Differential redox potential between the human cytosolic and mitochondrial branched-chain aminotransferase

The human branched-chain aminotransferase (hBCAT) isoenzymes are CXXC motif redox sensitive homodimers central to glutamate metabolism in the central nervous system. These proteins respond differently to oxidation by H(2)O(2), NO, and S-glutathionylation, suggesting that the redox potential is distinct between isoenzymes. Using various reduced to oxidized glutathione ratios (GSH:GSSG) to alter the redox environment, we demonstrate that hBCATc (cytosolic) has an overall redox potential that is 30 mV lower than hBCATm (mitochondrial). Furthermore, the CXXC motif of hBCATc was estimated to be 80 mV lower, suggesting that hBCATm is more oxidizing in nature. Western blot analysis revealed close correlations between hBCAT S-glutathionylation and the redox status of the assay environment, offering the hBCAT isoenzymes as novel biomarkers for cytosolic and mitochondrial oxidative stress.     

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.     

Human mitochondrial branched chain aminotransferase isozyme: structural role of the CXXC center in catalysis

Mammalian branched chain aminotransferases (BCATs) have a unique CXXC center. Kinetic and structural studies of three CXXC center mutants (C315A, C318A, and C315A/C318A) of human mitochondrial (hBCATm) isozyme and the oxidized hBCATm enzyme (hBCATm-Ox) have been used to elucidate the role of this center in hBCATm catalysis. X-ray crystallography revealed that the CXXC motif, through its network of hydrogen bonds, plays a crucial role in orienting the substrate optimally for catalysis. In all structures, there were changes in the structure of the beta-turn preceding the CXXC motif when compared with wild type protein. The N-terminal loop between residues 15 and 32 is flexible in the oxidized and mutant enzymes, the disorder greater in the oxidized protein. Disordering of the N-terminal loop disrupts the integrity of the side chain binding pocket, particularly for the branched chain side chain, less so for the dicarboxylate substrate side chain. The kinetic studies of the mutant and oxidized enzymes support the structural analysis. The kinetic results showed that the predominant effect of oxidation was on the second half-reaction rather than the first half-reaction. The oxidized enzyme was completely inactive, whereas the mutants showed limited activity. Model building of the second half-reaction substrate alpha-ketoisocaproate in the pyridoxamine 5'-phosphate-hBCATm structure suggests that disruption of the CXXC center results in altered substrate orientation and deprotonation of the amino group of pyridoxamine 5'-phosphate, which inhibits catalysis.     

Regulatory control of human cytosolic branched-chain aminotransferase by oxidation and S-glutathionylation and its interactions with redox sensitive neuronal proteins

Redox regulation of proteins through oxidation and S-thiolation are important regulatory processes, acting in both a protective and adaptive role in the cell. In the current study, we investigated the sensitivity of the neuronal human cytosolic branched-chain aminotransferase (hBCATc) protein to oxidation and S-thiolation, with particular attention focused on functionality and modulation of its CXXC motif. Thiol specific reagents showed significant redox cycling between the reactive thiols and the TNB anion, and using NEM, four of the six reactive thiols are critical to the functionality of hBCATc. Site-directed mutagenesis studies supported these findings where a reduced kcat (ranging from 50-70% of hBCATc) for C335S, C338S, C335/8S, and C221S, respectively, followed by a modest effect on C242S was observed. However, only the thiols of the CXXC motif (C335 and C338) were directly involved in the reversible redox regulation of hBCATc through oxidation (with a loss of 40-45% BCAT activity on air oxidation alone). Concurrent with these findings, under air oxidation, the X-ray crystallography structure of hBCATc showed a disulphide bond between C335 and C338. Further oxidation of the other four thiols was not evident until levels of hydrogen peroxide were elevated. S-thiolation experiments of hBCATc exposed to GSH provided evidence for significant recycling between GSH and the thiols of hBCATc, which implied that under reducing conditions GSH was operating as a thiol donor with minimal S-glutathionylation. Western blot analysis of WT hBCATc and mutant proteins showed that as the ratio of GSH:GSSG decreased significant S-glutathionylation occurred (with a further loss of 20% BCAT activity), preferentially at the thiols of the CXXC motif, suggesting a shift in function toward a more protective role for GSH. Furthermore, the extent of S-glutathionylation increased in response to oxidative stress induced by hydrogen peroxide potentially through a C335 sulfenic acid intermediate. Deglutathionylation of hBCATc-SSG using the GSH/glutaredoxin system provides evidence that this protein may play an important role in cellular redox regulation. Moreover, redox associations between hBCATc and several neuronal proteins were identified using targeted proteomics. Thus, our data provides strong evidence that the reactive thiol groups, in particular the thiols of the CXXC motif, play an integral role in redox regulation and that hBCATc has redox mediated associations with several neuronal proteins involved in G-protein cell signaling, indicating a novel role for hBCATc in cellular redox control.     

Distribution of the branched chain aminotransferase proteins in the human brain and their role in glutamate regulation

The branched chain aminotransferase enzymes (BCAT) serve as nitrogen donors for the production of 30% of de novo glutamate synthesis in rat brain. Despite the importance of this major metabolite and excitatory neurotransmitter, the distribution of BCAT proteins in the human brain (hBCAT) remains unreported. We have studied this and report, for the first time, that the mitochondrial isoform, hBCATm is largely confined to vascular endothelial cells, whereas the cytosolic hBCATc is restricted to neurons. The majority of hBCATc‐labelled neurons were either GABA‐ergic or glutamatergic showing both cell body and axonal staining indicating a role for hBCATc in both glutamate production and glutamate release during excitation. Strong staining in hormone secreting cells suggests a further role for the transaminases in hormone regulation potentially similar to that proposed for insulin secretion. Expression of hBCATm in the endothelial cells of the vasculature demonstrates for the first time that glutamate could be metabolized by aminotranferases in these cells. This has important implications given that the dysregulation of glutamate metabolism, leading to glutamate excitotoxicity, is an important contributor to the pathogenesis of several neurodegenerative conditions, where the role of hBCATm in metabolizing excess glutamate may factor more prominently.     

Roles for cysteine residues in the regulatory CXXC motif of human mitochondrial branched chain aminotransferase enzyme

The redox-active dithiol/disulfide C315-Xaa-Xaa-C318 center has been implicated in the regulation of the human mitochondrial branched chain aminotransferase isozyme (hBCATm) [Conway, M. E., Yennawar, N., Wallin, R., Poole, L. B., and Hutson, S. M. (2002) Biochemistry 41, 9070-9078]. To explore further the mechanistic details of this CXXC center, mutants of the Cys residues at positions 315 and 318 of hBCATm were individually and in combination converted to alanine or serine by site-directed mutagenesis (C315A, C315S, C318A, C318S, C315/318A, and C315/318S). The effects of these mutations on cofactor absorbance, secondary structures, steady-state kinetics, and sensitivity toward hydrogen peroxide (H(2)O(2)) treatment were examined. Neither the UV-visible spectroscopic studies nor the circular dichroism data showed any major perturbations in the structure of the mutants. Kinetic analyses of the CXXC mutant proteins indicated primarily a modest reduction in k(cat) with no significant change in K(m). The largest effect on the steady-state kinetics was observed with the C315 single mutants, in which substitution of the thiol group resulted in a reduced k(cat) (to 26-33% of that of wild-type hBCATm). Moreover, the C315 single mutants lost their sensitivity to oxidation by H(2)O(2). The kinetic parameters of the C318 mutants were largely unaffected by the substitutions, and as with wild-type hBCATm, reaction of the C318A mutant protein with H(2)O(2) resulted in the complete loss of activity. In the case of oxidized C318A, this loss was largely irreversible on incubation with dithiothreitol. Mass spectrometry and dimedone modification results revealed overoxidation of the thiol group at position 315 to sulfonic acid occurring via a sulfenic acid intermediate in the H(2)O(2)-treated C318A enzyme. Thus, C315 appears to be the sensor for redox regulation of BCAT activity, whereas C318 acts as the "resolving cysteine", allowing for reversible formation of a disulfide bond.     

Human mitochondrial branched chain aminotransferase: structural basis for substrate specificity and role of redox active cysteines

Crystal structures of the fold type IV pyridoxal phosphate (PLP)-dependent human mitochondrial branched chain aminotransferase (hBCATm) reaction intermediates have provided a structural explanation for the kinetically determined substrate specificity of hBCATm. The isoleucine side chain in the ketimine intermediate occupies a hydrophobic binding pocket that can be defined by three surfaces. Modeling of amino acids on the ketimine structure shows that the side chains of nonsubstrate amino acids such as the aromatic amino acids, alanine, or aspartate either are unable to interact through van der Waals' interactions or have steric clashes. The structural and biochemical basis for the sensitivity of the mammalian BCAT to reducing agents has also been elucidated. Two cysteine residues in hBCATm, Cys315 and Cys318 (CXXC), are part of a redox-controlled mechanism that can regulate hBCATm activity. The residues surrounding Cys315 and Cys318 show considerable sequence conservation in the prokaryotic and eukaryotic BCAT sequences, however, the CXXC motif is found only in the mammalian proteins. The results suggest that the BCAT enzymes may join the list of enzymes that can be regulated by redox status.     

Crystal structures of human mitochondrial branched chain aminotransferase reaction intermediates: ketimine and pyridoxamine phosphate forms

The three-dimensional structures of the isoleucine ketimine and the pyridoxamine phosphate forms of human mitochondrial branched chain aminotransferase (hBCATm) have been determined crystallographically at 1.9 A resolution. The hBCATm-catalyzed transamination can be described in molecular terms together with the earlier solved pyridoxal phosphate forms of the enzyme. The active site lysine, Lys202, undergoes large conformational changes, and the pyridine ring of the cofactor tilts by about 18 degrees during catalysis. A major determinant of the enzyme's substrate and stereospecificity for L-branched chain amino acids is a group of hydrophobic residues that form three hydrophobic surfaces and lock the side chain in place. Short-chain aliphatic amino acid side chains are unable to interact through van der Waals contacts with any of the surfaces whereas bulky aromatic side chains would result in significant steric hindrance. As shown by modeling, and in agreement with previous biochemical data, glutamate but not aspartate can form hydrogen bond interactions. The carboxylate group of the bound isoleucine is on the same side as the phosphate group of the cofactor. These active site interactions are largely retained in a model of the human cytosolic branched chain aminotransferase (hBCATc), suggesting that residues in the second tier of interactions are likely to determine the specificity of hBCATc for the drug gabapentin. Finally, the structures reveal a unique role for cysteine residues in the mammalian BCAT. Cys315 and Cys318, which immediately follow a beta-turn (residues 311-314) and are located just outside the active site, form an unusual thiol-thiolate hydrogen bond. This beta-turn positions Thr313 for its interaction with the pyridoxal phosphate oxygens and substrate alpha-carboxylate group.     

Immunocytochemical localization of beta-methylcrotonyl-CoA carboxylase in astroglial cells and neurons in culture

Astroglia-rich primary cultures and brain slices rapidly metabolize branched-chain amino acids (BCAAs), in particular leucine, as energy substrates. To allocate the capacity to degrade leucine oxidatively in neural cells, we have purified beta-methylcrotonyl-CoA carboxylase (beta-MCC) from rat liver as one of the enzymes unique for the irreversible catabolic pathway of leucine. Polyclonal antibodies raised against beta-MCC specifically cross-reacted with both enzyme subunits in liver and brain homogenates. Immunocytochemical examination of astroglia-rich rat primary cultures demonstrated the presence of beta-MCC in astroglial cells, where the enzyme was found to be located in the mitochondria, the same organelle that the mitochondrial isoform of the BCA(A) aminotransferase (BCAT) is located in. This colocalization of the two enzymes supports the hypothesis that mitochondrial BCAT is the isoenzyme that in brain energy metabolism prepares the carbon skeleton of leucine for irreversible degradation in astrocytes. Analysis of neuron-rich primary cultures revealed also that the majority of neurons contained beta-MCC. The presence of beta-MCC in most neurons demonstrates their ability to degrade the alpha-ketoisocaproate that could be provided by neighboring astrocytes or could be generated locally from leucine by the action of the cytosolic isoform of BCAT that is known to occur in neurons.     

Crystal Structures of Complexes of the Branched-Chain Aminotransferase from Deinococcus radiodurans with α-Ketoisocaproate and l-Glutamate Suggest the Radiation Resistance of This Enzyme for Catalysis

Branched-chain aminotransferases (BCAT), which utilize pyridoxal 5′-phosphate (PLP) as a cofactor, reversibly catalyze the transfer of the α-amino groups of three of the most hydrophobic branched-chain amino acids (BCAA), leucine, isoleucine, and valine, to α-ketoglutarate to form the respective branched-chain α-keto acids and glutamate. The BCAT from Deinococcus radiodurans (DrBCAT), an extremophile, was cloned and expressed in Escherichia coli for structure and functional studies. The crystal structures of the native DrBCAT with PLP and its complexes with l-glutamate and α-ketoisocaproate (KIC), respectively, have been determined. The DrBCAT monomer, comprising 358 amino acids, contains large and small domains connected with an interdomain loop. The cofactor PLP is located at the bottom of the active site pocket between two domains and near the dimer interface. The substrate (l-glutamate or KIC) is bound with key residues through interactions of the hydrogen bond and the salt bridge near PLP inside the active site pocket. Mutations of some interaction residues, such as Tyr71, Arg145, and Lys202, result in loss of the specific activity of the enzymes. In the interdomain loop, a dynamic loop (Gly173 to Gly179) clearly exhibits open and close conformations in structures of DrBCAT without and with substrates, respectively. DrBCAT shows the highest specific activity both in nature and under ionizing radiation, but with lower thermal stability above 60°C, than either BCAT from Escherichia coli (eBCAT) or from Thermus thermophilus (HB8BCAT). The dimeric molecular packing and the distribution of cysteine residues at the active site and the molecular surface might explain the resistance to radiation but small thermal stability of DrBCAT.     

The design and synthesis of human branched-chain amino acid aminotransferase inhibitors for treatment of neurodegenerative diseases

The inhibition of the cytosolic isoenzyme BCAT that is expressed specifically in neuronal tissue is likely to be useful for the treatment of neurodegenerative and other neurological disorders where glutamatergic mechanisms are implicated. Compound 2 exhibited an IC50 of 0.8 microM in the hBCATc assays; it is an active and selective inhibitor. Inhibitor 2 also blocked calcium influx into neuronal cells following inhibition of glutamate uptake, and demonstrated neuroprotective efficacy in vivo. SAR, pharmacology, and the crystal structure of hBCATc with inhibitor 2 are described.     

Identification of a peroxide-sensitive redox switch at the CXXC motif in the human mitochondrial branched chain aminotransferase

The human mitochondrial branched chain aminotransferase isoenzyme (hBCATm) must be stored in a reducing environment to remain active. Oxidation or labeling of hBCATm with sulfhydryl reagents results in enzyme inhibition. In this study, we investigated both the structural and biochemical basis for the sensitivity of hBCATm to these reagents. In its native form, hBCATm has two reactive cysteine residues which were identified as Cys315 and Cys318 using iodinated beta-(4-hydroxyphenyl)ethyl maleimide. These are located in the large domain of the homodimer, about 10 A from the active site. The crystal structures show evidence for a thiol-thiolate hydrogen bond between Cys315 and Cys318. Under oxidizing conditions, these cysteine residues can reasonably form a disulfide bond because of the short distance between the sulfur atoms (3.09-3.46 A), requiring only a decrease of 1.1-1.5 A. In addition to Cys315 playing a structural role by anchoring Tyr173, which in the ketimine form increases access to the active site, our evidence indicates that these cysteine residues act as a redox switch in hBCATm. Electrospray ionization mass spectrometry analysis and UV-Vis spectroscopic studies of 5,5'-dithiobis(2-nitrobenzoic acid) labeled hBCATm showed that during labeling, an intrasubunit disulfide bond was formed in a significant portion of the protein. Furthermore, it was established that reaction of hBCATm with H2O2 abolished its activity and resulted in the formation of an intrasubunit disulfide bond between Cys315 and Cys318. Addition of dithiothreitol completely reversed the oxidation and restored activity. Therefore, the results demonstrate that there is redox-linked regulation of hBCATm activity by a peroxide sensitive CXXC center. Future studies will determine if this center has an in vivo role in the regulation of branched chain amino acid metabolism.     

Role of Branched-Chain Aminotransferase Isoenzymes and Gabapentin in Neurotransmitter Metabolism

Abstract: Because it is well known that excess branched-chain amino acids (BCAAs) have a profound influence on neurological function, studies were conducted to determine the impact of BCAAs on neuronal and astrocytic metabolism and on trafficking between neurons and astrocytes. The first step in the metabolism of BCAAs is transamination with α-ketoglutarate to form the branched-chain α-keto acids (BCKAs). The brain is unique in that it expresses two separate branched-chain aminotransferase (BCAT) isoenzymes. One is the common peripheral form [mitochondrial (BCATm)], and the other [cytosolic (BCATc)] is unique to cerebral tissue, placenta, and ovaries. Therefore, attempts were made to define the isoenzymes' spatial distribution and whether they might play separate metabolic roles. Studies were conducted on primary rat brain cell cultures enriched in either astroglia or neurons. The data show that over time BCATm becomes the predominant isoenzyme in astrocyte cultures and that BCATc is prominent in early neuronal cultures. The data also show that gabapentin, a structural analogue of leucine with anticonvulsant properties, is a competitive inhibitor of BCATc but that it does not inhibit BCATm. Metabolic studies indicated that BCAAs promote the efflux of glutamine from astrocytes and that gabapentin can replace leucine as an exchange substrate. Studying astrocyte-enriched cultures in the presence of [U-¹⁴C]glutamate we found that BCKAs, but not BCAAs, stimulate glutamate transamination to α-ketoglutarate and thus irreversible decarboxylation of glutamate to pyruvate and lactate, thereby promoting glutamate oxidative breakdown. Oxidation of glutamate appeared to be largely dependent on the presence of an α-keto acid acceptor for transamination in astrocyte cultures and independent of astrocytic glutamate dehydrogenase activity. The data are discussed in terms of a putative BCAA/BCKA shuttle, where BCATs and BCAAs provide the amino group for glutamate synthesis from α-ketoglutarate via BCATm in astrocytes and thereby promote glutamine transfer to neurons, whereas BCATc reaminates the amino acids in neurons for another cycle.     

Branched-Chain Aminotransferases Control TORC1 Signaling in Saccharomyces cerevisiae

The conserved target of rapamycin complex 1 (TORC1) integrates nutrient signals to orchestrate cell growth and proliferation. Leucine availability is conveyed to control TORC1 activity via the leu-tRNA synthetase/EGOC-GTPase module in yeast and mammals, but the mechanisms sensing leucine remain only partially understood. We show here that both leucine and its α-ketoacid metabolite, α-ketoisocaproate, effectively activate the yeast TORC1 kinase via both EGOC GTPase-dependent and -independent mechanisms. Leucine and α-ketoisocaproate are interconverted by ubiquitous branched-chain aminotransferases (BCAT), which in yeast are represented by the mitochondrial and cytosolic enzymes Bat1 and Bat2, respectively. BCAT yeast mutants exhibit severely compromised TORC1 activity, which is partially restored by expression of Bat1 active site mutants, implicating both catalytic and structural roles of BCATs in TORC1 control. We find that Bat1 interacts with branched-chain amino acid metabolic enzymes and, in a leucine-dependent fashion, with the tricarboxylic acid (TCA)-cycle enzyme aconitase. BCAT mutation perturbed TCA-cycle intermediate levels, consistent with a TCA-cycle block, and resulted in low ATP levels, activation of AMPK, and TORC1 inhibition. We propose the biosynthetic capacity of BCAT and its role in forming multicomplex metabolons connecting branched-chain amino acids and TCA-cycle metabolism governs TCA-cycle flux to activate TORC1 signaling. Because mammalian mitochondrial BCAT is known to form a supramolecular branched-chain α-keto acid dehydrogenase enzyme complex that links leucine metabolism to the TCA-cycle, these findings establish a precedent for understanding TORC1 signaling in mammals.     

Sheep cytosolic branched-chain amino acid aminotransferase: cDNA cloning, primary structure and molecular modelling and its unique expression in muscles

This paper presents the cloning and the molecular modelling of the cytosolic branched-chain amino acid aminotransferase (BCATc) from sheep brain. The sheep BCATc cDNA (3 kb) encodes a mature polypeptide of 385 amino acids with a calculated molecular mass of 43072.93 Da. The sequence of the sheep BCATc cDNA is more similar to other mammalian BCATc cDNAs (53-87% identical) than to the sheep mitochondrial branched-chain amino acid aminotransferase (52%). Sheep BCATc belongs to the IV Folding class of pyridoxal-5'-phosphate-depending enzymes. Based on the known structure of the branched-chain amino acid aminotransferase (BCAT) from Escherichia coli, a molecular model of sheep BCATc (amino acid residues 62-385) was built. This is the first three-dimensional model of any mammalian BCAT. It suggests that the enzymatic mechanism of sheep BCATc and likely of all mammalian BCAT is very similar to the mechanism of the E. coli BCAT and confirms the hypotheses regarding to the substrate binding sites of E. coli BCAT. Sheep skeletal muscle, which is the main in vivo site for transamination of branched-chain amino acids, exhibits an unique expression of BCATc.     

A continuous 96-well plate spectrophotometric assay for branched-chain amino acid aminotransferases

A new, continuous 96-well plate spectrophotometric assay for the branched-chain amino acid aminotransferases is described. Transamination of L-leucine with alpha-ketoglutarate results in formation of alpha-ketoisocaproate, which is reductively aminated back to L-leucine by leucine dehydrogenase in the presence of ammonia and NADH. The disappearance of absorbance at 340 nm due to NADH oxidation is measured continuously. The specific activities obtained by this procedure for the highly purified human mitochondrial and cytosolic isoforms of BCAT compare favorably with those obtained by a commonly used radiochemical procedure, which measures transamination between alpha-ketoiso[1-14C]valerate and L-isoleucine. Due to the presence of glutamate dehydrogenase substrates (alpha-ketoglutarate, ammonia, and NADH) and L-leucine (an activator of glutamate dehydrogenase) in the standard assay mixture, interference with the measurement of BCAT activity in tissue homogenates by glutamate dehydrogenase is observed. However, by limiting the amount of ammonia and including the inhibitor GTP in the assay mixture, the interference from the glutamate dehydrogenase reaction is minimized. By comparing the rate of loss of absorbance at 340 nm in the modified spectrophotometric assay mixture containing leucine dehydrogenase to that obtained in the modified spectrophotometric assay mixture lacking leucine dehydrogenase, it is possible to measure BCAT activity in microliter amounts of rat tissue homogenates. The specific activities of BCAT in homogenates of selected rat tissues obtained by this method are comparable to those obtained previously by the radiochemical procedure.     

Branched-chain amino acid catabolism: unique segregation of pathway enzymes in organ systems and peripheral nerves

We have examined the localization of the first two enzymes in the branched-chain amino acid (BCAA) catabolic pathway: the branched-chain aminotransferase (BCAT) isozymes (mitochondrial BCATm and cytosolic BCATc) and the branched-chain alpha-keto acid dehydrogenase (BCKD) enzyme complex. Antibodies specific for BCATm or BCATc were used to immunolocalize the respective isozymes in cryosections of rat tissues. BCATm was expressed in secretory epithelia throughout the digestive tract, with the most intense expression in the stomach. BCATm was also strongly expressed in secretory cells of the exocrine pancreas, uterus, and testis, as well as in the transporting epithelium of convoluted tubules in kidney. In muscle, BCATm was located in myofibrils. Liver, as predicted, was not immunoreactive for BCATm. Unexpectedly, BCATc was localized in elements of the autonomic innervation of the digestive tract, as well as in axons in the sciatic nerve. The distributions of BCATc and BCATm did not overlap. BCATm-expressing cells also expressed the second enzyme of the BCAA catabolic pathway, BCKD. In selected monkey and human tissues examined by immunoblot and/or immunohistochemistry, BCATm and BCATc were distributed in patterns very similar to those found in the rat. The results show that BCATm is in a position to regulate BCAA availability as protein precursors and anabolic signals in secretory portions of the digestive and other organ systems. The unique expression of BCATc in neurons of the peripheral nervous system, without coexpression of BCKD, raises new questions about the physiological function of this BCAT isozyme.     

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.     

Structures of Escherichia coli branched-chain amino acid aminotransferase and its complexes with 4-methylvalerate and 2-methylleucine: induced fit and substrate recognition of the enzyme.

The following three-dimensional structures of three forms of Escherichia coli branched-chain amino acid aminotransferase (eBCAT) have been determined by the X-ray diffraction method: the unliganded pyridoxal 5'-phosphate (PLP) form at a 2.1 A resolution, and the two complexes with the substrate analogues, 4-methylvalerate (4-MeVA) as the Michaelis complex model and 2-methylleucine (2-MeLeu) as the external aldimine model at 2.4 A resolution. The enzyme is a trimer of dimers, and each subunit consists of small and large domains, and the interdomain loop. The active site is formed by the residues at the domain interface and those from two loops of the other subunit of the dimer unit, and binds one PLP with its re-face directed toward the protein side. Upon binding of a substrate, Arg40 changes its side-chain direction to interact with the interdomain loop, and the loop, which is disordered in the unliganded form, shows its ordered structure on the active-site cavity, interacts with the hydrophobic side chain of the substrate, and shields it from the solvent region. The substrate binds to the active-site pocket with its alpha-hydrogen toward the protein side, its side-chain on the side of O3 of PLP, and its alpha-carboxylate on the side of the phosphate group of PLP. The hydrophobic side-chain of the substrate is recognized by Phe36, Trp126, Tyr129, Tyr164, Tyr31*, and Val109*. The alpha-carboxylate of the substrate binds to the unique site constructed by three polar groups (two main-chain NH groups of the beta-turn at Thr257 and Ala258 and the hydroxy group of Tyr95) which are activated by the access of Arg40 to the main-chain C=O group of the beta-turn and the coordination of Arg97 to the hydroxy group. Since Arg40 is the only residue that significantly changes its side-chain conformation and directly interacts with the interdomain loop and the beta-turn, the residue plays important roles in the induced fit of the interdomain loop and the alpha-carboxylate recognition of the substrate.     

Expression of cytosolic branched chain aminotransferase (BCATc) mRNA in the developing mouse brain

Branched-chain aminotransferase (BCAT) catalyzes the transamination of essential branched-chain amino acids (BCAAs: leucine, isoleucine and valine) with alpha-ketoglutarate. Through this reaction, BCAAs provide nitrogen for the synthesis of glutamate, the predominant excitatory neurotransmitter. Two BCAT isoforms have been identified: one cytosolic (BCATc) and one mitochondrial (BCATm). In adult rodents, BCATc is expressed in a wide variety of structures of the central nervous system (CNS), in neurons. So far, no data were available about the expression of BCATc in the developing CNS. Here, we analyse the expression profile of BCATc mRNA in the mouse brain from embryonic day 12.5 to adult age. BCATc mRNA gradually appears in different brain regions starting from early stages of neural development, and is maintained until adulthood. BCATc mRNA is predominantly present in the cerebral cortex, hippocampus, thalamus, ventral midbrain, raphe, cerebellum and precerebellar system. This study represents the first detailed analysis of BCATc mRNA expression in the developing mouse brain.