Biochemical characterization of Anabaena sp. strain PCC 7120 non-specific 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 inhibitor from Anabuena sp. strain PCC 7120, in order to characterize these proteins in detail to spectroscopy revealed that NucA has a similar secondary-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 NuiA 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 stable than its target NucA with Delta Delta(H2O)(0) = 46.3 J mol(residue)(-1). The nuclease requires divalent metal ions as cofactors. the optimum concentration bring around 5 mM for Mn2+ or Mg2+. The order of effectiveness of various divalent cations to function as cofactors for the hydrolytic activity of NucA is Mn2+ = CO2+ > Mg2+ greater than or equal to Ni2+ much greater than Ca2+ = Cd2+ at a concentration of 5 mM. Nuclease activity decreases with increasing concentration of monovalent salt. The activity of NncA shows a pH optimum at pH 5.5-7.5. The temperature optimum is around 35 degrees C, the activation energy was calculated to be 53 kJ mol(-1), The specific activity of the nuclease towards high molecular-mass DNA is 8.4 x 10(6) Kunitz-units.mg(-1), which means that NucA is one of the most active nucleases known. Kinetic constants for the cleavage of various DNA and RNA substrates by NucA are 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 inhibitor. An ultracentrifugation analysis showed that NucA rind NuiA form a 1:1 complex. The interaction of NucA with NuiA was also investigated by CD spectroscopy and revealed no major conformational changes upon complex formation of the two proteins.