Intron-encoded endonuclease I-PpoI
I-PpoI is a homing endonuclease of the His-Cys family. It is encoded by the nuclear 26S rRNA gene of Physarum polycephalum and cleaves a 16bp homing site to generate four-base 3' overhangs. It requires divalent metal cations, but a wide range are accommodated.
Reference Protein and Structure
- Sequence
-
Q94702
(3.1.-.-)
(Sequence Homologues)
(PDB Homologues)
- Biological species
-
Physarum polycephalum (Slime mold)
- PDB
-
1cz0
- INTRON ENCODED HOMING ENDONUCLEASE I-PPOI/DNA COMPLEX LACKING CATALYTIC METAL ION
(2.1 Å)
- Catalytic CATH Domains
-
3.90.75.10
(see all for 1cz0)
- Cofactors
- Zinc(2+) (1)
Enzyme Mechanism
Introduction
The metal (likely to be magnesium in the native enzyme but is shown as Zinc in this pdb) is bound by Asn 119, a non-bridging oxygen of the scissile phosphate group, and the bridging 3' oxygen of the scissile bond. Zn(II) forces the DNA into a strained conformation. A water molecule, activated by His 98 acting as a base, performs inline attack on the scissile phosphate. The backbone of Cys 105 may increase the pKa of His 98 by hydrogen bonding to the protonated nitrogen of His 98. Zn(II) and His 98 stabilise the charge on the pentacovalent transition state. The transition state collapses with the 3' hydroxylate leaving group being stabilised by Zn(II) and Arg 61. Zn(II) acidifies a bound water molecule which can then donate a proton to the 3' hydroxylate product.
Catalytic Residues Roles
| UniProt | PDB* (1cz0) | ||
| Asn119 | Asn119(118)A(C) | Binds Metal ion (in this pdb it is Zinc) | metal ligand |
| His98 | His98(97)A(C) | His 98 activates the water molecule which performs nucleophilic, inline attack on the scissile phosphate group. His 98 also stabilises the charge build-up in the transition state. |
proton acceptor |
| Cys105 (main-C) | Cys105(104)A(C) (main-C) | The main chain carbonyl of Cys 105 hydrogen bonds to His 98, increasing the pKa of His 98. | electrostatic stabiliser |
| Arg61 | Arg61(60)A(C) | Arg 61 appears to stabilise the cleavage products, driving equilibrium over to the product side and preventing religation. | electrostatic stabiliser |
Chemical Components
proton transfer, bimolecular nucleophilic addition, coordination to a metal ion, intermediate formation, overall reactant used, unimolecular elimination by the conjugate base, decoordination from a metal ion, intermediate collapse, overall product formedReferences
- Galburt EA et al. (1999), Nat Struct Biol, 6, 1096-1099. A novel endonuclease mechanism directly visualized for I-PpoI. DOI:10.1038/70027. PMID:10581547.
Step 1. His98 abstracts a proton from a water which activates it to nucleophilically attack the phosphate.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Arg61(60)A(C) | electrostatic stabiliser |
| Cys105(104)A(C) (main-C) | electrostatic stabiliser |
| Asn119(118)A(C) | metal ligand |
| His98(97)A(C) | proton acceptor |
Chemical Components
proton transfer, ingold: bimolecular nucleophilic addition, coordination to a metal ion, intermediate formation, overall reactant used
Step 2. The 3' oxygen is protonated by a zinc bound water which initiates an elimination from the oxyanion which results in the cleavage of the sugar-phosphate bond.
Download: Image, Marvin FileCatalytic Residues Roles
| Residue | Roles |
|---|---|
| Arg61(60)A(C) | electrostatic stabiliser |
| Cys105(104)A(C) (main-C) | electrostatic stabiliser |
| Asn119(118)A(C) | metal ligand |
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