Metalloenzyme-guided discovery of new RiPP natural products

Project Summary/Abstract Rising antibiotic resistance and the emergence of multi-drug resistant pathogens requires the discovery of new drug targets and therapeutics. Currently, the most prominent source of new medicines is natural products, or secondary metabolites. The proposed research involves the investigation of biosynthetic gene clusters (BGCs) relevant to pathogenesis and utilizes genome mining of natural product BGCs containing metal-dependent enzymes as a query to uncover natural products with novel chemical structures. Metalloenzymes are responsible for many unusual chemical reactions in natural product biosynthesis. We recently identified a copper-binding natural product from nontypeable Haemophilus influenzae (NTHi), which we termed oxazolin. Since oxazolin is required for NTHi to infect human cells, its biosynthesis may serve as an ideal drug target. The core enzyme in oxazolin biosynthesis is a multinuclear non- heme iron-dependent oxidase (MNIO), which belongs to a poorly understood family of enzymes that produce ribosomally synthesized, post-translationally modified peptide (RiPP) natural products. We hypothesize that oxazolin serves a protective role for NTHi, chelating toxic levels of copper that human cells employ to stave off infecting pathogens. Aim 1 (K99 Phase) of the proposed research involves structural and functional characterization of copper-bound oxazolin and investigation of another oxazolin from the increasingly multi-drug resistant pathogen Pseudomonas aeruginosa. The latter half of this proposal, which will take place during the R00 Phase, builds on initial genome mining for metalloenzyme-containing RiPP BGCs to uncover novel chemical transformations in natural product biosynthesis. Genome-mining for other MNIOs revealed an operon from Streptomyces laurentii encoding two MNIOs and a secreted natural product; Aim 2 involves determining the function of each enzyme in this BGC and the structure of the natural product it produces. At least one of the MNIOs is predicted to carry out a new chemical reaction for this family of enzymes. A second growing family of iron-dependent enzymes in natural product biosynthesis is the heme oxygenase-like dimetal oxidases (HDOs). Mining for RiPP BGCs with HDOs identified an operon in Paraburkholderia diazotrophica encoding three HDOs of unknown function and an efflux pump, which likely facilitates secretion of the final product, perhaps to serve as an antimicrobial. Aim 3 includes characterization of each enzyme in the biosynthetic pathway and structure-function investigation of the HDOs. Not only will this line of inquiry reveal new natural products, but it will also provide biochemical insight into two families of iron-dependent enzymes with high biosynthetic value.