Finally, we report nucleotide-free and nucleotide-bound crystal structures for the YcaO proteins from M. Using these proteins, we demonstrate the basis for substrate recognition and regioselectivity of thioamide formation based on extensive mutagenesis, biochemical, and binding studies. We also reconstitute the thioamidation activity of two TfuA-independent YcaOs from the hyperthermophilic methanogenic archaea Methanopyrus kandleri and Methanocaldococcus jannaschii. Like other reported YcaO proteins, this reaction is ATP-dependent but requires an external sulfide source. Herein, we report more » the in vitro reconstitution of ribosomal peptide thioamidation using heterologously expressed and purified YcaO and TfuA proteins from M. Modification to thioGly has been postulated to stabilize the active site structure of MCR. We have recently demonstrated by genetic deletion and mass spectrometry that the tfuA and ycaO genes of Methanosarcina acetivorans are involved in thioamidation of Gly465 in the MCR active site. The α-subunit of this enzyme (McrA) contains several unusual posttranslational modifications, including the only known naturally occurring example of protein thioamidation. MCR catalyzes a reversible reaction involved in the production and consumption of the potent greenhouse gas methane. Methyl-coenzyme M reductase (MCR) is an essential enzyme found strictly in methanogenic and methanotrophic archaea. These studies also provide the foundational knowledge to impact our mechanistic understanding of additional RiPP biosynthetic classes. The data presented within provide a detailed molecular framework for understanding this family of enzymes, which reconcile several decades more » of prior data on RiPP cyclodehydratases. A bioinformatic survey of all YcaOs highlights the diverse sequence space in azoline-forming YcaOs and suggests their early divergence from a common ancestor. We also show that thioamide-forming YcaOs can carry out the cyclodehydration of a related peptide substrate, which underscores the mechanistic conservation across the YcaO family and allows for the extrapolation of mechanistic details to azoline-forming YcaOs involved in RiPP biosynthesis. The structural data are leveraged to identify and test the residues involved in substrate binding and catalysis by site-directed mutagenesis. Here, we present the first structure of any YcaO enzyme bound to its peptide substrate in the active site, specifically that from Methanocaldococcus jannaschii which is involved in the thioamidation of the α-subunit of methyl-coenzyme M reductase (McrA). However, there are major knowledge gaps in the mechanistic and structural underpinnings that govern each of the known YcaO-mediated modifications. These enzymes are found in multiple biosynthetic pathways, including those for several different classes of ribosomally synthesized and post-translationally modified peptides (RiPPs). Our results facilitate access to this class of peptide derivatives and inform the use of backbone N-hydroxylation as a tool in the design of constrained peptidomimetics.YcaO enzymes are known to catalyze the ATP-dependent formation of azoline heterocycles, thioamides, and (macro)lactamidines on peptide substrates. An enhancement in β-hairpin stability was observed for a di- N-hydroxylated variant.
In contrast to N-methyl substituents, backbone N-hydroxy groups are accommodated in the β-strand region of the hairpin without energetic penalty. Based on previous work demonstrating the β-sheet-stabilizing effect of α-hydrazino acids, we carried out an analogous study with N-hydroxy-α-amino acids using a model β-hairpin fold. Here, we describe a versatile method to prepare N-hydroxy peptide on solid support and evaluate the impact of backbone N-hydroxylation on secondary structure stability. Although uncommon relative to N-alkyl substituents, peptides harboring main-chain N-hydroxy groups exhibit unique conformational preferences and biological activities. Peptide backbone amide substitution can dramatically alter the conformational and physiochemical properties of native sequences.