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PubMed1993

Solid-state NMR determination of intra- and intermolecular 31P-13C distances for shikimate 3-phosphate and [1-13C]glyphosate bound to enolpyruvylshikimate-3-phosphate synthase.

Rotational-echo, double-resonance (REDOR) 31P NMR was used to obtain internuclear distances for shikimate 3-phosphate (S3P) and N-(phosphonomethyl)-[1-13C]glycine (glyphosate) bound to 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, a 46-kDa enzyme essential for the synthesis of aromatic amino acids in plants and microorganisms. An intermolecular 31P-13C distance of 7.2 A was measured between the phosphate of S3P and the labeled carbon of glyphosate. This means that S3P and glyphosate are in proximity in the binding site of the enzyme. An intramolecular 31P-13C distance of 5.6 A was measured between the phosphonate 31P and the labeled carbon of glyphosate. This distance can be achieved only if glyphosate is completely extended when bound to EPSP synthase.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗
PubMed2002

Di-tert-butyl phosphate as synthon for metal phosphate materials via single-source coordination polymers [M(dtbp)(2)](n) (M = Mn, Cu) and [Cd(dtbp)(2)(H2O)](n) (dtbp-H = (tBuO)(2)P(O)OH).

Reaction of M(OAc)(2).xH(2)O (M = Mn, Cu, or Cd) with di-tert-butyl phosphate (dtbp-H) in a 1:2 molar ratio in methanol followed by slow crystallization of the resultant solid in MeOH/THF medium results in the formation of three new polymeric metal phosphates [M(dtbp)(2)](n)() [M = Mn, 1 (beige); M = Cu, 2 (blue)] and [Cd(dtbp)(2)(H(2)O)](n)(), 3 (colorless)] in good yields. The formation of [Mn(dtbp)(2)](n) (1) proceeds via tetrameric manganese phosphate [Mn(4)(O)(dtbp)(6)] (4), which has been isolated in an analytically pure form. Perfectly air- and moisture-stable compounds 1-4 were characterized with the aid of analytical, thermoanalytical, and spectroscopic techniques. The molecular structures of 1-3 were further established by single-crystal X-ray diffraction studies. Crystal data for 1: C(32)H(72)Mn(2)O(16)P(4), monoclinic, P2(1)/c, a = 19.957(4) A, b = 13.419(1) A, c = 18.083(2) A, beta = 91.25(2) degrees, Z = 4. Crystal data for 2: C(16)H(36)CuO(8)P(2), orthorhombic, Pccn, a = 23.777(2) A, b = 10.074(1) A, c = 10.090(1) A, Z = 4. Crystal data for 3: C(48)H(114)Cd(3)O(27)P(6), triclinic, P1, a = 12.689(3) A, b = 14.364(3) A, c = 22.491(5) A, alpha = 84.54(3) degrees, beta = 79.43(3) degrees, gamma = 70.03(3) degrees, Z = 2. The diffraction studies reveal three different structural forms for the three compounds investigated, each possessing a one-dimensional coordination polymeric structure. While alternating triple and single dtbp bridges are found between the adjacent Mn(2+) ions in 1, uniform double dtbp bridges across the adjacent Cu(2+) ions are present in 2. The cadmium ions in the structure of 3 are pentacoordinated. Thermal analysis (TGA and DSC) indicates that compounds 1-3 convert to the corresponding crystalline metaphosphate materials M(PO(3))(2), in each case at temperatures below 500 degrees C. Similarly, the thermal decomposition of 4 results in the formation of Mn(PO(3))(3) and Mn(2)P(2)O(7). The final materials obtained by independent thermal decomposition of bulk samples have been characterized using IR spectroscopic, powder diffraction, and N(2) adsorption studies.

Journal Article↗
PubMed1997

Carboxy and Phosphate Esters Cleavage with Mono- and Dinuclear Zinc(II) Macrocyclic Complexes in Aqueous Solution. Crystal Structure of [Zn(2)L1(&mgr;-PP)(2)(MeOH)(2)](ClO(4))(2) (L1 = [30]aneN(6)O(4), PP(-) = Diphenyl Phosphate).

The ligand [30]aneN(6)O(4) (L1) binds two Zn(II) in aqueous solution. The stability constants of the L1 complexes have been determined at 308.1 K by means of potentiometric measurements. Dinuclear monohydroxo [Zn(2)L1OH](3+) and dihydroxo [Zn(2)L1(OH)(2)](2+) complexes are formed in aqueous solution from neutral to alkaline pH. The kinetics of promoted hydrolysis of p-nitrophenyl acetate (NA) was studied. Both hydroxo species promote p-nitrophenyl acetate (NA) hydrolysis at 298.1 K with second-order kinetics. The activity of these species in NA hydrolysis is similar to that found for the mononuclear L2-Zn-OH(+) complex (L2 = [15]aneN(3)O(2)), indicating that the hydrolytic process takes place via a simple bimolecular mechanism. The hydrolysis rate of bis(p-nitrophenyl) phosphate (BNP) was measured in aqueous solution at 308.1 K in the presence of the L1and L2 zinc complexes. The hydrolysis rate of BNP is increased almost 10-fold by the dinuclear [Zn(2)L1(OH)(2)](2+) complex with respect to the mononuclear L2-Zn-OH(+) one. This result indicates a cooperative role of the two metals in the hydrolytic mechanism. A bridging coordination of the phosphate ester to the two Zn(II) ions can be suggested. The crystal structure of [Zn(2)L1(&mgr;-PP)(2)(MeOH)(2)](ClO(4))(2) (PP(-) = diphenyl phosphate) (space group P&onemacr;, a = 10.681(5) Å, b = 12.042(1) Å, c = 13.191(3) Å, alpha = 74.63(2) degrees, beta = 71.74(3) degrees, gamma = 68.41(2) degrees, V = 1476.4(8) Å(3), Z = 1, R = 0.0472, R(w)(2) = 0.1166) strongly supports this hypothesis, since in the [Zn(2)L1(&mgr;-PP)(2)(MeOH)(2)](2+) cation the diphosphate anions bridge the two metals. The dinuclear Zn(II) complexes of L1 provide a simple model system for hydrolytic dizinc enzymes.

Journal Article↗
PubMed1984

Bis(m-nitrophenyl) and bis(p-nitrophenyl) esters and the phosphorodiamidate of thymidine 5'-phosphate as potential sources of intracellular thymidine 5'-phosphate in mouse cells in culture.

Thymidine 5'-phosphate (TMP) derivatives with masked phosphate groups were synthesized in tritiated form from [methyl-3H]thymidine. They were of interest as models for 5' nucleotide derivatives that might be able to permeate mammalian cells and then liberate intracellular antimetabolite 5' nucleotides by loss of the masking groups. Mouse L fibroblasts were grown in vitro in the presence of 1 mM 5'-amino-5'-deoxythymidine, which was found to suppress greater than 99% of cellular thymidine kinase activity while inhibiting the rate of cell division by only 30%. The TMP derivatives were less effective than thymidine in labeling the deoxyribonucleic acid (DNA) of the L cells. The labeling was inhibited 95-99% by 5'-amino-5'-deoxythymidine, indicating that it represented incorporation into DNA of [3H]thymidine formed from degradation of the test compounds. No evidence was obtained that the compounds acted as sources of intracellular TMP by cell permeation followed by loss of phosphate blocking groups. Similar studies yielded no evidence that the bis(m-nitrophenyl) ester of TMP produced intracellular TMP by that route in the LM(TK-) strain of L cells that are genetically deficient in thymidine kinase.

Amides↗
PubMed2005

Synthesis, structure, and acidic properties of MCM-41 functionalized with phosphate and titanium phosphate groups.

Mesoporous molecular sieves Si-MCM-41 (purely siliceous) and Ti-MCM-41 (partly covered with a surface layer of TiO2) were functionalized with phosphate groups by treatment with POCl3 (denoted -MCM-41(P)and Ti-MCM-41(P), respectively). With the use of TEM, X-ray diffraction, and N2 adsorption, it was shown that the initial hexagonal structure, the high specific surface area, and porosity are retained in the functionalized materials but are not as good as in the starting materials. 1H MAS NMR and 31P MAS NMR revealed that the surface of Si-MCM-41(P) consists of silicon phosphate and pyrophosphate species. That of Ti-MCM-41(P) additionally contains titanium dihydro-, hydro-, and pyrophosphate species, the latter being predominant. TPD of adsorbed ammonia for Si-MCM-41(P) and Ti-MCM-41(P) showed that functionalization leads to the creation of moderate and strong acid sites. A combination of mesoporous structure with acidic properties makes the MCM-41 functionalized with phosphate groups promising for use as solid acid catalysts.

Journal Article↗
PubMed1970

The role of nicotinamide-adenine dinucleotide phosphate-dependent malate dehydrogenase and isocitrate dehydrogenase in the supply of reduced nicotinamide-adenine dinucleotide phosphate for steroidogenesis in the superovulated rat ovary.

1. Superovulated rat ovary was found to contain high activities of NADP-malate dehydrogenase and NADP-isocitrate dehydrogenase. The activity of each enzyme was approximately four times that of glucose 6-phosphate dehydrogenase and equalled or exceeded the activities reported to be present in other mammalian tissues. Fractionation of a whole tissue homogenate of superovulated rat ovary indicated that both enzymes were exclusively cytoplasmic. The tissue was also found to contain pyruvate carboxylase (exclusively mitochondrial), NAD-malate dehydrogenase and aspartate aminotransferase (both mitochondrial and cytoplasmic) and ATP-citrate lyase (exclusively cytoplasmic). 2. The kinetic properties of glucose 6-phosphate dehydrogenase, NADP-malate dehydrogenase and NADP-isocitrate dehydrogenase were determined and compared with the whole-tissue concentrations of their substrates and NADPH; NADPH is a competitive inhibitor of all three enzymes. The concentrations of glucose 6-phosphate, malate and isocitrate in incubated tissue slices were raised at least tenfold by the addition of glucose to the incubation medium, from the values below to values above the respective K(m) values of the dehydrogenases. Glucose doubled the tissue concentration of NADPH. 3. Steroidogenesis from acetate is stimulated by glucose in slices of superovulated rat ovary incubated in vitro. It was found that this stimulatory effect of glucose can be mimicked by malate, isocitrate, lactate and pyruvate. 4. It is concluded that NADP-malate dehydrogenase or NADP-isocitrate dehydrogenase or both may play an important role in the formation of NADPH in the superovulated rat ovary. It is suggested that the stimulatory effect of glucose on steroidogenesis from acetate results from an increased rate of NADPH formation through one or both dehydrogenases, brought about by the increases in the concentrations of malate, isocitrate or both. Possible pathways involving the two enzymes are discussed.

Acetates↗
PubMed1971

The active centre of rabbit muscle triose phosphate isomerase. The site that is labelled by glycidol phosphate.

1. Glycidol (2,3-epoxypropanol) phosphate is a specific irreversible inhibitor of rabbit muscle triose phosphate isomerase (EC 5.3.1.1); the site of attachment has now been studied. 2. The labelled enzyme was digested with pepsin and a modified peptide isolated. The sequence of the peptide is: Ala-Tyr-Glu-Pro-Val-Trp. 3. It is the glutamic acid residue in this peptide that is labelled: the peptide is thus a gamma-glutamyl ester derived from glycerol phosphoric acid. The same site is labelled by a mixture of glycidol and inorganic phosphate. 4. Kinetic and stereochemical features of these reactions are discussed.

1-Propanol↗
PubMed1972

Active-site labelling of triose phosphate isomerase. The reaction of bromohydroxyacetone phosphate with a unique glutamic acid residue and the migration of the label to tyrosine.

Triose phosphate isomerase from chicken muscle reacts stoicheiometrically with the active-site-directed irreversible inhibitor bromohydroxyacetone phosphate with concomitant loss of all catalytic activity. The primary site of attachment has been shown to be a unique glutamic acid residue in the sequence Ala-Tyr-Glu-Pro-Val-Trp. Unless the inhibitor-enzyme bond is stabilized by reduction of the C-2 carbonyl group with borohydride, the phosphate group is lost and the label migrates to the adjacent tyrosine residue. It is suggested that the gamma-carboxylate group of the glutamic acid residue may be the base responsible for primary proton abstraction from substrate in the catalysis. The failure of this reagent specifically to inactivate either muscle or yeast aldolase, and the use of the reagent in preparing isomerase-free glycolytic enzymes, is discussed.

Acetone↗
PubMed1973

A membrane-bound activity catalysing phosphatidylinositol breakdown to 1,2-diacylglycerol, D-myoinositol 1:2-cyclic phosphate an D-myoinositol 1-phosphate. Properties and subcellular distribution in rat cerebral cortex.

1. Breakdown of phosphatidylinositol was studied in homogenates and subcellular fractions of rat cerebral cortex by using both membrane-bound and externally added [(32)P]phosphatidylinositol as substrate. 2. In the presence of deoxycholate breakdown followed first-order kinetics at low substrate concentrations ([unk]1mm) and zero-order kinetics at higher concentrations (6-9mm). 3. Maximum breakdown by cerebral-cortex homogenates was approximately 0.5mumol/h per mg of protein and occurred at pH7.0 in the presence of 8mm-phosphatidylinositol, 2mm-CaCl(2) and 2mg of deoxycholate/ml. Activity was abolished by 1mm-ethanedioxybis(ethylamine)tetra-acetate. 4. The products of phosphatidylinositol breakdown were 1,2-diacylglycerol and a mixture of d-myoinositol 1:2-cyclic phosphate (55%) and d-myoinositol 1-phosphate (45%). The two phosphate esters appeared to be produced together and in constant proportions. 5. Some 51% of the activity was particle-bound, with the highest activities in small nerve endings, microsomal material and two synaptic membrane fractions (fractions Mic(20), Mic(100), M(1) 1.0 and M(1) 0.9 respectively), all of which were also rich in acetylcholinesterase and which have been shown to be rich in other surface-membrane enzymes. Much of the particle-bound activity therefore appears to be present in cerebral-cortex plasma membranes. 6. The results are discussed in relation to previously described soluble activities that catalyse the same reaction, and to a possible role of the membrane-bound enzyme in enhanced phosphatidylinositol turnover in externally stimulated cells.

Acetylcholinesterase↗
PubMed1976

Dehydrogenation of the phosphonate analogue of glucose 6-phosphate by glucose 6-phosphate dehydrogenase.

6,7 -Dideoxy-alpha-D-gluco-heptose 7-phosphonic acid, the isosteric phosphonate analogue of glucose 6-phosphate, was synthesized in six steps from the readily available precursor benzyl 4,6-O-benzylidene-alpha-D-glucopyranoside. The analogue is a substrate for yeast glucose 6-phosphate dehydrogenase, showing Michaelis-Menten kinetics at pH7.5 and 8.0. At both pH values the Km values of the analogue are 4-5 fold higher and the values approx. 50% lower than those of the natural substrate. The product of enzymic dehydrogenation of the phosphonate analogue at pH8.5 is itself a substrate for gluconate 6-phosphate dehydrogenase.

Deoxy Sugars↗
PubMed2003

Ceramide 1-phosphate increases intracellular free calcium concentrations in thyroid FRTL-5 cells: evidence for an effect mediated by inositol 1,4,5-trisphosphate and intracellular sphingosine 1-phosphate.

Sphingolipid (SP) derivatives have diverse effects on the regulation of intracellular free calcium concentrations ([Ca2+]i) in a multitude of non-excitable cells. In the present investigation, the effect of C2-ceramide 1-phosphate (C1P) on [Ca2+]i was investigated in thyroid FRTL-5 cells. C1P evoked a concentration-dependent increase in [Ca2+]i, both in a calcium-containing and a calcium-free buffer. A substantial part of the C1P-evoked increase in [Ca2+]i was due to calcium entry. The effect of C1P was attenuated by overnight pretreatment of the cells with pertussis toxin. Similar results were obtained with C8-ceramide 1-phosphate, although the magnitude of the responses was smaller than with C1P. The phospholipase C inhibitor U73122 attenuated the effect of C1P. C1P invoked a small, but significant, increase in inositol 1,4,5-trisphosphate (IP3). However, the effect of C1P on [Ca2+]i was inhibited by neither Xestospongin C, 2-aminoethoxydiphenylborate nor neomycin. C1P mobilized calcium from an IP3-sensitive calcium store, as C1P did not increase [Ca2+]i in cells pretreated with thapsigargin. The effect of C1P on [Ca2+]i was potently attenuated by dihydrosphingosine and dimethylsphingosine, two inhibitors of sphingosine kinase, but not by the inactive SP-derivative N -acetyl sphingosine. Stimulating the cells with C1P evoked an increase in the production of intracellular sphingosine 1-phosphate. C1P did not modulate DNA synthesis or the forskolin-evoked production of cAMP. The results indicate that C1P may be an important SP participating in cellular signalling.

Animals↗
PubMed1989

2-Aminoethylarsonic acid as an analogue of ethanolamine phosphate. Endowment of ethanolamine-phosphate cytidylyltransferase with CTP pyrophosphatase activity.

2-Aminoethylarsonic acid was tested for its ability to act as a substrate for ethanolamine-phosphate cytidylytransferase as a cytidylyl acceptor in place of ethanolamine phosphate. The expected product, like all mixed anhydrides of arsonic acids, should hydrolyse spontaneously with regeneration of the substrate analogue and CMP formation; such CMP production was observed. The limiting velocity with aminoethylarsonic acid is about 90% that with ethanolamine phosphate, and the Michaelis constant is below 20 mM.

Arsenicals↗
PubMed1999

A type 5 acid phosphatase gene from Arabidopsis thaliana is induced by phosphate starvation and by some other types of phosphate mobilising/oxidative stress conditions.

Low phosphorous availability, a common condition of many soils, is known to stimulate phosphatase activity in plants; however, the molecular details of this response remain mostly unknown. We purified and sequenced the N-terminal region of a phosphate starvation induced acid phosphatase (AtACP5) from Arabidopsis thaliana, and cloned its cDNA and the corresponding genomic DNA. The nucleotide sequence of the cDNA predicted that AtACP5 is synthesised as a 338 amino acid-long precursor with a signal peptide. AtACP5 was found to be related to known purple acid phosphatases, especially to mammal type 5 acid phosphatases. Other similarities with purple acid phosphatases, which contain a dinuclear metal centre, include the conservation of all residues involved in metal ligand binding and resistance to tartrate inhibition. In addition, AtACP5, like other type 5 acid phosphatases, displayed peroxidation activity. Northern hybridisation experiments, as well as in situ glucuronidase (GUS) activity assays on transgenic plants harbouring AtACP5:GUS translational fusions, showed that AtACP5 is not only responsive to phosphate starvation but also to ABA and salt stress. It is also expressed in senescent leaves and during oxidative stress induced by H2O2, but not by paraquat or salicylic acid. Given its bifunctionality, as it displays both phosphatase and peroxidation activity, we propose that AtACP5 could be involved in phosphate mobilisation and in the metabolism of reactive oxygen species in stressed or senescent parts of the plant.

Acid Phosphatase↗
PubMed2006

Identification of class 2 1-deoxy-D-xylulose 5-phosphate synthase and 1-deoxy-D-xylulose 5-phosphate reductoisomerase genes from Ginkgo biloba and their transcription in embryo culture with respect to ginkgolide biosynthesis.

Diterpenoid ginkgolides having potent platelet-activating factor antagonist activity are major active ingredients of ginkgo extract. Class 2-type 1-deoxy-D-xylulose 5-phosphate synthase (GbDXS2) and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (GbDXR), the first two enzymes in 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway, operating in the earlier step of ginkgolide biosynthesis, were cloned from embryonic roots of Ginkgo biloba through a homology-based polymerase chain reaction for role assessment of the enzymes. Plasmids harboring each gene rescued the respective knockout E. coli mutants. The levopimaradiene synthase gene (LPS), responsible for the first committed step in ginkgolide biosynthesis, and GbDXS2 were transcribed exclusively in embryonic root, suggesting a specific role of GbDXS2 in ginkgolide biosynthesis. GbDXR retained a higher transcription level in roots than in leaves, whereas class 1 DXS (GbDXS1) showed 30 to 50 % higher level in leaves. Ginkgolides and bilobalide were found both in leaves and roots from an earlier stage of the embryo culture. Exclusive transcription of ginkgolide biosynthesis-specific LPS and GbDXS2 in roots and the appearance of ginkgolides in leaves was consistent with translocation of the compounds from roots to leaves.

Aldose-Ketose Isomerases↗
PubMed2003

Immediate and delayed effects of antenatal corticosteroids on fetal heart rate: a randomized trial that compares betamethasone acetate and phosphate, betamethasone phosphate, and dexamethasone.

OBJECTIVE: The purpose of this study was to evaluate the role of different formulations and different administration kinetics of antenatal corticosteroid therapy on fetal heart rate. STUDY DESIGN: One hundred five patients who were at high risk for preterm delivery were assigned randomly to receive two injections of betamethasone acetate + phosphate, four injections of betamethasone phosphate, or four injections of dexamethasone phosphate. Computerized fetal heart rate was recorded daily through day 4. RESULTS: The three formulations did not differ in their effect on fetal heart rate, which varied significantly as a function of the time of injection. During treatment (day 0-day 1), fetal heart rate variability increased (+9%, P <.05), as did the number of fetal movements felt by the mother (+60% at day 0, P <.001). After treatment (day 2-day 3), variability fell significantly (-14%, P <.01), as did accelerations (-35% at day 2, P <.01). No modifications were still detectable on day 4. CONCLUSION: Antenatal corticotherapy is responsible for two different phases of fetal heart rate modifications that do not vary according to the corticosteroid or the dosage regimen.

Betamethasone↗
PubMed2000

Mechanisms for high affinity mannose 6-phosphate ligand binding to the insulin-like growth factor II/mannose 6-phosphate receptor.

The two mannose 6-phosphate (Man-6-P) binding domains of the insulin-like growth factor II/mannose 6-phosphate receptor (Man-6-P/IGF2R), located in extracytoplasmic repeats 1-3 and 7-9, are capable of binding Man-6-P with low affinity and glycoproteins that contain more than one Man-6-P residue with high affinity. High affinity multivalent ligand binding sites could be formed through two possible mechanisms: the interaction of two Man-6-P binding domains within one Man-6-P/IGF2R molecule or by receptor oligomerization. To discriminate between these mechanisms, truncated FLAG epitope-tagged Man-6-P/IGF2R constructs, containing one or both of the Man-6-P binding domains, were expressed in 293T cells, and characterized for binding of pentamannose phosphate-bovine serum albumin (PMP-BSA), a pseudoglycoprotein bearing multiple Man-6-P residues. A construct containing all 15 repeats of the Man-6-P/IGF2R extracytoplasmic domain bound PMP-BSA with the same affinity as the full-length receptor (K(d) = 0.54 nm) with a curvilinear Scatchard plot. The presence of excess unlabeled PMP-BSA increased the dissociation rate of pre-formed (125)I-PMP-BSA/receptor complexes, suggesting negative cooperativity in multivalent ligand binding and affirming the role of multiple Man-6-P/IGF2R binding domains in forming high affinity binding sites. Truncated receptors containing only one Man-6-P binding domain and mutant receptor constructs, containing an Arg(1325) --> Ala mutation that eliminates binding to the repeats 7-9 binding domain, formed high affinity PMP-BSA binding, but with reduced stoichiometries. Collectively, these observations suggest that alignment of Man-6-P binding domains of separate Man-6-P/IGF2R molecules is responsible for the formation of high affinity Man-6-P binding sites and provide functional evidence for Man-6-P/IGF2R oligomerization.

Cell Line↗
PubMed2000

Dissection of the bifunctional Escherichia coli N-acetylglucosamine-1-phosphate uridyltransferase enzyme into autonomously functional domains and evidence that trimerization is absolutely required for glucosamine-1-phosphate acetyltransferase activity and cell growth.

The bifunctional N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) enzyme catalyzes both the acetylation of glucosamine 1-phosphate and the uridylation of N-acetylglucosamine 1-phosphate, two subsequent steps in the pathway for UDP-N-acetylglucosamine synthesis in bacteria. In our previous work describing its initial characterization in Escherichia coli, we proposed that the 456-amino acid (50.1 kDa) protein might possess separate uridyltransferase (N-terminal) and acetyltransferase (C-terminal) domains. In the present study, we confirm this hypothesis by expression of the two independently folding and functional domains. A fragment containing the N-terminal 331 amino acids (Tr331, 37.1 kDa) has uridyltransferase activity only, with steady-state kinetic parameters similar to the full-length protein. Further deletion of 80 amino acid residues at the C terminus results in a 250-amino acid fragment (28.6 kDa) still exhibiting significant uridyltransferase activity. Conversely, a fragment containing the 233 C-terminal amino acids (24.7 kDa) exhibits acetyltransferase activity exclusively. None of these individual domains could complement a chromosomal glmU mutation, indicating that each of the two activities is essential for cell viability. Analysis of truncated GlmU proteins by gel filtration further localizes regions of the protein involved in its trimeric organization. Interestingly, overproduction of the truncated Tr331 protein in a wild-type strain results in a rapid depletion of endogenous acetyltransferase activity, an arrest of peptidoglycan synthesis and cell lysis. It is shown that the acetyltransferase activity of the full-length protein is abolished once trapped within heterotrimers formed in presence of the truncated protein, suggesting that this enzyme activity absolutely requires a trimeric organization and that the catalytic site involves regions of contact between adjacent monomers. Data are discussed in connection with the recently obtained crystal structure of the truncated Tr331 protein.

Acetyltransferases↗
PubMed2004

Identification of a low affinity mannose 6-phosphate-binding site in domain 5 of the cation-independent mannose 6-phosphate receptor.

The 300-kDa cation-independent mannose 6-phosphate receptor (CI-MPR) and the 46-kDa cation-dependent MPR (CD-MPR) are type I integral membrane glycoproteins that play a critical role in the intracellular delivery of newly synthesized mannose 6-phosphate (Man-6-P)-containing acid hydrolases to the lysosome. The extracytoplasmic region of the CI-MPR contains 15 contiguous domains, and the two high affinity ( approximately 1 nm) Man-6-P-binding sites have been mapped to domains 1-3 and 9, with essential residues localized to domains 3 and 9. Domain 5 of the CI-MPR exhibits significant sequence homology to domains 3 and 9 as well as to the CD-MPR. A structure-based sequence alignment was performed that predicts that domain 5 contains the four conserved key residues (Gln, Arg, Glu, and Tyr) identified as essential for carbohydrate recognition by the CD-MPR and domains 3 and 9 of the CI-MPR, but lacks two cysteine residues predicted to form a disulfide bond within the binding pocket. To determine whether domain 5 harbors a carbohydrate-binding site, a construct that encodes domain 5 alone (Dom5His) was expressed in Pichia pastoris. Microarray analysis using 30 different oligosaccharides demonstrated that Dom5His bound specifically to a Man-6-P-containing oligosaccharide (pentamannosyl 6-phosphate). Frontal affinity chromatography showed that the affinity of Dom5His for Man-6-P was approximately 300-fold lower (K(i) = 5.3 mm) than that observed for domains 1-3 and 9. The interaction affinity for the lysosomal enzyme beta-glucuronidase was also much lower (K(d) = 54 microm) as determined by surface plasmon resonance analysis. Taken together, these results demonstrate that the CI-MPR contains a third Man-6-P recognition site that is located in domain 5 and that exhibits lower affinity than the carbohydrate-binding sites present in domains 1-3 and 9.

Amino Acid Sequence↗