DNA helicase

 

PcrA DNA helicase is a 3'-5' DNA helicase. It is a single-stranded DNA (ssDNA)-dependent ATPase that uses the free energy of ATP hydrolysis to unwind duplex DNA. PcrA is known to be essential in some species, such as Bacillus subtilis and Staphylococcus aureus, with roles in DNA repair and rolling circle replication. This annotation covers the ATPase activity of PcrA.

The conformational changes brought about through ATP binding, hydrolysis and subsequent release allow PcrA to bind to DNA and unwind the helix. In particular a Glutamine residue that binds to the gamma phosphate of ATP is part of the motif III switch which bridges the ATPase domain and the DNA binding domain. So the binding of the gamma phosphate and its cleavage from the ATP in the ATPase domain can result in conformational changes in the DNA binding domain which enables PcrA to bind to DNA.

 

Reference Protein and Structure

Sequence
P56255 UniProt (3.6.4.12) IPR005751 (Sequence Homologues) (PDB Homologues)
Biological species
Geobacillus stearothermophilus (Bacteria) Uniprot
PDB
1qhg - STRUCTURE OF DNA HELICASE MUTANT WITH ADPNP (2.5 Å) PDBe PDBsum 1qhg
Catalytic CATH Domains
3.40.50.300 CATHdb (see all for 1qhg)
Cofactors
Magnesium(2+) (1)
Click To Show Structure

Enzyme Reaction (EC:3.6.4.12)

ATP(4-)
CHEBI:30616ChEBI
+
water
CHEBI:15377ChEBI
→
ADP(3-)
CHEBI:456216ChEBI
+
hydron
CHEBI:15378ChEBI
+
hydrogenphosphate
CHEBI:43474ChEBI
Alternative enzyme names: 3' to 5' DNA helicase, 3'-5' DNA helicase, 3'-5' PfDH, 5' to 3' DNA helicase, AvDH1, BACH1 helicase, BcMCM, BLM protein, BRCA1-associated C-terminal helicase, CeWRN-1, Dbp9p, DmRECQ5, DNA helicase 120, DNA helicase A, DNA helicase E, DNA helicase II, DNA helicase III, DNA helicase RECQL5', DNA helicase VI, DnaB, DnaB helicase E1, Helicase HDH IV, Hel E, Helicase DnaB, Helicase domain of bacteriophage T7 gene 4 protein helicase, PcrA helicase, UvrD, HHcsA, Hmi1p, HPif1, MCM helicase, MCM protein, MER3 helicase, MER3 protein, MPH1, PcrA, PDH120, PfDH A, Pfh1p, PIF1,

Enzyme Mechanism

Introduction

PcrA uses Glu 224 as a general base to deprotonate the water molecule that attacks the gamma phosphate of ATP. In addition, Lys 37,Arg 387, Arg 610, Gln 254 and an Mg2+ ion contact the triphosphate chain of ATP and stabilise negative charge in the transition state.

Catalytic Residues Roles

UniProt PDB* (1qhg)
Asp223, Thr38 Asp223A, Thr38A Form Mg2+ binding site electrostatic stabiliser
Lys37 Lys37A Interacts with the beta phosphate of ATP; stabilises negative charge in the transition state. electrostatic stabiliser
Glu224 Glu224A Deprotonates the water molecule that attacks the gamma phosphate of ATP. proton acceptor
Gln254, Arg610 Gln254A, Arg610A Polarises the gamma phosphate of ATP, stabilising negative charge in the transition state. 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, native state of enzyme is not regenerated

References

  1. Soultanas P et al. (1999), J Mol Biol, 290, 137-148. DNA binding mediates conformational changes and metal ion coordination in the active site of PcrA helicase. DOI:10.1006/jmbi.1999.2873. PMID:10388562.
  2. Mhashal AR et al. (2016), J Mol Model, 22, 54-. Probing the ATP-induced conformational flexibility of the PcrA helicase protein using molecular dynamics simulation. DOI:10.1007/s00894-016-2922-3. PMID:26860503.
  3. Caruthers JM et al. (2002), Curr Opin Struct Biol, 12, 123-133. Helicase structure and mechanism. DOI:10.1016/S0959-440X(02)00298-1.
  4. Geourjon C et al. (2001), Trends Biochem Sci, 26, 539-544. A common mechanism for ATP hydrolysis in ABC transporter and helicase superfamilies. DOI:10.1016/S0968-0004(01)01907-7.
  5. Velankar SS et al. (1999), Cell, 97, 75-84. Crystal Structures of Complexes of PcrA DNA Helicase with a DNA Substrate Indicate an Inchworm Mechanism. DOI:10.1016/s0092-8674(00)80716-3. PMID:10199404.
  6. Dillingham MS et al. (1999), Nucleic Acids Res, 27, 3310-3317. Site-directed mutagenesis of motif III in PcrA helicase reveals a role in coupling ATP hydrolysis to strand separation. DOI:10.1093/nar/27.16.3310.

Step 1. A water molecule is activated for nucleophilic attack through deprotonation by Glu 224. The nucleophile then attacks the gamma phosphate resulting in the cleavage of the phosphoanhydride bond.

Download: Image, Marvin File

Catalytic Residues Roles

Residue Roles
Lys37A electrostatic stabiliser
Arg610A electrostatic stabiliser
Asp223A electrostatic stabiliser
Gln254A electrostatic stabiliser
Thr38A metal ligand
Glu224A proton acceptor

Chemical Components

proton transfer, ingold: bimolecular nucleophilic substitution, overall reactant used, overall product formed, native state of enzyme is not regenerated
Download: Image, Marvin File

Step 1. A water molecule is activated for nucleophilic attack through deprotonation by Glu 224. The nucleophile then attacks the gamma phosphate resulting in the cleavage of the phosphoanhydride bond.

Products.

Contributors

Steven Smith, Gemma L. Holliday, Charity Hornby