Y.H. Kim

A novel inorganic pyrophosphatase in Thermococcus onnurineus NA1

The TON_0002 gene, which is in close proximity to the DNA polymerase locus in Thermococcus onnurineus NA1, has been shown to encode an inorganic pyrophosphatase. Its genomic position and function suggest a role for pyrophosphate hydrolysis during DNA polymerization. This is the first report of an inorganic pyrophosphatase belonging to the haloacid dehalogenase superfamily, in which unique residues in motif I and II have been replaced with Trp and Gly, respectively. The optimum pyrophosphatase activity of the recombinant enzyme occurred at pH 6, and it displayed an absolute dependence on divalent metal ions, among which Ni(2+) was the most efficient. The site-specific mutation of the Gly residue in motif II to Ala or Ser residue exhibited only a slight change in the enzymatic activity and the K(m) value.

Enrichment and proteome analysis of a hyperthermostable protein set of archaeon Thermococcus onnurineus NA1

Thermococcus onnurineus NA1 is a hyperthermophilic archaeon that can be used for the screening of thermophilic enzymes. Previously, we characterized the metabolic enzymes of the cytosolic proteome by two-dimensional electrophoresis/tandem mass spectrometry (2-DE/MS–MS). In this study, we identified a subset of hyperthermostable proteins in the cytosolic proteome using enrichment by in vitro heat treatment and protein identification. After heat treatment at 100°C for 2xa0h, 13 and 149 proteins were identified from the soluble proteome subset by 2-DE/MS–MS and 1-DE/MS–MS analysis, respectively. Representative proteins included intracellular protease I, thioredoxin reductase, triosephosphate isomerase, putative hydroperoxide reductase, proteasome, and translation initiation factors. Intracellular protease, deblocking aminopeptidases, and fructose-1,6-bisphosphatase were overexpressed in Escherichia coli and biological activity above 85°C was confirmed. The folding transition temperature (Tm) of identified proteins was analyzed using the in silico prediction program TargetStar. The proteins enriched with the heat treatment have higher Tm than the homologous proteins from mesophilic strains. These results suggested that the heat-stable protein set of hyperthermophilic T. onnurineus NA1 can be effectively fractionated and enriched by in vitro heat treatment.

Characterization of a dITPase from the hyperthermophilic archaeon Thermococcus onnurineus NA1 and its application in PCR amplification.

In this study, we found that deoxyinosine triphosphate (dITP) could inhibit polymerase chain reaction (PCR) amplification of various family B-type DNA polymerases, and 0.93% dITP was spontaneously generated from deoxyadenosine triphosphate during PCR amplification. Thus, it was hypothesized that the generated dITP might have negative effect on PCR amplification of family B-type DNA polymerases. To overcome the inhibitory effect of dITP during PCR amplification, a dITP pyrophosphatase (dITPase) from Thermococcus onnurineus NA1 was applied to PCR amplification. Genomic analysis of the hyperthermophilic archaeon T. onnurineus NA1 revealed the presence of a 555-bp open reading frame with 48% similarity to HAM1-like dITPase from Methanocaldococcus jannaschii DSM2661 (NP_247195). The dITPase-encoding gene was cloned and expressed in Escherichia coli. The purified protein hydrolyzed dITP, not deoxyuridine triphosphate. Addition of the purified protein to PCR reactions using DNA polymerases from T. onnurineus NA1 and Pyrococcus furiosus significantly increased product yield, overcoming the inhibitory effect of dITP. This study shows the first representation that removing dITP using a dITPase enhances the PCR amplification yield of family B-type DNA polymerase.