Comprehensive exploration of the sequence-structure-function relationships within the nitroreductase superfamily.

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Background: The remarkable ability of enzymes to effectively catalyze diverse chemical reactions is one of the critical foundations of biology; more than that, it offers an abundance of biotechnological applications. At this point, however, we understand only “the tip of the iceberg” in terms of the vast functional diversity found in nature, and as such the exploration of uncharacterized enzymes is extremely important. The nitroreductase (NTR) superfamily is a historically neglected, but biologically and biotechnologically important, group of evolutionarily related enzymes. Despite the biological importance of these enzymes, historical research efforts have focused on only a few functional families, while the vast majority of enzymes remain uncharacterized.******Research Aims: In this research program, I aim to substantially build our knowledge of functional diversity and sequence-structure-function relationships of NTR enzymes. To this end, I have established two state-of-the-art large-scale enzyme characterization approaches that will provide both “global” and “high-resolution” insights. The integration of these two complementary approaches will reveal the “molecular blueprints” for NTR enzymes, and improve our ability to predict, design and engineer synthetic proteins that accomplish the functions of NTR enzymes.***Aim 1. Perform large-scale profiling of enzyme activity for more than 600 enzymesacross 22 NTR subgroups, as well as bioinformatically reconstructed ancestral NTR sequences. We will reveal the “global” sequence-structure-function relationships of enzymes in the NTR superfamily, and identify the key molecular signatures that result in functional transitions. In addition, we will identify, verify and discover NTR functions, including those from as yet uncharacterized families. ***Aim 2. Conduct extraordinarily deep mutational analysis on representative NTR enzymes. We will generate mutational libraries of key NTR enzymes, and characterize the functional effect of each mutation using high-throughput screening and sequencing technologies. This will enable us to analyze “high-resolution” molecular architecture and will reveal the residues and mutations responsible for evolutionary functional transitions.******Impacts: Our proposed program will have impacts that are both significant and broad: First, we will generate invaluable information that advances our knowledge of the biological and chemical functions and evolutionary divergence of NTR enzymes. Second, we will develop an “NTR toolbox” which will have applications in diverse industrial and biomedical settings. Finally, our novel and innovative approach characterizing superfamily-wide functions will be a milestone in the field and aid those seeking similar levels of comprehensive understanding for other protein superfamilies.