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At least 955 records · Page 53Linked to original sources
PubMed2001

Fructose 2,6-bisphosphate activates pyrophosphate: fructose-6-phosphate 1-phosphotransferase and increases triose phosphate to hexose phosphate cycling in heterotrophic cells.

The aim of this work was to establish the influence of fructose 2,6-bisphosphate (Fru-2,6-P2) on non-photosynthetic carbohydrate metabolism in plants. Heterotrophic callus lines exhibiting elevated levels of Fru-2,6-P2 were generated from transgenic tobacco (Nicotiana tabacum L.) plants expressing a modified rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase. Lines containing increased amounts of Fru-2,6-P2 had lower levels of hexose phosphates and higher levels of 3-phosphoglycerate than the untransformed control cultures. There was also a greater redistribution of label into the C6 position of sucrose and fructose, following incubation with [1-13C]glucose, in the lines possessing the highest amounts of Fru-2,6-P2, indicating a greater re-synthesis of hexose phosphates from triose phosphates in these lines. Despite these changes, there were no marked differences between lines in the metabolism of 14C-substrates, the rate of oxygen uptake, carbohydrate accumulation or nucleotide pool sizes. These data provide direct evidence that physiologically relevant changes in the level of Fru-2,6-P2 can affect pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) activity in vivo, and are consistent with PFP operating in a net glycolytic direction in the heterotrophic culture. However, the results also show that activating PFP has little direct effect on heterotrophic carbohydrate metabolism beyond increasing the rate of cycling between hexose phosphates and triose phosphates.

Animals↗
PubMed2002

PC and PE synthesis: mixed micellar analysis of the cholinephosphotransferase and ethanolaminephosphotransferase activities of human choline/ethanolamine phosphotransferase 1 (CEPT1).

The human choline/ethanolamine phosphotransferase 1 (CEPT1) gene codes for a dual-specificity enzyme that catalyzes the de novo synthesis of the two major phospholipids through the transfer of a phosphobase from CDP-choline or CDP-ethanolamine to DAG to form PC and PE. We used an expression system devoid of endogenous cholinephosphotransferase and ethanolaminephosphotransferase activities to assess the diradylglycerol specificity of CEPT1. A mixed micellar assay was used to ensure that the diradylglycerols delivered were not affecting the membrane environment in which CEPT1 resides. The CEPT1 enzyme displayed an apparent Km of 36 microM for CDP-choline and 4.2 mol% for di-18:1 DAG with a Vmax of 14.3 nmol min(-1) mg(-1). When CDP-ethanolamine was used as substrate, the apparent Km was 98 microM for CDP-ethanolamine and 4.3 mol% for di-18:1 DAG with a Vmax of 8.2 nmol min(-1) mg(-1). The preferred diradylglycerol substrates used by CEPT1 with CDP-choline as the phosphobase donor were di-18:1 DAG, di-16:1 DAG, and 16:0/18:1 DAG. A major difference between previous emulsion-based assay results and the mixed micelle results was a complete inability to use 16:0(O)/2:0 as a substrate for the de novo synthesis of platelet-activating factor when the mixed micelle assay was used. When CDP-ethanolamine was used as the phosphobase donor, 16:0/18:1 DAG, di-18:1 DAG, and di-16:1 DAG were the preferred substrates. The mixed micelle assay also allowed the lipid activation of CEPT to be measured, and both the cholinephosphotransferase and ethanolaminephosphotransferase activities displayed the unusual property of product activation at 5 mol%, implying that specific lipid activation binding sites exist on CEPT1. The protein kinase C inhibitor chelerythrine and the human DAG kinase inhibitor R59949 both inhibited CEPT1 activity with IC50 values of 40 microM.

Cell Membrane↗
PubMed1977

Release of the phosphodiesterase activator by cyclic AMP-dependent ATP:protein phosphotransferase from subcellular fractions of rat brain.

The subcellular distribution of the endogenous phosphodiesterase activator and its release from membranes by a cyclic AMP-dependent ATP:protein phosphotransferase was studied in fractions and subfractions of rat brain homogenate. These fractions were obtained by differential centrifugation and sucrose density gradient; their identity was ascertained by electron microscopy and specific enzyme markers. In the subcellular particulate fractions, the concentration of activator is highest in the microsomal fraction, followed by the mitochondrial and nuclear fractions. Gradient centrifugation of the main mitochondrial subfraction revealed that activator was concentrated in those fractions containing mainly synaptic membranes. Activator was releasted from membranes by a cyclic AMP-dependent phosphorylation of membrane protein. The release of activator occurred mainly from the mitochondrial subfractions containing synaptic membranes and synaptic vesicles. The data support the view that a release of activator from membranes may be important in normalizing the elevated concentration of cyclic AMP following persistent transsynaptic activation of adenylate cyclase.

Animals↗
PubMed2005

Enhanced catalytic efficiency of aminoglycoside phosphotransferase (3')-IIa achieved through protein fragmentation and reassembly.

Many monomeric proteins can be split into two fragments, yet the two fragments can associate to make an active heterodimer. However, for most locations in a protein such a conversion is not feasible, presumably due to inefficient assembly or improper folding of the fragments. For some locations, this can be overcome by fusion of the fragments to dimerization domains that facilitate correct assembly. A variety of heterodimers of aminoglycoside phosphotransferase (3')-IIa (Neo) were created in which the Neo fragments required fusion to a pair of leucine zippers for activity in vivo. However, the ability of these heterodimers to confer kanamycin resistance to Escherichia coli cells was impaired compared to wild-type Neo, primarily due to poor production of soluble protein. The mutations R177S and V198E restored the kanamycin resistance to wild-type levels while maintaining the dependence on leucine zippers for activity. These mutations restored high levels of kanamycin resistance not through an improvement in the production of soluble protein but rather by conferring a large improvement in k(cat)/K(m), surpassing that of Neo. Furthermore, whereas R177S and V198E served to improve k(cat)/K(m) 60-fold in the context of the heterodimer, the same mutations in the context of wild-type Neo had a ninefold negative effect on k(cat)/K(m). This demonstrates the possibility that enzymes with improved catalytic properties can be created through a process involving fragmentation and fusion to domains that facilitate assembly of the fragments.

Amino Acid Sequence↗
PubMed2004

Adenylate kinase and GTP:AMP phosphotransferase of the malarial parasite Plasmodium falciparum. Central players in cellular energy metabolism.

For coping with energetic and synthetic challenges, parasites require high activities of adenylate kinase (AK; ATP + AMP <==> 2 ADP) and GTP:AMP phosphotransferase (GAK; GTP + AMP <==> GDP + ADP). These enzymes were identified in erythrocytic stages of Plasmodium falciparum. The genes encoding PfAK and PfGAK are located on chromosomes 10 and 4, respectively. Molecular cloning and heterologous expression in E. coli yielded enzymatically active proteins of 28.9 (PfAK) and 28.0 kDa (PfGAK). Recombinant PfAK resembles authentic PfAK in its biochemical characteristics including the possible association with a stabilizing protein and the high specificity for AMP as the mononucleotide substrate. Specificity is less stringent for the triphosphate, with ATP as the best substrate (75 U/mg; kcat = 2160 min(-1) at 25 degrees C). PfAK contains the sequence of the amphiphatic helix that is known to mediate translocation of the cytosolic protein into the mitochondrial intermembrane space. PfGAK exhibits substrate preference for GTP and AMP (100 U/mg; kcat = 2800 min(-1) at 25 degrees C); notably, there is no detectable activity with ATP. In contrast to its human orthologue (AK3), PfGAK contains a zinc finger motif and binds ionic iron. The dinucleoside pentaphosphate compounds AP5A and GP5A inhibited PfAK and PfGAK, respectively, with Ki values of approximately 0.2 microM which is more than 250-fold lower than the KM values determined for the nucleotide substrates. The disubstrate inhibitors are useful for studying the enzymatic mechanism of PfAK and PfGAK as well as their function in adenine nucleotide homeostasis; in addition, the chimeric inhibitors represent interesting lead compounds for developing nucleosides to be used as antiparasitic agents.

Adenylate Kinase↗
PubMed2005

Allosteric inhibition of aminoglycoside phosphotransferase by a designed ankyrin repeat protein.

Aminoglycoside phosphotransferase (3')-IIIa (APH) is a bacterial kinase that confers antibiotic resistance to many pathogenic bacteria and shares structural homology with eukaryotic protein kinases. We report here the crystal structure of APH, trapped in an inactive conformation by a tailor-made inhibitory ankyrin repeat (AR) protein, at 2.15 A resolution. The inhibitor was selected from a combinatorial library of designed AR proteins. The AR protein binds the C-terminal lobe of APH and thereby stabilizes three alpha helices, which are necessary for substrate binding, in a significantly displaced conformation. BIAcore analysis and kinetic enzyme inhibition experiments are consistent with the proposed allosteric inhibition mechanism. In contrast to most small-molecule kinase inhibitors, the AR proteins are not restricted to active site binding, allowing for higher specificity. Inactive conformations of pharmaceutically relevant enzymes, as can be elucidated with the approach presented here, represent powerful starting points for rational drug design.

Allosteric Regulation↗
PubMed2000

Cross-talk between posttranscriptionally silenced neomycin phosphotransferase II transgenes.

Tobacco plants containing a transgene locus with two chimeric neomycin phosphotransferase II (nptII) genes in tail-to-tail orientation (locus 1) show posttranscriptional gene silencing. The silenced nptII transgenes of locus 1 can downregulate the expression of homologous nptII transgenes in hybrid plants. The 3' region of the silenced nptII genes located in the center of the inverted repeat locus 1 is extensively methylated. Moreover, 3' segments of in trans-inactivated transgenes also become methylated, suggesting cross-talk between homologous posttranscriptionally silenced genes. Our results are in accordance with the hypothesis that this cross-talk can be mediated by specially featured RNAs.

DNA Methylation↗
PubMed2001

Carbohydrate transporters of the bacterial phosphoenolpyruvate: sugar phosphotransferase system (PTS).

The glucose transporter of Escherichia coli couples translocation with phosphorylation of glucose. The IICB(Glc) subunit spans the membrane eight times. Split, circularly permuted and cyclized forms of IICB(Glc) are described. The split variant was 30 times more active when the two proteins were encoded by a dicistronic mRNA than by two genes. The stability and activity of circularly permuted forms was improved when they were expressed as fusion proteins with alkaline phosphatase. Cyclized IICB(Glc) and IIA(Glc) were produced in vivo by RecA intein-mediated trans-splicing. Purified, cyclized IIA(Glc) and IICB(Glc) had 100% and 30% of wild-type glucose phosphotransferase activity, respectively. Cyclized IIA(Glc) displayed increased stability against temperature and GuHCl-induced unfolding.

Amino Acid Sequence↗
PubMed2001

A Streptomyces rimosus aphVIII gene coding for a new type phosphotransferase provides stable antibiotic resistance to Chlamydomonas reinhardtii.

Although Chlamydomonas reinhardtii serves as the most popular algal model system, no efficient enzymatic selection marker for the nuclear transformation of wild-type cells is available. We sequenced an aminoglycoside 3'-phosphotransferase gene (aph) from Streptomyces rimosus. Though the derived protein sequence is homologous to members of APH type V, it constitutes a new type, named APHVIII. Since the aphVIII gene has a codon bias similar to that of the nuclear genome of green algae, the aphVIII coding sequence was fused to the 5'- and 3'-untranslated regions of the C. reinhardtii rbcS2 gene. C. reinhardtii transformants were capable of inactivating the antibiotics paromomycin, kanamycin, and neomycin, to which wild-type cells are sensitive. After addition of the 5'-region of hsp70A as a second promoter and insertion of the rbcS2 intron I, the transformation rate increased to two transformants per 1 x 10(5) cells, which is close to the efficiency of transforming auxotrophic strains with the homologous marker arg7. Transformation with the promoter-less aphVIII led to random gene fusion at high frequency. In an aphVIII-based reporter gene assay we have found a so far unknown promoter activity of the 3'-untranslated region of rbcS2, that may promote antisense RNA synthesis from the rbcS2 gene in vivo. We conclude that the aphVIII gene is a useful marker for nuclear transformation and promoter tagging of C. reinhardtii wild-type and probably other green algae.

5' Untranslated Regions↗
PubMed1998

The use of neamine as a molecular template: inactivation of bacterial antibiotic resistance enzyme aminoglycoside 3'-phosphotransferase type IIa.

Aminoglycoside 3'-phosphotransferase type IIa [APH(3')-IIa] is a member of the family of bacterial aminoglycoside-modifying enzymes. Bacteria that harbor these enzymes are resistant to aminoglycoside antibiotics. Four aminoglycoside-based affinity inactivators were synthesized and were shown to be both substrates and inactivators for APH(3')-IIa. These affinity inactivators are N-bromoacetylated derivatives of neamine, an aminoglycoside antibiotic, where the bromoacetyl moiety in each was introduced regiospecifically at a different amine of the parent compound.

Bacteria↗
PubMed1998

The use of neamine as a molecular template: identification of active site residues in the bacterial antibiotic resistance enzyme aminoglycoside 3'-phosphotransferase type IIa by mass spectroscopy.

Four novel aminoglycoside-based affinity inactivators were shown to covalently modify the active site of aminoglycoside 3'-phosphotransferase type IIa (APH(3')-IIa), an important resistance factor in bacteria for aminoglycoside antibiotics. Standard peptide mapping techniques failed with this enzyme. A novel mass spectroscopic analysis which combines protease digestion on the instrument probe, followed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is described which permitted rapid identification of the sites of protein modification. By this new technique, Glu-3 and Asp-23 were identified as active-site residues, the side chains of which potentially may serve as counter ions for the ammonium functionalities at positions 6', and 1 and 3 of the antibiotic substrates, respectively. These findings contradict previous assertions that the C-terminal third of the enzyme should form the active site, by placing the active site clearly in the N-terminal portion of the enzyme.

Aspartic Acid↗
PubMed1999

Aminoglycoside antibiotic phosphotransferases are also serine protein kinases.

BACKGROUND: Bacterial resistance to aminoglycoside antibiotics occurs primarily through the expression of modifying enzymes that covalently alter the drugs by O-phosphorylation, O-adenylation or N-acetylation. Aminoglycoside phosphotransferases (APHs) catalyze the ATP-dependent phosphorylation of these antibiotics. Two particular enzymes in this class, APH(3')-IIIa and AAC(6')-APH(2"), are produced in gram-positive cocci and have been shown to phosphorylate aminoglycosides on their 3' and 2" hydroxyl groups, respectively. The three-dimensional structure of APH (3')-IIIa is strikingly similar to those of eukaryotic protein kinases (EPKs), and the observation, reported previously, that APH(3')-IIIa and AAC(6')-APH(2") are effectively inhibited by EPK inhibitors suggested the possibility that these aminoglycoside kinases might phosphorylate EPK substrates. RESULTS: Our data demonstrate unequivocally that APHs can phosphorylate several EPK substrates and that this phosphorylation occurs exclusively on serine residues. Phosphorylation of Ser/Thr protein kinase substrates by APHs was considerably slower than phosphorylation of aminoglycosides under identical assay conditions, which is consistent with the primary biological roles of the enzymes. CONCLUSIONS: These results demonstrate a functional relationship between aminoglycoside and protein kinases, expanding on our previous observations of similarities in protein structure, enzyme mechanism and sensitivity to inhibitors, and suggest an evolutionary link between APHs and EPKs.

Amino Acid Sequence↗
PubMed2002

Novel sugar phosphotransferase system applicable to the efficient labeling of the compounds synthesized via the non-mevalonate pathway in Escherichia coli.

It is known that 1-deoxy-D-xylulose taken up by Escherichia coli is used as the precursor of the compounds synthesized via the non-mevalonate pathway, such as isoprenoids, probably after conversion into 1-deoxy-D-xylulose 5-phosphate. In this report, we show that a novel phospho(enol) pyruvate-dependent phosphotransferase system catalyzes the uptake and phosphorylation of 1-deoxy-D-xylulose.

Journal Article↗
PubMed1986

The Tetrahymena intervening sequence ribonucleic acid enzyme is a phosphotransferase and an acid phosphatase.

A shortened form of the Tetrahymena intervening sequence (IVS) RNA acts as an enzyme, catalyzing nucleotidyl transfer and hydrolysis reactions with oligo(cytidylic acid) substrates [Zaug, A. J., & Cech, T. R. (1986) Science (Washington, D.C.) 231, 470-475]. These reactions involve phosphodiester substrates. We now show that the same enzyme has activity toward phosphate monoesters. The 3'-phosphate of C5p or C6p is transferred to the 3'-terminal guanosine of the enzyme. The pH dependence of the reaction (optimum at pH 5) indicates that the enzyme has activity toward the dianion and much greater activity toward the monoanion form of the 3'-phosphate of the substrate. Phosphorylation of the enzyme is reversible by C5-OH and other oligo(pyrimidines) such as UCU-OH. Thus, the RNA enzyme acts as a phosphotransferase, transferring the 3'-terminal phosphate of C5p to UCU-OH with multiple turnover. At pH 4 and 5, the phosphoenzyme undergoes slow hydrolysis to yield inorganic phosphate. Thus, the enzyme has acid phosphatase activity. The RNA enzyme dephosphorylates oligonucleotide substrates with high sequence specificity, which distinguishes it from known protein enzymes.

Acid Phosphatase↗
PubMed1990

Analysis of sequence homologies in plant and bacterial pyruvate phosphate dikinase, enzyme I of the bacterial phosphoenolpyruvate: sugar phosphotransferase system and other PEP-utilizing enzymes. Identification of potential catalytic and regulatory motifs.

In this paper we report the amino acid sequence of pyruvate phosphate dikinase (PPDK) from Bacteroides symbiosus as determined from the nucleotide sequence of the PPDK gene. Comparison of the B. symbiosus PPDK amino acid sequence with that of the maize PPDK [Matsuoka, M., Ozeki, Y., Yamamoto, N., Hirano, H., Kamo-Murakami, Y., & Tanaka, Y. (1988) J. Biol. Chem. 263, 11080] revealed long stretches of homologous sequence (greater than 70% identity), which contributed to an overall sequence identity of 53%. The circular dichrosim spectra, hydropathy profiles, and calculated secondary structural elements of the two dikinases suggest that they may have very similar tertiary structures as well. A comparison made between the amino acid sequence of the maize and B. symbiosus dikinase with other known protein sequences revealed homology, concentrated in three stretches of sequences, to a mechanistically related enzyme, enzyme I of the Escherichia coli PEP: sugar phosphotransferase system [Saffen, D. W., Presper, K. A., Doering, T. L., Roseman, S. (1987) J. Biol. Chem. 262, 16241]. It is proposed that (i) these three stretches of sequence constitute the site for PEP binding and catalysis and a possible site for the regulation of enzymatic activity and (ii) the conserved sequences exist in a third mechanistically related enzyme, PEP synthase.

Amino Acid Sequence↗
PubMed2001

Structural analyses of nucleotide binding to an aminoglycoside phosphotransferase.

3',5"-Aminoglycoside phosphotransferase type IIIa [APH(3')-IIIa] is a bacterial enzyme that confers resistance to a range of aminoglycoside antibiotics while exhibiting striking homology to eukaryotic protein kinases (ePK). The structures of APH(3')-IIIa in its apoenzyme form and in complex with the nonhydrolyzable ATP analogue AMPPNP were determined to 3.2 and 2.4 A resolution, respectively. Furthermore, refinement of the previously determined ADP complex was completed. The structure of the apoenzyme revealed alternate positioning of a flexible loop (analogous to the P-loop of ePK's), occupying part of the nucleotide-binding pocket of the enzyme. Despite structural similarity to protein kinases, there was no evidence of domain movement associated with nucleotide binding. This rigidity is due to the presence of more extensive interlobe interactions in the APH(3')-IIIa structure than in the ePK's. Differences between the ADP and AMPPNP complexes are confined to the area of the nucleotide-binding pocket. The position of conserved active site residues and magnesium ions remains unchanged, but there are differences in metal coordination between the two nucleotide complexes. Comparison of the di/triphosphate binding site of APH(3')-IIIa with that of ePK's suggests that the reaction mechanism of APH(3")-IIIa and related aminoglycoside kinases will closely resemble that of eukaryotic protein kinases. However, the orientation of the adenine ring in the binding pocket differs between APH(3')-IIIa and the ePK's by a rotation of approximately 40 degrees. This alternate binding mode is likely a conserved feature among aminoglycoside kinases and could be exploited for the structure-based drug design of compounds to combat antibiotic resistance.

Adenosine Diphosphate↗