There are currently over 14,000 Structural Genomics (SG) protein structures deposited in the PDB by protein structure initiatives. However, most of these SG proteins have unknown or putative function annotations. This accumulated structural information represents a tremendous contribution to structural biology and genomics. Still, the addition of accurate functional annotations for these SG proteins would add substantial value to this information. Our approach to functional annotation and validation incorporates predicting functional assignments through structure-based computed chemical properties followed by biochemical validation. This research focuses on the Crotonase Superfamily, an enzyme superfamily containing numerous SG proteins and multiple diverse functional families whose members exhibit some functional promiscuity. First, Partial Order Optimum Likelihood (POOL) is used to predict computationally the catalytically important residues in the protein structures. Next, Structurally Aligned Local Sites of Activity (SALSA) develops spatially-localized consensus signatures for the proteins of known function in each functional family based on POOL-predicted residues and functionally characterized residues of importance. Finally, the POOL-predicted residues for each SG protein are compared to each consensus signature and scored to determine their degree of similarity at the local active site. Once the sets of computationally-predicted functions are established, we test the predictions biochemically. Currently in the Crotonase Superfamily, most SG proteins are annotated as putative enoyl-CoA hydratases. While some of these annotations are correct, we predict that most are incorrect. In some cases, we can provide alternative functional annotations for the SG proteins. To date, our biochemical data support our computational predictions and identify cases of promiscuity in the superfamily. For example, SG protein probable enoyl-CoA hydratase from <i>Thermus thermophilus </i>(PDB 1WZ8) is predicted by our methods to be a hydrolase, and we have demonstrated this activity as well as weaker hydratase activity through biochemical assays. Other SG proteins have been assayed for different functions in the superfamily to test our predictions. The goal of this project is to provide a validated approach to functional annotation to enable wide applications, including drug discovery, alternative energy technology, and biofuel production. Supported by NSF-CHE-1305655.

Characterizing the functions of structural genomics proteins through computed chemical properties and biochemical validation

254th National Meeting (2017)

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