Journal Article

Homology models guide discovery of diverse enzyme specificities among dipeptide epimerases in the enolase superfamily

Fedorov, Alexander A, Jacobson, Matthew P, Hillerich, Brandan, Imker, Heidi J, Sakai, Ayano, Song, Ling, Brown, Shoshana D, Babbitt, Patricia C, Seidel, Ronald, Vetting, Matthew W, Gerlt, John A, Kalyanaraman, Chakrapani, Fedorov, Elena V, Lukk, Tiit, Patskovsky, Yury, Almo, Steven C, Nair, Satish K, Toro, Rafael

2012

Overview

The rapid advance in genome sequencing presents substantial challenges for protein functional assignment, with half or more of new protein sequences inferred from these genomes having uncertain assignments. The assignment of enzyme function in functionally diverse superfamilies represents a particular challenge, which we address through a combination of computational predictions, enzymology, and structural biology. Here we describe the results of a focused investigation of a group of enzymes in the enolase superfamily that are involved in epimerizing dipeptides. The first members of this group to be functionally characterized were Ala-Glu epimerases in Eschericiha coli and Bacillus subtilis, based on the operon context and enzymological studies; these enzymes are presumed to be involved in peptidoglycan recycling. We have subsequently studied more than 65 related enzymes by computational methods, including homology modeling and metabolite docking, which suggested that many would have divergent specificities;, i.e., they are likely to have different (unknown) biological roles. In addition to the Ala-Phe epimerase specificity reported previously, we describe the prediction and experimental verification of: (i) a new group of presumed Ala-Glu epimerases; (ii) several enzymes with specificity for hydrophobic dipeptides, including one from Cytophaga hutchinsonii that epimerizes D-Ala-D-Ala; and (iii) a small group of enzymes that epimerize cationic dipeptides. Crystal structures for certain of these enzymes further elucidate the structural basis of the specificities. The results highlight the potential of computational methods to guide experimental characterization of enzymes in an automated, large-scale fashion.

• Publisher: National Academy of Sciences,National Acad Sciences,National Academy of Sciences, Washington, DC (United States)
• Subject: Active sites / amino acid sequences / Amino acids / Bacillus subtilis / Bacteria / Biochemistry / Biological Sciences / Catalytic Domain / Cations / chemistry / Cluster Analysis / Computational Biology / Crystal structure / Crystallography, X-Ray / Cytophaga / Cytophaga hutchinsonii / dipeptides / Dipeptides - metabolism / Enzyme substrates / Enzymes / enzymology / Escherichia coli / genome / Hydrophobic and Hydrophilic Interactions / Kinetics / metabolism / Metabolites / Modeling / Models, Molecular / Multigene Family / Peptides / Phosphopyruvate Hydratase / Phosphopyruvate Hydratase - metabolism / prediction / Proteins / Racemases and Epimerases / Racemases and Epimerases - chemistry / Racemases and Epimerases - metabolism / Recycling / Sequence Homology, Amino Acid / Substrate Specificity / Waste management