This dissertation describes the use and engineering of flavin-dependent halogenases (FDHs) as mild, selective halogenation catalysts. Unlike conventional aromatic halogenation that relies on substrate electronic properties to govern site selectivity, FDHs use catalyst control to halogenate at relatively less electronically activated sites. Moreover, FDH bioconversions can be conducted using water as solvent, NaCl or NaBr as a halide source, and oxygen as the terminal oxidant – making them a milder, greener alternative. Prior to the work in this dissertation, structural and mechanistic work had provided insight into this class of enzymes; however, their use in biocatalysis was largely limited to analytical-scale reactions on native substrates. Furthermore, protein engineering efforts using FDHs was minimal. Herein, the first directed evolution of an FDH for altered site selectivity is described., Chapter 1 provides a background of aromatic halogenation, FDHs, and directed evolution. Although critical in the development of pharmaceutical and agrochemicals, aromatic halogenation methods often suffer from harsh reaction conditions and poor site selectivity. Tryptophan-FDHs (Trp-FDHs) overcome limitations of substrate-controlled selectivity by using catalyst control. Thus, Trp-FDHs are able to halogenate the benzo positions of L-tryptophan in the presence of more electronically activated pyrrolo position. The mechanism by which this selectivity is conferred is thought to arise from positioning substrate within the active site proximal to a reactive halenium species. Although much structural and mechanistic work has developed our understanding of this class of enzyme, the limited success of protein engineering efforts using targeted point mutations reveals the limitations to this understanding. Directed evolution is a powerful approach to protein engineering which does not require previous knowledge of enzyme structure or mechanism. , Chapter 2 discusses the development of robust protocols for the use of 7-Trp-FDH RebH in biocatalysis. Bioconversions were conducted using these protocols to establish site selectivity of halogenation using RebH on a variety of aromatic substrates. In addition to the native substrate L-tryptophan, many other indole derivatives were chlorinated by RebH at benzo positions. Subsequently, directed evolution was used to increase the thermostability of RebH. Three rounds of mutagenesis and screening produced variants with melting temperatures up to 18 °C higher than that of RebH. After further characterization, these variants were found to have increased catalyst lifetimes, making them valuable biocatalysts., Chapter 3 describes the development of mutagenesis and screening methods that can be applied to FDH directed evolution efforts. A new targeted, combinatorial codon mutagenesis method is developed and used to generate high quality libraries for several enzyme targets. In addition, a colorimetric screen used to rapidly detect increased chlorination is described and used in an evolution campaign. Lastly, a MALDI MS screen using deuterium-substituted probes was developed as a means of directly observing changes in site selectivity of FDH halogenation in a high-throughput manner. , The MALDI MS screen detailed in Chapter 3 was used in an evolution campaign to alter the site selectivity of RebH on the substrate tryptamine. This campaign, which led to RebH variants capable of chlorinating substituted indoles ortho-, meta-, and para- to the indole nitrogen, is described in Chapter 4. The x-ray structures of these variants were elucidated and have provided insight into the mechanism by which site selectivity was altered., Chapter 5 describes the generation and analysis of substrate activity profiles for 4 native FDHs and 4 engineered variants on 93 low molecular weight aromatic compounds. These profiles provided fundamental insights into how substrate class, functional group substitution, electronic activation, and binding impact FDH activity and selectivity. Together, these data, provide a useful framework for understanding and exploiting FDH reactivity in organic synthesis., Chapter 6 briefly introduces an ongoing project that aims to discover new, natural FDHs through a genome mining approach. Selection of putative FDH genes, screening methods, and initial results are described within this chapter.