Glucosamine-6-phosphate Deaminase Research Articles

Novel chemical- and protein-mediated methods for glucosamine detection

We describe to novel approaches for the determination of glucosamine (GlcN). The first approach is based on the chemical derivatization of GlcN with the non-fluorophor 1,3-diphenyl-1,3-propanedione (DPPD), which results in a condensation product with interesting fluorescent properties. The obtained compound was isolated by silica-gel chromatography and its structure elucidated by NMR and mass spectrometry. The second approach is based on a previously undescribed sensitivity of the enzyme glucosamine-6-phosphate deaminase (GPDA) towards GlcN, which resulted in the precipitation of the enzyme. Using a rational enzyme engineering approach and both liquid-based and plate-based screening methods, mutational GPDA variants with significantly improved precipitation properties were identified and characterized. These novel glucosamine detection methods may be a useful addition to the repertoire of currently available glucosamine detection sensors.

Functional characterization of the LdNAGD gene in Leishmania donovani

The N-acetyl glucosamine catabolic pathway has been well established as a critically essential pathway for the survival and pathogenesis of several intracellular pathogens. The intracellular form of Leishmania donovani resides inside the parasitophorous vacuole of macrophages. Recent studies have shown that amino sugars, such as N-acetyl glucosamine, are released from the turnover of host macromolecules, such as glycosaminoglycans, glycoproteins, and proteoglycans, inside the parasitophorous vacuole. Three enzymes, hexokinase (Hxk), N-acetyl glucosamine-6-phosphate deacetylase (NAGD) and glucosamine-6-phosphate deaminase (GND), are sequentially involved in the catabolism of GlcNAc. The Leishmania donovani genome encodes all enzymes of the GlcNAc catabolic pathway. Here, we investigated the role of the GlcNAc catabolic pathway in the proliferation and survival of L. donovani by characterizing the NAGD gene of this pathway. Recombinant LdNAGD displayed deacetylation activity and was localized inside the glycosomes. LdNAGD gene deletion impaired GlcNAc catabolism and was indispensable for the viability of L. donovani in media containing GlcNAc as the sole carbon source. Furthermore, these Δnagd cells showed attenuated virulence in THP-1 cells and a significantly reduced proliferation rate compared to wild type (WT) cells inside THP-1 cells. Our data suggested that LdNAGD is important for the intracellular proliferation of L. donovani and may represent a potential drug target.

Atomic-Level Investigation of Reactant Recognition Mechanism and Thermodynamic Property in Glucosamine 6-Phosphate Deaminase Catalysis.

Glucosamine 6-phosphate deaminase (NagB) influences the direction of N-acetylglucosamine (GlcNAc) metabolism, facilitating the conversion of D-glucosamine 6-phosphate (GlcN6P) to D-fructose 6-phosphate (Fru6P) with the release of ammonia. Here, extensive molecular dynamics simulations combined with various techniques were performed to study the recognition and delivery process of GlcN6P by SmuNagB, due to its guidance of subsequent enzymatic reaction. The key residues Lys194, His130, Arg127, Thr38, and Ser37 stabilize GlcN6P in the active site by hydrogen bond interactions, therein electrostatic and polar solvent effects provide the primary traction. Four delivery channels were identified, with GlcN6P most likely to enter the active site of NagB through a “door” comprising residues 6–10, 122–136, and 222–233. The corresponding mechanism and thermodynamic properties were investigated. An exothermic recognition and delivery process were detected, accompanied by the flipping of GlcN6P and changes in key direct and indirect hydrogen bond interactions, which provide the driving force for the chemical reaction to occur. Furthermore, “the lid motif” was identified that remain open in alkaline condition with different extent of opening at each stage of transfer that induced GlcN6P to move the active site of NagB. The work will assist in the elucidation of the catalytic mechanism of action of NagB, allowing inhibitors to be designed with superior dynamic behavior.

Transcriptomic analysis of gills provides insights into the molecular basis of molting in Chinese mitten crab (Eriocheir sinensis).

Chinese mitten crab (Eriocheir sinensis) is an economically important freshwater aquaculture species and is a model species for research on the mechanism of molting. This study aimed to identify important candidate genes associated with the molting process and to determine the role of gills in the regulation of molting with the help of transcriptomic analysis. The transcriptomes of crabs at different molting stages—postmolt (PoM), intermolt (InM), premolt (PrM) and ecdysis (E)—were de novo assembled to generate 246,232 unigenes with a mean length of 851 bp. A total of 86,634 unigenes (35.18% of the total unigenes) were annotated against reference databases. Significantly upregulated genes were identified in postmolt compared to intermolt (1,475), intermolt compared to premolt (65), premolt compared to ecdysis (1,352), and ecdysis compared to postmolt (153), and the corresponding numbers of downregulated genes were 1,276, 32, 1,573 and 171, respectively. Chitin synthase, endochitinase, chitinase A, chitinase 3, chitinase 6 and chitin deacetylase 1 were upregulated during the postmolt and ecdysis stages, while phosphoglucomutase 3 (PGM3), glucosamine 6-phosphate deaminase (GNPDA) and glucosamine glycoside hydrolase (nagZ) were upregulated during the intermolt and premolt stages compared to the other stages. The upregulated genes were enriched in several lipid-related metabolic pathways, such as “fatty acid elongation”, “glycerophospholipid metabolism” and “sulfur metabolism”. Meanwhile, three signaling pathways, including the “phosphatidylinositol signaling system”, the “calcium signaling pathway” and the “GnRH signaling pathway” were also enriched. Tetraspanin-18, an important effector gene in the lysosomal pathway involved in cell apoptosis, up-regulate with the beginning of molting (in premolt stage) and reach the top in the ecdysis stage, and barely expressed in the intermolt stage. The expression variations in the tetraspanin-18 gene indicated that it may play an important role in the beginning of molting cycle, which might be regulated by the stress of salinity. This study revealed that the gills could participate in chitin degradation, in reestablishment of the exoskeleton and the signaling process. Based on transcriptomic analysis of the gills, we not only explored novel molecular mechanisms of molting in E. sinensis but also acquired foundational genetic data for E. sinensis.

The Nitrogen Regulatory PII Protein (GlnB) and N-Acetylglucosamine 6-Phosphate Epimerase (NanE) Allosterically Activate Glucosamine 6-Phosphate Deaminase (NagB) in Escherichia coli.

Amino sugars are good sources of both ammonia and fructose-6-phosphate, produced by the glucosamine 6-phosphate deaminase, NagB. NagB is known to be allosterically regulated by N-acetylglucosamine 6-phosphate (GlcNAc-6P) and the phosphocarrier protein of the bacterial phosphotransferase system, HPr, in Escherichia coli We provide evidence that NanE, GlcNAc-6P epimerase, and the uridylylated PII protein (U-PII) also allosterically activate NagB by direct protein-protein interactions. NanE is essential for neuraminic acid (NANA) and N-acetylmannosamine (ManNAc) utilization, and PII is known to be a central metabolic nitrogen regulator. We demonstrate that uridylylated PII (but not underivatized PII) activates NagB >10-fold at low concentrations of substrate, whereas NanE increases NagB activity >2-fold. NanE activates NagB in the absence or presence of GlcNAc-6P, but HPr and U-PII activation requires the presence of GlcNAc-6P. Activation of NagB by HPr and uridylylated PII, as well as by NanE and HPr (but not by NanE and U-PII), is synergistic, and the modeling, which suggests specific residues involved in complex formation, provides possible explanations. Specific physiological functions for the regulation of NagB by its three protein activators are proposed. Each regulatory agent is suggested to mediate signal transduction in response to a different stimulus.IMPORTANCE The regulation of amino sugar utilization is important for the survival of bacteria in a competitive environment. NagB, a glucosamine 6-phosphate deaminase in Escherichia coli, is essential for amino sugar utilization and is known to be allosterically regulated by N-acetylglucosamine 6-phosphate (GlcNAc-6P) and the histidine-phosphorylatable phosphocarrier protein, HPr. We provide evidence here that NanE, GlcNAc-6P epimerase, and the uridylylated PII protein allosterically activate NagB by direct protein-protein interactions. NanE is essential for N-acetylneuraminic acid (NANA) and N-acetylmannosamine (ManNAc) utilization, and the PII protein is known to be a central metabolic nitrogen regulator. Regulatory links between carbon and nitrogen metabolism are important for adaptation of metabolism to different growth conditions.

Synthesis, antibacterial activity, and docking studies of some novel N′-benzylidene-2-(2,4,5-trifluorophenyl)acetohydrazides

An improved method was developed for synthesis of 2,4,5-trifluorobenzohydrazide derivatives through condensation of trifluorophenylacetohydrazide with different aryl aldehydes in ethanol with 70% perchloric acid as catalyst. The developed method is efficient in terms of shorter reaction time, easy workup, and purity of final products. We synthesized a total of 25 molecules and screened them for antibacterial activity. Among the synthesized compounds, 4q showed good antibacterial activity against Streptococcus mutans MTCC 497 and Staphylococcus aureus MTCC 737 [50% inhibition concentration (IC50): 30 and 35 µg/mL, respectively], followed by compound 4c (IC50: 45 µg/mL) against Streptococcus mutans MTCC 497 as well as Salmonella enterica MTCC 3858. Molecular docking study employing glucosamine 6-phosphate deaminase (NagB) enzyme (PDB: 2RI1) revealed that the Glide scores of the present ligands played a role. Compounds with 2-NO2, 4-(NCH3)2, and 3,4-di-OH functional groups on the benzylidene ring (4e, i, m) exhibited significant Glide score of −8.2 kcal/mol.

A QM/MM MD study of the pH-dependent ring-opening catalysis and lid motif flexibility in glucosamine 6-phosphate deaminase.

The glucosamine 6-phosphate deaminase (NagB), which catalyzes the conversion of D-glucosamine 6-phosphate (GlcN6P) into d-fructose 6-phosphate (F6P) and ammonia, determines the final metabolic fate of N-acetylglucosamine (GlcNAc). Here using state-of-the-art ab initio QM/MM MD simulations, we have explored the plausible mechanisms for the enzymatic ring-opening of GlcN6P in the basic environment. Two different proton-shuttle mechanisms have been proposed. Calculations show that the protonated state of the amino group in the substrate dominates the concerted and stepwise catalytic pathways and a catalytic triad plays an important role in mediating the proton transfer and the resulting ring-opening process. The free energy barrier for the rate-determining step in the low-energy stepwise reaction is 17.9 kcal mol(-1). In acidic solution, the lid motif prefers a closed state while it always stays in the open state in basic solution upon substrate binding, which is basically dominated by the protonated state of the residue His145.

Evidence That Intracellular Stages of Leishmania major Utilize Amino Sugars as a Major Carbon Source

Intracellular parasites, such as Leishmania spp, must acquire suitable carbon sources from the host cell in order to replicate. Here we present evidence that intracellular amastigote stages of Leishmania exploit amino sugars in the phagolysosome of mammalian macrophages as a source of carbon and energy. L. major parasites are capable of using N-acetylglucosamine and glucosamine as primarily carbon sources and contain key enzymes required for conversion of these sugars to fructose-6-phosphate. The last step in this pathway is catalyzed by glucosamine-6-phosphate deaminase (GND), which was targeted to glycosomes via a canonical C-terminal targeting signal when expressed as a GFP fusion protein. Mutant parasites lacking GND were unable to grow in medium containing amino sugars as sole carbohydrate source and rapidly lost viability, concomitant with the hyper-accumulation of hexosamine-phosphates. Expression of native GND, but not a cytosolic form of GND, in Δgnd parasites restored hexosamine-dependent growth, indicating that toxicity is due to depletion of glycosomal pools of ATP. Non-lethal increases in hexosamine phosphate levels in both Δgnd and wild type parasites was associated with a defect in promastigote metacyclogenesis, suggesting that hexosamine phosphate levels may influence parasite differentiation. Promastigote and amastigote stages of the Δgnd mutant were unable to replicate within macrophages and were either completely cleared or exhibited reduced lesion development in highly susceptible Balb/c mice. Our results suggest that hexosamines are a major class of sugars in the macrophage phagolysosome and that catabolism of scavenged amino sugars is required to sustain essential metabolic pathways and prevent hexosamine toxicity.

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.

The role of glucosamine-6-phosphate deaminase at the early stages of Aspergillus niger growth in a high-citric-acid-yielding medium

Glucosamine-6-phosphate (GlcN6P) deaminase seems to be the main enzyme in Aspergillus niger cells responsible for rapid glucosamine accumulation during the early stages of growth in a high-citric-acid-yielding medium. By determining basic kinetic parameters on the isolated enzyme, a high affinity toward fructose-6-phosphate (Fru6P) was measured, while in the reverse direction the K(m) value for glucosamine-6-phosphate was lower than deaminases from other organisms measured so far. The enzyme characteristics of GlcN6P deaminase suggest it must compete with 6-phosphofructo-1-kinase (PFK1) for the common substrate-Fru6P in A. niger cells. Glucosamine accumulation seems therefore to remove an intermediate from the glycolytic flux, a situation which is reflected in slower citric acid accumulation and a specific growth rate after the germination of spores. When ammonium ions are depleted from the medium, one of the substrates for GlcN6P deaminase becomes limiting and Fru6P can be catabolised by PFK1 which enhances glycolytic flux. Other enzymatic features of GlcN6P deaminase such as pH optima for both aminating and deaminating reactions might play a significant role in rapid glucosamine accumulation during the early phase of fermentation and a slow consumption of aminosugar during the citric-acid-producing phase.

Protein preparation and preliminary X-ray crystallographic analysis of a putative glucosamine 6-phosphate deaminase from Streptococcus mutants.

The SMU.636 protein from Streptococcus mutans is a putative glucosamine 6-phosphate deaminase with 233 residues. The smu.636 gene was PCR-amplified from S. mutans genomic DNA and cloned into the expression vector pET-28a(+). The resultant His-tagged fusion protein was expressed in Escherichia coli and purified to homogeneity in two steps. Crystals of the fusion protein were obtained by the hanging-drop vapour-diffusion method. The crystals diffracted to 2.4 A resolution and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 53.83, b = 82.13, c = 134.70 A.

Site-Directed Fluorescence Labeling Reveals Differences on the R-Conformer of Glucosamine 6-Phosphate Deaminase of Escherichia coli Induced by Active or Allosteric Site Ligands at Steady State

Engineered glucosamine 6-phosphate deaminase of Escherichia coli with unique reactive cysteines at positions 164 or 206 was created by site-directed mutagenesis to monitor the allosteric transition in solution by the fluorescence emission of the bimane or dansyl-amidoethyl groups attached to the indicated residues. The selection of both positions was due to the differential interaction of these residues between T- and R-conformers at the interface of two trimers that form the hexameric structure; in the T-conformer, residue 164 or 206 presents only intrasubunit contacts, but in the R-conformer, new intersubunit contacts are established. As in the wild-type enzyme, fluorescent-labeled mutants show no modification on the allosteric activation of the K-system, only the kcat was reduced to a value of 72 s(-1) (approximately 50% of wild-type). With these preparations, conformational changes were detected by the fluorescence emission spectra at steady state when the active site or the allosteric site ligands were titrated. Despite the similar changes in the fluorescence spectra that were correlated with the induction of the R-state, differences were observed at the maximal change in the fluorescence spectra and in the relative solvent polarities at the positions labeled. These data suggested structural differences in the conformation of the R-state when it is induced from the active site or from the allosteric site, which is not consistent with the two-state structural model proposed by previous crystallographic studies of this enzyme.

Characterization of a Novel Glucosamine-6-Phosphate Deaminase from a Hyperthermophilic Archaeon

A key step in amino sugar metabolism is the interconversion between fructose-6-phosphate (Fru6P) and glucosamine-6-phosphate (GlcN6P). This conversion is catalyzed in the catabolic and anabolic directions by GlcN6P deaminase and GlcN6P synthase, respectively, two enzymes that show no relationship with one another in terms of primary structure. In this study, we examined the catalytic properties and regulatory features of the glmD gene product (GlmD(Tk)) present within a chitin degradation gene cluster in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. Although the protein GlmD(Tk) was predicted as a probable sugar isomerase related to the C-terminal sugar isomerase domain of GlcN6P synthase, the recombinant GlmD(Tk) clearly exhibited GlcN6P deaminase activity, generating Fru6P and ammonia from GlcN6P. This enzyme also catalyzed the reverse reaction, the ammonia-dependent amination/isomerization of Fru6P to GlcN6P, whereas no GlcN6P synthase activity dependent on glutamine was observed. Kinetic analyses clarified the preference of this enzyme for the deaminase reaction rather than the reverse one, consistent with the catabolic function of GlmD(Tk). In T. kodakaraensis cells, glmD(Tk) was polycistronically transcribed together with upstream genes encoding an ABC transporter and a downstream exo-beta-glucosaminidase gene (glmA(Tk)) within the gene cluster, and their expression was induced by the chitin degradation intermediate, diacetylchitobiose. The results presented here indicate that GlmD(Tk) is actually a GlcN6P deaminase functioning in the entry of chitin-derived monosaccharides to glycolysis in this hyperthermophile. This enzyme is the first example of an archaeal GlcN6P deaminase and is a structurally novel type distinct from any previously known GlcN6P deaminase.

Structure and Kinetics of a Monomeric Glucosamine 6-Phosphate Deaminase: MISSING LINK OF THE NagB SUPERFAMILY?

Glucosamine 6-phosphate is converted to fructose 6-phosphate and ammonia by the action of the enzyme glucosamine 6-phosphate deaminase, NagB. This reaction is the final step in the specific GlcNAc utilization pathway and thus decides the metabolic fate of GlcNAc. Sequence analyses suggest that the NagB "superfamily" consists of three main clusters: multimeric and allosterically regulated glucosamine-6-phosphate deaminases (exemplified by Escherichia coli NagB), phosphogluconolactonases, and monomeric hexosamine-6-phosphate deaminases. Here we present the three-dimensional structure and kinetics of the first member of this latter group, the glucosamine-6-phosphate deaminase, NagB, from Bacillus subtilis. The structures were determined in ligand-complexed forms at resolutions around 1.4 Angstroms. BsuNagB is monomeric in solution and as a consequence is active (k(cat) 28 s(-1), K(m(app)) 0.13 mM) without the need for allosteric activators. A decrease in activity at high substrate concentrations may reflect substrate inhibition (with K(i) of approximately 4 mM). The structure completes the NagB superfamily structural landscape and thus allows further interrogation of genomic data in terms of the regulation of NagB and the metabolic fate(s) of glucosamine 6-phosphate.

Why Does Escherichia coli Grow More Slowly on Glucosamine than on N -Acetylglucosamine? Effects of Enzyme Levels and Allosteric Activation of GlcN6P Deaminase (NagB) on Growth Rates

Wild-type Escherichia coli grows more slowly on glucosamine (GlcN) than on N-acetylglucosamine (GlcNAc) as a sole source of carbon. Both sugars are transported by the phosphotransferase system, and their 6-phospho derivatives are produced. The subsequent catabolism of the sugars requires the allosteric enzyme glucosamine-6-phosphate (GlcN6P) deaminase, which is encoded by nagB, and degradation of GlcNAc also requires the nagA-encoded enzyme, N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase. We investigated various factors which could affect growth on GlcN and GlcNAc, including the rate of GlcN uptake, the level of induction of the nag operon, and differential allosteric activation of GlcN6P deaminase. We found that for strains carrying a wild-type deaminase (nagB) gene, increasing the level of the NagB protein or the rate of GlcN uptake increased the growth rate, which showed that both enzyme induction and sugar transport were limiting. A set of point mutations in nagB that are known to affect the allosteric behavior of GlcN6P deaminase in vitro were transferred to the nagB gene on the Escherichia coli chromosome, and their effects on the growth rates were measured. Mutants in which the substrate-induced positive cooperativity of NagB was reduced or abolished grew even more slowly on GlcN than on GlcNAc or did not grow at all on GlcN. Increasing the amount of the deaminase by using a nagC or nagA mutation to derepress the nag operon improved growth. For some mutants, a nagA mutation, which caused the accumulation of the allosteric activator GlcNAc6P and permitted allosteric activation, had a stronger effect than nagC. The effects of the mutations on growth in vivo are discussed in light of their in vitro kinetics.

Transcription regulation is demonstrated for five key enzymes in Giardia intestinalis cyst wall polysaccharide biosynthesis.

The cyst wall of Giardia intestinalis contains proteins and a novel N-acetylgalactosamine (GalNAc) polysaccharide, which is its major constituent. GalNAc is not present in growing trophozoites, but is synthesized during encystment via an inducible pathway of enzymes that produce UDP-GalNAc from fructose 6-phosphate. This report focuses on the regulation of these enzymes and thus the genes for glucosamine 6-phosphate N-acetyltransferase (GNA), phosphoacetylglucosamine mutase (AGM), UDP-N-acetylglucosamine pyrophosphorylase (UAP), and UDP-N-acetylglucosamine 4-epimerase (UAE) were cloned and expressed in Escherichia coli. Each of these expressed enzymes had the predicted activity and was used to generate antibodies. Northern and Western blot analyses demonstrated that both the mRNA and protein levels for all of these enzymes increase during encystment. Nuclear run-on assays of these and the previously analyzed glucosamine 6-phosphate deaminase (GNP; glucosamine 6-P isomerase) showed that all of the genes responsible for UDP-GalNAc synthesis during encystment are induced at the transcription level.

Characterization of testicular mouse glucosamine 6-phosphate deaminase (GNPDA)

Mammalian glucosamine 6-phosphate deaminase (GNPDA) was first detected in hamster spermatozoa. To further elucidate its role, we have cloned mouse GNPDA and produced a polyclonal rabbit anti-GNPDA antibody. This antibody recognized a 33 kDa protein in soluble extracts from mouse brain, liver, kidney, muscle, ovary, testis and sperm. Immunofluorescent analysis of the localization of GNPDA in male reproductive tissue revealed its presence in spermatids and in spermatozoa. In spermatids, GNPDA localized close to the developing acrosome vesicle and in spermatozoa close to the acrosomal region. Following the induction of the acrosome reaction, GNPDA fluorescence in spermatozoa was either reduced or GNPDA was absent. These data suggest that GNPDA might play a role in the acrosome reaction.

The allosteric transition of glucosamine-6-phosphate deaminase: the structure of the T state at 2.3 A resolution.

The allosteric hexameric enzyme glucosamine-6-phosphate deaminase from Escherichia coli catalyses the regulatory step of N-acetylglucosamine catabolism, which consists of the isomerisation and deamination of glucosamine 6-phosphate (GlcN6P) to form fructose 6-phosphate (Fru6P) and ammonia. The reversibility of the catalysis and its rapid-equilibrium random kinetic mechanism, among other properties, make this enzyme a good model for studying allosteric processes. Here we present the structure of P6(3)22 crystals, obtained in sodium acetate, of GlcN6P deaminase in its ligand-free T state. These crystals are very sensitive to X-ray radiation and have a high (78%) solvent content. The activesite lid (residues 162-185) is highly disordered in the T conformer; this may contribute significantly to the free-energy change of the whole allosteric transition. Comparison of the structure with the crystallographic coordinates of the R conformer (Brookhaven Protein Data Bank entry 1 dea) allows us to describe the geometrical changes associated with the allosteric transition as the movement of two rigid entities within each monomer. The active site, located in a deep cleft between these two rigid entities, presents a more open geometry in the T conformer than in the R conformer. The differences in active-site geometry are related to alterations in the substrate-binding properties associated with the allosteric transition. The rigid nature of the two mobile structural units of each monomer seems to be essential in order to explain the observed kinetics of the deaminase hexamer. The triggers for both the homotropic and heterotropic allosteric transitions are discussed and particular residues are assigned to these functions. A structural basis for an entropic term in the allosteric transition is an interesting new feature that emerges from this study.

Tyr254 hydroxyl group acts as a two-way switch mechanism in the coupling of heterotropic and homotropic effects in Escherichia coli glucosamine-6-phosphate deaminase.

The involvement of tyrosine residues in the allosteric function of the enzyme glucosamine 6-phosphate deaminase from Escherichia coli was first proposed on the basis of a theoretical analysis of the sequence and demonstrated by spectrophotometric experiments. Two tyrosine residues, Tyr121 and Tyr254, were indicated as involved in the mechanism of cooperativity and in the allosteric regulation of the enzyme [Altamirano et al. (1994) Eur. J. Biochem. 220, 409-413]. Tyr121 replacement by threonine or tryptophan altered the symmetric character of the T --> R transition [Altamirano et al. (1995) Biochemistry 34, 6074-6082]. From crystallographic data of the R allosteric conformer, Tyr254 has been shown to be part of the allosteric pocket [Oliva et al. (1995) Structure 3, 1323-1332]. Although it is not directly involved in binding the allosteric activator, N-acetylglucosamine 6-phosphate, Tyr 254 is hydrogen bonded through its phenolic hydroxyl to the backbone carbonyl from residue 161 in the neighboring polypeptide chain. Kinetic and binding experiments with the mutant form Tyr254-Phe of the enzyme reveal that this replacement caused an uncoupling of the homotropic and heterotropic effects. Homotropic cooperativity diminished and the allosteric activation pattern changed from one of the K-type in the wild-type deaminase to a mixed K-V pattern. On the other hand, Tyr254-Trp deaminase is kinetically closer to a K-type enzyme and it has a higher catalytic efficiency than the wild-type protein. These results show that the interactions of Tyr254 are fundamental in coupling binding in the active site to events occurring in the allosteric pocket of E. coli glucosamine 6-P deaminase.

Molecularly cloned mammalian glucosamine-6-phosphate deaminase localizes to transporting epithelium and lacks oscillin activity.

Glucosamine-6-phosphate deaminase (GNPDA) catalyzes the conversion of glucosamine-6-phosphate to fructose-6-phosphate, a reaction that under physiological conditions proceeds to the formation of fructose-6-phosphate. Though first identified in mammalian tissues in 1956, the enzyme has not previously been molecularly characterized in mammalian tissues, although a bacterial GNPDA has been cloned. Recently, a protein displaying similarity to bacterial GNPDA was purified and cloned from sperm extract. It was proposed that this protein was the factor, found in sperm extracts, that causes calcium oscillations in cells; thus, the protein was named 'oscillin.' We demonstrate that oscillin is the mammalian form of glucosamine 6-phosphate deaminase by showing that cloned oscillin has a robust GNPDA activity and can account for all such activity in mammalian tissues extracts. In situ hybridization and immunohistochemistry localize GNPDA selectively to tissues with high energy requirements such as the apical zone of transporting epithelia in the proximal convoluted tubules of the kidney and the small intestine; to neurons (but not glia) and especially to nerve terminals in the brain; and to motile sperm. Recombinant GNPDA and GNPDA purified to homogeneity from hamster sperm fail to elevate intracellular calcium when injected into mouse eggs over a wide range of concentrations under conditions in which sperm extracts elicit pronounced calcium oscillations. Thus, the calcium-releasing or oscillin activity of sperm extracts is due to a substance other than GNPDA. Since GNPDA is the sole enzyme linking hexosamine systems with glycolytic pathways, we propose that it provides a source of energy in the form of phosphosugar derived from the catabolism of hexosamines found in glycoproteins, glycolipids, and sialic acid-containing macromolecules. Evidence that GNPDA can regulate hexosamine stores comes from our observation that transfection of GNPDA into HEK-293 cells reduces cellular levels of sialic acid.