Enzyme Function Prediction, Discovery, and Characterization in Undergraduate Biochemistry Teaching and Research Labs

Abstract

The Structural Genomics Initiative was an effort by consortiums to solve as many unique protein structures as possible; the Protein Data Bank contains a number of enzymes whose structures have been solved, but for which no enzymatic activity has been determined. The Enzyme Function Initiative is an effort to determine as many unique enzyme functions as possible. There are a number of putative NUDIX Hydrolase superfamily members whose structures have been solved, but for which no enzymatic activity has been determined. We have catalogued the structurally-determined enzymes within the NUDIX Hydrolase superfamily using BLAST, Dali, SCOP, and PDB. We then began to characterize these enzymes in the biochemistry teaching laboratory and are finishing their characterization in the research lab. In the biochemistry lab course, the students have expressed his-tagged Nudix Hydrolases (PDB entries 2AZW, 2PQV, 3QSJ, and 3R03) from plasmids obtained from DNASU, purified the enzymes using nickel affinity chromatography, and then did enzyme assays to determine the substrates. From the assays, the students discovered Nudix Hydrolase activities for these enzymes, which we have finished characterizing in the research lab.