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 the following chemical reaction
- The 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 of Sortase A
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
- ↑ Ton-That, H., Liu, G., Mazmanian, S.K., Faull, K.F. and Schneewind, O. (1999). "Purification and characterization of sortase, the transpeptidase that cleaves surface proteins of Staphylococcus aureus at the LPXTG motif". Proc. Natl. Acad. Sci. USA 96: 12424–12429. doi:10.1073/pnas.96.22.12424. PMID 10535938.
- ↑ Zong, Y., Bice, T.W., Ton-That, H., Schneewind, O. and Narayana, S.V. (2004). "Crystal structures of Staphylococcus aureus sortase A and its substrate complex". J. Biol. Chem. 279: 31383–31389. doi:10.1074/jbc.m401374200. PMID 15117963.
- ↑ Race, P.R., Bentley, M.L., Melvin, J.A., Crow, A., Hughes, R.K., Smith, W.D., Sessions, R.B., Kehoe, M.A., McCafferty, D.G. and Banfield, M.J. (2009). "Crystal structure of Streptococcus pyogenes sortase A: implications for sortase mechanism". J. Biol. Chem. 284: 6924–6933. doi:10.1074/jbc.m805406200. PMID 19129180.
- ↑ Suree, N., Liew, C.K., Villareal, V.A., Thieu, W., Fadeev, E.A., Clemens, J.J., Jung, M.E., Clubb, R.T. (2009). "The structure of the Staphylococcus aureus sortase-substrate complex reveals how the universally conserved LPXTG sorting signal is recognized". J. Biol. Chem. 284: 24465–24477. doi:10.1074/jbc.M109.022624. PMC 2782039. PMID 19592495.
- ↑ Popp, MW>, Antos J.M., Ploegh, H.L. (2009). "Site-specific protein labeling via sortase-mediated transpeptidation; Chapter 15". Curr PRotoc Protein Sci. doi:10.1002/0471140864.ps1503s56. PMID 19365788.
- ↑ Popp, M.W., Ploeugh, H.L. (2011). "Making and Breaking Peptide Bonds: Protein Engineering Using Sortase". Angew. Chem. Int. Ed. 50: 5024–5032. doi:10.1002/anie.201008267.
External links
- Sortase A at the US National Library of Medicine Medical Subject Headings (MeSH)
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