Evolution of energy metabolism and its compartmentation in Kinetoplastida

Kinetoplastida are protozoan organisms that probably diverged early in evolution from other eukaryotes. They are characterized by a number of unique features with respect to their energy and carbohydrate metabolism. These organisms possess peculiar peroxisomes, called glycosomes, which play a central role in this metabolism; the organelles harbour enzymes of several catabolic and anabolic routes, including major parts of the glycolytic and pentosephosphate pathways. The kinetoplastid mitochondrion is also unusual with regard to both its structural and functional properties.     

Functional proteomics for the discovery of carbohydrate-related enzyme activities

Carbohydrate metabolism is central to the growth and development of organisms. Thousands of genes that are believed to code for proteins that build and remodel saccharides have been uncovered by genome sequencing projects, thereby necessitating higher-throughput methods to delineate the chemical and biological functions of all these proteins. Recent methods discussed in this review have begun to address this problem by the design of new activity-based probes and mass-differentiated carbohydrate libraries. In addition, a comparative view of carbohydrate-related enzymes from all three branches of life has led to the discovery of unusual enzymes to simplify the synthesis of carbohydrates such as the sugar nucleotides required by glycosyltransferases.     

A model of oxidative stress management: moderation of carbohydrate metabolizing enzymes in SOD1-null Drosophila melanogaster

The response to oxidative stress involves numerous genes and mutations in these genes often manifest in pleiotropic ways that presumably reflect perturbations in ROS-mediated physiology. The Drosophila melanogaster SOD1-null allele (cSODn108) is proposed to result in oxidative stress by preventing superoxide breakdown. In SOD1-null flies, oxidative stress management is thought to be reliant on the glutathione-dependent antioxidants that utilize NADPH to cycle between reduced and oxidized form. Previous studies suggest that SOD1-null Drosophila rely on lipid catabolism for energy rather than carbohydrate metabolism. We tested these connections by comparing the activity of carbohydrate metabolizing enzymes, lipid and triglyceride concentration, and steady state NADPH:NADP(+) in SOD1-null and control transgenic rescue flies. We find a negative shift in the activity of carbohydrate metabolizing enzymes in SOD1-nulls and the NADP(+)-reducing enzymes were found to have significantly lower activity than the other enzymes assayed. Little evidence for the catabolism of lipids as preferential energy source was found, as the concentration of lipids and triglycerides were not significantly lower in SOD1-nulls compared with controls. Using a starvation assay to impact lipids and triglycerides, we found that lipids were indeed depleted in both genotypes when under starvation stress, suggesting that oxidative damage was not preventing the catabolism of lipids in SOD1-null flies. Remarkably, SOD1-nulls were also found to be relatively resistant to starvation. Age profiles of enzyme activity, triglyceride and lipid concentration indicates that the trends observed are consistent over the average lifespan of the SOD1-nulls. Based on our results, we propose a model of physiological response in which organisms under oxidative stress limit the production of ROS through the down-regulation of carbohydrate metabolism in order to moderate the products exiting the electron transport chain.     

Enzymes of intermediary carbohydrate metabolism in Ureaplasma urealyticum and Mycoplasma mycoides subsp. mycoides

Cell extracts of Ureaplasma urealyticum and Mycoplasma mycoides were examined for enzymes of intermediary carbohydrate metabolism using a sensitive radiochemical assay procedure. For M. mycoides, the enzyme activities detected were supporting evidence for the existence of a glycolytic pathway giving lactate anaerobically and acetate aerobically. U. urealyticum also had activities of many glycolytic enzymes. Enzymes of the pentose phosphate pathway occurred in both M. mycoides and U. urealyticum. Their presence allowed the proposal of a sequence for the synthesis from glycolytic pathway intermediates of ribose 5-phosphate, and hence phosphoribosyl diphosphate, for the synthesis of nucleotides. Pathways for the further metabolism of deoxyribose 1-phosphate and ribose 1-phosphate produced from nucleoside phosphorylase reactions operated in extracts from both organisms.     

Fructose metabolism in four Pseudomonas species

1. ATP-Dependent phosphorylation of fructose could not be detected in extracts of fructose-grown cells of Pseudomonas extorquens strain 16, Pseudomonas 3A2, Pseudomonas acidovorans and Pseudomonas fluorescens. Instead, phosphorylation of fructose to fructose-1-phosphate was found to occur when cell-free extracts were incubated with fructose and phosphoenolpyruvate. Such an activity could not be detected in cell-free extracts of succinate-grown cells. 2. High levels of 1-phosphofructokinase were found in extracts of the above organisms when growth on fructose. 3. Mutants of Pseudomonas extorquens strain 16 lacking 1-phosphofructokinase were unable to grow on fructose. Revertants to growth on fructose had regained the capacity to synthesize this enzyme, indicating its necessary involvement in fructose metabolism. 4. A survey has been carried out of enzymes involved in carbohydrate metabolism in the species listed above.     

Diurnal rhythms in liver carbohydrate metabolism. Comparative aspects and critical review

Literature data on the diurnal rhythms of blood glucose, liver glycogen levels and key hepatic enzyme activities of glycolysis, gluconeogenesis, glycogen metabolism and lipogenesis in animals are reviewed. Materials on the diurnal rhythms of the activities of other enzymes involved in carbohydrate metabolism and related pathways such as the equilibrium glycolytic enzymes are also given. Interspecies comparison and analysis of the results and their interpretation are given.     

Role and regulation of the ortho and meta pathways of catechol metabolism in pseudomonads metabolizing naphthalene and salicylate.

The enzymes of naphthalene metabolism are induced in Pseudomonas putida ATCC 17484, PpG7, NCIB 9816, and PG and in Pseudomonas sp. ATCC 17483 during growth on naphthalene or salicylate; 2-aminobenzoate is a gratuitous inducer of these enzymes. The meta-pathway enzymes of catechol metabolism are induced in ATCC 17483 and PPG7 during growth on naphthalene or salicylate or during growth in the presence of 2-aminobenzoate, but in ATCC 17484 and NCIB 9816 the ortho-pathway enzymes of catechol metabolism are induced during growth on naphthalene or salicylate. 2-Aminobenzoate does not induce any enzymes of catechol metabolism in the latter two organisms. In Pseudomonas PG the meta-pathway enzymes are present at high levels under all conditions of growth, but this organism and PpG7 can induce ortho-pathway enzymes during naphthalene or salicylate metabolism. Salicylate appears to be the inducer of the enzymes of naphthalene metabolism in all of the organisms studied and, where they are inducible, of the meta-pathway enzymes, but the properties of Pseudomonas PG suggest that separate, regulatory systems may exist.     

A comparison of the effects of duodenal glucose infusion on carbohydrate and lipid metabolism in liver, adipose tissue and small intestinal mucosa in sheep

The effects of duodenal glucose infusion on the specific activities of some enzymes of carbohydrate and lipid metabolism in the liver, perinephric adipose tissue and small intestinal mucosa of sheep were examined. Lipogenic enzyme activity was generally greatest in adipose tissue and lowest in liver and the response of these enzymes to glucose infusion was similarly greatest in adipose tissue. Glycolytic enzyme activity was significantly increased in all three tissues following duodenal glucose infusion. The effects of increasing carbohydrate availability in the small intestine in relation to tissue metabolism in sheep are discussed.     

Organismal Carbohydrate and Lipid Homeostasis

All living organisms maintain a high ATP:ADP ratio to drive energy-requiring processes. They therefore need mechanisms to maintain energy balance at the cellular level. In addition, multicellular eukaryotes have assigned the task of storing energy to specialized cells such as adipocytes, and therefore also need a means of intercellular communication to signal the needs of individual tissues and to maintain overall energy balance at the whole body level. Such signaling allows animals to survive periods of fasting or starvation when food is not available and is mainly achieved by hormonal and nervous communication. Insulin, adipokines, epinephrine, and other agonists thus stimulate pathways that regulate the activities of key enzymes involved in control of metabolism to integrate organismal carbohydrate and lipid metabolism. Overnutrition can dysregulate these pathways and have damaging consequences, causing insulin resistance and type 2 diabetes.     

Metabolism of mycobacteria

The metabolism of mycobacteria have been studied with reference to carbohydrate, lipids, nitrogen metabolism and oxidative phosphorylation. Some of the enzymes of glycolytic pathway, tricarboxylic acid cycle and lypogenic enzymes were purified, characterized and their kinetic properties investigated. The effect of age of the culture and environmental factors on different aspects of metabolism of mycobacteria were also studied. A comparison of lipid profile in various species of mycobacteria grown in different culture conditions were made. The metabolism of spheroplasts isolated from mycobacteria has been established with respect to their energy charge and to synthesize peptidoglycan using D-alanine as the precursor     

Enzymes of Intermediary Carbohydrate Metabolism in the Obligate Autotrophs Thiobacillus thioparus and Thiobacillus neapolitanus

Levels of enzymes operative in the Embden-Meyerhof-Parnas (glycolytic) pathway, pentose phosphate cycle, citric acid cycle, and certain other phases of intermediary carbohydrate metabolism have been compared in Thiobacillus thioparus and T. neapolitanus. All enzymes of the glycolytic pathway except phosphofructokinase were demonstrated in both organisms. There were some striking quantitative differences between the two organisms with respect to the activities of the individual enzymes of the glycolytic pathway and the citric acid cycle. Qualitative differences were also found: the isocitrate dehydrogenase activity of T. thioparus is strictly nicotinamide adenine dinucleotide phosphate (NADP)-dependent, whereas that of T. neapolitanus is primarily nicotinamide adenine dinucleotide-dependent, activity with NADP being low; the glucose-6-phosphate dehydrogenase of T. thioparus is particulate, whereas that of T. neapolitanus is partly soluble and partly particulate; the 6-phosphogluconate dehydrogenase of T. thioparus is soluble, that of T. neapolitanus is partly soluble and partly particulate. All enzymes which function in the carbon reduction cycle were present at very high levels. In contrast, enzymes which operate exclusively in cycles other than the carbon reduction cycle were present at low levels. Of the enzymes not operative in the carbon reduction cycle that were examined, isocitric dehydrogenase had the highest specific activity. Both organisms possessed reduced nicotinamide adenine dinucleotide dehydrogenase activity. The qualitative and quantitative aspects of the data are discussed in relation to possible biochemical explanations of obligate autotrophy.     

Metabolic priorities during starvation: enzyme sparing in liver and white muscle of Atlantic cod,Gadus morhua L

Atlantic cod, Gadus morhua, respond to starvation first by mobilising hepatic lipids, then muscle and hepatic glycogen and finally muscle proteins. The dual role of proteins as functional elements and energetic reserves should lead to a temporal hierarchy of mobilisation where the nature of a function dictates its conservation during starvation. We examined (1) whether lysosomal and anti-oxidant enzymes in liver and white muscle are spared during prolonged starvation, (2) whether the responses of these enzymes in muscle vary longitudinally. Hepatic contents of lysosomal proteases decreased with starvation, whereas those of catalase (CAT) increased and lysosomal enzymes of carbohydrate metabolism and glutathione S-transferase (GST) did not change. In white muscle, starvation decreased the specific activity of lysosomal enzymes of carbohydrate degradation and doubled that of cathepsin D (CaD). The activity of anti-oxidant enzymes and acid phosphatase in muscle was unchanged with starvation. In white muscle neither lysosomal enzymes nor anti-oxidant enzymes varied significantly with sampling position. In cod muscle, antioxidant enzymes, CaD and acid phosphatase are spared during a period of starvation that decreases lysosomal enzymes of carbohydrate metabolism and decreases glycolytic enzyme activities. In cod liver, the anti-oxidant enzymes, CAT and GST, were also spared during starvation.     

Systemic use of “limping” enzymes in plant cell walls

Diversity of proteins and enzymes engaged in carbohydrate metabolism is vast. This is related to the fact that plants contain the greater part of biospheric carbohydrates, whose structures are extremely diverse as well. In plant genomes, proteins involved in carbohydrate metabolism are grouped into numerous families, and each of them may include tens of sequences. This is especially typical of enzymes modifying polysaccharides of the cell wall. Expression of genes encoding such proteins is finely tuned. It may differ in different tissues and organs and depends on stages of development of the entire plant and its particular cells. However, certain genes, including highly expressive ones, encode enzymes with “limping” catalytic centers, which may be unable to conduct reactions characteristic of the particular enzymatic family. The review surveys examples of such proteins and discusses causes of their origin and possible functions.     

Hydrolysis of carbohydrates in roach (Rutilus rutilus (L.)) at different levels of mercury accumulation

It has been shown that chronic exposure to dietary mercury results in the intensive accumulation of this metal in roach yearlings. The concentrations of accumulated mercury in their organisms were proportional to the amount of metal added to water of experimental tanks. Increased Hg content in the organisms (0.05–0.16 mg/kg) of developing juvenile roaches decreases the activity of digestive carbohydrases and the affinity of enzymes to substrate with subsequent retardation of rates of initial stages of digestion. The accumulation of mercury in the organism is accompanied by an increase in the body length and weight in the exposed fish compared to the control. The possible mechanisms responsible for such an increase in fish with high Hg body burdens, and with a simultaneous decrease in carbohydrate metabolism rate, are discussed in the paper.     

Enzyme-catalyzed detoxication reactions: mechanisms and stereochemistry

Enzyme catalyzed detoxication reactions are one of the primary defenses organisms have against chemical insult. This article reviews current chemical approaches to understanding the cooperative role of enzymes in the metabolism of foreign compounds. Emphasis is placed on chemical and stereochemical studies which help elucidate the mechanism of action and active-site topologies of the detoxication enzymes. The stereoselectivity of the cytochromes P-450 and flavin containing monooxygenases as well as the role of hemoglobin and lipid peroxidation in the primary metabolism of xenobiotics is discussed. Current knowledge of the mechanism and stereoselectivity of epoxide hydrolase is also presented. Three enzymes involved in secondary metabolism of xenobiotics, UDP-glucuronosyltransferase, sulfotransferase and glutathione S-transferase are discussed with particular emphasis on active site topology and cooperative participation with the enzymes of primary metabolism.     

Polysaccharide lyases: recent developments as biotechnological tools

Polysaccharide lyases, which are polysaccharide cleavage enzymes, act mainly on anionic polysaccharides. Produced by prokaryote and eukaryote organisms, these enzymes degrade (1,4) glycosidic bond by a beta elimination mechanism and have unsaturated oligosaccharides as major products. New polysaccharides are cleaved only by their specific polysaccharide lyases. From anionic polysaccharides controlled degradations, various biotechnological applications were investigated. This review catalogues the degradation of bacterial, plant and animal polysaccharides (neutral and anionic) by this family of carbohydrate acting enzymes.     

In vitro grown potato microtubers are a suitable system for the study of primary carbohydrate metabolism

The aim of this work was to determine the suitability of tissue culture microtubers for the study of primary carbohydrate metabolism in potato plants (Solanum tuberosum L. cv. Desirée). We have determined the levels of key enzymes and intermediates in the pathways of sucrose metabolism, starch metabolism and glycolysis in wild type microtubers. Comparison of the metabolite levels in microtubers with a range of published studies on soil-grown developing tubers showed that the two systems were similar both in the absolute levels and in the ratios between metabolites, despite some differences in the maximum catalytic activities of some glycolytic enzymes. We conclude that in vitro grown microtubers are an adequate model system for studying primary carbohydrate metabolism in developing potato tubers.     

Carbohydrate metabolism of the Saccharolytic alkaliphilic anaerobes Halonatronum saccharophilum, Amphibacillus fermentum, and Amphibacillus tropicus

The saccharolytic anaerobic bacteria Halonatronum saccharophilum, Amphibacillus fermentum, and Amphibacillus tropicus produce formate the main fermentation product. In the alkaliphilic community, formate is used as the preferential substrate for sulfate reduction. To reveal the pathways of carbohydrate fermentation by these bacteria, the activity of the key enzymes of carbohydrate metabolism and their pH dependence was studied. It was established that H. saccharophilum utilized glucose by the fructose bisphosphate and hexose monophosphate pathways, and A. tropicus, by the fructose bisphosphate and Entner-Doudoroff pathways. The activity of the key enzymes of all three pathways of glucose metabolism was detected in Amphibacillus fermentum. According to the data obtained, the glucose catabolism in H. saccharophilum. A. fermentum, and A. tropicus mainly proceeds via the fructose bisphosphate pathway. The pH optima of the key enzymes of the glucose metabolism of the alkaliphiles are shifted to alkaline values. In A. tropicus, formate is formed from pyruvate under the action of pyruvate formate-lyase; and in the haloanaerobe H. saccharophilum, formate dehydrogenase is involved in formate metabolism.