Expression and biochemical characterization of α-ketoglutarate decarboxylase from cyanobacterium Synechococcus sp. PCC7002.

α-Ketoglutarate decarboxylase (α-KGD), one member of α-keto acid decarboxylases, catalyzing non-oxidative decarboxylation of α-ketoglutarate to form succinic semialdehyde, was proposed to play critical role in completing tricarboxylic acid (TCA) cycle of cyanobacteria. Although the catalytic function of α-KGD from Synechococcus sp. PCC7002 was demonstrated previously, there was no detailed biochemical characterization of α-KGD from Synechococcus sp. PCC7002 yet. In this study, the gene encoding α-KGD from Synechococcus sp. PCC7002 was amplified and soluble expression of recombinant α-KGD was achieved by coexpressing with pTf16 chaperone plasmid in E. coli BL21 (DE3). Kinetic analysis showed that the activity of α-KGD was dependent on cofactors of thiamine pyrophosphate and divalent cation. Meanwhile this α-KGD was specific for α-ketoglutarate with respect to the decarboxylation activity despite of the pretty low activity of acetolactate synthase. The catalytic efficiency of α-KGD (the values of kcat and kcat/Km for α-ketoglutarate were 1.2s-1 and 6.3×103M-1s-1, respectively) might provide evidence for its physiological role in TCA cycle of Synechococcus sp. PCC7002.

The combined use of photoaffinity labeling and surface plasmon resonance-based technology identifies multiple salicylic acid-binding proteins.

Salicylic acid (SA) is a small phenolic molecule that not only is the active ingredient in the multi-functional drug aspirin, but also serves as a plant hormone that affects diverse processes during growth, development, responses to abiotic stresses and disease resistance. Although a number of SA-binding proteins (SABPs) have been identified, the underlying mechanisms of action of SA remain largely unknown. Efforts to identify additional SA targets, and thereby elucidate the complex SA signaling network in plants, have been hindered by the lack of effective approaches. Here, we report two sensitive approaches that utilize SA analogs in conjunction with either a photoaffinity labeling technique or surface plasmon resonance-based technology to identify and evaluate candidate SABPs from Arabidopsis. Using these approaches, multiple proteins, including the E2 subunit of α-ketoglutarate dehydrogenase and the glutathione S-transferases GSTF2, GSTF8, GSTF10 and GSTF11, were identified as SABPs. Their association with SA was further substantiated by the ability of SA to inhibit their enzymatic activity. The photoaffinity labeling and surface plasmon resonance-based approaches appear to be more sensitive than the traditional approach for identifying plant SABPs using size-exclusion chromatography with radiolabeled SA, as these proteins exhibited little to no SA-binding activity in such an assay. The development of these approaches therefore complements conventional techniques and helps dissect the SA signaling network in plants, and may also help elucidate the mechanisms through which SA acts as a multi-functional drug in mammalian systems.

Characterization of novel 2-oxoglutarate dependent dioxygenases converting L-proline to cis-4-hydroxy-l-proline.

Hydroxyprolines are valuable chiral building blocks for organic synthesis of pharmaceuticals. Several microorganisms producing L-proline trans-4- and cis-3-hydroxylase were discovered and these enzymes were applied to the industrial production of trans-4- and cis-3-hydroxy-L-proline, respectively. Meanwhile, other hydroxyproline isomers, cis-4- and trans-3-hydroxy-L-proline, were not easily available because the corresponding hydroxylase have not been discovered. Herein we report novel L-proline cis-4-hydroxylases converting free L-proline to cis-4-hydroxy-L-proline. Two genes encoding uncharacterized proteins from Mesorhizobium loti and Sinorhizobium meliloti were cloned and overexpressed in Escherichia coli, respectively. The functions of purified proteins were investigated in detail, and consequently we detected L-proline cis-4-hydroxylase activity in both proteins. Likewise L-proline trans-4-, cis-3-hydroxylase and prolyl hydroxylase, these enzymes belonged to a 2-oxoglutarate dependent dioxygenase family and required a non-heme ferrous ion. Although their reaction mechanisms were similar to other hydroxylases, the amino acid sequence homology was not observed (less than 40%).

Novel isoenzyme of 2-oxoglutarate dehydrogenase is identified in brain, but not in heart.

2-Oxoglutarate dehydrogenase (OGDH) is the first and rate-limiting component of the multienzyme OGDH complex (OGDHC) whose malfunction is associated with neurodegeneration. The essential role of this complex in the degradation of glucose and glutamate, which have specific significance in brain, raises questions about the existence of brain-specific OGDHC isoenzyme(s). We purified OGDHC from extracts of brain or heart mitochondria using the same procedure of poly(ethylene glycol) fractionation, followed by size-exclusion chromatography. Chromatographic behavior and the insufficiency of mitochondrial disruption to solubilize OGDHC revealed functionally significant binding of the complex to membrane. Components of OGDHC from brain and heart were identified using nano-high performance liquid chromatography electrospray tandem mass spectrometry after trypsinolysis of the electrophoretically separated proteins. In contrast to the heart complex, where only the known OGDH was determined, the band corresponding to the brain OGDH component was found to also include the novel 2-oxoglutarate dehydrogenase-like (OGDHL) protein. The ratio of identified peptides characteristic of OGDH and OGDHL was preserved during purification and indicated comparable quantities of the two proteins in brain. Brain OGDHC also differed from the heart complex in the abundance of the components, lower apparent molecular mass and decreased stability upon size-exclusion chromatography. The functional competence of the novel brain isoenzyme and different regulation of OGDH and OGDHL by 2-oxoglutarate are inferred from the biphasic dependence of the overall reaction rate versus 2-oxoglutarate concentration. OGDHL may thus participate in brain-specific control of 2-oxoglutarate distribution between energy production and synthesis of the neurotransmitter glutamate.

Phosphonate analogues of alpha-ketoglutarate inhibit the activity of the alpha-ketoglutarate dehydrogenase complex isolated from brain and in cultured cells.

The alpha-ketoglutarate dehydrogenase complex (KGDHC), a control point of the tricarboxylic acid cycle, is partially inactivated in brain in many neurodegenerative diseases. Potent and specific KGDHC inhibitors are needed to probe how the reduced KGDHC activity alters brain function. Previous studies showed that succinyl phosphonate (SP) effectively inhibits muscle and Escherichia coli KGDHC [Biryukov, A. I., Bunik, V. I., Zhukov, Yu. N., Khurs, E. N., and Khomutov, R. M. (1996) FEBS Lett. 382, 167-170]. To identify the phosphonates with the highest affinity toward brain KGDHC and with the greatest effect in living cells, we investigated the ability of SP and several of its ethyl esters to inhibit brain KGDHC, other alpha-keto acid-dependent enzymes, and KGDHC in intact cells. At a concentration of 0.01 mM, SP and its phosphonoethyl (PESP) and carboxyethyl (CESP) esters completely inhibited isolated brain KGDHC even in the presence of a 200-fold higher concentration of its substrate [alpha-ketoglutarate (KG)], while the diethyl (DESP) and triethyl (TESP) esters were ineffective. In cultured human fibroblasts, 0.01 mM SP, PESP, or CESP produced 70% inhibition of KGDHC. DESP and TESP were also inhibitory in the cell system, but only after preincubation, suggesting the release of their charged groups by cellular esterases. Thus, SP and its monoethyl esters target cellular KGDHC directly, while the di- and triethyl esters are activated in intact cells. When tested on other enzymes that bind KG or related alpha-keto acids, SP had minimal effects and its two esters (CESP and TESP) were ineffective even at a concentration (0.1 mM) 1 order of magnitude higher than that which inhibited cellular KGDHC activity. The high specificity in targeting KGDHC, penetration into cells, and minimal transformation by cellular enzymes indicate that SP and its esters should be useful in studying the effects of reduced KGDHC activity on neuronal and brain function.

2-Oxoglutarate:NADP(+) oxidoreductase in Azoarcus evansii: properties and function in electron transfer reactions in aromatic ring reduction.

The conversion of [(14)C]benzoyl-coenzyme A (CoA) to nonaromatic products in the denitrifying beta-proteobacterium Azoarcus evansii grown anaerobically on benzoate was investigated. With cell extracts and 2-oxoglutarate as the electron donor, benzoyl-CoA reduction occurred at a rate of 10 to 15 nmol min(-1) mg(-1). 2-Oxoglutarate could be replaced by dithionite (200% rate) and by NADPH ( approximately 10% rate); in contrast NADH did not serve as an electron donor. Anaerobic growth on aromatic compounds induced 2-oxoglutarate:acceptor oxidoreductase (KGOR), which specifically reduced NADP(+), and NADPH:acceptor oxidoreductase. KGOR was purified by a 76-fold enrichment. The enzyme had a molecular mass of 290 +/- 20 kDa and was composed of three subunits of 63 (gamma), 62 (alpha), and 37 (beta) kDa in a 1:1:1 ratio, suggesting an (alphabetagamma)(2) composition. The native enzyme contained Fe (24 mol/mol of enzyme), S (23 mol/mol), flavin adenine dinucleotide (FAD; 1.4 mol/mol), and thiamine diphosphate (0.95 mol/mol). KGOR from A. evansii was highly specific for 2-oxoglutarate as the electron donor and accepted both NADP(+) and oxidized viologens as electron acceptors; in contrast NAD(+) was not reduced. These results suggest that benzoyl-CoA reduction is coupled to the complete oxidation of the intermediate acetyl-CoA in the tricarboxylic acid cycle. Electrons generated by KGOR can be transferred to both oxidized ferredoxin and NADP(+), depending on the cellular needs. N-terminal amino acid sequence analysis revealed that the open reading frames for the three subunits of KGOR are similar to three adjacently located open reading frames in Bradyrhizobium japonicum. We suggest that these genes code for a very similar three-subunit KGOR, which may play a role in nitrogen fixation. The alpha-subunit is supposed to harbor one FAD molecule, two [4Fe-4S] clusters, and the NADPH binding site; the beta-subunit is supposed to harbor one thiamine diphosphate molecule and one further [4Fe-4S] cluster; and the gamma-subunit is supposed to harbor the CoA binding site. This is the first study of an NADP(+)-specific KGOR. A similar NADP(+)-specific pyruvate oxidoreductase, which contains all domains in one large subunit, has been reported for the mitochondrion of the protist Euglena gracilis and the apicomplexan Cryptosporidium parvum.

Characterization of rainbow trout branched-chain alpha-keto acid dehydrogenase complex: inter-domain segments of the E2 component affect the overall activity.

Branched-chain alpha-keto acid dehydrogenase complex (BCKADH) contains decarboxylase (E1), dihydrolipoyl transacylase (E2), and dihydrolipoyl dehydrogenase (E3) as catalytic components. BCKADH purified from rainbow trout (Oncorhynchus mykiss) liver was comparable with mammalian BCKADH in various enzymatic characteristics, but less efficient in catalyzing the overall reaction. The trout E2 subunit was larger than the mammalian subunit and rather similar to the chicken one in relative molecular mass on SDS-PAGE, whereas the E1 component was similar between trout and mammalian both in relative molecular mass of its alpha and beta subunits and in the catalytic activity. Trout E2 cDNA cloning and nucleotide sequencing revealed that the mature trout E2 subunit consists of 435 residues, and possesses 14 additional residues compared with mammalian E2. Eleven of these are localized in two interdomain segments as two sequences with two and nine residues, respectively. Trout E2 was inferior to rat E2 in the capacity for binding the E1 component, similar to chicken E2. Thus, it appears that non-mammalian BCKADH E2 is distinct from that in mammals in the structure of interdomain segments, resulting in reduction of overall activity of the enzyme complex.

The cytotoxic lipid peroxidation product, 4-hydroxy-2-nonenal, specifically inhibits decarboxylating dehydrogenases in the matrix of plant mitochondria.

4-Hydroxy-2-nonenal (HNE), a cytotoxic product of lipid peroxidation, inhibits O(2) consumption by potato tuber mitochondria. 2-Oxoglutarate dehydrogenase (OGDC), pyruvate dehydrogenase complex (PDC) (both 80% inhibited) and NAD-malic enzyme (50% inhibited) are its major targets. Mitochondrial proteins identified by reaction with antibodies raised to lipoic acid lost this antigenicity following HNE treatment. These proteins were identified as acetyltransferases of PDC (78 kDa and 55 kDa), succinyltransferases of OGDC (50 kDa and 48 kDa) and glycine decarboxylase H protein (17 kDa). The significance of the effect of these inhibitions on the impact of lipid peroxidation and plant respiratory functions is discussed.

Kinetic properties of the 2-oxoglutarate dehydrogenase complex from Azotobacter vinelandii evidence for the formation of a precatalytic complex with 2-oxoglutarate.

The 2-oxoglutarate dehydrogenase complex was purified from Azotobacter vinelandii. The complex consists of three components, 2-oxoglutarate dehydrogenase/decarboxylase (E1o), lipoate succinyltransferase (E2o) and lipoamide dehydrogenase (E3). Upon purification, the E3 component dissociates partially from the complex. From reconstitution experiments, the Kd for E3 was found to be 26 nM, about 30 times higher than that for the pyruvate dehydrogenase complex. The Km values for the substrates 2-oxoglutarate, CoA and NAD+ were found to be 0.15, 0.014 and 0.17 mM, respectively. The system has a high specificity for 2-oxoglutarate, which is determined by the action of both E1o and E2o. Above 4 mM substrate inhibition is observed. From steady-state inhibition experiments with substrate analogs, two substrate-binding modes are revealed at different degrees of saturation of the enzyme with 2-oxoglutarate. At low substrate concentrations (10(-6) to 10(-5) M), the binding mainly depends on the interaction of the enzyme with the substrate carboxyl groups. At a higher degree of substrate saturation (10(-4) to 10(-3) M) the relative contribution of the 2-oxo group in the binding increases. A kinetic analysis points to a single binding site for a substrate analog under steady state conditions. Saturation of this site with an analog indicates that two kinetically different complexes are formed with 2-oxoglutarate in the course of catalysis. From competition studies with analogs it is concluded that one of these complexes is formed at the site that is sterically identical to the substrate inhibition site. The data obtained are represented by a minimal scheme that considers formation of a precatalytic complex SE between the substrate and E1o before the catalytic complex ES, in which the substrate is added to the thiamin diphosphate cofactor, is formed. The incorrect orientation of the substrate molecule in SE or the occupation of this site by analogs is supposed to cause substrate or analog inhibition, respectively.

In organello formaldehyde crosslinking of proteins to mtDNA: identification of bifunctional proteins.

The segregating unit of mtDNA is a protein-DNA complex called the nucleoid. In an effort to understand how nucleoid proteins contribute to mtDNA organization and inheritance, we have developed an in organello formaldehyde crosslinking procedure to identify proteins associated with mtDNA. Using highly purified mitochondria, we observed a time-dependent crosslinking of protein to mtDNA as determined by sedimentation through isopycnic cesium chloride gradients. We detected approximately 20 proteins crosslinked to mtDNA and identified 11, mostly by mass spectrometry. Among them is Abf2p, an abundant, high-mobility group protein that is known to function in nucleoid morphology, and in mtDNA transactions. In addition to several other proteins with known DNA binding properties or that function in mtDNA maintenance, we identified other mtDNA-associated proteins that were not anticipated, such as the molecular chaperone Hsp60p and a Krebs cycle protein, Kgd2p. Genetic experiments indicate that hsp60-ts mutants have a petite-inducing phenotype at the permissive temperature and that a kgd2Delta mutation increases the petite-inducing phenotype of an abf2Delta mutation. Crosslinking and DNA gel shift experiments show that Hsp60p binds to single-stranded DNA with high specificity for the template strand of a putative origin of mtDNA replication. These data identify bifunctional proteins that participate in the stability of rho(+) mtDNA.

Plant mitochondrial 2-oxoglutarate dehydrogenase complex: purification and characterization in potato.

The 2-oxoglutarate dehydrogenase complex (OGDC) in potato (Solanum tuberosum cv. Romano) tuber mitochondria is largely associated with the membrane fraction of osmotically ruptured organelles, whereas most of the other tricarboxylic acid cycle enzymes are found in the soluble matrix fraction. The purification of OGDC from either membrane or soluble matrix fractions resulted in the increasing dependence of its activity on the addition of dihydrolipoamide dehydrogenase (E3). A 30-fold purification of OGDC to apparent homogeneity and with a specific activity of 4.6 micromol/min per mg of protein in the presence of exogenously added E3 was obtained. SDS/PAGE revealed that the purified complex consisted of three major polypeptides with apparent molecular masses of 48, 50 and 105 kDa. Before the gel-filtration purification step, E3 polypeptides of 57 and 58 kDa were identified by immunoreaction as minor proteins associated with OGDC. The N-terminal sequence of the 57 kDa protein was identical with that previously purified as the E3 component of the pyruvate dehydrogenase complex from potato. The 105 kDa protein was identified as the 2-oxoglutarate dehydrogenase subunit of OGDC by N-terminal sequencing. The N-terminal sequences of the 50 and 48 kDa proteins shared 90-95% identity over 20 residues and were identified by sequence similarity as dihydrolipoamide succinyltransferases (OGDC-E2). The incubation of OGDC with [U-(14)C]2-oxoglutarate resulted in the reversible succinylation of both the 48 and the 50 kDa protein bands. Proteins previously reported as subunits of complex I of the respiratory chain from Vicia faba and Solanum tuberosum are proposed to be OGDC-E2 and the possible basis of this association is discussed.

Selective inactivation of alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase: reaction of lipoic acid with 4-hydroxy-2-nonenal.

Previous research has established that 4-hydroxy-2-nonenal (HNE), a highly toxic product of lipid peroxidation, is a potent inhibitor of mitochondrial respiration. HNE exerts its effects on respiration by inhibiting alpha-ketoglutarate dehydrogenase (KGDH). Because of the central role of KGDH in metabolism and emerging evidence that free radicals contribute to mitochondrial dysfunction associated with numerous diseases, it is of great interest to further characterize the mechanism of inhibition. In the present study, treatment of rat heart mitochondria with HNE resulted in the selective inhibition of KGDH and pyruvate dehydrogenase (PDH), while other NADH-linked dehydrogenases and electron chain complexes were unaffected. KGDH and PDH are structurally and catalytically similar multienzyme complexes, suggesting a common mode of inhibition. To determine the mechanism of inhibition, the effects of HNE on purified KGDH and PDH were examined. These studies revealed that inactivation by HNE was greatly enhanced in the presence of substrates that reduce the sulfur atoms of lipoic acid covalently bound to the E2 subunits of KGDH and PDH. In addition, loss of enzyme activity induced by HNE correlated closely with a decrease in the availability of lipoic acid sulfhydryl groups. Use of anti-lipoic acid antibodies indicated that HNE modified lipoic acid in both purified enzyme preparations and mitochondria and that this modification was dependent upon the presence of substrates. These results therefore identify a potential mechanism whereby free radical production and subsequent lipid peroxidation lead to specific modification of KGDH and PDH and inhibition of NADH-linked mitochondrial respiration.

Subunit interactions in the mammalian alpha-ketoglutarate dehydrogenase complex. Evidence for direct association of the alpha-ketoglutarate dehydrogenase and dihydrolipoamide dehydrogenase components.

Selective tryptic proteolysis of the mammalian alpha-ketoglutarate dehydrogenase complex (OGDC) leads to its rapid inactivation as a result of a single cleavage within the N-terminal region of its alpha-ketoglutarate dehydrogenase (E1) component, which promotes the dissociation of the dihydrolipoamide dehydrogenase (E3) enzyme and also a fully active E1' fragment. Similarities between the N-terminal region of E1 and the dihydrolipoamide acetyltransferase (E2) and E3-binding components (E3BP) of the pyruvate dehydrogenase complex are highlighted by the specific cross-reactivities of subunit-specific antisera. Analysis of the pattern of release of E1 and E1' polypeptides from the OGDC during tryptic inactivation suggests that both polypeptide chains of individual E1 homodimers must be cleaved to permit the dissociation of the E1 and E3 components. A new protocol has been devised that promotes E1 dissociation from the oligomeric dihydrolipoamide succinyltransferase (E2) core in an active state. Significant levels of overall OGDC reconstitution could also be achieved by re-mixing the constituent enzymes in stoichiometric amounts. Moreover, a high affinity interaction has been demonstrated between the homodimeric E1 and E3 components, which form a stable subcomplex comprising single copies of these two enzymes.

Some regulatory properties of the 2-oxoglutarate dehydrogenase complex from European bison heart.

Basic regulatory properties of the 2-oxoglutarate dehydrogenase complex (OGDC) isolated and purified from the heart muscle of European bison (Bison bonasus) were studied. Kinetic studies have shown that in the absence of phosphate ions OGDC exhibits kinetic attributes of negative cooperativity with respect to 2-oxoglutarate. ADP and phosphate lower S0.5 value of OGDC for 2-oxoglutarate without changing the maximum reaction rate. NADH inhibits OGDC versus both 2-oxoglutarate and NAD+. Moreover, bison heart OGDC shows negative kinetic cooperativity for NAD+ and positive kinetic cooperativity for CoA at low CoA concentrations. The latter property has not been observed in earlier studies on OGDC from bovine and pig heart and other tissues of these animals.

Alpha-ketoglutarate dehydrogenase in the in vitro-grown Mycobacterium lepraemurium.

The Hawaiian and Kumato strains of Mycobacterium lepraemurium were cultivated on Ogawa egg-yolk medium, and the alpha-ketoglutarate dehydrogenase activity was investigated in cell-free preparations of this mycobacterium. The enzymatic activity was mainly localized in the particulate fraction (150,000 x g pellet), and extremely low activity was found in the soluble fraction (150,000 x g supernatant). alpha-Ketoglutarate dehydrogenase was not stable; the activity was lost completely when the enzyme was kept at 45 degrees C for 1 hr or stored at -70 degrees C. The enzyme reduced only NAD+ but not NADP+ by alpha-ketoglutarate, indicating the presence of NAD(+)-dependent alpha-ketoglutarate dehydrogenase in cultivated M. lepraemurium.

Purification and molecular cloning of succinyltransferase of the rat alpha-ketoglutarate dehydrogenase complex. Absence of a sequence motif of the putative E3 and/or E1 binding site.

Full-length cDNA clones for succinyltransferase of the rat alpha-ketoglutarate dehydrogenase complex were isolated from rat heart cDNA libraries in lambda gt11. The cDNA clones were identified as those for rat succinyltransferase by the identity of their predicted amino acid sequence with the NH2-terminal amino acid sequence of rat succinyltransferase determined by protein chemical analysis and the known amino acid sequence of bovine succinyltransferase. The clone with the longest cDNA consisted of 2747 base pairs and coded for a leader peptide of 56 amino acid residues and a mature protein of 386 amino acid residues. The primary structure of rat succinyltransferase showed close similarity to Escherichia coli and Azotobacter vinelandii succinyltransferases, in the COOH-terminal part forming the lipoyl-binding domain and the NH2-terminal part forming the inner core-catalytic domain. However, the rat succinyltransferase did not contain a sequence motif that has been found as an E3- and/or E1-binding site in the dihydrolipoamide acyltransferases of three alpha-ketoacid dehydrogenase complexes (Hummel, K. B., Litwer, S., Bradford, A. P., Aitken, A., Danner, D. J., and Yeaman, S. J. (1988) J. Biol. Chem. 263, 6165-6168, Reed, L. J., and Hackert, M. L. (1990) J. Biol. Chem. 265, 8971-8974). The absence of this sequence was confirmed by direct sequencing of the polymerase chain reaction product of rat heart mRNA and by computer analysis. These results show that the rat succinyltransferase does not have the sequence motif of the putative E3- and/or E1-binding site.

In vivo assembly of yeast mitochondrial alpha-ketoglutarate dehydrogenase complex.

The assembly of alpha-ketoglutarate dehydrogenase complex (KGDC) has been studied in wild-type Saccharomyces cerevisiae and in respiratory-deficient strains (pet) with mutations in KGD1 and KGD2, the structural genes for alpha-ketoglutarate dehydrogenase (KE1) and dihydrolipoyl transsuccinylase (KE2) components, respectively. Mutants unable to express KE1 or KE2 form partial complexes similar to those reported in earlier studies on the resolution and reconstitution of bacterial and mammalian KGDC. Thus mutants lacking KE1 assemble a high-molecular-weight subcomplex consisting of a KE2 core particle with bound dihydrolipoyl dehydrogenase (E3). Similarly, mitochondrial extracts of mutants lacking KE2 contain dimeric KE1 and E3. These components, however, are not associated with each other. The partial complexes detected in the mutants are capable of reconstituting normal KGDC when supplied with the missing subunit. Complete restoration of overall alpha-ketoglutarate dehydrogenase activity is achieved by mixing appropriate ratios of mitochondrial extracts from mutants deficient in KE1 and KE2. The reconstitution of enzymatic activity correlates with binding of KE1 to the KE2-E3 particle to form a complex with the same sedimentation properties as wild-type KGDC. Overexpression of KE2 relative to KE1 results in a preponderance of incompletely assembled complexes with substoichiometric contents of KE1. Formation of a complex with a full complement of KE1 therefore depends on a balanced output of KE1 and KE2 from their respective genes. Biochemical screens of a pet mutant collection have led to the identification of a new gene required for the expression of enzymatically active KGDC. Mitochondria of the mutant have all of the catalytic subunits of KGDC. Sedimentation analysis of these components indicates that while the mutant has a stable KE2-E3 subcomplex, the interaction of KE1 with KE2 core is much weaker in the mutant than in the wild type. The gene product responsible for this phenotype, therefore, appears to function at a late stage of assembly of KGDC, most likely by posttranslational modification of one of the subunits.

Genetic characterization of Bacillus subtilis odhA and odhB, encoding 2-oxoglutarate dehydrogenase and dihydrolipoamide transsuccinylase, respectively.

The 2-oxoglutarate dehydrogenase complex consists of three different subenzymes, the E1o (2-oxoglutarate dehydrogenase) component, the E2o (dihydrolipoyl transsuccinylase) component, and the E3 (dihydrolipoamide dehydrogenase) component. In Bacillus subtilis, the E1o and E2o subenzymes are encoded by odhA and odhB, respectively. A plasmid with a 6.8-kilobase-pair DNA fragment containing odhA and odhB was isolated. Functional E1o and E2o are expressed from this plasmid in Escherichia coli. Antisera generated against B. subtilis E1o and E2o expressed in E. coli reacted with antigens of the same size from B. subtilis. The nucleotide sequence of odhB and the terminal part of odhA was determined. The deduced primary sequence of B. subtilis E2o shows striking similarity to the corresponding E. coli protein, which made it possible to identify the lipoyl-binding lysine residue as well as catalytic histidine and aspartic acid residues. An mRNA of 4.5 kilobases hybridizing to both odhA and odhB probes was detected, indicating that odhA and odhB form an operon.