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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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THEP1s contain Walker A and Walker B motifs. CLUSTALW analysis of all THEP1s. The positions of the Walker A and Walker B motifs are indicated. The shown sequences are from Archaeoglobus fulgidus (AF0814), Methanopyrus kandleri AV19 (MK0827), Methanosarcina acetivorans (MA3402), Methanothermobacter thermautotrophicus (MTH1068), Aeropyrum pernix (APE0781), Pyrococcus horikoshii (PH0792), Pyrococcus abyssi (PAB1537), Methanococcus jannaschii (MJ1559), Pyrobaculum aerophilum (PAE3292), Sulfolobus solfataricus (SSO2171), Aquifex aeolicus (aq_1292), Thermotoga maritima ( TM0036), Thermoplasma acidophilum (Ta0998), and Thermoplasma volcanium (TVN0737).
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Figure 1: THEP1s contain Walker A and Walker B motifs. CLUSTALW analysis of all THEP1s. The positions of the Walker A and Walker B motifs are indicated. The shown sequences are from Archaeoglobus fulgidus (AF0814), Methanopyrus kandleri AV19 (MK0827), Methanosarcina acetivorans (MA3402), Methanothermobacter thermautotrophicus (MTH1068), Aeropyrum pernix (APE0781), Pyrococcus horikoshii (PH0792), Pyrococcus abyssi (PAB1537), Methanococcus jannaschii (MJ1559), Pyrobaculum aerophilum (PAE3292), Sulfolobus solfataricus (SSO2171), Aquifex aeolicus (aq_1292), Thermotoga maritima ( TM0036), Thermoplasma acidophilum (Ta0998), and Thermoplasma volcanium (TVN0737).

Mentions: CLUSTALW analysis of all THEP1s led to the design of suitable experiments to assay catalytic in vitro function of aaTHEP1. All proteins contain both the Walker A (GxxxxGK [ST]) and Walker B motif (4 × hydrophobic [DE]xxG) indicating that aaTHEP1 is a P-loop NTPase (figure 1). Since the distal [NT]KxD motif responsible for guanine specificity [8] is absent, aaTHEP1 probably do not belong to the GTPase superclass of the P-loop NTPases. Cort et al. stated that THEP1s "probably represent a novel family of ATPases in both sequence and structural terms" [6]. The authors determined the structure of MTH538 from Methanobacterium thermoautotrophicum by NMR and based on extensive sequence and structure comparisons proposed that the relationship between MTH583 and THEP1s might be similar to that between CheY and ATPase/phosphatase members of the HAD family as suggested by Ridder and Dijkstra [9].


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)

THEP1s contain Walker A and Walker B motifs. CLUSTALW analysis of all THEP1s. The positions of the Walker A and Walker B motifs are indicated. The shown sequences are from Archaeoglobus fulgidus (AF0814), Methanopyrus kandleri AV19 (MK0827), Methanosarcina acetivorans (MA3402), Methanothermobacter thermautotrophicus (MTH1068), Aeropyrum pernix (APE0781), Pyrococcus horikoshii (PH0792), Pyrococcus abyssi (PAB1537), Methanococcus jannaschii (MJ1559), Pyrobaculum aerophilum (PAE3292), Sulfolobus solfataricus (SSO2171), Aquifex aeolicus (aq_1292), Thermotoga maritima ( TM0036), Thermoplasma acidophilum (Ta0998), and Thermoplasma volcanium (TVN0737).
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Related In: Results  -  Collection

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

Figure 1: THEP1s contain Walker A and Walker B motifs. CLUSTALW analysis of all THEP1s. The positions of the Walker A and Walker B motifs are indicated. The shown sequences are from Archaeoglobus fulgidus (AF0814), Methanopyrus kandleri AV19 (MK0827), Methanosarcina acetivorans (MA3402), Methanothermobacter thermautotrophicus (MTH1068), Aeropyrum pernix (APE0781), Pyrococcus horikoshii (PH0792), Pyrococcus abyssi (PAB1537), Methanococcus jannaschii (MJ1559), Pyrobaculum aerophilum (PAE3292), Sulfolobus solfataricus (SSO2171), Aquifex aeolicus (aq_1292), Thermotoga maritima ( TM0036), Thermoplasma acidophilum (Ta0998), and Thermoplasma volcanium (TVN0737).
Mentions: CLUSTALW analysis of all THEP1s led to the design of suitable experiments to assay catalytic in vitro function of aaTHEP1. All proteins contain both the Walker A (GxxxxGK [ST]) and Walker B motif (4 × hydrophobic [DE]xxG) indicating that aaTHEP1 is a P-loop NTPase (figure 1). Since the distal [NT]KxD motif responsible for guanine specificity [8] is absent, aaTHEP1 probably do not belong to the GTPase superclass of the P-loop NTPases. Cort et al. stated that THEP1s "probably represent a novel family of ATPases in both sequence and structural terms" [6]. The authors determined the structure of MTH538 from Methanobacterium thermoautotrophicum by NMR and based on extensive sequence and structure comparisons proposed that the relationship between MTH583 and THEP1s might be similar to that between CheY and ATPase/phosphatase members of the HAD family as suggested by Ridder and Dijkstra [9].

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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