Functional Characterization of Structural Genomics Proteins in the Crotonase Superfamily

Abstract

There are over 13,000 Structural Genomics (SG) proteins deposited in the PDB. However, most of these SG proteins are of unknown or uncertain biochemical function. Although many SG proteins have putative functional assignments, they are often incorrect. The Crotonase Superfamily consists of five diverse functional subgroups that are well characterized structurally and functionally, representing different types of reactivity: hydratase, isomerase, and dehalogenase activities. This superfamily also contains at least 70 SG proteins, so it is ideal to test predictions of protein function. Our approach is based on local structure matching at the computationally predicted active site. First, Partial Order Optimum Likelihood (POOL) is used to predict the functionally important residues of each SG protein and of the proteins of known function in the superfamily. Next, Structurally Aligned Local Site of Activity (SALSA) is used to align the predicted catalytic residues of the well characterized members in the superfamily. From this analysis we generate chemical signatures for each functional subgroup and compare them to the sets of catalytic residues predicted for the SG proteins. We demonstrate based on these computational methods that the majority of the putative annotations in this superfamily are likely incorrect. Currently, biochemical assays are being used to test these predictions. Preliminary biochemical results show that one SG protein classified as a probable enoyl-CoA hydratase possesses hydrolase activity as predicted by our methods. The outcome of this project will be to successfully classify the biochemical functions of SG proteins based on their local structure at the predicted active sites. This work is supported by NSF-CHE-1305655.