R. Max Wynn

Crystal structure of human branched-chain α-ketoacid dehydrogenase and the molecular basis of multienzyme complex deficiency in maple syrup urine disease

Abstract Background: Mutations in components of the extraordinarily large α-ketoacid dehydrogenase multienzyme complexes can lead to serious and often fatal disorders in humans, including maple syrup urine disease (MSUD). In order to obtain insight into the effect of mutations observed in MSUD patients, we determined the crystal structure of branched-chain α-ketoacid dehydrogenase (E1), the 170 kDa α 2 β 2 heterotetrameric E1b component of the branched-chain α-ketoacid dehydrogenase multienzyme complex. Results: The 2.7 A resolution crystal structure of human E1b revealed essentially the full α and β polypeptide chains of the tightly packed heterotetramer. The position of two important potassium (K + ) ions was determined. One of these ions assists a loop that is close to the cofactor to adopt the proper conformation. The second is located in the β subunit near the interface with the small C-terminal domain of the α subunit. The known MSUD mutations affect the functioning of E1b by interfering with the cofactor and K + sites, the packing of hydrophobic cores, and the precise arrangement of residues at or near several subunit interfaces. The Tyr→Asn mutation at position 393-α occurs very frequently in the US population of Mennonites and is located in a unique extension of the human E1b α subunit, contacting the β′ subunit. Conclusions: Essentially all MSUD mutations in human E1b can be explained on the basis of the structure, with the severity of the mutations for the stability and function of the protein correlating well with the severity of the disease for the patients. The suggestion is made that small molecules with high affinity for human E1b might alleviate effects of some of the milder forms of MSUD.

Autoantibodies to BCOADC‐E2 in patients with primary biliary cirrhosis recognize a conformational epitope

Primary biliary cirrhosis (PBC) is an autoimmune disease of liver associated with a unique serologic response to mitochondrial autoantigens. Many of the autoantigens recognized by autoantibodies in PBC are members of the 2-oxo-acid dehydrogenase complex. The two major autoantigens are the E2 component of the pyruvate dehydrogenase complex (PDC-E2) and the E2 component of the branched chain 2-oxo-acid dehydrogenase complex (BCOADC-E2). The autoantibody response to PDC-E2 has been mapped to one immunodominant epitope, which consists of both linear and conformational components. The presence of a single immunodominant epitope in PDC-E2 is unusual when contrasted to the immune response to autoantigens in other human autoimmune diseases. We have mapped the epitope recognized by antimitochondrial autoantibodies (AMA) specific to BCOADC-E2 in patients with PBC by taking advantage of the full-length bovine BCOADC-E2 complementary DNA (cDNA) and a series of expression clones spanning the entire molecule. Reactivity to the various expression clones was studied by immunoblotting, enzyme-linked immunosorbent assay (ELISA), as well as selective absorption of patient sera by expressed protein fragments. Autoantibodies to BCOADC-E2 map within peptides spanning amino acid residues 1 to 227 of the mature protein; our data demonstrate that the epitope is dependent on conformation and includes the lipoic acid binding region. However, only the full-length clone (amino acid residue 1 to 421) is sufficient to remove all detectable BCOADC-E2 reactivity. Moreover, the absence of lipoic acid on the recombinant polypeptides used in this study indicates that antibody binding to BCOADC-E2 is not dependent on the presence of lipoic acid.

A Novel Branched-chain Amino Acid Metabolon PROTEIN-PROTEIN INTERACTIONS IN A SUPRAMOLECULAR COMPLEX

The catabolic pathways of branched-chain amino acids have two common steps. The first step is deamination catalyzed by the vitamin B6-dependent branched-chain aminotransferase isozymes (BCATs) to produce branched-chain α-keto acids (BCKAs). The second step is oxidative decarboxylation of the BCKAs mediated by the branched-chain α-keto acid dehydrogenase enzyme complex (BCKD complex). The BCKD complex is organized around a cubic core consisting of 24 lipoate-bearing dihydrolipoyl transacylase (E2) subunits, associated with the branched-chain α-keto acid decarboxylase/dehydrogenase (E1), dihydrolipoamide dehydrogenase (E3), BCKD kinase, and BCKD phosphatase. In this study, we provide evidence that human mitochondrial BCAT (hBCATm) associates with the E1 decarboxylase component of the rat or human BCKD complex with a KD of 2.8 μm. NADH dissociates the complex. The E2 and E3 components do not interact with hBCATm. In the presence of hBCATm, kcat values for E1-catalyzed decarboxylation of the BCKAs are enhanced 12-fold. Mutations of hBCATm proteins in the catalytically important CXXC center or E1 proteins in the phosphorylation loop residues prevent complex formation, indicating that these regions are important for the interaction between hBCATm and E1. Our results provide evidence for substrate channeling between hBCATm and BCKD complex and formation of a metabolic unit (termed branched-chain amino acid metabolon) that can be influenced by the redox state in mitochondria.

Lessons from Genetic Disorders of Branched-Chain Amino Acid Metabolism

Genetic disorders of BCAA metabolism produce amino acidopathies and various forms of organic aciduria with severe clinical consequences. A metabolic block in the oxidative decarboxylation of BCAA caused by mutations in the mitochondrial branched-chain alpha-keto acid dehydrogenase complex (BCKDC) results in Maple Syrup Urine Disease (MSUD) or branched-chain ketoaciduria. There are presently five known clinical phenotypes for MSUD, i.e., classic, intermediate, intermittent, thiamin-responsive, and dihydrolipoamide dehydrogenase (E3)-deficient, based on severity of the disease, response to thiamin therapy, and the gene locus affected. Reduced glutamate, glutamine, and gamma-aminobutyrate concentrations induced by the accumulation of branched-chain alpha-ketoacids in the brain cortex of affected children and neonatal polled Hereford calves are considered the cause of MSUD encephalopathies. The long-term restriction of BCAA intake in diets and orthotopic liver transplantation have proven effective in controlling plasma BCAA levels and mitigating some of the above neurological manifestations. To date, approximately 100 mutations have been identified in four (branched-chain alpha-ketoacid decarboxylase/dehydrogenasealpha [E1alpha], E1beta, dihydrolipoyl transacylase [E2], and E3) of the six genes that encode the human BCKDC catalytic machine. We have documented a strong correlation between the presence of mutant E2 proteins and the thiamin-responsive MSUD phenotype. We show that the normal E1 component possesses residual decarboxylase activity, which is augmented by the binding to a mutant E2 protein in the presence of the E1 cofactor thiamin diphosphate. Our results provide a biochemical model for the effectiveness of thiamin therapy to thiamin-responsive MSUD patients.

Crystal structure of pyruvate dehydrogenase kinase 3 bound to lipoyl domain 2 of human pyruvate dehydrogenase complex.

The human pyruvate dehydrogenase complex (PDC) is regulated by reversible phosphorylation by four isoforms of pyruvate dehydrogenase kinase (PDK). PDKs phosphorylate serine residues in the dehydrogenase (E1p) component of PDC, but their amino‐acid sequences are unrelated to eukaryotic Ser/Thr/Tyr protein kinases. PDK3 binds to the inner lipoyl domains (L2) from the 60‐meric transacetylase (E2p) core of PDC, with concomitant stimulated kinase activity. Here, we present crystal structures of the PDK3–L2 complex with and without bound ADP or ATP. These structures disclose that the C‐terminal tail from one subunit of PDK3 dimer constitutes an integral part of the lipoyl‐binding pocket in the N‐terminal domain of the opposing subunit. The two swapped C‐terminal tails promote conformational changes in active‐site clefts of both PDK3 subunits, resulting in largely disordered ATP lids in the ADP‐bound form. Our structural and biochemical data suggest that L2 binding stimulates PDK3 activity by disrupting the ATP lid, which otherwise traps ADP, to remove product inhibition exerted by this nucleotide. We hypothesize that this allosteric mechanism accounts, in part, for E2p‐augmented PDK3 activity.

Structure of rat BCKD kinase: Nucleotide-induced domain communication in a mitochondrial protein kinase

Mitochondrial protein kinases (mPKs) are molecular switches that down-regulate the oxidation of branched-chain α-ketoacids and pyruvate. Elevated levels of these metabolites are implicated in disease states such as insulin-resistant Type II diabetes, branched-chain ketoaciduria, and primary lactic acidosis. We report a three-dimensional structure of a member of the mPK family, rat branched-chain α-ketoacid dehydrogenase kinase (BCK). BCK features a characteristic nucleotide-binding domain and a four-helix bundle domain. These two domains are reminiscent of modules found in protein histidine kinases (PHKs), which are involved in two-component signal transduction systems. Unlike PHKs, BCK dimerizes through direct interaction of two opposing nucleotide-binding domains. Nucleotide binding to BCK is uniquely mediated by both potassium and magnesium. Binding of ATP induces disorder–order transitions in a loop region at the nucleotide-binding site. These structural changes lead to the formation of a quadruple aromatic stack in the interface between the nucleotide-binding domain and the four-helix bundle domain, where they induce a movement of the top portion of two helices. Phosphotransfer induces further ordering of the loop region, effectively trapping the reaction product ADP, which explains product inhibition in mPKs. The BCK structure is a prototype for all mPKs and will provide a framework for structure-assisted inhibitor design for this family of kinases.

Regulation of Nuclear Import/Export of Carbohydrate Response Element-binding Protein (ChREBP) INTERACTION OF AN α-HELIX OF ChREBP WITH THE 14-3-3 PROTEINS AND REGULATION BY PHOSPHORYLATION

Carbohydrate response element-binding protein (ChREBP) is a glucose-responsive transcription factor that plays a critical role in the glucose-mediated induction of gene products involved in hepatic glycolysis and lipogenesis. Glucose affects the activity of ChREBP largely through post-translational mechanisms involving phosphorylation-dependent cellular localization. In this work we show that the N-terminal region of ChREBP (residues 1-251) regulates its subcellular localization via an interaction with 14-3-3. 14-3-3 binds an α-helix in this region (residues 125-135) to retain ChREBP in the cytosol, and binding of 14-3-3 is facilitated by phosphorylation of nearby Ser-140 and Ser-196. Phosphorylation of ChREBP at these sites was essential for its interaction with CRM1 for export to the cytosol, whereas nuclear import of ChREBP requires dephosphorylated ChREBP to interact with importin α. Notably, 14-3-3 appears to compete with importin α for ChREBP binding. 14-3-3β bound to a synthetic peptide spanning residues 125-144 and bearing a phosphate at Ser-140 with a dissociation constant of 1.1 μm, as determined by isothermal calorimetry. The interaction caused a shift in the fluorescence maximum of the tryptophan residues of the peptide. The corresponding unphosphorylated peptide failed to bind 14-3-3β. These results suggest that interactions with importin α and 14-3-3 regulate movement of ChREBP into and out of the nucleus, respectively, and that these interactions are regulated by the ChREBP phosphorylation status.

Catabolic Defect of Branched-Chain Amino Acids Promotes Heart Failure.

Background— Although metabolic reprogramming is critical in the pathogenesis of heart failure, studies to date have focused principally on fatty acid and glucose metabolism. Contribution of amino acid metabolic regulation in the disease remains understudied. Methods and Results— Transcriptomic and metabolomic analyses were performed in mouse failing heart induced by pressure overload. Suppression of branched-chain amino acid (BCAA) catabolic gene expression along with concomitant tissue accumulation of branched-chain &agr;-keto acids was identified as a significant signature of metabolic reprogramming in mouse failing hearts and validated to be shared in human cardiomyopathy hearts. Molecular and genetic evidence identified the transcription factor Krüppel-like factor 15 as a key upstream regulator of the BCAA catabolic regulation in the heart. Studies using a genetic mouse model revealed that BCAA catabolic defect promoted heart failure associated with induced oxidative stress and metabolic disturbance in response to mechanical overload. Mechanistically, elevated branched-chain &agr;-keto acids directly suppressed respiration and induced superoxide production in isolated mitochondria. Finally, pharmacological enhancement of branched-chain &agr;-keto acid dehydrogenase activity significantly blunted cardiac dysfunction after pressure overload. Conclusions— BCAA catabolic defect is a metabolic hallmark of failing heart resulting from Krüppel-like factor 15–mediated transcriptional reprogramming. BCAA catabolic defect imposes a previously unappreciated significant contribution to heart failure.

Branched-chain Amino Acid Metabolon: INTERACTION OF GLUTAMATE DEHYDROGENASE WITH THE MITOCHONDRIAL BRANCHED-CHAIN AMINOTRANSFERASE (BCATm)*

The catabolic pathway for branched-chain amino acids includes deamination followed by oxidative decarboxylation of the deaminated product branched-chain α-keto acids, catalyzed by the mitochondrial branched-chain aminotransferase (BCATm) and branched-chain α-keto acid dehydrogenase enzyme complex (BCKDC). We found that BCATm binds to the E1 decarboxylase of BCKDC, forming a metabolon that allows channeling of branched-chain α-keto acids from BCATm to E1. The protein complex also contains glutamate dehydrogenase (GDH1), 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1, pyruvate carboxylase, and BCKDC kinase. GDH1 binds to the pyridoxamine 5′-phosphate (PMP) form of BCATm (PMP-BCATm) but not to the pyridoxal 5′-phosphate-BCATm and other metabolon proteins. Leucine activates GDH1, and oxidative deamination of glutamate is increased further by addition of PMP-BCATm. Isoleucine and valine are not allosteric activators of GDH1, but in the presence of 5′-phosphate-BCATm, they convert BCATm to PMP-BCATm, stimulating GDH1 activity. Sensitivity to ADP activation of GDH1 was unaffected by PMP-BCATm; however, addition of a 3 or higher molar ratio of PMP-BCATm to GDH1 protected GDH1 from GTP inhibition by 50%. Kinetic results suggest that GDH1 facilitates regeneration of the form of BCATm that binds to E1 decarboxylase of the BCKDC, promotes metabolon formation, branched-chain amino acid oxidation, and cycling of nitrogen through glutamate.

Phenylbutyrate therapy for maple syrup urine disease

Therapy with sodium phenylacetate/benzoate or sodium phenylbutyrate in urea cycle disorder patients has been associated with a selective reduction in branched-chain amino acids (BCAA) in spite of adequate dietary protein intake. Based on this clinical observation, we investigated the potential of phenylbutyrate treatment to lower BCAA and their corresponding α-keto acids (BCKA) in patients with classic and variant late-onset forms of maple syrup urine disease (MSUD). We also performed in vitro and in vivo experiments to elucidate the mechanism for this effect. We found that BCAA and BCKA are both significantly reduced following phenylbutyrate therapy in control subjects and in patients with late-onset, intermediate MSUD. In vitro treatment with phenylbutyrate of control fibroblasts and lymphoblasts resulted in an increase in the residual enzyme activity, while treatment of MSUD cells resulted in the variable response which did not simply predict the biochemical response in the patients. In vivo phenylbutyrate increases the proportion of active hepatic enzyme and unphosphorylated form over the inactive phosphorylated form of the E1α subunit of the branched-chain α-keto acid dehydrogenase complex (BCKDC). Using recombinant enzymes, we show that phenylbutyrate prevents phosphorylation of E1α by inhibition of the BCKDC kinase to activate BCKDC overall activity, providing a molecular explanation for the effect of phenylbutyrate in a subset of MSUD patients. Phenylbutyrate treatment may be a valuable treatment for reducing the plasma levels of neurotoxic BCAA and their corresponding BCKA in a subset of MSUD patients and studies of its long-term efficacy are indicated.