Thursday, October 27, 2011: 6:50 PM
Room A1 (San Jose Convention Center)
A significant challenge in chemical biology is the correct functional prediction of newly sequenced genomes. Herein the focus is to develop and integrate strategies to correctly determine the function of proteins in COG3964, within the amidohydrolase superfamily. Atu3266 from Agrobacterium tumefaciens is annotated as a dihydroorotase, and Ef0837 from Enterococcus faecalis has been given the function of adenine deaminase. Both enzymes fail to demonstrate the activity of their annotation when experimentally tested. The X-ray crystal structures of Atu3266 (PDB: 2OGJ) and Ef0837 (PDB: 2ICS) have been determined to 2.6Å and 2.3Å, respectively. By focusing on these two enzymes, we have constructed a substrate profile for Atu3266 starting from initial screens of a library of modified aminoacids (i.e. N-acetyl-D/L-Xaa) and dipeptides. We have progressively moved from an initial hit with N-acetyl-D-Ser/Thr having a kcat/Km = 2-3 M-1s-1, to acetyl-D-mandelate with a kcat/Km = 1 x 105 M-1s-1 with Atu3266 and a kcat/Km = 200 M-1s-1 with Ef0837. Modifications to acetyl-D-mandelate have established the requirement for a carboxyl group at C1 of the substrate, D-stereochemistry at C2, the preference for the hydrolysis of ester over amide bonds, and faster rates with a phenyl substituent at C2. The phosphonate analog of the substrate inhibits the enzyme with a Ki = 35 uM. The current protocols for protein function annotation lack reliability; the integration of computational (high-energy intermediate docking, bioinformatics and operon analysis) and experimental (in-vitro screening) analysis, have served as tools to develop a system that can lead to reliable protein function annotation.