Cargando…

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

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 mesophil...

Descripción completa

Detalles Bibliográficos
Autores principales: Klinger, Claudia, Roßbach, Michael, Howe, Rebecca, Kaufmann, Michael
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2003
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC222928/
https://www.ncbi.nlm.nih.gov/pubmed/14503925
http://dx.doi.org/10.1186/1471-2091-4-12
Descripción
Sumario: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°C, aaTHEP1 shows enzymatic activity in hydrolyzing ATP to ADP + P(i )with k(cat )= 5 × 10(-3 )s(-1 )and K(m )= 5.5 × 10(-6 )M. In addition, the enzyme exhibits GTPase activity (k(cat )= 9 × 10(-3 )s(-1 )and K(m)= 45 × 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.