ABSTRACT
A gene encoding DNA ligase (ligTk
) from a hyperthermophilic archaeon, Thermococcus kodakaraensisKOD1, has been cloned and sequenced, and its protein product has been characterized. ligTk
consists of 1,686 bp, corresponding to a polypeptide of 562 amino acids with a predicted molecular mass of 64,079 Da. Sequence comparison with previously reported DNA ligases and the presence of conserved motifs suggested that Lig
Tk
was an ATP-dependent DNA ligase. Phylogenetic analysis indicated that Lig
Tk
was closely related to the ATP-dependent DNA ligase fromMethanobacterium thermoautotrophicum ΔH, a moderate thermophilic archaeon, along with putative DNA ligases fromEuryarchaeota and Crenarchaeota. We expressedligTk
in Escherichia coli and purified the recombinant protein. Recombinant Lig
Tk
was monomeric, as is the case for other DNA ligases. The protein displayed DNA ligase activity in the presence of ATP and Mg2+. The optimum pH of Lig
Tk
was 8.0, the optimum concentration of Mg2+, which was indispensable for the enzyme activity, was 14 to 18 mM, and the optimum concentration of K+ was 10 to 30 mM. Lig
Tk
did not display single-stranded DNA ligase activity. At enzyme concentrations of 200 nM, we observed significant DNA ligase activity even at 100°C. Unexpectedly, Lig
Tk
displayed a relatively small, but significant, DNA ligase activity when NAD+ was added as the cofactor. Treatment of NAD+ with hexokinase did not affect this activity, excluding the possibility of contaminant ATP in the NAD+ solution. This unique cofactor specificity was also supported by the observation of adenylation of Lig
Tk
with NAD+. This is the first biochemical study of a DNA ligase from a hyperthermophilic archaeon.
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