Structural and functional characterization of a novel phosphodiesterase from Methanococcus jannaschii

Methanococcus jannaschii MJ0936 is a hypothetical protein of unknown function with over 50 homologs found in many bacteria and Archaea. To help define the molecular ( biochemical and biophysical) function of MJ0936, we determined its crystal structure at 2.4-Angstrom resolution and performed a series of biochemical screens for catalytic activity. The overall fold of this single domain protein consists of a four-layered structure formed by two beta-sheets flanked by alpha-helices on both sides. The crystal structure suggested its biochemical function to be a nuclease, phosphatase, or nucleotidase, with a requirement for some metal ions. Crystallization in the presence of Ni2+ or Mn2+ produced a protein containing a binuclear metal center in the putative active site formed by a cluster of conserved residues. Analysis of MJ0936 against a panel of general enzymatic assays revealed catalytic activity toward bis-p-nitrophenyl phosphate, an indicator substrate for phosphodiesterases and nucleases. Significant activity was also found with two other phosphodiesterase substrates, thymidine 5'-monophosphate p-nitrophenyl ester and p-nitrophenylphosphorylcholine, but no activity was found for cAMP or cGMP. Phosphodiesterase activity of MJ0936 had an absolute requirement for divalent metal ions with Ni2+] and Mn2+ being most effective. Thus, our structural and enzymatic studies have identified the biochemical function of MJ0936 as that of a novel phosphodiesterase.