Table 2.
Activity of Sortase Enzymes In Vitro
| Sortase | Primary Substrate (Km mM) | Secondary Substrate (Km µM) |
Cleavage kcat (s−1) | Transpeptidation kcat (s−1) |
|---|---|---|---|---|
| Class A | ||||
| SaSrtAΔ24 (Frankel, Kruger, Robinson, Kelleher, & McCafferty, 2005) | Abz-LPETG-Dap(Dnp) (7.33 ± 1.01) | Gly5 (196 ± 64) | 0.086 ± 0.015 | 0.28 ± 0.02 |
| SaSrtAΔ24 (Kruger, Dostal, & McCafferty, 2004) | Abz-LPETG-Dap(Dnp) (5.5 mM) | Gly5 (140) | NR | 0.27 |
| SaSrtAΔ24 (Bentley, Lamb, & McCafferty, 2008) | Abz-LPETGG-Dap(Dnp) (8.76 ± 0.78)a | Gly5 (NR) | NR | 1.10 ± 0.06a |
| SaSrtAΔ59 (Chen, Dorr, & Liu, 2011) | Abz-LPETGK-(Dnp) (7.6 ± 0.5) | Gly3 (140 ± 30) | NR | 1.5 ± 0.2 |
| SaSrtAΔ59 Evolved tetramutant (Chen et al., 2011) | Abz-LPETGK-(Dnp) (0.17 ± 0.03) | Gly3 (4800 ± 700) | NR | 4.8 ± 0.8 |
| SpySrtAΔ81 (Race et al., 2009) | Abz-LPETGG-Dap(Dnp) (0.83 ± 0.11) | Ala2 (NR) | NR | 0.0136 ± 0.0011 |
| BaSrtAΔ56 (Weiner, Robson, Marohn, & Clubb, 2010) | Abz-LPETG-Dap(Dnp) (0.038 ± 4)b | m-DAPc | 0.0004 ± 0.0001b | c |
| BaSrtAΔ56 (Chan et al., 2015) | Abz-LPETG-Dap(Dnp) (0.306 ± 0.023) | m-DAPc | 3.6 ± 0.2 × 10−5 | c |
| BaSrtAΔ64 (Chan et al., 2015) | Abz-LPETG-Dap(Dnp) (0.173 ± 0.011) | m-DAPc | 5.7 ± 0.2 × 10−5 | c |
| SmutSrtAΔ40 (Wallock-Richards et al., 2015) | Dabcyl-QALPETGEE-Edans (0.0904 ± 0.0047)b | NR | Yes | NR |
| Class B | ||||
| SaSrtBΔ21 (Bentley, Gaweska, Kielec, & McCafferty, 2007) | Abz-KVENPQTNAGT-Dap(DNP) (7.8 ± 2) | Gly5 (NR) | NR | 5.4 ± 0.5 × 10−4 |
| SaSrtBΔ31 (Jacobitz et al., 2014) | SNKDKVENPQTNAGT (1.8) | Gly5 (NR) | NR | 1.010−4 |
| BaSrtBΔ37 (Maresso, Chapa, & Schneewind, 2006) | Abz -KTDNPKTGDEA-Dap(DNP) | NR | Yes | NR |
| CdSrtBΔ26 (van Leeuwen et al., 2014) | KIVKSPKTGDETQLMK KPPVPPKTGDSTTIGK | Gly4/5 or Ala-d-Glu-DAP | NR | NR |
| Class C | ||||
| SpnSrtC117–228 (Manzano et al., 2008) | IPQTG in RrgB30–633 | YPKN in RrgB30–633 | No | Yes |
| SagSrtC143–254 from PI-2a (Cozzi et al., 2011) | Dabcyl-KKVTIPQTGGIGT-Edans (0.0138) | NR | Yes | NR |
| SagSrtC142–305 from PI-1 (Cozzi et al., 2012) | Dabcyl-RPPGVFPKTGGIG-Edans (0.0 1358 ± 0.00063) | NR | 1.16 ± 0.044 × 10−3 | NR |
| Dabcyl-RPSIPNTGGIG-Edans (0.03100 ± 0.00462) | NR | 1.77 ± 0.101 × 10−3 | NR | |
| Dabcyl-RGGLIPKTGEQQ-Edans (0.01639 ± 0.00250) | NR | 0.77 ± 0.038 × 10−3 | NR | |
| SagSrtC242–283 from PI-1 (Cozzi et al., 2012) | Dabcyl-RPPGVFPKTGGIG-Edans (0.006385 ± 0.00142) | NR | 1.04 ± 0.058 × 10−3 | NR |
| Dabcyl-RGGLIPKTGEQQ-Edans (0.02733 ± 0.00435) | NR | 4.36 ± 0.256 × 10−4 | NR | |
| Dabcyl-RPSIPNTGGIG-Edans (0.05715 ± 0.00354) | NR | 5.56 ± 0.174 × 10−3 | NR | |
| Class D | ||||
| BaSrtDΔ55 (Robson, Jacobitz, Phillips, & Clubb, 2012) | VQGEKLPNTASNN | m-DAP (NR) | Yes | NR |
| BaSrtDΔ55 (Marraffini & Schneewind, 2006) | Abz-GEKLPNTASNN-Dnp | m-DAP (NR) | Yes | NR |
| CpSrtDΔ23–187 (Suryadinata, Seabrook, Adams, Nuttall, & Peat, 2015) | Aβ1–16-LPQTGS | NR | Yes | NR |
a
Values reported from fluorescence assay and subject to inner filter effect and are likely underestimates of true parameters.
b
These values calculated assuming a hydrolytic shunt mechanism.
c
The enzyme reportedly does not perform this reaction in vitro.
Sorting signals for all substrates are highlighted in bold.
Errors are reported where published.
“Yes” indicates the reaction was performed in vitro but kinetics parameters were not reported.
NR, not reported.
m-DAP, mesodiaminopimelic acid.