Please note that these documents are being included by direct request from our administrative review process as they are marked as required by NIH Assist. These project summaries are the same as included in the Research Summary. Please disregard if not needed! Project Summaries PROJECT 1: Synthetic biology approaches to new fluorinated pharmaceuticals (1R01GM123181- 01): Fluorinated pharmaceuticals represent a rapidly expanding class of small molecule drugs that have become important in the treatment of diverse human health conditions ranging from cancer to high cholesterol. The focus of this project is to develop synthetic biology approaches to engineering enzymatic and living systems for the production of complex fluorinated compounds with bioactivity, with the long- term goal of developing new approaches to fluorinated drug discovery. Specific aims of this proposal include: (i) elucidating the molecular mechanism of naturally-occurring fluorine selectivity in enzymes from Streptomyces cattleya, one of the few known native organofluorine-producing organisms, in order to build a knowledgebase for engineering fluorine-selective enzymes, (ii) studying the mechanism of fluorinated extender unit usage in polyketide synthases, which produce a large family of medicinally- important natural products, and (iii) developing in vitro and in vivo methods for production of fluorinated natural products in the polyketide family. PROJECT 2: Discovery and application of new halogenases (R01GM134271): The rapid and modular generation of molecular diversity is key to the search for new chemical functions. One particularly useful functional group is the halogen (X = Cl, Br, I), which enables many selective and effective downstream strategies for creating structural complexity. In this regard, halogenase enzymes have provided an important and complementary approach to synthetic catalysts for regio- and stereoselective introduction of a halogen substituent on a complex scaffold. While many families of halogenases exist, the radical halogenases provide the greatest potential for reaction diversity, as they are competent to replace unactivated C-H bonds with a halogen unlike those that operate by electrophilic or nucleophilic mechanisms. However, the substrate scope of these enzymes has been limited to date to either protein- bound substrates or large late-stage natural product intermediates. Our group has discovered a new clade of radical halogenases capable of reacting with small molecule substrates. We now seek to take advantage of this discovery to develop new tools for in vitro and in vivo synthesis. Specific aims of this proposal include: (i) elucidating the structure and mechanism of these new radical halogenases, which will provide important insight into their engineering, (ii) investigating and engineering selectivity in halogenases.