Enzymatic reconstitution of ribosomal peptide backbone thioamidation
Enzymatic reconstitution of ribosomal peptide backbone thioamidation
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DOI：
10.1073/pnas.1722324115
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发表时间：
2018-03-20
影响因子：
11.1
通讯作者：
Mitchell, Douglas A.
中科院分区：
文献类型：
--
作者：
Mahanta, Nilkamal;Liu, Andi;Mitchell, Douglas A.
甲基辅酶M还原酶（methylcoenzymeM reductase，MCR）是一种严格存在于产甲烷古菌和嗜甲烷古菌中的必需酶。MCR催化一种可逆反应，该反应涉及强效温室气体甲烷的生产和消耗。这种酶的α-亚基（McrA）含有几种不寻常的翻译后修饰，包括唯一已知的天然存在的蛋白质硫代酰胺化的例子。我们最近通过基因缺失和质谱分析证明，醋酸甲烷八叠球菌的tfuA和ycaO基因参与MCR活性位点Gly 465的硫代酰胺化。假定对硫代Gly的修饰可稳定MCR的活性位点结构。在此，我们报告了在体外重组核糖体肽硫代酰胺化异源表达和纯化的YcaO和TfuA蛋白从M。食醋动物。与其他报道的YcaO蛋白一样，该反应是ATP依赖性的，但需要外部硫化物源。我们还重建了两个TfuA-独立的YcaOs从超嗜热产甲烷古菌Methanopyrus kandleri和Methanocaldococcus jannaschii的硫代酰胺化活性。使用这些蛋白质，我们证明了广泛的诱变，生物化学和结合研究的基础上形成的硫代酰胺的底物识别和区域选择性的基础。最后，我们报告的YcaO蛋白从mM的无核苷酸和核苷酸结合的晶体结构。kandleri。序列和结构指导的诱变与随后的生化评价，使我们能够分配的作用，参与硫代酰胺化的残基，并确认反应通过骨架O-磷酸化进行。这些数据分配一个新的生化反应YcaO超家族和铺平了道路，为进一步表征额外的肽骨架翻译后修饰。
Methyl-coenzymeM 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 a-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 fromthe 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 fromM. 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.