Characterization of a highly thermostable alkaline phosphatase from the euryarchaeon Pyrococcus abyssi

This work reports the first isolation and characterization of an alkaline p hosphatase (AP) from a hyperthermophilic archaeon. An AP gene from Pyrococc us abyssi, a euryarchaeon isolated from a deep-sea hydrothermal vent, was c loned and the enzyme expressed in Escherichia coli. Analysis of the sequenc e showed conservation of the active site and structural elements of the E. coli AP. The recombinant AP was purified and characterized. Monomeric and h omodimeric active forms were detected, with a monomer molecular mass of 54 kDa. Apparent optimum pH and temperature were estimated at 11.0 and 70 degr eesC, respectively. Thus far, P. abyssi AP has been demonstrated to be the most thermostable AP, with half-lives at 100 and 105 degreesC of 18 and 5 h , respectively. Enzyme activity was found to be dependent on divalent catio ns: metal ion chelators inhibited activity, whereas the addition of exogeno us Mg(II), Zn(II), and Co(II) increased activity. The enzyme was inhibited by inorganic phosphate, but not by molybdate and vanadate. Strong inhibitor y effects were observed in the presence of thiol-reducing agents, although cysteine residues of the P. abyssi AP were not found to be incorporated wit hin intra-or interchain disulfide bonds. In addition, P. abyssi AP was demo nstrated to dephosphorylate linear DNA fragments with dephosphorylation eff iciencies of 93.8 and 84.1 % with regard to cohesive and blunt ends, respec tively.