<i>Oa</i> AEP1-Mediated Ligation of Mirror-Image Peptides via Acyl Donor Substrate Engineering

d-proteins are important synthetic molecules needed for nonproteolytic d-peptide drug discovery and the development of mirror-image life. The chemical synthesis of such molecules primarily relies on chemical ligation methods; however, enzymatic ligation cannot be readily applied due to the inherent chiral selectivity of native enzymes. Here, we demonstrate that acyl donor substrate engineering enables the <i>Oa</i>AEP1-mediated ligation of mirror-image peptides. In this approach, an acyl donor d-peptide with a C-terminal asparaginyl thioester is directly recognized by <i>Oa</i>AEP1, which is ligated to a d-peptide acyl acceptor bearing an N-terminal Gx- motif (x = l, i, v, c, G, etc.). This method enables efficient d-peptides ligation under nondenaturing conditions (PBS buffer, pH 7.0), and its effectiveness and practicality are exemplified by the synthesis of the d-cyclopeptides, d-mini-proteins, and mirror-image K6-ubiquitinated α-synuclein (1-17) comprising 102 amino acids. This work demonstrates that substrate engineering is a powerful strategy for harnessing the hidden potential of natural enzymes.