This study presents the first three-dimensional structures of human cytosolic branched-chain aminotransferase (hBCATc) isozyme complexed with the neuroactive drug gabapentin, the hBCATc Michaelis complex with the substrate analog, 4-methylvalerate, and the mitochondrial isozyme (hBCATm) complexed with gabapentin. The branched-chain aminotransferases (BCAT) reversibly catalyze transamination of the essential branched-chain amino acids (leucine, isoleucine, valine) to ␣-ketoglutarate to form the respective branched-chain ␣-keto acids and glutamate. The cytosolic isozyme is the predominant BCAT found in the nervous system, and only hBCATc is inhibited by gabapentin. Pre-steady state kinetics show that 1.3 mM gabapentin can completely inhibit the binding of leucine to reduced hBCATc, whereas 65.4 mM gabapentin is required to inhibit leucine binding to hBCATm. Structural analysis shows that the bulky gabapentin is enclosed in the activesite cavity by the shift of a flexible loop that enlarges the active-site cavity. The specificity of gabapentin for the cytosolic isozyme is ascribed at least in part to the location of the interdomain loop and the relative orientation between the small and large domain which is different from these relationships in the mitochondrial isozyme. Both isozymes contain a CXXC center and form a disulfide bond under oxidizing conditions. The structure of reduced hBCATc was obtained by soaking the oxidized hBCATc crystals with dithiothreitol. The close similarity in active-site structures between cytosolic enzyme complexes in the oxidized and reduced states is consistent with the small effect of oxidation on pre-steady state kinetics of the hBCATc first half-reaction. However, these kinetic data do not explain the inactivation of hBCATm by oxidation of the CXXC center. The structural data suggest that there is a larger effect of oxidation on the interdomain loop and residues surrounding the CXXC center in hBCATm than in hBCATc. Pyridoxal 5Ј-phosphate (PLP)3 -dependent branched-chain aminotransferases (BCAT) reversibly catalyze the transfer of the ␣-amino group of the hydrophobic branched-chain amino acids (leucine, isoleucine, valine) to ␣-ketoglutarate to form the respective branched-chain ␣-keto acids and glutamate (1, 2). BCAT enzymes are found in both bacteria and higher organisms (3, 4). Mammals have a mitochondrial (BCATm) and a cytosolic (BCATc) form of the enzyme (5, 6), whereas bacteria have a single BCAT enzyme (7). Human mitochondrial BCATm (hBCATm) is expressed ubiquitously in body tissues, whereas human cytosolic BCATc (hBCATc) is found primarily in the nervous system (5, 8 -10). In fact, hBCATc is the predominant isozyme in brain, accounting for 60 -70% of total brain BCAT activity (8,11, 12). In the rat nervous system BCATc is expressed in selected populations of glutamatergic and ␥-aminobutyric acid (GABAergic) neurons (13), and Hutson and co-workers (14 -16) have postulated the existence of a glial-neuron nitrogen shuttle for the BCATs and branched-chain amino acids involved i...
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.
Serine palmitoyltransferase (SPT) is a key enzyme of sphingolipid biosynthesis and catalyses the pyridoxal 5'-phosphate (PLP)-dependent decarboxylative condensation reaction of l-serine with palmitoyl-CoA to generate 3-ketodihydrosphingosine. The crystal structure of SPT from Sphingobacterium multivorum GTC97 complexed with l-serine was determined at 2.3 A resolution. The electron density map showed the Schiff base formation between l-serine and PLP in the crystal. Because of the hydrogen bond formation with His138, the orientation of the Calpha-H bond of the PLP-l-serine aldimine was not perpendicular to the PLP-Schiff base plane. This conformation is unfavourable for the alpha-proton abstraction by Lys244 and the reaction is expected to stop at the PLP-l-serine aldimine. Structural modelling of the following intermediates indicated that His138 changes its hydrogen bond partner from the carboxyl group of l-serine to the carbonyl group of palmitoyl-CoA upon the binding of palmitoyl-CoA, making the l-serine Calpha-H bond perpendicular to the PLP-Schiff base plane. These crystal and model structures well explained the observations on bacterial SPTs that the alpha-deprotonation of l-serine occurs only in the presence of palmitoyl-CoA. This study provides the structural evidence that directly supports our proposed mechanism of the substrate synergism in the SPT reaction.
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 -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 ␣-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. Branched chain aminotransferases (BCATs)4 (EC 2.6.1.42) catalyze transamination of the branched chain amino acids, leucine, isoleucine, and valine, to their respective ␣-keto acids, ␣-ketoisocaproate, ␣-keto--methylvalerate, and ␣-ketoisovalerate. In humans, there are two isozymes, a mitochondrial (hBCATm) and a cytosolic (hBCATc) form (1-6), whereas bacteria contain only a single BCAT (6). The BCAT and transamination reactions of other pyridoxal 5Ј-phosphate (PLP)-dependent enzymes follow a ping-pong Bi-Bi reaction (7,8).Amino acid 1 ϩ ␣-keto acid 2^␣ -keto acid 1 ϩ amino acid 2 REACTION 1The reaction is accompanied by interconversion of the cofactor between the PLP and the pyridoxamine 5Ј-phosphate (PMP) forms (7-12). In the first half-reaction, the PLP form of BCAT reacts with the branched chain amino acid, and the reaction proceeds through a Michaelis complex, an external aldimine, quinonoid intermediate, ketimine, and finally the PMP form of the BCAT and the branched chain ␣-keto acid product. In the Michaelis complex, the ␣-amino group of the substrate must be deprotonated to form a new Schiff base with PLP. The migration of the proton on the ␣-amino group of the substrate to the short contact pair between the ␣-carboxylate and the phosphate of PLP reduces the electrostatic repulsion between the pair and renders the ␣-amino group of the substrate deprotonated (6). The second half-reaction is the reverse of the first half-reaction.Structurally PLP-dependent enzymes are c...
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.
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