Micrococcal nuclease

 

Staphyloccal nuclease is a Ca2+ activated phosphodiesterase which catalyses the hydrolysis of both DND and RNA at the 5' position of the phophodiester bond to yield a 3'-mononucleotides and polynucleotides.

 

Reference Protein and Structure

Sequence
P00644 UniProt (3.1.31.1) IPR016071 (Sequence Homologues) (PDB Homologues)
Biological species
Staphylococcus aureus (Bacteria) Uniprot
PDB
2sns - STAPHYLOCOCCAL NUCLEASE. PROPOSED MECHANISM OF ACTION BASED ON STRUCTURE OF ENZYME-THYMIDINE 3(PRIME),5(PRIME)-BIPHOSPHATE-CALCIUM ION COMPLEX AT 1.5-ANGSTROMS RESOLUTION (1.5 Å) PDBe PDBsum 2sns
Catalytic CATH Domains
2.40.50.90 CATHdb (see all for 2sns)
Cofactors
Calcium(2+) (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:3.1.31.1)

water
CHEBI:15377ChEBI
+
single-stranded DNA
CHEBI:9160ChEBI
→
5'-end 2'-deoxyribonucleotide(2-) residue
CHEBI:136412ChEBI
+
hydron
CHEBI:15378ChEBI
+
2'-deoxynucleoside 3'-monophosphate(2-)
CHEBI:131705ChEBI
Alternative enzyme names: Staphylococcus aureus nuclease, Staphylococcus aureus nuclease B, Micrococcal endonuclease, Nuclease T, Nuclease T', Ribonucleate (deoxynucleate) 3'-nucleotidohydrolase, Spleen endonuclease, Spleen phosphodiesterase, Staphylococcal nuclease, Thermonuclease,

Enzyme Mechanism

Introduction

The hydrolysis proceeds via direct nucleophilic attack on phosphate with formation of a five-coordinate, trigonal bipyramidal transition state or meta-stable intermediate followed by breakdown to from the product. The attacking nucleophile is a water molecule coordinated to the Ca2+ ion. Ca2+ facilitates the generation of hydroxide ion to allow its nucleophilic attack on the phosphate group. It also stabilises the transition state. Arg35 and Arg87 stabilise the transition state via bidentate hydrogen bonding. Arg87 is also the general acid that protonates the 5'-hydroxyl leaving group.

Catalytic Residues Roles

UniProt PDB* (2sns)
Asp122, Asp103, Thr123 (main-N) Asp40A, Asp21A, Thr41A (main-N) Coordinate the calcium ion. metal ligand
Arg169 Arg87A Stabilises the transition state via bidentate hydrogen bonding and acts as an acid to protonate the 5'-hydroxyl leaving group. hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor, electrostatic stabiliser
Glu125 Glu43A Glu43 is acting as a general Acid/Base hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Arg117 Arg35A Stabilises the negatively charged transition state via bidentate hydrogen bonding. hydrogen bond donor, electrostatic stabiliser
*PDB label guide - RESx(y)B(C) - RES: Residue Name; x: Residue ID in PDB file; y: Residue ID in PDB sequence if different from PDB file; B: PDB Chain; C: Biological Assembly Chain if different from PDB. If label is "Not Found" it means this residue is not found in the reference PDB.

Chemical Components

proton transfer, bimolecular nucleophilic substitution, overall reactant used, overall product formed, hydrolysis, rate-determining step, native state of enzyme regenerated, inferred reaction step

References

  1. Weber DJ et al. (1992), Proteins, 13, 275-287. NMR docking of a substrate into the X-ray structure of staphylococcal nuclease. DOI:10.1002/prot.340130402. PMID:1518799.
  2. Libson AM et al. (1994), Biochemistry, 33, 8007-8016. Crystal structures of the binary Ca2+ and pdTp complexes and the ternary complex of the Asp21-->Glu mutant of staphylococcal nuclease. Implications for catalysis and ligand binding. DOI:10.2210/pdb1enc/pdb. PMID:8025105.
  3. Hale SP et al. (1993), Biochemistry, 32, 7479-7487. Mechanism of the reaction catalyzed by staphylococcal nuclease: Identification of the rate-determining step. DOI:10.1021/bi00080a020. PMID:8338846.
  4. Pourmotabbed T et al. (1990), Biochemistry, 29, 3677-3683. Kinetic and conformational effects of lysine substitutions for arginines 35 and 87 in the active site of staphylococcal nuclease. DOI:10.1021/bi00467a013. PMID:2111164.
  5. Serpersu EH et al. (1987), Biochemistry, 26, 1289-1300. Kinetic and magnetic resonance studies of active-site mutants of staphylococcal nuclease: factors contributing to catalysis. DOI:10.1021/bi00379a014. PMID:3567171.

Step 1. Glu43 deprotonates the calcium activated water molecule, which then attacks the DNA phosphate bond in a nucleophilic substitution. The 3' DNA hydroxyl protonates from Arg87.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu43A hydrogen bond acceptor
Arg35A hydrogen bond donor, electrostatic stabiliser
Arg87A hydrogen bond donor, electrostatic stabiliser
Asp21A metal ligand
Asp40A metal ligand
Thr41A (main-N) metal ligand
Glu43A proton acceptor
Arg87A proton donor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic substitution, overall reactant used, overall product formed, hydrolysis, rate-determining step

Step 2. Arg87 deprotonates water, and water deprotonates Glu43 in an inferred step.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu43A hydrogen bond donor
Arg87A hydrogen bond acceptor
Glu43A proton donor
Arg87A proton acceptor

Chemical Components

proton transfer, native state of enzyme regenerated, inferred reaction step
Download: Image, Marvin File

Step 1. Glu43 deprotonates the calcium activated water molecule, which then attacks the DNA phosphate bond in a nucleophilic substitution. The 3' DNA hydroxyl protonates from Arg87.

Step 2. Arg87 deprotonates water, and water deprotonates Glu43 in an inferred step.

Products.

Contributors

Gemma L. Holliday, Daniel E. Almonacid, Mei Leung, James Willey