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.

甲基辅酶M还原酶（MCR）是严格存在于产甲烷和甲烷营养古细菌中的必需酶。MCR催化与有效温室气体甲烷的产生和消耗有关的可逆反应。这种酶（McrA）的α亚基含有几种不同寻常的翻译后修饰，包括唯一已知的自然发生的蛋白质硫酰胺化实例。最近，我们通过遗传删除和质谱法证明了乙草甲烷菌的tfuA和ycaO基因在MCR活性位点参与Gly465的硫酰胺化。已假定对thioGly进行修饰以稳定MCR的活性位点结构。在这里，我们报道了使用重组表达的和纯化的来自乙酸乙型支原体的YcaO和TfuA蛋白，体外重组核糖体肽硫酰胺化反应。像其他报道的YcaO蛋白一样，该反应是ATP依赖性的，但需要外部硫化物源。我们还从嗜热产甲烷古生菌Methanopyrus kandleri和Methanocaldococcus jannaschii重构了两个不依赖TfuA的YcaO的硫酰胺化活性。。使用这些蛋白质，我们基于广泛的诱变，生化和结合研究证明了硫酰胺形成的底物识别和区域选择性的基础。最后，我们报告了来自M. kandleri的YcaO蛋白的无核苷酸和核苷酸结合的晶体结构。通过序列和结构指导的诱变以及随后的生化评估，我们得以确定参与硫酰胺化反应的残基的作用，并确认反应是通过骨架O-磷酸化进行的。这些数据为YcaO超家族赋予了新的生化反应，并为进一步表征其他肽主链翻译后修饰铺平了道路。