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• W2037635640 abstract "Maple syrup urine disease (MSUD) results from mutations affecting different subunits of the mitochondrial branched-chain α-ketoacid dehydrogenase complex. In this study, we identified seven novel mutations in MSUD patients from Israel. These include C219W-α (TGC to TGG) in the E1α subunit; H156Y-β (CAT to TAT), V69G-β (GTT to GGT), IVS 9 del[-7:-4], and 1109 ins 8bp (exon 10) in the E1β subunit; and H391R (CAC to CGC) and S133stop (TCA to TGA) affecting the E2 subunit of the branched-chain α-ketoacid dehydrogenase complex. Recombinant E1 proteins carrying the C219W-α or H156Y-β mutation show no catalytic activity with defective subunit assembly and reduced binding affinity for cofactor thiamin diphosphate. The mutant E1 harboring the V69G-β 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. Maple syrup urine disease (MSUD) results from mutations affecting different subunits of the mitochondrial branched-chain α-ketoacid dehydrogenase complex. In this study, we identified seven novel mutations in MSUD patients from Israel. These include C219W-α (TGC to TGG) in the E1α subunit; H156Y-β (CAT to TAT), V69G-β (GTT to GGT), IVS 9 del[-7:-4], and 1109 ins 8bp (exon 10) in the E1β subunit; and H391R (CAC to CGC) and S133stop (TCA to TGA) affecting the E2 subunit of the branched-chain α-ketoacid dehydrogenase complex. Recombinant E1 proteins carrying the C219W-α or H156Y-β mutation show no catalytic activity with defective subunit assembly and reduced binding affinity for cofactor thiamin diphosphate. The mutant E1 harboring the V69G-β 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. Maple syrup urine disease (MSUD) 1The abbreviations used are: MSUD, maple syrup urine disease; BCAA, branched-chain amino acid; BCKA, branched-chain α-ketoacid; BCKD, branched-chain α-ketoacid dehydrogenase; DCPIP, 2,6-dichlo-rophenolindophenol; DD, di-domain; lip-DD, lipoylated di-domain; E1, branched-chain α-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. 1The abbreviations used are: MSUD, maple syrup urine disease; BCAA, branched-chain amino acid; BCKA, branched-chain α-ketoacid; BCKD, branched-chain α-ketoacid dehydrogenase; DCPIP, 2,6-dichlo-rophenolindophenol; DD, di-domain; lip-DD, lipoylated di-domain; E1, branched-chain α-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. or branched-chain α-ketoaciduria is an autosomal recessive metabolic disorder caused by deficiency in the mitochondrial branched-chain α-ketoacid dehydrogenase (BCKD) complex. The BCKD complex catalyzes the oxidative decarboxylation (Reaction 1) of three branched-chain α-ketoacids (BCKAs) derived from branched-chain amino acids (BCAAs) leucine, isoleucine, and valine (1Chuang D.T. Chuang J.L. Wynn R.M. Song J.-L. Creighton T.E. Encyclopedia of Molecular Medicine. Vol. 5. John Wiley & Sons, Inc., New York2001: 393-396Google Scholar). R-CO-COOH+CoA-SH+NAD+→R-CO-S-CoA+CO2↑+NADH+H+REACTION 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) (2Chuang D.T. Shih V.E. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Vogelstein K.B. Childs B. The Metabolic and Molecular Basis of Inherited Disease. 8th Ed. McGraw-Hill Inc., New York2001: 1971-2006Google Scholar). The classic form with the most severe phenotype, was originally reported by Menke et al. in 1954 (3Menke J.H. Hurst P.L. Craig J.M. Pediatrics. 1954; 14: 462-466PubMed Google Scholar) 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 (1Chuang D.T. Chuang J.L. Wynn R.M. Song J.-L. Creighton T.E. Encyclopedia of Molecular Medicine. Vol. 5. John Wiley & Sons, Inc., New York2001: 393-396Google Scholar). 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 (2Chuang D.T. Shih V.E. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Vogelstein K.B. Childs B. The Metabolic and Molecular Basis of Inherited Disease. 8th Ed. McGraw-Hill Inc., New York2001: 1971-2006Google Scholar). 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 α-ketoglutarate dehydrogenase complexes. Patients with E3 deficiency display a combined dysfunction of the three α-ketoacid dehydrogenase complexes (4Taylor J. Robinson B.H. Sherwood W.G. Pediatr. Res. 1978; 12: 60-62Crossref PubMed Scopus (37) Google Scholar). Two prevailing mutations (G229C and Y335stop) in the E3 gene have been identified in the Ashkenazi Jewish community in Israel (5Shaag A. Saada A. Berger I. Mandel H. Joseph A. Feigenbaum A. Elpelg O.N. Am. J. Med. Genet. 1999; 82: 177-182Crossref PubMed Scopus (77) Google Scholar). 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 × 106-dalton metabolic machine organized around a cubic core comprising 24 lipoate-bearing dihydrolipoyl transacylase (E2) subunits, to which multiple copies of branched-chain α-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 (6Harris R.A. Hawes J.W. Popov K.M. Zhao Y. Shimomura Y. Sato J. Jaskiewicz J. Hurley T.D. Adv. Enzyme Regul. 1997; 37: 271-293Crossref PubMed Scopus (92) Google Scholar). The E1 component is a thiamin diphosphate (ThDP)-dependent enzyme consisting of two E1α and two E1β 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 (E1α, E1β, E2, and E3) have been described in MSUD patients (2Chuang D.T. Shih V.E. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Vogelstein K.B. Childs B. The Metabolic and Molecular Basis of Inherited Disease. 8th Ed. McGraw-Hill Inc., New York2001: 1971-2006Google Scholar). Genetic subtypes of MSUD have been proposed to indicate the altered subunit in the BCKD complex (2Chuang D.T. Shih V.E. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Vogelstein K.B. Childs B. The Metabolic and Molecular Basis of Inherited Disease. 8th Ed. McGraw-Hill Inc., New York2001: 1971-2006Google Scholar). These include type IA MSUD affecting the E1α subunit, type IB affecting the E1β 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 (2Chuang D.T. Shih V.E. Scriver C.R. Beaudet A.L. Sly W.S. Valle D. Vogelstein K.B. Childs B. The Metabolic and Molecular Basis of Inherited Disease. 8th Ed. McGraw-Hill Inc., New York2001: 1971-2006Google Scholar, 7Fisher C.W. Chuang J.L. Griffin T.A. Lau K.S. Cox R.P. Chuang D.T. J. Biol. Chem. 1989; 264: 3448-3453Abstract Full Text PDF PubMed Google Scholar, 8Chuang J.L. Cox R.P. Chuang D.T. J. Clin. Invest. 1997; 100: 736-744Crossref PubMed Scopus (23) Google Scholar) and E3-deficient phenotypes (4Taylor J. Robinson B.H. Sherwood W.G. Pediatr. Res. 1978; 12: 60-62Crossref PubMed Scopus (37) Google Scholar), 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 (9Reed L.J. Acc. Chem. Res. 1974; 7: 40-46Crossref Scopus (651) Google Scholar), are as follows. R-CO-COOH+E1-ThDP→E1-R-COH=ThDP+CO2↑E1-R-COH=ThDP+E2-[lipS2]→E2-[R-CO-S-lipSH]+E1-ThDPE2-[R-CO-S-lipSH]+CoA-SH→R-CO-S-CoA+E2-[lip(SH)2]E2-[lip(SH)2]+E3-[FAD-S2]→E2-[lipS2]+E3-[FAD-(SH)2]E3-[FAD-(SH)2]+NAD+→E3-[FAD-S2]+NADH+H+Reactions 2–6 The E1 component binds ThDP and catalyzes a ThDP-mediated decarboxylation of α-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” (10Perham R.N. Annu. Rev. Biochem. 2000; 69: 961-1004Crossref PubMed Scopus (474) Google Scholar) 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 (11Wynn R.M. Chuang J.C. Sansaricq C. Mandel H. Chuang D.T. J. Biol. Chem. 2001; 276: 36550-36556Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). The incidence of MSUD in Israel is relatively high, presumably as a result of consanguinity (12Chitayat D. Balbul A. Hani V. Mamer O.A. Clow C. Scriver C.R. J. Inherit. Metab. Dis. 1992; 15: 198-203Crossref PubMed Scopus (18) Google Scholar). 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 E1α (type IA), E1β (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 (13Griffin T.S. Chuang D.T. J. Biol. Chem. 1990; 265: 13174-13180Abstract Full Text PDF PubMed Google Scholar, 14Meng M. Chuang D.T. Biochemistry. 1994; 33: 12879-12885Crossref PubMed Scopus (15) Google Scholar). 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. 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 α-keto[1-14C]isovalerate by intact cells as described previously (15Chuang J.L. Chuang D.T. Methods Enzymol. 2000; 324: 413-424Crossref PubMed Google Scholar). Nucleotide Sequencing for MSUD Mutations—The first-strand E1α cDNA was synthesized from the total RNA prepared from patients' cells using the primer 5′-GAAGACAGTGGTGTGCTGTC-3′ (cDNA sequence 1461-1442) and the Omniscript™ 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 E1β 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 E1β 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 E1β 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 E1β 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™ 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 (16Wynn R.M. Davie J.R. Chuang J.L. Cote C.D. Chuang D.T. J. Biol. Chem. 1998; 273: 13110-13118Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). The same plasmids carrying MSUD mutations were produced using the QuikChange™ 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 (17Wynn R.M. Davie J.R. Song J.L. Chuang J.L. Chuang D.T. Methods Enzymol. 2000; 324: 179-191Crossref PubMed Google Scholar, 18Wynn R.M. Davie J.R. Cox R.P. Chuang D.T. J. Biol. Chem. 1992; 267: 12400-12403Abstract Full Text PDF PubMed Google Scholar). His6-tagged wild-type and mutant E1 were purified with Ni2+-nitrilotriacetic acid resin and Resource Q and Superdex-200 columns (17Wynn R.M. Davie J.R. Song J.L. Chuang J.L. Chuang D.T. Methods Enzymol. 2000; 324: 179-191Crossref PubMed Google Scholar) 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 (19Chuang J.L. Davie J.R. Wynn R.M. Chuang D.T. Methods Enzymol. 2000; 324: 192-200Crossref PubMed Google Scholar). 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 (20Chuang J.L. Wynn R.M. Chuang D.T. J. Biol. Chem. 2002; 277: 36905-36908Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Lipoylated LBD2 (lip-LBD2) and DD (lip-DD) were prepared as described above. To produce the SBD, a tobacco-etch virus protease site (LENLYFQ↓S) 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 (19Chuang J.L. Davie J.R. Wynn R.M. Chuang D.T. Methods Enzymol. 2000; 324: 192-200Crossref PubMed Google Scholar). 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 α-ketoisovalerate. The oxidative decarboxylation of α-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 α-ketoisovalerate as a substrate in the presence of an artificial electron acceptor 2,6-dichlorophenolindophenol (DCPIP) (Re-action 7) as described previously (21Lau K.S. Cooper A.J.L. Chuang D.T. Biochim. Biophys. Acta. 1990; 1038: 360-366Crossref PubMed Scopus (9) Google Scholar). R-CO-COOH+DCPIP(oxidized)+H2O→R-COOH+CO2↑+DCPIP(reduced)+H+Reaction 7 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. [1-14C]R-CO-S-CoA+Lip(SH)2⇄[1-14C]R-S-Lip-SH+CoAReaction 8 The assay mixture contained 100 mm MOPS, pH 7.5, 2.5 mmdl-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 (22Chuang D.T. Methods Enzymol. 1988; 166: 146-154Crossref PubMed Scopus (10) Google Scholar). 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 E1α and E1β 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 (15Chuang J.L. Chuang D.T. Methods Enzymol. 2000; 324: 413-424Crossref PubMed Google Scholar). Binding Measurements Based on Tryptophan Fluorescence Quenching—Steady-state fluorescence quenching upon ThDP binding (23Hennig J. Kern G. Neef H. Bisswanger H. Hubner G. Bisswanger H. Schellenberger A. Biochemistry and Physiology of Thiamin Diphosphate Enzymes. A. u. C. Intemann, Prien, Germany1966: 243-251Google Scholar) to wild-type and mutant E1 was measured using a Perkin-Elmer Life Sciences LS50 B luminescence spectrometer as described previously (24Wynn R.M. Machius M. Chuang J.L. Li J. Tomchick D.R. Chuang D.T. J. Biol. Chem. 2003; 278: 43402-43410Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). 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 (25Nemeria N. Yan Y. Zhang Z. Brown A.M. Arjunan P. Furey W. Guest J.R. Jordan F. J. Biol. Chem. 2001; 276: 45969-45978Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), (-ΔF/F0)=(-ΔFmax/F0)×[ThDP]/(Kd+[ThDP])(Eq. 1) where ΔF represents the fluorescence change corrected for dilution and inner filter effects (26Lakowicz J.R. Maliwal B.P. Cherek H. Balter A. Biochemistry. 1983; 22: 1741-1752Crossref PubMed Scopus (173) Google Scholar), F0 is the fluorescence intensity prior to the addition of ThDP, ΔFmax is the maximal fluorescence change, Kd is the dissociation constant, and [ThDP] is the concentration of ThDP in the cuvette. The parameters determined by the fitting procedure were ΔFmax and Kd. 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. 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 E1α and E1β subunits) or E2 as a probe. Cells from the classic patient B.R. showed reduced levels of both the E1α and the E1β subunit, with the level of the E2 subunit in the normal range. Levels of both E1α and E1β 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 E1α or the E1β 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 E1α or E1β subunit usually results in reduced levels or the absence of both subunits in cells from MSUD patients (7Fisher C.W. Chuang J.L. Griffin T.A. Lau K.S. Cox R.P. Chuang D.T. J. Biol. Chem. 1989; 264: 3448-3453Abstract Full Text PDF PubMed Google Scholar). 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-α (TGC → TGG) is present in classic patient B.R. Two homozygous type IB mutations, H156Y-β (CAT → TAT) and V69G-β (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-β. 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.Table INovel mutations in subunits of the BCKD complex in Israeli MSUD patientsPatientGenetic subtypeAltered subunitResidual proteinMSUD alleleGene/protein alterationGenotypeClinical phenotypeB.R.IAE1aYesC219W-αTGC to TGG−/−ClassicW.A.IBE1bNoH156Y-βCAT to TAT−/−ClassicD.M.IBE1bNoV69G-βGTT to GGT−/−ClassicK.Y.IBE1bNoIVS 9 del [−7:−4]aIVS, intervening sequence or intron; intronic deletion beginning at 7 bases and ending at 4 bases upstream of the intron 9/exon 10 junction (see Ref. 30 for nomenclature).Exon 10 deleted+/−Classic1,109 ins 8bp (exon 10)bins, insertion; 8-bp insertion at base 1,109 of the human E1β cDNA sequence corresponding to exon 10 (GenBank accession number U51015).Frameshift at Glu304+/−ClassicH.C.IIE2YesH391RCAC to CGC−/−Thiamin-responsiveH.S.IIE2YesH391RCAC to CGC−/−Thiamin-responsiveT.G.IIE2NoS133stopTCA to TGA−/−ClassicC.R.IIE2NoS133stopTCA to TGA−/−Classica IVS, intervening sequence or intron; intronic deletion beginning at 7 bases and ending at 4 bases upstream of the intron 9/exon 10 junction (see Ref. 30Ad Hoc Committee on Mutation NomenclatureHum. Mutat. 1996; 8: 197-202Crossref PubMed Scopus (77) Google Scholar for nomenclature).b ins, insertion; 8-bp insertion at base 1,109 of the human E1β cDNA sequence corresponding to exon 10 (GenBank accession number U51015). Open table in a new tab Activity Measurement of MSUD Mutant Prot" @default.
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• W2037635640 date "2004-04-01" @default.
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• W2037635640 title "Structural and Biochemical Basis for Novel Mutations in Homozygous Israeli Maple Syrup Urine Disease Patients" @default.
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• W2037635640 doi "https://doi.org/10.1074/jbc.m313879200" @default.
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