Butelase 1: A Versatile Ligase for Peptide and Protein Macrocyclization

Abstract

Macrocyclization is a valuable tool for drug design and protein engineering. Although various methods have been developed to prepare macrocycles, a general and efficient strategy is needed. Here we report a highly efficient method using butelase 1 to macrocyclize peptides and proteins ranging in sizes from 26 to >200 residues. We achieved cyclizations that are 20,000 times faster than sortase A, the most widely used ligase for protein cyclization. The reactions completed within minutes with up to 95% yields.

Crystal Structure of Plant Legumain Reveals a Unique Two-Chain State with pH-Dependent Activity Regulation

“…While this pH dependence is consistent with the ligase activity found here and in Clitoria ternatea (butelase-1) or human legumain (Nguyen et al, 2015;Dall and Brandstetter, 2016;Dall et al, 2015a) , it is also the pH-regime where the plant-specific two-chain activation state described herein is stabilized and the access to the active site is tightly controlled by the α6-helix, which may be a paradox at first sight. However, an efficient peptide ligase needs to suppress premature hydrolysis of ligation intermediates, otherwise initiated ligation reactions would be futile.…”

“…Although the authors did not report on the functional relevance of dimerization or on the existence of the two-chain state of OaAEP1, structural comparison suggests that the here derived activity and stability regulation principles similarly apply to OaAEP1, including a two-chain form of OaAEP1. In fact, the conformational stability of the two-chain form at neutral pH and the role of the α6-helix as a pH-dependent master switch, will cast light on the enigmatic dual protease and ligase activities of legumain isoforms (Nguyen et al, 2015), not only restricted to A. thaliana.…”

“…Plant legumains convert functionally cryptic linear polypeptides into active cyclic compounds with diverse biological activities, as exemplified by the sunflower trypsin inhibitor 1 and the insecticidal cyclotide kalata B1, among many others (Nguyen et al, 2015;Nguyen, Giang K T et al, 2014;Mylne et al, 2011;Bernath-Levin et al, 2015a). Cyclic peptides, like the cyclotides, combine fold robustness with a large sequence variability, making them unique scaffolds for diverse biotechnological and medical applications, including multivalent drugs against HIV or cancer (Gould et al, 2011;Lesner et al, 2011;Ireland et al, 2008;Gerlach et al, 2010).…”

Recent advances in sortase-catalyzed ligation methodology

“…A final notable development, apart from sortase, has been the emergence of the ligase butelase-1 isolated from Clitoria ternatea [58•, 59, 60]. First described in 2014, butelase-1 has been shown to promote cyclizations and intermolecular ligations analogous to those catalyzed by sortases.…”

Rapid protein immobilization for thin film continuous flow biocatalysis

“…In the laboratory, enzymes can perform a wide range of transformations including reductions, 3,4 oxidations, 5,6 cyclization, 7,8 aziridinations 9 and nitration reactions. 10 Improving the performance of these enzymes typically relies on directed evolution 11,12 and computational design.…”