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J. Biol. Chem., Vol. 279, Issue 17, 17792-17800, April 23, 2004

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Structural and Biochemical Basis for Novel Mutations in Homozygous Israeli Maple Syrup Urine Disease Patients

A PROPOSED MECHANISM FOR THE THIAMIN-RESPONSIVE PHENOTYPE^*

Jacinta L. Chuang, R. Max Wynn¶, Clint C. Moss, Jiu-li Song, Jun Li, Nibal Awad||, Hanna Mandel||**, and David T. Chuang¶**

From the Departments of Biochemistry and ^¶Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390 and ^||Metabolic Unit, Meyers Children's Hospital, Rambam Medical Center, Technion Faculty of Medicine, Haifa 31906, Israel

Received for publication, December 18, 2003 , and in revised form, January 23, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Maple syrup urine disease (MSUD)¹ or branched-chain -ketoaciduria is an autosomal recessive metabolic disorder caused by deficiency in the mitochondrial branched-chain -ketoacid dehydrogenase (BCKD) complex. The BCKD complex catalyzes the oxidative decarboxylation (Reaction 1) of three branched-chain -ketoacids (BCKAs) derived from branched-chain amino acids (BCAAs) leucine, isoleucine, and valine (1).

The metabolic block in the BCKD complex results in the inability of MSUD patients to degrade BCKA. The elevated BCKA levels produce severe clinical consequences including often-fatal ketoacidosis, mental retardation, and neurological impairment. There are presently five known MSUD clinical phenotypes (i.e. classic, intermediate, intermittent, thiamin-responsive, and E3-deficient forms) (2). The classic form with the most severe phenotype, was originally reported by Menke et al. in 1954 (3) and manifests within the first 2 weeks of life by poor feeding, lethargy, seizures, coma, and death if left untreated. The classic form accounts for 75% of MSUD patients (1). Intermediate MSUD is associated with progressive mental retardation and developmental delay without a history of catastrophic illness. An intermittent form of MSUD has normal levels of BCAA as well as normal intelligence and development until a stress such as infection precipitates metabolic decompensation with ketoacidosis. Thiamin-responsive MSUD is similar to the intermediate or intermittent phenotype but responds to pharmacological doses of thiamin with normalization of the BCAA levels (2). The E3-deficient MSUD is caused by defects in the dihydrolipamide dehydrogenase (E3) component of the BCKD complex, which is common to that of the pyruvate and -ketoglutarate dehydrogenase complexes. Patients with E3 deficiency display a combined dysfunction of the three -ketoacid dehydrogenase complexes (4). Two prevailing mutations (G229C and Y335stop) in the E3 gene have been identified in the Ashkenazi Jewish community in Israel (5). Most of the Israeli patients who were homozygous for the G229C E3 mutation presented in early childhood or later with recurrent episodes of vomiting, encephalopathy, and prolonged prothrombin time, occasionally associated with lactic acidosis and ketoacidosis. Patients who were compound-heterozygous for these two mutations presented neonatally with more severe sequelae.

The mammalian BCKD multienzyme complex is a 4 x 10⁶-dalton metabolic machine organized around a cubic core comprising 24 lipoate-bearing dihydrolipoyl transacylase (E2) subunits, to which multiple copies of branched-chain -ketoacid decarboxylase (E1), E3, a specific kinase, and a specific phosphatase are attached through ionic interactions. The kinase and the phosphatase are responsible for the regulation of BCKD complex by a reversible phosphorylation (inactivation)/dephosphorylation (activation) cycle (6). The E1 component is a thiamin diphosphate (ThDP)-dependent enzyme consisting of two E1 and two E1 subunits. The E3 component is a homodimeric flavoprotein. There are in total six genetic loci that encode subunits of the BCKD complex. Mutations in the four different catalytic subunits (E1, E1, E2, and E3) have been described in MSUD patients (2). Genetic subtypes of MSUD have been proposed to indicate the altered subunit in the BCKD complex (2). These include type IA MSUD affecting the E1 subunit, type IB affecting the E1 subunit, type II affecting the E2 subunit, and type III affecting the E3 subunit. Types IV and V, which have not been reported, are reserved for MSUD in which the kinase and the phosphatase, respectively, are affected. Except for type II and III MSUD, which are linked to the thiamin-responsive (2, 7, 8) and E3-deficient phenotypes (4), respectively, a tight correlation between a specific genetic subtype and a particular clinical phenotype of MSUD has not been demonstrated.

Reaction steps catalyzed by the three enzyme components, based largely on those elucidated for the related pyruvate dehydrogenase complex (9), are as follows.

The E1 component binds ThDP and catalyzes a ThDP-mediated decarboxylation of -ketoacids (Reaction 2), and subsequent reduction of the lipoyl moiety, which is covalently attached to E2 (Reaction 3). The lipoyl-bearing domain (LBD) carrying the S-acyldihydrolipoamide serves as a "swinging arm" (10) to transfer the acyl group from E1 to the E2 active site giving rise to acyl-CoA (Reaction 4). Finally, the E3 component with a tightly bound FAD moiety reoxidizes the dihydrolipoyl residue on E2 (Reaction 5) with NAD⁺ as the ultimate electron acceptor (Reaction 6). The overall reaction is the production of branched-chain acyl-CoA, CO₂, and NADH from BCKAs (Reaction 1).

We previously described two novel missense mutations in MSUD patients from the non-Jewish Druze kindred in Israel (11). The incidence of MSUD in Israel is relatively high, presumably as a result of consanguinity (12). Toward the goal of providing a wider spectrum of MSUD mutations in this region, we continue to analyze cell cultures derived from Israeli patients. In the present study, we report seven additional MSUD mutations in the Israeli population, which affect the E1 (type IA), E1 (type IB), and E2 (type II) subunits of the BCKD complex. Among them are novel type IA and type IB homozygous missense mutations that impede subunit assembly of the E1 component and nullify ThDP-mediated decarboxylation of BCKAs. A type II homozygous point mutation H391R in the E2 subunit involves the key catalytic residue that functions as a general base in the acyltransfer reaction catalyzed by E2 (13, 14). A biochemical mechanism is proposed to explain the unequivocal thiamin-responsive phenotype presented by the Israeli type II MSUD patients. The genetic and biochemical information presented here provides new insights into structure and function of the human BCKD complex, and will facilitate DNA-based detection of these MSUD alleles in the Israeli population.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell Lines and Cell Cultures—Blood samples (15 ml) were withdrawn from classic MSUD patients B.R, W.A., D.M., K.Y., T.G., and C.R. as well as Druze thiamin-responsive patients H.C. and H.S., all from Israel. Lymphoblasts were prepared from blood samples by infection with the Epstein-Barr virus, and lymphoblastoid cell cultures were grown and assayed for the decarboxylation of -keto[1-¹⁴C]isovalerate by intact cells as described previously (15).

Nucleotide Sequencing for MSUD Mutations—The first-strand E1 cDNA was synthesized from the total RNA prepared from patients' cells using the primer 5'-GAAGACAGTGGTGTGCTGTC-3' (cDNA sequence 1461-1442) and the Omniscript^TM reverse transcriptase from Qiagen (Chatsworth, CA). The subsequent PCR amplification was carried out using the forward primer 5'-CGGACCGCTGAGTGGTTG-3' (positions 1-18) and the reverse primer 5'-CTTAGAGTGGGGCTACCTCTCG-3' (positions 1425-1404). For the first-strand E1 cDNA synthesis, the primer 5'-GTAGAACTTTTCAGCCAATATCATGATGG-3' (positions 1367-1339) was employed. The first round PCR was carried out using the forward primer 5'-GTGCGGCTGCATAGCCTGAG-3' (positions 8-27) and the reverse primer: 5'-AAAAGAGGTAAGTCGGAGGA-3' (positions 1312-1293). To amplify the 5' segment of the E1 cDNA, a second round PCR was performed using the forward primer 5'-ATGGCGGTTGTAGCGGC-3' (positions 48-64) and the reverse primer 5'-CCAGGCAACTAGAGTAACATC-3' (positions 878-858). The 3' region of the E1 cDNA was amplified in a parallel second round PCR utilizing the forward primer 5'-ATACCCCATTGTGTGAACAAGGAATTGTTG-3' (positions 409-438) and as a reverse primer the above first-stand primer for the E1 cDNA. To synthesize the first-strand E2 cDNA, the primer 5'-CAGCTAGGGTTTACATACTC-3' (positions 1523-1504) was utilized. The ensuing PCR amplification was performed with the forward primer 5'-TCCGGGGTAAGATGGCTG-3' (positions 5-22) and the reverse primer 5'-GCTCAAAAAGTTCAAGAATGTCTTATCAGT-3' (positions 1496-1467). PCR products were sequenced with an ABI Prism^TM model 377 automated DNA sequencer manufactured by Applied Biosystems (Foster City, CA).

Production of Wild-type and Mutant Proteins—The pTrcHisB expression plasmid (Invitrogen) for N-terminally His₆-tagged wild-type E1 was constructed as described previously (16). The same plasmids carrying MSUD mutations were produced using the QuikChange^TM site-directed mutagenesis kit from Stratagene (La Jolla, CA) according to the manufacturer's instructions. Wild-type and mutant E1 proteins were expressed in Escherichia coli by co-transformation with the pGroESL plasmid over expressing chaperonins GroEL and GroES, as described previously (17, 18). His₆-tagged wild-type and mutant E1 were purified with Ni²⁺-nitrilotriacetic acid resin and Resource Q and Superdex-200 columns (17) and used for biochemical studies without removal of the His₆ tag. The E2 24-mer was expressed in XL1-Blue cells and lipoylated in vitro with bacterial lipoyl ligase as described previously (19). C-terminal His₆-tagged E2 domain constructs LBD2 (residues 1-99) containing the lipoyl domain and the C-terminal linker and the di-domain (DD) (residues 1-167) containing the LBD and subunit-binding domain (SBD) were prepared as described previously (20). Lipoylated LBD2 (lip-LBD2) and DD (lip-DD) were prepared as described above. To produce the SBD, a tobacco-etch virus protease site (LENLYFQS) with a nucleotide sequence of 5'-ctcgagaatctttattttcaatca-3' was inserted into the linker between the lipoyl-bearing and subunit binding domains. The purified C-terminally His₆-tagged DD was digested with the tobacco-etch virus protease, and the SBD was extracted by Ni²⁺-nitrilotriacetic acid resin.

Assay for Activity of the Reconstituted BCKD Complex—The BCKD complex was reconstituted with E1, lipoylated E2 (lip-E2), and E3 at a molar ratio of 12:1:55, in which lip-E2 exists as a 24-mer as described previously (19). The assay mixture contained 30 mM potassium phosphate, pH 7.5, 100 mM NaCl, 3 mM NAD⁺, 0.4 mM CoA, 2 mM MgCl₂, 2 mM dithiothreitol, 0.1% Triton X-100, 2 mM ThDP, and 4 mM sodium -ketoisovalerate. The oxidative decarboxylation of -ketoisovalerate catalyzed by the reconstituted complex (Reaction 1) at 30 °C was monitored by the increase in absorbance at 340 nm.

Assays for Activities of Individual E1 and E2 Components—The decarboxylation catalyzed by the isolated E1 component (Reaction 2) was assayed with -ketoisovalerate as a substrate in the presence of an artificial electron acceptor 2,6-dichlorophenolindophenol (DCPIP) (Re-action 7) as described previously (21).

The assay mixture contained 100 mM potassium phosphate, pH 7.5, 2.0 mM MgCl₂, 0.2 mM ThDP, and 0.1 mM DCPIP. The rate of decarboxylation at 30 °C was measured by monitoring the reduction of the dye at 600 nm. The acyltransferase activity (Reaction 4) of E2 was assayed using a model reaction (Reaction 8) with [1-¹⁴C]isovaleryl-CoA and dihydrolipoamide (Lip(SH)₂) as substrates.

The assay mixture contained 100 mM MOPS, pH 7.5, 2.5 mM DL-dihydrolipoamide, and 2.5 mM [1-¹⁴C]isovaleryl-CoA. The reaction product S-[1-¹⁴C]isovaleryl dihydrolipoamide (R-S-Lip-SH) was extracted with benzene as also described previously (22).

Western Blotting of Cell Lysates—Homogenates from cultured lymphoblasts were subjected to SDS-PAGE, followed by transfer to Immobilon-P membranes (Millipore Corp.). After blocking with a phosphate-buffered saline containing 5% (w/v) nonfat dry milk, these membranes were probed with either anti-E2 or anti-E1 (with titers against both E1 and E1 subunits) antibodies each at a 1:500 dilution. Unbound antibodies were removed by washing the membranes with the same buffer containing 0.05% (v/v) Tween 20 and 0.05% (v/v) Nonidet P-40. The bound antibodies were detected with ¹²⁵I-protein A as described previously (15).

Binding Measurements Based on Tryptophan Fluorescence Quenching—Steady-state fluorescence quenching upon ThDP binding (23) to wild-type and mutant E1 was measured using a Perkin-Elmer Life Sciences LS50 B luminescence spectrometer as described previously (24). Fluorescence intensities were recorded at 25 °C at an excitation wavelength of 290 nm and an emission wavelength of 335 nm. Slit widths were set at 5 nm for both excitation and emission. A 290-nm cut-off emission filter was installed to reduce light scattering effects. Protein concentrations for E1 (A₂₈₀ = 1.14 mg^-1 ml·cm^-1) and ThDP (A₂₃₅ = 11,300 M^-1 cm^-1,pH >7.0) were determined spectrophotometrically. The concentration for all protein samples was 0.23 µM (as heterotetramers) in 50 mM potassium phosphate buffer, pH 7.5, 200 mM KCl, and 1 mM MgCl₂. The binding data were fitted by nonlinear regression using the program KaleidaGraph (Synergy Software, Essex Junction, VT) according to Equation 1 describing a bimolecular reaction (25),

(Eq. 1)

where F represents the fluorescence change corrected for dilution and inner filter effects (26), F₀ is the fluorescence intensity prior to the addition of ThDP, F_max is the maximal fluorescence change, K_d is the dissociation constant, and [ThDP] is the concentration of ThDP in the cuvette. The parameters determined by the fitting procedure were F_max and K_d.

Treatment of Mutant E1 Proteins with Natural Osmolyte Trimethylamine N-Oxide—Wild type or mutant E1 proteins (200 µg/ml) were incubated with different concentrations of trimethylamine N-oxide (TMAO) at 23 °C for 16 h in buffer A that contained 50 mM potassium phosphate, pH 7.5, 100 mM KCl, 2 mM ThDP, 2 mM MgCl₂, 10 mM dithiothreitol, 0.6 M lysine, and a mixture of protease inhibitors (Roche Applied Science, Indianapolis, IN). Activity of the BCKD complex reconstituted with TMAO-treated wild-type or mutant E1 was assayed as described above.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Clinical Phenotypes—B.R., W.A., D.M., and K.Y. of Israeli origin manifested the classic MSUD phenotype as described above. T.G. and C.R. were from the Druze kindred in Israel, who suffered also from the classic MSUD phenotype with a turbulent neonatal presentation. Both exhibited mild to moderate developmental retardation with leukodystrophy in brain MRI. Furthermore, both patients had recurrent encephalopathic episodes during viral infections or suspected dietary noncompliance. T.G. underwent hemodialysis at age 2 and 4, which may have also resulted from dietary noncompliance.

H.C. is the third child of first generation Arabs. He was delivered uneventfully after a normal pregnancy. The parents reported irritability of the child since the first week of life. Brain MRI reveals leukodystrophy, which in combination with elevated levels of BCAAs led to the diagnosis of MSUD. The patient underwent hemodialysis and was placed on a MSUD formula supplemented with thiamin at the dosage of 100 mg/day. The BCAA levels decreased to normal after 3 days. On follow-ups, the plasma BCAA levels remained in the normal range (50-100 nmol/ml) for the next 4 years. The patient consumed 4 times the leucine levels that were allowed for MSUD patients, but during acute infections his BCAA and alloisoleucine levels rose only slightly above the normal range. At age 5, he had spastic cerebral palsy and his IQ was 80. H.S. was also born uneventfully after a normal pregnancy. Parents of H.S. and H.C. were first-degree cousins; therefore, dietary restriction was instituted for H.S after birth. She was diagnosed with MSUD at 2 days of age, and thiamin supplement (100 mg/day) was added to the restricted diet. At 1 week of age, H.S. was discharged and given breast milk at home. Three days later, she was readmitted with vomiting, apathy, and encephalopathy with the plasma leucine level at 1,800 nmol/ml, and underwent hemodialysis. Since this episode, the regiment of restricted diet with thiamin supplements was reinstated and maintained. At 18 months of age, H.C. showed normal growth and development.

Identification of the Affected BCKD Subunit in MSUD Patients—Since the human BCKD complex consisted of six different subunits, it was necessary for mutational analysis to identify which subunit was affected in the MSUD patient of interest. Homogenates of cultured lymphoblasts from the eight Israeli patients were subjected to Western blotting using antibodies against E1 (both E1 and E1 subunits) or E2 as a probe. Cells from the classic patient B.R. showed reduced levels of both the E1 and the E1 subunit, with the level of the E2 subunit in the normal range. Levels of both E1 and E1 subunits were below the detection limit in cells from classic patients W.A., D.M., and K.Y. The data strongly suggested that either the E1 or the E1 locus was affected in these Israeli MSUD patients. Individual E1 subunits are not stable in cells, and a mutation that impedes the stability of either the E1 or E1 subunit usually results in reduced levels or the absence of both subunits in cells from MSUD patients (7). Levels of both E1 subunits were normal in thiamin-responsive patients H.C. and H.S. as well as classic patients T.G. and C.R. In contrast, the E2 protein level was reduced in H.S. and H.C. compared with the wild type and was absent in T.G. and C.R., implicating that the E2 locus was affected in these MSUD patients.

Novel MSUD Mutations in the BCKD Complex—Samples of total RNA isolated from cells of MSUD patients were amplified by reverse transcriptase-PCR using primers corresponding to the cDNA sequence of the affected subunit. Nucleotide sequencing identified mutations in different subunits of the BCKD complex. All mutations in mRNA were confirmed by sequencing of the corresponding gene of the human BCKD complex in these patients. Table I shows seven novel MSUD mutations (types IA, IB, and II) identified in the eight Israeli patients investigated. A homozygous C to G type IA missense mutation C219W- (TGC TGG) is present in classic patient B.R. Two homozygous type IB mutations, H156Y- (CAT TAT) and V69G- (GTT GGT), occur in classic patients W.A. and D.M., respectively. Another classic patient, K.Y., harbors two compound heterozygous type IB alleles: a 4-base pair deletion in intron 9 (IVS9 del[-7:-4], resulting in the deletion of entire exon 10, and an 8-base pair insertion in exon 10 (1,109 ins 8bp) that causes a frameshift beginning at residue Glu³⁰⁴-. Two type II missense mutations were also detected. The homozygous H391R substitution (CAC CGC) in the E2 subunit is present in related thiamin-response patients H.C. and H.S. The homozygous S133stop (TCA TGA) in the E2 subunit is detected in classic patients T.G. and C.R.

The activity of the BCKD complex (Reaction 1) reconstituted with stoichiometric amounts of E1, E2, and E3 components was measured using -keto[1-¹⁴C]isovalerate as a substrate. The wild-type human BCKD complex when reconstituted with normal E1, E2 and E3 exhibits a decarboxylation rate of 196 min^-1. A substitution of the wild-type E1 with a mutant carrying the C219W- or H156Y- mutation does not produce BCKD activity in the presence of normal E2 and E3. Direct measurements of E1 component activity using 2,6-dichlorophe-nolindophenol as an electron acceptor (Reaction 7) confirmed the absence of E1 activity in the C219W- and H156Y- E1 variants (data not shown). Similarly, no BCKD activity was reconstituted when the wild-type E2 was replaced by a mutant containing the H391R alteration. The activity of the E2 component alone, based on the transfer of the radiolabeled acyl moiety from [1-¹⁴C]isovaleryl-CoA to dihydrolipoamide (Reaction 8), was measured. The wild-type E2 showed a specific activity of 1.97 nmol/min/mg, whereas the mutant E2 harboring the H391R substitution exhibited no acyltransferase activity. The above data establish that the C219W- and H156Y- substitutions are the cause of E1-deficient or type IA MSUD, whereas the H391R-E2 mutation is the culprit for E2-deficient or type II MSUD. The remaining three mutations in Table I (i.e. intron 9 (IVS9 del[-7:-4]) and exon 10 (1,109 ins 8bp) type IB gene alterations and S133stop type II subunit truncation) are obligatory null mutants and therefore also responsible for MSUD.

Assembly State of Wild-type and Mutant E1 Proteins—We have shown previously that the folded E1 and E1 subunits assemble into a heterodimeric intermediate that subsequently dimerizes to form a functional native E1 heterotetramer (16, 27). To dissect the effect of these novel MSUD mutations on E1 assembly, purified wild-type and mutant E1 proteins were subjected to a 10-30% sucrose density gradient centrifugation, and collected fractions were analyzed by SDS-PAGE coupled with Coomassie Blue staining. Both and subunits of wild-type E1 migrate as a single species consistent with a 170-kDa heterotetramer (Fig. 1). The mutant E1 carrying the C219W- substitution exists as two major species, one corresponding to the heterotetramer and the other migrating as a soluble high molecular weight form. A small amount of heterodimeric species that sediments close to the top of the gradient is also present in this mutant. The H156Y- mutant E1 occurs in solution in multiple species ranging from heterodimers to heterotetramers to the higher molecular weight form.

View larger version (33K): [in this window] [in a new window]   FIG. 1. Assembly state of wild-type and mutant E1 analyzed by sucrose density gradient centrifugation. Wild-type and mutant E1 (50 µg) carrying MSUD mutations in the E1 or the E1 subunit were applied to a 10-30% sucrose density gradient. Centrifugation was carried out at 210,000 x g at 4 °C for 15 h. Fractions collected were analyzed by SDS-PAGE and stained with Coomassie Blue. The heterodimer migrates in fractions 3-5, and the 22 heterotetramer sediments in fractions 6-8. Molecular weight markers with mass shown in parenthesis were as follows: ovalbumin (44 kDa), bovine serum albumin (66 kDa), dihydrolipoyl dehydrogenase (110 kDa), aldolase (158 kDa), catalase (232 kDa), and GroEL (840 kDa).

View this table: [in this window] [in a new window]   TABLE II Kd values for the binding of cofactor ThDP to wild-type, C219W-, and H156Y-' E1 as determined by fluorescence quenching

View larger version (13K): [in this window] [in a new window]   FIG. 2. Activation of mutant E1 carrying MSUD mutations by trimethylamine N-oxide. Wild-type and mutant E1 proteins (200 µg/ml) were incubated with increasing concentrations of TMAO for 16 h in the presence of 0.5 M lysine. BCKD activity was measured spectrophotometrically using the reconstituted assay (Reaction 1). Both H156Y- and C219W- mutants exhibit no BCKD activity in the absence of TMAO. Activation of mutant H156Y- () E1 in the presence of TMAO is expressed as percentage of wild-type BCKD activity, which remains relatively constant at 196 min-1 over the TMAO concentration range. No activation by TMAO was obtained with the mutant C219W- () E1.

View larger version (12K): [in this window] [in a new window]   FIG. 3. Activation of E1 activity by wild-type and mutant E2 protein/domains. A, three separate domains of the full-length E2 chain. The wild-type E2 chain (residues 1-421) contains (from N to C termini) an LBD, an SBD, and an inner core domain that are linked by flexible hinge regions. The inner core domain confers the 24-meric structure for the full-length E2. Lys44 (K44) is the lipoylation site, and His391 (H391) is the catalytic residue that serves as a general base in the acyltransferase reaction (Reaction 4). B, E1 activity was measured with purified wild-type E1 (15 µg) in the absence and presence of wild-type and mutant E2 protein/domains according to Reaction 7. The assays were carried out at nonsaturating ThDP concentration (10 µM). The reduction DCPIP was monitored spectrophotometrically at 600 nm. The molar ratio of E1 to E2 domain was 1:1, and the molar ratio of E1 to full-length E2 was 12:1 at the fixed E1 concentration. The basal activity for E1 alone was 11.9 min-1. The activation of E1 activity by wild-type and H391R E2 protein/domain is indicated by the -fold increase over the basal activity.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The crystal structure of human E1 was previously determined at 2.7-Å resolution (29). The overall structure is organized in a tetrahedral arrangement with each lobe roughly corresponding to one of the four subunits. The two active sites that accommodate cofactor ThDP are at the interfaces of -' and '- subunits. Novel structural K⁺ ion-binding pockets are present in both and subunits. Our laboratory has recently refined the human E1 structure to 1.8-Å resolution (24). This high resolution structure is used to provide a structural basis for the present novel MSUD mutations in Israeli patients. As shown in Fig. 4A, the Mn²⁺ ion in the ThDP-binding pocket coordinates to side chains of Asn²²²- and Glu¹⁹³- and the main-chain carbonyl oxygen atom of Tyr²²⁴- (not shown) as well as two phosphate oxygen atoms of cofactor ThDP. The Cys²¹⁹- residue is in the immediate vicinity of the ThDP-binding pocket. The replacement of Cys²¹⁹- by a large bulky Trp residue in the C219W- mutation is likely to produce a clash with Glu¹⁹³- and Asn²²²-, thereby disrupting the octahedral coordination of the Mn²⁺ ion. This explains the significant reduction in the affinity of this mutant for cofactor ThDP (Table II). The strained conformation surrounding the ThDP-binding site probably accounts for the loss of E1 activity in the C219W- variant. However, the binding of ThDP cannot correct this structural defect, as indicated by the absence of activity for the BCKD complex reconstituted with this mutant and measured at a saturating cofactor concentration. Since this mutation is away from the subunit interface, the structural basis for the appearance of high molecular weight species in addition to the heterotetramer in this E1 mutant is not immediately clear.

View larger version (25K): [in this window] [in a new window]   FIG. 4. Structural basis for type IA and type IB mutations in homozygous Israeli MSUD patients. The coordinates for wild-type human E1 at 1.8-Å resolution (code 1OLS [PDB] in the Protein Data Bank) (24) were used to model mutant E1 structures. Molecular graphics were generated using the programs Bobscript 2.6a (41) rendered with Pov-Ray Team 3.5 (available on the World Wide Web at www.povray.org). The wild-type residue is overlaid with its mutant counterpart (in whitish gray) at the affected amino acid position. Carbon atoms are in greenish gray, oxygen atoms in red, and nitrogen atoms in blue. Hydrogen bonds and salt bridges are indicated by red dotted lines. A, C219W- mutation; B, H156Y- mutation; C, V69G- mutation. For more detail, see "Discussion."

The V69G- alteration occurs on strand c in the interior of the subunit (Fig. 4C). Strand c precedes helix 3 in the E1 active site, which carries the invariant Glu⁷⁶- that serves as the electron-withdrawing residue during ThDP-mediated decarboxylation. The Val⁶⁹- residue coordinates to Ile⁴³- on strand b, Leu⁶⁰- on helix 2, and Val⁴⁸- on the loop between strand b and helix 2 through hydrophobic interactions. The replacement of a valine by a glycine residue may disrupt these interactions as well as produce a kink in strand c. These combined effects could impart instability in the structure surrounding helix 3 that carries the invariant catalytic residue Glu⁷⁶-. The net result is the misfolding of the subunit, which explains the inability to express the mutant E1 carrying the V69G- substitution.

We proposed previously that the invariant His³⁹¹ in E2 is the essential catalytic residue that functions as a general base for the E2-mediated transacylation reaction (13, 14). A replacement of His³⁹¹ by an asparagine, glutamine, or alanine results in a nearly complete inactivation of E2 activity. The structure of the E2 component of human BCKD complex has not been determined. Based on the available structure of the cognate transacetylase of the pyruvate dehydrogenase complex from Azotobacter vinelandii (33), a simplified reaction scheme involving His³⁹¹ and the conserved Ser³³⁸ is shown in Fig. 5. The N-3 atom on the imidazole ring of His³⁹¹ on E2 abstracts a proton from the reactive thiol group of acyl-CoA. The nucleophilic sulfur atom, strengthened by the proton abstraction, then attacks the carbonyl carbon of S-acyldihydrolipoamide on the lipoyl-bearing domain of the same subunit, forming the putative tetrahedral intermediate. The free energy of the transition state is significantly lowered by Ser³³⁸ that confers a hydrogen bond to the charged transition state. The acyltransfer reaction is completed following the breakdown of the tetrahedral intermediate. It is unclear whether the formation or the breakdown step of the tetrahedral intermediate is rate-limiting in the E2-catalyzed acyltransfer reaction. A substitution of arginine for His³⁹¹ in the H391R E2 mutation in the Israeli MSUD patients prevents the formation of the tetrahedral intermediate, thereby thwarting the E2-mediated acyltransferase reaction.

View larger version (9K): [in this window] [in a new window]   FIG. 5. His391 in the E2 subunit as a general base in the acyltransfer reaction catalyzed by E2. The proposed reaction scheme involving His391 and Ser338 is based on the crystal structure of dihydrolipoyl acetyltransferase from A. vinelandii (33). The main-chain carbonyl oxygen from His391 forms a hydrogen bond to the N-1 nitrogen atom of its own side chain to increase the basicity of the N-3 atom in the same imidazole ring. The nucleophilic attack by the N-3 nitrogen atom on the thiol group of CoA produces a tetrahedral intermediate involving S-acyldihydrolipoamide carried by the E2 subunit, with the intermediate stabilized by Ser338 through hydrogen bonding. The abstraction of the N-3 atom of His391 by the tetrahedral intermediate results in the production of dihydrolipoamide on E2 and free acyl-CoA.

On the other hand, type II MSUD patients in the present study (H.C. and H.S.) carrying the homozygous H391R mutation in the full-length E2 subunit exhibit an unequivocal thiamin-responsive phenotype. These two patients presented with neonatal encephalopathy, which was completely eradicated by thiamin supplements at 100 mg/day while on a restrictive diet. On this regimen, the patients have been asymptomatic, and their plasma BCAA levels remained in the normal range for the next 4 years. The overriding feature of these two Israeli thiamin-responsive patients are 1) the presence of a obligatory wild-type E1 and a full-length mutant E2 protein and 2) the display of significant residual decarboxylation activity by intact cells incubated with -keto[1-¹⁴C]isovalerate. These criteria are met by an additional four established thiamin-responsive patients that we studied previously (8) including the first patient (WG-34) described by Scriver et al. (35), who was found to contain one allele expressing a full-length F215C mutant E2 (36) (Table III). In contrast, the nine type II patients that manifest the classic MSUD phenotype including the two in this study do not contain at least one allele that produces a full-length mutant E2 protein. These null mutations, as defined by the absence of a full-length E2 protein, are accompanied by the complete absence of residual decarboxylation activity in intact cells derived from these patients.

View larger version (32K): [in this window] [in a new window]   FIG. 6. A biochemical model for the thiamin-responsive MSUD phenotype. A, basal E1 activity in null type II (E2-deficient) MSUD. In the absence of E2 due to a null or truncated (e.g. S133stop) mutation, E1 is dephosphorylated, since the BCKD kinase is inactive. Thiamin supplements convert the E1 active site from partially occupied by cofactor ThDP to fully occupied. The thin curved arrow denotes the basal E1 activity that decarboxylates BCKAs to branched-chain fatty acids (Reaction 7), with DCPIP replaced by an unidentified naturally occurring electron acceptor. The basal E1 activity at 11.9 min-1 is 6% of the oxidative decarboxylation catalyzed by the BCKD complex (Reaction 1). In vivo, the basal E1 activity is too low to significantly degrade BCKAs, resulting in the classic MSUD phenotype. B, augmented E1 activity in the presence of a mutant (e.g. H391R) 24-meric E2. Binding of BCKD kinase to the lip-LBD domain of the mutant full-length E2 renders the kinase active. This results in a partially phosphorylated E1 (indicated by circled P) bound to the SBD domain. Thiamin supplements result in complete occupation of the E1 active site by the cofactor and the complete dephosphorylation of E1. More significantly, conformational changes in the E1 active site induced by binding to SBD result in a 4-fold increase in the rate of BCKA decarboxylation as indicated by the thick curved arrow. The stimulated E1 activity may account for significant residual activity of BCKA decarboxylation in thiamin-responsive MSUD cells. The dotted circle in the E2 inner core domain depicts the E2 active site that contains the H391R type II MSUD mutation.

	FOOTNOTES

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

These authors contributed equally to this work.

^** To whom correspondence may be addressed. E-mail: david.chuang{at}utsouthwestern.edu; or h_mandel{at}rambam.health.gov.il.

¹ The abbreviations used are: MSUD, maple syrup urine disease; BCAA, branched-chain amino acid; BCKA, branched-chain -ketoacid; BCKD, branched-chain -ketoacid dehydrogenase; DCPIP, 2,6-dichlo-rophenolindophenol; DD, di-domain; lip-DD, lipoylated di-domain; E1, branched-chain -ketoacid decarboxylase; E2, dihydrolipoyl transacylase; E3, dihydrolipoamide dehydrogenase; LBD, lipoyl-bearing domain; lip-LBD, lipoylated lipoyl-bearing domain; lip-E2, lipoylated E2; SBD, subunit-binding domain; ThDP, thiamin diphosphate; TMAO, trimethylamine N-oxide; MOPS, 4-morpholinepropanesulfonic acid.

	ACKNOWLEDGMENTS

 
We are indebted to Karthikeyan Subramanian for the preparation of molecular graphics for mutant E1 structures.

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