Featured Article - October 2014
Short description: A search for a plant peptidase ends with the discovery of one of the fastest peptide ligases.
Macrocyclic peptides can be important research tools and leads for drug discovery. They present several advantages over smaller molecules, such as a large surface area that is well suited to inhibiting difficult targets like protein-protein interactions. On the other hand, this large size also creates problems in synthesis, as joining the ends of a peptide chain is entropically disfavored. A variety of methods have been developed to enable macrocycle formation, but a general and efficient strategy is still needed.
To address this gap, Tam and colleagues searched for a ligase from the plant Clitoria ternatea, known to produce cyclic peptides. The authors were expecting to find an ortholog of legumain or an asparaginyl endopeptidase, two classes of enzymes implicated in cyclic peptide formation. Analyses of plant extracts indicated that the active fraction did not process a standard legumain substrate mimic, yet surprisingly the isolated enzyme—purified based on its ability to cyclize peptide substrate kalata B1—had 70% sequence identity with two legumains. The enzyme was termed butelase 1, and also shared 38% sequence identity with human legumain, the crystal structure of which was then used for homology modeling.
Butelase 1 can process a variety of peptide substrates, requiring a C-terminal NHV or DHV sequence, from which the HV residues are cleaved during cyclization. The enzyme formed cyclic dimers at high substrate concentrations, which led the authors to test intermolecular ligation as well: this was successful using a panel of sequences with varying N-terminal amino acids. The kinetics of the reaction are much faster than existing soluble peptide ligases, with a turnover of seconds rather than hours or days. Indeed, butelase 1 is comparable to the fastest ligases known, which are thioesterase domains that exist within specialized protein machineries and are thus impractical for synthetic purposes.
It is currently unclear why butelase 1 acts as fast as it does, or how it allows such extensive substrate variability and strongly favors peptide bond formation rather than hydrolysis. Future research should answer these questions and inform searches for other related enzymes. In the meantime, the discovery of butelase 1 can create new opportunities in the synthesis and application of peptide macrocycles.
G.K.T. Nguyen et al. Butelase 1 is an Asx-specific ligase enabling peptide macrocyclization and synthesis.
Nat. Chem. Biol. 10, 732-738 (2014). doi:10.1038/nchembio.1586