d-chiro-Inositol Ribophostin: A Highly Potent Agonist of d-myo-Inositol 1,4,5-Trisphosphate Receptors: Synthesis and Biological Activities.

Analogues of the Ca2+-releasing intracellular messenger d-myo-inositol 1,4,5-trisphosphate [1, Ins(1,4,5)P3] are important synthetic targets. Replacement of the α-glucopyranosyl motif in the natural product mimic adenophostin 2 by d-chiro-inositol in d-chiro-inositol adenophostin 4 increased the potency. Similar modification of the non-nucleotide Ins(1,4,5)P3 mimic ribophostin 6 may increase the activity. d-chiro-Inositol ribophostin 10 was synthesized by coupling as building blocks suitably protected ribose 12 with l-(+)-3-O-trifluoromethylsulfonyl-6-O-p-methoxybenzyl-1,2:4,5-di-O-isopropylidene-myo-inositol 11. Separable diastereoisomeric 3-O-camphanate esters of (±)-6-O-p-methoxy-benzyl-1,2:4,5-di-O-isopropylidene-myo-inositol allowed the preparation of 11. Selective trans-isopropylidene deprotection in coupled 13, then monobenzylation gave separable regioisomers 15 and 16. p-Methoxybenzyl group deprotection of 16, phosphitylation/oxidation, then deprotection afforded 10, which was a full agonist in Ca2+-release assays; its potency and binding affinity for Ins(1,4,5)P3R were similar to those of adenophostin. Both 4 and 10 elicited a store-operated Ca2+ current ICRAC in patch-clamped cells, unlike Ins(1,4,5)P3 consistent with resistance to metabolism. d-chiro-Inositol ribophostin is the most potent small-molecule Ins(1,4,5)P3 receptor agonist without a nucleobase yet synthesized.

Kinetics mechanism and regulation of native human hepatic thymidine phosphorylase.

Thymidine phosphorylase (TP; EC 2.4.2.4) catalyzes the reversible phosphorolysis of thymidine, deoxyuridine, and their analogues to their respective nucleobases and 2-deoxy-α-d-ribose-1-phosphate (dRib-1-P). TP is a key enzyme in the pyrimidine salvage pathways. Activity of the enzyme is crucial in angiogenesis, cancer chemotherapy, radiotherapy, and tumor imaging, Nevertheless, a complete set of kinetic parameters has never been reported for any human TP. This study describes the kinetic mechanism and regulation of native human hepatic TP. The liver is a main site of pyrimidine metabolism and contains high levels of TP. Initial velocity and product inhibition studies demonstrated that the basic mechanism of this enzyme is a sequential random bi-bi mechanism. Initial velocity studies showed an intersecting pattern, consistent with substrate-enzyme-co-substrate complex formation, and a binding pattern indicating that the binding of the substrate interferes with the binding of the co-substrate and vice versa. Estimated kinetic parameters were KThymidine = 284 ± 55, KPi = 5.8 ± 1.9, KThymine = 244 ± 69, and KdRib-1-P = 90 ± 33 µM. Thymine was a product activator, but becomes a substrate inhibitor at concentrations eight times higher than its Km. dRib-1-P was a non-competitive product inhibitor of the forward reaction. It bounded better to the Enzyme●Pi complex than the free enzyme, but had better affinity to the free enzyme than the Enzyme●Thymidine complex. In the reverse reaction, dRib-1-P enhanced the binding of thymine. The enhancement of the thymine binding along with the fact that dRib-1-P was a non-competitive product inhibitor suggests the presence of another binding site for dRib-1-P on the enzyme.

Direct Activation of NADPH Oxidase 2 by 2-Deoxyribose-1-Phosphate Triggers Nuclear Factor Kappa B-Dependent Angiogenesis.

AIMS: Deoxyribose-1-phosphate (dRP) is a proangiogenic paracrine stimulus released by cancer cells, platelets, and macrophages and acting on endothelial cells. The objective of this study was to clarify how dRP stimulates angiogenic responses in human endothelial cells. RESULTS: Live cell imaging, electron paramagnetic resonance, pull-down of dRP-interacting proteins, followed by immunoblotting, gene silencing of different NADPH oxidases (NOXs), and their regulatory cosubunits by small interfering RNA (siRNA) transfection, and experiments with inhibitors of the sugar transporter glucose transporter 1 (GLUT1) were utilized to demonstrate that dRP acts intracellularly by directly activating the endothelial NOX2 complex, but not NOX4. Increased reactive oxygen species generation in response to NOX2 activity leads to redox-dependent activation of the transcription factor nuclear factor kappa B (NF-κB), which, in turn, induces vascular endothelial growth factor receptor 2 (VEGFR2) upregulation. Using endothelial tube formation assays, gene silencing by siRNA, and antibody-based receptor inhibition, we demonstrate that the activation of NF-κB and VEGFR2 is necessary for the angiogenic responses elicited by dRP. The upregulation of VEGFR2 and NOX2-dependent stimulation of angiogenesis by dRP were confirmed in excisional wound and Matrigel plug vascularization assays in vivo using NOX2-/- mice. INNOVATION: For the first time, we demonstrate that dRP acts intracellularly and stimulates superoxide anion generation by direct binding and activation of the NOX2 enzymatic complex. CONCLUSIONS: This study describes a novel molecular mechanism underlying the proangiogenic activity of dRP, which involves the sequential activation of NOX2 and NF-κB and upregulation of VEGFR2. Antioxid. Redox Signal. 28, 110-130.

Synthesis and biological evaluation of phosphoramidate prodrugs of two analogues of 2-deoxy-d-ribose-1-phosphate directed to the discovery of two carbasugars as new potential anti-HIV leads.

2-Deoxy-α-d-ribose-1-phosphate is of great interest as it is involved in the biosynthesis and/or catabolic degradation of several nucleoside analogues of biological and therapeutic relevance. However due to the lack of a stabilising group at its 2-position, it is difficult to synthesize stable prodrugs of this compound. In order to overcome this lack of stability, the synthesis of carbasugar analogues of 2-deoxyribose-1-phosphate was envisioned. Herein the preparation of a series of prodrugs of two carbocyclic analogues of 2-deoxyribose-1-phosphate using the phosphoramidate ProTide technology, along with their biological evaluation against HIV and cancer cell proliferation, is reported.

Nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase is phosphorylated in wheat endosperm at serine-404 by an SNF1-related protein kinase allosterically inhibited by ribose-5-phosphate.

Nonphosphorylating glyceraldehyde-3-phosphate dehydrogenase (np-Ga3PDHase) is a cytosolic unconventional glycolytic enzyme of plant cells regulated by phosphorylation in heterotrophic tissues. After interaction with 14-3-3 proteins, the phosphorylated enzyme becomes less active and more sensitive to regulation by adenylates and inorganic pyrophosphate. Here, we acknowledge that in wheat (Triticum aestivum), np-Ga3PDHase is specifically phosphorylated by the SnRK (SNF1-related) protein kinase family. Interestingly, only the kinase present in heterotrophic tissues (endosperm and shoots, but not in leaves) was found active. The specific SnRK partially purified from endosperm exhibited a requirement for Mg(2+) or Mn(2+) (being Ca(2+) independent), having a molecular mass of approximately 200 kD. The kinase also phosphorylated standard peptides SAMS, AMARA, and SP46, as well as endogenous sucrose synthase, results suggesting that it could be a member of the SnRK1 subfamily. Concurrently, the partially purified wheat SnRK was recognized by antibodies raised against a peptide conserved between SnRK1s from sorghum (Sorghum bicolor) and maize (Zea mays) developing seeds. The wheat kinase was allosterically inhibited by ribose-5-phosphate and, to a lesser extent, by fructose-1,6-bisphosphate and 3-phosphoglycerate, while glucose-6-phosphate (the main effector of spinach [Spinacia oleracea] leaves, SnRK1) and trehalose-6-phosphate produced little or no effect. Results support a distinctive allosteric regulation of SnRK1 present in photosynthetic or heterotrophic plant tissues. After in silico analysis, we constructed two np-Ga3PDHase mutants, S404A and S447A, identifying serine-404 as the target of phosphorylation. Results suggest that both np-Ga3PDHase and the specific kinase could be under control, critically affecting the metabolic scenario involving carbohydrates and reducing power partition and storage in heterotrophic plant cells.

Imidazoleacetic acid-ribotide induces depression of synaptic responses in hippocampus through activation of imidazoline receptors.

Imidazole-4-acetic acid-ribotide (IAA-RP), an endogenous agonist at imidazoline receptors (I-Rs), is a putative neurotransmitter/regulator in mammalian brain. We studied the effects of IAA-RP on excitatory transmission by performing extracellular and whole cell recordings at Schaffer collateral-CA1 synapses in rat hippocampal slices. Bath-applied IAA-RP induced a concentration-dependent depression of synaptic transmission that, after washout, returned to baseline within 20 min. Maximal decrease occurred with 10 µM IAA-RP, which reduced the slope of field extracellular postsynaptic potentials (fEPSPs) to 51.2 ± 5.7% of baseline at 20 min of exposure. Imidazole-4-acetic acid-riboside (IAA-R; 10 µM), the endogenous dephosphorylated metabolite of IAA-RP, also produced inhibition of fEPSPs. This effect was smaller than that produced by IAA-RP (to 65.9 ± 3.8% of baseline) and occurred after a further 5- to 8-min delay. The frequency, but not the amplitude, of miniature excitatory postsynaptic currents was decreased, and paired-pulse facilitation (PPF) was increased after application of IAA-RP, suggesting a principally presynaptic site of action. Since IAA-RP also has low affinity for α(2)-adrenergic receptors (α(2)-ARs), we tested synaptic depression induced by IAA-RP in the presence of α(2)-ARs, I(1)-R, or I(3)-R antagonists. The α(2)-AR antagonist rauwolscine (100 nM), which blocked the actions of the α(2)-AR agonist clonidine, did not affect either the IAA-RP-induced synaptic depression or the increase in PPF. In contrast, efaroxan (50 µM), a mixed I(1)-R and α(2)-AR antagonist, abolished the synaptic depression induced by IAA-RP and abolished the related increase in PPF. KU-14R, an I(3)-R antagonist, partially attenuated responses to IAA-RP. Taken together, these data support a role for IAA-RP in modulating synaptic transmission in the hippocampus through activation of I-Rs.

Complexes of the enzyme phosphomannomutase/phosphoglucomutase with a slow substrate and an inhibitor.

Two complexes of the enzyme phosphomannomutase/phosphoglucomutase (PMM/PGM) from Pseudomonas aeruginosa with a slow substrate and with an inhibitor have been characterized by X-ray crystallography. Both ligands induce an interdomain rearrangement in the enzyme that creates a highly buried active site. Comparisons with enzyme-substrate complexes show that the inhibitor xylose 1-phosphate utilizes many of the previously observed enzyme-ligand interactions. In contrast, analysis of the ribose 1-phosphate complex reveals a combination of new and conserved enzyme-ligand interactions for binding. The ability of PMM/PGM to accommodate these two pentose phosphosugars in its active site may be relevant for future efforts towards inhibitor design.

Imidazoline receptors, novel agents and therapeutic potential.

The initial realization that agents containing an imidazoline structure may interact with a distinct class of receptors, has led to a major class of cardiovascular agents, which now has the potential to enter a third generation. There is now general acceptance that there are three main imidazoline receptor classes, the I(1) imidazoline receptor which mediates the sympatho-inhibitory actions to lower blood pressure, the I(2) receptor which is an important allosteric binding site of monoamine oxidase and the I(3) receptor which regulates insulin secretion from pancreatic beta cells. Thus all three represent important targets for cardiovascular research. Interestingly, an I(1)- receptor candidate has been cloned (IRAS, imidazoline receptor antisera selected) which is a homologue of the mouse cell adhesion integrin binding protein Nischarin. There has been range of new agonists and antagonists with very high selectivity for I(1), I(2) and I(3) receptors developed. Three different endogenous ligands have been characterized including agmatine (decarboxylated arginine), a range of beta-carbolines including harman and harmane, and more recently imidazoleacetic acid-ribotide. The imidazoline field has recently seen an enormous diversification with discoveries that I(1) and I(2) receptors also play a role in cell proliferation, regulation of body fat, neuroprotection, inflammation and some psychiatric disorders such as depression. This diversification has continued with the addition of effective agents with imidazoline affinity in the fields of cancer, pain and opioid addiction, stress, cell adhesion, epilepsy and appetite. The imidazoline field has maturated considerably with a range of highly selective leader molecules, candidate receptors and endogenous ligands. We are therefore only at the threshold of an exciting new era as we begin to understand the diverse and complex nature of their function.

Imidazoleacetic acid-ribotide: an endogenous ligand that stimulates imidazol(in)e receptors.

We identified the previously unknown structures of ribosylated imidazoleacetic acids in rat, bovine, and human tissues to be imidazole-4-acetic acid-ribotide (IAA-RP) and its metabolite, imidazole-4-acetic acid-riboside. We also found that IAA-RP has physicochemical properties similar to those of an unidentified substance(s) extracted from mammalian tissues that interacts with imidazol(in)e receptors (I-Rs). ["Imidazoline," by consensus (International Union of Pharmacology), includes imidazole, imidazoline, and related compounds. We demonstrate that the imidazole IAA-RP acts at I-Rs, and because few (if any) imidazolines exist in vivo, we have adopted the term "imidazol(in)e-Rs."] The latter regulate multiple functions in the CNS and periphery. We now show that IAA-RP (i) is present in brain and tissue extracts that exhibit I-R activity; (ii) is present in neurons of brainstem areas, including the rostroventrolateral medulla, a region where drugs active at I-Rs are known to modulate blood pressure; (iii) is present within synaptosome-enriched fractions of brain where its release is Ca(2+)-dependent, consistent with transmitter function; (iv) produces I-R-linked effects in vitro (e.g., arachidonic acid and insulin release) that are blocked by relevant antagonists; and (v) produces hypertension when microinjected into the rostroventrolateral medulla. Our data also suggest that IAA-RP may interact with a novel imidazol(in)e-like receptor at this site. We propose that IAA-RP is a neuroregulator acting via I-Rs.

The charged milieu: a major player in fertilization reactions.

In previous studies, we have found that negatively charged, but not uncharged, amino acids and sugars block sea urchin fertilization. These studies were developed from modeling work in non-living systems using derivatized agarose beads that suggested that charge-charge bonding may control at least some adhesive interactions. In the present study, the effects of positively charged, negatively charged and uncharged molecules were examined in the sea urchin sperm-egg system in over 300 individual trials. The results indicate that depending on the specific molecules utilized, both sperm and egg are exquisitely sensitive to charged but not uncharged molecules and to pH changes in sea water caused by some of the charged molecules. It is shown that egg activation, as well as sperm motility and sperm-egg interactions, can be affected by charged molecules. One compound, fructose-1-phosphate blocked fertilization in S. purpuratus sea urchins but not in Lytechinus pictus sea urchins. These findings indicate that charge alone cannot explain all the results. In this case, the presence of a ketone instead of an aldehyde group indicates that species-specific components may control fertilization reactions. The present study is a comprehensive survey of the effects of charge, pH and molecular structure on the fertilization activation continuum in a model system of sea urchins.

Thymidine phosphorylase induces angiogenesis in vivo and in vitro: an evaluation of possible mechanisms.

1 Thymidine phosphorylase (TP) is elevated in the plasma of cancer patients, and has been implicated in pathophysiological angiogenesis. However, the downstream signals underlying this implication remain obscure. The purpose of the present study was to examine the effects of TP on the neovascularisation response in vitro and in vivo. 2 Both TP and its catalytic product, 2-deoxy-D-ribose-1-phosphate, and downstream 2-deoxy-D-ribose (2-DDR) promoted endothelial tubulogenesis in vitro, and the regeneration of a wounded monolayer of endothelial cells without exerting any mitogenic effect. In vivo, both TP and 2-DDR promoted the development of functional vasculature into an avascular sponge. A TP inhibitor, 6-amino-5-chlorouracil, was able to partially reverse the effects of TP, but had no effect on the 2-DDR-induced angiogenesis. 3 Enhanced monolayer regeneration was observed with TP-cDNA-transfected bladder carcinoma cells. The transfection of TP-cDNA, however, did not confer any proliferative advantage. The regeneration of TP overexpressing cells was associated with a time-dependent expression of the enzyme haeme-oxygenase (HO-1). 4 The present study demonstrates that both TP and its ribose-sugar metabolites induce angiogenesis by mediating a cohesive interplay between carcinoma and endothelial cells. The induction of HO-1 in TP-transfected cells suggests that it could be a possible downstream signal for the angiogenic effects of TP. Furthermore, reducing sugars have been shown to induce oxidative stress, and ribose could be a possible cause for the upregulation of HO-1, which has been implicated in the release of angiogenic factors. Therefore, we postulate that 2-DDR could be mediating the angiogenic effects of TP possibly through an oxidative stress mechanism and additionally getting integrated in the endothelial metabolic machinery.

Mechanisms by which tumor cells and monocytes expressing the angiogenic factor thymidine phosphorylase mediate human endothelial cell migration.

The angiogenic factor thymidine phosphorylase (TP) is highly expressed in many human solid tumors, and the level of its expression is associated with tumor neovascularization, invasiveness, and metastasis and with shorter patient survival time. TP promotes endothelial cell (EC) migration in vitro and angiogenesis in vivo, and these have been linked to its enzymatic activity. The mechanism by which TP stimulates EC migration was investigated using human umbilical vein ECs (HUVECs). TP induced concentration-dependent HUVEC migration, which required a TP gradient and thymidine and which was abrogated by the TP inhibitor CIMU (5-chloro-6(1-imidazolylmethyl)uracil). The chemotactic actions of TP plus thymidine were duplicated by the TP metabolite, 2-deoxyribose-1-phosphate (dR-1-P), and 10-fold more potently by its subsequent metabolite, 2-deoxyribose (2dR). Migration induced by dR-1-P, but not 2dR, was blocked by an alkaline phosphatase inhibitor, suggesting that the actions of dR-1-P first required its conversion to 2dR. In the migration assay, [5'-3H]dThd was metabolized to dR-1-P (96%) and 2dR (3.8%), and a gradient of both metabolites was maintained between the lower and upper chambers over the entire 5-h assay. TP expression in human solid tumors occurs in both tumor epithelial cells and in tumor-associated macrophages. The migration assay was adapted to use TP-transfected carcinoma cells to stimulate HUVEC migration, and they were found to induce more migration than did control vector-transfected cells. Human monocyte cells U937 and THP1, which constitutively expressed high levels of TP, also strongly induced HUVEC migration in the coculture assay. CIMU inhibited tumor-cell and monocyte-induced migration. In contrast, a neutralizing antibody to TP had no effect on cell-stimulated HUVEC migration, even though it completely blocked the migration mediated by purified TP. Thus, the intracellular actions of TP were sufficient to stimulate HUVEC chemotaxis. In contrast to purified TP, when incubated with [5'-3H]-thymidine, cells expressing TP released up to 20-fold more 2dR into the medium than dR-1-P. These studies demonstrate that TP-expressing cells mediate EC migration via the intracellular metabolism of thymidine and subsequent extracellular release of 2dR, which forms a chemotactic gradient.

Acceptor substrate inhibits transketolase competitively with respect to donor substrate.

Two substrates of the transketolase reaction are known to bind with the enzyme according to a ping-pong mechanism [1]. It is shown in this work that high concentrations of ribose-5-phosphate (acceptor substrate) compete with xylulose-5-phosphate (donor substrate), suppressing the transketolase activity (Ki = 3.8 mM). However, interacting with the donor-substrate binding site on the protein molecule, the acceptor substrate, unlike the donor substrate, does not cause any change in the active site of the enzyme. The data are interesting in terms of studying the regulatory mechanism of the transketolase activity and the structure of the enzyme-substrate complex.

Glycation of aspartate aminotransferase and conformational flexibility.

Glycation of proteins alters biological function and changes cellular processes. Our study investigated the conformational changes that accompany glycation using the cardiac aspartate aminotransferase (cAAT). We examined the effects of brief and prolonged exposure of cAAT to glyceraldehyde (Glyc) and ribose 5-phosphate (R5P). When cAAT was briefly incubated (3.5 h) with Glyc (500 microM) or R5P (5 mM) at 37 degrees C, cAAT activity and 1-anilinonaphthalene 8-sulfonate (ANS) binding increased relative to control. After prolonged incubation (64 h) with Glyc (500 microM) or R5P (5 mM) at 37 degrees C, activity and ANS binding decreased relative to control. Furthermore, upon prolonged incubation of cAAT with 500 microM Glyc (14.5 h) or 2 mM R5P (64.25 h) at 37 degrees C, the denaturation curves shifted to the right relative to control. We conclude that upon brief incubation with Glyc and R5P, cAAT exhibited a more open and flexible structure and upon prolonged incubation, a more rigid structure.

Influence of transketolase substrates on its conformation.

Dynamics stimulation of the holotransketolase molecule revealed that the enzyme's conformation in crystal was different from that in solution. It was shown also that dissolved holotransketolase can bind aldose (the acceptor substrate) even in the absence of ketose (the donor substrate). The holotransketolase conformation did not change upon aldose binding unlike in the case of ketose binding/cleavage. Therefore the conformation of a catalytic complex of holotransketolase with an intermediate-i.e., a glycolaldehyde residue formed upon binding and subsequent cleavage of ketose-differed, at least in solution, from the conformation of both the free and aldose-complexed holotransketolase. Some structural peculiarities of the holotransketolase with the intermediate were established by means of molecular dynamics stimulation.

Steady state kinetic model for the binding of substrates and allosteric effectors to Escherichia coli phosphoribosyl-diphosphate synthase.

A steady state kinetic investigation of the P(i) activation of 5-phospho-d-ribosyl alpha-1-diphosphate synthase from Escherichia coli suggests that P(i) can bind randomly to the enzyme either before or after an ordered addition of free Mg(2+) and substrates. Unsaturation with ribose 5-phosphate increased the apparent cooperativity of P(i) activation. At unsaturating P(i) concentrations partial substrate inhibition by ribose 5-phosphate was observed. Together these results suggest that saturation of the enzyme with P(i) directs the subsequent ordered binding of Mg(2+) and substrates via a fast pathway, whereas saturation with ribose 5-phosphate leads to the binding of Mg(2+) and substrates via a slow pathway where P(i) binds to the enzyme last. The random mechanism for P(i) binding was further supported by studies with competitive inhibitors of Mg(2+), MgATP, and ribose 5-phosphate that all appeared noncompetitive when varying P(i) at either saturating or unsaturating ribose 5-phosphate concentrations. Furthermore, none of the inhibitors induced inhibition at increasing P(i) concentrations. Results from ADP inhibition of P(i) activation suggest that these effectors compete for binding to a common regulatory site.

Purification and characterization of the DeoR repressor of Bacillus subtilis.

Transcription of the Bacillus subtilis dra-nupC-pdp operon is repressed by the DeoR repressor protein. The DeoR repressor with an N-terminal His tag was overproduced with a plasmid under control of a phage T5 promoter in Escherichia coli and was purified to near homogeneity by one affinity chromatography step. Gel filtration experimental results showed that native DeoR has a mass of 280 kDa and appears to exist as an octamer. Binding of DeoR to the operator DNA of the dra-nupC-pdp operon was characterized by using an electrophoretic gel mobility shift assay. An apparent dissociation constant of 22 nM was determined for binding of DeoR to operator DNA, and the binding curve indicated that the binding of DeoR to the operator DNA was cooperative. In the presence of low-molecular-weight effector deoxyribose-5-phosphate, the dissociation constant was higher than 1,280 nM. The dissociation constant remained unchanged in the presence of deoxyribose-1-phosphate. DNase I footprinting exhibited a protected region that extends over more than 43 bp, covering a palindrome together with a direct repeat to one half of the palindrome and the nucleotides between them.

Ribose 1-phosphate and inosine activate uracil salvage in rat brain.

The purpose of this study was to determine the mechanism by which inosine activates pyrimidine salvage in CNS. The levels of cerebral inosine, hypoxanthine, uridine, uracil, ribose 1-phosphate and inorganic phosphate were determined, to evaluate the Gibbs free energy changes (deltaG) of the reactions catalyzed by purine nucleoside phosphorylase and uridine phosphorylase, respectively. A deltaG value of 0.59 kcal/mol for the combined reaction inosine+uracil <==> uridine+hypoxanthine was obtained, suggesting that at least in anoxic brain the system may readily respond to metabolite fluctuations. If purine nucleoside phosphorolysis and uridine phosphorolysis are coupled to uridine phosphorylation, catalyzed by uridine kinase, whose activity is relatively high in brain, the three enzyme activities will constitute a pyrimidine salvage pathway in which ribose 1-phosphate plays a pivotal role. CTP, presumably the last product of the pathway, and, to a lesser extent, UTP, exert inhibition on rat brain uridine nucleotides salvage synthesis, most likely at the level of the kinase reaction. On the contrary ATP and GTP are specific phosphate donors.

Identification and characterization of a DeoR-specific operator sequence essential for induction of dra-nupC-pdp operon expression in Bacillus subtilis.

The deoR gene located just upstream the dra-nupC-pdp operon of Bacillus subtilis encodes the DeoR repressor protein that negatively regulates the expression of the operon at the level of transcription. The control region upstream of the operon was mapped by the use of transcriptional lacZ fusions. It was shown that all of the cis-acting elements, which were necessary for full DeoR regulation of the operon, were included in a 141-bp sequence just upstream of dra. The increased copy number of this control region resulted in titration of the DeoR molecules of the cell. By using mutagenic PCR and site-directed mutagenesis techniques, a palindromic sequence located from position -60 to position -43 relative to the transcription start point was identified as a part of the operator site for the binding of DeoR. Furthermore, it was shown that a direct repeat of five nucleotides, which was identical to the 3' half of the palindrome and was located between the -10 and -35 regions of the dra promoter, might function as a half binding site involved in cooperative binding of DeoR to the regulatory region. Binding of DeoR protein to the operator DNA was confirmed by a gel electrophoresis mobility shift assay. Moreover, deoxyribose-5-phosphate was shown to be a likely candidate for the true inducer of the dra-nupC-pdp expression.