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[Isolation and purification of acetolactate synthase and acetolactate decarboxylase from a Lactococcus lactis culture].

Enzymes catalyzing the synthesis and subsequent transformation of alpha-acetolactate (AcL)--acetolactate synthase (AcLS) and acetolactate decarboxylase (AcLDC)--were isolated and partially purified from the cells of lactic acid bacteria Lactococcus lactis ssp. lactis biovar. diacetylactis strain 4. The preparation of AcLS, purified 560-fold, had a specific activity of 358,300 U/mg protein (9% yield). The preparation of AcLDC, purified 4828-fold, had a specific activity of 140 U/mg protein (4.8% yield). The enzymes exhibited optimum activity at pH 6.5 and 6.0, respectively (medium, phosphate buffer). The values of apparent Km, determined for AcLS and AcLDC with pyruvate and AcL, respectively, were equal to 70 mM and 20 mM. AcLS appeared as an allosteric enzyme with low affinity for the substrate and a sigmoid dependence of the activity on the substrate concentration. In the case of AcLDC, this dependence was hyperbolic, and the affinity of the enzyme for its substrate was high (Km = 20 mM). Leucine, valine, and isoleucine were shown to be activators of AcDLC.

Acetolactate Synthase↗
PubMed2000

Tools for discovery of inhibitors of the 1-deoxy-D-xylulose 5-phosphate (DXP) synthase and DXP reductoisomerase: an approach with enzymes from the pathogenic bacterium Pseudomonas aeruginosa.

Two Pseudomonas aeruginosa genes encoding the enzymes 1-deoxy-D-xylulose 5-phosphate (DXP) synthase and DXP reductoisomerase, both involved in the mevalonate-independent biosynthesis of isoprenoids, have been expressed as recombinant enzymes in Escherichia coli. The purified P. aeruginosa DXP reductoisomerase was inhibited by submicromolar concentrations of the antibiotics fosmidomycin and FR-900098 in a well established method. A novel and convenient spectrophotometric assay was developed to determine activity and inhibition of P. aeruginosa DXP synthase. Fluoropyruvate is described as a first inhibitor of DXP synthase.

Aldose-Ketose Isomerases↗
PubMed1990

Analysis of sequence homologies in plant and bacterial pyruvate phosphate dikinase, enzyme I of the bacterial phosphoenolpyruvate: sugar phosphotransferase system and other PEP-utilizing enzymes. Identification of potential catalytic and regulatory motifs.

In this paper we report the amino acid sequence of pyruvate phosphate dikinase (PPDK) from Bacteroides symbiosus as determined from the nucleotide sequence of the PPDK gene. Comparison of the B. symbiosus PPDK amino acid sequence with that of the maize PPDK [Matsuoka, M., Ozeki, Y., Yamamoto, N., Hirano, H., Kamo-Murakami, Y., & Tanaka, Y. (1988) J. Biol. Chem. 263, 11080] revealed long stretches of homologous sequence (greater than 70% identity), which contributed to an overall sequence identity of 53%. The circular dichrosim spectra, hydropathy profiles, and calculated secondary structural elements of the two dikinases suggest that they may have very similar tertiary structures as well. A comparison made between the amino acid sequence of the maize and B. symbiosus dikinase with other known protein sequences revealed homology, concentrated in three stretches of sequences, to a mechanistically related enzyme, enzyme I of the Escherichia coli PEP: sugar phosphotransferase system [Saffen, D. W., Presper, K. A., Doering, T. L., Roseman, S. (1987) J. Biol. Chem. 262, 16241]. It is proposed that (i) these three stretches of sequence constitute the site for PEP binding and catalysis and a possible site for the regulation of enzymatic activity and (ii) the conserved sequences exist in a third mechanistically related enzyme, PEP synthase.

Amino Acid Sequence↗
PubMed2000

Effects of the combined thromboxane receptor antagonist and synthase inhibitor DTTX-30 on intestinal O2-exchange and energy metabolism during hyperdynamic porcine endotoxemia.

Sepsis may lead to deranged thromboxane-prostacyclin ratio with consecutive organ dysfunction. Because of the suggested role of the gut in the pathogenesis of septic shock and multiple organ failure, we investigated the effects of the novel dual thromboxane synthase inhibitor and receptor antagonist DTTX-30 (TRASI) on intestinal tissue perfusion, O2 kinetics, and energy metabolism over 24 h of hyperdynamic, normotensive porcine endotoxemia. Before, 12, 18, and 24 h after starting continuous i.v. endotoxin (LPS), we measured portal venous (PV) blood flow, intestinal oxygen extraction (iO2ER), intracapillary hemoglobin O2 saturation (HbO2%) of the ileal wall, intramucosal ileal PCO2, PV lactate-pyruvate (L-P) ratio, and plasma levels of thromboxane and prostacyclin. Treatment with TRASI (0.12 mg/kg i.v. bolus injection followed by an infusion of 0.29 mg/kg/h) initiated after 12 h of LPS infusion markedly reduced the plasma thromboxane levels and attenuated the LPS-induced fall in systemic vascular resistance, resulting in hemodynamic stabilization. TRASI did not influence the LPS-induced increase in PV blood flow nor intracapillary HbO2%, thus reflecting unchanged microcirculatory O2 availability and decreased iO2ER, possibly because of reduced O2 requirements. Nevertheless, TRASI prevented the LPS-induced increase in the PV L-P ratio, attenuated the progression of the ileal mucosal-arterial PCO2 gap, and tended to attenuate the gradual fall of PV pH. Hence, compounds like TRASI may beneficially influence LPS-related derangements of gut energy metabolism.

Animals↗
PubMed1994

Aliphatic alcohols stabilize an alternative conformation of the tryptophan synthase alpha 2 beta 2 complex from Salmonella typhimurium.

The conformational changes that accompany association of the tryptophan synthase alpha and beta 2 subunits are probed by investigating the effects of solvents on the catalytic and spectroscopic properties of tryptophan synthase. Low concentrations of ethanol, propanol, and butanol inhibit conversion of L-serine and indole to L-tryptophan by the alpha 2 beta 2 complex. Inhibition depends on the concentration, chain length, and hydrophobicity of the alcohol. In contrast, these alcohols increase the rates of conversion of beta-chloro-L-alanine and indole to L-tryptophan and of L-serine to pyruvate. Thus, alcohols alter the substrate specificity and reaction specificity of the alpha 2 beta 2 complex. These altered specificities are similar to those of the free beta 2 subunit in aqueous solution. The spectroscopic properties of enzyme-substrate intermediates formed by the alpha 2 beta 2 complex in 3 M ethanol and 0.5 M 1-butanol also resemble the intermediates formed by the free beta 2 subunit in aqueous solution. Experiments using gel-filtration, membrane ultrafiltration, and limited proteolysis show that the alpha 2 beta 2 complex does not dissociate at low concentrations of alcohols. Our results provide evidence that alcohols can stabilize an alternative conformation of the alpha 2 beta 2 complex which is similar to the open conformation of the free beta 2 subunit in aqueous solution.

1-Butanol↗
PubMed1992

Cloning, sequencing and heterologous expression of a Klebsiella pneumoniae gene encoding an FAD-independent acetolactate synthase.

The gene encoding the valine-resistant and FAD-independent acetolactate synthase of Klebsiella pneumoniae was isolated and expressed in Escherichia coli. The nucleotide sequence of this gene was determined and it exhibited an open reading frame of 1680 bp in length. In vivo expression of the acetolactate synthase-encoding gene in E. coli revealed a single 60-kDa protein which is consistent with the molecular weight calculated from the deduced amino acid sequence of the gene product. The gene product shares about 20-30% homology with the acetolactate synthases of E. coli, yeast and higher plants.

Acetolactate Synthase↗
PubMed2006

The activity of a thermostable lipoyl synthase from Sulfolobus solfataricus with a synthetic octanoyl substrate.

The protein lipoyl synthase (LipA) is essential for lipoic acid biosynthesis via sulfur insertions into a protein-bound octanoyl group. We have developed an in vitro assay for LipA using a synthetic tetrapeptide substrate, containing an N(epsilon)-octanoyl lysine residue, corresponding in sequence to the lipoyl binding domain of the E2 subunit of pyruvate dehydrogenase. A putative LipA from the hypothermophilic archaea Sulfolobus solfataricus was expressed in Escherichia coli and purified, and the activity was measured using this novel assay. The optimal temperature for the S. solfataricus LipA-dependent formation of the lipoyl group was found to be 60 degrees C.

Base Sequence↗
PubMed1988

Functional residues at the active site of horse liver phosphopantothenoylcysteine decarboxylase.

Horse liver phosphopantothenoylcysteine decarboxylase (EC 4.1.1.36) is rapidly inactivated by N-acetoacetylation with diketene following a pseudo-first-order kinetics: the presence of substrate quantitatively protects against this inactivation. Histidine photo-oxidation with methylene blue or rose bengal brings about the total loss of activity. These results indicate the presence of functional lysyl and histidyl groups at the active site of the enzyme. The substrate sulphydryl group is essential for enzyme activity. Enzymatic decarboxylation is proposed to result from a combined action of the keto group of the enzyme-bound pyruvate protonated by an essential histidine and a protonated amino group of a lysine.

Animals↗
PubMed1998

Effects of short-term submaximal training in humans on muscle metabolism in exercise.

Muscle metabolism, including the role of pyruvate dehydrogenase (PDH) in muscle lactate (Lac-) production, was examined during incremental exercise before and after 7 days of submaximal training on a cycle ergometer [2 h daily at 60% peak O2 uptake (VO2 max)]. Subjects were studied at rest and during continuous steady-state cycling at three stages (15 min each): 30, 65, and 75% of the pretraining VO2 max. Blood was sampled from brachial artery and femoral vein, and leg blood flow was measured by thermodilution. Biopsies of the vastus lateralis were obtained at rest and during steady-state exercise at the end of each stage. VO2 max, leg O2 uptake, and the maximum activities of citrate synthase and PDH were not altered by training; muscle glycogen concentration was higher. During rest and cycling at 30% VO2 max, muscle Lac- concentration ([Lac-]) and leg efflux were similar. At 65% VO2 max, muscle [Lac-] was lower (11.9 +/- 3.2 vs. 20.0 +/- 5.8 mmol/kg dry wt) and Lac- efflux was less [-0.22 +/- 0.24 (one leg) vs. 1.42 +/- 0.33 mmol/min] after training. Similarly, at 75% VO2 max, lower muscle [Lac-] (17.2 +/- 4.4 vs. 45.2 +/- 6.6 mmol/kg dry wt) accompanied less release (0.41 +/- 0.53 vs. 1.32 +/- 0.65 mmol/min) after training. PDH in its active form (PDHa) was not different between conditions. Calculated pyruvate production at 75% VO2 max fell by 33%, pyruvate reduction to lactate fell by 59%, and pyruvate oxidation fell by 24% compared with before training. Muscle contents of coenzyme A and phosphocreatine were higher during exercise after training. Lower muscle lactate production after training resulted from improved matching of glycolytic and PDHa fluxes, independently of changes in muscle O2 consumption, and was associated with greater phosphorylation potential.

Adenine Nucleotides↗
PubMed1995

Effects of chronic renal failure on enzymes of energy metabolism in individual human muscle fibers.

In order to improve knowledge about the mechanisms underlying the alterations of energy metabolism recently observed in the skeletal muscle of patients suffering from chronic renal failure, this study was designed to test (1) whether changes in the activity of key enzymes of energy metabolism occur in the muscle of these patients, and if so (2) whether the different muscle fiber types are equally altered in their metabolic machinery. For this, the maximum activities of 14 enzymes were measured in individual muscle fibers microdissected from biopsies of rectus abdominis muscle obtained from seven normal subjects and seven patients with end-stage renal failure before renal replacement therapy. A large decrease in the activities of beta-hydroxyacyl-coenzyme A dehydrogenase, a key enzyme of the beta-oxidation pathway, of citrate synthase, which initiates the tricarboxylic acid cycle, and of fructose-1,6-bisphosphatase, which contributes to the synthesis of glycogen from lactate, was observed in the three fiber types (slow-twitch oxidative, fast-twitch oxidative-glycolytic, and fast-twitch glycolytic). A smaller reduction of the activities of phosphofructokinase and/or pyruvate kinase, two key enzymes of glycolysis, was also observed in slow-twitch oxidative and/or fast-twitch oxidative-glycolytic fibers. These results demonstrate that the abnormalities of muscle energy metabolism observed in patients with chronic renal failure are due, at least in part, to intrinsic changes in the key enzymes of major energy-providing pathways; they also offer a satisfactory explanation for the defect of oxidative metabolism recently demonstrated in the muscle of these patients.

Adult↗
PubMed1969

Utilization of L-threonine by a species of Arthrobacter. A novel catabolic role for "aminoacetone synthase".

1. A species of Arthrobacter (designated Arthrobacter 9759) was isolated from soil by its ability to grow aerobically on l-threonine as sole source of carbon atoms, nitrogen atoms and energy; the organism also grew well on other sources of carbon atoms including glycine, but no growth was obtainable on aminoacetone or dl-1-aminopropan-2-ol. 2. During growth on threonine, (14)C from l-[U-(14)C]threonine was rapidly incorporated into glycine and citrate, and thereafter into serine, alanine, aspartate and glutamate. 3. With extracts of threonine-grown cells supplied with l-[U-(14)C]threonine, evidence was obtained of the NAD and CoA-dependent catabolism of l-threonine to produce acetyl-CoA plus glycine. Short-term incorporation studies in which [2-(14)C]acetate and [2-(14)C]glycine were supplied (a) to cultures growing on threonine, and (b) to extracts of threonine-grown cells, showed that the acetyl-CoA was metabolized via the tricarboxylic acid cycle and glyoxylate cycle whereas the glycine was converted into pyruvate via the folate-dependent ;serine pathway'. 4. The threonine-grown organism contained ;biosynthetic' threonine dehydratase and a potent NAD-linked l-threonine dehydrogenase but possessed no l-threonine aldolase activity. 5. Evidence was obtained that the acetyl-CoA and glycine produced from l-threonine had their immediate origin in the alpha-amino-beta-oxobutyrate formed by the threonine dehydrogenase; the CoA-dependent cleavage of this compound was catalysed by an alpha-amino-beta-oxobutyrate CoA-ligase, which was identified with ;aminoacetone synthase'. A continuous spectrophotometric assay of this enzyme was developed, and it was found to be inducibly synthesized only during growth on threonine and not during growth on acetate plus glycine. 6. By using a reconstituted mixture of separately purified l-threonine dehydrogenase and alpha-amino-beta-oxobutyrate CoA-ligase (i.e. ;aminoacetone synthase'), l-[U-(14)C]threonine was broken down to [(14)C]glycine plus [(14)C]acetyl-CoA (trapped as [(14)C]citrate). 7. There was no evidence of aminoacetone metabolism by Arthrobacter 9759 even though a small amount of this amino ketone appeared in the culture medium during growth on threonine.

Acetates↗
PubMed1992

Glucose stimulation of lipogenic enzyme gene expression in cultured white adipose tissue. A role for glucose 6-phosphate.

The expression of fatty acid synthase (FAS) and acetyl-CoA carboxylase (ACC) is low in the adipose tissue of suckling rats and increases markedly at weaning to a high carbohydrate diet. We have studied in vitro the factors regulating this phenomenon. Inguinal adipose tissue pieces from 19-day-old suckling rats were cultured for 6 or 24 h in minimal essential medium. Insulin (100 nM) added in the presence of lactate and pyruvate did not stimulate the expression of FAS and ACC. Glucose (20 mM) alone resulted in a 5-7-fold increase of FAS and ACC mRNA. Insulin potentiated the effect of glucose. 3-O-Methylglucose, a glucose analog that is transported into the cell but not metabolized, had no effect on FAS and ACC mRNA accumulation. However, 2-deoxyglucose (1 mM), a glucose analog which is phosphorylated to 2-deoxyglucose 6-phosphate, stimulated the expression of FAS and ACC to the same extent as 20 mM glucose. Glucose 6-phosphate concentrations in adipose tissue pieces cultured in various conditions changed in parallel with the FAS and ACC mRNA levels. We conclude that glucose 6-phosphate could be the metabolite involved in the stimulation of lipogenic enzyme gene expression in response to glucose.

3-O-Methylglucose↗
PubMed1968

Comparative intermediary metabolism of vegetative cells and microcysts of Myxococcus xanthus.

Crude extracts of both vegetative cells and glycerol-induced microcysts of Myxococcus xanthus contained the following enzyme activities: phosphofructokinase, phosphoglucoisomerase, fructose-1,6-diphosphatase, fructosediphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphopyruvate carboxylase, citrate synthase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate dehydrogenase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, phosphoglucomutase, and uridine diphosphate glucose pyrophosphorylase. With the exception of isocitrate dehydrogenase, which was present at a fivefold higher concentration in microcysts, all activities in extracts from both types of cells were essentially equal. Hexokinase and pyruvate kinase could not be detected in extracts from either type of cell. Microcysts metabolized acetate at a lower rate than did vegetative cells. Most of this decrease was reflected in a substantial decrease in ability of microcysts to oxidize acetate to CO(2). In addition, microcysts and vegetative cells showed a different distribution of (14)C-label from incorporated acetate.

Acetates↗
PubMed1998

Insulin resistance in obesity: metabolic mechanisms and measurement methods.

In obesity several mechanisms contribute to produce insulin resistance. Elevation of plasma FFA increases the concentration of cytoplasmic long-chain-CoA (LC-CoA) and mitochondrial acetyl-CoA. The latter inhibits pyruvate dehydrogenase (PDH) and, therefore, glucose oxidation. LC-CoA exerts an array of effects, some mediated by peroxisome proliferator-activated receptors, including modulation of gene expression of enzymes of glycolipid metabolism, thus inhibiting glucose utilization and potentiating FFA oxidation. Enhanced availability of glucose plus insulin forces glucose utilization (activation of PDH and glycogen synthase) and leads to increased production of malonyl-CoA (via citrate), which inhibits carnitine palmitoyl transferase 1 and therefore FFA beta-oxidation. In obesity there is often enhanced availability of both FFA and glucose plus insulin. The latter, by increasing malonyl-CoA, may limit FFA beta-oxidation. This, however, leads to further increases in LC-CoA, which worsens insulin resistance. All these mechanisms occur through both short-term and long-term effects. Therefore, when insulin sensitivity is measured with the hyperinsulinemic clamp, which artificially suppresses FFA levels, the FFA short-term effects are lost. More physiological methods are those utilizing OGTT data, allowing calculation of an Insulin Sensitivity Index for glycemia, or ISI(gly), through the formula: 2/((INSp x GLYp)+1), where INSp and GLYp are the measured insulin and glycemic areas expressed by taking mean normal value as 1. The corresponding Insulin Resistance Index, or IRI(gly), can be obtained through the formula: 2/((1/(INSp x GLYp))+1). Substitution of glycemic (GLYp) with FFA (FFAp) values allows the calculation of indices of insulin sensitivity and resistance for FFA, i.e., ISI(ffa) and IRI(ffa).

Animals↗
PubMed2002

Metabolic engineering of lactic acid bacteria, the combined approach: kinetic modelling, metabolic control and experimental analysis.

Everyone who has ever tried to radically change metabolic fluxes knows that it is often harder to determine which enzymes have to be modified than it is to actually implement these changes. In the more traditional genetic engineering approaches 'bottle-necks' are pinpointed using qualitative, intuitive approaches, but the alleviation of suspected 'rate-limiting' steps has not often been successful. Here the authors demonstrate that a model of pyruvate distribution in Lactococcus lactis based on enzyme kinetics in combination with metabolic control analysis clearly indicates the key control points in the flux to acetoin and diacetyl, important flavour compounds. The model presented here (available at http://jjj.biochem.sun.ac.za/wcfs.html) showed that the enzymes with the greatest effect on this flux resided outside the acetolactate synthase branch itself. Experiments confirmed the predictions of the model, i.e. knocking out lactate dehydrogenase and overexpressing NADH oxidase increased the flux through the acetolactate synthase branch from 0 to 75% of measured product formation rates.

Base Sequence↗
PubMed1981

Induction of glyconeogenic enzymes by gibberellin a(3) in endosperm of castor bean seedlings.

Seedlings of castor bean (Ricinus communis cv. Hale) were exposed to gibberellin A(3) (GA(3)) (100 micromolar) for periods up to 20 hours. Endosperm homogenates were fractionated on linear sucrose gradients and enzymes in mitochondria, glyoxysome, and cytosol fractions were assayed. Gibberellin treatment resulted in increases in the activities of enzymes in all three compartments. There were also enzymes in all three compartments which were not affected by exogenous applications of GA(3). The isozymes of l-asparate-alpha-ketoglutarate aminotransferase in both mitochondria and glyoxysomes were induced coordinately, whereas the isozymes of citrate synthase and malate dehydrogenase were not. All gluconeogenic enzymes in glyoxysomes are induced by GA(3). With the exception of the mitochondrial malate dehydrogenase isozyme, all enzymes of the tricarboxylic acid cycle believed to participate in glyconeogenesis were increased. The cytosolic enzymes malate dehydrogenase, phosphoenolpyruvate carboxykinase, and fructose bisphosphatase were induced, but the levels of pyruvate kinase and enolase were not affected by GA(3) treatment.

Journal Article↗