The researchers had sought to understand the biosynthetic roles of the proteins in a RiPP biosynthetic gene cluster (BGC) that was recently identified by a bioinformatic approach. For this purpose, the orthologous spy BGC from Streptomyces sp. NRRL F-5065 was heterologously expressed in Escherichia coli including its precursor peptide and the encoded proteins SpyC, SpyE, SpyD and SpyB. A peptide resulting from enzymatic conversion of the precursor was found to have a mass loss of 20 Da, which would be consistent with the formation of an azole moiety at a serine or threonine residue, but this was surprisingly not confirmed by further experiments. For easier characterization, the researchers produced a shortened version of the peptide, which enabled them to identify that the modification of the precursor occurs at an asparagine residue. Moreover, nuclear magnetic resonance spectroscopy showed the formation of an uracil-like six-membered pyrimidone ring structure. Based on biochemical analyses, the researchers proposed a two-step pathway (pictured), where a YcaO/RRE/dehydrogenase complex (consisting of SpyC, SpyD and SpyB) assisted by the histidine residue of the substrate converts the unreactive asparagine residue in the peptide to the pyrimidone ring. Subsequently, the C-terminal follower peptide is cleaved by a protease (SpyE) generating the final peptide–nucleobase hybrid product.

The hybrid molecule reported in this work will likely motivate future studies to address questions that arise by its discovery. While the enzymes involved in the nucleobase formation have been identified, different catalytic mechanistic scenarios are possible. Moreover, the biological function of the hybrid product is unclear. Finally, the potential of this pathway in bioengineering and how widely this biosynthetic logic with respect to peptide–nucleobase hybrids is applied in nature are yet to be explored.

研究人员试图了解蛋白质在最近通过生物信息学方法鉴定的 RiPP 生物合成基因簇 （BGC） 中的生物合成作用。为此，来自链霉菌属 NRRL F-5065 的直系同源间谍 BGC 在大肠杆菌中异源表达，包括其前体肽和编码蛋白 SpyC、SpyE、SpyD 和 SpyB。发现前体酶促转化产生的肽具有 20 Da 的质量损失，这与在丝氨酸或苏氨酸残基处形成唑基团一致，但令人惊讶的是，进一步的实验并未证实这一点。为了更容易地表征，研究人员制作了肽的缩短版本，这使他们能够确定前体的修饰发生在天冬酰胺残基处。此外，核磁共振波谱显示形成类似尿嘧啶的六元嘧啶酮环结构。基于生化分析，研究人员提出了一种两步途径（如图），其中 YcaO/RRE/脱氢酶复合物（由 SpyC、SpyD 和 SpyB 组成）在底物的组氨酸残基的辅助下将肽中的未反应性天冬酰胺残基转化为嘧啶酮环。随后，C 端跟随肽被蛋白酶 （SpyE） 裂解，生成最终的肽-核碱基杂交产物。

这项工作中报道的杂交分子可能会激励未来的研究解决其发现所带来的问题。虽然已经确定了参与核碱基形成的酶，但不同的催化机制方案是可能的。此外，杂交产物的生物学功能尚不清楚。最后，该途径在生物工程中的潜力以及这种关于肽-核碱基杂交的生物合成逻辑在自然界中的应用范围还有待探索。