Limits...
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.

Show MeSH
aaTHEP1 consists of a single domain. SDS-PAGE after limited proteolysis by trypsin and endoproteinase Glu-C. aaTHEP1 is shown on lanes 1, 3 and 5. As a control, the β-subunit of tryptophan synthase from E. coli is run on lanes 2, 4, and 6. Lanes 1 and 2 show the native proteins without the addition of proteases. Proteolysis by trypsin is seen on lanes 3 and 4 whereas proteolysis by endoproteinase Glu-C is shown on lanes 5 and 6.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC222928&req=5

Figure 10: aaTHEP1 consists of a single domain. SDS-PAGE after limited proteolysis by trypsin and endoproteinase Glu-C. aaTHEP1 is shown on lanes 1, 3 and 5. As a control, the β-subunit of tryptophan synthase from E. coli is run on lanes 2, 4, and 6. Lanes 1 and 2 show the native proteins without the addition of proteases. Proteolysis by trypsin is seen on lanes 3 and 4 whereas proteolysis by endoproteinase Glu-C is shown on lanes 5 and 6.

Mentions: A possible multidomain structure of aaTHEP1 was probed by limited proteolysis. As a control, the β-subunit of E. coli tryptophan synthase as a typical protein composed of distinct domains connected by a hinge region [10] was also proteolyzed. In contrast to β-tryptophan synthase, aaTHEP1 is resistant to proteolytic cleavage by both trypsin and endoproteinase Glu-C (figure 10). Compared to published experiments on the proteolytic cleavage of β-tryptophan synthase [10,11], the experimental conditions were chosen in a way that fragmented β-tryptophan synthase (~30 and ~10 kDa fragments) already were further degraded. Even under these conditions, aaTHEP1 remains stable although it contains 36 (20 × K + 16 × R) possible trypsin and 17 (17 × E) possible endoproteinase Glu-C cleavage sites as predicted from the sequence.


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)

aaTHEP1 consists of a single domain. SDS-PAGE after limited proteolysis by trypsin and endoproteinase Glu-C. aaTHEP1 is shown on lanes 1, 3 and 5. As a control, the β-subunit of tryptophan synthase from E. coli is run on lanes 2, 4, and 6. Lanes 1 and 2 show the native proteins without the addition of proteases. Proteolysis by trypsin is seen on lanes 3 and 4 whereas proteolysis by endoproteinase Glu-C is shown on lanes 5 and 6.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 10: aaTHEP1 consists of a single domain. SDS-PAGE after limited proteolysis by trypsin and endoproteinase Glu-C. aaTHEP1 is shown on lanes 1, 3 and 5. As a control, the β-subunit of tryptophan synthase from E. coli is run on lanes 2, 4, and 6. Lanes 1 and 2 show the native proteins without the addition of proteases. Proteolysis by trypsin is seen on lanes 3 and 4 whereas proteolysis by endoproteinase Glu-C is shown on lanes 5 and 6.
Mentions: A possible multidomain structure of aaTHEP1 was probed by limited proteolysis. As a control, the β-subunit of E. coli tryptophan synthase as a typical protein composed of distinct domains connected by a hinge region [10] was also proteolyzed. In contrast to β-tryptophan synthase, aaTHEP1 is resistant to proteolytic cleavage by both trypsin and endoproteinase Glu-C (figure 10). Compared to published experiments on the proteolytic cleavage of β-tryptophan synthase [10,11], the experimental conditions were chosen in a way that fragmented β-tryptophan synthase (~30 and ~10 kDa fragments) already were further degraded. Even under these conditions, aaTHEP1 remains stable although it contains 36 (20 × K + 16 × R) possible trypsin and 17 (17 × E) possible endoproteinase Glu-C cleavage sites as predicted from the sequence.

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