Enzymatic C‐to‐C Protein Ligation

Abstract

Transpeptidase‐catalyzed protein and peptide modifications have been widely utilized for generating conjugates of interest for biological investigation or therapeutic applications. However, all known transpeptidases are constrained to ligating in the N‐to‐C orientation, limiting the scope of attainable products. Here, we report that an engineered asparaginyl ligase accepts diverse incoming nucleophile substrate mimetics, particularly when a means of selectively quenching the reactivity of byproducts released from the recognition sequence is employed. In addition to directly catalyzing formation of l‐/d‐ or α‐/β‐amino acid junctions, we find C‐terminal Leu‐ethylenediamine (Leu‐Eda) motifs to be bona fide mimetics of native N‐terminal Gly‐Leu sequences. Appending a C‐terminal Leu‐Eda to synthetic peptides or, via an intein‐splicing approach, to recombinant proteins enables direct transpeptidase‐catalyzed C‐to‐C ligations. This work significantly expands the synthetic scope of enzyme‐catalyzed protein transpeptidation reactions.

Biocompatible strategies for peptide macrocyclisation

“…In addition to the primarily chemical strategies discussed in this perspective, there has been signicant interest in enzymemediated macrocyclisation, 260,261 expressed protein ligation 262,263 and SICLOPPS for peptide macrocyclisation within living cells. 40,63 The eld of chemoenzymatic ligation has experienced rapid advancements, notably with non-ribosomal peptide synthetases, 264 asparaginyl endopeptidases 265 and subtiligase derivatives, 266 allowing for the cyclisation of various classes of peptides under physiological conditions.…”

OaAEP1 Ligase-Assisted Chemoenzymatic Synthesis of Full Cysteine-Rich Metal-Binding Cyanobacterial Metallothionein SmtA

“…1 Moreover, its improved variant, the OaAEP1_C247A mutant, is 160 times more active than the wild type, which makes it an attractive tool for protein semi-or total synthesis. 36 Furthermore, it has been shown that the enzyme can operate the substrates not only in the regular N-to-C orientation but also in C-to-C ligations, 40 which enlarges the synthetic scope of the enzyme. Despite the unique properties of OaAEP1 and its improved version, this enzyme has not yet been applied for the chemoenzymatic synthesis of any full-length protein.…”