Enzyme-catalyzed peptide ligation is a powerful tool that enables the assembly of peptides, proteins and protein conjugates. It is carried out by enzymes known as ‘peptide ligases’ which catalyze the formation of peptide bonds with site and substrate specificity. These peptide-bond staplers enable not only site-specific bonding of peptides and proteins but also polymers and chemicals, allowing a myriad of applications in basic research, diagnostics and the development of biomaterials. Examples include the assemble of protein domains, anchoring proteins to solid surfaces and the production of therapeutic peptides and protein conjugates (e.g. antibody-drug conjugates or post-translationally modified proteins) [1].

Over the past decades, the increasing demand for peptides and proteins as drugs or functional materials have speeded up the development of different methodologies for peptide/protein ligation or modification (for a recent review [2] ). Enzyme-mediated ligation technologies have attracted great interest since - compared to chemical approaches - they are highly site-specific and thus have been increasingly applied [3-5]. Although very few enzymes are known to catalyze these reactions, approaches such as protein engineering and genome mining have been applied to increase this number. The recent advancements in this field include the use of several peptide ligases including sortases, butelases, trypsiligase and subtilisins, which will be briefly reviewed in this article.

Sortases are peptide ligases that have demonstrated extraordinary value over the years for manipulating peptides and proteins. In nature, these enzymes are responsible for the post-translational tethering of surface proteins to the cell wall of Gram-positive bacteria, a vital process for colonization and pathogenesis [6].

Among this family, the most widely used enzyme is sortase A from Staphylococcus aureus (SrtA_staph). This well characterized transpeptidase has been adopted for a broad range of applications such as introduction of phophorylated residues [7], semi-synthesis of proteins [8], N- and C-terminal labeling of proteins [9, 10], antibody-peptide vaccine [11], cell-surface modification [12], generation of non-natural protein dimers and cyclic proteins [13], immobilization of proteins to solid surfaces [14] and the production of homogeneous antibody drug conjugates (ADCs) [15].

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Figure 1. Schematic representation of Sortase mediated protein/peptide modification.

During catalysis, sortase A recognizes a C-terminal LPXTG motif within its substrate and then cleaves the bond between the Thr-Gly (TG) residues. The acyl-enzyme intermediate can react with a variety of N-terminal poly-G payloads to couple two proteins or peptides of interest (Figure 1). One major disadvantage of sortase A is that the product of the SrtA reaction also carries the LPXTG sequence, and thus it is also a substrate for the enzyme. This result in the reversibility of the reaction.

Sortase A can be easily obtained commercially or from recombinant expression with high yields [16]. However, its use has been hampered by its poor catalytic efficiency, which translates in the need of extremely high concentrations of enzyme and extended coupling times (up to one day) to achieve the desired result. Moreover, it requires high concentrations of Ca⁺² which limits in vivo and cell-based applications. The reversibility of the enzyme on the other hand, requires high concentrations of the nucleophile to drive the equilibrium of the reaction [17], as well as robust purification protocols, which do not represent economically attractive features.

Despite these drawbacks, significant progress have been made to surpass the limitations of SrtA_staph. A great example is the creation of an optimized pentamutant (“5M”) with a 140-fold increased activity over wild-type enzyme [18]. Also, several groups have generated heptamutant variants (“7M SrtA”) by combining mutations on the 5M to obtain Ca⁺²-independent versions [19-21] and more recently the generation of a 7M SrtA with enhanced activity for both N- and C-terminal labeling, as well as for cell surface modification under physiological conditions [22]. As for the reversibility of the sortase reaction, several approaches have led to its reaction irreversible. One example is the use of depsipeptide substrates which are poor nucleophiles for the reverse reaction [23, 24]. For example, Gibson BA et al incubated 22 μM purified BRD4, 50 μM Cy5-labeled depsipeptide, 3 μM eSrtA, and 10 mM CaCl2 at 4 C overnight to label BRD4 with Cy5 [25]. Thompson RE et al [8] incorporated an intein contruct into the protein product to drive the reaction forward and greatly improved the reaction efficiency and yield [8].

Butelase-1 is a naturally occurring cyclase involved in the biosynthesis of cyclotides - a family of plant cyclic cysteine-rich peptides [26]. Another group of enzymes, asparaginyl endopeptidase, also catalyze the cyclization of plant peptides [27]. In contrast to sortase, Butelase-1 recognizes a much simpler motif at the C-terminus: a D/N-HV tripeptide. This enzyme catalyzes the peptide backbone cyclization by cleaving the HV sequence and ligation D/N to the N-terminal residue to form a macrocycle. So far, it is the fastest ligase known capable of catalyzing peptide cyclization and therefore it has gained a great deal of attention in recent years. Its unpaired catalytic efficiency is reflected in a k_cat/K_M reaching as high as 1340000 L mol^-1 s^-1 for medium-sized peptides and incredible cyclation rates that are >10000 times faster than those of sortases. Hemu X et al has identified the ligase-activity structural determinants in butelase 1 homologs from cyclic peptide-producing plants Viola yedoensis and Viola canadensis [28].

The production of butelase via recombinant expression have not been successful so far, thus studies have been performed using butelase 1 derived from plants. Also - similar to sortase A - the intrinsic reversibility of the ligation reaction catalyzed by butelase requires an excess of substrate to reach reaction yields >50%. These obstacles currently limit this enzyme’s potential biotechnological applications.

This quadruple mutant of trypsin is used for the selective modification of N- and C-terminal residues of several proteins with different reagents. It has a high ligation activity and is highly specific for the tripeptide motif Y-RH [29]. One of the most interesting features of trypsiligase is that it adopts a zymogen-like conformation i.e. it acts like a proenzyme that is activated upon ligand binding. Thus, as it is active exclusively in the presence of the Y-RH tripeptide motif, it effectively minimizes proteolytic side reactions . This advantage has enabled its use for selective C-terminal modification of several proteins including antibody fragments [30].

Nevertheless, only a tiny fraction of proteins (0.5% of all protein sequences in the SwissProt database) contain the Y-RH recognition motif, thus the use of trypsiligase is still very restricted. However, the introduction of the Y-RH tag at the N- or C-terminal region of target proteins overcomes this issue.

Derived from the name of the bacterial species Bacillus subtilis, from which this enzyme was first isolated, subtilisins are a family of extracellular nonspecific serine proteases. As a result of the long-standing interests of using serine proteases in peptide synthesis, a mutant of Bacillus amyloliquefaciens subtilisin BPN’ was designed. This mutant termed subtiligase, has an alternative mechanism in which the aminolysis is favored over peptidase activity, thereby leading to peptide ligation [31].

Unlike sortase and butelase, subtilisin does not require a recognition motif at the termini of the reaction partners. However, the sequence of the substrates greatly influence its catalytic performance, therefore each target is usually modified to optimize the yields, but this is a tedious work. Subtiligase also requires a large excess of one ligation partner and the presence of Ca²⁺ in the ligation process. To this end, a novel Ca²⁺-independent and stable subtilisin mutant, termed peptiligase, has been shown extremely high ligation yield (>98%) with only a slight excess of one of the reaction partners [32].

Peptiligase-mediated ligations do not rely on transpeptidation, as other ligases. It is exceptionally thermostable (T_M = 66°C) and tolerates the presence of organic co-solvents, therefore enabling the ligation of poorly soluble and folded peptides. Interestingly, several peptiligase variants with a broad substrate scope have been engineered, such as omniligase-1, which have been applied in gram-scale peptide synthesis, making them viable industrial enzymes [33].

Peptide ligases are powerful tools that had enabled the efficient synthesis of highly complex peptides, proteins and protein conjugates that have served many purposes both in biology and medicine. Over the past decade, advances in this field have provided innovative solutions to improve their enzymatic properties and even to find new members in nature. This has allowed the existence of a large enzyme toolbox which is currently available and that can be further improved. To date, there is no a single peptide ligase that can be considered the gold standard. Therefore, pros and cons of each one of them should be considered before choosing the enzyme to be used (Table 1). Whether it is going to be used for protein ligation/cyclization, labeling, or whether it is for in vitro or in vivo applications, different enzymes would certainly be required. Also, whether they are commercially or recombinantly available is an important fact to take into account. However, as sometimes the choice might not be straightforward, several approaches should be explored.