Sortase A
{{Infobox enzyme
| Name = Sortase A
| EC_number = 3.4.22.70
| CAS_number =
| GO_code =
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Sortase A ({{EC number|3.4.22.70}}, SrtA, SrtA protein, SrtA sortase) is an enzyme.{{cite journal | vauthors = Ton-That H, Liu G, Mazmanian SK, Faull KF, Schneewind O | title = Purification and characterization of sortase, the transpeptidase that cleaves surface proteins of Staphylococcus aureus at the LPXTG motif | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 96 | issue = 22 | pages = 12424–9 | date = October 1999 | pmid = 10535938 | pmc = 22937 | doi = 10.1073/pnas.96.22.12424 | bibcode = 1999PNAS...9612424T | doi-access = free }}{{cite journal | vauthors = Zong Y, Bice TW, Ton-That H, Schneewind O, Narayana SV | title = Crystal structures of Staphylococcus aureus sortase A and its substrate complex | journal = The Journal of Biological Chemistry | volume = 279 | issue = 30 | pages = 31383–9 | date = July 2004 | pmid = 15117963 | doi = 10.1074/jbc.m401374200 | doi-access = free }}{{cite journal | vauthors = Race PR, Bentley ML, Melvin JA, Crow A, Hughes RK, Smith WD, Sessions RB, Kehoe MA, McCafferty DG, Banfield MJ | title = Crystal structure of Streptococcus pyogenes sortase A: implications for sortase mechanism | journal = The Journal of Biological Chemistry | volume = 284 | issue = 11 | pages = 6924–33 | date = March 2009 | pmid = 19129180 | pmc = 2652338 | doi = 10.1074/jbc.m805406200 | doi-access = free }} 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.{{cite journal | vauthors = Suree N, Liew CK, Villareal VA, Thieu W, Fadeev EA, Clemens JJ, Jung ME, Clubb RT | title = The structure of the Staphylococcus aureus sortase-substrate complex reveals how the universally conserved LPXTG sorting signal is recognized | journal = The Journal of Biological Chemistry | volume = 284 | issue = 36 | pages = 24465–77 | date = September 2009 | pmid = 19592495 | pmc = 2782039 | doi = 10.1074/jbc.M109.022624 | doi-access = free }}
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.{{cite journal | vauthors = Popp MW, Antos JM, Ploegh HL | title = Site-specific protein labeling via sortase-mediated transpeptidation | journal = Current Protocols in Protein Science | volume = Chapter 15 | pages = 15.3.1–15.3.9 | date = April 2009 | pmid = 19365788 | doi = 10.1002/0471140864.ps1503s56 | pmc = 5551486 }}
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.{{cite journal | vauthors = Popp MW, Ploegh HL | title = Making and breaking peptide bonds: protein engineering using sortase | journal = Angewandte Chemie | volume = 50 | issue = 22 | pages = 5024–32 | date = May 2011 | doi = 10.1002/anie.201008267 | pmid = 21538739 | bibcode = 2011ACIE...50.5024P }}
References
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External links
- {{MeshName|Sortase+A}}
{{Cysteine proteases}}
{{Enzymes}}
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