different splicing types of first exons per locus partly produced by the PAPs, suggesting that a wide variety of transcripts can be achieved by this mechanism. Our findings suggest that use of alternate promoters and consequent alternative use of first exons should play a pivotal role in generating the complexity required for the highly elaborated molecular systems in humans.

    Genome research 2006;16;1;55-65

  • The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC).

    Gerhard DS, Wagner L, Feingold EA, Shenmen CM, Grouse LH, Schuler G, Klein SL, Old S, Rasooly R, Good P, Guyer M, Peck AM, Derge JG, Lipman D, Collins FS, Jang W, Sherry S, Feolo M, Misquitta L, Lee E, Rotmistrovsky K, Greenhut SF, Schaefer CF, Buetow K, Bonner TI, Haussler D, Kent J, Kiekhaus M, Furey T, Brent M, Prange C, Schreiber K, Shapiro N, Bhat NK, Hopkins RF, Hsie F, Driscoll T, Soares MB, Casavant TL, Scheetz TE, Brown-stein MJ, Usdin TB, Toshiyuki S, Carninci P, Piao Y, Dudekula DB, Ko MS, Kawakami K, Suzuki Y, Sugano S, Gruber CE, Smith MR, Simmons B, Moore T, Waterman R, Johnson SL, Ruan Y, Wei CL, Mathavan S, Gunaratne PH, Wu J, Garcia AM, Hulyk SW, Fuh E, Yuan Y, Sneed A, Kowis C, Hodgson A, Muzny DM, McPherson J, Gibbs RA, Fahey J, Helton E, Ketteman M, Madan A, Rodrigues S, Sanchez A, Whiting M, Madari A, Young AC, Wetherby KD, Granite SJ, Kwong PN, Brinkley CP, Pearson RL, Bouffard GG, Blakesly RW, Green ED, Dickson MC, Rodriguez AC, Grimwood J, Schmutz J, Myers RM, Butterfield YS, Griffith M, Griffith OL, Krzywinski MI, Liao N, Morin R, Morrin R, Palmquist D, Petrescu AS, Skalska U, Smailus DE, Stott JM, Schnerch A, Schein JE, Jones SJ, Holt RA, Baross A, Marra MA, Clifton S, Makowski KA, Bosak S, Malek J and MGC Project Team

    The National Institutes of Health's Mammalian Gene Collection (MGC) project was designed to generate and sequence a publicly accessible cDNA resource containing a complete open reading frame (ORF) for every human and mouse gene. The project initially used a random strategy to select clones from a large number of cDNA libraries from diverse tissues. Candidate clones were chosen based on 5'-EST sequences, and then fully sequenced to high accuracy and analyzed by algorithms developed for this project. Currently, more than 11,000 human and 10,000 mouse genes are represented in MGC by at least one clone with a full ORF. The random selection approach is now reaching a saturation point, and a transition to protocols targeted at the missing transcripts is now required to complete the mouse and human collections. Comparison of the sequence of the MGC clones to reference genome sequences reveals that most cDNA clones are of very high sequence quality, although it is likely that some cDNAs may carry missense variants as a consequence of experimental artifact, such as PCR, cloning, or reverse transcriptase errors. Recently, a rat cDNA component was added to the project, and ongoing frog (Xenopus) and zebrafish (Danio) cDNA projects were expanded to take advantage of the high-throughput MGC pipeline.

    Funded by: PHS HHS: N01-C0-12400

    Genome research 2004;14;10B;2121-7

  • Characterization of a lipoyl domain-independent B-cell autoepitope on the human branched-chain acyltransferase in primary biliary cirrhosis and overlap syndrome with autoimmune hepatitis.

    Csepregi A, Obermayer-Straub P, Kneip S, Kayser A, Loges S, Schmidt E, Nemesánszky E, Szalay F, Manns MP and Strassburg CP

    Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Carl-Neuberg-Street 1, 30625 Hannover, Germany.

    Antimitochondrial antibodies (AMA) which recognize pyruvate acetyltransferase (PDC-E2) represent a highly diagnostic feature of primary biliary cirrhosis (PBC). The analysis of immunofluorescence (IF)-AMA-positive sera in PBC patients indicates a conformational epitope located within the lipoyl binding domain of bovine branched-chain acyltransferase (BCKADC-E2) alone or in combination with AMA directed against PDC-E2 the significance of which is presently unclear. In the present study, immunoreactivities and disease associations of AMA against BCKADC-E2 were analyzed. B-cell autoepitopes on BCKADC-E2 were mapped by immunoprecipitation assay.

    Methods: Sera of 96 IF-AMA-positive patients with serological evidence of anti-BCKADC-E2 alone (n = 26), anti-PDC-E2 alone (n = 15), and both anti-BCKADC-E2 and anti-PDC-E2 (n = 55) were analyzed by Western blot and ELISA in addition to an analysis of B cell autoepitopes on BCKADC-E2 by immunoprecipitation using in vitro translated, unmodified human proteins. Ninety-four patients without IF-AMA [blood donors (n = 30), rheumatoid arthritis (n = 40), autoimmune hepatitis (AIH)(n = 10) and primary sclerosing cholangitis (PSC) (n = 14) served as controls.

    Results: Eighty of 81 (99%) sera positive for BCKADC-E2 recognized the full length, mature protein, while only 2/10 AIH sera and none of the other controls showed reactivity. Of the 68 PBC sera 58 (85%) recognized the N-terminus consisting of aa 1-144 representing the lipoyl domain. Surprisingly, C-terminal sequences (aa 143-421) were recognized by 46 out of 68 sera (68%). Three PBC sera reacted with the C-terminus only. Only 1/7 serum from patients with an "overlap syndrome of PBC and AIH" was reactive with C-terminal sequences.

    Conclusions: Our analysis of BCKADC-E2-positive PBC sera identified a novel B cell epitope on the C-terminal part of the human protein. Our data indicate that a distinct subset of AMA recognize sequence(s) on BCKADC-E2 which located outside of the lipoyl binding domain. The absence of immunoreactivity against C-terminal sequences may serve as a marker differentiating patients with PBC and overlap syndrome of PBC with AIH.

    Clinical & developmental immunology 2003;10;2-4;173-81

  • Solution structure and dynamics of the lipoic acid-bearing domain of human mitochondrial branched-chain alpha-keto acid dehydrogenase complex.

    Chang CF, Chou HT, Chuang JL, Chuang DT and Huang TH

    Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan 11529, Republic of China.

    The lipoyl-bearing domain (LBD) of the transacylase (E2) subunit of the branched-chain alpha-keto acid dehydrogenase complex plays a central role in substrate channeling in this mitochondrial multienzyme complex. We have employed multidimensional heteronuclear NMR techniques to determine the structure and dynamics of the LBD of the human branched-chain alpha-keto acid dehydrogenase complex (hbLBD). Similar to LBD from other members of the alpha-keto acid dehydrogenase family, the solution structure of hbLBD is a flattened beta-barrel formed by two four-stranded antiparallel beta-sheets. The lipoyl Lys(44) residue resides at the tip of a beta-hairpin comprising a sharp type I beta-turn and the two connecting beta-strands 4 and 5. A prominent V-shaped groove formed by a surface loop, L1, connecting beta 1- and beta 2-strands and the lipoyl lysine beta-hairpin constitutes the functional pocket. We further applied reduced spectral density functions formalism to extract dynamic information of hbLBD from (15)N-T(1), (15)N-T(2), and ((1)H-(15)N) nuclear Overhauser effect data obtained at 600 MHz. The results showed that residues surrounding the lipoyl lysine region comprising the L1 loop and the Lys(44) beta-turn are highly flexible, whereas beta-sheet S1 appears to display a slow conformational exchange process.

    Funded by: NIDDK NIH HHS: DK-26758

    The Journal of biological chemistry 2002;277;18;15865-73

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

    Edelmann L, Wasserstein MP, Kornreich R, Sansaricq C, Snyderman SE and Diaz GA

    Department of Human Genetics, Mount Sinai School of Medicine, New York, NY 10029, USA.

    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.

    Funded by: NICHD NIH HHS: 5 P30 HD 28822

    American journal of human genetics 2001;69;4;863-8

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

    Wynn RM, Chuang JL, Sansaricq C, Mandel H and Chuang DT

    Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

    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.

    Funded by: NIDDK NIH HHS: DK26758

    The Journal of biological chemistry 2001;276;39;36550-6

  • Molecular basis of intermittent maple syrup urine disease: novel mutations in the E2 gene of the branched-chain alpha-keto acid dehydrogenase complex.

    Tsuruta M, Mitsubuchi H, Mardy S, Miura Y, Hayashida Y, Kinugasa A, Ishitsu T, Matsuda I and Indo Y

    Department of Pediatrics, Kumamoto University School of Medicine, Japan.

    The E2 gene of the branched-chain alpha-keto acid dehydrogenase (BCKDH) complex was studied at the molecular level in three patients with intermittent maple syrup urine disease (MSUD). All three patients had higher BCKDH activity than did those with the classical phenotype. In the first patient, a single base substitution from A to G in intron 8 created a new 5' splice site and caused an insertion of 126 nucleotides between exons 8 and 9 by activating an upstream cryptic 3' splice site in the same intron. The predicted mRNA encoded a truncated protein with 282 amino acids including 4 novel ones at the carboxyl terminus, compared with the normal protein with 421 amino acids. In vitro, the region from the patient but not from a normal control was recognized and was recovered as a novel exon, indicating that the single substitution was responsible for incorporation of the region into mRNA. This mutation probably supports an exon definition model in which the spliceosome recognizes a 3' splice site and then scans downstream for an acceptable 5' splice site, thereby defining an exon. The second patient was homozygous for a G to T transversion at nucleotide 1463 in exon 11, which predicted a substitution of the termination codon by a leucine residue and the addition of 7 extra amino acids at the carboxyl terminus. For each mutation, these two patients were homozygous and their parents were heterozygous. The third patient was a compound heterozygote for a C to G transversion at nucleotide 309 in exon 4 and a G to A transition at nucleotide 1165 in exon 9, causing an Ile-to-Met substitution at amino acid 37 and a Gly-to-Ser substitution at amino acid 323, respectively. Taken together, these results indicate that the molecular basis of intermittent phenotype MSUD in some patients can be due to mutations in the E2 gene, giving rise to a low but significant residual activity of the BCKDH complex.

    Journal of human genetics 1998;43;2;91-100

  • Mammalian alpha-keto acid dehydrogenase complexes: gene regulation and genetic defects.

    Patel MS and Harris RA

    Department of Biochemistry, School of Medicine and Biomedical Sciences, State University of New York at Buffalo 14214, USA.

    Recent application of the tools of molecular biology has provided insight with respect to the structure, regulation, and defects of the genes encoding the PDC and the BCKADC. cDNAs for the alpha-KGDC have only recently been cloned. Evidence for long-term regulation of the expression of the subunits of the PDC and the BCKADC has been obtained. Detailed analyses of the promoter-regulatory regions of the genes encoding the individual subunits of these complexes have provided leads as to molecular mechanisms involved. Evidence for coordinated regulation of expression of the subunits of the complexes has been obtained under some conditions but not others. Except for the PDC E1 alpha (X) promoter, the other promoters of the genes encoding PDC and BCKADC components are TATA-less with characteristics of housekeeping genes. Most cases of PDC deficiency, which produces lactic acidosis, are caused by defects in the coding region of the E1 alpha subunit. In contrast, gene defects that cause maple syrup urine disease are more heterogeneous, involving errors in any one of the subunits of the BCKADC. Alpha-KGDC deficiency has been reported but specific mutations have not been identified. Future work is expected to include detailed studies of the genes encoding the protein kinases and phosphatases that specifically regulate the PDC and the BCKADC by covalent modification.

    Funded by: NIDDK NIH HHS: DK19259, DK20478, DK42885; ...

    FASEB journal : official publication of the Federation of American Societies for Experimental Biology 1995;9;12;1164-72

  • Differential processing of human and rat E1 alpha precursors of the branched-chain alpha-keto acid dehydrogenase complex caused by an N-terminal proline in the rat sequence.

    Wynn RM, Kochi H, Cox RP and Chuang DT

    Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235.

    The N-terminal sequences of the E1 alpha, E1 beta and E2 subunits of the human branched-chain alpha-keto acid dehydrogenase complex have been determined by microsequencing. The N-terminal of human E1 beta and E2 subunits (Val and Gly, respectively) are identical to those of the corresponding rat and bovine subunits. However, the N-terminus of the human E1 alpha subunit (Ser) is identical to bovine, but differs from the rat E1 alpha (Phe) subunit. Comparison of the N-terminal sequences of human and rat E1 alpha subunits shows that the serine residue at the +1 position in the human sequence is replaced by a proline residue in the rat sequence. The presence of the proline residue apparently causes a 5'-shift by one residue in the cleavage site by the mitochondrial processing peptidase in the rat sequence, when compared to the human sequence. The results provide evidence that the mitochondrial processing peptidase cannot cleave an X-Pro bond, similar to trypsin, chymotrypsin and microsomal signal peptidases.

    Funded by: NIDDK NIH HHS: DK-26758, DK-37373

    Biochimica et biophysica acta 1994;1201;1;125-8

  • 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.

    Fisher CW, Fisher CR, Chuang JL, Lau KS, Chuang DT and Cox RP

    Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235-8889.

    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.

    Funded by: NIDDK NIH HHS: DK-26758

    American journal of human genetics 1993;52;2;414-24

  • Structure of the gene encoding dihydrolipoyl transacylase (E2) component of human branched chain alpha-keto acid dehydrogenase complex and characterization of an E2 pseudogene.

    Lau KS, Herring WJ, Chuang JL, McKean M, Danner DJ, Cox RP and Chuang DT

    Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235.

    We have determined the structural organization of the dihydrolipoyl transacylase (E2) gene of the human branched chain alpha-keto acid dehydrogenase complex. The single copy E2 gene spans approximately 68 kilobases of genomic DNA. The complete coding region consisting of the 5'- and 3'-untranslated regions, the mitochondrial targeting sequence (61 amino acids), and the mature E2 sequence (421 amino acids) are encoded by 11 exons ranging from 62 to 2239 base pairs. All the donor and acceptor splice sites conform to the gt-ag rule. Sequence analysis of the promoter-regulatory region showed the presence of a "CAAT box"-like sequence 537 bases upstream of the transcription initiation site. The "TATA box"-like sequence is absent. Also located in this region are sequences resembling glucocorticoid-responsive and cAMP-responsive elements, fat-specific elements, and Sp1- and AP-2-binding sites. Several sets of direct and inverted repeats are also present. Promoter assays using human hepatoma cells (Hep-G2) and Swiss mouse preadipocytes (3T3-L1) showed that a 4.1-kilobase PstI fragment upstream of the transcription start site confers high expression of the luciferase reporter gene. Moreover, an intronless E2 pseudogene was isolated. It corresponds to the complete mitochondrial presequence and the lipoyl-bearing domain that are encoded by exons I through IV of the functional E2 gene. However, the E2 pseudogene contains multiple base changes, deletions, and insertions, and is flanked by short direct repeats. The data indicate that the E2 pseudogene is a retroposon.

    Funded by: NIDDK NIH HHS: DK-26758, DK-37373

    The Journal of biological chemistry 1992;267;33;24090-6

  • The complete cDNA sequence for dihydrolipoyl transacylase (E2) of human branched-chain alpha-keto acid dehydrogenase complex.

    Lau KS, Chuang JL, Herring WJ, Danner DJ, Cox RP and Chuang DT

    Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235-9038.

    We have determined the complete nucleotide sequence for the cDNA encoding human dihydrolipoyl transacylase (E2) using the rapid amplification of cDNA ends (RACE) procedure. The full-length E2 cDNA is 3535 nucleotides in length. The coding region spans 1446 bp and the 3'-noncoding region spans 2074 bp. The latter contains three Alu repetitive sequences and two transcription termination sites.

    Funded by: NIDDK NIH HHS: DK26758, DK37373, DK38320

    Biochimica et biophysica acta 1992;1132;3;319-21

  • Branched-chain alpha-ketoacid dehydrogenase kinase. Molecular cloning, expression, and sequence similarity with histidine protein kinases.

    Popov KM, Zhao Y, Shimomura Y, Kuntz MJ and Harris RA

    Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis 46202-5122.

    A cDNA for branched-chain alpha-ketoacid dehydrogenase kinase was cloned from a rat heart cDNA library. The cDNA had an open reading frame encoding a protein of 382 amino acid residues with a calculated molecular weight of 43,280. The clone codes for the branched-chain alpha-ketoacid dehydrogenase kinase based on the following: 1) the deduced amino acid sequence contained the partial sequence of the kinase determined by direct sequencing; 2) expression of the cDNA in Escherichia coli resulted in synthesis of a 43,000-Da protein that was recognized specifically by kinase antibodies; and 3) enzyme activity that phosphorylated and inactivated the branched-chain alpha-ketoacid dehydrogenase complex was found in extracts of E. coli expressing the protein. Northern blot analysis indicated the mRNA for the branched-chain alpha-ketoacid dehydrogenase kinase was more abundant in rat heart than in rat liver, as expected from the relative amounts of kinase activity expressed in these tissues. The deduced sequence of the kinase aligned with a high degree of similarity within subdomains characteristic of procaryotic histidine protein kinases. This first mitochondrial protein kinase to be cloned appears more closely related in sequence to procaryotic histidine protein kinases than to eucaryotic serine/threonine protein kinases.

    Funded by: NIADDK NIH HHS: AM 20542; NIDDK NIH HHS: DK 19259

    The Journal of biological chemistry 1992;267;19;13127-30

  • Regional assignment of two genes of the human branched-chain alpha-keto acid dehydrogenase complex: the E1 beta gene (BCKDHB) to chromosome 6p21-22 and the E2 gene (DBT) to chromosome 1p31.

    Zneimer SM, Lau KS, Eddy RL, Shows TB, Chuang JL, Chuang DT and Cox RP

    Department of Pathology, University of Texas Southwestern Medical Center, Dallas 75235.

    Maple syrup urine disease (MSUD) is caused by the deficiency of the mitochondrial branched-chain alpha-keto acid dehydrogenase complex. The multienzyme complex is a macromolecule (Mr 4 X 10(6] consisting of at least six distinct subunits. In this study, the human E1 beta gene (BCKDHB) has been localized to human chromosome 6 by hybrid somatic cell analysis, and regionally assigned to chromosome bands 6p21-22 by in situ hybridization. The E2 gene (DBT), which was previously localized to chromosome 1, is regionally assigned to the chromosome band 1p31 also by in situ hybridization. Localization of the E1 beta gene to chromosome 6p21-22 assigns another major human disease locus to a region that contains several important genes, including the major histocompatability complex, tumor necrosis factor, and heat-shock protein HSP70. Mapping of the E1 beta and the E2 genes may provide information for the linkage analysis of MSUD families with mutations in these two loci.

    Funded by: NICHD NIH HHS: HD 05196; NIDDK NIH HHS: DK 26758; NIGMS NIH HHS: GM 20454

    Genomics 1991;10;3;740-7

  • Maple syrup urine disease caused by a partial deletion in the inner E2 core domain of the branched chain alpha-keto acid dehydrogenase complex due to aberrant splicing. A single base deletion at a 5'-splice donor site of an intron of the E2 gene disrupts the consensus sequence in this region.

    Mitsubuchi H, Nobukuni Y, Akaboshi I, Indo Y, Endo F and Matsuda I

    Department of Pediatrics, Kumamoto University Medical School, Japan.

    We have studied the molecular bases of maple syrup urine disease by analyzing the activity, subunit structure, mRNA sequence, and the genome of the affected enzyme. The branched chain alpha-keto acid dehydrogenase (BCKDH) activity in the patient was 4.2-4.5% of the control level. Immunoblot analysis revealed that the E2 subunit of BCKDH (Mr 52,000) was absent and another protein band with an Mr of 49,000 was present. We amplified the cDNA of the E2 subunit obtained from the patient's cell using the polymerase chain reaction method, then sequenced the amplified cDNA, in which a 78-bp deletion was identified. The consanguineous parents and a sister had two species of mRNA; the one corresponding to the normal E2 subunit and the other with a 78-bp deletion, whereas findings in a brother were normal. The molecular size of the translation products as deduced from the abnormal mRNA sequence was compatible with an abnormal protein band (Mr 49,000) detected in the patient's cells by immunoblot analysis. Analysis of genomic DNA of BCKDH-E2 subunit revealed that the 78-bp deletion in the mRNA was caused by an exon skipping due to a single base deletion in the 5'-splice donor site. As a result of the mutation, part of the inner E2 core domain was omitted. The specified region of the inner E2 core domain was highly homologous to the region of the E2 subunit of pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase. These observations imply the biological importance of the region in the inner E2 core domain of BCKDH to maintain normal function of the activity.

    The Journal of clinical investigation 1991;87;4;1207-11

  • Premature termination of transcription and alternative splicing in the human transacylase (E2) gene of the branched-chain alpha-ketoacid dehydrogenase complex.

    Lau KS, Lee J, Fisher CW, Cox RP and Chuang DT

    Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235-9038.

    We have isolated a human genomic clone hgE2-14 containing exons 5, 6, 7 and 8 of the branched-chain alpha-ketoacid dehydrogenase E2 transacylase gene. Sequencing of exon 8 and its surrounding intronic sequences reveals complete identity with the previously reported truncated E2 cDNA (hE2-1) sequence between nucleotides 938 and 1521. We have identified consensus splice site junctions flanking exon 8 and also a cryptic 3' splice site 370 bases upstream from the start of exon 8 in the gene. In addition, two polyadenylation signals located in the hE2-1 cDNA are also present in the intronic sequence downstream of exon 8 which promote termination of transcription. The data indicate that shortened human liver E2 transcripts undergo alternative splicing to yield mRNA of the hE2-1 type.

    Funded by: NIDDK NIH HHS: DK26758, DK37373

    FEBS letters 1991;279;2;229-32

  • Maple syrup urine disease: domain structure, mutations and exon skipping in the dihydrolipoyl transacylase (E2) component of the branched-chain alpha-keto acid dehydrogenase complex.

    Chuang DT, Fisher CW, Lau KS, Griffin TA, Wynn RM and Cox RP

    Department of Biochemistry, University of Texas, Southwestern Medical Center, Dallas 75235.

    Maple syrup urine disease (MSUD) is an autosomal recessive disorder in the oxidative decarboxylation of the branched-chain alpha-keto acids derived from leucine, isoleucine and valine. The enzyme deficient in MSUD, the branched-chain alpha-keto acid dehydrogenase (BCKAD) complex, is a mitochondrial multienzyme complex consisting of at least six distinct subunits. MSUD is genetically heterogeneous as manifested by lesions in different subunits of the BCKAD complex among unrelated patients. To approach the biochemical basis of MSUD involving the dihydrolipoyl transacylase (E2) subunit, the domain structure of this polypeptide from human and bovine livers has been defined by limited proteolysis and cDNA cloning. The assembly of 24 E2 subunits into a cubic structure, forming the core of the mammalian BCKAD complex, was established by electron microscopy and sedimentation equilibrium analysis. Highly assembled bovine E2 devoid of prosthetic lipoic acid has been overexpressed in Escherichia coli. Studies carried out with this bacterial expression system have provided insights into the lipoylation process of E2, and the involvement of the His391 residue in the transacylation reaction. At the genetic level, the human E2 gene (DBT) has been regionally assigned to chromosome 1p31, and a related E2 pseudogene to chromosome 3q24 by in situ hybridization. Genomic cloning has shown that the human E2 gene undergoes premature transcriptional termination and alternate splicing as normal events, although its functional significance is unknown. Through the use of the polymerase chain reaction and other recombinant DNA methods, several compound heterozygous mutations at the E2 locus have been identified in classical as well as thiamine-responsive MSUD patients. These mutations would appear to be useful genetic models, which will facilitate investigations into macromolecular organization and protein-protein interactions. Moreover, an array of precise single and multiple exon deletions has been observed in the amplified mutant E2 transcripts. The results represent unexpected secondary effects that are apparently associated with the above primary mutations in the E2 gene.

    Funded by: NIDDK NIH HHS: DK-26758, DK-37373

    Molecular biology & medicine 1991;8;1;49-63

  • Molecular genetic basis of maple syrup urine disease in a family with two defective alleles for branched chain acyltransferase and localization of the gene to human chromosome 1.

    Herring WJ, Litwer S, Weber JL and Danner DJ

    Department of Pediatrics, Emory University School of Medicine, Atlanta, GA.

    Maple syrup urine disease in humans results from inherited defects in branched chain alpha-ketoacid dehydrogenase, a mitochondrial multienzyme complex. A variety of genetic changes may produce this phenotype by affecting the function of any of the three complex-specific subunits. The varied clinical expression observed in patients may be partially explained by the defects in the involved subunit. Here we report localization of the gene for the branched chain acyltransferase component of the complex to human chromosome 1 and describe a proband who is a compound heterozygote at this locus. One allele, inherited from the father, produces transcripts with 124 nucleotides deleted from the coding region. The deletion is not found in the branched chain acyltransferase gene, implying that the deleted transcripts arise by an error in transcript processing. Cells from the patient's mother contain 50% of the normal amount of mRNA for the subunit, and the proband has inherited this nonexpressing allele from her. As a result, the proband produces no acyltransferase protein and therefore has greatly impaired complex activity. A phenotypically normal sibling is shown to be genetically similar to the mother having inherited the mother's nonexpressing allele and the father's normal allele.

    Funded by: NCRR NIH HHS: MO1 RR00039; NIDDK NIH HHS: DK38320

    American journal of human genetics 1991;48;2;342-50

  • 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.

    Fisher CW, Lau KS, Fisher CR, Wynn RM, Cox RP and Chuang DT

    Department of Biochemistry, University of Texas, Southwestern Medical School, Dallas 75235.

    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.

    Funded by: NIDDK NIH HHS: DK-26758

    Biochemical and biophysical research communications 1991;174;2;804-9

  • Complete primary structure of the transacylase (E2b) subunit of the human branched chain alpha-keto acid dehydrogenase complex.

    Nobukuni Y, Mitsubuchi H, Endo F and Matsuda I

    Department of Pediatrics, Kumamoto University Medical School, Japan.

    We isolated from a placental cDNA library by immunoscreening a cDNA clone encoding the transacylase (E2b) precursor of the human branched chain alpha-keto acid dehydrogenase (BCKDH) complex. The cDNA insert consists of 2,649 base pairs with an open reading frame of 1,431 base pairs which can be translated into 477 amino acids and a 3'-untranslated region of 1,205 base pairs. The deduced amino acid sequence includes a leader peptide of 56 amino acid residues, a lipoyl-bearing domain, a E3-binding domain and an inner core domain. A mature human E2b subunit is likely to contain 421 amino acid residues with a calculated Mr 46,322. The nucleotide sequence of the open reading frame and the deduced amino acid sequence of the human E2b shows 91.6% and 92.0% homology with those of the bovine E2b subunit, respectively.

    Biochemical and biophysical research communications 1989;161;3;1035-41

  • Construction and nucleotide sequence of a cDNA encoding the full-length preprotein for human branched chain acyltransferase.

    Danner DJ, Litwer S, Herring WJ and Pruckler J

    Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia 30322.

    A cDNA (1.6 kilobases) for branched chain acyltransferase (E2b) isolated from a human liver library encoded only the amino-terminal half of the protein (Hummel, K. B., Litwer, S., Bradford, A. P., Aitken, A., Danner, D. J., and Yeaman, S. J. (1988) J. Biol. Chem. 263, 6165-6168). Here we report the isolation of other cDNAs which encode the carboxyl-terminal half of E2b and the construction of a cDNA which encodes the entire pre-E2b. cDNA from the original clone encoding the leader sequence, lipoate binding domain, and E3 binding domain was ligated to the cDNA from a clone which by restriction maps contained an additional 3' sequence. Both cDNAs used in the construct made a fusion protein in their original phage isolate recognized by antibodies to E2b. The nucleotide sequence of the constructed cDNA was determined, and the 1431 base pairs in the open reading frame encoded a protein of 477 amino acids. In vitro transcription and translation of this cDNA produced a 57-kDa protein recognized by E2b-specific antibodies. Mouse liver mitochondria imported and processed the 57-kDa protein to a 52-kDa antigenic protein which co-migrated with E2b isolated from tissue. Comparing the protein structure of this human pre-E2b protein with that for other acyltransferase proteins showed a similarity in structure throughout all the proteins suggesting evolutionary conservation. Branched chain acyltransferase from Pseudomonas putida showed the most similarity to human E2b.

    Funded by: NIDDK NIH HHS: DK38320

    The Journal of biological chemistry 1989;264;13;7742-6

  • Mitochondrial import and processing of an in vitro synthesized human prebranched chain acyltransferase fragment.

    Litwer S and Danner DJ

    Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322.

    A 1.6-kb cDNA for human liver branched-chain acyltransferase [E2b] was placed in a transcription vector under the control of the SP6 promoter. In vitro translation of transcripts from this vector produced a pre-E2b fragment of Mr 39,000. Following import into mitochondria, this protein was processed to a protein with an Mr of 36,000. The processed protein was fully protected from trypsin digestion. Import and processing did not occur in the presence of rhodamine 123 or carbonyl cyanide m-chlorophenyl hydrazone, suggesting that membrane potential and coupled respiration were required. Uptake and processing were species and tissue independent, since both mouse-liver and human-lymphoblast mitochondria converted the human pre-E2b protein fragment. Mitochondria from patient cells that lack E2b through an inherited defect were able to import and process the in vitro-made protein, suggesting that the inherited defect was in the gene for E2b and not in the organelle-structure function. This system now provides additional methods for investigation of mechanisms responsible for the human inherited disorders affecting the branched-chain alpha-ketoacid dehydrogenase complex.

    Funded by: NIDDK NIH HHS: DK 38320

    American journal of human genetics 1988;43;5;764-9

  • Nucleotide sequence of a cDNA for branched chain acyltransferase with analysis of the deduced protein structure.

    Hummel KB, Litwer S, Bradford AP, Aitken A, Danner DJ and Yeaman SJ

    Department of Pediatrics, Emory University, Atlanta, Georgia 30322.

    Nucleotide sequence was determined for a 1.6-kilobase human cDNA putative for the branched chain acyltransferase protein of the branched chain alpha-ketoacid dehydrogenase complex. Translation of the sequence reveals an open reading frame encoding a 315-amino acid protein of molecular weight 35,759 followed by 560 bases of 3'-untranslated sequence. Three repeats of the polyadenylation signal hexamer ATTAAA are present prior to the polyadenylate tail. Within the open reading frame is a 10-amino acid fragment which matches exactly the amino acid sequence around the lipoate-lysine residue in bovine kidney branched chain acyltransferase, thus confirming the identity of the cDNA. Analysis of the deduced protein structure for the human branched chain acyltransferase revealed an organization into domains similar to that reported for the acyltransferase proteins of the pyruvate and alpha-ketoglutarate dehydrogenase complexes. This similarity in organization suggests that a more detailed analysis of the proteins will be required to explain the individual substrate and multienzyme complex specificity shown by these acyltransferases.

    Funded by: NIADDK NIH HHS: AM 38320

    The Journal of biological chemistry 1988;263;13;6165-8

  • Conservation of primary structure in the lipoyl-bearing and dihydrolipoyl dehydrogenase binding domains of mammalian branched-chain alpha-keto acid dehydrogenase complex: molecular cloning of human and bovine transacylase (E2) cDNAs.

    Lau KS, Griffin TA, Hu CW and Chuang DT

    Department of Medicine, Veterans Administration Medical Center, Cleveland, Ohio 44106.

    The subunit structures and conservation of the dihydrolipoyl transacylase (E2) components of bovine and human branched-chain alpha-keto acid dehydrogenase complexes were investigated by Western blotting, peptide sequencing, and cDNA cloning methods. Rabbit antiserum prepared against the sodium dodecyl sulfate (SDS) denaturated bovine E2 subunit recognized the inner E2 core, and the first hinge region of the E2 chain, but failed to react with the lipoyl-bearing domain as determined by Western blot analysis. The lack of antigenicity in the lipoyl-bearing domain was confirmed with antibodies directed against the native E2 component. A human E2 cDNA (1.6 kb) was isolated from a human liver cDNA library in lambda gt11 with a combination of the above anti-native and anti-SDS-denatured E2 immunoglobulin G's as a probe. The fidelity of the human E2 cDNA was established by nucleotide sequencing which showed the determined peptide sequences of the amino terminus and tryptic fragments of bovine E2. A bovine E2 cDNA (0.7 kb) was also isolated from a bovine liver cDNA library in lambda ZAP with the human E2 cDNA as a probe. Northern blot analysis using the human E2 cDNA probe showed that E2 mRNAs in bovine liver and human kidney mesangial cells are 3.3 and 4.6 kb in size, respectively. Primary structures derived from human and bovine E2 cDNAs show leader sequences including the initiator methionine and the homologous mature peptides consisting of complete lipoyl-bearing and dihydrolipoyl dehydrogenase (E3) binding domains and two hinge regions. In addition, the human E2 cDNA contains a portion of the inner E2 core sequence, a 3'-untranslated region, and a poly(A+) tail. Deduced amino acid sequences of the mammalian E2's were compared with those of Escherichia coli transacetylase and transsuccinylase and bovine kidney transacetylase. The results indicate a high degree of conservation in the sequence flanking the lipoyl-attachment site and in the E3-binding domain. Models are presented to discuss implications for the conserved structure-function relationship in the lipoyl-bearing and E3-binding domains of alpha-keto acid dehydrogenase complexes.

    Funded by: NIDDK NIH HHS: DK 26758, DK 37373; NIGMS NIH HHS: GM 07250

    Biochemistry 1988;27;6;1972-81

  • Identification of a cDNA clone in lambda gt11 for the transacylase component of branched chain ketoacid dehydrogenase.

    Litwer S and Danner DJ

    Two cDNA clones for the transacylase protein of the branched chain ketoacid dehydrogenase complex [E.C.] have been isolated from a human fetal liver cDNA expression library in lambda gt11 using antibody selection. By selective antibody elution from nitrocellulose filters containing the fusion proteins, it was determined that these inserts represent the transacylase protein. These data support the hypothesis that this protein is synthesized in the cytosol on transcripts independent of the other proteins of the branched chain ketoacid dehydrogenase complex.

    Biochemical and biophysical research communications 1985;131;2;961-7

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

    Chuang DT, Niu WL and Cox RP

    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.

    Funded by: NIADDK NIH HHS: AM 26758

    The Biochemical journal 1981;200;1;59-67

Gene lists (4)

Gene List Source Species Name Description Gene count
L00000009 G2C Homo sapiens Human PSD Human orthologues of mouse PSD adapted from Collins et al (2006) 1080
L00000010 G2C Homo sapiens Human mitochondria Human orthologues of mouse mitochondria adapted from Collins et al (2006) 91
L00000016 G2C Homo sapiens Human PSP Human orthologues of mouse PSP adapted from Collins et al (2006) 1121
L00000069 G2C Homo sapiens BAYES-COLLINS-HUMAN-PSD-FULL Human cortex biopsy PSD full list 1461
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EUROSPIN (FP7-HEALTH-241498), SynSys (FP7-HEALTH-242167) and GENCODYS (FP7-HEALTH-241995).

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