H+-transporting two-sector ATPase (V-type)

 

The homing endonuclease from S. cerevisiae, PI-SceI is able to catalyse the specific cleavage of double stranded DNA in order to allow the incorporation of a mobile intron into the allele. This enables non-mendelian propagation of certain introns without harming the target allele, as the introns are spliced out before functional mRNA is produced. It belongs to a large family of endonucleases all with the sequence LADLIDADG, and contains two active sites to allow simultaneous cleavage of both strands of DNA. The homing endonucleases in general are highly specific, and PI-SceI thus recognises a 31 base pair DNA sequence as its substrate, the longest specific sequence for a DNA binding protein yet discovered. This entry covers the active site which cleaves the top strand, running 5' to 3'.

 

Reference Protein and Structure

Sequence
P17255 UniProt (3.1.-.-, 7.1.2.2) IPR022878 (Sequence Homologues) (PDB Homologues)
Biological species
Saccharomyces cerevisiae S288c (Baker's yeast) Uniprot
PDB
1lws - Crystal structure of the intein homing endonuclease PI-SceI bound to its recognition sequence (3.5 Å) PDBe PDBsum 1lws
Catalytic CATH Domains
2.170.16.10 CATHdb 3.10.28.10 CATHdb (see all for 1lws)
Cofactors
Calcium(2+) (2)
Click To Show Structure

Enzyme Reaction (EC:7.1.2.2)

hydron
CHEBI:15378ChEBI
+
water
CHEBI:15377ChEBI
+
ATP(4-)
CHEBI:30616ChEBI
→
hydron
CHEBI:15378ChEBI
+
ADP(3-)
CHEBI:456216ChEBI
+
hydrogenphosphate
CHEBI:43474ChEBI
Alternative enzyme names: ATP synthase, F(1)-ATPase, F(o)F(1)-ATPase, H(+)-transporting ATPase, Bacterial Ca(2+)/Mg(2+) ATPase, Chloroplast ATPase, Coupling factors (F(o), F(1) and CF(1)), Mitochondrial ATPase, F(0)F(1)-ATPase, H(+)-transporting ATP synthase,

Enzyme Mechanism

Introduction

Protein splicing begins with an acyl rearrangement at the N-terminal junction where Cys 1 attacks the carbonyl carbon of the preceding residue forming a thioester intermediate. The nucleophilicity of the cysteine residue is increased by polarisation by Asn 76, which acts as a general base catalyst, and the transition state is stabilised by the backbone nitrogen of Gly 433. His 79 protonates the amide nitrogen of Cys 1 to promote breakdown of the tetrahedral intermediate to a thioester. Cys 455, activated by a zinc ion, nucleophilically attacks the same carbonyl carbon as Cys 1 causing release of Cys 1. Cyclisation of Asn 454 follows, with nucleophilic attack by the nitrogen activated by the carbonyl oxygen of Ile 434 on the carbonyl carbon, with the transition state stabilised by the zinc ion. On cleavage of the peptide bond His 453, activated by Zn, can act as a proton donor to Cys 455. Stabilisation is also provided by Asn 76 and Thr 78.

Catalytic Residues Roles

UniProt PDB* (1lws)
Gly716 (main-N) Gly433A(C) (main-N) Stabilises the transition state in attack of Cys 1 on the preceding residue. electrostatic stabiliser
Asn737 Asn454A(C) Nucleophilic attack involved in cyclisation. covalent catalysis
Ile717 (main-C) Ile434A(C) (main-C) Activates nitrogen of Asn 454 for cyclisation. activator
Lys686 Lys403A(C) Protonates the leaving group, facilitating collapse of the pentavalent phosphate transition state. proton shuttle (general acid/base)
Cys284 Cys1A(C) Attacks the carbonyl carbon of the preceding residue nucleophilically. This is later cleaved by the action of Cys455 (not in the representative crystal structure) covalent catalysis
His362 His79A(C) Protonates Cys 1 to facilitate bond cleavage. proton shuttle (general acid/base)
Thr361, Asn359 Thr78A(C), Asn76A(C) Involved in stabilisation. electrostatic stabiliser
Lys584 Lys301A(C) Protonates the leaving group to enable collapse of the pentavalent phosphate transition state liberating the product. proton shuttle (general acid/base)
*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

References

  1. Poland BW et al. (2000), J Biol Chem, 275, 16408-16413. Structural Insights into the Protein Splicing Mechanism of PI-SceI. DOI:10.1074/jbc.275.22.16408. PMID:10828056.
  2. Moure CM et al. (2002), Nat Struct Biol, 9, 764-770. Crystal structure of the intein homing endonuclease PI-SceI bound to its recognition sequence. DOI:10.1038/nsb840. PMID:12219083.
  3. Schöttler S et al. (2000), Biochemistry, 39, 15895-15900. Identification of Asp218 and Asp326 as the Principal Mg2+Binding Ligands of the Homing Endonuclease PI-SceI†,‡. DOI:10.1021/bi001775n. PMID:11123916.
  4. Christ F et al. (1999), EMBO J, 18, 6908-6916. The monomeric homing endonuclease PI-SceI has two catalytic centres for cleavage of the two strands of its DNA substrate. DOI:10.1093/emboj/18.24.6908. PMID:10601013.
  5. Chong S et al. (1996), J Biol Chem, 271, 22159-22168. Protein Splicing Involving the Saccharomyces cerevisiae VMA Intein. THE STEPS IN THE SPLICING PATHWAY, SIDE REACTIONS LEADING TO PROTEIN CLEAVAGE, AND ESTABLISHMENT OF AN IN VITRO SPLICING SYSTEM. DOI:10.1074/jbc.271.36.22159. PMID:8703028.
  6. Xu MQ et al. (1994), EMBO J, 13, 5517-5522. Protein splicing: an analysis of the branched intermediate and its resolution by succinimide formation. PMID:7988548.

Step 1.

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Catalytic Residues Roles

Residue Roles
Lys403A(C) proton shuttle (general acid/base)
Lys301A(C) proton shuttle (general acid/base)
Asn76A(C) electrostatic stabiliser
Thr78A(C) electrostatic stabiliser
Ile434A(C) (main-C) activator
Asn454A(C) covalent catalysis
Cys1A(C) covalent catalysis
His79A(C) proton shuttle (general acid/base)
Gly433A(C) (main-N) electrostatic stabiliser

Chemical Components

Step 1.

Contributors

Peter Sarkies, Gemma L. Holliday