Enzymatic reconstitution of ribosomal peptide backbone thioamidation

Abstract

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 and genes of 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 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 and 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 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-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.

Uncovering the unexplored diversity of thioamidated ribosomal peptides in Actinobacteria using the RiPPER genome mining tool

“…was previously determined that a distantly related pair of homologs catalyze thioamidation of methylcoenzyme M reductase in archaea. 32,33 The relatively simple thiovarsolin pathway therefore represents a promising system for future biochemical studies of this reaction in the context of RiPP biosynthesis. Unexpectedly, genes conserved across multiple homologous var-like pathways (varS, varP and varL, Figure 5B) were not required for thiovarsolin biosynthesis.…”

“…This protein pair has been identified elsewhere in nature, including in archaea, where they are involved in the ATP-dependent thioamidation of a glycine residue of methyl-coenzyme M reductase. 32,33 We therefore hypothesized that the identification of tfuA-like genes could be employed as a rational criterion for the identification of BGCs responsible for the production of novel thioamidated RiPPs in bacteria. Peptide similarity networking is then used to identify putative RiPP families.…”

Genome Editing Reveals Novel Thiotemplated Assembly of Polythioamide Antibiotics in Anaerobic Bacteria

“…Communications adenine nucleotide hydrolases (AANH;6-thioguanine [10] and thio-tRNAb iosynthesis [2b] ), YcaO proteins (thioviridamide biosynthesis [2a] and methyl-coenzyme Mr eductase maturation [11] ), and MbnB/MbnC proteins (methanobactin biosynthesis [7,12] ). However,t he R. cellulolyticum genome encoded too many AANH homologs and no YcaO or MbnB/MbnC homologs,preventing identification of acandidate CTAgene cluster.T herefore,weconsidered enzymes for the biosynthesis of p-hydroxybenzoic acid (PHBA), ap otential building block of CTA.…”

Functional interactions between post-translationally modified amino acids of methyl-coenzyme M reductase inMethanosarcina acetivorans

“…The core modified amino acids are widely conserved and include N 1 -methylhistidine (or 3-methylhistidine), 5-(S)-methylarginine, and thioglycine (Figure 1B). The widespread conservation of these core modifications is exemplified in the recently discovered genes responsible for installation of the thioglycine and 5-(S)-methylarginine modifications, which were originally designated as methanogenesis marker genes because of their universal occurrence in the genomes of sequenced methanogens (26–29). Given that 5-(S)-methylarginine and thioglycine are extremely rare in Nature, yet universally conserved in MCR, it was speculated that these residues must be essential for catalysis (24).…”