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 Anabaena sp. strain PCC 7120, in order to characterize these proteins in detail. CD 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 deltaG0H2O = 63.4 J x mol(-1)residue, being slightly more stable than its target NucA with delta deltaG0H2O = 46.3 J x mol(-1)residue. The nuclease requires divalent metal ions as cofactors, the optimum concentration being 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+ > or = Ni2+ >> or Ca2+ = Cd2+ at a concentration of 5 mM. Nuclease activity decreases with increasing concentration of monovalent salt. The activity of NucA 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 x 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 Km < or = 0.1 mg x ml(-1) and k(cat) approximately 1000 s(-1). As other non-specific nucleases, NucA exhibits sequence preferences, similar to the related Serratia nuclease, NucA avoids cleavage of d(A) x d(T) tracts. The nucleolytic activity of NucA is completely inhibited at equimolar concentrations of nuclease and inhibitor. An ultracentrifugation analysis showed that NucA and 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.
Meiss
G
G
Institut für Biochemie, Justus-Liebig-Universität Giessen, Germany. Gregor.Meiss@chemie.bio.uni-giessen.de
Franke
I
I
Gimadutdinow
O
O
Urbanke
C
C
Pingoud
A
A
D000595:Amino Acid Sequence; D017033:Anabaena / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D001483:Base Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D002942:Circular Dichroism; D019976:Cloning, Organism; D017931:DNA Primers; D004720:Endonucleases; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D010726:Phosphodiesterase Inhibitors / Q000737:chemistry / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012705:Serratia / Q000201:enzymology; D013379:Substrate Specificity
D001426:Bacterial Proteins; D002413:Cations, Divalent; D017931:DNA Primers; C106206:NuiA protein, Anabaena; D010726:Phosphodiesterase Inhibitors; D011994:Recombinant Proteins; D004720:Endonucleases; C085858:sugar-nonspecific nuclease; D010727:Phosphoric Diester Hydrolases