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Enzymatic reconstitution of ribosomal peptide backbone thioamidation

Significance Thioamidation as a posttranslational modification is exceptionally rare, with only one protein example known (methyl-coenzyme M reductase, MCR), as well as a few ribosomal natural products. The genes involved in MCR thioamidation have recently been elucidated, but the enzymes have yet to be characterized. Herein, we report the in vitro reconstitution and substrate preferences of peptidic thioamidation using peptides and enzymes derived from methanogenic archaea. We demonstrate that MCR thioamidation requires an ATP-dependent YcaO enzyme and a sulfide source. Our results shed light on the biosynthesis of other thioamide-containing compounds, which bioinformatics surveys predict to be considerably more numerous than currently appreciated, thus laying a foundation for assigning biological functions for this posttranslational modification. Methyl-coenzyme M reductase (MCR) is an essential enzyme found strictly in methanogenic and methanotrophic archaea. MCR catalyzes a reversible reaction involved in the production and consumption of the potent greenhouse gas methane. The α-subunit of this enzyme (McrA) contains several unusual posttranslational modifications, including the only known naturally occurring example of protein thioamidation. 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. Modification to thioGly has been postulated to stabilize the active site structure of MCR. Herein, we report the in vitro reconstitution of ribosomal peptide thioamidation using heterologously expressed and purified YcaO and TfuA proteins from M. acetivorans. Like other reported YcaO proteins, this reaction is ATP-dependent but requires an external sulfide source. We also reconstitute the thioamidation activity of two TfuA-independent YcaOs from the hyperthermophilic methanogenic archaea Methanopyrus kandleri and Methanocaldococcus jannaschii. Using these proteins, we demonstrate the basis for substrate recognition and regioselectivity of thioamide formation based on extensive mutagenesis, biochemical, and binding studies. Finally, we report nucleotide-free and nucleotide-bound crystal structures for the YcaO proteins from M. kandleri. Sequence and structure-guided mutagenesis with subsequent biochemical evaluation have allowed us to assign roles for residues involved in thioamidation and confirm that the reaction proceeds via backbone O-phosphorylation. These data assign a new biochemical reaction to the YcaO superfamily and paves the way for further characterization of additional peptide backbone posttranslational modifications.