Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To study subdomain organization of the potato virus X (PVX) movement protein (MP) encoded by the first gene in the triple gene block (TGB), we mutated the 25-kDa TGBp1 protein. The N-terminal deletion of the helicase motifs I, IA, and II resulted in loss of the ATPase activity and RNA binding. A frameshift mutation truncating the C-terminal motifs V and VI gave rise to increase of the TGBp1 ATPase activity and had little effect on RNA binding in vitro. Fusions of the green fluorescent protein with 25-kDa MP and its derivative lacking motifs V-VI exhibited similar fluorescence patterns in epidermal cells of Nicotiana benthamiana leaves. Cell-to-cell movement of the 25K-deficient PVX genome was not complemented by the TGBp1 of Plantago asiatica mosaic potexvirus (PlAMV) but was efficiently complemented by a chimeric TGBp1 consisting of the N-terminal part of PlAMV protein (motifs I-IV) and the PVX-specific C-terminal part (motifs V-VI). These results suggest that NTP hydrolysis, RNA binding, and targeting to the specific cellular compartment(s) are associated with the N-terminal domain of the TGBp1 including the helicase motifs I-IV and that the C-terminal domain is involved in specific interactions with other virus proteins.
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PMID:Evidence for two nonoverlapping functional domains in the potato virus X 25K movement protein. 1040 56

The nonstructural protein NSP2 is a component of rotavirus replication intermediates and accumulates in cytoplasmic inclusions (viroplasms), sites of genome RNA replication and the assembly of subviral particles. To better understand the structure and function of the protein, C-terminally His-tagged NSP2 was expressed in bacteria and purified to homogeneity. In its purified form, the protein did not exist as a monomer but rather was present as an 8S-10S homomultimer consisting of 6 +/- 2 subunits of recombinant NSP2 (rNSP2). As shown by gel mobility shift assays, the rNSP2 multimers bound to RNA in discrete cooperative steps to form higher-order RNA-protein complexes. The RNA-binding activity of the rNSP2 multimers was determined to be nonspecific and to have a strong preference for single-stranded RNA over double-stranded RNA, for which it displayed little affinity. Enzymatic analysis revealed that rNSP2 possessed an associated nucleoside triphosphatase (NTPase) activity in vitro, which in the presence of Mg(2+) catalyzed the hydrolysis of each of the four NTPs to NDPs with equal efficiency. Evidence indicating that the hydrolysis of NTP resulted in the covalent linkage of the gamma-phosphate to rNSP2 was obtained. Additional experiments showed that NSP2 expressed transiently in MA014 cells is phosphorylated. We propose that NSP2 functions as a molecular motor, catalyzing the packaging of viral mRNA into core-like replication intermediates through the energy derived from its NTPase activity.
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PMID:Multimers formed by the rotavirus nonstructural protein NSP2 bind to RNA and have nucleoside triphosphatase activity. 1055 6

The RNA-stimulated nucleoside triphosphatase (NTPase) and helicase of hepatitis C virus (HCV) consists of three domains with highly conserved NTP binding motifs located in the first domain. The ATP-binding domain was obtained by limited proteolysis of a greater fragment of the HCV polyprotein, and it was purified to homogenity by column chromatography. The identity of the domain, comprising amino acids 1203 to 1364 of the HCV polyprotein, was confirmed by N- and C-terminal sequencing and by its capability to bind 5'-fluorosulfonylbenzoyladenosine (FSBA). The analyses of the kinetics of ATP binding revealed a single class of binding site with the Kd of 43.6 microM. The binding is saturable and dependent on Mn2+ or Mg2+ ions. Poly(A) and poly(dA) show interesting properties as regulators of the ATP-binding capacity of the domain. Polynucleotides bind to the domain and enhance its affinity for ATP. In addition, ATP enhances the affinity of the domain for the polynucleotides. Different compounds, which are known to interact with nucleotide binding sites of various classes of enzymes, were tested for their ability to inhibit the binding of ATP to the domain. Of the compounds tested, two agents behaved as inhibitors: paclitaxel, which inhibits the ATP binding competitively (IC50 = 22 microM), and trifluoperazine, which inhibits the ATP binding by a noncompetitive mechanism (IC50 = 98 microM). Kinetic experiments with the NTPase/helicase indicate that both compounds inhibit the NTPase activity of the holoenzyme by interacting with its ATP-binding domain.
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PMID:Biochemical properties of a minimal functional domain with ATP-binding activity of the NTPase/helicase of hepatitis C virus. 1058 65

Ribosomal protein L3 (L3) has been demonstrated to participate in formation of the peptidyltransferase center and is essential for its catalytic activity. In the present study we show that L3 is able to bind nucleotide triphosphates with high and specific affinity in vitro. L3 was serendipitously identified by screening of a genomic phage library from a primitive kinetoplastid flagellate Trypanoplasma borreli with the ATPase domain of the topoisomerase II gene as a probe. The cloned gene was overexpressed and purified as a his-tag fusion protein in E. coli. Radioligand binding experiments, using [gamma-35S]ATP, showed that L3 is able to bind ATP but also GTP and UTP with similar high affinity (IC50 50-100 nM), while it has no ATPase activity. Furthermore, we showed that L3 has more than 500-fold higher affinity for nucleotide triphosphates compared to the corresponding nucleotide monophosphates and diphosphates. Molecular genetic and biochemical analyses allowed us to localize the NTP binding domain of L3 to the N-terminal 296 residues. Suramin, a polysulfonated naphthylamine derivative of urea, known for its chemotherapeutic effects completely inhibited the binding of [gamma-35S]ATP at subclinical levels. Results obtained with surface plasmon resonance technology showed that suramin both forms weak multimolecular complexes with L3 and binds strongly to L3 in nearly stoichiometric amounts.
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PMID:Suramin blocks nucleotide triphosphate binding to ribosomal protein L3 from Trypanoplasma borreli. 1071 4

To identify splicing factors in proximity of the 5' splice site (5'SS), we followed a crosslinking profile of site-specifically modified, photoreactive RNA substrates. Upon U4/U5/U6 snRNP addition, the 5'SS RNA crosslinks in an ATP-dependent manner to U6 snRNA, an unidentified protein p27, and the 100-kDa U5 snRNP protein, a human ortholog of an ATPase/RNA helicase yPrp28p. The 5'SS:hPrp28p crosslink maps to the highly conserved TAT motif in proximity of the ATP-binding site in hPrp28p. We propose that hPrp28p acts as a helicase to unwind the 5'SS:U1 snRNA duplex, and at the same time as a 5'SS translocase, which, upon NTP-dependent conformational change, positions the 5'SS for pairing with U6 snRNA within the spliceosome. This repositioning of the 5'SS takes place regardless of whether the 5'SS is originally duplexed with U1 snRNA.
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PMID:The 100-kda U5 snRNP protein (hPrp28p) contacts the 5' splice site through its ATPase site. 1123 76

Plasmid R64 pilQ gene is essential for the formation of thin pilus, a type IV pilus. The pilQ product contains NTP binding motifs and belongs to the PulE-VirB11 family of NTPases. The pilQ gene was overexpressed with an N-terminal His tag, and PilQ protein was purified. Purified His tag PilQ protein displayed ATPase activity with a V(max) of 0.71 nmol/min/mg of protein and a K(m) of 0.26 mm at pH 6.5. By gel filtration chromatography, PilQ protein was eluted at the position corresponding to 460 kDa, suggesting that PilQ protein forms a homooctamer. To analyze the relationship between structure and function of PilQ protein, amino acid substitutions were introduced within several conserved motifs. Among 11 missense mutants, 7 mutants exhibited various levels of reduced DNA transfer frequencies in liquid matings. Four mutant genes (T234I, K238Q, D263N, and H328A) were overexpressed with a His tag. The purified mutant PilQ proteins contained various levels of reduced ATPase activity. Three mutant PilQ proteins formed stable multimers similar to wild-type PilQ, whereas the PilQ D263N multimer was unstable. PilQ D263N monomer exhibited low ATPase activity, while PilQ D263N multimer did not. These results indicate that ATPase activity of the PilQ multimer is essential for R64 thin pilus biogenesis.
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PMID:Atpase activity and multimer formation of Pilq protein are required for thin pilus biogenesis in plasmid R64. 1127 18

The 464-amino acid baculovirus Lef4 protein is a bifunctional mRNA capping enzyme with triphosphatase and guanylyltransferase activities. The hydrolysis of 5'-triphosphate RNA and free NTPs by Lef4 is dependent on a divalent cation cofactor. RNA triphosphatase activity is optimal at pH 7.5 with either magnesium or manganese, yet NTP hydrolysis at neutral pH is activated only by manganese or cobalt. Here we show that Lef4 possesses an intrinsic magnesium-dependent ATPase with a distinctive alkaline pH optimum and a high K(m) for ATP (4 mm). Lef4 contains two conserved sequences, motif A ((8)IEKEISY(14)) and motif C ((180)LEYEF(184)), which define the fungal/viral/protozoal family of metal-dependent RNA triphosphatases. We find by mutational analysis that Glu(9), Glu(11), Glu(181), and Glu(183) are essential for phosphohydrolase chemistry and likely comprise the metal-binding site of Lef4. Conservative mutations E9D and E183D abrogate the magnesium-dependent triphosphatase activities of Lef4 and transform it into a strictly manganese-dependent RNA triphosphatase. Limited proteolysis of Lef4 and ensuing COOH-terminal deletion analysis revealed that the NH(2)-terminal 236-amino acid segment of Lef4 constitutes an autonomous triphosphatase catalytic domain.
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PMID:Mutational analysis of baculovirus capping enzyme Lef4 delineates an autonomous triphosphatase domain and structural determinants of divalent cation specificity. 1155 38

Cells of Actinobacillus actinomycetemcomitans, a gram-negative pathogen responsible for an aggressive form of juvenile periodontitis, form tenaciously adherent biofilms on solid surfaces. The bacteria produce long fibrils of bundled pili, which are required for adherence. Mutations in flp-1, which encodes the major subunit of the pili, or any of seven downstream tad genes (tadABCDEFG) cause defects in fibril production, autoaggregation, and tenacious adherence. We proposed that the tad genes specify part of a novel secretion system for the assembly and transport of Flp pili. The predicted amino acid sequence of TadA (426 amino acids, 47,140 Da) contains motifs for nucleotide binding and hydrolysis common among secretion NTP hydrolase (NTPase) proteins. In addition, the tadA gene is the first representative of a distinct subfamily of potential type IV secretion NTPase genes. Here we report studies on the function of TadA. The tadA gene was altered to express a modified version of TadA that has the 11-residue epitope (T7-TAG) fused to its C terminus. The TadA-T7 protein was indistinguishable from the wild type in its ability to complement the fibril and adherence defects of A. actinomycetemcomitans tadA mutants. Although TadA is not predicted to have a transmembrane domain, the protein was localized to the inner membrane and cytoplasmic fractions of A. actinomycetemcomitans cells, indicating a possible peripheral association with the inner membrane. TadA-T7 was purified and found to hydrolyze ATP in vitro. The ATPase activity is stimulated by Triton X-100, with maximal stimulation at the critical micellar concentration. TadA-T7 forms multimers that are stable during sodium dodecyl sulfate-polyacrylamide gel electrophoresis in nonreducing conditions, and electron microscopy revealed that TadA-T7 can form structures closely resembling the hexameric rings of other type IV secretion NTPases. Site-directed mutagenesis was used to substitute Ala and Gln residues for the conserved Lys residue of the Walker A box for nucleotide binding. Both mutants were found to be defective in their ability to complement tadA mutants. We suggest that the ATPase activity of TadA is required to energize the assembly or secretion of Flp pili for tight adherence of A. actinomycetemcomitans.
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PMID:Nonspecific adherence and fibril biogenesis by Actinobacillus actinomycetemcomitans: TadA protein is an ATPase. 1156 92

Sequences and available structures were compared for all the widely distributed representatives of the P-loop GTPases and GTPase-related proteins with the aim of constructing an evolutionary classification for this superclass of proteins and reconstructing the principal events in their evolution. The GTPase superclass can be divided into two large classes, each of which has a unique set of sequence and structural signatures (synapomorphies). The first class, designated TRAFAC (after translation factors) includes enzymes involved in translation (initiation, elongation, and release factors), signal transduction (in particular, the extended Ras-like family), cell motility, and intracellular transport. The second class, designated SIMIBI (after signal recognition particle, MinD, and BioD), consists of signal recognition particle (SRP) GTPases, the assemblage of MinD-like ATPases, which are involved in protein localization, chromosome partitioning, and membrane transport, and a group of metabolic enzymes with kinase or related phosphate transferase activity. These two classes together contain over 20 distinct families that are further subdivided into 57 subfamilies (ancient lineages) on the basis of conserved sequence motifs, shared structural features, and domain architectures. Ten subfamilies show a universal phyletic distribution compatible with presence in the last universal common ancestor of the extant life forms (LUCA). These include four translation factors, two OBG-like GTPases, the YawG/YlqF-like GTPases (these two subfamilies also consist of predicted translation factors), the two signal-recognition-associated GTPases, and the MRP subfamily of MinD-like ATPases. The distribution of nucleotide specificity among the proteins of the GTPase superclass indicates that the common ancestor of the entire superclass was a GTPase and that a secondary switch to ATPase activity has occurred on several independent occasions during evolution. The functions of most GTPases that are traceable to LUCA are associated with translation. However, in contrast to other superclasses of P-loop NTPases (RecA-F1/F0, AAA+, helicases, ABC), GTPases do not participate in NTP-dependent nucleic acid unwinding and reorganizing activities. Hence, we hypothesize that the ancestral GTPase was an enzyme with a generic regulatory role in translation, with subsequent diversification resulting in acquisition of diverse functions in transport, protein trafficking, and signaling. In addition to the classification of previously known families of GTPases and related ATPases, we introduce several previously undetected families and describe new functional predictions.
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PMID:Classification and evolution of P-loop GTPases and related ATPases. 1191 78

Site-directed mutagenesis was performed on Glu143, an essential amino acid in Lactobacillus casei folylpolyglutamate synthetase (FPGS) and the structurally equivalent residue, Glu146, in Escherichia coli FPGS. Glu143 is positioned near the P-loop and interacts with the Mg(2+) of Mg NTP-binding proteins. We have solved the structure of the E143A mutant of L. casei FPGS in the presence of AMPPCP and Mg(2+). The structure showed a water molecule at the place where Mg(2+) bound to the wild type enzyme. Mutant proteins E143A, and even E143D and E143Q with conservative mutations, lacked enzyme activity and failed to complement the methionine auxotrophy of the E. coli folC mutant SF4, showing that Glu143 is an essential residue. Both the L. casei and the E. coli FPGS mutant proteins bound methylene-tetrahydrofolate diglutamate and dihydropteroate normally. The E. coli E146Q mutant FPGS bound ADP with the same affinity as the wild type enzyme but bound ATP with much lower affinity and had higher ATPase activity than the wild type enzyme. The mutant enzyme was defective in forming the acyl-phosphate reaction intermediate from ATP and dihydropteroate. The E. coli FPGS requires activation by dihydropteroate or tetrahydrofolate binding to allow full activity. In the absence of a pteroate substrate, only 30% of the total enzyme binds ATP. We suggest that dihydropteroate causes a conformational change to allow increased ATP binding. The mutant enzyme was similarly activated by dihydropteroate resulting in increased ADP binding.
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PMID:Mutation of an essential glutamate residue in folylpolyglutamate synthetase and activation of the enzyme by pteroate binding. 1205 87


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