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:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Aspartic acid residues comprising the -D-(aa) n -Y-L-D-D- DNA polymerase active site triad of reverse transcriptase from the Saccharomyces cerevisiae long terminal repeat-retrotransposon Ty3 (Asp151, Asp213 and Asp214) were evaluated via site-directed mutagenesis. An Asp151-->Glu substitution showed a dramatic decrease in catalytic efficiency and a severe translocation defect following initiation of DNA synthesis. In contrast, enzymes harboring the equivalent alteration at Asp213 and Asp214 retained DNA polymerase activity. Asp151-->Asn and Asp213-->Asn substitutions eliminated both polymerase activities. However, while Asp214 of the triad could be replaced by either Asn or Glu, introducing Gln seriously affected processivity. Mutants of the carboxylate triad at positions 151 and 213 also failed to catalyze pyrophosphorolysis. Finally, alterations to the DNA polymerase active site affected RNase H activity, suggesting a close spatial relationship between these N- and C-terminal catalytic centers. Taken together, our data reveal a critical role for Asp151 and Asp213 in catalysis. In contrast, the second carboxylate of the Y-L-D-D motif (Asp214) is not essential for catalysis, and possibly fulfills a structural role. Although Asp214 was most insensitive to substitution with respect to activity of the recombinant enzyme, all alterations at this position were lethal for Ty3 transposition.
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PMID:Functional roles of carboxylate residues comprising the DNA polymerase active site triad of Ty3 reverse transcriptase. 1564

We reported previously that a novel dipeptide alcohol, l-homoserylaminoethanol (Hse-Gly-ol), is a selective inhibitor of eukaryotic DNA polymerase epsilon (pol epsilon) [Bioorg. Med. Chem.2004, 12, 957-962]. The discovery suggests that the dipeptide structure could be a chemical frame for a DNA polymerase inhibitor. Therefore, we chemically synthesized 27 different species of dipeptide alcohols, and tested this inhibitory capability. Compound 6 (l-aspartylaminoethanol, Asp-Gly-ol) was found to be the strongest pol alpha inhibitor. Compound 6 did not influence the activities of other replicative DNA polymerases such as delta and epsilon, and had no effect on the activities of prokaryotic DNA polymerases, nor DNA metabolic enzymes such as human immunodeficiency virus type 1 reverse transcriptase, T7 RNA polymerase and bovine deoxyribonuclease I. The inhibitory effect of compound 6 on pol alpha was dose-dependent, and 50% inhibition was observed at a concentration of 33.5 microM. Compound 6-induced inhibition of pol alpha activity was non-competitive with both the DNA template-primer and the dNTP substrate. This is the first report on a water-soluble pol alpha-specific inhibitor, sought for precise biochemical studies of pol alpha. The relationships between the structures of dipeptide alcohols and the inhibition of eukaryotic DNA polymerases are discussed.
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PMID:Dipeptide alcohol-based inhibitors of eukaryotic DNA polymerase alpha. 1572 71

The coumarins represent a unique class of non-nucleoside reverse transcriptase inhibitors (NNRTIs) that were isolated from tropical plants. (+)-Calanolide A, the most potent compound of this class, selects for the T139I resistance mutation in HIV-1 reverse transcriptase (RT). Seven RTs mutated at amino acid position 139 (Ala, Lys, Tyr, Asp, Ile, Ser, and Gln) were constructed by site-directed mutagenesis. The mutant T139Q enzyme retained full catalytic activity compared with wild-type RT, whereas the mutant T139I, T139S, and T139A RTs retained only 85 to 50% of the activity. Mutant T139K, T139D, and T139Y RTs had seriously impaired catalytic activities. The mutations in the T139I and T139D RTs were shown to destabilize the RT heterodimer. (+)-Calanolide A lost inhibitory activity (up to 20-fold) against the mutant T139Y, T139Q, T139K, and T139I enzymes. All of the mutant enzymes retained marked susceptibility toward the other NNRTIs, including nevirapine, delavirdine, efavirenz, thiocarboxanilide UC-781, quinoxaline GW867420X, TSAO [[2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro-5''-(4''-amino-1'',2''-oxathiole-2'',2''-dioxide)] derivatives, and the nucleoside inhibitor, ddGTP. The fact that the T139I RT 1) proved to be resistant to (+)-calanolide A, 2) represents a catalytically efficient enzyme, and 3) requires only a single transition point mutation (ACA-->ATA) in codon 139 seems to explain why mutant T139I RT virus strains, but not virus strains containing other amino acid changes at this position, predominantly emerge in cell cultures under (+)-calanolide A pressure.
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PMID:The role of Thr139 in the human immunodeficiency virus type 1 reverse transcriptase sensitivity to (+)-calanolide A. 1596 74

Telomerase is a special reverse transcriptase that extends one strand of the telomere repeat by using a template embedded in an RNA subunit. Like other polymerases, telomerase is believed to use a pair of divalent metal ions (coordinated by a triad of aspartic acid residues) for catalyzing nucleotide addition. Here we show that, in the presence of manganese, both yeast and human telomerase can switch to a template- and RNA-independent mode of DNA synthesis, acting in effect as a terminal transferase. Even as a terminal transferase, yeast telomerase retains a species-dependent preference for GT-rich, telomere-like DNA on the 5' end of the substrate. The terminal transferase activity of telomerase may account for some of the hitherto unexplained effects of telomerase overexpression on cell physiology.
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PMID:Telomerase can act as a template- and RNA-independent terminal transferase. 1599 30

Single-base deletions at nucleotide runs or -1 frameshifting by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) result from template slippage during polymerization. In crystal structures of HIV-1 RT complexed with DNA-DNA template-primer, the palm subdomain in the template cleft contacts the template backbone near the proposed site of slippage via the Glu(89) side chain. We investigated the role of Glu(89) in frameshifting by perturbing this interaction. Substitutions with Asp, Gly, Ala, Val, Ser, Thr, Asn, or Lys were created in recombinant HIV RT, and frameshift frequencies of the resulting mutant RTs were measured. All substitutions led to reduced -1 frameshifting by HIV-1 RT (2-40-fold). Interestingly, the suppression of -1 frameshifting frequently coincided with an enhancement of +1 frameshifting (3-47-fold) suggesting that Glu(89) can influence the slippage of both strands. Glu(89) substitutions also led to reduced rates of dNTP misincorporation that paralleled reductions in -1 frameshifting, suggesting a common structural mechanism for both classes of RT error. Our results reveal a major influence of Glu(89) on slippage-mediated errors and dNTP incorporation fidelity. The crystal structure of HIV-1 RT reveals a salt bridge between Glu(89) and Lys(154), which may facilitate -1 frameshifting; this concept is supported by the observed reduction in -1 frameshifting for K154A and K154R mutants.
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PMID:Structural determinants of slippage-mediated mutations by human immunodeficiency virus type 1 reverse transcriptase. 1642 28

Crimean-Congo haemorrhagic fever (CCHF) is a potentially fatal viral disease. In this study, the aim was to investigate the prognostic factors affecting the patient's survival and risk factors to fatality. At Ondokuz Mayis University Faculty of Medicine, a tertiary referral centre near the CCHF epidemic region, patients with typical clinical findings and indicative microbiological results for IgM and/or reverse transcriptase-polymerase chain reaction of CCHF virus were enrolled in the study, from 2004 to 2007. Patients were divided into two subgroups according to their survival outcomes; group I (n = 44) survived patients and group II (n = 6) consisted of fatal cases. The median platelet count was significantly lower in the fatal group (11000/mm(3)) when compared to the survived group (49500/mm(3)). Aspartate transferase and alanine transferase (ALT) levels were significantly higher in group II, when compared to group I. Also, the median range of serum lactic dehydrogenase (LDH) and creatinine phosphokinase (CPK) levels were much more elevated, and prothrombin time (PT) and activated partial thromboplastin time (aPTT) were prolonged in fatal cases. There was also a significant difference in median age of these two groups. Advanced age, late admission, low platelet count, increased AST, ALT, CPK and LDH levels, and prolonged PT and aPTT could be an early indicator of poor prognosis in patients with CCHF.
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PMID:Risk factors for fatality in patients with Crimean-Congo haemorrhagic fever. 1953 53

L-Aspartic acid has recently been found to be a good leaving group during HIV reverse transcriptase catalyzed incorporation of deoxyadenosine monophosphate (dAMP) in DNA. This showed that L-Asp is a good mimic for the pyrophosphate moiety of deoxyadenosine triphosphate. The present work explores the thermochemistry and mechanism for hydrolysis of several models for L-aspartic-dAMP using B3LYP/DGDZVP, MP2/6-311++G** and G3MP2 level of theory. The effect of the new compound is gradually investigated: starting from a simple methyl amine leaving group up to the aspartic acid leaving group. The enzymatic environment was mimicked by involving two Mg(2+) ions and some important active site residues in the reaction. All reactions are compared to the corresponding O-coupled leaving group, which is methanol for methyl amine and malic acid for aspartic acid. With methyl amine as a leaving group a tautomeric associative or tautomeric dissociative mechanism is preferred and the barrier is lower than the comparable mechanism with methanol as a leaving group. The calculations on the aspartic acid in the enzymatic environment show that qualitatively the mechanism is the same as for triphosphate but the barrier for hydrolysis by the associative mechanism is higher for L-aspartic-dAMP than for L-malic-dAMP and pyrophosphate.
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PMID:Hydrolysis of aspartic acid phosphoramidate nucleotides: a comparative quantum chemical study. 1967 39

Nucleoside reverse transcriptase inhibitors (NRTIs) are employed in first line therapies for the treatment of human immunodeficiency virus (HIV) infection. They generally lack a 3'-hydroxyl group, and thus when incorporated into the nascent DNA they prevent further elongation. In this report we show that 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), a nucleoside analog that retains a 3'-hydroxyl moiety, inhibited HIV-1 replication in activated peripheral blood mononuclear cells with an EC(50) of 0.05 nm, a potency several orders of magnitude better than any of the current clinically used NRTIs. This exceptional antiviral activity stems in part from a mechanism of action that is different from approved NRTIs. Reverse transcriptase (RT) can use EFdA-5'-triphosphate (EFdA-TP) as a substrate more efficiently than the natural substrate, dATP. Importantly, despite the presence of a 3'-hydroxyl, the incorporated EFdA monophosphate (EFdA-MP) acted mainly as a de facto terminator of further RT-catalyzed DNA synthesis because of the difficulty of RT translocation on the nucleic acid primer possessing 3'-terminal EFdA-MP. EFdA-TP is thus a translocation-defective RT inhibitor (TDRTI). This diminished translocation kept the primer 3'-terminal EFdA-MP ideally located to undergo phosphorolytic excision. However, net phosphorolysis was not substantially increased, because of the apparently facile reincorporation of the newly excised EFdA-TP. Our molecular modeling studies suggest that the 4'-ethynyl fits into a hydrophobic pocket defined by RT residues Ala-114, Tyr-115, Phe-160, and Met-184 and the aliphatic chain of Asp-185. These interactions, which contribute to both enhanced RT utilization of EFdA-TP and difficulty in the translocation of 3'-terminal EFdA-MP primers, underlie the mechanism of action of this potent antiviral nucleoside.
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PMID:Mechanism of inhibition of HIV-1 reverse transcriptase by 4'-Ethynyl-2-fluoro-2'-deoxyadenosine triphosphate, a translocation-defective reverse transcriptase inhibitor. 1983 73

Excessive energy intake greatly contributes to the development of nonalcoholic fatty liver disease (NAFLD) in modern society. To better understand the comprehensive mechanisms of NAFLD development, we investigated the metabolic alterations of rats with NAFLD induced by high-fat diet (HFD). Male Wistar rats were fed a HFD or standard chow for control. After 16 weeks, rat serum was collected for biochemical measurement. The rats' livers were resected and subjected to histology inspection and gene expression analysis with complementary DNA microarray and metabolic analysis with gas chromatography-mass spectroscopy. In HFD rats, the serum cholesterol, triglycerides, glucose, and insulin contents were increased; and the total cholesterol and triglycerides in the livers were also significantly increased. Complementary DNA microarray analysis revealed that 130 genes were regulated by HFD. Together with real-time reverse transcriptase polymerase chain reaction, lipid metabolism regulatory members like sterol regulatory element binding factor 1 and stearoyl-coenzyme A desaturase 1 had up-regulation, whereas others like peroxisome proliferator-activated receptor, carnitine palmitoyltransferase 1, and 3-hydroxy-3-methylglutaryl-coenzyme A reductase had repressed expression, in HFD rat livers. Metabolomic analysis showed that tetradecanoic acid, hexadecanoic acid, and oleic acid had elevation and arachidonic acid and eicosapentaenoic acid had decreased content in HFD rat livers. Amino acids including glycine, alanine, aspartic acid, glutamic acid, and proline contents were decreased. The integrative results from transcriptomic and metabolomic studies revealed that, in HFD rat livers, fatty acid utilization through beta-oxidation was inhibited and lipogenesis was enhanced. These observations facilitated our understanding of the pathways involved in the development of NAFLD induced by HFD.
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PMID:Analysis of transcriptome and metabolome profiles alterations in fatty liver induced by high-fat diet in rat. 1991 42

Protobothrops flavoviridis (Crotalinae) venom gland phospholipase A(2) (PLA(2)) isozyme genes have evolved in an accelerated manner to acquire diverse physiological activities in their products. For elucidation of the multiplication mechanism of PLA(2) genes, a 25,026 bp genome segment harboring five PLA(2) isozyme genes was obtained from Amami-Oshima P. flavoviridis liver and sequenced. The gene PfPLA 2 encoded [Lys(49)]PLA(2) called BPII, the gene PfPLA 4 neurotoxic [Asp(49)]PLA(2) called PLA-N, the gene PfPLA 5 basic [Asp(49)]PLA(2) called PLA-B, and PfPLA 1(psi) and PfPLA 3(psi) were the inactivated genes. The 5' truncated reverse transcriptase (RT) elements, whose intact forms constitute long interspersed nuclear elements (LINEs), were found in close proximity to the 3' end of PLA(2) genes and named PLA(2) gene-coupled RT fragments (PcRTFs). The facts that PcRTFs have the stem-loop and repetitive sequence in the 3' untranslated region (UTR) which is characteristic of CR1 LINEs suggest that PcRTFs are the debris of P. flavoviridis ancestral CR1 LINEs, denoted as PfCR1s. Since the associated pairs of PLA(2) genes and PcRTFs are arranged in tandem in the 25,026 bp segment, it is thought that an ancestral PLA(2) gene-PfCR1 unit (PfPLA-PfCR1) which was produced by retrotransposition of PfCR1 by itself to the 3' end of PLA(2) gene duplicated several times to form a multimer of PfPLA-PfCR1, a cluster of PLA(2) genes, in the period after Crotalinae and Viperinae snakes branched off. Recombinational hot spot of a 37bp segment, named Scomb, was found in the region 548 bp upstream from the TATA box of PLA(2) genes. Thus, it could be assumed that multiplication of PfPLA-PfCR1 occurred by unequal crossing over of the segment, -Scomb-PfPLA-PfCR1-Scomb-. The PfCR1 moieties were afterward disrupted in the 5' portion to PcRTFs. The detection of two types of PcRTFs different in length which were produced by elimination of two definitive sequences in PfCR1 moiety possibly by gene conversion clearly supports such process but not multiplication of the PLA(2) gene-PcRTF unit.
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PMID:Unique structural characteristics and evolution of a cluster of venom phospholipase A2 isozyme genes of Protobothrops flavoviridis snake. 2040 71


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