The Nudix Hydrolase Ndx1 from Thermus thermophilus HB8 Is a Diadenosine Hexaphosphate Hydrolase with a Novel Activity
The ndx1 gene, which encodes a Nudix protein, was cloned from the extremely thermophilic bacterium Thermus thermophilus HB8. This gene encodes a 126-amino acid protein that includes the characteristic Nudix motif conserved among Nudix proteins. Ndx1 was overexpressed in Escherichia coli and purified...
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Veröffentlicht in: | The Journal of biological chemistry 2004-05, Vol.279 (21), p.21732-21739 |
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Zusammenfassung: | The ndx1 gene, which encodes a Nudix protein, was cloned from the extremely thermophilic bacterium Thermus thermophilus HB8. This gene encodes a 126-amino acid protein that includes the characteristic Nudix motif conserved among Nudix proteins.
Ndx1 was overexpressed in Escherichia coli and purified. Ndx1 was stable up to 95 °C and at extreme pH. Size exclusion chromatography indicated that Ndx1 was monomeric
in solution. Ndx1 specifically hydrolyzed (di)adenosine polyphosphates but not ATP or diadenosine triphosphate, and it always
generated ATP as the product. Diadenosine hexaphosphate (Ap 6 A), the most preferred substrate, was hydrolyzed to produce two ATP molecules, which is a novel hydrolysis mode for Ap 6 A, with a K m of 1.4 μ m and a k cat of 4.1 s â1 . These results indicate that Ndx1 is a (di)adenosine polyphosphate hydrolase. Ndx1 activity required the presence of the
divalent cations Mn 2+ , Mg 2+ , Zn 2+ , and Co 2+ , whereas Ca 2+ , Ni 2+ , and Cu 2+ were not able to activate Ndx1. Fluoride ion inhibited Ndx1 activity via a non-competitive mechanism. Optimal activity for
Ap 6 A was observed at around pH 8.0 and about 70 °C. We found two important residues with p K a values of 6.1 and 9.6 in the free enzyme and p K a values of 7.9 and 10.0 in the substrate-enzyme complex. Kinetic studies of proteins with amino acid substitutions suggested
that Glu-46 and Glu-50 were conserved residues in the Nudix motif and were involved in catalysis. Trp-26 was likely involved
in enzyme-substrate interactions based on fluorescence measurements. Based on these results, the mechanism of substrate recognition
and catalysis are discussed. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.M312018200 |