In vitro complementation of Bacillus subtilis and Escherichia coli. 2-oxoglutarate dehydrogenase complex mutants and genetic mapping of B. subtilis citK and citM mutations

Abstract The 2-oxoglutarate dehydrogenase complex of the tricarboxylic acid cycle (TCA) consists of multiple copies of 3 different subenzymes; E₁, E₂ and E₃. The E₃ subenzyme is also a component of the pyruvate dehydrogenase complex. Bacillus subtilis 2-oxoglutarate dehydrogenase mutants were studied. The mutants defective in E₁, E₂ and E₃ subenzyme activity, respectively, could be separated into 3 groups by biochemical complementation analyses. The groups correspond to the citK, citM and citL genes. A B. subtilis subenzyme defect, probably E₁, could be complemented with the corresponding Escherichia coli wild-type subenzyme and vice versa. Mutations in citK and citM are closely linked. The gene order kauA——citK-citM was determined from 3-factor transformation crosses. It is concluded that the gene organization and the subenzyme structure of the 2-oxoglutarate dehydrogenase complex are similar in B. subtilis and E. coli .     

Isolation of a third lipoamide dehydrogenase from Pseudomonas putida.

Pseudomonads are the only organisms so far known to produce two lipoamide dehydrogenases (LPDs), LPD-Val and LPD-Glc. LPD-Val is the specific E3 component of branched-chain oxoacid dehydrogenase, and LPD-Glc is the E3 component of 2-ketoglutarate and possibly pyruvate dehydrogenases and the L-factor of the glycine oxidation system. Three mutants of Pseudomonas putida, JS348, JS350, and JS351, affected in lpdG, the gene encoding LPD-Glc, have been isolated; all lacked 2-ketoglutarate dehydrogenase, but two, JS348 and JS351, had normal pyruvate dehydrogenase activity. The pyruvate and 2-ketoglutarate dehydrogenases of the wild-type strain of P. putida were both inhibited by anti-LPD-Glc, but the pyruvate dehydrogenase of the lpdG mutants was not inhibited, suggesting that the mutant pyruvate dehydrogenase E3 component was different from that of the wild type. The lipoamide dehydrogenase present in one of the lpdG mutants, JS348, was isolated and characterized. This lipoamide dehydrogenase, provisionally named LPD-3, differed in molecular weight, amino acid composition, and N-terminal amino acid sequence from LPD-Glc and LPD-Val. LPD-3 was clearly a lipoamide dehydrogenase as opposed to a mercuric reductase or glutathione reductase. LPD-3 was about 60% as effective as LPD-Glc in restoring 2-ketoglutarate dehydrogenase activity and completely restored pyruvate dehydrogenase activity in JS350. These results suggest that LPD-3 is a lipoamide dehydrogenase associated with an unknown multienzyme complex which can replace LPD-Glc as the E3 component of pyruvate and 2-ketoglutarate dehydrogenases in lpdG mutants.     

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.     

Mitochondrial protein lipoylation does not exclusively depend on the mtKAS pathway of de novo fatty acid synthesis in Arabidopsis

The photorespiratory Arabidopsis (Arabidopsis thaliana) mutant gld1 (now designated mtkas-1) is deficient in glycine decarboxylase (GDC) activity, but the exact nature of the genetic defect was not known. We have identified the mtkas-1 locus as gene At2g04540, which encodes beta-ketoacyl-[acyl carrier protein (ACP)] synthase (mtKAS), a key enzyme of the mitochondrial fatty acid synthetic system. One of its major products, octanoyl-ACP, is regarded as essential for the intramitochondrial lipoylation of several proteins including the H-protein subunit of GDC and the dihydrolipoamide acyltransferase (E2) subunits of two other essential multienzyme complexes, pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase. This view is in conflict with the fact that the mtkas-1 mutant and two allelic T-DNA knockout mutants grow well under nonphotorespiratory conditions. Although on a very low level, the mutants show residual lipoylation of H protein, indicating that the mutation does not lead to a full functional knockout of GDC. Lipoylation of the pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase E2 subunits is distinctly less reduced than that of H protein in leaves and remains unaffected from the mtKAS knockout in roots. These data suggest that mitochondrial protein lipoylation does not exclusively depend on the mtKAS pathway of lipoate biosynthesis in leaves and may occur independently of this pathway in roots.     

Purification of a new dihydrolipoamide dehydrogenase from Escherichia coli.

I purified a new dihydrolipoamide dehydrogenase from a lpd mutant of Escherichia coli deficient in the lipoamide dehydrogenase (EC 1.6.4.3) common to the pyruvate dehydrogenase (EC 1.2.4.1) and 2-oxoglutarate dehydrogenase complexes. The occurrence of the new lipoamide dehydrogenase in lpd mutants, including a lpd deletion mutant and the immunological properties of the enzyme, showed that it is different from the lpd gene product. The new dihydrolipoamide dehydrogenase had a molecular weight of 46,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was expressed in low amounts. It catalyzed the NAD+-dependent reduction of dihydrolipoamide with a maximal activity of 20 mumol/min per mg of protein and exhibited a hyperbolic dependence of catalytic activity on the concentration of both dihydrolipoamide and NAD+. The possible implication of the new dihydrolipoamide in the function of 2-oxo acid dehydrogenase complexes is discussed, as is its relation to binding protein-dependent transport.     

Biosynthesis of o-succinylbenzoic acid in Bacillus subtilis: identification of menD mutants and evidence against the involvement of the alpha-ketoglutarate dehydrogenase complex.

The biosynthesis of o-succinylbenzoic acid (OSB), the first aromatic intermediate involved in the biosynthesis of menaquinone (vitamin K2) is demonstrated for the first time in the gram-positive bacterium Bacillus subtilis. Cell extracts were found to contain isochorismate synthase, 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid (SHCHC) synthase-alpha-ketoglutarate decarboxylase and o-succinylbenzoic acid synthase activities. An odhA mutant which lacks the decarboxylase component (usually termed E1, EC 1.2.4.2, oxoglutarate dehydrogenase [lipoamide]) of the alpha-ketoglutarate dehydrogenase complex was found to synthesize SHCHC and form succinic semialdehyde-thiamine pyrophosphate. Thus, the presence of an alternate alpha-ketoglutarate decarboxylase activity specifically involved in menaquinone biosynthesis is established for B. subtilis. A number of OSB-requiring mutants were also assayed for the presence of the various enzymes involved in the biosynthesis of OSB. All mutants were found to lack only the SHCHC synthase activity.     

Biochemical genetics of the alpha-keto acid dehydrogenase complexes of Escherichia coli K12: isolation and biochemical properties of deletion mutants.

Mutants of Escherichia coli K12 with deletions in the nadC-lpd region of the chromosome were obtained for use in studies on the expression of the ace (pyruvate dehydrogenase complex, specific components) and lpd (lipomide dehydrogenase) genes. These were isolated by selecting spontaneous aroP mutants (lacking the general aromatic amino-acid permease and thus resistant to inhibitory aromatic amino-acid analogues) and screening for auxotrophy due to deletions extending into neighbouring genes. From 2892 isolates tested, the AroP- phenotypes of 2322 were confirmed and, of these, 28 stable and independently-derived auxotrophos were designated as deletion mutants. Six nutritionally-distinct categories were recognized: Nad- (8 strains); Nad-Ace-(7): Nad-'Ace-' (3); Ace- (8); 'Ace-' (I); Lpd-(I). The Ace- phenotypes of four isolates designated 'Ace-' were leaky and enzymological studies confirmed that they had less than 7% of parental pyruvate dehydrogenase complex activity. Enzymological studies showed that the 15 Ace- or Nad-Ace- strains all lacked the pyruvate dehydrogenase complex and pyruvate dehydrogenase (EIp) activities and only three retained detectable dihydrolipoamide acetyltransferase (E2p). The one Lpd- strain lacked pyruvate dehydrogenase, dihydrolipoamide acetyltransferase and lipoamide dehydrogenase (E3) activities as well as the activities of the pyruvate and alpha-ketoglutarate dehydrogenase complexes. The results confirmed the gene order nadC-aroP-aceE-aceF-lpd and indicated that no other essential functions are determined by genes within the nadC-lpd region. Resistance to lactate during growth of pps mutants on acetate was directly related to the specific activity of the pyruvate dehydrogenase complex. None of the deletions promoted the high degree of resistance characteristically associated with constitutive expression of the dehydrogenase complex. Six pps mutants having Ace+ or 'Ace-' phenotypes were more sensitive than the parental strains and expression of their ace operons appeared to be affected; most sensitive were the Ace- strains which lacked pyruvate dehydrogenase complex and phosphoenolpyruvate synthetase activities. The lipoamide dehydrogenase activities of the deletion strains (Lpd+) ranged between 30% and 100% of parental levels indicating that expression of their ace operons appeared to be affected; most sensitive were the Ace- strains which lacked pyruvate dehydrogenase complex and phosphoenolpyruvate synthetase activities. The lipoamide dehydrogenase activities of the deletion strains (Lpd+) ranged between 30% and 100% of parental levels indicating that expression of the lpd gene may be affected by the ace operon but can be independent.     

alpha-Ketoglutarate dehydrogenase complex of Escherichia coli. A hybrid complex containing pyruvate dehydrogenase subunits from pyruvate dehydrogenase complex

The alpha-ketoglutarate dehydrogenase complex of Escherichia coli utilizes pyruvate as a poor substrate, with an activity of 0.082 units/mg of protein compared with 22 units/mg of protein for alpha-ketoglutarate. Pyruvate fully reduces the FAD in the complex and both alpha-keto[5-14C]glutarate and [2-14C]pyruvate fully [14C] acylate the lipoyl groups with approximately 10 nmol of 14C/mg of protein, corresponding to 24 lipoyl groups. NADH-dependent succinylation by [4-14C]succinyl-CoA also labels the enzyme with approximately 10 nmol of 14C/mg of protein. Therefore, pyruvate is a true substrate. However, the pyruvate and alpha-ketoglutarate activities exhibit different thiamin pyrophosphate dependencies. Moreover, 3-fluoropyruvate inhibits the pyruvate activity of the complex without affecting the alpha-ketoglutarate activity, and 2-oxo-3-fluoroglutarate inhibits the alpha-ketoglutarate activity without affecting the pyruvate activity. 3-Fluoro[1,2-14C]pyruvate labels about 10% of the E1 components (alpha-ketoacid dehydrogenases). The dihydrolipoyl transsuccinylase-dihydrolipoyl dehydrogenase subcomplex (E2E3) is activated as a pyruvate dehydrogenase complex by addition of E. coli pyruvate dehydrogenase, the E1 component of the pyruvate dehydrogenase complex. All evidence indicates that the alpha-ketoglutarate dehydrogenase complex purified from E. coli is a hybrid complex containing pyruvate dehydrogenase (approximately 10%) and alpha-ketoglutarate dehydrogenase (approximately 90%) as its E1 components.     

Identification of specific subunits of highly purified bovine liver branched-chain ketoacid dehydrogenase

Branched-chain alpha-ketoacid dehydrogenase has been purified to homogeneity from bovine liver mitochondria. The isolated complex has a specific activity of 5-8 mumol of reduced nicotinamide adenine dinucleotide min-1 (mg of protein)-1 as isolated and does not require the addition of exogenous lipoamide dehydrogenase for activity. Addition of porcine heart lipoamide dehydrogenase stimulated complex activity by no more than 20%. Four subunits copurify with the complex with molecular weights by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 55 000, 52 000, 46 500, and 37 500. Here we show that the 52 000-dalton subunit is the lipoyl-containing transacylase component of the complex. Data are presented to support the hypothesis that the branched-chain ketoacid dehydrogenase complex is physically and catalytically similar to, but separate from, the pyruvate and alpha-ketoglutarate dehydrogenase complexes. The transacylase of the branched-chain ketoacid dehydrogenase complex has an exposed trypsin-sensitive region. Proteolytic action of trypsin separates a lipoyl-containing component from the remainder of the protein. Data from our laboratory presented here and elsewhere define a specific function for three of the four subunits.     

Inhibition of glycine oxidation by pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids in rat liver mitochondria: presence of interaction between the glycine cleavage system and alpha-keto acid dehydrogenase complexes

Pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids which were transaminated products of valine, leucine, and isoleucine inhibited glycine decarboxylation by rat liver mitochondria. However, glycine synthesis (the reverse reaction of glycine decarboxylation) was stimulated by those alpha-keto acids with the concomitant decarboxylation of alpha-keto acid added in the absence of NADH. Both the decarboxylation and the synthesis of glycine by mitochondrial extract were affected similarly by alpha-ketoglutarate and branched-chain alpha-keto acids in the absence of pyridine nucleotide, but not by pyruvate. This failure of pyruvate to have an effect was due to the lack of pyruvate oxidation activity in the mitochondrial extract employed. It indicated that those alpha-keto acids exerted their effects by providing reducing equivalents to the glycine cleavage system, possibly through lipoamide dehydrogenase, a component shared by the glycine cleavage system and alpha-keto acid dehydrogenase complexes. On the decarboxylation of pyruvate, alpha-ketoglutarate, and branched-chain alpha-keto acids in intact mitochondria, those alpha-keto acids inhibited one another. In similar experiments with mitochondrial extract, decarboxylations of alpha-ketoglutarate and branched-chain alpha-keto acid were inhibited by branched-chain alpha-keto acid and alpha-ketoglutarate, respectively, but not by pyruvate. NADH was unlikely to account for the inhibition. We suggest that the lipoamide dehydrogenase component is an indistinguishable constituent among alpha-keto acid dehydrogenase complexes and the glycine cleavage system in mitochondria in nature, and that lipoamide dehydrogenase-mediated transfer of reducing equivalents might regulate alpha-keto acid oxidation as well as glycine oxidation.     

Bacillus subtilis citM, the structural gene for dihydrolipoamide transsuccinylase: cloning and expression in Escherichia coli

The 2-oxoglutarate dehydrogenase multienzyme complex is composed of three different subenzymes: 2-oxoglutarate dehydrogenase (E1o), dihydrolipoamide transsuccinylase (E2o), and dihydrolipoamide dehydrogenase (E3). Bacillus subtilis E1o and E2o are encoded by the citK and citM genes, respectively. A 3.4-kb BamHI DNA fragment containing citK and citM markers was isolated from a library of B. subtilis DNA in Escherichia coli. Functional E2o was expressed from the cloned DNA both in B. subtilis and E. coli. E2o had an apparent Mr of 60,000 when expressed in E. coli. The B. subtilis E2o component complemented an E. coli E2o-defective mutant in vivo and in vitro. It is concluded that functional B. subtilis E2o can be produced in E. coli and can interact with E. coli and E1o and E3 to form an active chimeric enzyme complex.     

Biochemical genetics of the alpha-keto acid dehydrogenase complexes of Escherichia coli K12: genetic characterization and regulatory properties of deletion mutants.

Twenty-eight spontaneous auxotrophic aroP mutants with deletions in the azi--nadC--aroP--aceE--aceF--lpd region of the Escherichia coli K12 chromosome were characterized genetically with respect to various azi, nadC, ace and lpd markers by P1-mediated transduction. One mutant (Kdelta18; aroP--lpddelta) had a deletion which extended through the aceE and aceF genes to end within the lpd gene. The polarity of the ace operon (aceE to aceF) was confirmed. It was concluded that 10 out of 15 deletions generating a strict requirement for acetate terminated in the aceE gene. Of the ten, three mutants (Kdelta22, Cdelta41 and Cdelta41) synthesized detectable dihydrolipoamide acetyltransferase (the aceF gene product) and seven were assumed to possess deletions generating polar effects on aceF gene expression. Five deletions appeared to extend into the aceF gene. A further five deletions, which limited the expression of the ace operon without generating an Ace- phenotype or a complete Ace- phenotype, ended closest to the aroP-proximal aceE markers. The opposite ends of all these deletions appeared to terminate before (10), within (2) or extend beyond (9) the nadC gene. There was no obvious correlation between the deletion end-points and the corresponding lipoamide dehydrogenase activities, which ranged from 30 to 95% of parental levels in different deletion strains. The remaining seven deletions simply extended between the aroP and nadC genes (nad--aroPdelta) without affecting expression of the ace operon. Regulation of the synthesis of the pyruvate and alpha-ketoglutarate dehydrogenase complexes was investigated in some of the parental and deletion strains under different physiological conditions including thiamin-deprivation. The results indicate that the syntheses of the two dehydrogenase complexes are independently regulated. Expression of the lpd gene appears to be coupled to complex synthesis but can be dissociated under some conditions. Mechanisms for regulating lpd gene expression are discussed and an autogenous mechanism involving uncomplexed lipoamide dehydrogenase functioning as a negatively acting repressor at the operator site of an independent lpd gene is proposed as the simplest mechanism which is consistent with all available information.     

The metabolic acclimation of Arabidopsis thaliana to arsenate is sensitized by the loss of mitochondrial LIPOAMIDE DEHYDROGENASE2, a key enzyme in oxidative metabolism

Mitochondrial lipoamide dehydrogenase is essential for the activity of four mitochondrial enzyme complexes central to oxidative metabolism. The reduction in protein amount and enzyme activity caused by disruption of mitochondrial LIPOAMIDE DEHYDROGENASE2 enhanced the arsenic sensitivity of Arabidopsis thaliana. Both arsenate and arsenite inhibited root elongation, decreased seedling size and increased anthocyanin production more profoundly in knockout mutants than in wild‐type seedlings. Arsenate also stimulated lateral root formation in the mutants. The activity of lipoamide dehydrogenase in isolated mitochondria was sensitive to arsenite, but not arsenate, indicating that arsenite could be the mediator of the observed phenotypes. Steady‐state metabolite abundances were only mildly affected by mutation of mitochondrial LIPOAMIDE DEHYDROGENASE2. In contrast, arsenate induced the remodelling of metabolite pools associated with oxidative metabolism in wild‐type seedlings, an effect that was enhanced in the mutant, especially around the enzyme complexes containing mitochondrial lipoamide dehydrogenase. These results indicate that mitochondrial lipoamide dehydrogenase is an important protein for determining the sensitivity of oxidative metabolism to arsenate in Arabidopsis.     

Kinetic advantages of hetero-enzyme complexes with glutamate dehydrogenase and the alpha-ketoglutarate dehydrogenase complex

We have found previously (Fahien, L.A., Kmiotek, E.H., MacDonald, M. J., Fibich, B., and Mandic, M. (1988) J. Biol. Chem. 263, 10687-10697) that glutamate-malate oxidation can be enhanced by cooperative binding of mitochondrial aspartate aminotransferase and malate dehydrogenase to the alpha-ketoglutarate dehydrogenase complex. The present results demonstrate that glutamate dehydrogenase, which forms binary complexes with these enzymes, adds to this ternary complex and thereby increases binding of the other enzymes. Kinetic evidence for direct transfer of alpha-ketoglutarate and NADH, within these complexes, has been obtained by measuring steady-state rates of E2 when most of the substrate or coenzyme is bound to the aminotransferase or glutamate dehydrogenase (E1). Rates significantly greater than those which can be accounted for by the concentration of free ligand, calculated from the measured values of the E1-ligand dissociation constants, require that the E1-ligand complex serve as a substrate for E2 (Srivastava, D. K., and Bernhard, S. A. (1986) Curr. Tops. Cell Regul. 28, 1-68). By this criterion, NADH is transferred directly from glutamate dehydrogenase to malate dehydrogenase and alpha-ketoglutarate is channeled from the aminotransferase to both glutamate dehydrogenase and the alpha-ketoglutarate dehydrogenase complex. Similar evidence indicates that GTP bound to an allosteric site on glutamate dehydrogenase functions as a substrate for succinic thiokinase. The potential physiological advantages to channeling of activators and inhibitors as well as substrates within multienzyme complexes organized around the alpha-ketoglutarate dehydrogenase complex are discussed.     

Analysis of Sporulation Mutants II. Mutants Blocked in the Citric Acid Cycle

Sporulation mutants that were unable to incorporate uracil during the developmental period recovered this capacity with the addition of ribose and in most cases with the addition of glutamate. Of the mutants that responded to both ribose and glumate, all but three also responded to citrate, and all but five responded to acetate. One of the exceptional strains was deficient in aconitase and another one in aconitase and isocitrate dehydrogenase; both required glutamate for growth. For the mutants which did not respond to glutamate, the products made from (14)C-glutamate were determined by thin-layer chromatography. Significant differences were found which enabled the identification of mutant blocks. The deficiency of the corresponding enzyme activity was verified. Several mutants were deficient in alpha-ketoglutarate dehydrogenase, and one lacked succinic dehydrogenase. These mutants could still grow on glucose as sole carbon source, but not on glutamate. The intact Krebs cycle is therefore not required for vegetative growth of aerobic Bacillis subtilis, but it is indispensable for sporulation.     

Purification, resolution, and reconstitution of rat liver branched-chain alpha-keto acid dehydrogenase complex

Branched-chain alpha-keto acid dehydrogenase (BCKADH) was solubilized as an enzyme complex from rat liver mitochondria by sonic treatment. Dehydrogenase (E1) and dihydrolipoyltransacylase (E2) components of the complex were purified in an associated form and resolved into individual components in the presence of 1 M NaCl, while lipoamide dehydrogenase (E3) component was dissociated from the complex during purification. Analysis by gel electrophoresis in dodecyl sulfate revealed the E1 comprised two different subunits with apparent molecular weights of 36,000 and 45,500, presumably in an equal molar ratio, while E2 consisted of a single subunit with an apparent molecular weight of 51,000. The BCKADH complex was reconstituted by combining E1, E2, and E3, and the formation of the complex was confirmed by analysis by sucrose density gradient centrifugation. The reconstituted enzyme complex oxidized not only alpha-ketoisovalerate (KIV), alpha-ketoisocaproate (KIC), and alpha-keto-beta-methylvalerate (KMV), but also pyruvate and alpha-ketoglutarate. Apparent Km values were 10-12 microM for the branched-chain alpha-keto acids, 2.2 mM for pyruvate, and 2.5 mM for alpha-ketoglutarate.     

The lpd gene product functions as the L protein in the Escherichia coli glycine cleavage enzyme system.

The lpd-encoded lipoamide dehydrogenase, common to the pyruvate and 2-oxoglutarate dehydrogenase multienzyme complexes, also functions as the lipoamide dehydrogenase (L protein) in the Escherichia coli glycine cleavage (GCV) enzyme complex. Inducible GCV enzyme activity was not detected in an lpd deletion mutant; lpd+ transductants had normal levels of inducible GCV enzyme activity. A serA lpd double mutant was unable to utilize glycine as a serine source and lacked detectable GCV enzyme activity, the phenotype of a serA gcv mutant. Transformation of the double mutant with a plasmid encoding a functional lpd gene restored the ability of the mutant to use glycine as a serine source and restored inducible GCV enzyme activity to normal levels. The presence of acetate and succinate in the growth medium of a strain wild type for lpd and gcv resulted in a 50% reduction in inducible GCV enzyme activity. Enzyme levels were restored to normal under these growth conditions when the strain was transformed with a plasmid encoding a functional lpd gene.     

Purification and primary amino acid sequence of the L subunit of glycine decarboxylase. Evidence for a single lipoamide dehydrogenase in plant mitochondria.

In order to purify the lipoamide dehydrogenase associated with the glycine decarboxylase complex of pea leaf mitochondria, the activity of free lipoamide dehydrogenase has been separated from those of the pyruvate and 2-oxoglutarate dehydrogenase complexes under conditions in which the glycine decarboxylase dissociates into its component subunits. This free lipoamide dehydrogenase which is normally associated with the glycine decarboxylase complex has been further purified and the N-terminal amino acid sequence determined. Positive cDNA clones isolated from both a pea leaf and embryo lambda gt11 expression library using an antibody raised against the purified lipoamide dehydrogenase proved to be the product of a single gene. The amino acid sequence deduced from the open reading frame included a sequence matching that determined directly from the N terminus of the mature protein. The deduced amino acid sequence shows good homology to the sequence of lipoamide dehydrogenase associated with the pyruvate dehydrogenase complex from Escherichia coli, yeast, and humans. The corresponding mRNA is strongly light-induced both in etiolated pea seedlings and in the leaves of mature plants following a period of darkness. The evidence suggests that the mitochondrial enzyme complexes: pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, and glycine decarboxylase all use the same lipoamide dehydrogenase subunit.     

Structural alterations by five disease-causing mutations in the low-pH conformation of human dihydrolipoamide dehydrogenase (hLADH) analyzed by molecular dynamics – Implications in functional loss and modulation of reactive oxygen species generation by pathogenic hLADH forms

Human dihydrolipoamide dehydrogenase (hLADH) is a flavoenzyme component (E3) of the human alpha-ketoglutarate dehydrogenase complex (α-KGDHc) and few other dehydrogenase complexes. Pathogenic mutations of hLADH cause severe metabolic diseases (atypical forms of E3 deficiency) that often escalate to cardiological or neurological presentations and even premature death; the pathologies are generally accompanied by lactic acidosis. hLADH presents a distinct conformation under acidosis (pH 5.5–6.8) with lower physiological activity and the capacity of generating reactive oxygen species (ROS). It has been shown by our laboratory that selected pathogenic mutations, besides lowering the physiological activity of hLADH, significantly stimulate ROS generation by hLADH, especially at lower pH, which might play a role in the pathogenesis of E3-deficiency in respective cases. Previously, we generated by molecular dynamics (MD) simulation the low-pH hLADH structure and analyzed the structural changes induced in this structure by eight of the pathogenic mutations of hLADH. In the absence of high resolution mutant structures these pieces of information are crucial for the mechanistic investigation of the molecular pathogeneses of the hLADH protein. In the present work we analyzed by molecular dynamics simulation the structural changes induced in the low-pH conformation of hLADH by five pathogenic mutations of hLADH; the structures of these disease-causing mutants of hLADH have never been examined before.