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

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

  1. 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.
  2. 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.
  3. 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.
  4. 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.
  5. 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.
  6. 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

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