Impaired Assembly of E1 Decarboxylase of the Branched-chain alpha -Ketoacid Dehydrogenase Complex in Type IA Maple Syrup Urine Disease*

R. Max Wynn, James R. DavieDagger , Jacinta L. Chuang, Cynthia D. Cote, and David T. Chuang§

From the Departments of Biochemistry and Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9038

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The E1 decarboxylase component of the human branched-chain ketoacid dehydrogenase complex comprises two E1alpha (45.5 kDa) and two E1beta (37.5 kDa) subunits forming an alpha 2beta 2 tetramer. In patients with type IA maple syrup urine disease, the E1alpha subunit is affected, resulting in the loss of E1 and branched-chain ketoacid dehydrogenase catalytic activities. To study the effect of human E1alpha missense mutations on E1 subunit assembly, we have developed a pulse-chase labeling protocol based on efficient expression and assembly of human (His)6-E1alpha and untagged E1beta subunits in Escherichia coli in the presence of overexpressed chaperonins GroEL and GroES. Assembly of the two 35S-labeled E1 subunits was indicated by their co-extraction with Ni2+-nitrilotriacetic acid resin. The nine E1alpha maple syrup urine disease mutants studied showed aberrant kinetics of assembly with normal E1beta in the 2-h chase compared with the wild type and can be classified into four categories of normal (N222S-alpha and R220W-alpha ), moderately slow (G245R-alpha ), slow (G204S-alpha , A240P-alpha , F364C-alpha , Y368C-alpha , and Y393N-alpha ), and no (T265R-alpha ) assembly. Prolonged induction in E. coli grown in the YTGK medium or lowering of induction temperature from 37 to 28 °C (in the case of T265R-alpha ), however, resulted in the production of mutant E1 proteins. Separation of purified E1 proteins by sucrose density gradient centrifugation showed that the wild-type E1 existed entirely as alpha 2beta 2 tetramers. In contrast, a subset of E1alpha missense mutations caused the occurrence of exclusive alpha beta dimers (Y393N-alpha and F364C-alpha ) or of both alpha 2beta 2 tetramers and lower molecular weight species (Y368C-alpha and T265R-alpha ). Thermal denaturation at 50 °C indicated that mutant E1 proteins aggregated more rapidly than wild type (rate constant, 0.19 min-1), with the T265R-alpha mutant E1 most severely affected (rate constant, 4.45 min-1). The results establish that the human E1alpha mutations in the putative thiamine pyrophosphate-binding pocket that are studied, with the exception of G204S-alpha , have no effect on E1 subunit assembly. The T265R-alpha mutation adversely impacts both E1alpha folding and subunit interactions. The mutations involving the C-terminal aromatic residues impede both the kinetics of subunit assembly and the formation of the native alpha 2beta 2 structure.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The mammalian mitochondrial branched-chain alpha -ketoacid dehydrogenase (BCKD)1 complex catalyzes the oxidative decarboxylation of the branched-chain alpha -ketoacids derived from the branched-chain amino acids, leucine, isoleucine, and valine (1). This multienzyme complex is organized around a dihydrolipoyl transacylase (E2) core, to which a branched-chained alpha -ketoacid decarboxylase (E1), a dihydrolipoamide dehydrogenase (E3), a specific kinase, and a specific phosphatase are attached through ionic interactions (2, 3). E1 is a thiamine pyrophosphate (TPP)-dependent enzyme comprising two E1alpha and two E1beta subunits that assemble into an alpha 2beta 2 tetramer. E2 is a 24-meric protein consisting of identical lipoic acid-bearing subunits arranged on octahedral 4,3,2-point group symmetry. Each E2 polypeptide contains three independently folded domains (i.e. lipoyl-bearing, E1/E3-binding, and inner core) that are highly conserved among E2 proteins of the alpha -ketoacid dehydrogenase complexes (4). These complexes include pyruvate dehydrogenase, alpha -ketoglutarate dehydrogenase, and BCKD complexes (3). E3 is a homodimeric flavoprotein that is common to members of the alpha -ketoacid dehydrogenase complexes (3). The kinase and the phosphatase are specific for the BCKD complex and regulate its activity through a reversible phosphorylation (inactivation) and dephosphorylation (activation) cycle (5).

In patients with maple syrup urine disease (MSUD) or branched-chain ketoaciduria, the activity of the BCKD complex is deficient. This leads to clinical manifestations including often fatal ketoacidosis, neurological derangements, and mental retardation (1). The molecular genetics of MSUD are heterogeneous as mutations in the E1alpha , E1beta , E2, and E3 genes have been described (1, 6). Based on the locus affected, genetic subtypes of MSUD have been proposed, with type IA referring to mutations in the E1alpha gene, type IB to the E1beta gene, type II to the E2 gene, and type III to the E3 gene (1). It has been suggested that certain type IA MSUD missense mutations, for example Y393N-alpha (7) and Y368C-alpha (8), may impede the assembly of mutant E1alpha with normal E1beta subunit, resulting in the degradation of E1 subunits in patient's cells.

We have recently established that chaperonins GroEL and GroES are essential for efficient folding and assembly of the E1 tetramer in Escherichia coli (9) and the E2 24-mer in vitro (10). To gain insight into the biochemical basis of the apparently impaired assembly of E1 in type IA MSUD, we have co-expressed both mature mutant E1alpha and normal E1beta in E. coli co-transformed with a second plasmid overproducing chaperonins GroEL and GroES. Pulse-chase labeling of both E1 subunits was carried out to measure the kinetics of assembly of the mutant E1alpha with normal E1beta in the bacterial cell. The results showed a marked reduction in the rate of E1 assembly in certain E1alpha mutants compared with normal. It was also found that a subset of E1alpha mutations affect the assembly state of mutant E1 after the prolonged induction in E. coli. Thermostability and protease digestion studies further indicated these slowly assembled mutant E1 proteins had less stable conformations than the wild type. These results define the residues that are critical for subunit interactions and stability of E1 and have implications for understanding chaperonin-mediated biogenesis of hetero-oligomeric structures.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Cell Lines and Cell Cultures-- Fibroblasts of classic MSUD patients were kindly provided by the following physicians: B.A. by Dr. David Valle, John F. Kennedy Institute, Baltimore, MD; C.Q. by Dr. Robin Casey, University of Saskatchewan, Canada; and K.U. by Dr. Selma Snyderman, New York University Medical Center, New York, NY. Fibroblasts of classic MSUD patient L.C. were obtained from Cell Repository of McGill Medical Center, Montreal, Canada. Amniocytes of the fetus-at-risk for classic MSUD (F.J.) were kindly provided by Dr. Frederico Mariona of Hutzel Hospital, Detroit, MI. Cells were cultured in Waymouth medium containing 15% fetal calf serum as described previously (11).

Construction of pHisT-E1 Prokaryotic Expression Vector-- The 5' portion of the mature human E1alpha cDNA sequence was amplified from the pMAL-c2-hE1alpha expression vector (12) using an internal 22-mer antisense primer with sequence 5'-GTAACAGATATCGACCCTGTT-3', and a 49-mer sense primer with sequence 5'-GGCTCTAGACTCGAGAATCTTTATTTtcaatcatctctggatgacaagc-3' to yield a 601-bp product. The sense primer adds exogenous sequence (shown in uppercase) to the 5' terminus of the mature E1alpha open reading frame (shown in lowercase). This exogenous sequence includes an XbaI restriction site (shown in bold), followed by sequence encoding the first six amino acids specific for the tobacco etch virus (TEV) protease cleavage (shown underlined). The seventh required amino acid for the TEV protease cleavage is supplied by the N-terminal serine of the mature E1alpha sequence.

To generate the pHisT-E1alpha expression vector, the 601-bp amplification product was cut with XbaI and NarI to yield a 454-bp fragment encoding the TEV cleavage site and the 5' portion of the E1alpha open reading frame. The pEBO-hbE1alpha expression vector (7) was cleaved with NarI and XhoI to yield a 1064-bp fragment that encoded the 3' portion of the mature E1alpha open-reading frame. Both fragments were ligated into the pTrcHisB expression vector (Invitrogen, Carlsbad, CA) digested with NheI and XhoI to yield the pHisT-E1alpha expression vector. To generate the pHisT-E1 expression vector, a BamHI/ScaI fragment (2,555 bp) comprising the trc promoter and the mature human E1beta open reading frame was isolated from expression vector pKK-hE1beta (13) and ligated into the corresponding sites in the host pHisT-E1alpha expression vector, yielding the pHisT-E1 expression vector. Mutant E1alpha variants of the pHisT-E1 expression vectors were constructed identically, except for the substitution of 1064-bp NarI/XhoI fragments isolated from variant pEBO-hbE1alpha plasmids (7) harboring the desired E1alpha mutation.

Expression and Purification of Recombinant E1 Proteins-- E. coli strain CG712 (ESts) and the plasmid pGroESL, which overexpresses chaperonins GroEL and GroES (14), were kind gifts of Dr. Anthony Gatenby of DuPont Experimental Station, Wilmington, DE. CG712 cells co-transformed with GroESL and the wild-type or mutant pHisT-E1 plasmids were grown as an overnight culture at 37 °C in YTGK medium. The medium was modified from the 2× YT medium (15), and contained the following per liter: 16 g of yeast extract, 10 g of bacto-tryptone, 5 g of NaCl, 10 ml of glycerol, and 0.75 g of KCl; 100 mg of ampicillin and 12.5 mg of tetracycline. Antibiotics were added to maintain the expression plasmid (Ampr) and the F' plasmid (Tetr) that carried the lacI transcriptional repressor gene. The overnight culture was diluted 3:1000 into 1 liter of YTGK medium and grown with shaker aeration at 37 °C to a measured A550 of 0.6. The expression of (His)6-E1 was induced with 1 mM IPTG at 37 °C for 15-20 h.

E. coli cells were collected by centrifugation and resuspended in 5 cell-pellet volumes of a wash buffer (50 mM Tris·HCl, 100 mM NaCl, 5 mM EGTA, 5 mM EDTA, pH 7.5). Rinsed cells were pelleted and resuspended in a lysis buffer (50 mM KPi, 100 mM NaCl, 2 mM MgCl2, 0.2 TPP, 0.1% (v/v) Triton X-100, 0.01% (w/v) NaN3, 0.1 mM EDTA, 0.25 mM dithiothreitol, pH 7.0) with protease inhibitors (1 mM PMSF and 1 mM benzamidine) at 10 ml of buffer per g wet cell weight. Resuspended cells were sonicated in an ice-water bath with a Bronson model 350 sonicator to eliminate most viscosity. Cell lysates clarified by centrifugation were treated with Ni2+-NTA (Qiagen, Chatsworth, CA) derivatized Sephacryl resin by batch mixing for 2 h, and the resin was packed into a column. The column was washed with 10 bed volumes of an elution buffer (100 mM KPi, pH 7.5, 500 mM KCl, 0.2% Tween 20, 2 mM beta -mercaptoethanol, 0.1 mM MgCl2, and 0.1 mM TPP) containing 15 mM imidazole. (His)6-E1 was eluted in the same buffer with a 15-250 mM imidazole gradient. Column fractions were analyzed by SDS-PAGE. E1 activity was assigned spectrophotometrically (16) or radiochemically (17) using 2,6-dichlorophenol indophenol as an artificial electron acceptor.

Pulse-Chase Labeling to Determine Kinetics of E1alpha and E1beta Subunit Assembly-- CG712 cells were transformed with the pGroESL plasmid and pHisT-hE1 expression vectors carrying the normal mature E1beta cDNA and either normal or mutant His-tagged mature E1alpha cDNA. Cells were grown at 42 °C to an A595 of 0.8 in C2 broth minimal media. The C2 broth minimal medium was modified from the low sulfate and low amino acid content C broth medium described by Guzman-Verduzco and Kupersztoch (18) and contained the following per liter: 2 g of NH4Cl, 6 g of Na2HPO4·7H2O, 3 g of KH2PO4, 3 g of NaCl, 6 g of yeast extract, 40 µmol of TPP, 50 mg of carbenicillin, and 50 mg of chloramphenicol. Cells were pelleted and resuspended in one-fifth original volume of the same media without antibiotics and allowed to recover with shaking for 5 min at 37 °C. Cells were subsequently induced with 2 mM IPTG for 5 min, pulsed with 50 µCi/ml [35S]Cys/[35S]Met (ICN Radiochemicals, Costa Mesa, CA) for 1 min, and chased with 3 volumes of the same media (without antibiotics) supplemented with 8 mg/ml each of non-radioactive L-cysteine and L-methionine. At specified time points following the chase, cell samples (1.8 ml) were taken and quickly frozen in liquid N2. Thawed samples were lysed by sonication, and supernatants after microcentrifugation were treated batchwise with an excess (15 µl) of Ni2+-NTA resin. The resin was washed three times (total volume: 2.4 ml) with 15 mM imidazole in 100 mM KPi, pH 7.5, containing 2 mM MgCl2, 0.1 mM EDTA, 0.1 mM EGTA, 0.2 mM TPP, and 2 mM beta -mercaptoethanol. Bound (His)6-tagged E1alpha and assembled untagged E1beta polypeptides were eluted with 30 µl of Laemmli SDS sample buffer (19) containing 50 mM EDTA. Eluted labeled polypeptides were analyzed by SDS-PAGE, and autoradiograms were obtained by storage phosphorimaging.

Analysis of Assembly State by Sucrose Density Gradient Centrifugation-- Wild-type and mutant E1 proteins were fractionated on a 10-25% sucrose density gradient (10 ml). Gradients were poured in 50 mM potassium phosphate, pH 7.5, 250 mM KCl, and 0.2 mM EDTA. Proteins (25-200 µg) were applied to each gradient following elution from Ni2+-NTA resin and fractionation on Sephacryl S-100 HR column. The gradients were run at 41,000 rpm (210,000 × g) in a Beckman SW-41 rotor for 18 h. Gradients were fractionated (700 µl/fraction) from top to bottom. Each fraction was treated with 10 µl of 12.5 mg/ml deoxycholate and 700 µl of 15% trichloroacetic acid. Samples were incubated on ice for 20 min and then spun in a microcentrifuge for 15 min. Each pellet was washed with 700 µl of acetone (80%), followed by a 5-min microcentrifuge spin and evacuation of the supernatant by vacuum. Pellets were resuspended in 10 µl of H2O and 5 µl of SDS-PAGE sample buffer, and the entire volume was applied to the gels for analysis by SDS-PAGE.

Thermal Denaturation of Normal and Mutant E1 Proteins-- Thermal aggregation was monitored by measuring absorbance at 360 nm versus time in a Gilford response spectrophotometer equipped with a Peltier heating device as described previously (20). Wild-type or mutant E1 proteins (1.2 µM, final concentration) were added to a buffer preheated to 50 °C, which contained 50 mM KPi, 250 mM KCl, 0.5 mM beta -mercaptoethanol, 0.2 mM EDTA, and 10% glycerol at pH 7.5 in a final volume of 0.5 ml. The temperature of samples in glass cuvettes (2 mm in width and 10 mm in light path length) was measured using a small-bead thermocouple. The effects of cofactors on thermal denaturation and aggregation was studied by adding 2 mM TPP and 1 mM MgCl2 to the incubation mixture.

Thermal denaturation curves were analyzed as follows. The aggregation for each sample was allowed to proceed until no additional absorbance changes were detected. The final absorbance was taken as the 100% denaturation point. All the earlier data points relative to the final end point were used to calculate percentage of the protein that remained soluble. The log of these values were plotted against the incubation time, and the slopes of the lines were determined. The plots appeared to be pseudo-first order decays. The rate constant, kobsd, was calculated from these slopes using CA-Cricket Graph III version 1.01 for the Macintosh.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Identification of E1alpha Mutations in Type IA MSUD Patients-- Type IA (E1alpha -deficient) MSUD was initially suggested by a reduced level or absence of the E1-alpha subunit in cells from the patients. Missense mutations were identified by DNA sequencing with subclones of the RT-PCR product or by direct sequencing of that product. Y393N-alpha (codon TACright-arrowAAC), F364C-alpha (TTCright-arrowTGC), Y368C-alpha (TATright-arrowTGT), and G245R-alpha (GGGright-arrowAGG) mutations were reported previously (8, 21, 22). The N222S-alpha (AATright-arrowAGT) is present in a homozygous classic MSUD patient F.J. G204S-alpha (GGGright-arrowAGG), R222W-alpha (CGGright-arrowTGG), and A240P-alpha (GCAright-arrowCCA) are present in one allele of classic patients L.C., B.A., and K.U., respectively. The other allele in these three patients is the prevalent Y393N-alpha mutation previously reported to occur in homozygous Mennonite MSUD patients (23, 24). The T265R-alpha mutation (ACAright-arrowAGA) is in one allele of a classic patient C.Q. The second allele in this patient is a single C nucleotide insertion in exon 2 (the 144insC allele) that we reported previously (8). The four novel missense mutations (G204S-alpha , R220W-alpha , N222S-alpha , and A240P-alpha ) as a cause of MSUD were studied by transfection of the full-length human E1alpha cDNA carrying one of these mutations into type IA MSUD lymphoblasts (22). Transfected cells were unable to decarboxylate the alpha -keto-[1-14C]isovalerate (data not shown). As a positive control, decarboxylation activity in type IA MSUD cells was complemented by transfection with the normal pEBOhbE1-alpha plasmid (7).

Expression and Purification of Normal (His)6-Human E1-- In our earlier study of chaperonin-augmented expression of mammalian E1, a maltose-binding protein (MBP) ligand was fused to the N terminus of the E1alpha subunit. The presence of the MBP sequence increases the solubility of MBP-E1 and facilitates its purification by amylose resin affinity chromatography (12). In the present study, the MBP ligand is replaced with a (His)6-tag, which is linked to the N terminus of the mature E1alpha subunit through a TEV-protease recognition sequence (LENLYFQ). Co-expression of (His)6-E1alpha and untagged E1beta (the pHis T-E1 plasmid) was carried out in an E. coli CG712 host, which contained a second plasmid GroESL that overproduced chaperonins GroEL and GroES. The cells grown in the YTGK medium were heat-shocked at 42 °C for 4 h, followed by induction at 37 °C with IPTG for 16 h. The cell lysate was purified by Ni2+-NTA column chromatography. Fig. 1 (lower panel) shows the elution profile with an imidazole gradient. The upper panel shows the SDS-PAGE profile of column fractions stained with Coomassie Blue. The appearance of the (His)6-E1 tetramer coincides with the presence of E1 activity as measured by a spectrophotometric assay with 2,6-dichlorophenol indophenol as an electron acceptor. The earlier fractions corresponding to the GroEL-E1alpha -E1beta complex may represent a folding intermediate (see "Discussion").


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Fig. 1.   Purification of recombinant human (His)6-E1 by Ni2+-NTA affinity chromatography. E. coli strain CG712 (ESts) was co-transformed with the pHisT-E1 plasmid (co-expressing human (His)6-E1alpha and untagged E1beta subunits) and the pGroESL plasmid (overproducing chaperonins GroEL and GroES). Expression of the human (His)6-E1 tetramer in CG712 cells grown in the YTGK medium was induced by IPTG at 37 °C for 16 h. Cell lysates prepared by sonication were clarified by centrifugation and mixed batchwise with Ni2+-NTA-derivatized Sephacryl resin for 2 h. The resin was packed in a column and washed with the phosphate buffer at pH 7.5 containing 500 mM KCl and 15 mM imidazole (see "Experimental Procedures"). (His)6-E1 was eluted with an imidazole gradient of 15-250 mM. Column fractions (2.0 ml each) were analyzed by SDS-PAGE. E1 activity in each fraction was assayed by a spectrophotometric method, in which the reduction of 2,6-dichlorophenol indophenol was monitored at 600 nm. The upper panel is the Coomassie Blue staining of the SDS-PAGE profile. The lower panel depicts the activity and A280 elution profiles.

Measurements of E1 Assembly Kinetics by Pulse-Chase Labeling-- Our previous data suggested that the Y393N-alpha mutation in MSUD impedes the assembly of the mutant E1alpha subunit with normal E1beta , resulting in the preferential degradation of the latter subunit in patient's cells (7). In the present study, we set out to measure the kinetics of assembly of normal and the nine MSUD mutant E1alpha subunits, including Y393N-alpha , with normal E1beta by pulse-chase labeling. The method was based on the efficient expression of (His)6-E1 in the presence of excess chaperonins GroEL and GroES as described above. CG712 cells co-expressing (His)6-E1alpha , untagged E1beta , GroEL, and GroES were grown in the C2 broth minimal medium and heat-shocked at 42 °C for 4 h, followed by induction with IPTG for 5 min at 37 °C. The cells were pulsed with [35S]cysteine/[35S]methionine for 1 min and then chased with unlabeled amino acids from 2 to 120 min. Lysates prepared from cells harvested at different times were purified by Ni2+-NTA affinity chromatography. The eluted radiolabeled polypeptides were separated by SDS-PAGE, and autoradiograms were obtained by storage phosphorimaging. Since the E1beta subunit was untagged, the co-purification of this subunit with the (His)6-E1alpha subunit by Ni2+-NTA indicated assembly of the two polypeptides synthesized during the 1-min pulse. Fig. 2 shows that the assembly of normal E1beta with normal (His)6-E1alpha occurs as early as 10 min in the chase and reaches a plateau at 30 min. Similar results were obtained with N222S-alpha (second panel from the top) and R220W-alpha (data not shown). These two mutations are in the category of a normal assembly with the E1beta subunit. A second group of mutations represented by the G245R-alpha showed that significant assembly with E1beta did not occur until 30 min into the chase and plateaued at 60 min. Mutations that produce this sluggish assembly kinetics belong to the category of moderately slow assembly. A third group of E1alpha mutations comprising G204S-alpha , A240P-alpha , F364C-alpha , Y368C-alpha and Y393N-alpha did not generate detectable assembly with normal E1beta during the 2-h chase. This is indicated by the absence of the E1beta subunit in the autoradiogram. This group of mutations are classified as slow assembly, as they produce the assembled mutant E1 only after a prolonged growth of the transformed cells at 37 °C for 16 h (see below). The fourth category of no assembly is represented by the T265R-alpha subunit, which is not soluble when cells are grown at 37 °C, as indicated by the rapid disappearance of the mutant E1alpha subunit in the chase. This resulted in a complete absence of assembly with the normal E1beta subunit, even after a prolonged growth at 37 °C. However, a significant amount of the assembled T265R-alpha E1 was produced, when the induction temperature was lowered from 37 to 28 °C (see below).


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Fig. 2.   Kinetics of normal and aberrant E1 subunit assembly analyzed by pulse-chase labeling in E. coli. CG712 cells were co-transformed with the pGroESL plasmid and the pHisT-E1 plasmid carrying the (His)6-tagged wild-type or mutant E1alpha cDNA and the untagged normal E1beta cDNA. Cells grown in the C2 broth minimal medium were induced for expression of E1 subunits with IPTG for 5 min and pulsed with [35S]cysteine/[35S]methionine (50 µCi/ml) for 1 min. The cells were then chased with 8 mg/ml non-radioactive cysteine and methionine for up to 120 min. Lysates prepared from cells harvested at different chase times were mixed batchwise with Ni2+-NTA resin. 35S-Labeled (His)6-E1alpha subunits assembled with untagged E1beta subunits or GroEL were eluted with the SDS sample buffer (19). The eluted radiolabeled polypeptides were analyzed by SDS-PAGE, and autoradiograms were obtained by storage phosphorimaging. The aberrant assembly of mutant (His)6-E1alpha subunits with the normal E1beta subunit is classified into four categories based on the kinetics of appearance of the co-purified E1beta subunit: normal assembly (10-20 min), moderately slow assembly (30-40 min), slow assembly (>2 h), and no assembly (indefinite time).

Measurements of Total and Soluble E1alpha and E1beta Subunits-- The levels of total recombinant E1alpha and E1beta polypeptides in E. coli cells were measured by Western blotting. Cells co-transformed with pHisT-E1 and pGroESL plasmids and grown in the C2 broth minimal medium were heat-shocked as described above and induced with IPTG for 12 h. Cells were harvested and total lysates prepared by sonication followed by solubilization in an SDS-PAGE sample buffer. After SDS-PAGE, the samples were subjected to Western blotting using the antibody to E1alpha or E1beta as a probe. Fig. 3A shows that the levels of normal and mutant E1alpha are comparable. More significantly, the levels of normal E1beta in cells expressing normal or mutant E1alpha are relatively constant. The data rule out the possibility that the reduced level or absence of normal E1beta assembled with the mutant E1alpha subunits (Fig. 2) is caused by aberrant E1beta expression. Fig. 3B shows Western blotting of total soluble E1alpha (normal or mutant) and E1beta (normal) subunits in E. coli cells when co-expressed at 37 °C. Five (G204S-alpha , R220W-alpha , N222S-alpha , A240P-alpha , and G245R-alpha ) of the nine E1alpha mutants studied are associated with wild-type levels of soluble E1alpha and E1beta subunits. In contrast, the level of T265R-alpha is markedly reduced with a concomitant near-absence of the normal E1beta subunit. The results support the conclusion drawn from pulse-chase labeling (Fig. 2), which indicates that the mutant T265R-alpha is largely insoluble at 37 °C and fails to assemble with the normal E1beta subunit. The unassembled E1beta subunit, while expressed at a normal rate (Fig. 3A), became aggregated and was removed from the supernatant after centrifugation. As for the F364C-alpha , Y368C-alpha , and Y393N-alpha mutants (Fig. 3B), the levels of both soluble mutant E1alpha subunits and the soluble E1beta subunit are decreased, compared with the wild-type E1alpha .


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Fig. 3.   Western blot analysis of the levels of normal and mutant E1 subunits expressed in E. coli. The expression of the normal or mutant (His)6-E1alpha subunit and the normal E1beta subunit in CG712 (ESts) cells co-transformed with the wild-type or mutant pHisT-E1 plasmid and the pGroESL plasmid was induced with IPTG in the C2 broth minimal medium at 37 °C for 12 h. Crude lysates were prepared by sonication, and the supernatant was obtained by removing the pellet after centrifugation for 2 h at 29,000 × g. Samples of both crude lysates and the supernatant were subjected to SDS-PAGE, and separated polypeptides were electroblotted to polyvinylidene difluoride membranes. The blots were probed with anti-E1alpha , stripped of the antibody, and reprobed with anti-E1beta . A, crude lysates. B, supernatants.

Expression and Assembly State of Wild-type and Mutant E1 Proteins-- The co-purification of (His)6-E1alpha with untagged E1beta cannot discern the assembly state of assembled E1 subunits. To address this question, E. coli cells in the C2 broth medium which expressed wild-type E1 subunits were grown for 20 h. E1 subunits in cells harvested at different induction times were purified by Ni2+-NTA resin and subsequently subjected to size fractionation on a TSK-G3000SWXL column by HPLC. The molecular weight of assembled E1 subunits species is inversely proportional to the retention time on HPLC. Fig. 4A shows that (His)6-E1alpha and untagged E1beta subunits form mostly alpha beta dimers with molecular mass in the 80-kDa range at the 1-h induction time. The Ni2+-NTA extractable alpha beta dimers are the predominant species with the appearance of a minor species of alpha 2beta 2 tetramers (165 kDa) when induced for 2 h. Conversely, at the 3- and 20-h induction times the major species is alpha 2beta 2 tetramers and the minor species alpha beta dimers. The data indicate that (His)6-E1alpha and E1beta subunits initially form the alpha beta dimers which later slowly dimerize to produce alpha 2beta 2 tetramers. The fractions collected at different retention times from HPLC were analyzed for E1 activity using an assay mixture containing added excess lipoylated recombinant E2 and recombinant E3, which allowed one to measure reconstituted BCKD activity. This radiochemical assay for BCKD activity is 15-fold more sensitive than the one measuring the E1 component activity alone. As shown in Fig. 4B, the fraction collected at the 9-min retention time, which corresponds to the alpha 2beta 2 tetramer in the 20-h induction lysate, contains the peak enzyme activity. The fractions corresponding to alpha beta dimers with a retention time of 10.3 min in 2- and 3 h-induction lysates do not have enzyme activity.


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Fig. 4.   Assembled wild-type E1 species during 20 h induction in the C2 broth minimal medium. CG712 E. coli cells co-transformed with the pHis T-E1 and the pGroESL plasmids were induced for the expression of wild-type (His)6-E1alpha and E1beta subunits with IPTG in the C2 broth minimal medium. Cells were harvested at different times during the 20 h induction. Assembled E1 subunits were extracted from the cell lysate with Ni2+-NTA resin. Purified E1 species in 50 mM potassium phosphate, pH 7.5, 0.2 mM EDTA, and 250 mM KCl were subjected to size fractionation by HPLC on a TSK-G3000SWXL column. Fractions collected at different retention times were analyzed by SDS-PAGE and Coomassie Blue staining. E1 activity in eluted fractions was assayed using a reconstituted system with addition of recombinant E2 and recombinant E3. Radiolabeled alpha -keto-[1-14C]isovalerate was used as a substrate, and enzyme activity of the BCKD complex was measured. A, elution profile of assembled E1 species. The molecular mass markers (indicated on top) are GroEL (840 kDa), thyroglobulin (669 kDa), catalase (232 kDa), aldolase (158 kDa), and GroES (70 kDa). B, BCKD activity in fractions collected at different retention times. black-square, reconstituted E1 activity after 2 h induction; black-triangle after 3 h induction; bullet , after 20 h induction.

Despite the slow assembly of mutant E1alpha subunits with normal E1beta as determined by pulse-chase labeling, prolonged induction with IPTG in E. coli grown in the YTGK medium resulted in the production of mutant E1 proteins. Bacterial lysates prepared from E. coli cells after the 16-h induction at 37 or 28 °C were purified by Ni2+-NTA column, followed by gel filtration on Sephacryl S-100 column. The purified wild-type and mutant E1 proteins were subjected to sucrose density gradient centrifugation to analyze their subunit assembly state. Fig. 5 depicts the sedimentation profiles of E1 proteins as determined by SDS-PAGE analysis of gradient fractions after the centrifugation. The wild-type E1 protein induced at 37 °C migrated as a tetrameric species of 165 kDa with the alpha 2beta 2 structure (fractions 7-9). The trace amounts of E1alpha and E1beta subunits at the bottom of the gradient were the result of slight aggregation that occurred during the centrifugation. In contrast, the Y393N-alpha mutant E1 expressed at 37 °C migrated as an alpha beta dimeric species with a molecular mass of 83 kDa (fractions 3-6). The mutant E1 with the F364C-alpha mutation also occurred entirely as alpha beta dimers in sucrose density gradient centrifugation (data not shown). Interestingly, the mutant T265R-alpha , when expressed at 28 °C, was able to remain soluble and assemble with the normal E1beta subunit. The sedimentation profile indicated that T265R-alpha mutant E1 migrated predominantly as tetramers, although lesser amounts of lower molecular weight species were present and sedimented in early gradient fractions. Similarly, the mutant E1 bearing the Y368C-alpha mutation sedimented as approximately equal amounts of both tetramers and lower molecular weight species when expressed at 37 °C. The mutant E1 proteins carrying the remaining E1alpha mutations (R220W-alpha , N222S-alpha , G245R-alpha , and A240P-alpha ) were present only as tetramers (Table I). The assembly state of the above wild-type and mutant E1 proteins was confirmed by the elution profiles from TSK-G3000SWXL sizing column on HPLC and Sephacryl S-100 column (data not shown).


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Fig. 5.   Assembly state of wild-type and mutant E1 proteins as analyzed by sucrose density centrifugation. Wild-type and mutant E1 carrying different mutations in the E1 subunits were expressed in co-transformed CG712 cells and purified by Ni2+-NTA extraction, followed by gel filtration on Sephacryl S-100 HR column. Purified wild-type and mutant E1 protein were fractionated on 10-25% 10 ml of sucrose density gradient, which was spun at 210,000 × g in a Beckman SW-41 rotor for 18 h. Fractions (0.7 ml each) were collected from top to bottom and analyzed by SDS-PAGE and Coomassie Blue staining. The molecular mass markers used were as follows: bovine serum albumin (68 kDa), E3 (110 kDa), E1 tetramers (165 kDa), and GroEL (840 kDa).

                              
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Table I
Assembly state and specific activities of wild-type and mutant E1 proteins
Wild-type and mutant E1 proteins were expressed at 37 °C in E. coli grown on the YTGK medium, except for the T265R-alpha E1, which was expressed at 28 °C. Recombinant proteins were purified by Ni2+-NTA affinity column and gel filtration on Sephacryl S-100 column. The purified wild-type and mutant E1 proteins were analyzed for the subunit assembly state by sucrose density gradient centrifugation as described in the legend to Fig. 5. E1 activity was assayed using the radiochemical assay with 2,6-dichlorophenol indophenol as an electron acceptor.

Activity Levels and Stability of Wild-type and Mutant E1 Proteins-- Wild-type and mutant E1 proteins produced in E. coli grown on the YTGK medium were purified by Ni2+-NTA affinity column and gel filtration on Sephacryl S-100 column. The E1 activity of normal and mutant proteins was assayed by the radiochemical method with 2,6-dichlorophenol indophenol as an electron acceptor. As shown in Table I, only the mutant E1 carrying N222S-alpha or G245R-alpha has residual E1 activity (1.37 and 2.66% of normal activity, respectively). The mutant E1 proteins containing each of the remaining seven E1alpha mutations do not have detectable E1 catalytic activity.

The thermal stability of the above purified normal and mutant E1 proteins was studied by heat denaturation at 50 °C. Light scattering at 360 nm as a result of protein aggregation was monitored. The fraction of soluble proteins was calculated from the progress curve and expressed as log % values versus the incubation time. Fig. 6 shows that the wild-type E1 at 1.21 µM in the presence of 2 mM TPP is most stable with a denaturation rate constant of 0.08 min-1. Similar denaturation rate constants were obtained at 0.97, 0.48, 0.24, and 0.12 µM concentrations of E1 (data not shown). The data support the thesis that the aggregation of E1 is caused by heat-induced conformational changes rather than high concentrations of E1. In the absence of TPP, the denaturation rate constant of wild-type E1 increased to 0.19 min-1. TPP had no effect on the thermal stability of mutant E1 proteins. Among the nine mutant E1 proteins studied, the one containing N222S-alpha was more stable than wild-type E1 (in the absence of TPP) with a denaturation rate constant of 0.14 min-1. The mutant E1 protein carrying the remaining E1alpha mutations are less stable than the wild-type E1 (±TPP). The mutant E1 that harbors the T265R-alpha mutation is the least stable with a denaturation rate constant of 4.45 min-1.


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Fig. 6.   Rates of thermal aggregation of wild-type and mutant E1 proteins at 50 °C. Purified wild-type and mutant E1 proteins (100 µg) were added to 0.5 ml of 50 mM KPi, pH 7.5, 250 mM KCl, 10% glycerol, 0.5 mM beta -mercaptoethanol, and 0.2 mM EDTA preheated to 50 °C in a 2 × 10-mm cuvette. Aggregation at 50 °C was monitored by light scattering at 360 nm wavelength as a function of time. The plateau of the progressive curve is considered 100% aggregation and was used to calculate percent soluble E1 at a given time point. The slope of percent soluble E1 versus time was used to calculate the rate constant, kobsd. bullet , wild type (+TPP); open circle , wild type (no addition); ×, G204S; black-square, R220W; square , N222S; black-triangle, A240P; triangle , T265R; down-triangle, G245R; black-down-triangle , Y393N.

The susceptibility of wild-type E1 tetramers and Y393N-alpha E1 dimers to proteolysis was also studied. The E1 proteins were incubated with different concentrations of trypsin (protein/trypsin = 250 to 25,000:1, w/w) at 0 °C for 20 min, followed by termination of the digestion with 10 mM PMSF. Fig. 7 shows that Y393N-alpha dimers are markedly more susceptible to the tryptic digestion than wild-type tetramers as analyzed by SDS-PAGE.


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Fig. 7.   Susceptibility of wild-type E1 tetramer and Y393N-alpha E1 dimers to tryptic digestion. Purified wild-type E1 tetramers or Y393N-alpha E1 dimers at 50 µg/0.1 ml were incubated with trypsin for 20 min on ice at the trypsin-to-E1 ratio (w/w) indicated. The digestion was terminated by addition of PMSF (10 mM final concentration). The sample was analyzed by SDS-PAGE and Coomassie Blue staining.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The major focus of this investigation is to characterize the effect of human E1alpha mutations in type IA MSUD on the assembly and stability of mutant E1 proteins. For these studies, we have developed efficient bacterial expression systems for folding and assembly of E1 alpha 2beta 2 tetramers. We showed previously that co-expression of mature MBP-E1alpha and E1beta sequences of the human BCKD complex in the same E. coli cells is essential for MBP-E1 assembly; however, the yield was very low (12). Co-transformation of a second plasmid that overexpressed GroEL and GroES into the same E. coli cell resulted in a 500-fold increase in the yield of active MBP-E1 tetramers (9). In the present study, a (His)6 affinity tag is fused to the mature E1alpha N terminus through a TEV protease recognition site. Co-transformation with the pGroESL plasmid was also found necessary and sufficient for productive folding and assembly of (His)6-E1. The results argue against the suggestion that the dependence of human E1 on chaperonins for a high yield is due to the presence of the MBP sequence in the chimeric E1alpha polypeptide (23). Our recent in vitro refolding results indicate that the reconstitution of untagged E1, MBP-E1, and (His)6-E1 show the same chaperonin-dependent kinetics.2 The findings further established that productive folding and assembly of mature human E1 have an absolute requirement for enrichment for chaperonins GroEL and GroES and are not affected by the presence of affinity tags. Thus, the pulse-chase labeling protocol developed in this study provides the first approximation of the rate of E1 subunit assembly under optimal conditions through the augmentation of bacterial chaperonins that are homologue of mitochondrial chaperonins Hsp60 and Hsp10, respectively.

The wild-type and mutant human E1alpha and the wild-type E1beta subunits are expressed at relatively equal efficiencies and are stable, as indicated by Western blotting of the total crude lysates prepared 12 h after induction (Fig. 3A). This allows one to follow the fates of E1alpha and E1beta subunits synthesized during the 1-min window of pulse-labeling. The presence of the (His)6-tag in the wild-type and mutant E1alpha subunits facilitates the isolation of the 35S-labeled subunit. One can measure the kinetics of the E1beta assembly with the wild-type and mutant E1alpha subunits by the co-purification of the untagged E1beta with (His)6-E1alpha with the Ni2+-NTA resin as a function of time. The equally strong signals of E1alpha and E1beta subunits during the 2-h chase (Fig. 2A) indicate that both subunits are efficiently synthesized. The total numbers of cysteine and methionine residues in the E1alpha and E1beta subunits are similar, which are 16 and 15, respectively. The autoradiogram of the co-purified E1 subunits as separated by SDS-PAGE cannot discern the assembly state of the associated subunits. However, size fractionation of the pulse-chase labeled products by HPLC show that the wild-type E1alpha and E1beta assemble during the 2-h chase occur predominantly as inactive alpha beta dimers, which are later converted to active alpha 2beta 2 tetramers (Fig. 4, A and B). It is noteworthy that a significant amount of the wild-type alpha beta dimeric intermediate was observed only when E. coli cells were grown on the C2 broth minimal medium. When the bacterial cells were cultured on the YTGK medium, wild-type E1 was expressed predominantly as alpha 2beta 2 tetramers (Fig. 5) with little or no accumulation of alpha beta dimers during the 16-h induction period (data no shown). The factors responsible for the apparent effects of culture media on the accumulation of the wild-type dimeric intermediate during E1 assembly are currently unknown. The major differences between the two bacterial culture media lie in the fact that the C2 minimal medium is low in the content of SO42- and amino acids when compared with the YTGK medium (18). Possible effects of these ingredients on the dimerization of wild-type alpha beta dimers are under investigation.

It is of interest that a weak GroEL signal co-purifies with wild-type (His)6-E1alpha and E1beta at 120 min into the chase (Fig. 2A). This apparent ternary complex is also observed in the early fractions of the Imidazole gradient during purification of wild-type (His)6-E1 (Fig. 1). The GroEL-E1alpha -E1beta ternary complex is a productive intermediate at a later step of the chaperonin-mediated assembly of E1 alpha 2beta 2 tetramers.2 Only a GroEL-E1alpha binary complex is observed in F364C-alpha because the assembly of E1beta with mutant E1alpha did not occur within the 2-h chase. The weak and sub-stoichiometric signal of GroEL relative to E1 subunits is a result of isotopic dilution by the overabundance of unlabeled GroEL in E. coli.

The assembled mutant alpha beta dimers were produced in E. coli grown on the YTGK medium after the 16-h induction with "slow assembly" E1alpha mutants including Y393N-alpha and F364C-alpha . The results indicate that this group of mutations not only reduces the rate of the assembly of the mutant E1alpha with normal E1beta but also prevents conversion of the dimeric assembly intermediate into the stable alpha 2beta 2 structure of wild-type E1. The production of these mutant alpha beta dimers apparently is not affected by growth media, as the expression of mutant E1 carrying these mutations in E. coli grown in the C2 broth minimal medium also resulted in the expression of exclusive dimers. In vitro reconstitution of the 6 M urea-denatured mutant Y393N-alpha E1 in the presence of chaperonins GroEL and GroES also resulted exclusively in mutant alpha beta dimers.2 The unstable Y393N-alpha dimeric intermediate as demonstrated by its propensity for thermal aggregation and proteolytic digestion compared with the wild-type tetramer explains the markedly reduced levels E1alpha and E1beta subunits in cells from Mennonite MSUD patients homozygous for the Y393N-alpha mutation (24, 25). The present study establishes that C-terminal aromatic residues (F364-alpha , Y368-alpha , and Y393-alpha ) in the E1alpha subunit are crucial for proper E1 assembly. The important roles of the C terminus in subunit assembly and protein interactions have been demonstrated. For example, the C-terminal 25 amino acid residues of the herpes simplex virus type 1 UL26.5 protein are required for the assembly of the icosahedral capsid shell (26). In the case of the E2 core of the related pyruvate dehydrogenase complex from Azotobacter vinelandii, the C-terminal residues 632-637 comprise a 310-like helix (H6) which acts as a "hydrophobic knob" that fits into a "hole" in the 2-fold related subunit to produce the 24-mer cubic assembly (27). Introduction of a polyhistidine tag into the C terminus of BCKD-E2, which is highly homologous to the bacterial pyruvate dehydrogenase-E2, results in the formation of stable trimers instead of the native 24-mer structure (data not shown).

The T265R-alpha subunit, when expressed at 37 °C, was largely insoluble even in the presence of excess chaperonins GroEL and GroES. This was indicated by the rapid disappearance of the mutant E1alpha signal in the soluble fraction during the 2-h chase (Fig. 2A); however, the level of the T265R-alpha subunits in total crude lysates was comparable to that of wild type (Fig. 3A). These results strongly suggest that the T265R-alpha residue is important for proper folding of the E1alpha subunit. It is also of interest that lowering of the induction temperature from 37 to 28 °C resulted in the production of a significant amount of assembled mutant E1 protein carrying the T265R-alpha mutation. The yield of the mutant E1 was 5 mg/liter culture at 28 °C compared with 20-40 mg/liter culture for the wild-type E1 expressed at 37 °C (data not shown). The finding is consistent with the thesis that lowering the expression temperature slows the folding kinetics of the nascent peptide, thereby reducing the probability of the off-pathway folding reactions, as demonstrated by the expression of rabbit muscle phosphorylase (28) and the human E1beta (13). However, the assembled T265R-alpha mutant E1 has a grossly altered conformation, which renders it very unstable as indicated by its most rapid thermal aggregation at 50 °C among the nine E1alpha mutants studied. This unstable conformation is also manifested by the apparent dissociation of the mutant tetramers to lower molecular weight species as detected by sucrose density gradient centrifugation. The current data indicate that the T265R-alpha residue also plays a key role in subunit interactions and are consistent with the location of this residue at the putative subunit-interaction site conserved between BCKD and pyruvate dehydrogenase E1 proteins (29).

The crystal structure of the E1 alpha 2beta 2 has not been solved, but structures are known for the related TPP-dependent proteins transketolase (30) and pyruvate decarboxylase (31) from Saccharomyces cerevisiae and pyruvate oxidase from Lactobacillus plantarum (32). The transketolase is a homodimer, whereas the human E1 is a tetramer made up of two non-identical subunits. Sequence alignment between the two enzymes shows that the highly conserved TPP-binding pocket in E1 is composed of residues from both E1alpha and E1beta subunits (33, 34). Aromatic residues from E1beta form a hydrophobic pocket to accommodate the pyrimidium and thiazolium rings of cofactor TPP. On the other hand, the highly conserved TPP-binding motif GDG(X)22-28NN, which was first described by Hawkins et al. (35) and is essential for binding the pyrophosphate moiety, is located in the E1alpha subunit (Fig. 8). It should be mentioned that a D440E mutation introduced via mutagenesis into this motif in pyruvate decarboxylase from Zymomonas mobilis yielded a homodimeric enzyme with reduced affinity for TPP, in contrast to the wild-type enzyme which exists as a homotetramer (36). It was proposed that deficient TPP binding may have caused a failure in the conversion of the mutant dimeric forms into native tetramers. However, the occurrence of alpha beta dimers in the mutant E1 carrying Y393N-alpha and F364C-alpha substitutions is likely through a different mechanism, since these residues are not involved in TPP binding and are located in the C-terminal region. G204S-alpha , R220W-alpha , and N222S-alpha that are affected in type IA MSUD are residues within the TPP-binding motif (Fig. 8). Specifically, N222S-alpha aligns with Asn-187 in the yeast transketolase and provides a ligand to this pentameric coordination involving the Mg2+ cation. The N222S-alpha mutation conceivably disrupts the pentameric coordination, resulting in the inability of E1 to bind the pyrophosphate moiety of TPP and the loss of E1 catalytic function. However, the N222S-alpha mutation is without effect on the assembly of the mutant E1alpha with normal E1beta , as determined by pulse-chase labeling (Fig. 2A). The R220W-alpha mutation, which is also located in the pyrophosphate moiety binding site, also has no adverse effect on E1 subunit assembly. In contrast, G204S-alpha mutation, which is presumably located at the interface between the two non-identical subunits of E1, based on the yeast transketolase structure (37), impedes the assembly of the mutant E1alpha subunit with the normal E1beta unit.


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Fig. 8.   Sequence alignment of the GDG (X)22-28 NN motif in different TPP-dependent enzymes. Sequences of the GDG(X)22-28 NN motif for binding the pyrophosphate moiety of cofactor TPP in the nine selected cofactor-dependent enzymes were searched and aligned using an advanced version of BLASTP 2.0.3 employing the SWISS-PROT protein data base (38). Residue numbers for the N and C termini of the motif in each sequence are indicated. Residues that are boxed are absolutely conserved in all TPP-dependent enzymes. The abbreviations used are: BDH, branched-chain alpha -ketoacid dehydrogenase; KDH, alpha -ketoglutarate dehydrogenase; TNK, transketolase; PDC, pyruvate decarboxylase; PDH, pyruvate dehydrogenase; POX, pyruvate oxidase.

    FOOTNOTES

* This work was supported in part by Grant DK26758 from the National Institutes of Health, Grant I-1286 from the Welch Foundation, and Grant 95G-074 from American Heart Association, Texas Affiliate.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Medical Scientist Trainee supported by National Institutes of Health Grant 5-P32 GM08014 and by the Perot Family Foundation.

§ To whom correspondence should be addressed: Dept. of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9038. Tel.: 214-648-2457; Fax: 214-648-8856; E-mail: chuang01{at}utsw.swmed.edu.

1 The abbreviations used are: BCKD, branched-chain alpha -ketoacid dehydrogenase; E1, branched-chain alpha -ketoacid decarboxylase; E2, dihydrolipoyl transacylase; E3, dihydrolipoamide dehydrogenase; HPLC, high performance liquid chromatography; Hsp, heat shock protein; IPTG, isopropyl beta -D-thiogalactopyranoside; KPi, potassium phosphate; MBP, maltose-binding protein; MSUD, maple syrup urine disease; NTA, nitrilotriacetic acid; PMSF, phenylmethylsulfonyl fluoride; PAGE, polyacrylamide gel electrophoresis; TEV, tobacco etch virus; TPP, thiamine pyrophosphate.

2 J. L. Chuang, R. M. Wynn, and D. T. Chuang, manuscript in preparation.

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Top
Abstract
Introduction
Procedures
Results
Discussion
References

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