Nuclease

 

Serratia endonuclease is of particular interest because of its broad specificity, high activity and chemical stability. In addition antiviral and anti-tumour properties have been attributed to this enzyme.

It acts without any apparent base preference and cleaves both single- and double - stranded DNA and RNA. Functionally, the enzyme is similar to DNase I as they are both magnesium-dependent endonucleases that catalyse the cleavage of 3'O-P bond. Although functional similarities exist, Serratia nuclease structure contrasts sharply with the protein fold observed in DNase I.

Magnesium is important for activity, absence of magnesium significantly reduces the activity of the nuclease. The nuclease DNA-binding site is located between two main amino and carboxy-terminal structural domains of the protein, the DNA binding cleft is flanked by two rows of positively charged amino acids that could interact with about one full turn of DNA B-form. This cleft contains among other cationic amino acids, the invariant catalytic residues of ARG 87 and ARG 131, and also contains HIS 89 and GLU 127.

 

Reference Protein and Structure

Sequence
P13717 UniProt (3.1.30.2) IPR001604 (Sequence Homologues) (PDB Homologues)
Biological species
Serratia marcescens (Bacteria) Uniprot
PDB
1ql0 - Sm Endonuclease from Seratia marcenscens at atomic resolution (1.1 Å) PDBe PDBsum 1ql0
Catalytic CATH Domains
3.40.570.10 CATHdb (see all for 1ql0)
Cofactors
Magnesium(2+) (1), Water (1) Metal MACiE
Click To Show Structure

Enzyme Reaction (EC:3.1.30.2)

water
CHEBI:15377ChEBI
+
single-stranded DNA
CHEBI:9160ChEBI
→
2'-deoxynucleoside 3'-monophosphate(2-)
CHEBI:131705ChEBI
+
hydron
CHEBI:15378ChEBI
+
5'-end 2'-deoxyribonucleotide(2-) residue
CHEBI:136412ChEBI
Alternative enzyme names: Barley nuclease, Endonuclease (Serratia marcescens), Nucleate endonuclease, Plant nuclease I,

Enzyme Mechanism

Introduction

HIS 89 and GLU 127 are both potential participants in the nuclease reaction. It has been postulated that HIS 89 serves as a general acid and GLU 127 serves as either a nucleophile or a general base. Phosphodiesters are hydrolysed via nucleophilic attack by a water molecule forming a penta-coordinated trigonal bipyramid intermediate.

Catalytic Residues Roles

UniProt PDB* (1ql0)
His110 His89(85)A Acts as a general acid/base, deprotonates the nucleophilic water hydrogen bond acceptor, hydrogen bond donor, proton acceptor, proton donor
Asn140 Asn119(115)A Binds the Mg(II) ion and helps stabilise the negatively charged transition state. activator, hydrogen bond acceptor, hydrogen bond donor, metal ligand
Glu148 Glu127(123)A Part of the Mg(II) binding site and acts as a general acid/base. hydrogen bond acceptor, metal ligand, proton acceptor, proton donor, electrostatic stabiliser
Arg78 Arg57(53)A Stabilizes the transition state and the leaving group as well electrostatic stabiliser
Asn131 Asn110(106)A Activates and stabilises the general acid/base His89 in the DNA bound state. In the apo enzyme, Asn110 is replaced with Arg87 increase basicity, electrostatic stabiliser, increase acidity
*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

bimolecular nucleophilic addition, overall reactant used, intermediate formation, proton transfer, rate-determining step, unimolecular elimination by the conjugate base, intermediate collapse, intermediate terminated, overall product formed, proton relay, native state of enzyme regenerated, inferred reaction step

References

  1. Shlyapnikov SV et al. (2000), Acta Crystallogr D Biol Crystallogr, 56, 567-572. Atomic structure of theSerratia marcescensendonuclease at 1.1 Å resolution and the enzyme reaction mechanism. DOI:10.1107/s090744490000322x. PMID:10771425.
  2. Chen C et al. (2009), J Phys Chem B, 113, 511-521. Advantage of Being a Dimer for Serratia marcescens Endonuclease. DOI:10.1021/jp8057838. PMID:19053714.
  3. Koziołkiewicz M et al. (2001), Bioorg Med Chem, 9, 2403-2409. Stereochemistry of cleavage of internucleotide bonds by Serratia marcescens endonuclease. PMID:11553482.
  4. Miller MD et al. (1999), J Mol Biol, 288, 975-987. The active site of Serratia endonuclease contains a conserved magnesium-water cluster. DOI:10.1006/jmbi.1999.2729. PMID:10329193.
  5. Lunin VY et al. (1997), FEBS Lett, 412, 217-222. Three-dimensional structure ofSerratiamarcescensnuclease at 1.7 Å resolution and mechanism of its action. DOI:10.1016/s0014-5793(97)00512-7. PMID:9257723.
  6. Klabunde T et al. (1996), J Mol Biol, 259, 737-748. Mechanism of Fe(III) – Zn(II) Purple Acid Phosphatase Based on Crystal Structures. DOI:10.1006/jmbi.1996.0354. PMID:8683579.
  7. Miller MD et al. (1994), Nat Struct Biol, 1, 461-468. 2.1 Å structure of Serratia endonuclease suggests a mechanism for binding to double-stranded DNA. DOI:10.1038/nsb0794-461. PMID:7664065.

Step 1. His89 deprotonates water, which initiates a nucleophilic attack on the phosphate of the DNA, forming a pentavalent intermediate in an addition reaction.

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Catalytic Residues Roles

Residue Roles
Glu127(123)A hydrogen bond acceptor
Asn119(115)A hydrogen bond acceptor, hydrogen bond donor, activator
His89(85)A hydrogen bond acceptor
Asn110(106)A increase basicity
Arg57(53)A electrostatic stabiliser
Asn119(115)A metal ligand
Glu127(123)A metal ligand
His89(85)A proton acceptor

Chemical Components

ingold: bimolecular nucleophilic addition, overall reactant used, intermediate formation, proton transfer, rate-determining step

Step 2. The pentavalent intermediate collapses, eliminating the 3'-hydroxyl of the DNA, which deprotonates water, which in turn deprotonates Glu127.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Glu127(123)A hydrogen bond acceptor, electrostatic stabiliser
Asn119(115)A hydrogen bond acceptor, hydrogen bond donor
His89(85)A hydrogen bond donor
Asn119(115)A metal ligand
Glu127(123)A metal ligand
Asn110(106)A electrostatic stabiliser
Arg57(53)A electrostatic stabiliser
Glu127(123)A proton donor

Chemical Components

ingold: unimolecular elimination by the conjugate base, proton transfer, intermediate collapse, intermediate terminated, overall product formed, proton relay, native state of enzyme regenerated

Step 3. Inferred return step in which Glu abstracts a proton from water, and water abstracts a proton from His.

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Catalytic Residues Roles

Residue Roles
Asn119(115)A metal ligand
Glu127(123)A metal ligand
Asn110(106)A increase acidity
His89(85)A proton donor
Glu127(123)A proton acceptor

Chemical Components

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

Step 1. His89 deprotonates water, which initiates a nucleophilic attack on the phosphate of the DNA, forming a pentavalent intermediate in an addition reaction.

Step 2. The pentavalent intermediate collapses, eliminating the 3'-hydroxyl of the DNA, which deprotonates water, which in turn deprotonates Glu127.

Step 3. Inferred return step in which Glu abstracts a proton from water, and water abstracts a proton from His.

Products.

Contributors

Gemma L. Holliday, Gail J. Bartlett, Daniel E. Almonacid, Christian Drew, Craig Porter