Publications

• Gene ytkD of Bacillus subtilis encodes an atypical nucleoside triphosphatase member of the Nudix hydrolase superfamily.

  Xu W., Jones C.R., Dunn C.A., Bessman M.J.

  Gene ytkD of Bacillus subtilis, a member of the Nudix hydrolase superfamily, has been cloned and expressed in Escherichia coli. The purified protein has been characterized as a nucleoside triphosphatase active on all of the canonical ribo- and deoxyribonucleoside triphosphates. Whereas all other n ... >> More

  Gene ytkD of Bacillus subtilis, a member of the Nudix hydrolase superfamily, has been cloned and expressed in Escherichia coli. The purified protein has been characterized as a nucleoside triphosphatase active on all of the canonical ribo- and deoxyribonucleoside triphosphates. Whereas all other nucleoside triphosphatase members of the superfamily release inorganic pyrophosphate and the cognate nucleoside monophosphate, YtkD hydrolyses nucleoside triphosphates in a stepwise fashion through the diphosphate to the monophosphate, releasing two molecules of inorganic orthophosphate. Contrary to a previous report, our enzymological and genetic studies indicate that ytkD is not an orthologue of E. coli mutT. << Less

  J. Bacteriol. 186:8380-8384(2004) [PubMed] [EuropePMC]

  This publication is cited by 32 other entries.

• Purification and properties of human placental ATP diphosphohydrolase.

  Christoforidis S., Papamarcaki T., Galaris D., Kellner R., Tsolas O.

  ATP diphosphohydrolase activity (ATP-DPH) has been previously identified in the particulate fraction of human term placenta [Papamarcaki, T. & Tsolas, O. (1990) Mol. Cell. Biochem. 97, 1-8]. In the present study we have purified to homogeneity and characterized this activity. A 260-fold purificati ... >> More

  ATP diphosphohydrolase activity (ATP-DPH) has been previously identified in the particulate fraction of human term placenta [Papamarcaki, T. & Tsolas, O. (1990) Mol. Cell. Biochem. 97, 1-8]. In the present study we have purified to homogeneity and characterized this activity. A 260-fold purification has been obtained by solubilization of the particulate fraction and subsequent chromatography on DEAE Sepharose CL-6B and 5'-AMP Sepharose 4B. The preparation has been shown to be free of alkaline phosphatase even though the placental extract is rich in this activity. The purified enzyme is a glycoprotein and migrates as a single broad band of 82 kDa on SDS/PAGE. The same band is obtained after photoaffinity labeling of the enzyme with 8-azido-[alpha-32P]ATP. The enzyme has a broad substrate specificity, hydrolyzing triphosphonucleosides and diphosphonucleosides but not monophosphonucleosides or other phosphate esters. The activity is dependent on the addition of divalent cations Ca2+ or Mg2+. The Km values for ATP and ADP were determined to be 10 microM and 20 microM, respectively. Maximum activity was found at pH 7.0-7.5 with ATP as substrate, and pH 7.5-8.0 with ADP. The enzymic activity is inhibited by NaN3, NaF, adenosine 5'-[beta,gamma-imido]triphosphate and adenosine 5'-[alpha,beta-methylene]triphosphate. Protein sequence analysis showed ATP-DPH to be N-terminally blocked. Partial internal amino acid sequence information was obtained after chymotryptic cleavage and identified a unique sequence with no significant similarity to known proteins. ATP-DPH activity has been reported to be implicated in the prevention of platelet aggregation, hydrolysing ADP to AMP and thus preventing blood clotting. << Less

  Eur. J. Biochem. 234:66-74(1995) [PubMed] [EuropePMC]

  This publication is cited by 12 other entries.

• Mitochondrial translation requires folate-dependent tRNA methylation.

  Morscher R.J., Ducker G.S., Li S.H., Mayer J.A., Gitai Z., Sperl W., Rabinowitz J.D.

  Folates enable the activation and transfer of one-carbon units for the biosynthesis of purines, thymidine and methionine. Antifolates are important immunosuppressive and anticancer agents. In proliferating lymphocytes and human cancers, mitochondrial folate enzymes are particularly strongly upregu ... >> More

  Folates enable the activation and transfer of one-carbon units for the biosynthesis of purines, thymidine and methionine. Antifolates are important immunosuppressive and anticancer agents. In proliferating lymphocytes and human cancers, mitochondrial folate enzymes are particularly strongly upregulated. This in part reflects the need for mitochondria to generate one-carbon units and export them to the cytosol for anabolic metabolism. The full range of uses of folate-bound one-carbon units in the mitochondrial compartment itself, however, has not been thoroughly explored. Here we show that loss of the catalytic activity of the mitochondrial folate enzyme serine hydroxymethyltransferase 2 (SHMT2), but not of other folate enzymes, leads to defective oxidative phosphorylation in human cells due to impaired mitochondrial translation. We find that SHMT2, presumably by generating mitochondrial 5,10-methylenetetrahydrofolate, provides methyl donors to produce the taurinomethyluridine base at the wobble position of select mitochondrial tRNAs. Mitochondrial ribosome profiling in SHMT2-knockout human cells reveals that the lack of this modified base causes defective translation, with preferential mitochondrial ribosome stalling at certain lysine (AAG) and leucine (UUG) codons. This results in the impaired expression of respiratory chain enzymes. Stalling at these specific codons also occurs in certain inborn errors of mitochondrial metabolism. Disruption of whole-cell folate metabolism, by either folate deficiency or antifolate treatment, also impairs the respiratory chain. In summary, mammalian mitochondria use folate-bound one-carbon units to methylate tRNA, and this modification is required for mitochondrial translation and thus oxidative phosphorylation. << Less

  Nature 554:128-132(2018) [PubMed] [EuropePMC]

  This publication is cited by 2 other entries.

• G-domain dimerization orchestrates the tRNA wobble modification reaction in the MnmE/GidA complex.

  Meyer S., Wittinghofer A., Versees W.

  MnmE and GidA are involved in the modification of wobble uridine to carboxymethylaminomethyl uridine in certain tRNAs. Malfunctioning of the human orthologs has been implicated in mitochondrial diseases. MnmE is a conserved G protein activated by dimerization. Here, we show that complex formation ... >> More

  MnmE and GidA are involved in the modification of wobble uridine to carboxymethylaminomethyl uridine in certain tRNAs. Malfunctioning of the human orthologs has been implicated in mitochondrial diseases. MnmE is a conserved G protein activated by dimerization. Here, we show that complex formation between MnmE and GidA involves large conformational changes that induce G-domain dimerization of MmnE and that GidA co-stimulates GTP hydrolysis on MnmE. Starting from a structural model of the complex, we identify interface mutations disrupting complex formation or communication. Although GidA does not directly contact the G-domains, conformational changes in MnmE, induced by G-domain dimerization in the triphosphate state, regulate the affinity for GidA. We developed a tRNA modification assay and demonstrate for the first time in vitro that the MnmE/GidA complex catalyzes incorporation of glycine into tRNA. An intact MnmE/GidA complex rather than their sequential action is crucial for in vitro modification. Since only GTP, but not GDP or non-hydrolyzable GTP analogs, drives the MnmE/GidA-catalyzed modification reaction, we conclude that GTP hydrolysis is essential for activity. We finally show that an active GTPase, an intact MnmE/GidA communication, and dimerization of G-domains are necessary for in vivo functioning since mutations disrupting either result in a respiratory deficient phenotype in yeast. << Less

  J Mol Biol 392:910-922(2009) [PubMed] [EuropePMC]

• The Escherichia coli trmE (mnmE) gene, involved in tRNA modification, codes for an evolutionarily conserved GTPase with unusual biochemical properties.

  Cabedo H., Macian F., Villarroya M., Escudero J.C., Martinez-Vicente M., Knecht E., Armengod M.-E.

  The evolutionarily conserved 50K protein of Escherichia coli, encoded by o454, contains a consensus GTP-binding motif. Here we show that 50K is a GTPase that differs extensively from regulatory GTPases such as p21. Thus, 50K exhibits a very high intrinsic GTPase hydrolysis rate, rather low affinit ... >> More

  The evolutionarily conserved 50K protein of Escherichia coli, encoded by o454, contains a consensus GTP-binding motif. Here we show that 50K is a GTPase that differs extensively from regulatory GTPases such as p21. Thus, 50K exhibits a very high intrinsic GTPase hydrolysis rate, rather low affinity for GTP, and extremely low affinity for GDP. Moreover, it can form self-assemblies. Strikingly, the 17 kDa GTPase domain of 50K conserves the guanine nucleotide-binding and GTPase activities of the intact 50K molecule. Therefore, the structural requirements for GTP binding and GTP hydrolysis by 50K are without precedent and justify a separate classification in the GTPase superfamily. Immunoelectron microscopy reveals that 50K is a cytoplasmic protein partially associated with the inner membrane. We prove that o454 is allelic with trmE, a gene involved in the biosynthesis of the hypermodified nucleoside 5-methylaminomethyl-2-thiouridine, which is found in the wobble position of some tRNAs. Our results demonstrate that 50K is essential for viability depending on the genetic background. We propose that combination of mutations affecting the decoding process, which separately do not reveal an obvious defect in growth, can give rise to lethal phenotypes, most likely due to synergism. << Less

  EMBO J. 18:7063-7076(1999) [PubMed] [EuropePMC]

• Structural and functional characterization of mycobacterial PhoH2 and identification of potential inhibitor of its enzymatic activity.

  Shivangi X., Khan Y., Ekka M.K., Meena L.S.

  Mycobacterium tuberculosis is composed of a cumbersome signaling and protein network which partakes in bacterial survival and augments its pathogenesis. Mycobacterial PhoH2 (Mt-PhoH2) is a signaling element and a predictive phosphate starvation protein that works in an ATP-dependent manner. Here, ... >> More

  Mycobacterium tuberculosis is composed of a cumbersome signaling and protein network which partakes in bacterial survival and augments its pathogenesis. Mycobacterial PhoH2 (Mt-PhoH2) is a signaling element and a predictive phosphate starvation protein that works in an ATP-dependent manner. Here, we elaborated the characterization of Mt-PhoH2 through biophysical, biochemical, and computational methods. In addition to its intrinsic ATPase activity, the biochemical experiments revealed its GTPase activity and both activities are metal ion dependent. Magnesium, manganese, copper, iron, nickel, zinc, cesium, calcium, and lithium were examined for their effect on activity, and the optimum activity was found with 10 mM of Mg²⁺ ions. The kinetic parameters of 3 µM Mt-PhoH2 were observed as K_m 4.873 ± 0.44 µM, V_max 12.3817 ± 0.084 µM/min/mg, K_cat 0.0075 ± 0.00005 s^-1, and K_cat/K_m 0.0015 ± 0.000001 µM^-1 s^-1 with GTP. In the case of GTP as a substrate, a 20% decrease in enzymatic activity and a 50% increase in binding affinity of Mt-PhoH2 were observed. The substrates ADP and GDP inhibit the ATPase and GTPase activity of Mt-PhoH2. CD spectroscopy showed the dominance of alpha helix in the secondary structure of Mt-PhoH2, and this structural pattern was altered upon addition of ATP and GTP. In silico inhibitor screening revealed ML141 and NAV_2729 as two potential inhibitors of the catalytic activity of Mt-PhoH2. Mt-PhoH2 is essential for mycobacterial growth as its knockdown strain showed a decreased growth effect. Overall, the present article emphasizes the factors essential for the proper functioning of Mt-PhoH2 which is a participant in the toxin-antitoxin machinery and may also play an important role in phosphate starvation. << Less

  Braz. J. Microbiol. 0:0-0(2024) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• The human tRNA taurine modification enzyme GTPBP3 is an active GTPase linked to mitochondrial diseases.

  Peng G.X., Zhang Y., Wang Q.Q., Li Q.R., Xu H., Wang E.D., Zhou X.L.

  GTPBP3 and MTO1 cooperatively catalyze 5-taurinomethyluridine (τm5U) biosynthesis at the 34th wobble position of mitochondrial tRNAs. Mutations in tRNAs, GTPBP3 or MTO1, causing τm5U hypomodification, lead to various diseases. However, efficient in vitro reconstitution and mechanistic study of τm5 ... >> More

  GTPBP3 and MTO1 cooperatively catalyze 5-taurinomethyluridine (τm5U) biosynthesis at the 34th wobble position of mitochondrial tRNAs. Mutations in tRNAs, GTPBP3 or MTO1, causing τm5U hypomodification, lead to various diseases. However, efficient in vitro reconstitution and mechanistic study of τm5U modification have been challenging, in part due to the lack of pure and active enzymes. A previous study reported that purified human GTPBP3 (hGTPBP3) is inactive in GTP hydrolysis. Here, we identified the mature form of hGTPBP3 and showed that hGTPBP3 is an active GTPase in vitro that is critical for tRNA modification in vivo. Unexpectedly, the isolated G domain and a mutant with the N-terminal domain truncated catalyzed GTP hydrolysis to only a limited extent, exhibiting high Km values compared with that of the mature enzyme. We further described several important pathogenic mutations of hGTPBP3, associated with alterations in hGTPBP3 localization, structure and/or function in vitro and in vivo. Moreover, we discovered a novel cytoplasm-localized isoform of hGTPBP3, indicating an unknown potential noncanonical function of hGTPBP3. Together, our findings established, for the first time, the GTP hydrolysis mechanism of hGTPBP3 and laid a solid foundation for clarifying the τm5U modification mechanism and etiology of τm5U deficiency-related diseases. << Less

  Nucleic Acids Res. 49:2816-2834(2021) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.