Serratia endonucleases

Table I. Amino acid alignment of nucleases homologous to the Serratia endonuclease.

The Serratia endonuclease is well suited for X-ray studies. Crystals can be grown from PEG or ammonium sulfate and diffract to very high resolution (beyond 0.88 A at 100 K). The nuclease crystallizes in space group P2i2i2 with one homodimer (490 amino acids / 53.4 kDa) in the asymmetric unit of e cell (a = 106.7 A, b = 74.5 A, c = 68.9 A). 

Figure 2. Precession photograph of the OKL zone from crystals of Serratia endonuclease demonstrating strong pseudo-centering.

Inspection of the Serratia endonuclease structure reveals that its fold differs from other nucleases whose structures are known (28). In the SCOP protein classification system it is listed as the only member of its family (29) In the CATH protein classification system, it is listed as the only member of its tertiary group (20). The dimeric enzyme is almost cylindrical with overall dimensions of 30 A X 35 A X 90 A (Fig. 3). Each monomer consists of a central six-stranded anti-parallel (3-sheet flanked by a-helices on both sides. One long helix, termed... 

Getting to this point in our study of Serratia endonuclease represented a significant advance to us because we thought we imderstood the structural basis... 

Like the Serratia endonuclease, the enzyme cleaves the 3 0-P bond in DNA. Also like the Serratia endonuclease, the enzyme prefers magnesium over other divalent metal ions. VPpol endonuclease is a domain swapped dimer with 163 amino acids per monomer. In the absence of salt, its kcat/KM appraoches 10. This number is dominated by the Kii/value which is close to 1 x 10 M. The kcat value is highly dependent on ionic strength, but without salt it has been reported to be 0.046 min (39). 

Figure 11. Superposition of the Serratia endonuclease with FPpoI endonuclease (a) Overall view showing both dimers and the FPpoI cleaved DMA oligomer, (b) Stereo view of the active site including the bound Mg - water cluster and essential histidine and asparagine residues. (Adaptedfrom reference 21)...

Miller, M. D. Cai, J. Krause, K. L., The active site of Serratia endonuclease contains a conserved magnesium- water cluster. J. Mol Biol 1999,288, 975-87. 

Miller, M. Krause, K., Identification of the Serratia endonuclease dimer structural basis and implications for catalysis. Protein Sci 1996, 5,24-33. 

As phosphorothioate oligonucleotides are widely used as antisense agents, there have been a number of reports on their mode of action. Using Rp and Sp phosphorothioate internucleotide linkages in ODNs, it was shown that Serratia marcescens endonuclease hydrolyses the Rp phosphorothioate bond with inversion of configuration at phosphorus. The presence of an Sp phosphorothioate... 

The final MALDI TOF analysis of 35 showed the presence of two ions m/z 1626 and 1628 in the expected ratio, thus indicating the retention of isotopic enrichment. This observation provided evidence that the degradation of 33 by Serratia marcescens endonuclease proceeds with inversion of configuration at the phosphorus atom [41]. 

Friedhoff, P. Gimadutdinow, O. Pingoud, A., Identification of catalytically relevant amino acids of the extracellular Serratia marcescens endonuclease by alignment-guided mutagenesis. Nucleic Acids Res 1994,22, 3280-7. 

Friedhoff, P. Franke, I. Krause, K L. Pingoud, A., Cleavage experiments with deoxythymidine 3 -5 -bis-(p-nitrophenyl phosphate) suggest that the homing endonuclease l-Ppol follows the same mechanism of phosphodiester bond hydrolysis as the non-specific Serratia nuclease. FEBS Lett 1999,443,209-214. 

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