Investigating the structure of a novel RiPP in Thermobifida fusca

Abstract

In recent decades, scientific interest has increased in a new class of natural products: ribosomally synthesized and post-translationally modified peptides (RiPPs). RiPPs begin as precursor peptides, or linear chains of amino acids, that are modified by maturation enzymes to form a structurally diverse group of natural products. The stability conferred by the post-translational modifications make these molecules an attractive source of therapeutic drugs. Microviridins are tricyclic RiPPs that constitute one of the >20 sub-classes of RiPPs identified today. The two lactone rings and single lactam ring that characterize microviridins are synthesized by two novel ATP-grasp enzymes. These ATP-grasp enzymes may have roles in other microviridin-like RiPPs. This study aimed to investigate the role of the ATP-grasp enzyme in Thermobifida fusca in potentially modifying the precursor peptide into a novel microviridin-like RiPP. Initial co-expression of the precursor protein and ATP-grasp enzyme resulted in significant amounts of purified protein. While the protein was determined by LC-MS to be largely unmodified, additional experiments with longer expression times and higher temperatures showed the disappearance of this unmodified precursor protein band. This seemed to coincide with the gradual appearance of an upper band identified in the SDS-PAGE gels. While many different experiments were performed in order to analyze the significance of this putative transformation, further studies are needed to elucidate the identity of the upper band.