OsSQD1 at the crossroads of phosphate and sulfur metabolism affects plant morphology and lipid composition in response to phosphate deprivation.

In phosphate (Pi)-deprived Arabidopsis (Arabidopsis thaliana), phosphatidylglycerol (PG) is substituted by sulfolipid for maintaining Pi homeostasis. Sulfoquinovosyl diacylglycerol1 (AtSQD1) encodes a protein, which catalyzes uridine diphosphate glucose (UDPG) and sulfite (SO32- ) to UDP-sulfoquinovose, which is a key component in the sulfolipid biosynthetic pathway. In this study, a reverse genetics approach was employed to decipher the function of the AtSQD1 homolog OsSQD1 in rice. Differential expressions of OsSQD1 in different tissue and response to -P and -S also detected, respectively. The in vitro protein assay and analysis suggests that OsSQD1 is a UDP-sulfoquinovose synthase. Transient expression analysis showed that OsSQD1 is located in the chloroplast. The analyses of the knockout (ossqd1) and knockdown (Ri1 and Ri2) mutants demonstrated reductions in Pi and total P concentrations, 32 Pi uptake rate, expression levels of Pi transporters and altered developmental responses of root traits, which were accentuated during Pi deficiency. The inhibitory effects of the OsSQD1 mutation were also evident in the development of reproductive tissue. Furthermore, OsSQD1 differently affects lipid composition under different Pi regime affects sulfur (S) homeostasis. Together, the study revealed that OsSQD1 affects Pi and S homeostasis, and lipid composition in response to Pi deprivation.

UDP-sugars activate P2Y14 receptors to mediate vasoconstriction of the porcine coronary artery.

AIMS: UDP-sugars can act as extracellular signalling molecules, but relatively little is known about their cardiovascular actions. The P2Y14 receptor is a Gi/o-coupled receptor which is activated by UDP-glucose and related sugar nucleotides. In this study we sought to investigate whether P2Y14 receptors are functionally expressed in the porcine coronary artery using a selective P2Y14 receptor agonist, MRS2690, and a novel selective P2Y14 receptor antagonist, PPTN (4,7-disubstituted naphthoic acid derivative). METHODS AND RESULTS: Isometric tension recordings were used to evaluate the effects of UDP-sugars in porcine isolated coronary artery segments. The effects of the P2 receptor antagonists suramin and PPADS, the P2Y14 receptor antagonist PPTN, and the P2Y6 receptor antagonist MRS2578, were investigated. Measurement of vasodilator-stimulated phosphoprotein (VASP) phosphorylation using flow cytometry was used to assess changes in cAMP levels. UDP-glucose, UDP-glucuronic acid UDP-N-acetylglucosamine (P2Y14 receptor agonists), elicited concentration-dependent contractions of the porcine coronary artery. MRS2690 was a more potent vasoconstrictor than the UDP-sugars. Concentration dependent contractile responses to MRS2690 and UDP-sugars were enhanced in the presence of forskolin (activator of cAMP), where the level of basal tone was maintained by addition of U46619, a thromboxane A2 mimetic. Contractile responses to MRS2690 were blocked by PPTN, but not by MRS2578. Contractile responses to UDP-glucose were also attenuated by PPTN and suramin, but not by MRS2578. Forskolin-induced VASP-phosphorylation was reduced in porcine coronary arteries exposed to UDP-glucose and MRS2690, consistent with P2Y14 receptor coupling to Gi/o proteins and inhibition of adenylyl cyclase activity. CONCLUSIONS: Our data support a role of UDP-sugars as extracellular signalling molecules and show for the first time that they mediate contraction of porcine coronary arteries via P2Y14 receptors.

Clostridium difficile toxin glucosyltransferase domains in complex with a non-hydrolyzable UDP-glucose analogue.

Clostridium difficile is the leading cause of hospital-acquired diarrhea and pseudomembranous colitis worldwide. The organism produces two homologous toxins, TcdA and TcdB, which enter and disrupt host cell function by glucosylating and thereby inactivating key signalling molecules within the host. As a toxin-mediated disease, there has been a significant interest in identifying small molecule inhibitors of the toxins' glucosyltransferase activities. This study was initiated as part of an effort to identify the mode of inhibition for a small molecule inhibitor of glucosyltransferase activity called apigenin. In the course of trying to get co-crystals with this inhibitor, we determined five different structures of the TcdA and TcdB glucosyltransferase domains and made use of a non-hydrolyzable UDP-glucose substrate. While we were able to visualize apigenin bound in one of our structures, the site was a crystal packing interface and not likely to explain the mode of inhibition. Nevertheless, the structure allowed us to capture an apo-state (one without the sugar nucleotide substrate) of the TcdB glycosyltransferase domain that had not been previously observed. Comparison of this structure with structures obtained in the presence of a non-hydrolyzable UDP-glucose analogue have allowed us to document multiple conformations of a C-terminal loop important for catalysis. We present our analysis of these five new structures with the hope that it will advance inhibitor design efforts for this important class of biological toxins.

Catalytic Promiscuity of O-GlcNAc Transferase Enables Unexpected Metabolic Engineering of Cytoplasmic Proteins with 2-Azido-2-deoxy-glucose.

O-GlcNAc transferase (OGT) catalyzes the installation of N-acetylglucosamine (GlcNAc) O-linked to nucleocytoplasmic proteins (O-GlcNAc) within multicellular eukaryotes. OGT shows surprising tolerance for structural changes in the sugar component of its nucleotide sugar donor substrate, uridine diphosphate N-acetylglucosamine (UDP-GlcNAc). Here, we find that OGT uses UDP-glucose to install O-linked glucose (O-Glc) onto proteins only 25-fold less efficiently than O-GlcNAc. Spurred by this observation, we show that OGT transfers 2-azido-2-deoxy-d-glucose (GlcAz) in vitro from UDP-GlcAz to proteins. Further, feeding cells with per-O-acetyl GlcAz (AcGlcAz), in combination with inhibition or inducible knockout of OGT, shows OGT-dependent modification of nuclear and cytoplasmic proteins with O-GlcAz as detected using microscopy, immunoblot, and proteomics. We find that O-GlcAz is reversible within cells, and an unidentified cellular enzyme exists to cleave O-Glc that can also process O-GlcAz. We anticipate that AcGlcAz will prove to be a useful tool to study the O-GlcNAc modification. We also speculate that, given the high concentration of UDP-Glc within certain mammalian tissues, O-Glc may exist within mammals and serve as a physiologically relevant modification.

Sulfoquinovosyldiacylglycerol has an Essential Role in Thermosynechococcus elongatus BP-1 Under Phosphate-Deficient Conditions.

Anionic lipids, sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG), are major classes of the thylakoid membrane lipids in cyanobacteria and plant chloroplasts. PG is essential for growth and photosynthesis of cyanobacteria, algae and plants, but the requirement for SQDG differs even among cyanobacterial species. Although SQDG and PG can compensate each other in part presumably to maintain proper balance of anionic charge in lipid bilayers, the functional relationship of these lipids is largely unknown. In this study, we inactivated the sqdB gene, encoding a UDP-sulfoquinovose synthase and involved in SQDG biosynthesis, in Thermosynechococcus elongatus BP-1. In wild-type cells, PG accounted for only approximately 3.5 mol% of total membrane lipids, but its content was substantially increased along with complete loss of SQDG in the sqdB mutant. Under phosphate (Pi)-sufficient conditions, the growth rate and PSII activity were slightly lower in sqdB than in wild-type cells. In addition, the formation of PSI trimers and PSII dimers and energy transfer in phycobilisomes were perturbed in the mutant. Under Pi-deficient conditions, the growth of sqdB cells was severely impaired, with a decrease in PSII activity. PG supplementation could partially rescue the defective growth and PSII activity of Pi-deficient sqdB cells but fully recovered the impaired growth of the pgsA mutant of T. elongatus, which is deficient in PG biosynthesis. These data suggest that SQDG has a specific role in the growth and photosynthesis of T. elongatus, which cannot be complemented by PG, particularly under Pi-deficient conditions.

UDP-sulfoquinovose formation by Sulfolobus acidocaldarius.

The UDP-sulfoquinovose synthase Agl3 from Sulfolobus acidocaldarius converts UDP-D-glucose and sulfite to UDP-sulfoquinovose, the activated form of sulfoquinovose required for its incorporation into glycoconjugates. Based on the amino acid sequence, Agl3 belongs to the short-chain dehydrogenase/reductase enzyme superfamily, together with SQD1 from Arabidopsis thaliana, the only UDP-sulfoquinovose synthase with known crystal structure. By comparison of sequence and structure of Agl3 and SQD1, putative catalytic amino acids of Agl3 were selected for mutational analysis. The obtained data suggest for Agl3 a modified dehydratase reaction mechanism. We propose that in vitro biosynthesis of UDP-sulfoquinovose occurs through an NAD(+)-dependent oxidation/dehydration/enolization/sulfite addition process. In the absence of a sulfur donor, UDP-D-glucose is converted via UDP-4-keto-D-glucose to UDP-D-glucose-5,6-ene, the structure of which was determined by (1)H and (13)C-NMR spectroscopy. During the redox reaction the cofactor remains tightly bound to Agl3 and participates in the reaction in a concentration-dependent manner. For the first time, the rapid initial electron transfer between UDP-D-glucose and NAD(+) could be monitored in a UDP-sulfoquinovose synthase. Deuterium labeling confirmed that dehydration of UDP-D-glucose occurs only from the enol form of UDP-4-keto-glucose. The obtained functional data are compared with those from other UDP-sulfoquinovose synthases. A divergent evolution of Agl3 from S. acidocaldarius is suggested.

Efficient enzymatic systems for synthesis of novel α-mangostin glycosides exhibiting antibacterial activity against Gram-positive bacteria.

Two enzymatic systems were developed for the efficient synthesis of glycoside products of α-mangostin, a natural xanthonoid exhibiting anti-oxidant, antibacterial, anti-inflammatory, and anticancer activities. In these systems, one-pot reactions for the synthesis of UDP-α-D-glucose and UDP-α-D-2-deoxyglucose were modified and combined with a glycosyltransferase (GT) from Bacillus licheniformis DSM-13 to afford C-3 and C-6 position modified glucose and 2-deoxyglucose conjugated novel α-mangostin derivatives. α-Mangostin 3-O-ß-D-glucopyranoside, α-mangostin 6-O-ß-D-glucopyranoside, α-mangostin 3,6-di-O-ß-D-glucopyranoside, α-mangostin 3-O-ß-D-2-deoxyglucopyranoside, α-mangostin 6-O-ß-D-2-deoxyglucopyranoside, and α-mangostin 3,6-di-O-ß-D-2-deoxyglucopyranoside were successfully produced in practical quantities and characterized by high-resolution quadruple time-of-flight electrospray ionization-mass spectrometry (HR-QTOF ESI/MS), (1)H and (13)C NMR analyses. In excess of the substrate, the maximum productions of three α-mangostin glucopyranosides (4.8 mg/mL, 86.5 % overall conversion of α-mangostin) and three α-mangostin 2-deoxyglucopyronosides (4.0 mg/mL, 79 % overall conversion of α-mangostin) were achieved at 4-h incubation period. All the α-mangostin glycosides exhibited improved water solubility, and their antibacterial activity against three Gram-positive bacteria Micrococcus luteus, Bacillus subtilis, and Staphylococcus aureus was drastically enhanced by the glucosylation at C-3 position. In this study, diverse glycosylated α-mangostin were produced in significant quantities by using inexpensive starting materials and recycling co-factors within a reaction vessel without use of expensive NDP-sugars in the glycosylation reactions.

Novel vasocontractile role of the P2Y14 receptor: characterization of its signalling in porcine isolated pancreatic arteries.

BACKGROUND AND PURPOSE: The P2Y14 receptor is the newest member of the P2Y receptor family; it is G(i/o) protein-coupled and is activated by UDP and selectively by UDP-glucose and MRS2690 (2-thiouridine-5'-diphosphoglucose) (7-10-fold more potent than UDP-glucose). This study investigated whether P2Y14 receptors were functionally expressed in porcine isolated pancreatic arteries. EXPERIMENTAL APPROACH: Pancreatic arteries were prepared for isometric tension recording and UDP-glucose, UDP and MRS2690 were applied cumulatively after preconstriction with U46619, a TxA2 mimetic. Levels of phosphorylated myosin light chain 2 (MLC2) were assessed with Western blotting. cAMP concentrations were assessed using a competitive enzyme immunoassay kit. KEY RESULTS: Concentration-dependent contractions with a rank order of potency of MRS2690 (10-fold) > UDP-glucose ≥ UDP were recorded. These contractions were reduced by PPTN {4-[4-(piperidin-4-yl)phenyl]-7-[4-(trifluoromethyl)phenyl]-2-naphthoic acid}, a selective antagonist of P2Y14 receptors, which did not affect responses to UTP. Contraction to UDP-glucose was not affected by MRS2578, a P2Y6 receptor selective antagonist. Raising cAMP levels and forskolin, in the presence of U46619, enhanced contractions to UDP-glucose. In addition, UDP-glucose and MRS2690 inhibited forskolin-stimulated cAMP levels. Removal of the endothelium and inhibition of endothelium-derived contractile agents (TxA2, PGF(2α) and endothelin-1) inhibited contractions to UDP glucose. Y-27632, nifedipine and thapsigargin also reduced contractions to the agonists. UDP-glucose and MRS2690 increased MLC2 phosphorylation, which was blocked by PPTN. CONCLUSIONS AND IMPLICATIONS: P2Y14 receptors play a novel vasocontractile role in porcine pancreatic arteries, mediating contraction via cAMP-dependent mechanisms, elevation of intracellular Ca²âº levels, activation of RhoA/ROCK signalling and MLC2, along with release of TxA2, PGF(2α) and endothelin-1.

A cloning vector employing a versatile ß-glucosidase as an indicator for recombinant clones.

A mutant glucosidase, cpGluT, with activity toward chromogenic substrates (X-gal [5-bromo-4-chloro-3-idolyl-ß-d-galactoside] and indican) and a fluorogenic 4-methylumbeliferyl-ß-d-glucopyranoside (MUG) was constructed by replacing the monomeric ß-glucosidase region (E314-N326) with designed multiple cloning sites. When expressed in hosts (lacZ+ and lacZ-), a vector containing the cpGluT produced a colored or fluorescent phenotype according to the substrate supplemented on LB plates without any inducer. cpGluT is readily incorporable into customized vectors and does not require special hosts to detect recombinant plasmids, thereby making screening recombinants more effective and less expensive.

Biosynthesis and functions of the plant sulfolipid.

Higher-plant chloroplast membranes are composed primarily of four characteristic lipids, namely monogalactosyldiacylglycerol, digalactosyldiacylglycerol, sulfoquinovosyldiacylglycerol (SQDG), and phosphatidylglycerol. Among them, SQDG is the only sulfur-containing anionic glycerolipid and is the least prevalent component of photosynthetic membrane lipids. SQDG biosynthesis is mostly mediated by UDP-sulfoquinovose synthase (SQD1) and SQDG synthase (SQD2). Recently, another essential gene for SQDG synthesis, UGP3, was identified using transcriptome coexpression analysis and reverse genetics. UGP3 is a novel plastid UDP-glucose pyrophosphorylase that supplies UDP-glucose to SQD1 in plastids. In Arabidopsis, SQDG is dispensable under normal growth conditions but important in certain environments, particularly phosphate-depleted conditions. The function of SQDG under phosphate-limited growth conditions is highly correlated with the regulation of other plant glycerolipid biosyntheses. This review summarizes recent research defining the mechanism for SQDG biosynthesis and its biological function in higher plants, particularly under phosphate-starved conditions.

Chemoenzymatic synthesis of uridine 5'-diphospho-2-acetonyl-2-deoxy-alpha-D-glucose as C(2)-carbon isostere of UDP-GlcNAc.

2-ketoGlc, which is the C(2)-carbon isostere of GlcNAc, is a novel GlcNAc analogue with a ketone group. The corresponding glycosyltransferase donor substrate, UDP-2-ketoGlc, is necessary for synthesizing 2-ketoGlc-containing molecules and is thus highly important for metabolic polysaccharide remodeling and engineering. We report here the first chemoenzymatic synthesis of UDP-2-ketoGlc using our two-enzyme (NahK and GlmU) system in vitro.

UDP-glucose acting at P2Y14 receptors is a mediator of mast cell degranulation.

UDP-glucose (UDPG), a glycosyl donor in the biosynthesis of carbohydrates, is an endogenous agonist of the G protein-coupled P2Y(14) receptor. RBL-2H3 mast cells endogenously express a P2Y(14) receptor at which UDPG mediates degranulation as indicated by beta-hexosaminidase (HEX) release. Both UDPG and a more potent, selective 2-thio-modified UDPG analog, MRS2690 (diphosphoric acid 1-alpha-d-glucopyranosyl ester 2-[(2-thio)uridin-5''-yl] ester), caused a substantial calcium transient in RBL-2H3 cells, which was blocked by pertussis toxin, indicating the presence of the G(i)-coupled P2Y(14) receptor, supported also by quantitative detection of abundant mRNA. Expression of the closely related P2Y(6) receptor was over 100 times lower than the P2Y(14) receptor, and the P2Y(6) agonist 3-phenacyl-UDP was inactive in RBL-2H3 cells. P2Y(14) receptor agonists also induced [(35)S]GTPgammaS binding to RBL-2H3 cell membranes, and phosphorylation of ERK1/2, P38 and JNK. UDPG and MRS2690 concentration-dependently enhanced HEX release with EC(50) values of 1150+/-320 and 103+/-18nM, respectively. The enhancement was completely blocked by pertussis toxin and significantly diminished by P2Y(14) receptor-specific siRNA. Thus, mast cells express an endogenous P2Y(14) receptor, which mediates G(i)-dependent degranulation and is therefore a potential novel therapeutic target for allergic conditions.

Human P2Y(14) receptor agonists: truncation of the hexose moiety of uridine-5'-diphosphoglucose and its replacement with alkyl and aryl groups.

Uridine-5'-diphosphoglucose (UDPG) activates the P2Y(14) receptor, a neuroimmune system GPCR. P2Y(14) receptor tolerates glucose substitution with small alkyl or aryl groups or its truncation to uridine 5'-diphosphate (UDP), a full agonist at the human P2Y(14) receptor expressed in HEK-293 cells. 2-Thiouracil derivatives displayed selectivity for activation of the human P2Y(14) vs the P2Y(6) receptor, such as 2-thio-UDP 4 (EC(50) = 1.92 nM at P2Y(14), 224-fold selectivity vs P2Y(6)) and its beta-propyloxy ester 18. EC(50) values of the beta-methyl ester of UDP and its 2-thio analogue were 2730 and 56 nM, respectively. beta-tert-Butyl ester of 4 was 11-fold more potent than UDPG, but beta-aryloxy or larger, branched beta-alkyl esters, such as cyclohexyl, were less potent. Ribose replacement of UDP with a rigid North or South methanocarba (bicyclo[3.1.0]hexane) group abolished P2Y(14) receptor agonist activity. alpha,beta-Methylene and difluoromethylene groups were well tolerated at the P2Y(14) receptor and are expected to provide enhanced stability in biological systems. alpha,beta-Methylene-2-thio-UDP 11 (EC(50) = 0.92 nM) was 2160-fold selective versus P2Y(6). Thus, these nucleotides and their congeners may serve as important pharmacological probes for the detection and characterization of the P2Y(14) receptor.

Molecular recognition in the P2Y(14) receptor: Probing the structurally permissive terminal sugar moiety of uridine-5'-diphosphoglucose.

The P2Y(14) receptor, a nucleotide signaling protein, is activated by uridine-5'-diphosphoglucose 1 and other uracil nucleotides. We have determined that the glucose moiety of 1 is the most structurally permissive region for designing analogues of this P2Y(14) agonist. For example, the carboxylate group of uridine-5'-diphosphoglucuronic acid proved to be suitable for flexible substitution by chain extension through an amide linkage. Functionalized congeners containing terminal 2-acylaminoethylamides prepared by this strategy retained P2Y(14) activity, and molecular modeling predicted close proximity of this chain to the second extracellular loop of the receptor. In addition, replacement of glucose with other sugars did not diminish P2Y(14) potency. For example, the [5'']ribose derivative had an EC(50) of 0.24muM. Selective monofluorination of the glucose moiety indicated a role for the 2''- and 6''-hydroxyl groups of 1 in receptor recognition. The beta-glucoside was twofold less potent than the native alpha-isomer, but methylene replacement of the 1''-oxygen abolished activity. Replacement of the ribose ring system with cyclopentyl or rigid bicyclo[3.1.0]hexane groups abolished activity. Uridine-5'-diphosphoglucose also activates the P2Y(2) receptor, but the 2-thio analogue and several of the potent modified-glucose analogues were P2Y(14)-selective.

Novel derivatives of UDP-glucose: concise synthesis and fluorescent properties.

A series of novel 5-substituted UDP-glucose derivatives with interesting fluorescent properties and potential applications as sensors for carbohydrate-active enzymes is reported. An efficient synthesis of the target molecules was developed, centred around the Suzuki-Miyaura reaction of (hetero)arylboronic acids with 5-iodo UDP-glucose. Interestingly, the optimised cross-coupling conditions could also be applied successfully to 5-bromo UMP, but not to 5-bromo UDP-glucose.

A UDP-glucose derivative is required for vacuolar autophagic cell death.

Autophagic cell death in Dictyostelium can be dissociated into a starvation-induced sensitization stage and a death induction stage. A UDP-glucose pyrophosphorylase (ugpB) mutant and a glycogen synthase (glcS) mutant shared the same abnormal phenotype. In vitro, upon starvation alone mutant cells showed altered contorted morphology, indicating that the mutations affected the pre-death sensitization stage. Upon induction of cell death, most of these mutant cells underwent death without vacuolization, distinct from either autophagic or necrotic cell death. Autophagy itself was not grossly altered as shown by conventional and electron microscopy. Exogenous glycogen or maltose could complement both ugpB(-) and glcS(-) mutations, leading back to autophagic cell death. The glcS(-) mutation could also be complemented by 2-deoxyglucose that cannot undergo glycolysis. In agreement with the in vitro data, upon development glcS(-) stalk cells died but most were not vacuolated. We conclude that a UDP-glucose derivative (such as glycogen or maltose) plays an essential energy-independent role in autophagic cell death.

Synthesis of FUDP-N-acetylglucosamine and FUDP-glucose in Saccharomyces cerevisiae cells treated with 5-fluorouracil.

Saccharomyces cerevisiae cells (strain W303-1A) treated with 5-fluorouracil and grown in 2% (fermentative conditions) or in 0.1% glucose (oxidative conditions) accumulated two types of 5-fluoro-UDP-sugars (FUDP-sugars): FUDP-N-acetylglucosamine and FUDP-glucose. No difference was observed in both conditions of culture. The viability of yeast cells on treatment with 5-fluorouracil was also followed. Both FUDP-sugars were partially purified by column chromatography (on Hypersil ODS and Mono Q columns) and characterized by: (i) treatment with alkaline phosphatase (EC 3.1.3.1), snake venom phosphodiesterase (EC 3.1.4.1) and UDP-glucose dehydrogenase (EC 1.1.1.22); (ii) UV spectra; and (iii) matrix-assisted laser desorption/ionization-time of flight mass analysis and 1H-nuclear magnetic resonance spectrometry. The syntheses of both FUDP-sugars were inversely related to the concentration of uracil and directly related to the concentration of 5-fluorouracil in the culture medium. The strain W303-1A, requiring uracil for growth, was useful as a tool to analyze the effect of 5-fluorouracil on nucleotide metabolism.

Structure-activity relationship of uridine 5'-diphosphoglucose analogues as agonists of the human P2Y14 receptor.

UDP-glucose (UDPG) and derivatives are naturally occurring agonists of the Gi protein-coupled P2Y14 receptor, which occurs in the immune system. We synthesized and characterized pharmacologically novel analogues of UDPG modified on the nucleobase, ribose, and glucose moieties, as the basis for designing novel ligands in conjunction with modeling. The recombinant human P2Y14 receptor expressed in COS-7 cells was coupled to phospholipase C through an engineered Galpha-q/i protein. Most modifications of the uracil or ribose moieties abolished activity; this is among the least permissive P2Y receptors. However, a 2-thiouracil modification in 15 (EC50 49 +/- 2 nM) enhanced the potency of UDPG (but not UDP-glucuronic acid) by 7-fold. 4-Thio analogue 13 was equipotent to UDPG, but S-alkylation was detrimental. Compound 15 was docked in a rhodposin-based receptor homology model, which correctly predicted potent agonism of UDP-fructose, UDP-mannose, and UDP-inositol. The hexose moiety of UDPG interacts with multiple H-bonding and charged residues and provides a fertile region for agonist modification.

Cyclic uridine diphosphate glucose: a new pyrimidine analog of cyclic ADP ribose.

Novel compound 1, as the first example of cyclic ADP-ribose analogs containing a pyrimidine residue, was synthesized by a chemical strategy employing a Mitsunobu reaction for the condensation of the glucosyl moiety on protected uridine, and a Matsuda procedure for the cyclization step.

Neurospora crassa FKS protein binds to the (1,3)beta-glucan synthase substrate, UDP-glucose.

The essential fungal cell-wall polymer (1,3)beta-glucan is synthesized by the enzyme (1,3)beta-glucan synthase. This enzyme, which is the target of the echinocandin and pneumocandin families of fungicidal antibiotics, is a complex composed of at least two proteins, Rho1p and Fks1p. Homologs of the yeast FKS1 gene have been discovered in numerous fungi, and existing evidence points to, but has not yet proved, Fks1p being the catalytic subunit of (1,3)beta-glucan synthase. We have purified (1,3)beta-glucan synthase from Neurospora crassa approximately 400-fold enrichment and labeled the substrate-binding protein by using a UDP-glucose analog, 5-azido-[beta-(32)P]-UDP-glucose. UDP-glucose-binding proteins were photo-crosslinked to the substrate analog and identified from SDS-PAGE gels by Quadrupole time-of-flight mass spectrometry by sequencing the tryptic peptides. Two plasma membrane proteins were labeled FKS and H(+)-ATPase. These results suggest that FKS appears to be the substrate-binding subunit of (1,3)beta-glucan synthase.