Glucosamine metabolism in Drosophila virilis salivary glands: Ontogenetic changes of enzyme activities and metabolite synthesis

In Drosophila virilis salivary glands the in vitro activities of enzymes involved in the glucosamine pathway were examined during the third larval instar and in the prepupa. While glutamine-fructose-6-phosphate aminotransferase (EC 5.3.1.19) becomes inactive at the time of puparium formation, glucosamine-6-phosphate isomerase (EC 5.3.1.10) and glucosamine-6-phosphate N-acetyltransferase (EC 2.3.1.3) show maximal activities in the prepupal gland. The activity of UDP-N-acetylglucosamine pyrophosphorylase (EC 2.7.7.23) may also decrease prior to puparium formation. Incubation of larval and prepupal glands in medium containing [³H]glucose + [¹⁴C]-uridine or [¹⁴C]glucosamine and subsequent separation of intermediates of the glucosamine pathway by chromatographic procedures reveal that the capacity of the glands to incorporate the isotopes into these intermediates decreases significantly at the time of puparium formation. The results suggest that in D. virilis salivary glands the formation of aminosugars is mainly controlled by the activities of the two enzymes glutamine-fructose-6-phosphate aminotransferase and UDP-N-acetylglucosamine pyrophosphorylase.     

Further studies on the regulation of amino sugar metabolism in Bacillus subtilis

1. Glucosamine 6-phosphate deaminase [2-amino-2-deoxy-d-glucose 6-phosphate ketol-isomerase (deaminating), EC 5.3.1.10] of Bacillus subtilis has been partially purified. Its K_m is 3·0mm. 2. Extracts of B. subtilis contain N-acetylglucosamine 6-phosphate deacetylase (K_m 1·4mm), glucosamine 1-phosphate acetylase and amino sugar kinases (EC 2.7.1.8 and 2.7.1.9). 3. Glucosamine 6-phosphate synthetase (l-glutamine–d-fructose 6-phosphate aminotransferase, EC 2.6.1.16) is repressed by growth of B. subtilis in the presence of glucosamine, N-acetylglucosamine, N-propionylglucosamine or N-formylglucosamine. Glucosamine 6-phosphate deaminase and N-acetylglucosamine 6-phosphate deacetylase are induced by N-acetylglucosamine. Amino sugar kinases are induced by glucose, glucosamine and N-acetylglucosamine. The synthesis of glucosamine 1-phosphate acetylase is unaffected by amino sugars. 4. Glucose in the growth medium prevents the induction of glucosamine 6-phosphate deaminase and of N-acetylglucosamine 6-phosphate deacetylase caused by N-acetylglucosamine; glucose also alleviates the repression of glucosamine 6-phosphate synthetase caused by amino sugars. 5. Glucosamine 6-phosphate deaminase increases in bacteria incubated beyond the exponential phase of growth. This increase is prevented by glucose.     

The incorporation of labelled amino sugars by Bacillus subtilis

1. Glucosamine 6-phosphate deaminase [2-amino-2-deoxy-d-glucose 6-phosphate ketol-isomerase (deaminating), EC 5.3.1.10] of Bacillus subtilis has been partially purified. Its K_m is 3·0mm. 2. Extracts of B. subtilis contain N-acetylglucosamine 6-phosphate deacetylase (K_m 1·4mm), glucosamine 1-phosphate acetylase and amino sugar kinases (EC 2.7.1.8 and 2.7.1.9). 3. Glucosamine 6-phosphate synthetase (l-glutamine–d-fructose 6-phosphate aminotransferase, EC 2.6.1.16) is repressed by growth of B. subtilis in the presence of glucosamine, N-acetylglucosamine, N-propionylglucosamine or N-formylglucosamine. Glucosamine 6-phosphate deaminase and N-acetylglucosamine 6-phosphate deacetylase are induced by N-acetylglucosamine. Amino sugar kinases are induced by glucose, glucosamine and N-acetylglucosamine. The synthesis of glucosamine 1-phosphate acetylase is unaffected by amino sugars. 4. Glucose in the growth medium prevents the induction of glucosamine 6-phosphate deaminase and of N-acetylglucosamine 6-phosphate deacetylase caused by N-acetylglucosamine; glucose also alleviates the repression of glucosamine 6-phosphate synthetase caused by amino sugars. 5. Glucosamine 6-phosphate deaminase increases in bacteria incubated beyond the exponential phase of growth. This increase is prevented by glucose.     

Synthesis and evaluation of an N-acetylglucosamine biosynthesis inhibitor

The structural rationale, synthesis and evaluation of an inhibitor designed to block glucosamine synthesis by competitively inhibiting the action of glutamine: fructose-6-phosphate amidotransferase and subsequently reducing the transformation of any glucosamine-6-phosphate formed to UDP-N-acetylglucosamine are described. The inhibitor 2-acetamido-2,6-dideoxy-6-sulfo-d-glucose (d-glucosamine-6-sulfonate) is an analog of glucosamine-6-phosphate in which the phosphate group in the latter is replaced with a sulfonic acid group. The inhibitor is designed to function by three different modes which together reduce UDP-N-acetylglucosamine synthesis. This reduction was confirmed by evaluating the effect of the inhibitor on bacterial cell-wall synthesis and by demonstrating that it inhibits acetylation of glucosamine-6-phosphate competitively and by acting as a surrogate substrate. Inhibition of glucosamine production or suitably activated glucosamine in bacteria leads to disruption of the peptidoglycan structure, which results in softening, bulging, deformation, fragility and lysis of the cells. These modifications were documented by scanning electron microscopy for bacteria treated with the inhibitor. They were observed for inhibitor concentrations in the 20 mg/mL range for Escherichia coli and Bacillus subtilis and the 5 mg/mL range for Rhizobium trifolii.     

Conversion of N-acetylglucosamine to CMP-N-acetylneuraminic acid in a cell-free system of rat liver

A soluble fraction of rat liver converts glucosamine and N-acetylglucosamine in the presence of ATP and UTP to N-acetylneuraminic acid. This system, when supplemented with CTP, forms CMP-N-acetylneuraminic acid in high yield. Nicotinamide was found to enhance the synthesis of UDP-N-acetylglucosamine and N-acetylneuraminic acid. Kinetic analysis reveals N-acetylglucosamine 6-phosphate, UDP-N-acetylglucosamine, N-acetylmannosamine, N-acetylmannosamine 6-phosphate and N-acetylneuraminic acid 9-phosphate as intermediates. Under certain experimental conditions, however, an epimerisation of N-acetylglucosamine to N-acetylmannosamine was seen.     

Acetate-mediated pH-stat fed-batch cultivation of transconjugant <Emphasis Type="Italic">Enterobacter</Emphasis> sp. BL-2S over-expressing <Emphasis Type="Italic">glmS</Emphasis> gene for excretive production of microbial polyglucosamine PGB-1

A unique cationic polyglucosamine biopolymer PGB-1 comprising more than 95% D-glucosamine was excretively produced from a new bacterial strain Enterobacter sp. BL-2 under acetate-mediated culture conditions. Since the biopolymer PGB-1 could be synthesized from the UDP-N-acetylglucosamine monomer derived from the hexosamine pathway, three glmS, glmM, and glmU genes in the hexosamine pathway were cloned from Enterobacter sp. BL-2, and their molecular structures were elucidated. The cloned glmS, glmM, and glmU genes were reintroduced into the parent strain Enterobacter sp. BL-2 through a conjugative transformation for the overproduction of the biopolymer PGB-1. The biopolymer production increased 1.5-fold in the transconjugant Enterobacter sp. BL-2S over-expressing the first-step glmS gene encoding glucosamine-6-phosphate synthase. The transconjugant Enterobacter sp. BL-2S was cultivated pH-stat fed-batch widely, while intermittently feeding an acetate solution to maintain a constant pH level of 8.0 for 72 h, resulting in 1.15 g/L of the extracellular polyglucosamine biopolymer PGB-1.     

Engineering of an N-acetylneuraminic acid synthetic pathway in Escherichia coli

N-acetylneuraminic acid (NeuAc) has recently drawn much attention owing to its wide applications in many aspects. Besides extraction from natural materials, production of NeuAc was recently focused on enzymatic synthesis and whole-cell biocatalysis. In this study, we designed an artificial NeuAc biosynthetic pathway through intermediate N-acetylglucosamine 6-phosphate in Escherichia coli. In this pathway, N-acetylglucosamine 2-epimerase (slr1975) and glucosamine-6-phosphate acetyltransferase (GNA1) were heterologously introduced into E. coli from Synechocystis sp. PCC6803 and Saccharomyces cerevisiae EBY100, respectively. By derepressing the feedback inhibition of glucosamine-6-phosphate synthase, increasing the accumulation of N-acetylglucosamine and pyruvate, and blocking the catabolism of NeuAc, we were able to produce 1.62 g l^?1 NeuAc in recombinant E. coli directly from glucose. The NeuAc yield reached 7.85 g l^?1 in fed-batch fermentation. This process offered an efficient fermentative method to produce NeuAc in microorganisms using glucose as carbon source and can be optimized for further improvement.     

Characterisation of glutamine fructose-6-phosphate amidotransferase (EC 2.6.1.16) and <Emphasis Type="Italic">N</Emphasis>-acetylglucosamine metabolism in <Emphasis Type="Italic">Bifidobacterium</Emphasis>

Bifidobacterium bifidum, in contrast to other bifidobacterial species, is auxotrophic for N-acetylglucosamine. Growth experiments revealed assimilation of radiolabelled N-acetylglucosamine in bacterial cell walls and in acetate, an end-product of central metabolism via the bifidobacterial d-fructose-6-phosphate shunt. While supplementation with fructose led to reduced N-acetylglucosamine assimilation via the d-fructose-6-phosphate shunt, no significant difference was observed in levels of radiolabelled N-acetylglucosamine incorporated into cell walls. Considering the central role played by glutamine fructose-6-phosphate transaminase (GlmS) in linking the biosynthetic pathway for N-acetylglucosamine to hexose metabolism, the GlmS of Bifidobacterium was characterized. The genes encoding the putative GlmS of B. longum DSM20219 and B. bifidum DSM20082 were cloned and sequenced. Bioinformatic analyses of the predicted proteins revealed 43% amino acid identity with the Escherichia coli GlmS, with conservation of key amino acids in the catalytic domain. The B. longum GlmS was over-produced as a histidine-tagged fusion protein. The purified C-terminal His-tagged GlmS possessed glutamine fructose-6-phosphate amidotransferase activity as demonstrated by synthesis of glucosamine-6-phosphate from fructose-6-phosphate and glutamine. It also possesses an independent glutaminase activity, converting glutamine to glutamate in the absence of fructose-6-phosphate. This is of interest considering the apparently reduced coding potential in bifidobacteria for enzymes associated with glutamine metabolism. S. Foley and E. Stolarczyk contributed equally to this work     

A procedure for the synthesis of uridine-5'-diphospho-N-acetylglucosamine-14C

A complete procedure for the synthesis of 1-¹⁴C-glucosamine-labeled UDP-N-acetylglucosamine is described. Glucosamine is first phosphorylated with ATP and hexokinase to form glucosamine 6-phosphate. This is N-acetylated with acetic anhydride, and the product is converted to UDP-N-acetylglucosamine by incubation with a crude yeast extract. The sugar nucleotide is isolated from the incubation mixture by paper electrophoresis, and purified by paper chromatography.     

The metabolism of d-galactosamine and N-acetyl-d-galactosamine in rat liver

d-[1-¹⁴C]Galactosamine appears to be utilized mainly by the pathway of galactose metabolism in rat liver, as evidenced by the products isolated from the acid-soluble fraction of perfused rat liver. These products were eluted in the following order from a Dowex 1 (formate form) column and were characterized as galactosamine 1-phosphate, sialic acid, UDP-glucosamine, UDP-galactosamine, N-acetylgalactosamine 1-phosphate, N-acetylglucosamine 6-phosphate, UDP-N-acetylglucosamine, UDP-N-acetylgalactosamine and an unidentified galactosamine-containing compound. In addition, [1-¹⁴C]glucosamine was found in the glycogen, an incorporation previously shown to result from the substitution of UDP-glucosamine for UDP-glucose in the glycogen synthetase reaction. Analysis of the [1-¹⁴C]glucosamine-containing disaccharides released from glycogen by β-amylase provided additional evidence that they consist of a mixture of glucose and glucosamine in a 1:1 ratio, but with glucose predominating on the reducing end. UDP-N-acetylgalactosamine was shown to result from the reaction of UTP with N-acetylgalactosamine 1-phosphate in the presence of a rat liver extract.     

Core requirements for glmS ribozyme self-cleavage reveal a putative pseudoknot structure

The glmS ribozyme is a self-cleaving RNA catalyst that resides in the 5′-untranslated region of glmS mRNA in certain bacteria. The ribozyme is specifically activated by glucosamine-6-phosphate (GlcN6P), the metabolic product of the GlmS protein, and is thus proposed to provide a feedback mechanism of riboswitch regulation. Both phylogenetic and biochemical analyses of the glmS ribozyme have established a highly conserved core sequence and secondary structure required for GlcN6P-dependent self-cleavage. However, the high degree of nucleotide conservation offers few clues regarding the higher-order structural organization of the catalytic core. To further investigate core ribozyme structure, minimal ‘consensus-type’ glmS ribozymes that retain GlcN6P-dependent activity were produced. Mutational analyses of consensus-type glmS ribozymes support a model for core ribozyme folding through a pseudoknot structure formed by the interaction of two highly conserved sequence segments. Moreover, GlcN6P-dependent function is demonstrated for bimolecular constructs in which substrate interaction with the ribozyme is minimally comprised of sequence representing that involved in putative pseudoknot formation. These studies suggest that the glmS ribozyme adopts an intricate multi-strand catalytic core through the formation of a pseudoknot structure, and provide a refined model for further considering GlcN6P interaction and GlcN6P-dependent ribozyme function.     

Regulation of amino sugar utilization in Bacillus subtilis by the GntR family regulators,NagR and GamR

In Bacillus subtilis separate sets of genes are implicated in the transport and metabolism of the amino sugars, glucosamine and N‐acetylglucosamine. The genes for use of N‐acetylglucosamine (nagAB and nagP) are found in most firmicutes and are controlled by a GntR family repressor NagR (YvoA). The genes for use of glucosamine (gamAP) are repressed by another GntR family repressor GamR (YbgA). The gamR‐gamAP synton is only found in B. subtilis and a few very close relatives. Although NagR and GamR are close phylogenetically, there is no cross regulation between their operons. GlcN6P prevents all binding of GamR to its targets. NagR binds specifically to targets containing the previously identified dre palindrome but its binding is not inhibited by GlcN6P or GlcNAc6P. GamR‐like binding sites were also found in some other Bacilli associated with genes for use of chitin, the polymer of N‐acetylglucosamine, and with a gene for another GamR homologue (yurK). We show that GamR can bind to two regions in the chi operon of B. licheniformis and that GamR and YurK are capable of heterologous regulation. GamR can repress the B. licheniformis licH‐yurK genes and YurK can repress B. subtilis gamA.     

Schistosoma mansoni: patter of release of secretory products.

Adult Schistosoma mansoni were maintained in vitro for 1 hr with radioactively labeled precursors of protein, glycoprotein, and polysaccharides. The worms were then washed extensively and the supernates analyzed. The precursors N-acetylglucosamine, N-acetylgalactosamine, glucosamine, galactosamine, glucose, leucine, and fucose were incorporated into the worms and both large and small molecular weight products accumulated in the supernatant. For all the precursors except fucose, there was an initial rapid and then slower phase of release for both the large and small molecular weight materials. The amount of label retained by the worms as well as the proportion excreted as large molecular weight material was characteristic for the precursor used. In contrast, the products of fucose were released within 4 to 6 hr and therefore only exhibited the early secretory phase. There was no retention of fucose by the worms. Hydrolysis of large molecular weight products revealed that the N-acetylglucosamine-derived material was incorporated as amino sugars and fucose was incorporated as fucose. Therefore, N-acetylglucosamine and fucose precursors can specifically label secretory glycoproteins of schistosomes in a manner similar to that in mammalian systems.     

Chitinase is an inducible enzyme in Beauveria bassiana

Sterilization of chitin by autoclaving or boiling causes release of d-glucosamine and N-acetylglucosamine from the macromolecule and these solubilized components actually function as the inducers for synthesis of chitinase. The insoluble macromolecule is not an inducer of chitinase since sterilization by dry heat or chloroform will not bring about release of the amino sugars or induction of the enzyme. Free glucosamine, N-acetylglucosamine, and chitobiose are all good inducers of chitinase. Most sustained synthesis of the enzyme occurs in an autoclaved chitin-salts medium.     

Ring-opening mechanism revealed by crystal structures of NagB and its ES intermediate complex

Glucosamine 6-phosphate deaminase (NagB) catalyzes the conversion of d-glucosamine 6-phosphate (GlcN6P) to d-fructose 6-phosphate and ammonia. This reaction is the final step of N-acetylglucosamine utilization and decides its metabolic fate. The enzyme from Streptococcus mutans belongs to the monomeric subfamily of NagB. The crystal structure of the native SmuNagB (NagB from S. mutans) presented here, compared with the structures of its homologs BsuNagB (NagB from Bacillus subtilis) and EcoNagB (NagB from E. coli), implies a conformational change of the ‘lid’ motif in the activation of the monomeric NagB enzyme. We have also captured the enzyme-substrate intermediate complex of the NagB family at low pH, where a remarkable loss of the catalytic activity of SmuNagB was detected. The enzyme-substrate intermediate presents the initial step of the GlcN6P deaminase reaction. The structural evidence (1) supports the α-anomer of GlcN6P as the specific natural substrate of NagB; (2) displays the substrate-binding pocket at the active site; and (3) together with the site-directed mutagenesis studies, demonstrates the ring-opening mechanism of an Asn-His-Glu triad that performs the proton transfer from O1 to O5 to open the sugar ring.     

glmS核糖开关研究进展

glmS核酶是存在于革兰氏阳性细菌中,对葡糖胺-6-磷酸(GlcN6P)的合成起反馈抑制作用的核糖开关。同时,glmS核糖开关是一种位于glmS基因5′非翻译区的自剪切核酶。glmS核糖开关/核酶通过结合GlcN6P后自剪切抑制下游基因glmS的表达。对glmS核糖开关结构和功能的研究将有助于开发新的抗生素作用靶点。本文对glmS核糖开关的结构和功能进行阐述并介绍glmS核糖开关近年来的研究进展和应用。     

A Tn7-Like Transposon Is Present in the glmUS Region of the Obligately Chemoautolithotrophic Bacterium Thiobacillus ferrooxidans

The region downstream of the Thiobacillus ferrooxidans ATCC 33020 atp operon was examined, and the genes encoding N-acetylglucosamine-1-uridyltransferase (glmU) and glucosamine synthetase (glmS) were found. This atpEFHAGDC-glmUS gene order is identical to that of Escherichia coli. The T. ferrooxidans glmS gene was shown to complement E. coli glmS mutants for growth on minimal medium lacking glucosamine. A Tn7-like transposon, Tn5468, was found inserted into the region immediately downstream of the glmS gene in a manner similar to the site-specific insertion of transposon Tn7 within the termination region of the E. coli glmS gene. Tn5468 was sequenced, and Tn7-like terminal repeat sequences as well as several open reading frames which are related to the Tn7 transposition genes tnsA, tnsB, tnsC, and tnsD were found. Tn5468 is the closest relative of Tn7 to have been characterized to date. Southern blot hybridization indicated that a similar or identical transposon was present in three T. ferrooxidans strains isolated from different parts of the world but not in two Thiobacillus thiooxidans strains or a Leptospirillum ferrooxidans strain. Since T. ferrooxidans is an obligately acidophilic autotroph and E. coli is a heterotroph, ancestors of the Tn7-like transposons must have been active in a variety of physiologically different bacteria so that their descendants are now found in bacteria that occupy very different ecological niches.