The relationship of glycolytic/gluconeogenic intermediates in brewing yeast (Saccharomyces uvarum) fermentations to growth

Bibliography: pages 215-264.The objective of this study has been to understand the metabolic interrelationship between yeast growth, regulation of glycolytic/gluconeogenic flux and accumulation of glycosyl donors for polysaccharide synthesis in brewing yeast (Saccharomyces uvarum) fermentations. Loss of fermenting power of a brewing yeast population may be created by a condition that inhibits growth by limiting amino acid formation and protein synthesis. In commercial strains of S. uvarum this loss may be transitory, or, if not corrected, may ultimately lead to yeast degeneration. The potential industrial impact is realised for fermentation systems which may limit yeast growth, eg. continuous systems, use of pressure and, particularly, systems utilizing immobilised cells

Decreased expression of plastidial adenylate kinase in potato tubers results in an enhanced rate of respiration and a stimulation of starch synthesis that is attributable to post-translational redox-activation of ADP-glucose pyrophosphorylase

Adenine nucleotides are of general importance for many aspects of cell function, but their role in the regulation of biosynthetic processes is still unclear. It was previously reported that decreased expression of plastidial adenylate kinase, catalysing the interconversion of ATP and AMP to ADP, leads to increased adenylate pools and starch content in transgenic potato tubers. However, the underlying mechanisms were not elucidated. Here, it is shown that decreased expression of plastidial adenylate kinase in growing tubers leads to increased rates of respiratory oxygen consumption and increased carbon fluxes into starch. Increased rates of starch synthesis were accompanied by post-translational redox-activation of ADP-glucose pyrophosphorylase (AGPase), catalysing the key regulatory step of starch synthesis in the plastid, while there were no substantial changes in metabolic intermediates or sugar levels. A similar increase in post-translational redox-activation of AGPase was found after supplying adenine to wild-type potato tuber discs to increase adenine nucleotide levels. Results provide first evidence for a link between redox-activation of AGPase and adenine nucleotide levels in plants

Mechanistic studies on bovine brain hexokinase

The variation of kinetic parameters was examined for bovine brain hexokinase with glucose and MgATP as substrates. The -log(V(,1)) and -log(V(,1)/K(,m)) profiles for both substrates were examined and seen to decrease below pH 6.5. All profiles asymptotically approached slopes of -1, indicating that the loss of activity in each instance was due to the protonation of a single group on the enzyme. Analysis of the data indicated two ionizable groups are involved in the reaction. One functions in the binding of ATP and in catalysis while the other participates in the binding of glucose;The -log(V(,1)) profiles showed a hump attributed to a loss of activity in the pH region 7.5-5.5. This inhibition was found to be caused by aluminum present in commercial preparations of ATP and probably inhibits as an AlATP complex. In an effort to remove this ion, we developed an extraction procedure that eliminates more than 99% of the contaminant with no loss of nucleotide. This method involves repeated extractions of the nucleotide solution with 8-hydroxyquinoline in chloroform, followed by one extraction at higher pH, that removes the final 10% of the aluminum contaminant that seems to be sequestered in a slowly dissociating complex and is unavailable to the 8-hydroxyquinoline at low pH;Two mechanisms have been suggested to account for the regulation of brain hexokinase by glucose 6-phosphate. One mechanism places glucose-6-P at an allosteric site, remote from the active site, while the second describes glucose-6-P as a simple product inhibitor of the enzyme, binding at the (gamma)-phosphate subsite within the ATP locus of the active site. To distinguish between these possibilities, we have undertaken a study of the back reaction of hexokinase I. Our data indicate that glucose-6-P displays classical Michaelis-Menten kinetics with brain hexokinase. This finding is consistent only with the high affinity glucose-6-P site on the enzyme being the catalytic site. The dissociation constant, estimated from the initial-rate experiments, is approximately 25 (mu)M, a value that agrees well with the inhibition constant for glucose-6-P in the forward direction

Biochemical Characterization of a Novel Mammalian Polyphosphate Dependent Glucokinase

Hexokinases are a family of enzymes that catalyse the phosphorylation of glucose by transferring the γ- phosphoryl group from adenosine triphosphate (ATP) to the sixth position hydroxyl group of glucose to generate Glucose 6-phosphate (G6P). Until now, five isozymes of mammalian hexokinase (HK) have been described: types I, II, III and IV, all of them ATP dependent, and type V which is ADP-dependent. The present thesis describes a novel hexokinase, we have designated as PPGKm. The enzyme is strictly polyphosphate (pp) dependent, is correctly defined as a glucokinase (GK), by virtue of its kinetics and is present in mammalian tisues, at high activity in liver. The enzyme does not use ATP and ADP and indeed appears to be inhibited by them. As far as can be ascertained this is the first description of a mammalian enzyme using inorganic polyphosphate as a phosphoryl donor. Polyphosphate is used as phosphoryl donor in bacterial systems and is quite well characterized, although only two micro-organisms have been shown to be strictly pp-dependent like the one described here. This novel enzyme (PPGKm) also showed unique features, differing from the others hexokinases in having a longer half-life and can be stored for several months at -20˚C without loss of activity. However, thermal stability was lower than other hexokinases studied. The enzyme activity is concentrated in the hepatocyte nucleus has a higher molecular weight compared to Hexokinases 1, 2 and 3. The full biological significance of the enzyme is as yet unclear, and attempts to purify and sequence it have been only partly successful. Its specific role in cellular, and especailly nuclear metabolism remains unknown

Investigation of the regulation of carbohydrate metabolism in Arabidopsis thaliana using a genetic approach

The regulation of carbohydrate metabolism in Arabidopsis thaliana was investigated using a genetic approach. A new class of carbohydrate insensitive mutant (cai) was characterised in order to gain insight into the control of carbohydrate metabolism. Wild type seedlings germinated on media containing 100 mM sucrose and 0.1 mM nitrogen but their cotyledons did not expand and accumulated anthocyanins. After 1 week growth was arrested. The internal carbohydrate content increased accompanied by repression of photosynthetic genes and induction of chs gene expression, cai mutants germinated on agar media containing 100 mM sucrose and 0.1 mM nitrogen but their cotyledons expanded and greened. After initial characterisation of a number of the mutants, two were selected for further analysis. When germinated on a range of different carbon; nitrogen ratios cai 10 and cai 28 displayed a reduced sensitivity to the high carbohydrate and low nitrogen conditions, cai 10 also displayed a mannose insensitive (mig) phenotype compared to the post-germinative growth of wild type which was arrested by mannose. This growth arrest in the wild type on mannose correlates with phosphate sequestration, cai 10 metabolises mannose at a different rate and accumulates less hexose phosphate than the wild type when germinated on mannose, thus indicating that the mannose insensitive phenotype may be a consequence of a disruption in metabolism. Overexpression of Arabidopsis hexokinase 1 in cai 10 did not complement the cai 10 phenotype. In contrast to previous results by Jang et al., (1997), who found that plants overexpressing hexokinase were hypersensitive to sugars, our results indicate that they are less sensitive than wild type. This is not in agreement with the proposed model of hexokinase as a sugar sensor (Jang et al., 1997). Seeds of the hexokinase overexpressors germinated rapidly (within 18-20 h). The seeds also contained elevated levels of some amino acids, smaller lipid bodies and less lipid than the wild type. It is proposed that hexokinase overexpression increases glucose-6-phosphate concentration which activates phosphoenolpyravate carboxylase (PEPCase) and in so doing diverts carbon from lipid biosynthesis to amino acid synthesis

Grapefruit juice ameliorates nephropathy in streptozotocin induced diabetes.

M. Pharm. University of KwaZulu-Natal, Durban 2012.Background Diabetic Nephropathy (DN) is the leading cause of end stage renal disease and mortality in diabetic patients. Over the years, medicinal plants have been used to manage diabetes and its complications. Metformin, the synthetic analogue of galegine is used as first line therapy for Diabetes Mellitus (DM) but its use is contraindicated in patients with kidney dysfunction. The management of DN currently is limited to the use of anti-hypertensive agents; ACE inhibitors and angiotensin receptor blockers. Grapefruit juice (GFJ) has shown potential as an anti-diabetogenic agent because of its ability to ameliorate hyperglycaemia and dyslipidemia but its effect on fluid and electrolyte disturbance is not known. This study was designed to investigate the effect of GFJ on renal dysfunction in streptozotocin (STZ) induced male wistar rats. Materials and Methods Male wistar rats weighing between 250-300g were divided into 7 groups (n=7) and kept in cages for the treatment period of 8 weeks. Laboratory conditions of 12 hour light/dark cycle, temperature 25±20C and humidity 50-55 % were maintained throughout the study period. Non-diabetic animals group 1 (Control) were treated orally with 1.0 ml /Kg BW of distilled water, while group 2 (ND-GFJ) were treated orally with 3.0 ml /kg BW of GFJ. The diabetic groups 3, 4, 5, 6 and 7 were starved overnight in preparation for the STZ injection. Fasting blood glucose concentration was obtained via tail prick before 45 mg or 60 mg of STZ was administered via a single injection in the peritoneal cavity. STZ was prepared by dissolving it in 0.2 ml of 0.1 M Citrate buffer at pH 4.5. Groups 3, 4 and 7 received 60.0 mg/ Kg BW of STZ while group 5 and 6 received 45 mg/kg BW of STZ. Three days following STZ induction, the diabetic state was confirmed by measuring fasting blood glucose and animals with glucose concentration greater than 6 mmol/L were included in the study. Group 4 (INS- D60) and group 5 (INS- D45) were additionally treated with 4.0 U/kg BW of insulin via subcutaneous injection (S.C) twice a day while Group 6 (GFJ-D45) and group 7 (GFJ-D60) were treated orally with 3.0ml/Kg BW of GFJ. Group 3 (D-60) were similarly treated with 1.0 ml /Kg BW of distilled water. Fasting blood glucose (FBG) and glucose tolerance tests (GTT) were done on days 1 and 58 respectively in all the treatment groups. Urine was collected for a period of 24 hours on day 59 and on the last day animals were sacrificed by halothane overdose. Blood samples were obtained via cardiac puncture; kidney tissues were removed and preserved in formalin while the liver was snap frozen with liquid nitrogen and stored in a freezer (-800C) before analysis. Results Reduced plasma insulin was accompanied by decrease in body weight and an increase in FBG accompanied by polyuria, polydipsia and glucose intolerance in the non-treated diabetic animals compared to the control. Fasting blood glucose was significantly (p<0.0001) increased in the diabetic groups and treatment with GFJ or insulin lowered FBG in groups (GFJ-D45, GFJ-D60 & INS-D60) compared to the diabetic control (D60). GFJ significantly (p=0.0034) improved glucose intolerance in diabetic animals (GFJ-D60 & GFJ-D45) when compared to diabetic control groups D-60 & D-45 respectively. Hepatic glycogen content was reduced in diabetic animals (P=0.024) and treatment with GFJ significantly (P=0.00016) increased the glycogen concentration. In the non-diabetic group (GFJ-ND) treatment with GFJ significantly (P=0.0013) increased the glycogen concentration when compared to the control group. In the diabetic animals, decreased GFR was accompanied by Na+ retention accompanied by low urinary K+ and Cl- concentration. Treatment with GFJ significantly (p<0.05) increased urinary Na+ and K+ and Cl- in the diabetic group (GFJ-D60) but did not increase urinary Cl- in non-diabetic group and consequently improved GFR in the diabetic group. Renal pathology showed structural changes in the glomerulus and treatment with GFJ had some reno-protective effect. Conclusion GFJ lowered the fasting blood glucose and improved glucose tolerance in the STZ-induced diabetic rats in a comparable manner to insulin treated diabetic rats. GFJ decreased Na+ retention and increased GFR in the diabetic animals. This study suggests that GFJ could ameliorate nephropathy associated with diabetes mellitus. These results are the first to show that GFJ has renoprotective effects in STZ-Induced diabetic rats

Caractérisation biochimique et physiologique de la fonction catalytique de l'hexokinase dans la racine de pomme de terre (Solanum tuberosum)

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal

The metabolism of lactulose by Clostridium perfringens in batch and continuous culture

The aim of this project was to initiate studies on lactulose (4-O-[beta]-D-galactopyranosyl D-fructofuranose) metabolism by human intestinal bacteria. The organism of choice was Cl. perfringens, due to its rapid metabolism of the sugar. Work was conducted with cells grown anaerobically, in both batch and continuous culture. Use of the latter system permitted closer approximation of the in vivo situation.Initial work was concerned with the identification of the metabolic pathway(s) involved. Radiolabelled lactulose was not available, hence a variety of 'cold' procedures were employed; including fluorimetric determination of key metabolites, and assay of key enzymes.Growth in medium supplemented with lactulose resulted in the induction ofB-galactosidase, whereas phospho-B-galactosidase activity could not be detected. This suggested that lactulose was accumulated in the free form and that the first step in intracellular metabolism was hydrolysis to its constituent monosaccharides. This was confirmed by the detection of intracellular free galactose and fructose. The galactosyl moiety was found to be metabolised by the Leloir pathway: the enzymes galactokinase, galactose 1-phosphate uridylyl transferase and UDPgalactose 4-epimerase were present in lactulose-grown cells, and both galactose 1-phosphate and glucose 1-phosphate were detected. The fructosyl moiety was most likely metabolised by an ATP-dependent fructokinase activity, present in lactulose-grown cells.Measurement of lactulose utilisation in a buffered incubation system permitted the study of various transport system inhibitors. Utilisation was inhibited by both CCCP and DCCD, suggesting that transport requires a proton gradient; formed by the action of the membrane-bound ATPase activity. The presence of a proton gradient-dependent uptake system is consistent with the findings from the metabolic studies. A similar metabolic system was responsible for lactulose metabolism under conditions of carbon limitation in the chemostat.Galactokinase was further studied. Basal levels of this enzyme were repressed by inclusion of glucose in the culture medium. Glucose also prevented induction of galactokinase by lactulose or galactose. This latter 'glucose effect' could not be abolished by the addition of cyclic AMP, and appeared to be mediated via inducer exclusion. <p

Cellular Processes and Mechanisms in Saccharomyces cerevisiae Influencing Anaerobic Xylose Fermentation

In 2009 the EU approved two directives as a first initiative towards reducing greenhouse gas emissions and becoming independent of fossil fuels: the Renewable Energy Directive and the Fuel Quality Directive. As a result, the demand for biofuels will increase enormously over the next decade, both nationally and in the entire EU. This huge demand will require a more advanced type of biofuels, produced from cellulosic and lignocellulosic raw materials that do not compete with the supply of food crops. These biofuels are referred to as second generation (2G) fuels. The production of 2G bioethanol at a commercial scale requires yeast strains capable of producing ethanol at high yield and high productivity from all sugars (hexoses and pentoses) extracted from the raw material. The aim of the work presented in this thesis has been to increase the ethanol productivity of recombinant xylose-fermenting strains of the yeast Saccharomyces cerevisiae during batch fermentation of a glucose/xylose mixture. A parameter that has a big influence on productivity is cellular growth and the yeast strains currently used today grow rather poorly on xylose. Many of the signals cells use to regulate growth originate from changes in the concentrations of metabolites inside the cells. To increase our knowledge of xylose metabolism the dynamic changes in intracellular metabolite concentrations were measured during batch fermentation of a glucose/xylose mixture using LC-MS/MS. This study gave meaningful insights about important intracellular signals, biological phenomena and mechanism. The analysis of the metabolite data pointed toward limitations in the folding of proteins inside the ER, which might be the underlying cause of the slow growth on xylose. Another important factor is the regulation of expression of genes required for sugar transport and those related to fermentative metabolism. Hexokinase 2 (Hxk2p) is an important bi-functional protein that acts both as a catalytic enzyme and a global transcription factor. This protein plays a role in the regulation of the above mentioned genes and becomes inactivated in the presence of xylose. As a consequence it loses its’ regulatory function. In an effort to improve repression signals during xylose fermentation this protein was engineered to become immune towards inactivation by xylose. By combining methods for protein and genetic engineering with fermentation technology a mutation in the gene was identified which increased the catalytic activity by 64% in the presence of xylose. The new variant allowed faster glucose consumption in the presence of xylose, but had no obvious impact on xylose fermentation. These results indicate that Hxk2p does not act alone and other proteins are involved in the regulation. These proteins remain to be identified. This thesis describes the cellular processes required for balanced anaerobic microbial growth and the intracellular signals that regulate them. The aim has been to identify biochemical mechanisms that limit anaerobic growth of recombinant S. cerevisiae strains on xylose