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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% A helix and 20% β sheet, to the related Serratia nuclease, while NuiA represents a protein with a higher A-helical (29%) and β-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 ∆G 0 H2O ϭ 63.4 J · mol Ϫ1 residue , being slightly more stable than its target NucA with ∆∆G 0 H2O ϭ 46.3 J · mol Ϫ1residue . The nuclease requires divalent metal ions as cofactors, the optimum concentration being around 5 mM for Mn 2ϩ or Mg 2ϩ . The order of effectiveness of various divalent cations to function as cofactors for the hydrolytic activity of NucA is Mn 2ϩ ϭ Co 2ϩ Ͼ Mg 2ϩ у Ni 2ϩ ӷ Ca 2ϩ ϭ Cd 2ϩ 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 5.5Ϫ7.5. The temperature optimum is around 35°C, the activation energy was calculated to be 53 kJ mol Ϫ1 . The specific activity of the nuclease towards high molecularmass DNA is 8.4ϫ10 6 Kunitz-units · mg Ϫ1 , 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 р 0.1 mg · ml Ϫ1 and k cat Х 1000 s Ϫ1 . As 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 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.Keywords : nuclease ; steady-state kinetics ; nuclease inhibitor ; protein-protein interaction.NucA, a sugar non-specific nuclease of Anabaena sp. strain mologous nucleases can be found in Serratia marcescens [5, 6, 7], Streptococcus pneumoniae [8], in the yeast Saccharomyces PCC 7120 [1, 2] is a member of a family of highly active sugar non-specific endonucleases. These enzymes predominantly act cerevisiae [9, 10], in the mold Syncephalostrum racemosum [11], in Bos taurus [12] and in Mus musculus (Prats, E., Noel, as non-specific nucleases without exhibiting pronounced base preferences, although some of them cleave substrates in a se-M., Letourneau, J., Tiranti, V., Vaque, J., Debon, R., Zeviani, M., Cornudella, M. and Ruiz-Carrillo, A., unpublished results) quence-dependent and/or structure-dependent manner [3, 4] Schizosaccharomyces pombe (Murphy, L., McDoogall, R.,

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Alpha-aminoadipic and alpha-ketoadipic aciduria is an autosomal recessive inborn error of lysine, hydroxylysine, and tryptophan degradation. To date, DHTKD1 mutations have been reported in two alpha-aminoadipic and alpha-ketoadipic aciduria patients. We have now sequenced DHTKD1 in nine patients diagnosed with alpha-aminoadipic and alpha-ketoadipic aciduria as well as one patient with isolated alpha-aminoadipic aciduria, and identified causal mutations in eight. We report nine novel mutations, including three missense mutations, two nonsense mutations, two splice donor mutations, one duplication, and one deletion and insertion. Two missense mutations, one of which was reported before, were observed in the majority of cases. The clinical presentation of this group of patients was inhomogeneous. Our results confirm that alpha-aminoadipic and alpha-ketoadipic aciduria is caused by mutations in DHTKD1, and further establish that DHTKD1 encodes the E1 subunit of the alpha-ketoadipic acid dehydrogenase complex.

We have studied the mechanism of action of the Serratia nuclease using deoxythymidine 3',5'-bis+nitrophenyl-phosphate) as a substrate. A comparison of the activity with which the wild-type enzyme and several mutant enzymes attack this artificial substrate and herring sperm DNA, respectively, supports the suggestion that His89 is the general base and a Mg*+ ion bound to Glu'*' the general acid in the mechanism of phosphodiester bond hydrolysis by the Serratia nuclease, and that As@' directly participates in catalysis, for example by transition state stabilisation. Arg", A$' and Arg13', essential for nuclease activity, are not needed for cleavage of the artificial substrate, suggesting that they are involved in binding and positioning of nucleic acid substrates.

We show here that two nucleases, Serratia nuclease and I-PpoI, with contrasting specificities, i.e. non-specific vs. highly sequence specific, share a structurally similar active site region with conservation of the catalytically relevant histidine and asparagine residues. On the basis of a comparison of the available structures and biochemical data for wild type and mutant variants of Serratia nuclease and I-PpoI we propose that both enzymes have a common catalytic mechanism, a proposition that is supported by our finding that both enzymes accept deoxythymidine 3P,5P-bis-(p-nitrophenyl phosphate) as a substrate and cleave it in an identical manner. According to this mechanism a histidine residue functions as a general base and Mg P+ bound to an asparagine residue as a Lewis acid in phosphodiester bond cleavage.z 1999 Federation of European Biochemical Societies.

The extracellular endonucleases from Serratia marcescens and Anabaena sp. are members of a family of nonspecific endonucleases. In contrast to the monomeric Anabaena nuclease, the Serratia nuclease is a dimer of two identical subunits. To find out whether the two active sites of the Serratia nuclease function independently of each other and what the advantage of being a dimer for this enzyme might be, we produced (i) dimers in which the two subunits were cross-linked, (ii) heterodimers consisting of a wild type and an inactive mutant subunit which were also cross-linked, and (iii) monomeric variants which are unable to dimerize. The monomeric H184R variant and the cross-linked S140C variant exhibit the same activity as the wild type enzyme, while the cross-linked heterodimer with one inactive subunit shows only half of the activity of the wild type enzyme, demonstrating functional independence of the two subunits of the Serratia nuclease. On the other hand at low enzyme and substrate concentrations dimeric forms of the Serratia nuclease are relatively more active than monomeric forms or the monomeric Anabaena nuclease in cleaving polynucleotides, not, however, oligonucleotides, which is correlated with the ability of dimeric forms of the Serratia nuclease to form large enzyme-substrate networks with high molecular weight DNA and to cleave polynucleotides in a processive manner. We conclude that in the natural habitat of Serratia marcescens where the supply of nutrients may become growth limiting the dimeric nuclease can fulfil its nutritive function more efficiently than a monomeric enzyme.

The Serratia nuclease is a non-specific endonuclease which cleaves single-and double-stranded RNA and DNA. It is a member of a large family of related endonucleases, most of which are dimers of identical subunits, with the notable exception of the Anabaena nuclease which is a monomer. In order to find out whether the dimer state of the Serratia nuclease is essential for its function we have produced variants of this nuclease which based on the crystal structure (Miller, M.D. and Krause, K.L. (1996), Protein Science 5, 24^33) were expected to be unable to dimerise. We demonstrate here that these variants, H184A, H184N, H184T and H184R, are monomers and have the same secondary structure, stability towards chemical denaturation and activity as the wild-type enzyme. This allows to conclude that the dimeric state is not essential for the catalytic function of the Serratia nuclease. In contrast, the S179C variant which is also a monomer shows little activity, presumably because this amino acid substitution changes the structure of the enzyme.z 1998 Federation of European Biochemical Societies.