BIOCHEMICAL-CHARACTERIZATION OF ANABAENA SP. STRAIN PCC-7120 NONSPECIFIC NUCLEASE NUCA AND ITS INHIBITOR NUIA

We have established overexpression systems and purification protocols for NucA and NuiA, a sugar non-specific nuclease and its protein inhib itor from Anabuena sp. strain PCC 7120, in order to characterize these proteins in detail to spectroscopy revealed that NucA has a similar s econdary-structure composition. 13 % alpha helix and 20 % beta sheet, to the related Serratia nuclease. while NuiA represents a protein with a higher alpha-helical (29 %) and beta-sheet (24 %) content than NucA . Denaturation experiments showed that the stabilities of NucA and Nui A are in the typical range for proteins of mesophilic organisms, NuiA with Delta G(H2O)(n) = 63.4 j.mol(residue)(-1) being slightly more sta ble than its target NucA with Delta Delta(H2O)(0) = 46.3 J mol(residue )(-1). The nuclease requires divalent metal ions as cofactors. the opt imum concentration bring around 5 mM for Mn2+ or Mg2+. The order of ef fectiveness of various divalent cations to function as cofactors for t he hydrolytic activity of NucA is Mn2+ = CO2+ > Mg2+ greater than or e qual to Ni2+ much greater than Ca2+ = Cd2+ at a concentration of 5 mM. Nuclease activity decreases with increasing concentration of monovale nt salt. The activity of NncA shows a pH optimum at pH 5.5-7.5. The te mperature optimum is around 35 degrees C, the activation energy was ca lculated to be 53 kJ mol(-1), The specific activity of the nuclease to wards high molecular-mass DNA is 8.4 x 10(6) Kunitz-units.mg(-1), whic h means that NucA is one of the most active nucleases known. Kinetic c onstants for the cleavage of various DNA and RNA substrates by NucA ar e all in the range K-m less than or equal to 0.1 mg.ml(-1) and k(cat) congruent to 1000 s(-1). A other non-specific nucleases, NucA exhibits sequence preferences, similar to the related Serratia nuclease, NucA avoids cleavage of d(A).d(T) tracts. The nucleolytic activity of NucA is completely inhibited at equimolar concentrations of nuclease and in hibitor. An ultracentrifugation analysis showed that NucA rind NuiA fo rm a 1:1 complex. The interaction of NucA with NuiA was also investiga ted by CD spectroscopy and revealed no major conformational changes up on complex formation of the two proteins.