The 6-oxopurine phosphoribosyltransferase (HPRT, EC 2.4.2.8) from the hyperthermophile Pyrococcus horikoshii was expressed in Escherichia coli and purified. Steady-state kinetic studies indicated that the enzyme is able to use hypoxanthine, guanine and xanthine. The first two substrates showed similar catalytic efficiencies, and xanthine presented a much lower value (around 20 times lower), but the catalytic constant was comparable to that of hypoxanthine. The enzyme was notable to bind to GMP-agarose, but was able to bind the other reverse reaction substrate, inorganic pyrophosphate, with low affinity (K(d) of 4.7 +/- 0.1 mM). Dynamic light scattering and analytical gel filtration suggested that the enzyme exists as a homohexamer in solution. (C) 2008 Elsevier B.V. All rights reserved.17846953960