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.computational biology | enzymology | protein function

Prediction of function for the polyprenyl transferase subgroup in the isoprenoid synthase superfamily

“…However, when we allowed the side chains in the elongation cavity to adjust flexibly to the ligands, we correctly predicted the correct product chain lengths for 8 of the 10 structures. The incorrect predictions were generated for PDB 3KRF, which we predicted to be a C 15 -ase but experiments have shown to be primarily a C 10 -ase, and PDB 2E8W, for which we predicted a C 25 product rather than the experimentally determined C 20 . 3OYR proves to be a special case.…”

“…To visualize relationships between members of the subgroup, we generated sequence similarity networks using Pythoscape (16), where nodes represent sequences, and edges represent pairwise local alignments with BLAST e-values more significant than a specified cutoff, allowing a dynamic view of clustering patterns and sequence annotations. Sequence-similarity networks can handle thousands of sequences, are quick to compute and robust to missing data (17,18), and have been shown to correlate well with phylogenetic trees (19,20). Fig.…”

A Transient Receptor Potential Ion Channel in Chlamydomonas Shares Key Features with Sensory Transduction-Associated TRP Channels in Mammals

“…SSNs are based on all-against-all sequence comparisons and, like other networks, are very robust to missing data. Furthermore, they allow easy identification of clades and correlate well with phylogenetic trees (Atkinson et al, 2009;Brown and Babbitt, 2012;Lukk et al, 2012). The network was first built considering the 67 TRP channels annotated in the International Union of Basic and Clinical Pharmacology database encompassing TRP subfamilies A (ankyrin), C (classical or canonical), M (melastatin), ML (mucolipin), P (polycystin), and V (vanilloid) (http://www.guidetopharmacology.org/GRAC/ FamilyDisplayForward?familyId=78), with the addition of No mechanoreceptor potential C (NompC) from Drosophila belonging to the TRPN (NompC-like) subfamily (Cheng et al, 2010), as seed sequences.…”

Protein production from the structural genomics perspective: achievements and future needs

“…Over 11,000 protein-encoding constructs have been successfully cloned and small-scale expression tested, resulting in ~4000 expression validated constructs, >1700 purified protein targets, and 151 X-ray crystallographic structures in the past 24 months. Even more remarkable, this platform has been broadly successful, yielding structures from mechanistically diverse enzyme superfamilies [17], prokaryotic model systems such as the nitrogen-fixing organism Sinorhizobium meliloti (Figure 2), plus an extensive array of eukaryotic targets, encompassing constituents of large multicomponent assemblies involved in cell adhesive processes, nuclear pore function, and mammalian immunity (http://www.sbkb.org/kb/psi_centers.html). Importantly, these approaches are supporting large-scale efforts outside of the traditional structural genomics community.…”