Biology:Sortase A
| Sortase A | |||||||||
|---|---|---|---|---|---|---|---|---|---|
| Identifiers | |||||||||
| EC number | 3.4.22.70 | ||||||||
| Databases | |||||||||
| IntEnz | IntEnz view | ||||||||
| BRENDA | BRENDA entry | ||||||||
| ExPASy | NiceZyme view | ||||||||
| KEGG | KEGG entry | ||||||||
| MetaCyc | metabolic pathway | ||||||||
| PRIAM | profile | ||||||||
| PDB structures | RCSB PDB PDBe PDBsum | ||||||||
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Sortase A (EC 3.4.22.70, SrtA, SrtA protein, SrtA sortase) is an enzyme.[1][2][3] This enzyme catalyses a cell wall sorting reaction, in which a surface protein with a sorting signal containing a LPXTG motif, is cleaved between the Thr and Gly residue.
This enzyme belongs to the peptidase family C60.
Structure
Sortase A has an eight stranded β-barrel fold with a hydrophobic cleft formed by β7-β8 strands. This cleft is surrounded by β3-β4, β2-β3, β6-β7, and β7-β8 loops. The catalytic cysteine residue is found in this cleft and accepts subsequent binding of a nucleophilic agent. The β3-β4 loop contains a calcium binding site which binds calcium via coordination to a residue in the β6-β7 loop. Such binding slows down the motion of the β6-β7 loop, allowing the substrate of Sortase to bind and increase its activity eightfold.[4]
Use in protein engineering
Sortase A has been widely used as an in vitro tool to post-translationally modify proteins at the N- and C-termini with an appended label. These labels include biotin, fluorophores, crosslinkers, and multifunctional probes.[5]
In both cases, one molecule is engineered to contain a LPXTG motif at one end and another molecule is engineered to contain a (Gly)n motif at another end. Upon cleavage of the LPXTG motif, Sortase forms a thioester intermediate with the engineered molecule. This intermediate is then resolved by nucleophilic attack by the (Gly)n containing molecule to form a fusion between the two molecules with an intervening LPXT(Gly)n motif.
To achieve N-terminal labeling of a protein, the LPXTG motif is engineered to be at the C-terminus of the label. The protein is engineered to have an N-terminal (Gly)n. To achieve C-terminal labeling of the same protein, the LPXTG motif is engineered to be at the C-terminus of the protein. A (Gly)n molecule is engineered to contain the label at its C-terminus.
Finally, both N and C-termini of proteins can be labeled by using Sortases of different substrate specificity. For example, Sortase A from streptococcus pyogenes, recognizes and cleaves the LPXTA motif and accepts Ala-based nucleophiles. This SrtA also recognizes and cleaves the LPXTG motif with reduced efficiency. However, Staph. A. Sortase A does not recognize LPXTA substrates and thus are orthogonal to the LPXTA sequence.
In addition, Sortase A has also been used to piecewise create proteins, protein domains, and peptides.[6]
References
- ↑ "Purification and characterization of sortase, the transpeptidase that cleaves surface proteins of Staphylococcus aureus at the LPXTG motif". Proceedings of the National Academy of Sciences of the United States of America 96 (22): 12424–9. October 1999. doi:10.1073/pnas.96.22.12424. PMID 10535938. Bibcode: 1999PNAS...9612424T.
- ↑ "Crystal structures of Staphylococcus aureus sortase A and its substrate complex". The Journal of Biological Chemistry 279 (30): 31383–9. July 2004. doi:10.1074/jbc.m401374200. PMID 15117963.
- ↑ "Crystal structure of Streptococcus pyogenes sortase A: implications for sortase mechanism". The Journal of Biological Chemistry 284 (11): 6924–33. March 2009. doi:10.1074/jbc.m805406200. PMID 19129180.
- ↑ "The structure of the Staphylococcus aureus sortase-substrate complex reveals how the universally conserved LPXTG sorting signal is recognized". The Journal of Biological Chemistry 284 (36): 24465–77. September 2009. doi:10.1074/jbc.M109.022624. PMID 19592495.
- ↑ "Site-specific protein labeling via sortase-mediated transpeptidation". Current Protocols in Protein Science Chapter 15: 15.3.1–15.3.9. April 2009. doi:10.1002/0471140864.ps1503s56. PMID 19365788.
- ↑ "Making and breaking peptide bonds: protein engineering using sortase". Angewandte Chemie 50 (22): 5024–32. May 2011. doi:10.1002/anie.201008267. PMID 21538739.
External links
- Sortase+A at the US National Library of Medicine Medical Subject Headings (MeSH)
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