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

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.     

Subunit structure of the dihydrolipoyl transacylase component of branched-chain alpha-keto acid dehydrogenase complex from bovine liver. Characterization of the inner transacylase core

Limited proteolysis has been used to probe the subunit structure (Mr = 52,000) of the dihydrolipoyl transacylase (E2) component of the branched-chain alpha-keto acid dehydrogenase complex from bovine liver. Digestion of the complex at 0 degrees C with a low concentration of trypsin produces an inner E2 core that retains the activity for the transacylation reaction and is completely dissociated from the decarboxylase (E1) component. The trypsinized E2 maintains the highly assembled structure and migrates faster than the native E2 in the Sepharose 4B column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis shows that the inner E2 core consists of two lipoate-free tryptic fragments, i.e. fragment A and fragment B with Mr = 26,000 and 22,000, respectively. Both fragments apparently fail to bind the E1 component. Fragment A is converted into fragment B by increasing trypsin concentrations. Fragment B is a stable limit polypeptide containing the intersubunit-binding sites for E2. The assemblage of fragment B confers the cubelike appearance of the inner E2 core in electron micrographs. Activity measurements indicate that the larger fragment A, but not fragment B, possesses transacylation activity. It is likely that a critical portion of the active site is present in the 4,000-dalton fragment that is lost during the conversion of fragment A to B.     

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

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.     

Genetic reconstruction and characterization of the recombinant transacylase (E2b) component of bovine branched-chain alpha-keto acid dehydrogenase complex. Implication of histidine 391 as an active site residue

Genetically altered transacylase (E2b) proteins of the bovine branched-chain alpha-keto acid dehydrogenase complex were overexpressed in Escherichia coli and characterized. Deletion by PstI or Bal31 digestion of the amino-terminal region of the inner-core domain (residues 175-421) beyond residue 209 resulted in a complete loss of transacylase activity. The enzyme assay was carried out using [1-14C]isovaleryl-CoA and exogenous dihydrolipoamide as substrates. The removal of 4 residues (Thr-Ile-Pro-Ile) (residues 175-178) from the amino terminus of the inner-core domain significantly reduced the level of transacylase activity. The results establish that the segment between residues 175 and 209 is an integral part of the active site of E2b. The residue His-391 in the recombinant inner-core domain (E2b delta 167) was changed to Asn or Gln by site-directed mutagenesis. The wild-type and the two mutant inner-core domains were assembled into 24-mers as determined by gel filtration. However, both Asn and Gln mutations were accompanied by a complete loss of the enzymatic activity. Titration of the natural branched-chain alpha-keto dehydrogenase complex from pH 8 to 6 sharply reduced transacylase activity. The above data support the hypothesis that a conserved histidine residue in E2 acts as a general base for the transacylation reaction by analogy with E. coli chloramphenicol acetyltransferases.     

Subunit structure of the dihydrolipoyl transacylase component of branched-chain alpha-keto acid dehydrogenase complex from bovine liver. Mapping of the lipoyl-bearing domain by limited proteolysis

To characterize the lipoyl-bearing domain of the dihydrolipoyl transacylase (E2) component, purified branched-chain alpha-keto acid dehydrogenase complex from bovine liver was reductively acylated with [U-14C] alpha-ketoisovalerate in the presence of thiamin pyrophosphate and N-ethylmaleimide. Digestion of the modified complex with increasing concentrations of trypsin sequentially cleaved the E2 polypeptide chain (Mr = 52,000) into five radiolabeled lipoyl-containing fragments in the order of L1 (Mr = 28,000), L2 (Mr = 24,500), L3 (Mr = 21,000), L4 (Mr = 15,000) to L5 (Mr = 14,000) as determined by the autoradiography of sodium dodecyl sulfate-polyacrylamide gel. In addition, a lipoate-free inner E2 core consisting of fragment A (Mr = 26,000) and fragment B (Mr = 22,000) was produced. Fragment A contains the active site for transacylation reaction and fragment B is the subunit-binding domain. Fragment L5 and fragment B were stable and resistant to further tryptic digestion. Mouse antiserum against E2 reacted only with fragments L1, L2, and L3, and did not bind fragments L4, L5, A, and B as judged by immunoblotting analysis. The anti-E2 serum strongly inhibited the overall reaction catalyzed by the complex, but was without effect on the transacylation activity of E2. Measurement of incorporation of [1-14C]isobutyryl groups into the E2 subunit indicated the presence of 1 lipoyl residue/E2 chain. Based on the above data, a model is proposed in which the lipoyl-bearing domain is connected to the inner E2 core via a trypsin-sensitive hinge. The lipoyl-bearing domain contains five consecutive tryptic sites (L1 to L5), with the L1 site in the hinge region, and the L5 site next to the terminal lipoyl-binding sequence. An exposed and antigenic region is located between L1 and L4 tryptic sites of the lipoyl-bearing domain. The region accounts for about 24% of the E2 chain length. Binding of antibodies to this region probably impairs the mobility of the lipoyl-containing polypeptide, resulting in an interruption of the active-site interactions that are necessary for the overall reaction. The lack of antigenicity and resistance to tryptic digestion indicate a highly folded conformation for fragment L5, the limit polypeptide carrying the single lipoyl residue.     

Characterization and conservation of the inner E2 core domain structure of branched-chain alpha-keto acid dehydrogenase complex from bovine liver. Construction of a cDNA encoding the entire transacylase (E2b) precursor

A cDNA clone encoding the entire transacylase (E2b) precursor of the bovine branched-chain alpha-keto acid dehydrogenase complex has been constructed from two overlapping incomplete cDNA clones which were isolated from a lambda ZAP library prepared from bovine liver poly(A)+ RNA. Nucleotide sequencing indicates that this bovine E2b cDNA insert (bE2-11) is 2701 base pairs in length with an open reading frame of 1446 base pairs. The bE2-11 cDNA insert encodes a leader peptide of 61 residues and a mature E2b polypeptide of 421 amino acid residues with a calculated monomeric molecular mass of 46,518 daltons. The molecular mass of the native E2b component isolated from bovine liver is 1,110,000 daltons as determined by sedimentation equilibrium. This value establishes the 24-subunit octahedral model for the quaternary structure of bovine E2b. The amino-terminal sequences of two tryptic fragments (A and B) of the E2b protein have been determined. Fragment A comprises residues 175 to 421 of the E2b protein and is the inner E2 core domain which contains the transacylase active site. Fragment B, produced by further tryptic cleavage of fragment, comprises residues 205 to 421, but does not have transacylase activity. Both fragments A and B confer the highly assembled 24-mer structure. The primary structure of the inner E2 core domain of bovine E2b (fragment A) is very similar to those of three other E2 proteins (human E2p, Escherichia coli E2p, and E. coli E2k). These similarities suggest that these E2 proteins are structurally and evolutionarily related.     

Purification and characterization of human liver branched-chain alpha-keto acid dehydrogenase complex

Human liver BCKADH complex was purified. On SDS-polyacrylamide gel electrophoresis, the purified enzyme complex gave three major bands having molecular weights of 51,000, 46,000, and 36,000, and one minor band with a molecular weight of 55,000. The minor band corresponded in molecular weight to lipoamide oxidoreductase which was purified separately. The purified BCKADH represented only approximately 20% of the maximum activity when assayed without addition of exogenous lipoamide oxidoreductase, indicating that lipoamide oxidoreductase component was readily dissociable from the complex. The BCKADH effectively oxidized all of KIV, KIC, and KMV, yielding apparent Km values in the range of 14-17 microM for those alpha-keto acids. Vmax values obtained were 0.86, 0.61, and 0.51 mumole NADH produced/min/mg of protein for KIV, KIC, and KMV, respectively, in the presence of excess amount of lipoamide oxidoreductase. This ratio of Vmax values was practically identical to those of specific activities obtained with respective branched-chain alpha-keto acids at each purification step. The enzyme complex also oxidized pyruvate and alpha-ketoglutarate to a lesser extent. Kinetic experiments gave Km values of 0.98 and 2.9 mM for pyruvate and alpha-ketoglutarate, respectively, with Vmax of 0.43 and 0.08 mumole NADH produced/min/mg of protein. NAD and CoASH were absolutely required for the reaction. Km values for NAD and CoASH were estimated to be 47 and 25 microM, respectively.     

Expression and assembly of mature apotransacylase (E2b) of bovine branched-chain alpha-keto acid dehydrogenase complex in Escherichia coli. Demonstration of transacylase activity and modification by lipoylation

A cDNA clone encoding the entire transacylase (E2b) precursor of the bovine branched-chain alpha-keto acid dehydrogenase complex (Griffin, T. A., Lau, K. S., and Chuang, D. T. (1988) J. Biol. Chem. 263, 14008-14014) was used to construct a prokaryotic expression vector for recombinant mature E2b. The overexpression in Escherichia coli correlates with the presence near the 5'-terminus of the mature E2b coding region (nucleotides 20 to 28) of the sequence 5'-TCAAACT-CT-3'. It has been proposed that this sequence is involved in secondary mRNA recognition through interaction with the 5'-terminus of the bacterial 16 S rRNA. The mature E2b protein has transacylase activity when assayed with exogenous dihydrolipoamide and [1-14C] isovaleryl-CoA as substrates. However, the recombinant protein has no attached lipoic acid. This was established by the absence of radiolabel incorporation when transformed E. coli cells were grown in a medium containing DL-[2-3H]lipoic acid. The recombinant mature E2b protein was purified to greater than 95% purity in one step using Sepharose 4B column chromatography. The purified recombinant protein was shown to have a cubic 24-mer structure by electron microscopy and to possess a specific activity similar to that of the purified natural bovine E2b. The purified recombinant mature E2b was lipoylated in vitro in the presence of 2 mM ATP using a mitochondrial extract prepared from bovine liver. The above results provide the first evidence that the proper folding and assembly of mature bovine E2b is independent of the attachment of lipoyl moieties and that mammalian lipoylation activity is present in mitochondria.     

Evidence for the regulation of the branched chain alpha-keto acid dehydrogenase multienzyme complex by a phosphorylation/dephosphorylation mechanism

The regulation of the branched chain alpha-keto acid dehydrogenase complex by covalent modification was investigated in rat liver mitochondria. Depletion of intramitochondrial calcium and magnesium caused an inactivation of the branched chain alpha-keto acid dehydrogenase complex. Following inactivation of the branched chain complex, addition of calcium or magnesium ions separately to incubations of mitochondria only partially reactivated the enzyme complex. However, simultaneous addition of calcium and magnesium activated the branched chain enzyme complex rapidly and nearly completely. Mitochondrial incubations were performed in the presence of [32P]phosphate under conditions known to activate or to inactivate the branched chain alpha-keto acid dehydrogenase complex. Evidence demonstrating that [32P]-phosphate was incorporated into two major protein bands separated in sodium dodecyl sulfate-polyacrylamide gels of the mitochondrial incubations is presented. Migration of the labeled mitochondrial protein bands in the gel system corresponded exactly to the migration of the alpha subunit of the purified heart-derived pyruvate dehydrogenase (decarboxylase, E1) and the alpha subunit of the purified kidney-derived branched chain alpha-keto acid dehydrogenase (decarboxylase, E1). Furthermore, when the measured activity of the branched chain complex was minimized, the amount of [32P]phosphate incorporated into the alpha chain of the branched chain enzyme was maximal. Conversely, incubation conditions which activated maximally the enzyme complex minimized the [32P]phosphate incorporation into the alpha subunit of the branched chain dehydrogenase.     

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.     

Characterization of rainbow trout branched-chain alpha-keto acid dehydrogenase complex: inter-domain segments of the E2 component affect the overall activity

Branched-chain alpha-keto acid dehydrogenase complex (BCKADH) contains decarboxylase (E1), dihydrolipoyl transacylase (E2), and dihydrolipoyl dehydrogenase (E3) as catalytic components. BCKADH purified from rainbow trout (Oncorhynchus mykiss) liver was comparable with mammalian BCKADH in various enzymatic characteristics, but less efficient in catalyzing the overall reaction. The trout E2 subunit was larger than the mammalian subunit and rather similar to the chicken one in relative molecular mass on SDS-PAGE, whereas the E1 component was similar between trout and mammalian both in relative molecular mass of its alpha and beta subunits and in the catalytic activity. Trout E2 cDNA cloning and nucleotide sequencing revealed that the mature trout E2 subunit consists of 435 residues, and possesses 14 additional residues compared with mammalian E2. Eleven of these are localized in two interdomain segments as two sequences with two and nine residues, respectively. Trout E2 was inferior to rat E2 in the capacity for binding the E1 component, similar to chicken E2. Thus, it appears that non-mammalian BCKADH E2 is distinct from that in mammals in the structure of interdomain segments, resulting in reduction of overall activity of the enzyme complex.     

Sepharose-insolubilization of the dihydrolipoyl transacetylase core component of the pyruvate dehydrogenase complex: Preparation and characterization

The dihydrolipoyl transacetylase core component of the bovine kidney and heart pyruvate dehydrogenase complexes were covalently attached through the lipoyl moiety to Sepharose by the thiol-crosslinking reagent, N, N′-p-phenylenedimaleimide.In one approach, the N, N′-p-phenylenedimaleimide was allowed to react with glutathione which was in turn linked by its N-terminal to Sepharose CL-6B. In addition, we found the N, N′-p-phenylenedimaleimide would react directly with Sepharose CL-6B (at undetermined sites) and could be used as the sole bridge in forming a stable linkage of the transacetylase core to Sepharose. With the latter approach the extent of multiple-linkage of the 60-subunit core could more easily be controlled. This should be a generally useful approach for linking proteins with reactive surface thiol residues.Insolubilization of the core of the pyruvate dehydrogenase complex by these methods did not appear to significantly alter the binding of other protein components of the complex, but the catalytic activities of the complex requiring the lipoyl moiety were appreciably altered. Procedures for coupling the transacetylase core to various derivatives of phenylenedimaleimide-Sepharose and techniques described for studying the protein products should be useful in preparation of specialized matrices for both protein purification and the study of protein-protein interactions.     

Localization of the dihydrolipoamide branched-chain transacylase gene (DBT) of the human branched-chain keto acid dehydrogenase complex to chromosome 1

The gene coding for the transacylase subunit (DBT) of the human branched-chain keto acid dehydrogenase complex was localized to chromosome 1 by probing panels of human x mouse chromosome hybrids with an E2 cDNA amplified by the polymerase chain reaction. Additional data with two hybrids containing chromosome 1 fragments suggest that the DBT gene is located on the short arm (1pter----p21) of the chromosome.