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.     

Structural determinants for branched-chain aminotransferase isozyme-specific inhibition by the anticonvulsant drug gabapentin

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 alpha-ketoglutarate to form the respective branched-chain alpha-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 active-site 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.     

SAS solution structures of the apo and Mg2+/BeF3(-)-bound receiver domain of DctD from Sinorhizobium meliloti

Two-component signal transduction is the predominant information processing mechanism in prokaryotes and is also present in single-cell eukaryotes and higher plants. A phosphorylation-based switch is commonly used to activate as many as 40 different types of output domains in more than 6000 two-component response regulators that can be identified in the sequence databases. Previous biochemical and crystallographic studies showed that phosphorylation of the two-component receiver domain of DctD causes a switch between alternative dimeric forms, but it was unclear from the crystal lattice of the activated protein precisely which of four possible dimeric configurations is the biologically relevant one [Park, S., et al. (2002) FASEB J. 16, 1964-1966]. Here we report solution structures of the apo and activated DctD receiver domain derived from small angle scattering data. The apo dimer closely resembles that seen in the crystal structure, and the solution data for the activated protein eliminate two of the possible four dimeric conformations seen in the crystal lattice and strongly implicate one as the biologically relevant structure. These results corroborate the previously proposed model for how receiver domains regulate their downstream AAA+ ATPase domains.     

Characterization of PR-10 genes from eight Betula species and detection of Bet v 1 isoforms in birch pollen

Background  

Bet v 1 is an important cause of hay fever in northern Europe. Bet v 1 isoforms from the European white birch (Betula pendula) have been investigated extensively, but the allergenic potency of other birch species is unknown. The presence of Bet v 1 and closely related PR-10 genes in the genome was established by amplification and sequencing of alleles from eight birch species that represent the four subgenera within the genus Betula. Q-TOF LC-MS^E was applied to identify which PR-10/Bet v 1 genes are actually expressed in pollen and to determine the relative abundances of individual isoforms in the pollen proteome.     

Crystal structure of phosphotransacetylase from the methanogenic archaeon Methanosarcina thermophila

Phosphotransacetylase (Pta) [EC 2.3.1.8] is ubiquitous in the carbon assimilation and energy-yielding pathways in anaerobic prokaryotes where it catalyzes the reversible transfer of the acetyl group from acetyl phosphate to CoA forming acetyl CoA and inorganic phosphate. The crystal structure of Pta from the methane-producing archaeon Methanosarcina thermophila, representing the first crystal structure of any Pta, was determined by multiwavelength anomalous diffraction at 2.7 A resolution. In solution and in the crystal, the enzyme forms a homodimer. Each monomer consists of two alpha/beta domains with a cleft along the domain boundary, which presumably contains the substrate binding sites. Comparison of the four monomers present in the asymmetric unit indicates substantial variations in the relative orientation of the two domains and the structure of the putative active site cleft. A search for structural homologs revealed the NADP(+)-dependent isocitrate and isopropylmalate dehydrogenases as the only homologs with a similar two-domain architecture.     

Recent advances in ABE fermentation: hyper-butanol producing Clostridium beijerinckii BA101

This is an overview of the mutant strain Clostridium beijerinckii BA101 which produces solvents (acetone–butanol–ethanol, ABE) at elevated levels. This organism expresses high levels of amylases when grown on starch. C. beijerinckii BA101 hydrolyzes starch effectively and produces solvent in the concentration range of 27–29 g l⁻¹. C. beijerinckii BA101 has been characterized for both substrate and butanol inhibition. Supplementing the fermentation medium (MP2) with sodium acetate enhances solvent production to 33 g l⁻¹. The results of studies utilizing commercial fermentation medium and pilot plant-scale reactors are consistent with the results using small-scale reactors. Pervaporation, a technique to recover solvents, has been applied to fed-batch reactors containing C. beijerinckii BA101, and solvent production as high as 165 g l⁻¹ has been achieved. Immobilization of C. beijerinckii BA101 by adsorption and use in a continuous reactor resulted in reactor productivity of 15.8 g l⁻¹ h⁻¹. Recent economic studies employing C. beijerinckii BA101 suggested that butanol can be produced at US$0.20–0.25 lb⁻¹ by employing batch fermentation and distillative recovery. Application of new technologies such as pervaporation, fed-batch culture, and immobilized cell reactors is expected to further reduce these prices. Journal of Industrial Microbiology & Biotechnology (2001) 27, 287–291. Received 12 September 2000/ Accepted in revised form 27 January 2001     

The effect of diethyl- -fluoroglutarate on phosphates and glutamate in slices of rabbit cerebral cortex

Abstract— The diethyl ester of α-fluoroglutarate (DEFG), an inhibitor of glutamate dehydrogenase, was prepared, and its effect on glutamate and phosphates in slices of rabbit cerebral cortex was examined. The primary effect of the drug on cortical slices incubating in a Krebs-Ringer glucose medium was to decrease the tissue levels of glutamate in association with decreased levels and turnover of high-energy phosphates. Assimilation of exogenous glutamate by the slices was partially blocked in the presence of the drug and severely depressed oxidative phosphorylation resulted when glutamate and DEFG were both present in the incubation mixture. The results suggested a significant relationship between the activity of cerebral glutamate dehydrogenase and oxidative phosphorylation. During incubation in a Krebs-Ringer glucose medium the endogenous pool of free amino acids in the cortical slice partitioned with the medium. Little or no glutamate, aspartate or GABA was present in the medium after incubation, but glycine, alanine, threonine, serine and glutamine did partition to varying degrees, with over one-half of the glutamine present in the incubation medium. With the exception of ‘leakage’ of aspartate, the partitioning patterns were relatively unaffected by the presence of added glutamate or DEFG.