Russian Journal of Bioorganic Chemistry, Vol. 27, No. 6, 2001, đ . 417
X-Ray Analysis of the Magnesium-containing Endonuclease from Serratia marcescens
S. V. Shlyapnikov*, V. V. Lunin**, E. V. Blagova**, M. Pembrandt***, Ch. Betzel***, and A. M. Mikhailov** 1
*Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow, 117984 Russia, **Shubnikov Institute of Crystallography, Russian Academy of Sciences, Leninskii pr. 59, Moscow, 117333 Russia, ***Institute of Physiological Chemistry, Notkestrasse 85, Hamburg, 22603 Germany
Abstract: The three-dimensional crystal structure of the DNA/RNA nonspecific endonuclease from Serratia marcescens was refined at the resolution of 1.07 Å to R factor of 12.4% and R free factor of 15.3% using the anisotropic approximation. The structure includes 3924 non-hydrogen atoms, 715 protein-bound water molecules, and a Mg2+ ion in each binding site of each subunit of the nuclease homodimeric globular molecule. The 3D topological model of the enzyme was revealed, the inner symmetry of the monomers in its N- and C-termini was found, and the local environment of the magnesium cofactor in the nuclease active site was defined. Mg2+ ion was found to be bound to the Asn119 residue and surrounded by five associated water molecules that form an octahedral configuration. The coordination distances for the water molecules and the O 1 atom of Asn119 were shown to be within the range of 2.01–2.11 Å. The thermal factors for the magnesium ion in subunits are 7.08 and 4.60 Å2, and the average thermal factors for the surrounding water molecules are 11.14 and 10.30 Å2, respectively. The region of the nuclease subunit interactions was localized, and the alternative side chain conformations were defined for 51 amino acid residues of the nuclease dimer.
Key words: atomic structure of holoenzyme, Serratia marcescens extracellular endonuclease, x-ray analysis
Russian Journal of Bioorganic Chemistry, Vol. 28, No. 1, 2002 đ. 20
A Comparative Structure–Function Analysis and Molecular Mechanism of Action of Endonucleases from Serratia marcescens and Physarum polycephalum
S. V. Shlyapnikov*, V. V. Lunin**, E. V. Blagova**, L. V. Abaturov*, M. Perbandt***, Ch. Betzel***, and A. M. Mikhailov** 1
*Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow, 117984 Russia, **Shubnikov Institute of Crystallography, Russian Academy of Sciences, Leninskii pr. 59, Moscow, 117333 Russia ,***Institute of Physiological Chemistry, Notkestrasse 85, Hamburg, 22603 Germany
Abstract: Structural and functional characteristics were compared for wild-type nuclease from Serratia marcescens, which belongs to the family of DNA/RNA nonspecific endonucleases, its mutational forms, and the nuclease I-PpoI from Physarum polycephalum, which is a representative of the Cys-His box-containing subgroup of the superfamily of extremely specific intron-encoded homing DNases. Despite the lack of sequence homology and the overall different topology of the Serratia marcescens and I-PpoI nucleases, their active sites have a remarkable structural similarity. Both of them have a unique magnesium atom in the active site, which is a part of the coordinatively bonded water–magnesium complex involved in their catalytic acts. In the enzyme–substrate complexes, the Mg2+ ion is chelated by an Asp residue, coordinates two oxygen atoms of DNA, and stabilizes the transition state of the phosphate anion and 3'-OH group of the leaving nucleotide. A new mechanism of the phosphodiester bond cleavage, which is common for the Serratia marcescens and I-PpoI nucleases and differs from the known functioning mechanism of the restriction and homing endonucleases, was proposed. It presumes a His residue as a general base for the activation of a non-cluster water molecule at the nucleophilic in line displacement of the 3'-leaving group. A strained metalloenzyme–substrate complex is formed during hydrolysis and relaxes to the initial state after the reaction.
Key words: endonucleases, mechanism of action, properties, structure, Physarum polycephalum, Serratia marcescens