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 B(6)-dependent branched-chain aminotransferase isozymes (BCATs) to produce branched-chain alpha-keto acids (BCKAs). The second step is oxidative decarboxylation of the BCKAs mediated by the branched-chain alpha-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 alpha-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 K(D) of 2.8 microM. NADH dissociates the complex. The E2 and E3 components do not interact with hBCATm. In the presence of hBCATm, k(cat) 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.     

Two novel mutations in the BCKDHB gene (R170H, Q346R) cause the classic form of maple syrup urine disease (MSUD)

Maple syrup urine disease (MSUD) is an autosomal recessive metabolic disorder that is caused by mutations in the subunits of the branched-chain α-ketoacid dehydrogenase (BCKD) complex. BCKD is a mitochondrial complex encoded by four nuclear genes (BCKDHA, BCKDHB, DBT, and DLD) and is involved in the metabolism of branched-chain amino acids (BCAAs). In this study, we investigated the DNA sequences of BCKDHA, BCKDHB and DBT genes for mutations in a Chinese newborn with the classic form of MSUD and predicted the associated conformational changes using molecular modeling. We identified two previously unreported mutations in the BCKDHB gene, R170H (c.509G>A) in exon 5 and Q346R (c.1037 A>G) in exon 9. In silico analysis of the two novel missense mutations revealed that the mutation R170H-β alters the spatial orientation with both Y195-β' and S206-α, which results in unstable β-β' assembly and an unstable K(+) ion binding loop of the α subunit, respectively; The Q346R mutation is predicted to disrupt the spatial conformation between Q346-β and I357-β', which reduces the affinity of the β-β' subunits. These results indicate that R170-β and Q346-β are crucial for the activity of the E1 component. These two novel mutations, R170H and Q346R result in the patient's clinical manifestation of the classic form of MSUD.     

Controlled overexpression of BCKD kinase expression: metabolic engineering applied to BCAA metabolism in a mammalian system

A common metabolic complication of human disease is uncontrolled muscle protein breakdown or cachexia, which occurs in patients with chronic diseases such as cancer, AIDS, renal failure, and diabetes. Increased branched-chain amino acid catabolism is implicated as causal and has stimulated the investigation of methods to regulate the metabolism of these amino acids. Here we demonstrate doxycycline-controlled overexpression of a branched-chain alpha-ketoacid dehydrogenase (BCKD) kinase transgene in mammalian cell culture. This kinase functions to inactivate the BCKD complex by phosphorylation, thus preventing the catabolism of these essential, regulatory metabolites. In this study, doxycycline treatment leads to a 10-fold increase in BCKD kinase protein. The transgene-generated kinase is rapidly incorporated within mitochondria and functions correctly to inactivate the BCKD complex. The maximum reduction in basal BCKD activity achieved was 94%. Unexpectedly, total BCKD activity was also decreased by kinase overexpression despite no observable change in expression of the BCKD catalytic proteins. These results demonstrate that artificial regulation of branched-chain amino acid metabolism is possible through the controlled overexpression of a single endogenous enzyme and suggest the feasibility of clinical applications.     

Insulin increases branched-chain alpha-ketoacid dehydrogenase kinase expression in Clone 9 rat cells

The branched-chain amino acids (BCAA) are committed to catabolism by the activity of the branched-chain alpha-ketoacid dehydrogenase (BCKD) complex. BCKD activity is regulated through the action of the complex-specific BCKD kinase that phosphorylates two serine residues in the E1alpha subunit. Greater BCKD kinase expression levels result in a lower activity state of BCKD and thus a decreased rate of BCAA catabolism. Activity state varies among tissues and can be altered by diet, exercise, hormones, and disease state. Within individual tissues, the concentration of BCKD kinase reflects the activity state of the BCKD complex. Here we investigated the effects of insulin, an important regulator of hepatic metabolic enzymes, on BCKD kinase expression in Clone 9 rat cells. Insulin effected a twofold increase in message levels and a twofold increase in BCKD kinase protein levels. The response was completely blocked by treatment with LY-294002 and partially blocked by rapamycin, thus demonstrating a dependence on phosphatidylinositol 3-kinase and mTOR function, respectively. These studies suggest that insulin acts to regulate BCAA catabolism through stimulation of BCKD kinase expression.     

Molecular characterization of maple syrup urine disease patients from Tunisia

Maple syrup urine disease (MSUD) is a rare disorder of branched-chain amino acids (BCAA) metabolism caused by the defective function of branched-chain α-ketoacid dehydrogenase complex (BCKD). The disease causal mutations can occur either in BCKDHA, BCKDHB or DBT genes encoding respectively the E1α, E1β and E2 subunits of the complex. In this study we report the molecular characterization of 3 Tunisian patients with the classic form of MSUD. Two novel putative mutations have been identified: the alteration c.716A>G (p.Glu239Gly) in BCKDHB and a small deletion (c.1333_1336delAATG; p.Asn445X) detected in DBT gene.     

Obesity-related elevations in plasma leucine are associated with alterations in enzymes involved in branched-chain amino acid metabolism

Elevations in branched-chain amino acids (BCAAs) in human obesity were first reported in the 1960s. Such reports are of interest because of the emerging role of BCAAs as potential regulators of satiety, leptin, glucose, cell signaling, adiposity, and body weight (mTOR and PKC). To explore loss of catabolic capacity as a potential contributor to the obesity-related rises in BCAAs, we assessed the first two enzymatic steps, catalyzed by mitochondrial branched chain amino acid aminotransferase (BCATm) or the branched chain alpha-keto acid dehydrogenase (BCKD E1alpha subunit) complex, in two rodent models of obesity (ob/ob mice and Zucker rats) and after surgical weight loss intervention in humans. Obese rodents exhibited hyperaminoacidemia including BCAAs. Whereas no obesity-related changes were observed in rodent skeletal muscle BCATm, pS293, or total BCKD E1alpha or BCKD kinase, in liver BCKD E1alpha was either unaltered or diminished by obesity, and pS293 (associated with the inactive state of BCKD) increased, along with BCKD kinase. In epididymal fat, obesity-related declines were observed in BCATm and BCKD E1alpha. Plasma BCAAs were diminished by an overnight fast coinciding with dissipation of the changes in adipose tissue but not in liver. BCAAs also were reduced by surgical weight loss intervention (Roux-en-Y gastric bypass) in human subjects studied longitudinally. These changes coincided with increased BCATm and BCKD E1alpha in omental and subcutaneous fat. Our results are consistent with the idea that tissue-specific alterations in BCAA metabolism, in liver and adipose tissue but not in muscle, may contribute to the rise in plasma BCAAs in obesity.     

Metabolism of phytol to phytanic acid in the mouse, and the role of PPARalpha in its regulation

Phytol, a branched-chain fatty alcohol, is the naturally occurring precursor of phytanic and pristanic acid, branched-chain fatty acids that are both ligands for the nuclear hormone receptor peroxisome proliferator-activated receptor alpha (PPARalpha). To investigate the metabolism of phytol and the role of PPARalpha in its regulation, wild-type and PPARalpha knockout (PPARalpha-/-) mice were fed a phytol-enriched diet or, for comparison, a diet enriched with Wy-14,643, a synthetic PPARalpha agonist. After the phytol-enriched diet, phytol could only be detected in small intestine, the site of uptake, and liver. Upon longer duration of the diet, the level of the (E)-isomer of phytol increased significantly in the liver of PPARalpha-/- mice compared with wild-type mice. Activity measurements of the enzymes involved in phytol metabolism showed that treatment with a PPARalpha agonist resulted in a PPARalpha-dependent induction of at least two steps of the phytol degradation pathway in liver. Furthermore, the enzymes involved showed a higher activity toward the (E)-isomer than the (Z)-isomer of their respective substrates, indicating a stereospecificity toward the metabolism of (E)-phytol. In conclusion, the results described here show that the conversion of phytol to phytanic acid is regulated via PPARalpha and is specific for the breakdown of (E)-phytol.     

Influence of subunit transcript and protein levels on formation of a mitochondrial multienzyme complex

Constitutive expression of nuclear genes encoding mitochondrial proteins raises the question of whether these proteins are present in similar amounts in mitochondria of different tissues. We report that amounts of a single multienzyme complex can vary on a per mitochondrion basis depending on the number of mitochondria per cell. Human branched-chain α-keto acid dehydrogenase (BCKD) expression is used as a paradigm in these studies. Expression is compared and contrasted in HepG2 and DG75 cells in which mitochondrial content is twofold higher in the hepatocarcinoma line than in the lymphoblastoid line. Per cell, BCKD activity is equal in the two cell types, but BCKD protein concentration per mitochondrion is twofold higher in DG75 cells. Steady-state mRNA levels do not appear to be directly related to amounts of protein in the two cell lines. To test whether one subunit is limiting in formation of complex, overexpression of each BCKD subunit was elicited by plasmid transfection of the DG75 cells. Only overexpression of the β-subunit of the decarboxylase component induced more BCKD activity without apparent increase in mRNA for the other endogenously expressed subunits. This implies that free BCKD subunits exist in a cell and can be recruited into an active complex when the limiting subunit becomes available. © 1996 Wiley-Liss, Inc.     

Activation of hepatic branched-chain alpha-keto acid dehydrogenase complex by tumor necrosis factor-alpha in rats

Tumor necrosis factor-alpha (TNFalpha) promotes oxidation of branched-chain amino acids (BCAA). BCAA catabolism is regulated by branched-chain alpha-keto acid dehydrogenase (BCKDH) complex, which is regulated by phosphorylation-dephosphorylation of the E1alpha subunit at Ser293. BCKDH kinase is responsible for inactivation of the complex by phosphorylation. In the present study, we examined the effects of TNFalpha administration on hepatic BCKDH complex and kinase in rats. Rats were intravenously administered with 25 or 50 microg TNFalpha/kg body weight 4 h prior to sacrifice. The TNFalpha treatment at both doses elevated the activity state (percentage of the active form) of BCKDH complex from 22% to 69% and 86%, respectively, and the amount of phospho-Ser293 on the E1alpha subunit in each group of rats corresponded inversely to the activity state of BCKDH complex. The TNFalpha treatment of rats significantly decreased the activity as well as the bound form of BCKDH kinase. These results suggest that the decrease in the bound form of kinase is involved in the mechanism responsible for TNFalpha-induced activation of the BCKDH complex.     

Distribution of key enzymes of branched-chain amino acid metabolism in glial and neuronal cells in culture.

Transamination of branched-chain amino acids (BCAAs) catalyzed by the branched chain aminotransferase isoenzymes (BCATs) is believed to play an important role in nitrogen shuttling and excitatory neurotransmitter glutamate metabolism in brain. Recently, we have shown that the mitochondrial isoenzyme (BCATm) is the predominant form found in cultured astrocytes. In this study we used immunocytochemistry to examine the distribution of BCAT isoenzymes in cultured rat neurons and microglial cells. The cytoplasm of neurons displayed intense staining for the cytosolic isoenzyme (BCATc), whereas BCATm staining was not detectable in neurons. In contrast, microglial cells expressed BCATm in high concentration. BCATc appeared to be absent in this cell type. The second and committed step in the BCAA catabolic pathway is oxidative decarboxylation of the alpha-keto acid products of BCAT catalyzed by the branched-chain alpha-keto acid dehydrogenase (BCKD) enzyme complex. Because the presence of BCKD should provide an index of the ability of a cell to oxidize BCAA, we have also immunocytochemically localized BCKD in neuron and glial cell cultures from rat brain. Our results suggest ubiquitous expression of this BCKD enzyme complex in cultured brain cells. BCKD immunoreactivity was detected in neurons and in astroglial and microglial cells. Therefore, the expression of BCAT isoenzymes shows cell-specific localization, which is consistent with the operation of an intercellular nitrogen shuttle between neurons and astroglia. On the other hand, the ubiquitous expression of BCKD suggests that BCAA oxidation can probably take place in all types of brain cells and is most likely regulated by the activity state of BCKD rather than by its cell-specific localization.     

Natural osmolyte trimethylamine N-oxide corrects assembly defects of mutant branched-chain alpha-ketoacid decarboxylase in maple syrup urine disease

Maple syrup urine disease is caused by deficiency in the mitochondrial branched-chain alpha-ketoacid dehydrogenase (BCKD) complex. The clinical phenotype includes often fatal ketoacidosis, neurological derangement, and mental retardation. The type IA mutations Y393N-alpha, Y368C-alpha, and F364C-alpha, which occur in the E1alpha subunit of the decarboxylase (E1) component of the BCKD complex, impede the conversion of an alphabeta heterodimeric intermediate to a native alpha(2)beta(2) heterotetramer in the E1 assembly pathway. In the present study, we show that a natural osmolyte trimethylamine N-oxide (TMAO) at the optimal 1 m concentration restores E1 activity, up to 50% of the wild type, in the mutant E1 carrying the above missense mutations. TMAO promotes the conversion of otherwise trapped mutant heterodimers to active heterotetramers. This slow step does not involve dissociation/reassociation of the mutant heterodimers, which are preformed in the presence of chaperonins GroEL/GroES and Mg-ATP. The TMAO-stimulated mutant E1 activity is remarkably stable upon removal of the osmolyte, when cofactor thiamine pyrophosphate and the transacylase component of the BCKD complex are present. The above in vitro results offer the use of chemical chaperones such as TMAO as an approach to mitigate assembly defects caused by maple syrup urine disease mutations.     

支链氨基酸合成调控机制及菌种选育策略研究进展

提高国内支链氨基酸产生菌的高产菌株选育水平有助于缩短与国外生产之间的差距,满足国内市场需求。根据支链氨基酸生物合成途径及代谢调节,重点阐述了合成过程中关键酶的代谢调控,介绍了诱变育种、代谢工程、基因组改组及全局转录机器工程四种育种策略的研究进展。在支链氨基酸选育方面,全局转录机器工程育种目前虽无成功实例,但具有很大的潜力,而其他育种策略在氨基酸的选育中均发挥重要作用,可供国内相关育种工作者参考使用。     

A subset of Actinobacillus pleuropneumoniae in vivo induced promoters respond to branched-chain amino acid limitation

Actinobacillus pleuropneumoniae is the causative agent of a necrotizing hemorrhagic pleuropneumonia in swine. In this study, we investigate the possibility that the limitation of branched-chain amino acids is a stimulus that A. pleuropneumoniae will encounter during infection and will respond to by up-regulation of genes involved in branched-chain amino acid biosynthesis and virulence. Actinobacillus pleuropneumoniae genetic loci that are specifically induced during infection were screened in vitro for expression in response to limitation of branched-chain amino acids. Of 32 in vivo induced promoter clones screened in vitro, eight were induced on chemically defined medium without isoleucine, leucine and valine as compared to complete chemically defined medium. We identify the genomic context of each clone and discuss its relevance to branched-chain amino acid limitation and virulence. We conclude that limitation of branched-chain amino acids is a cue for expression of a subset in vivo induced genes, including not only genes involved in the biosynthesis of branched-chain amino acids, but also other genes that are induced during infection of the natural host. These results suggest that limitation of branched-chain amino acids may be one of an array of environmental cues responsible for the induction of virulence-associated genes in A. pleuropneumoniae.     

Roles of active site and novel K+ ion-binding site residues in human mitochondrial branched-chain alpha-ketoacid decarboxylase/dehydrogenase

The human mitochondrial branched-chain alpha-ketoacid decarboxylase/dehydrogenase (BCKD) is a heterotetrameric (alpha(2)beta(2)) thiamine diphosphate (TDP)-dependent enzyme. The recently solved human BCKD structure at 2.7 A showed that the two TDP-binding pockets are located at the interfaces between alpha and beta' subunits and between alpha' and beta subunits. In the present study, we show that the E76A-beta' mutation results in complete inactivation of BCKD. The result supports the catalytic role of the invariant Glu-76-beta' residue in increasing basicity of the N-4' amino group during the proton abstraction from the C-2 atom on the thiazolium ring. A substitution of His-146-beta' with Ala also renders the enzyme completely inactive. The data are consistent with binding of the alpha-ketoacid substrate by this residue based on the Pseudomonas BCKD structure. Alterations in Asn-222-alpha, Tyr-224-alpha, or Glu-193-alpha, which coordinates to the Mg(2+) ion, result in an inactive enzyme (E193A-alpha) or a mutant BCKD with markedly higher K(m) for TDP and a reduced level of the bound cofactor (Y224A-alpha and N222S-alpha). Arg-114-alpha, Arg-220-alpha, and His-291-alpha interact with TDP by directly binding to phosphate oxygens of the cofactor. We show that natural mutations of these residues in maple syrup urine disease (MSUD) patients (R114W-alpha and R220W-alpha) or site-directed mutagenesis (H291A-alpha) also result in an inactive or partially active enzyme, respectively. Another MSUD mutation (T166M-alpha), which affects one of the residues that coordinate to the K(+) ion on the alpha subunit, also causes inactivation of the enzyme and an attenuated ability to bind TDP. In addition, fluorescence measurements establish that Trp-136-beta in human BCKD is the residue quenched by TDP binding. Thus, our results define the functional roles of key amino acid residues in human BCKD and provide a structural basis for MSUD.     

Expression of 3-hydroxyisobutyrate dehydrogenase in cultured neural cells

The branched-chain amino acids (BCAAs) – isoleucine, leucine, and valine – belong to the limited group of substances transported through the blood–brain barrier. One of the functions they are thought to have in brain is to serve as substrates for meeting parenchymal energy demands. Previous studies have shown the ubiquitous expression of a branched-chain alpha-keto acid dehydrogenase among neural cells. This enzyme catalyzes the initial and rate-limiting step in the irreversible degradative pathway for the carbon skeleton of valine and the other two branched-chain amino acids. Unlike the acyl-CoA derivates in the irreversible part of valine catabolism, 3-hydroxyisobutyrate could be expected to be released from cells by transport across the mitochondrial and plasma membranes. This could indeed be demonstrated for cultured astroglial cells. Therefore, to assess the ability of neural cells to make use of this valine-derived carbon skeleton as a metabolic substrate for the generation of energy, we investigated the expression in cultured neural cells of the enzyme processing this hydroxy acid, 3-hydroxyisobutyrate dehydrogenase (HIBDH). To achieve this, HIBDH was purified from bovine liver to serve as antigen for the production of an antiserum. Affinity-purified antibodies against HIBDH specifically recognized the enzyme in liver and brain homogenates. Immunocytochemistry demonstrated the ubiquitous expression of HIBDH among cultured glial (astroglial, oligodendroglial, microglial, and ependymal cells) and neuronal cells. Using an RT-PCR technique, these findings were corroborated by the detection of HIBDH mRNA in these cells. Furthermore, immunofluorescence double-labeling of astroglial cells with antisera against HIBDH and the mitochondrial marker pyruvate dehydrogenase localized HIBDH to mitochondria. The expression of HIBDH in neural cells demonstrates their potential to utilize valine imported into the brain for the generation of energy.     

Metabolic modulation and cellular therapy of cardiac dysfunction and failure

At present the prevalence of heart failure rises along with aging of the population. Current heart failure therapeutic options are directed towards disease prevention via neurohormonal antagonism (β-blockers, angiotensin converting enzyme inhibitors and/or angiotensin receptor blockers and aldosterone antagonists), symptomatic treatment with diuretics and digitalis and use of biventricular pacing and defibrillators in a special subset of patients. Despite these therapies and device interventions heart failure remains a progressive disease with high mortality and morbidity rates. The number of patients who survive to develop advanced heart failure is increasing. These patients require new therapeutic strategies. In this review two of emerging therapies in the treatment of heart failure are discussed: metabolic modulation and cellular therapy. Metabolic modulation aims to optimize the myocardial energy utilization via shifting the substrate utilization from free fatty acids to glucose. Cellular therapy on the other hand has the goal to achieve true cardiac regeneration. We review the experimental data that support these strategies as well as the available pharmacological agents for metabolic modulation and clinical application of cellular therapy.     

Sequence conservation in the alpha and beta subunits of pyruvate dehydrogenase and its similarity to branched-chain alpha-keto acid dehydrogenase

Amino acid sequence comparison of 8 alpha and 6 beta subunits of the alpha-keto acid dehydrogenase (E1) component of the pyruvate dehydrogenase complex and branched-chain alpha-keto acid dehydrogenase complex form multiple species was performed by computer analysis. In addition to 2 previously recognized regions of homology in the alpha subunit, a 3rd region of extensive homology was identified in E1 alpha, and may be one of the sites involved in subunit interaction. E1 beta contains 4 regions of extensive homology. Region 1 contains 10 amino acids that are homologous to a 10-amino acid stretch in Escherichia coli E1. Regions 2 and 3 have sequence homologies with other dehydrogenases suggesting that these regions may be involved in catalysis.