S-adenosylhomocysteine hydrolase from the archaeon Pyrococcus furiosus: biochemical characterization and analysis of protein structure by comparative molecular modeling.

S-adenosylhomocysteine hydrolase (AdoHcyHD) is an ubiquitous enzyme that catalyzes the breakdown of S-adenosylhomocysteine, a powerful inhibitor of most transmethylation reactions, to adenosine and L-homocysteine. AdoHcyHD from the hyperthermophilic archaeon Pyrococcus furiosus (PfAdoHcyHD) was cloned, expressed in Escherichia coli, and purified. The enzyme is thermoactive with an optimum temperature of 95 degrees C, and thermostable retaining 100% residual activity after 1 h at 90 degrees C and showing an apparent melting temperature of 98 degrees C. The enzyme is a homotetramer of 190 kDa and contains four cysteine residues per subunit. Thiol groups are not involved in the catalytic process whereas disulfide bond(s) could be present since incubation with 0.8 M dithiothreitol reduces enzyme activity. Multiple sequence alignment of hyperthermophilic AdoHcyHD reveals the presence of two cysteine residues in the N-terminus of the enzyme conserved only in members of Pyrococcus species, and shows that hyperthermophilic AdoHcyHD lack eight C-terminal residues, thought to be important for structural and functional properties of the eukaryotic enzyme. The homology-modeled structure of PfAdoHcyHD shows that Trp220, Tyr181, Tyr184, and Leu185 of each subunit and Ile244 from a different subunit form a network of hydrophobic and aromatic interactions in the central channel formed at the subunits interface. These contacts partially replace the interactions of the C-terminal tail of the eukaryotic enzyme required for tetramer stability. Moreover, Cys221 and Lys245 substitute for Thr430 and Lys426, respectively, of the human enzyme in NAD-binding. Interestingly, all these residues are fairly well conserved in hyperthermophilic AdoHcyHDs but not in mesophilic ones, thus suggesting a common adaptation mechanism at high temperatures.