title: 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.
authors.affiliation: Department of Biochemistry, University of Illinois at Urbana Champaign, Urbana, IL 61801, USA.
authors.forename: Tiit
authors.initials: T
authors.lastname: Lukk
authors.forename: Ayano
authors.initials: A
authors.lastname: Sakai
authors.forename: Chakrapani
authors.initials: C
authors.lastname: Kalyanaraman
authors.forename: Shoshana D
authors.initials: SD
authors.lastname: Brown
authors.forename: Heidi J
authors.initials: HJ
authors.lastname: Imker
authors.forename: Ling
authors.initials: L
authors.lastname: Song
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Fedorov
authors.forename: Elena V
authors.initials: EV
authors.lastname: Fedorov
authors.forename: Rafael
authors.initials: R
authors.lastname: Toro
authors.forename: Brandan
authors.initials: B
authors.lastname: Hillerich
authors.forename: Ronald
authors.initials: R
authors.lastname: Seidel
authors.forename: Yury
authors.initials: Y
authors.lastname: Patskovsky
authors.forename: Matthew W
authors.initials: MW
authors.lastname: Vetting
authors.forename: Satish K
authors.initials: SK
authors.lastname: Nair
authors.forename: Patricia C
authors.initials: PC
authors.lastname: Babbitt
authors.forename: Steven C
authors.initials: SC
authors.lastname: Almo
authors.forename: John A
authors.initials: JA
authors.lastname: Gerlt
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Jacobson
mesh_terms: D020134:Catalytic Domain; D002412:Cations; D016000:Cluster Analysis; D019295:Computational Biology; D018360:Crystallography, X-Ray; D004151:Dipeptides / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008958:Models, Molecular; D005810:Multigene Family; D010751:Phosphopyruvate Hydratase / Q000378:metabolism*; D019998:Racemases and Epimerases / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D002412:Cations; D004151:Dipeptides; D010751:Phosphopyruvate Hydratase; D019998:Racemases and Epimerases