Mannitol 1-phosphate Research Articles

Purification of the mannitol-specific enzyme II of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system.

The inducible, mannitol-specific Enzyme II of the phosphoenolpyruvate:sugar phosphotransferase system has been purified approximately 230-fold from Escherichia coli membranes. The enzyme, initially solubilized with deoxycholate, was first subjected to hydrophobic chromatography on hexyl agarose and then purified by several ion exchange steps in the presence of the nonionic detergent, Lubrol PX. The purified protein appears homogeneous by several criteria and probably consists of a single kind of polypeptide chain with a molecular weight of 60,000 (+/- 5%). In addition to catalyzing phosphoenolpyruvate-dependent phosphorylation of mannitol in the presence of the soluble enzymes of the phosphotransferase system, the purified Enzyme II also catalyzes mannitol 1-phosphate:mannitol transphosphorylation in the absence of these components. A number of other physical and catalytic properties of the enzyme are described. The availability of a stable, homogeneous Enzyme II should be invaluable for studying the mechanism of sugar translocation and phosphorylation catalyzed by the bacterial phosphotransferase system.

Isoamyl phenylacetate

Cupriavidus necator H16 is one of the most researched carbon dioxide (CO2)-fixing bacteria. It can store carbon in form of the polymer polyhydroxybutyrate and generate energy by aerobic hydrogen oxidation under lithoautotrophic conditions, making C. necator an ideal chassis for the biological production of value-added compounds from waste gases. Despite its immense potential, however, the experimental evidence of C. necator utilisation for autotrophic biosynthesis of chemicals is limited. Here, we genetically engineered C. necator for the high-level de novo biosynthesis of the industrially relevant sugar alcohol mannitol directly from Calvin-Benson-Bassham (CBB) cycle intermediates. To identify optimal mannitol production conditions in C. necator, a mannitol-responsive biosensor was applied for screening of mono- and bifunctional mannitol 1-phosphate dehydrogenases (MtlDs) and mannitol 1-phosphate phosphatases (M1Ps). We found that MtlD/M1P from brown alga Ectocarpus siliculosus performed overall the best under heterotrophic growth conditions and was selected to be chromosomally integrated. Consequently, autotrophic fermentation of recombinant C. necator yielded up to 3.9 g/L mannitol, representing a substantial improvement over mannitol biosynthesis using recombinant cyanobacteria. Importantly, we demonstrate that at the onset of stationary growth phase nearly 100% of carbon can be directed from the CBB cycle into mannitol through the glyceraldehyde 3-phosphate and fructose 6-phosphate intermediates. This study highlights for the first time the potential of C. necator to generate sugar alcohols from CO2 utilising precursors derived from the CBB cycle.

D-Mannitol dehydrogenase from Absidia glauca. Steady-state kinetic properties and the inhibitory role of mannitol 1-phosphate

Steady-state kinetic studies including initial velocity for mannitol oxidation and fructose reduction and product inhibition for mannitol oxidation using fructose and reduced nicotinamide adenine dinucleotide (NADH) are in accord with a reaction mechanism best described as ordered Bi-Bi with NAD+ and NADH designated as the first substrate, last product, respectively at pH 8.8. All replots of slopes and intercepts from product inhibition studies were linear. Dead-end inhibition studies using mannitol 1-phosphate gave slope-parabolic, intercept-linear noncompetitive inhibition for both NAD+ and mannitol as substrates. The dead-end inhibitor is capable of binding multiply to the E, EA, and EQ forms of the enzyme to an extent that is controlled by the concentration of substrates. The EQ complex is inferred to undergo a conformational change, E'Q equilibrium EQ, since (V1/E1) greater than (KiqV2)/(KqE1), and no evidence for dead-end complex formation with NADH can be adduced. This is interpreted to mean that the release of fructose from the central complex is faster than the isomerization of the E-NADH complex. When mannitol is saturating, the noncompetitive inhibition against NAD+, as the variable substrate, becomes parabolic uncompetitive. A replot of the slopes of the parabola against mannitol 1-phosphate remains concave upward. This situation could arise if the conformational change we infer in the EQ complex opens up additional sites on the protein which can interact with the dead-end inhibitor.

Direct transfer of the phosphoryl moiety of mannitol 1-phosphate to [14C]mannitol catalyzed by the enzyme II complexes of the phosphoenolpyruvate: mannitol phosphotransferase systems in Spirochaeta aurantia and Salmonella typhimurium.

Spirochaeta aurantia possesses a phosphoenolpyruvate:mannitol phosphotransferase system which catalyzes the transmembrane transport and phosphorylation of mannitol. In vitro studies showed that both phosphoenolpyruvate and mannitol 1-phosphate could serve as phosphate donors. The phosphoenolpyruvate-dependent reaction required two soluble proteins, Enzyme SI and HPr, and an integral membrane complex, Enzyme SII. Only Enzyme SII was required for the mannitol 1-phosphate-dependent reaction. Enzyme II-dependent transphosphorylation of sugars was also demonstrated in eubacterial extracts. The results lead to the suggestion that the Enzyme II complexes of bacterial phosphotransferase systems possess nonoverlapping binding sites for sugar and sugar phosphate.

D-Mannitol dehydrogenase from Absidia glauca. Purification, metabolic role, and subunit interactions.

When Absidia glauca was grown in minimal media with D-mannitol as the only source of carbon, an NAD+ specific D-mannitol dehydrogenase (EC 1.1.1.67) was induced. The crude extract also gave evidence of mannitol kinase, mannitol-1-phosphate dehydrogenase, phosphofructokinase, and L-iditol dehydrogenase activity. The heat labile purified preparation was judged enzymically homogeneous based on evidence derived from substrate specificity studies and activity staining, following disc gel electrophoresis. The enzymic monomer, with a weight of about 67000 daltons, slowly polymerizes when stored at -20 degrees C, giving a multiplicity of protein bands on electrophoresis distributed predominantly across a spectrum from dimer to pentamer, with enzymic activity resident predominantly in even multiples of the monomer. Depolymerization occurred rapidly (hours) when a frozen preparation was brought to and held between 4 and 20 degrees C. Aggregate fragmentation with sodium dodecyl sulfate showed a time-temperature dependence, terminating in a subunit component of 13000 daltons. pH optimum for polyol oxidation occurs at 9.6 (NaOH-glycine buffer) while ketose reduction proceeded most rapidly at pH 7.0-7.2 (phosphate buffer). A regulatory role is suggested for this enzyme based on dead-end inhibition by mannitol 1-phosphate, multiple enzyme forms, and its locus at the initiation site for mannitol utilization. The physiological relevance of low-temperature aggregation to regulatory control remains to be established.

Glycolytic and related enzymes in clostridial classification.

The activities of 15 glycolytic and related enzymes were determined in clostridia. All contained 1-phosphofructokinase; three of them lacked 6-phosphofructokinase and mannitol 1-phosphate dehydrogenase. Glucose 6-phosphate dehydrogenase was found in six clostridia, thus demonstrating the presence of hexose monophosphate shunt. Only parts of the citric acid cycle were found to be present in most clostridia with an indication of the full cycle in Clostridium septicum. The intermediary enzyme activities were used to differentiate between the different clostridia.

D-mannitol metabolism by Aspergillus candidus.

Pathways of mannitol biosynthesis and utilization in Aspergillus candidus NRRL 305 were studied in cell-free extracts of washed mycelia prepared by sonic and French pressure cell treatments. A nicotinamide adenine dinucleotide-linked mannitol-1-phosphate (M1P) dehydrogenase was found in French pressure cell extracts of d-glucose-grown cells, whereas a specific mannitol-1-phosphatase was present in extracts prepared by both methods. The existence of these two enzymes indicated that mannitol may be synthesized in this organism by the reduction of fructose-6-phosphate. A specific nicotinamide adenine dinucleotide phosphate-linked mannitol dehydrogenase was also identified in both extracts. This enzyme may have been involved in mannitol utilization. However, the level of the mannitol dehydrogenase appeared to be substantially reduced in extracts from mannitol-grown cells, whereas the level of M1P dehydrogenase was increased. A hexokinase has been identified in this organism. Fructose-6-phosphatase, glucose isomerase, and mannitol kinase could not be demonstrated.

Glycolytic and Related Enzymes in Clostridial Classification

The activities of 15 glycolytic and related enzymes were determined in clostridia. All contained 1-phosphofructokinase; three of them lacked 6-phosphofructokinase and mannitol 1-phosphate dehydrogenase. Glucose 6-phosphate dehydrogenase was found in six clostridia, thus demonstrating the presence of hexose monophosphate shunt. Only parts of the citric acid cycle were found to be present in most clostridia with an indication of the full cycle in Clostridium septicum. The intermediary enzyme activities were used to differentiate between the different clostridia.

Molar growth yields as evidence for oxidative phosphorylation in Streptococcus faecalis strain 10Cl.

During the aerobic growth of Streptococcus faecalis strain 10C1, with limiting levels of glucose as the substrate, a molar growth yield (Y) of 58.2 g (dry weight) per mole of glucose was obtained. Under these conditions of growth, glucose was dissimilated primarily to acetate and CO(2). The incorporation of (14)C-glucose into cell material was no greater under aerobic conditions than during anaerobic growth. Assuming an adenosine triphosphate coefficient of 10.5, the aerobic Y cannot be explained solely on the basis of substrate phosphorylation and would appear to substantiate previous enzymatic evidence for oxidative phosphorylation in this cytochromeless species. With mannitol as the substrate, an aerobic Y of 64.6 was obtained. Extracts of mannitol-grown cells contained a nicotinamide adenine dinucleotide (NAD)-linked mannitol-1-phosphate (M-1-P) dehydrogenase. The difference in aerobic Y values with mannitol and glucose as substrates would indicate that the in vivo P/O ratio from the oxidation of reduced NAD generated by the oxidation of M-1-P approximates 0.6. The Y values with pyruvate and glycerol as substrates under aerobic conditions were 15.5 and 24.7, respectively.

Intracellular pH effect upon phosphoglucose isomerase in Escherichia coli

Abstract 1. 1. Whole cells of Escherichia coli hydrolyze fructose 1,6-diphosphate to fructose 6-phosphate, and when the cells are incubated in an unbuffered medium the product is not isomerized to glucose 6-phosphate. The reason for this lack of isomerization has been investigated. 2. Ubder specified conditions phosphoglucose isomerase ( d -glucose-6-phosphate ketol isomerase, EC 5.3.1.9) is inhibited, probably because of a low pH in the cells which is unfavourable for the phosphoglucose isomerase reaction, but which permits other enzymes in the glycolytic sequence to act. 3. 3. Aerobically incubated cells of E. coli contain more acids than what is found under anaerobic conditions. This may result in a pH inhibition of the phosphoglucose isomerase which is dependent upon the oxygen pressure. 4. 4. In suspensions of E. coli buffered at pH 5.8, hydrolysis in the 1 position is competing favourably woth glycolytic breakdown of fructose 1,6-diphosphate. 5. 5. The specific radioativity of mannitol 1-phosphate observed in experiments with 32 P-labelled orthophosphate, strongly suggests that E. coli contains an active mannitol kinase.

Metabolism of mannitol and induction of mannitol 1-phosphate dehydrogenase in Lactobacillus plantarum.

Metabolism of mannitol and induction of mannitol 1-phosphate dehydrogenase in Lactobacillus plantarum.

Mannitol 1-phosphate formation in Escherichia coli during glucose utilization

1. 1. Centrifuged cell-free extracts and intact cells of Escherichia coli were incubated in presence of glucose and 32P i under both aerobic and anaerobic c onditions. 2. 2. Radioactively labelled mannitol 1-phosphate and various sugar phosphates were isolated from the media. 3. 3. The concentration levels and labelling of the various compounds containing esterified phosphate, together with the fact that mannitol 1-phosphate accumulates, indicate that fructose 6-phosphate is being used for the reoxidation of reduced diphosphopyridine nucleotide formed in the triose phosphate dehydrogenase reaction.

The isolation of cytidine diphosphate glycerol, cytidine diphosphate ribitol and mannitol 1-phosphate from Lactobacillus arabinosus.

Research Article| December 01 1956 The isolation with cytidine diphosphate glycerol, cytidine diphosphate ribitol and mannitol 1-phosphate from Lactobacillus arabinosus J. Baddiley; J. Baddiley 1Department of Chemistry, University of Durham, King's College, Newcastle upon Tyne Search for other works by this author on: This Site PubMed Google Scholar J. G. Buchanan; J. G. Buchanan 1Department of Chemistry, University of Durham, King's College, Newcastle upon Tyne Search for other works by this author on: This Site PubMed Google Scholar B. Carss; B. Carss 1Department of Chemistry, University of Durham, King's College, Newcastle upon Tyne Search for other works by this author on: This Site PubMed Google Scholar A. P. Mathias; A. P. Mathias 1Department of Chemistry, University of Durham, King's College, Newcastle upon Tyne Search for other works by this author on: This Site PubMed Google Scholar A. R. Sanderson A. R. Sanderson 1Department of Chemistry, University of Durham, King's College, Newcastle upon Tyne Search for other works by this author on: This Site PubMed Google Scholar Biochem J (1956) 64 (4): 599–603. https://doi.org/10.1042/bj0640599 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation J. Baddiley, J. G. Buchanan, B. Carss, A. P. Mathias, A. R. Sanderson; The isolation with cytidine diphosphate glycerol, cytidine diphosphate ribitol and mannitol 1-phosphate from Lactobacillus arabinosus. Biochem J 1 December 1956; 64 (4): 599–603. doi: https://doi.org/10.1042/bj0640599 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Journal Search Advanced Search This content is only available as a PDF. © 1956 CAMBRIDGE UNIVERSITY PRESS1956 Article PDF first page preview Close Modal You do not currently have access to this content.