Dual role of a single multienzyme complex in the oxidative decarboxylation of pyruvate and branched-chain 2-oxo acids in Bacillus subtilis.

The pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase activities of Bacillus subtilis were found to co-purify as a single multienzyme complex. Mutants of B. subtilis with defects in the pyruvate decarboxylase (E1) and dihydrolipoamide dehydrogenase (E3) components of the pyruvate dehydrogenase complex were correspondingly affected in branched-chain 2-oxo acid dehydrogenase complex activity. Selective inhibition of the E1 or lipoate acetyltransferase (E2) components in vitro led to parallel losses in pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complex activity. The pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complexes of B. subtilis at the very least share many structural components, and are probably one and the same. The E3 component appeared to be identical for the pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complexes in this organism and to be the product of a single structural gene. Long-chain branched fatty acids are thought to be essential for maintaining membrane fluidity in B. subtilis, and it was observed that the ace (pyruvate dehydrogenase complex) mutant 61142 was unable rapidly to take up acetoacetate, unlike the wild-type, indicative of a defect in membrane permeability. A single pyruvate dehydrogenase and branched-chain 2-oxo acid dehydrogenase complex can be seen as an economical means of supplying two different sets of essential metabolites.     

A synthetic peptide-directed antibody as a probe of the phosphorylation site of pyruvate dehydrogenase

A synthetic peptide, corresponding to the 14-amino acid tryptic fragment containing phosphorylation sites one and two of bovine mitochondrial pyruvate dehydrogenase, was coupled to Limulus polyphemus hemocyanin and used to produce rabbit polyclonal antibodies. A positive signal was observed when Western blots of bovine, porcine, or yeast mitochondrial pyruvate dehydrogenase complexes were probed with the antibodies but not with blots of bacterial, cellular slime mold, plant mitochondrial, or plant plastid pyruvate dehydrogenase complexes. The antibodies also gave a positive signal when used to probe blots of the bovine kidney branched chain 2-oxo acid dehydrogenase complex. The ATP-dependent phosphorylation/inactivation of rat liver mitochondrial pyruvate dehydrogenase complex could be inhibited by prior incubation with the anti-peptide antibodies.     

Identification and analysis of the genes coding for the putative pyruvate dehydrogenase enzyme complex in Acholeplasma laidlawii.

A monospecific antibody recognizing two membrane proteins in Acholeplasma laidlawii identified a plasmid clone from a genomic library. The nucleotide sequence of the 4.6-kbp insert contained four sequential genes coding for proteins of 39 kDa (E1 alpha, N terminus not cloned), 36 kDa (E1 beta), 57 kDa (E2), and 36 kDa (E3; C terminus not cloned). The N termini of the cloned E2, E1 beta, and native A. laidlawii E2 proteins were verified by amino acid sequencing. Computer-aided searches showed that the translated DNA sequences were homologous to the four subenzymes of the pyruvate dehydrogenase complexes from gram-positive bacteria and humans. The plasmid-encoded 57-kDa (E2) protein was recognized by antibodies against the E2 subenzymes of the pyruvate and oxoglutarate dehydrogenase complexes from Bacillus subtilis. A substantial fraction of the E2 protein as well as part of the pyruvate dehydrogenase enzymatic activity was associated with the cytoplasmic membrane in A. laidlawii. In vivo complementation with three different Escherichia coli pyruvate dehydrogenase-defective mutants showed that the four plasmid-encoded proteins were able to restore pyruvate dehydrogenase enzyme activity in E. coli. Since A. laidlawii lacks oxoglutarate dehydrogenase and most likely branched-chain dehydrogenase enzyme complex activities, these results strongly suggest that the sequenced genes code for the pyruvate dehydrogenase complex.     

Glycolytic enzymes and intermediates in carbon catabolite repression mutants of Saccharomyces cerevisiae

Summary Glycolytic parameters were determined in recessive yeast mutants with partial defects in carbon catabolite repression. Specific activities of pyruvate kinase and pyruvate decarboxylase in glucose grown cells of all mutant and wild type stains were 4–5 times higher than in ethanol grown cells. Mutants of gene HEX1 had a reduced hexose phosphorylating activity on allmedia wheras those of gene HEX2 had elevated levels but only in glucose grown cells. Mutants of gene CAT80 were normal in this respect. All other glycolytic enzymes were normal in all mutants. This was also true for glycolytic intermediates. Only hexlmutants showed a reduced fermentation of repressing sugars. The three genes appear to be involved in catabolite repression of several but not of all repressible enzymes. Even though all three types of mutants show a limited overlap in their effects on certain enzymes, they still are distinctly different in their action spectra. Carbon catabolite repression apparently does not depend on the sole accumulation of glycolytic intermediales. The activity of the products of the three genes HEX1, HEX2 and CAT80 are required directly or indirectly for triggering carbon catabolite repression. Even a small segment of carbon catabolite repression is controlled by several genes with regulatory functions indicating that the entire regulatory circuit is highly complex.     

Synthesis of oxaloacetate in Bacillus subtilis mutants lacking the 2-ketoglutarate dehydrogenase enzymatic complex.

Bacillus subtilis mutants deficient in the 2-ketoglutarate dehydrogenase enzymatic complex required aspartate for growth at wild-type rates on carbon sources for which synthesis of the degradative enzymes is sensitive to catabolite repression (e.g., poor carbon sources), but did not require aspartate for growth on carbon sources which exert catabolite repression (e.g., good carbon sources). Measurement of metabolite pools in a mutant lacking the 2-ketoglutarate dehydrogenase active complex showed that the aspartate requirement for growth on poor carbon sources resulted from a deficiency in intracellular oxaloacetate pools even through pyruvate carboxylase was present at levels corresponding to those in wild-type cells. The oxaloacetate deficiency most likely resulted from the inability of the mutant to regenerate oxaloacetate from citrate due to the enzymatic block in the tricarboxylic acid cycle. Mutants in the enzymes of the dicarboxylic acid half of the citric acid cycle similarly required aspartate for wild-type growth in minimal medium. These results suggested that the complete turning of the tricarboxylic acid cycle is involved in the maintainance of oxaloacetate levels in B. subtilis. The ability of the mutants lacking the 2-ketoglutarate dehydrogenase enzymatic complex to grow at wild-type rates on media containing good carbon sources in the absence of exogenous aspartate is not understood.     

Catabolite repression-resistant mutants of Bacillus subtilis.

Mutants of Bacillus subtilis that are able to sporulate under the condition of catabolite repression were isolated by a simple selection technique. The mutants used in the present study were able to grow normally on minimal medium with ammonium sulphate as the nitrogen source and glucose as the carbon source. Studies carried out with these mutants show that there is no close relation between catabolite repression of an inducible enzyme, acetoin dehydrogenase, and that of sporulation. Certain mutants are able to sporulate in the presence of all the carbon sources tested but some mutants are resistant only to the carbon source used in isolation. It is suggested that several metabolic steps may be affected in catabolite repression of sporulation.     

Catabolite repressive effects of 5-thio-D-glucose on Saccharomyces cerevisiae

The effect of the glucose analogue 5-thio-D-glucose (5TG) on the yeast Saccharomyces cerevisiae was studied. Derepression of mitochondrial respiratory chain cytochromes, alcohol dehydrogenase (isoenzyme II), NADH dehydrogenase and maltase was inhibited by 0.5-2 mM-5TG. Growth rate was only slightly affected. Ethanol was efficiently produced with 2 mM-5TG in medium initially containing 0.25% glucose. Mutants resistant to the growth inhibitory effects of 5TG on glycerol medium showed resistance to the catabolite repressing effects of glucose. Other mutants, known to be catabolite repression resistant, showed resistance to 5TG. The analogue seems to inhibit derepression of glucose repressible enzymes with greater potency than glucose itself.     

Synthesis of the E. coli pyruvate dehydrogenase complex: non-dependence on 3'-5'-cyclic AMP

A determination of the level of the pyruvate dehydrogenase complex in a 3′–5′-c-AMP deficient mutant of $\text{E.}$$\text{coli}$ K12 has been carried out. The deficiency has no effect on specific activities for derivatives carrying either the inducible genes for two components of the complex or constitutive mutants. We conclude that synthesis of the complex is not sensitive to catabolite repression.     

Regulation of arginine and proline catabolism in Bacillus licheniformis

The enzymes in the arginine breakdown pathway (arginase, ornithine-delta-transaminase, and Delta'-pyrroline-5-carboxylate dehydrogenase) were found to be present in Bacillus licheniformis cells during exponential growth on glutamate. These enzymes could be coincidentally induced by arginine or ornithine to a very high level and their synthesis could be repressed by the addition of glucose, clearly demonstrating catabolite repression control of the arginine degradative pathway. The strongest catabolite repression control of arginase occurred when cells were grown on glucose and this control decreased when cells were grown on glycerol, acetate, pyruvate, or glutamate. The proline catabolite pathway was present in B. licheniformis during exponential growth on glutamate. The proline oxidation and the Delta'-pyrroline-5-carboxylate dehydrogenase in this breakdown pathway were induced by l-proline to a high level. The Delta'-pyrroline-5-carboxylate dehydrogenase was found to be under catabolite repression control. Arginase could be induced by proline and arginine addition induced proline oxidation, suggesting a common in vivo inducer for these convergent pathways.     

Phenotypes of pleiotropic-negative sporulation mutants of Bacillus subtilis

The phenotypic properties of representatives of the five genetic classes of pleiotropic-negative sporulation mutants have been investigated. Protease production, alkaline and neutral proteases, was curtailed in spoA mutants, but the remainder of mutant classes produced both proteases, albeit at reduced levels. The spoA and spoB mutants plaqued phi2 and phi15 at high efficiency, but the efficiency of plating of these phages on spoE, spoF, and spoH mutants was drastically reduced. Antibiotic was produced by the spoH mutants and to a degree by some spoF mutants, but the other classes did not produce detectable activity. The spoA mutants were less responsive to catabolite repression of histidase synthesis by glucose than was the wild type. Severe catabolite repression could be induced in spoA mutants by amino acid limitation, suggesting that the relaxation of catabolite repression observed is not due to a defect in the mechanism of catabolite repression. Although others have shown a perturbation in cytochrome regulation in spoA and spoB mutants, the primary dehydrogenases, succinate dehydrogenase and reduced nicotinamide adenine dinucleotide dehydrogenase, leading to these cytochromes are unimpaired in all mutant classes. A comparison of the structural components of cell walls and membranes of spoA and the wild type is made. The pleiotropic phenotypes of these mutants are discussed.     

A mitochondrial pyruvate dehydrogenase bypass in the yeast Saccharomyces cerevisiae.

Spheroplasts of the yeast Saccharomyces cerevisiae oxidize pyruvate at a high respiratory rate, whereas isolated mitochondria do not unless malate is added. We show that a cytosolic factor, pyruvate decarboxylase, is required for the non-malate-dependent oxidation of pyruvate by mitochondria. In pyruvate decarboxylase-negative mutants, the oxidation of pyruvate by permeabilized spheroplasts was abolished. In contrast, deletion of the gene (PDA1) encoding the E1alpha subunit of the pyruvate dehydrogenase did not affect the spheroplast respiratory rate on pyruvate but abolished the malate-dependent respiration of isolated mitochondria. Mutants disrupted for the mitochondrial acetaldehyde dehydrogenase gene (ALD7) did not oxidize pyruvate unless malate was added. We therefore propose the existence of a mitochondrial pyruvate dehydrogenase bypass different from the cytosolic one, where pyruvate is decarboxylated to acetaldehyde in the cytosol by pyruvate decarboxylase and then oxidized by mitochondrial acetaldehyde dehydrogenase. This pathway can compensate PDA1 gene deletion for lactate or respiratory glucose growth. However, the codisruption of PDA1 and ALD7 genes prevented the growth on lactate, indicating that each of these pathways contributes to the oxidative metabolism of pyruvate.     

Purification of 2-oxo acid dehydrogenase multienzyme complexes from ox heart by a new method.

A new method is described that allows the parallel purification of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes from ox heart without the need for prior isolation of mitochondria. All the assayable activity of the 2-oxo acid dehydrogenase complexes in the disrupted tissue is made soluble by the inclusion of non-ionic detergents such as Triton X-100 or Tween-80 in the buffer used for the initial extraction of the enzyme complexes. The yields of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes are many times greater than those obtained by means of previous methods. In terms of specific catalytic activity, banding pattern on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, sedimentation properties and possession of the regulatory phosphokinase bound to the pyruvate dehydrogenase complex, the 2-oxo acid dehydrogenase complexes prepared by the new method closely resemble those described by previous workers. The greatly improved yield of 2-oxo acid dehydrogenase complexes occasioned by the use of Triton X-100 or Tween-80 as solubilizing agent supports the possibility that the bulk of the pyruvate dehydrogenase complex is associated in some way with the mitochondrial inner membrane and is not free in the mitochondrial matrix space.     

Relaxation of catabolite repression in streptomycin-dependent Escherichia coli

Acetohydroxy acid synthetase, which is sensitive to catabolite repression in wild-type Escherichia coli B, was relatively resistant to this control in a streptomycin-dependent mutant. The streptomycin-dependent mutant was found to be inducible for beta-galactosidase in the presence of glucose, although repression of beta-galactosidase by glucose occurred under experimental conditions where growth of the streptomycin-dependent mutant was limited. Additional glucose-sensitive enzymes of wild-type E. coli B (citrate synthase, fumarase, aconitase and isocitrate dehydrogenase) were found to be insensitive to the carbon source in streptomycin-dependent mutants: these enzymes were formed by streptomycin-dependent E. coli B in equivalent quantities when either glucose or glycerol was the carbon source. Two enzymes, glucokinase and glucose 6-phosphate dehydrogenase, that are glucose-insensitive in wild-type E. coli B were formed in equivalent quantity on glucose or glycerol in both streptomycin-sensitive and streptomycin-dependent E. coli B. The results indicate a general decrease or relaxation of catabolite repression in the streptomycin-dependent mutant. The yield of streptomycin-dependent cells from glucose was one-third less than that of the streptomycin-sensitive strain. We conclude that the decreased efficiency of glucose utilization in streptomycin-dependent E. coli B is responsible for the relaxation of catabolite repression in this mutant.     

Regulation of acetohydroxy acid synthase in streptomycin-dependent Escherichia coli.

Growth of streptomycin-dependent mutants of Escherichia coli K-12 was insensitive to valine when dihydrostreptomycin was present in a nonlimiting concentration in glucose-salts medium. Acetohydroxy acid synthase was derepressed under these conditions, owing to relaxation of catabolite repression. Valine sensitivity and catabolite repression were restored when streptomycin-dependent E. coli K-12 mutants were grown with limiting dihydrostreptomycin. End product repression of acetohydroxy acid synthase under conditions of relaxed catabolite repression was effected by any two (or more) end products except the combination valine plus isoleucine, which caused derepression. Single end products had no detectable effect on acetohydroxy acid synthase formation.     

Immunological comparison of the pyruvate dehydrogenase complexes from pea mitochondria and chloroplasts

The pyruvate dehydrogenase complex (PDC) in pea (Pisum sativum L., cv. Little Marvel) was studied immunologically using antibodies to specific subunits of mammalian PDC. Pea mitochondria and chloroplasts were both found to contain PDC, but distinct differences were noted in the subunit relative molecular mass (M_r) values of the individual enzymes in the mitochondrial and chloroplast PDC complexes. In particular, the mitochondrial E3 enzyme (dihydrolipoamide dehydrogenase; EC 1.8.1.4) has a high subunit M_r value of 67 000, while the chloroplast E3 enzyme has a subunit M_r value of 52 000, similar in size to the prokaryotic, yeast ad mammalian E3 enzymes. In addition, component X (not previously noted in plant PDC) was also found to be present in two distinct forms in pea mitochondrial and chloroplast complexes. As in the case of E3, mitochondrial component X has a higher subunit M_r value (67 000) than component X from chloroplasts (48 000), which is similar in size to its mammalian counterpart. The subunit M_r value of E2 (dihydrolipoamide acetyltransferase; EC 2.3.1.12) in both mitochondria and chloroplasts (50 000) is lower than that of mammalian E2 (74 000) but similar to that of yeast E2 (58 000), and is consistent with the presence of only a single lipoyl domain. Neither mitochondria nor chloroplasts showed any appreciable cross-reactivity with antiserum to mammalian E1 (pyruvate dehydrogenase; EC 1.2.4.1). However, mitochondria cross-reacted strongly with antiserum to yeast E1, giving a single band (M_r 41 000) which is thought to be E1_a. Chloroplasts showed no cross-reactivity with yeast E1, indicating that the mitochondrial E1_a subunit and its chloroplast equivalent are antigenically distinct polypeptides.Abbreviations E1 pyruvate dehydrogenase - E2 dihydrolipoamide acetyltransferase - E3 dihydrolipoamide dehydrogenase - M_r relative molecular mass - PDC pyruvate dehydrogenase multienzyme complex - SDS sodium dodecyl sulphate The financial support of the Agricultural and Food Research Council is gratefully acknowledged. We thank Steve Hill (Department of Botany, University of Edinburgh, UK) for advice on mitochondrial isolation, and James Neagle (Department of Biochemistry, University of Glasgow) and Ailsa Carmichael for helpful discussion.     

Binding protein-dependent transports in 2-oxo acids dehydrogenase mutants of Escherichia coli

The binding protein-dependent transport of ribose, galactose and maltose are reduced in several 2-oxo acids dehydrogenase mutants of Escherichia coli. The results suggest an implication of the pyruvate dehydrogenase complex and to a lesser extent of the 2-oxoglutarate dehydrogenase complex in the energization of these transport systems.     

Absence of involvement of glutamine synthetase and of NAD-linked glutamate dehydrogenase in the nitrogen catabolite repression of arginase and other enzymes in Saccharomyces cerevisiae

The escape of several enzymes from “ammonia catabolite repression” in gdhA^? (NADP-linked glutamate-dehydrogenase-less) mutants, as well as in gdhCR mutants of Saccharomyces cerevisiae, does not involve glutamine synthetase, either as a positive or as a negative control element. A glutamine-synthetase-less mutant (gln^?) was used in this demonstration.In addition to its derepressing effect on the NAD-linked glutamate dehydrogenase, the gdhCR mutation releases “nitrogen catabolite repression” on arginase and allatoinase, as well as glutamine repression on glutamine synthetase. A gdhCS mutation was used to demonstrate that these effects are not mediated through the NAD-linked glutamate dehydrogenase.     

Branched-chain 2-oxo acid dehydrogenase interferes with the measurement of the activity and activity state of hepatic pyruvate dehydrogenase.

Oxidative decarboxylation of pyruvate by branched-chain 2-oxo acid dehydrogenase can result in overestimation of the expressed and total activity of hepatic pyruvate dehydrogenase. Pyruvate is a poor substrate for branched-chain 2-oxo acid dehydrogenase relative to the branched-chain oxo acids; however, the comparable total activities of the two complexes in liver, the much greater activity state of branched-chain 2-oxo acid dehydrogenase compared with pyruvate dehydrogenase in most physiological states, and the use of high pyruvate concentrations, explain the interference that can occur in conventional radiochemical or indicator-enzyme linked assays of pyruvate dehydrogenase. Goat antibody that specifically inhibited branched-chain 2-oxo acid dehydrogenase was used in this study to provide a more specific assay for pyruvate dehydrogenase.     

Resolution and reconstitution of bovine kidney branched-chain 2-oxo acid dehydrogenase complex.

Branched-chain 2-oxo acid dehydrogenase complex was resolved into component E1 and E2-kinase subcomplex by gel filtration in the presence of 1 M-NaC1. Essentially all the original activity of the complex can be regained after reconstitution of the component enzymes, reassociation being a rapid process. The specific activities of E1 and E2 were 25.1 and 19.0 units/mg respectively. Non-phosphorylated active E1 has an approx. 6-fold higher affinity for E2 than does phosphorylated E1. The components of the branched-chain 2-oxo acid dehydrogenase complex do not crossreact with the respective components from the pyruvate dehydrogenase complex. The significance of these results and of the tight association of the kinase with E2 are discussed.