SERRATIA MARCESCENS ENDONUCLEASE. REFINED STRUCTURE OF ENZYME AT 1.7A RESOLUTION AND MECHANIZM OF ITS ACTION

V.V.Lunin¹, V.Yu.Lunin², E.V.Blagova¹, S.V.Shlyapnikov³, V.M.Levdikov¹, A.M.Mikhailov1

¹Institute of Crystallography, Russian Academy of Sciences, Leninski pr. 59, 117333 Moscow, Russia
²Institute of Mathematical Problems in Biology, Russian Academy of Sciences, 142292 Pushchino, Moscow region, Russia
³Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov st. 32, 117984 Moscow, Russia

Keywords: endonuclease, X-ray structure

Serratia marcescens nuclease is an extracellular sugar non-specific endonuclease and catalyzes the splitting of the 3'O-P bonds of both single- and double-stranded DNA and RNA, producing mono-, di-, tri- and tetra-5'-monophosphate-terminated nucleotides. The nuclease is a dimer of identical subunits of 245 amino acid residues.

X-ray data do 1.7 A resolution were collected on the EMBL X11 beam line during the parasitic mode of operation of DORIS storage ring with use of imagine-plate scanner developed by J.Hendrix and A. Lentfer.

The model of Sm nuclease deposited to PDB by Miller et al. was used to obtain a start model. This model at 2.1A resolution was refined to 16.8% value of crystallographic R-factor. The start value of R-factor in the same resolution zone but calculated with new experimental data was 32.4% and the exclusion of water molecules increased the R-factor value up to 36%.

The model has been refined at 1.7A resolution to the R-factor of 17.3% and R-free of 22.2%, with RMS 0.011A for bond lengths (max. 0.07A), 1.8 for angles values (max. 11). The final model consists of 3678 non-hydrogen atoms and 443 water molecules.

The analysis of refined model shows the rich secondary structure in the second domain of enzyme, that earlier was called as «long disordered loop». Considering the final model enabled us to suggest a catalytic mechanism of enzyme action based on a concerted participation of the catalytically important amino acid residues of the enzyme active site.