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

Klinger C, Rossbach M, Howe R, Kaufmann M - BMC Biochem. (2003)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Neurobiochemistry, The Protein Chemistry Group, Witten/Herdecke University, Stockumer Str 10, 58448 Witten, Germany. cklinger@uni-wh.de

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.

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Recombinant aaTHEP1 can be expressed and purified. SDS-PAGE after different steps during purification of recombinant aaTHEP1. Lane 1: crude cell extract, lane 2: supernatant after heat treatment, lane 3: eluate after cation exchange chromatography, lane 4: eluate after hydrophobic interaction chromatography. To demonstrate the purity of the final preparation, lane 4 intentionally was overloaded.
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Figure 2: Recombinant aaTHEP1 can be expressed and purified. SDS-PAGE after different steps during purification of recombinant aaTHEP1. Lane 1: crude cell extract, lane 2: supernatant after heat treatment, lane 3: eluate after cation exchange chromatography, lane 4: eluate after hydrophobic interaction chromatography. To demonstrate the purity of the final preparation, lane 4 intentionally was overloaded.

Mentions: The bulk of E. coli proteins could be removed by heating the crude cell extract for 10 min at 75°C. As expected from the sequence-based theoretical pI of 9.88, aaTHEP1 binds to the matrix of a cation exchanger. Ion exchange followed by hydrophobic interaction chromatography finally resulted in a homogenous aaTHEP1 preparation that migrates slightly higher than the calculated molecular weight of 20,555 Da (figure 2).


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

Klinger C, Rossbach M, Howe R, Kaufmann M - BMC Biochem. (2003)

Recombinant aaTHEP1 can be expressed and purified. SDS-PAGE after different steps during purification of recombinant aaTHEP1. Lane 1: crude cell extract, lane 2: supernatant after heat treatment, lane 3: eluate after cation exchange chromatography, lane 4: eluate after hydrophobic interaction chromatography. To demonstrate the purity of the final preparation, lane 4 intentionally was overloaded.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC222928&req=5

Figure 2: Recombinant aaTHEP1 can be expressed and purified. SDS-PAGE after different steps during purification of recombinant aaTHEP1. Lane 1: crude cell extract, lane 2: supernatant after heat treatment, lane 3: eluate after cation exchange chromatography, lane 4: eluate after hydrophobic interaction chromatography. To demonstrate the purity of the final preparation, lane 4 intentionally was overloaded.
Mentions: The bulk of E. coli proteins could be removed by heating the crude cell extract for 10 min at 75°C. As expected from the sequence-based theoretical pI of 9.88, aaTHEP1 binds to the matrix of a cation exchanger. Ion exchange followed by hydrophobic interaction chromatography finally resulted in a homogenous aaTHEP1 preparation that migrates slightly higher than the calculated molecular weight of 20,555 Da (figure 2).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute for Neurobiochemistry, The Protein Chemistry Group, Witten/Herdecke University, Stockumer Str 10, 58448 Witten, Germany. cklinger@uni-wh.de

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.

Show MeSH
Related in: MedlinePlus