Atomic structure of theSerratia marcescensendonuclease at 1.1 Å resolution and the enzyme reaction mechanism

Abstract

The three-dimensional crystal structure of Serratia marcescens endonuclease has been refined at 1.1 A resolution to an R factor of 12.9% and an R(free) of 15.6% with the use of anisotropic temperature factors. The model contains 3694 non-H atoms, 715 water molecules, four sulfate ions and two Mg(2+)-binding sites at the active sites of the homodimeric protein. It is shown that the magnesium ion linked to the active-site Asn119 of each monomer is surrounded by five water molecules and shows an octahedral coordination geometry. The temperature factors for the bound Mg(2+) ions in the A and B subunits are 7.08 and 4.60 A(2), respectively, and the average temperature factors for the surrounding water molecules are 12.13 and 10.3 A(2), respectively. In comparison with earlier structures, alternative side-chain conformations are defined for 51 residues of the dimer, including the essential active-site residue Arg57. A plausible mechanism of enzyme function is proposed based on the high-resolution S. marcescens nuclease structure, the functional characteristics of the natural and mutational forms of the enzyme and consideration of its structural analogy with homing endo-nuclease I-PpoI.

A novel secreted endonuclease from Culex quinquefasciatussalivary glands

“…S. marcescens endonuclease is a well studied example of this family for which structural information is available (Miller et al, 1999;Shlyapnikov et al, 2000). CuquEndo shares some structural and enzymatic similarities with S. marcescens endonuclease.…”

“…His89 of S. marcescens (CuquEndoHis171) nuclease and the magnesium water cluster are directly involved in the phosphodiester bond cleavage of the DNA substrate. The metal ion could serve to activate the electrophile, stabilize the transition state, and protonate the leaving group (Shlyapnikov et al, 2000).…”

“…A Lys exists in the mosquito sequence within one residue of the missing Arg. The importance of these residues for catalysis has been previously studied in detail (Miller et al, 1999;Shlyapnikov et al, 2000).…”

“…According to Shlyapnikov et al (Shlyapnikov et al, 2000), the structure of S. marcescens endonuclease -which has been extensively (biochemically and structurally) studied -reveals essential inner symmetry. The protein secondary structure can be divided into three regions: (i) a central ␤ sheet formed by six antiparallel ␤ strands, which is flanked by (ii) an ␣ domain consisting of four short ␣ helices and joining the N-and C-terminal parts of the molecule on one side, and (iii) an ␣/␤ conformational domain forming the upper layer of the globule, which consists of a ds ␤ sheet embraced by a symmetrical system of eight helical fragments.…”

Rescue of the activity of HNH nuclease mutants - towards controlled enzymes for gene therapy

“…Examples are, e.g. the Vvn endonuclease (PDB ID 1OUP) [87], Sm endonuclease (PDB ID 1G8T) [127], Nuclease A (PDB ID 1ZM8) [86] NColE7 (PDB ID 3FBD) [108]. R57 has been identified by a mutational analysis in the Serratia nuclease to be essential for catalysis [74,128].…”