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

Abstract

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.

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  • Authors

    Lukk T, Sakai A, Kalyanaraman C, Brown SD, Imker HJ, Song L, Fedorov AA, Fedorov EV, Toro R, Hillerich B, Seidel R, Patskovsky Y, Vetting MW, Nair SK, Babbitt PC, Almo SC, Gerlt JA, Jacobson MP

    Source

    Proceedings of the National Academy of Sciences of the United States of America 109:11 2012 Mar 13 pg 4122-7

    MeSH

    Catalytic Domain
    Cations
    Cluster Analysis
    Computational Biology
    Crystallography, X-Ray
    Dipeptides
    Hydrophobic and Hydrophilic Interactions
    Kinetics
    Models, Molecular
    Multigene Family
    Phosphopyruvate Hydratase
    Racemases and Epimerases
    Sequence Homology, Amino Acid
    Substrate Specificity

    Pub Type(s)

    Journal Article
    Research Support, N.I.H., Extramural

    Language

    eng

    PubMed ID

    22392983