Uncovering New Metalloenzyme-Catalyzed Reactions from RiPP Natural Product Biosynthesis

Abstract

The combination of next-generation DNA sequencing technologies and bioinformatics have revitalized natural product discovery. The biosynthetic pathways of microbial natural products that can now be identified computationally provide a rich source of novel enzyme-catalyzed transformations and compounds to serve as leads for new antibiotics and other drugs. Using a bioinformatic search strategy, we recently identified an abundance of gene clusters for ribosomally synthesized and post-translationally modified peptides (RiPPs) that contain at least one radical S-adenosylmethionine (RaS) metalloenzyme and are regulated by quorum sensing. These grouped into 16 subfamilies and pointed to an unexplored biosynthetic landscape. Herein, we investigate several of these RiPP subfamilies and report additional examples of novel enzymatic transformations. In Chapter 2, we characterize a RaS enzyme from one such gene cluster, the TQQ subfamily from Streptococcus suis, and find that it installs an aliphatic ether cross-link. This reaction marks the first ether installed by a RaS enzyme. In Chapter 3, investigations of the KGR subfamily led to the discovery of the structure, biosynthesis, and function of another one of these natural product groups, that we term enteropeptins, from the gut microbe Enterococcus cecorum. We elucidated three novel reactions, each catalyzed by a different family of metalloenzymes. Biological assays with the mature product revealed bacteriostatic activity only against the producing strain, extending an emerging theme of self-inhibitory metabolites in microbiome firmicutes. In Chapter 4, we expand our original bioinformatics approach by focusing on the joint presence of a RaS enzyme and a transporter gene. This search revealed thousands of new RaS-RiPP biosynthetic loci across diverse bacterial phyla. As a proof of concept for this approach, in Chapter 5, we characterize the ITF cluster from a prevalent human microbiome strain, Bacteroides thetaiotamicron, and find that the RaS enzyme installs a carbon-carbon cross-link, joining a tryptophan and isoleucine. In Chapter 6, investigation of the VRM cluster from Eubacterium rectale shows the first in vitro reconstitution of a tyramine excision reaction. Each of these chapters details the discovery of a new enzymatic reaction and a new modification for RiPP natural products, underlining the utility of our bioinformatics approach.