3′-Phosphorylated Nucleotides Are Tight Binding Inhibitors of Nucleoside Diphosphate Kinase Activity

Schneider, Benoı̂t; Xu, Yingqiang; Janin, Joël; Véron, Michel; Deville‐Bonne, Dominique

doi:10.1074/jbc.273.44.28773

Cited by 34 publications

(31 citation statements)

References 45 publications

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“…As reflected in the IC 50 (the concentration of inhibitor resulting in 50% inhibition) values, AMP and UDP were potent inhibitors compared with cyclic AMP. However, unlike other NdKs (44,45), the phosphotransfer activity of mNdK (Table I) was not affected by either cromoglycate or 3Ј-phosphate nucleotides (PAP or PAPS). Thus our results highlighted the distinction in the behavior of mNdK compared with other bacterial homologues.…”

Section: Resultsmentioning

confidence: 75%

Amino Acid Residues Involved in Autophosphorylation and Phosphotransfer Activities Are Distinct in Nucleoside Diphosphate Kinase from Mycobacterium tuberculosis

Tiwari¹,

Kishan

Chakrabarti

et al. 2004

Journal of Biological Chemistry

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Nucleoside diphosphate kinase (NdK) is a ubiquitous enzyme in both prokaryotes and eukaryotes and is primarily involved in the maintenance of cellular nucleotide pools. We have cloned ndk from Mycobacterium tuberculosis strain H37Ra and expressed it in Escherichia coli as a fusion protein with glutathione S-transferase. The purified protein, following thrombin cleavage and gel permeation chromatography, was found to be hexameric with a monomeric unit molecular mass of ϳ16.5 kDa. The protein exhibited nucleotide binding, divalent cation-dependent autophosphorylation, and phosphate transfer ability from nucleoside triphosphate to nucleoside diphosphate. Although UDP inhibited the catalytic activity of the recombinant protein, the classic inhibitors, like cromoglycate, 5-adenosine 3-phosphate, and adenosine 3-phosphate 5-phosphosulfate, had no effect on the activity. Among three histidine residues in the protein, His-117 was found to be essential for autophosphorylation. However, in subsequent phosphate transfer, we observed that His-53 had a significant contribution. Consistent with this observation, substitution of His-53 with either Ala or Gln affected the ability of the recombinant protein to complement NdK function in Pseudomonas aeruginosa. Furthermore, mutational analysis established critical roles for Tyr-50 and Arg-86 of the M. tuberculosis protein in maintaining phosphotransfer ability.

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Section: Resultsmentioning

confidence: 75%

Amino Acid Residues Involved in Autophosphorylation and Phosphotransfer Activities Are Distinct in Nucleoside Diphosphate Kinase from Mycobacterium tuberculosis

Tiwari¹,

Kishan

Chakrabarti

et al. 2004

Journal of Biological Chemistry

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“…5). Because pAp acts as a competitive inhibitor of several enzymes (31,49,50), high concentrations of pAp are toxic for cells. RecJ-like proteins may degrade pAp to control the concentration in bacteria and archaea that do not have CysQ (30 -34) (Fig.…”

Section: Discussionmentioning

confidence: 99%

Role of RecJ-like Protein with 5′-3′ Exonuclease Activity in Oligo(deoxy)nucleotide Degradation

Wakamatsu

Kim

Uemura

et al. 2011

Journal of Biological Chemistry

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RecJ-like proteins belonging to the DHH family have been proposed to function as oligoribonucleases and 3-phosphoadenosine 5-phosphate (pAp) phosphatases in bacteria and archaea, which do not have Orn (oligoribonuclease) and CysQ (pAp phosphatase) homologs. In this study, we analyzed the biochemical and physiological characterization of the RecJ-like protein TTHA0118 from Thermus thermophilus HB8. TTHA0118 had high enzymatic activity as an oligodeoxyribonucleotide-and oligoribonucleotide-specific exonuclease and as pAp phosphatase. The polarity of degradation was 5 to 3, in contrast to previous reports about Bacillus subtilis NrnA, a RecJ-like protein. TTHA0118 preferentially hydrolyzed short oligodeoxyribonucleotides and oligoribonucleotides, whereas the RecJ exonuclease from T. thermophilus HB8 showed no such length dependence on oligodeoxyribonucleotide substrates. An insertion mutation of the ttha0118 gene led to growth reduction in minimum essential medium. Added 5-mononucleotides, nucleosides, and cysteine increased growth of the ttha0118 mutant in minimum essential medium. The RecJ-like protein Mpn140 from Mycoplasma pneumoniae M129, which cannot synthesize nucleic acid precursors de novo, showed similar biochemical features to TTHA0118. Furthermore, B. subtilis NrnA also hydrolyzed oligo(deoxy)ribonucleotides in a 5-3 direction. These results suggested that these RecJ-like proteins act in recycling short oligonucleotides to mononucleotides and in controlling pAp concentrations in vivo.Nucleases are essential for nucleic acid metabolism and cell viability. DNA repair and recombination require a variety of specific deoxyribonucleases (1, 2). RNA metabolism also requires various ribonucleases (RNases) 2 for a broad range of processes (3, 4). In mRNA turnover, mRNA decay is an important determinant of gene expression and is mediated by several RNases with endonuclease and exonuclease activity. Nucleases belonging to different superfamilies have been identified with endonuclease and exonuclease activities (5-8).In addition, genome sequences have revealed numerous ORFs annotated as putative nucleases.One of the phosphoesterase families is the DHH superfamily (9), which includes phosphodiesterase and exopolyphosphatase (10 -12). RecJ is a DNA exonuclease in the DHH protein superfamily, with DHH motifs I-IV and an associated DHHA1 motif (9), as described in the Pfam database (13). The RecJ protein is a Mg 2ϩ -or Mn 2ϩ -dependent single-stranded DNA (ssDNA)-specific 5Ј-3Ј exonuclease/deoxyribophosphodiesterase and plays a role in homologous recombination, mismatch repair, and base excision repair (14 -19). Although the ORFs annotated as RecJ-related proteins also have DHH and DHHA1 motifs, each RecJ-related protein has to be investigated for nuclease activity similar to RecJ protein. According to the SYSTERS database (20), proteins with DHH and DHHA1 motifs can be further divided into three main families. Representatives of each family are RecJ (N_155469 in SYSTERS), putative poly(A) polymerase (O_142140), and ex...

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“…The main enzyme involved in the phosphorylation of TDP to TTP is NDPK. However, the phosphorylation efficiencies of NDPK for AZTDP and D4TDP are at least 10 4 -fold less and 10 3 -fold less than those for naturally occurring TDP, respectively (40)(41)(42). Hsu et al observed that 4Ј-Ed4TDP can also be phosphorylated by PGK and pyruvate kinase (19).…”

Section: Discussionmentioning

confidence: 99%

Intracellular Metabolism and Persistence of the Anti-Human Immunodeficiency Virus Activity of 2′,3′-Didehydro-3′-Deoxy-4′-Ethynylthymidine, a Novel Thymidine Analog

Paintsil

Dutschman²,

Hu³

et al. 2007

Antimicrob Agents Chemother

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The therapeutic benefits of current antiretroviral therapy are limited by the evolution of drug-resistant virus and long-term toxicity. Novel antiretroviral compounds with activity against drug-resistant viruses are needed. 2,3-Didehydro-3-deoxy-4-ethynylthymidine (4-Ed4T), a novel thymidine analog, has potent anti-human immunodeficiency virus (HIV) activity, maintains considerable activity against multidrug-resistant HIV strains, and is less inhibitory to mitochondrial DNA synthesis in cell culture than its progenitor stavudine (D4T). We investigated the intracellular metabolism and anti-HIV activity of 4-Ed4T. The profile of 4-Ed4T metabolites was qualitatively similar to that for zidovudine (AZT), with the monophosphate metabolite as the major metabolite, in contrast to that for D4T, with relatively poor formation of total metabolites. The first phosphorylation step for 4-Ed4T in cells was more efficient than that for D4T but less than that for AZT. The amount of 4-Ed4T triphosphate (4-Ed4TTP) was higher than that of AZTTP at 24 h in culture. There was a dose-dependent accumulation of 4-Ed4T diphosphate and 4-Ed4TTP on up-regulation of thymidylate kinase and 3-phosphoglycerate kinase expression in Tet-On RKO cells, respectively. The anti-HIV activity of 4-Ed4T in cells persisted even after 48 h of drug removal from culture in comparison with AZT, D4T, and nevirapine (NVP). The order of increasing persistence of anti-HIV activity of these compounds after drug removal was 4-Ed4T > D4T > AZT > NVP. In conclusion, with the persistence of 4-Ed4TTP and persistent anti-HIV activity in cells, we anticipate less frequent dosing and fewer patient compliance issues than for D4T. 4-Ed4T is a promising antiviral candidate for HIV type 1 chemotherapy.

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3′-Phosphorylated Nucleotides Are Tight Binding Inhibitors of Nucleoside Diphosphate Kinase Activity

Abstract: The sulfate group is directed toward the protein surface. PAPS will be useful for the design of high affinity drugs targeted to NDP kinases.

Cited by 34 publications

References 45 publications

Amino Acid Residues Involved in Autophosphorylation and Phosphotransfer Activities Are Distinct in Nucleoside Diphosphate Kinase from Mycobacterium tuberculosis

Amino Acid Residues Involved in Autophosphorylation and Phosphotransfer Activities Are Distinct in Nucleoside Diphosphate Kinase from Mycobacterium tuberculosis

Role of RecJ-like Protein with 5′-3′ Exonuclease Activity in Oligo(deoxy)nucleotide Degradation

Intracellular Metabolism and Persistence of the Anti-Human Immunodeficiency Virus Activity of 2′,3′-Didehydro-3′-Deoxy-4′-Ethynylthymidine, a Novel Thymidine Analog

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