Post-translationally created hybrids

Abstract

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.