Thermophile-specific proteins: the gene product of aq_1292 from Aquifex aeolicus is an NTPase

BMC Biochem. 2003 Sep 23:4:12. doi: 10.1186/1471-2091-4-12.

Abstract

Background: To identify thermophile-specific proteins, we performed phylogenetic patterns searches of 66 completely sequenced microbial genomes. This analysis revealed a cluster of orthologous groups (COG1618) which contains a protein from every thermophile and no sequence from 52 out of 53 mesophilic genomes. Thus, COG1618 proteins belong to the group of thermophile-specific proteins (THEPs) and therefore we here designate COG1618 proteins as THEP1s. Since no THEP1 had been analyzed biochemically thus far, we characterized the gene product of aq_1292 which is THEP1 from the hyperthermophilic bacterium Aquifex aeolicus (aaTHEP1).

Results: aaTHEP1 was cloned in E. coli, expressed and purified to homogeneity. At a temperature optimum between 70 and 80 degrees C, aaTHEP1 shows enzymatic activity in hydrolyzing ATP to ADP + Pi with kcat = 5 x 10(-3) s(-1) and Km = 5.5 x 10(-6) M. In addition, the enzyme exhibits GTPase activity (kcat = 9 x 10(-3) s(-1) and Km= 45 x 10(-6) M). aaTHEP1 is inhibited competitively by CTP, UTP, dATP, dGTP, dCTP, and dTTP. As shown by gel filtration, aaTHEP1 in its purified state appears as a monomer. The enzyme is resistant to limited proteolysis suggesting that it consists of a single domain. Although THEP1s are annotated as "predicted nucleotide kinases" we could not confirm such an activity experimentally.

Conclusion: Since aaTHEP1 is the first member of COG1618 that is characterized biochemically and functional information about one member of a COG may be transferred to the entire COG, we conclude that COG1618 proteins are a family of thermophilic NTPases.

MeSH terms

  • Adenosine Triphosphate / metabolism
  • Amino Acid Motifs
  • Amino Acid Sequence
  • Bacteria / enzymology*
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Hot Temperature
  • Kinetics
  • Molecular Sequence Data
  • Nucleoside-Triphosphatase / chemistry
  • Nucleoside-Triphosphatase / genetics
  • Nucleoside-Triphosphatase / metabolism*
  • Nucleotides / pharmacology
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Temperature

Substances

  • Bacterial Proteins
  • Nucleotides
  • Recombinant Proteins
  • Adenosine Triphosphate
  • Nucleoside-Triphosphatase