Once bound, the YcaO protein then enzymatically modifies the C-terminal “core” region. With few exceptions ( 29, 30), the recognition sequence resides in the N-terminal “leader” region of the precursor peptide. These partner proteins harbor an ∼90-residue N-terminal RiPP precursor peptide recognition element (RRE) ( 28), which recognizes and binds a particular region of the precursor peptide. Two varieties of partner proteins are known, with one type resembling E1-ubiquitin–activating enzymes and the other referred to as “ocin-ThiF” proteins ( 12, 26, 27). Well-characterized YcaOs require a partner protein for efficient azoline formation, which is found either fused as a single polypeptide with the YcaO or encoded adjacently as a separate protein. This modification is found in several RiPP classes, including the linear azol(in)e-containing peptides, thiopeptides, and certain cyanobactins ( 12). YcaO enzymes catalyze the ATP-dependent backbone cyclodehydration of Cys, Ser, and Thr to the corresponding thiazoline and (methyl)oxazoline heterocycle ( Fig. The functions of these PTMs have yet to be elucidated although recent work has suggested a potential structural role for thioglycine ( 11). Among these, thioglycine, 5-methylarginine, and 3-methylhistidine are present in all methanogens examined thus far ( 9). These include thioglycine, 3-methylhistidine, S-methylcysteine, 5-methylarginine, 2-methylglutamine, and didehydroaspartate ( 8– 10). One remarkable feature of MCR is the posttranslational modification (PTM) of several active site residues in the α-subunit (McrA). MCR exhibits a hexameric (α 2β 2γ 2) architecture ( 2) with an essential nickel porphinoid coenzyme F 430 present in the active site ( 4– 7). This reaction plays a key role in the global carbon cycle by maintaining steady-state levels of atmospheric methane, a potent greenhouse gas ( 3). Methyl-coenzyme M reductase (MCR) catalyzes the reversible conversion of methyl-coenzyme M (2-methylmercaptoethanesulfonate, CoM) and coenzyme B (7-thioheptanoylthreonine-phosphate, CoB) to methane and CoB-CoM heterodisulfide and is found only in anaerobic archaea ( 1, 2). These data assign a new biochemical reaction to the YcaO superfamily and paves the way for further characterization of additional peptide backbone posttranslational modifications. 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. 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 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.
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