Back to Home

Type of Ligases

Peptide ligases can be classified into two major categories: transpeptidases and acyltransferases. Transpeptidases represent the majority of characterized ligases and include enzymes such as intein, sortases, prolyl oligopeptidase (POP)-like cyclases, trypsiligases, subtilisin-like ligase (PatG), peptide asparaginyl ligases (PALs), and connectases. In contrast, acyltransferases catalyze peptide bond formation by facilitating nucleophilic amine attack on pre-activated ester or thioester intermediates. This category includes subtilisin-derived engineered variants like subtiligases, and penicillin-binding protein(PBP)-like cyclases from NRPS biosynthetic pathways.

Type (Count)
Connectase (2)
Intein (5)
Other Ligase (12)
PAL (25)
PBP-like cyclase (1)
POP-like cyclase (3)
Sortase (9)
Subtiligase (5)
Subtilisin-like ligase (2)
Trypsiligase (1)

Connectase

Connectase is a unique peptide ligase derived from methanogenic archaea, functioning as a highly specific and efficient transpeptidase. Unlike traditional proteases or sortases, Connectase belongs to the proteasome-like N-terminal nucleophile (Ntn) hydrolase family but lacks hydrolytic activity. It catalyzes reversible peptide bond formation by recognizing a conserved recognition motif (KDPGA), ligating peptides or proteins through a native amide bond at the N-terminus. Notably, Connectase operates under mild conditions, exhibits strict sequence specificity, and avoids side reactions such as hydrolysis

Name	Host Organism	Substrate specificity
Connectase	Methanosarcina mazei	P2(K)-P1(D)-P1'(P)-P2'(G)-P3'(A)
MmCET	Methanococcus maripaludis	P2(K)-P1(D)-P1'(P)-P2'(G)-P3'(A)-P1''(A)-P2''(G)-P3''(A)

Intein

Inteins are naturally occurring self-splicing enzymes that excise themselves from a precursor protein while simultaneously ligating the remaining protein fragments, known as exteins. Inteins have garnered significant interest in the development of controllable protein assembly systems, protein purification methods, and tools for controlling gene expression. Engineering of inteins have focused on splicing efficiency and specificity, conditional splicing, minimization/splitting, and stability.

Name	Host Organism	Substrate specificity
Intein-VMA1	Saccharomyces cerevisiae	P1'(Cys/Thr/Ser)
Intein-RecA	Mycobacterium tuberculosis	P1'(Cys/Thr/Ser)
Intein-Mxe GyrA	Mycobacterium xenopi	P1'(Cys/Thr/Ser)
Npu DnaE split intein	Nostoc punctiforme	P1'(Cys/Thr/Ser)
Ssp DnaE split intein	Synechocystis sp.	P1'(Cys/Thr/Ser)

Other Ligase

Non-native peptide-bond-forming peptide ligases are conjugating enzymes that does not catalyze peptide ligation via an amide linkage on the backbone. Instead they catalyze formation of a side chain amido-linkage (such as transglutaminase, TG) or an isopeptide bond (such as the SpyCatcher-SpyTag pair). These enzymes are also highly useful engineering tools, which can be used independently or in combination with other conjugating tools.

Name	Host Organism	Substrate specificity
Transglutaminase 1	Homo sapiens	Gln-Lys isopeptide bond
Transglutaminase 2	Homo sapiens	Gln-Lys isopeptide bond
Transglutaminase 3	Homo sapiens	Gln-Lys isopeptide bond
Transglutaminase	Streptomyces mobaraensis	Gln-Lys isopeptide bond
Transglutaminase	Streptococcus suis	Gln-Lys isopeptide bond
SpyCatcher	Streptococcus pyogenes	SpyTag(AHIVMV-D-AYKPTK)-KTag(ATHIKFS-K-RD)
SpyLigase	Streptococcus pyogenes	SpyTag(AHIVMV-D-AYKPTK)
SpyCatcher002	Streptococcus pyogenes	SpyTag002(VPTIVMV-D-AYKRYK)
SpyCatcher003	Streptococcus pyogenes	SpyTag003(RGVPHIVMV-D-AYKRYK)
SnoopCatcher	Streptococcus pneumoniae	SnoopTag(KLGDIEFIKV-N-K)
SnoopLigase	Streptococcus pneumoniae	SnoopTagJr(KLGSIEFI-K-VNK)-DogTag(DIPATYEFTDGKHYIT-N-EPIPPK)
DogCatcher	Streptococcus pneumoniae	DogTag(DIPATYEFTDGKHYIT-N-EPIPPK)

PAL

Peptide Asparaginyl Ligases (PALs) are a class of ligases originally discovered in plants, known for their exceptional efficiency and specificity in catalyzing peptide bond formation at asparagine (Asn) or aspartic acid (Asp) residues. Unlike typical proteases from C13 family that favor hydrolysis, PALs catalyze transpeptidation reactions, forming natural amide bonds between a peptide substrate and an incoming nucleophile, such as the N-terminal amine of another peptide. A prominent example is butelase-1 from Clitoria ternatea, which is among the fastest peptide ligases known to date. PALs operate under mild, cofactor-free conditions and exhibit broad substrate tolerance, making them powerful tools for protein labeling, cyclization, drug conjugation, and synthetic biology.

Name	Host Organism	Substrate specificity
Butelase-1	Clitoria ternatea	P1(N/D)-P1'(X)-P2'(L/V/I/F)-P1''(X except Pro)-P2''(L/V/I/F)
OaAEP1b	Oldenlandia affinis	P1(N/D)-P1'(X)-P2'(L/I)
OaAEP1b-C247A	Oldenlandia affinis	P1(N/D)-P1'(X)-P2'(L/I)
OaAEP3	Oldenlandia affinis	P1(N/D)-P1'(X)-P2'(L/I)
OaAEP4	Oldenlandia affinis	P1(N/D)-P1'(X)-P2'(L/I)
OaAEP5	Oldenlandia affinis	P1(N/D)-P1'(X)-P2'(L/I)
VyPAL2	Viola yedoensis	P1(N/D)-P1'(X)-P2'(L/I/F)
VyPAL2-I244V	Viola yedoensis	P1(N/D)-P1'(X)-P2'(L/I/F)
VaPAL1	Viola albida	P1(N/D)-P1'(X)-P2'(L/I/F)
VaPAL2	Viola albida	P1(N/D)-P1'(X)-P2'(L/I/F)
VvPAL1	Viola verecunda	P1(N/D)-P1'(X)-P2'(L/I/F)
VvPAL2	Viola verecunda	P1(N/D)-P1'(X)-P2'(L/I/F)
VoPAL1	Viola orientalis	P1(N/D)-P1'(X)-P2'(L/I/F)
VuPAL1	Viola uliginosa	P1(N/D)-P1'(X)-P2'(L/I/F)
HaPAL1	Helianthus annuus	P1(N/D)-P1'(X)-P2'(L/I)
VyPAL1	Viola yedoensis	P1(N/D)-P1'(X)-P2'(L/I/F)
VyPAL4	Viola yedoensis	P1(N/D)-P1'(X)-P2'(L/I/F)
VyPAL5	Viola yedoensis	P1(N/D)-P1'(X)-P2'(L/I/F)
VbAEP1	Viola betonicifolia	P1(N/D)-P1'(X)-P2'(L/I/F)
HeAEP3	Hybanthus enneaspermus	P1(N/D)-P1'(X)-P2'(L/I)
Vc1c	Viola canadensis	P1(N/D)-P1'(X)-P2'(L/I/F)
ConPAL3	Artificial	P1(N/D)-P1'(X)-P2'(L/I/F)
VdiPAL1	Viola dissecta	P1(N/D)-P1'(X)-P2'(L/I/F)-P1''(X except Pro)-P2''(L/I/F/W/Y/M)
VyOpt1	Viola yedoensis	P1(N/D)-P1'(X)-P2'(L/I/F/Y/W/M)
VaPAL2*	Viola arcuata	P1(N/D)

PBP-like cyclase

Penicillin-binding protein (PBP)-like cyclase involved in the final step of macrocyclization in some NRPs. They act like transestrase (TE) domain of the NRPS complex but physically discrete from the megasynthetase, such as SurE in surugamide biosynthesis. SurE catalyzes peptide bond formation via a serine-dependent transacylation mechanism. It recognizes and cyclizes linear peptide substrates bearing a C-terminal thioester, forming a head-to-tail amide bond.

Name	Host Organism	Substrate specificity
SurE	Streptomyces albidoflavus	P1(dAA)-P1'(ester)-P1''(Hydrophobic)

POP-like cyclase

POP-like cyclases are a class of enzymes derived from prolyl oligopeptidases (POPs), repurposed to function as peptide macrocyclases in the biosynthesis of orbitides and other cyclic peptides. Unlike classical POPs, which act as serine proteases to hydrolyze internal proline-containing peptide bonds, POP-like cyclases have evolved to catalyze head-to-tail backbone cyclization of linear precursor peptides. These enzymes typically operate without the need for cofactors or accessory proteins, and their substrate flexibility and regioselectivity make them attractive tools for synthetic biology, especially in the generation of macrocyclic peptide libraries with drug-like properties.

Name	Host Organism	Substrate specificity
PCY1	Saponaria vaccaria	P1(P/A)-P1'(X)-P2'(Q)-P3'(A/T)
PhPCY3	Pseudostellaria heterophylla	P1(P)-P1'(aromaticAA)
GmPOPB	Galerina marginata	Core(IWGIGCNP)-Tail(WTAEHVDQTLASGNDIC

Sortase

Sortases are Cys transpeptidases found in Gram-positive bacteria, essential for anchoring virulence-associated surface proteins to the cell wall. They specifically recognize pentapeptide signals, such as LPXTG for SrtA, and catalyze peptide bond formation between the conserved Thr and the N-terminal Gly of peptidoglycan precursors. The enzymatic versatility of sortases, particularly SrtA, has been harnessed for a broad range of biotechnological applications. Recent engineering efforts have focused on enhancing the specificity, efficiency, and stability of sortase enzymes.

Name	Host Organism	Substrate specificity
Sortase A	Staphylococcus aureus	P4(L)-P3(P)-P2(X)-P1(T)-P1'(G)
ESrtA	Staphylococcus aureus	P4(L)-P3(P)-P2(X)-P1(T)-P1'(G)
ESrtA(2A-9)	Staphylococcus aureus	P4(L)-P3(A)-P2(X)-P1(T)-P1'(G)
ESrtA(4S-9)	Staphylococcus aureus	P4(L)-P3(P)-P2(X)-P1(S)-P1'(G)
SrtA7M	Staphylococcus aureus	P4(L)-P3(P)-P2(X)-P1(T)-P1'(G)-P2'(G)
SrtAβ	Staphylococcus aureus	P4(L)-P3(M)-P2(V)-P1(G)-P1'(G)
Sortase B	Staphylococcus aureus	P4(N)-P3(P)-P2(Q)-P1(T)-P1'(N)
Sortase C	Streptococcus pneumoniae	P4(L)-P3(P)-P2(X)-P1(T)-P1'(G)
Sortase D	Bacillus anthracis	P4(L)-P3(P)-P2(N)-P1(T)-P1'(A)-P2'(G)

Subtiligase

Subtiligases are engineered ligase variants derived from the subtilisin family of proteases. They function by ligating a peptide ester to the N-terminal α-amine of a protein or peptide, providing irreversible reactions and broader sequence tolerance compared to other transpeptidase-type ligation enzymes. Engineered versions of subtiligase have achieved better ligation/hydrolysis ratio, high tolerance to organic solvents, and tailored specificity, enhancing their utility in protein engineering and biochemical applications.

Name	Host Organism	Substrate specificity
Subtiligase	Bacillus amyloliquefaciens	P2(A/S/G)-P1(L/F/A/V)-P1'(Ester/thioester)-P1''(G/A/S)-P2''(A/G)
Peptiligase	Bacillus amyloliquefaciens	-
Thymoligase	Bacillus amyloliquefaciens	P4(I)-P3(T)-P2(T)-P1(K)-O-Cam-P2'(L)-P1''(D)-P2''(L)
Omniligase-1	Bacillus amyloliquefaciens	-
Aqualigase	Bacillus subtilis	P1(theoritically any residue) followed with a carboxylic acid terminus

Subtilisin-like ligase

The sole natural enzyme of this category is PatG, a subtilisin-like serine protease that functions as a macrocyclase in the biosynthesis of cyanobactins. Unlike typical proteases, PatG catalyzes head-to-tail cyclization of linear precursor peptides by mediating nucleophilic attack on a thioester intermediate, forming a natural amide bond. It has broad substrate tolerance and ability to generate cyclic peptides with diverse sequences.

Name	Host Organism	Substrate specificity
PatG	Prochloron didemni	P1(Pro/Thiazole/Thiazoline/Oxazoline)-P1'(A)-P2'(Y)-P3'(D)-P4'(G)-P5'(E)
PatGmac	Prochloron didemni	P1(P/Thiazolidine)-P1'(A)-P2'(Y)-P3'(D)-P4'(G)-P5'(E)

Trypsiligase

Trypsiligase is an engineered variant of trypsin designed to function as a peptide ligase rather than a protease. By introducing key mutations K60E/N143H/E151H/D189K, its hydrolytic activity is significantly reduced while retaining substrate recognition. This modification enables the enzyme to catalyze site-specific peptide bond formation through a transpeptidation mechanism, typically targeting peptide substrates with YRH recognition sequences at the C-terminus. Operating under mild, metal-free conditions and requiring no cofactors, Trypsiligase exhibits high efficiency and low levels of hydrolytic side reactions.

Name	Host Organism	Substrate specificity
Trypsiligase	Bos taurus	P2(Y)-P1(R)-P1'(H)