Structure of the Lasso Peptide Isopeptidase Identifies a Topology for Processing Threaded Substrates. Author Jonathan Chekan, Joseph Koos, Chuhan Zong, Mikhail Maksimov, A James Link, Satish Nair Publication Year 2016 Type Journal Article Abstract Lasso peptides are a class of bioactive ribosomally synthesized and post-translationally modified peptides (RiPPs), with a threaded knot structure that is formed by an isopeptide bond attaching the N-terminus of the peptide to a side chain carboxylate. Some lasso peptide biosynthetic clusters harbor an enzyme that specifically hydrolyzes the isopeptide bond to yield the linear peptide. We describe here the 2.4 Å resolution structure of a lasso peptide isopeptidase revealing a topologically novel didomain architecture consisting of an open β-propeller appended to an α/β hydrolase domain. The 2.2 Å resolution cocrystal structure of an inactive variant in complex with a lasso peptide reveals deformation of the substrate, and reorganization of the enzyme active site, which exposes and orients the isopeptide bond for hydrolysis. Structure-based mutational analysis reveals how this enzyme recognizes the lasso peptide substrate by shape complementarity rather than through sequence specificity. The isopeptidase gene can be used to facilitate genome mining, as a network-based mining strategy queried with this sequence identified 87 putative lasso peptide biosynthetic clusters, 65 of which have not been previously described. Lastly, we validate this mining approach by heterologous expression of two clusters encoded within the genome of Asticcaucalis benevestitus, and demonstrate that both clusters produce lasso peptides. Keywords Protein Processing, Post-Translational, Peptides, Carbon-Nitrogen Lyases, Protein Domains Journal J Am Chem Soc Volume 138 Issue 50 Pages 16452-16458 Date Published 2016 Dec 21 ISSN Number 1520-5126 DOI 10.1021/jacs.6b10389 Alternate Journal J Am Chem Soc PMCID PMC5245167 PMID 27998080 PubMedPubMed CentralGoogle ScholarBibTeXEndNote X3 XML