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

Department of Biochemistry, University of Illinois at Urbana Champaign, Urbana, IL 61801, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 03/2012; 109(11):4122-7. DOI: 10.1073/pnas.1112081109
Source: PubMed

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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Available from: Matthew Vetting, Sep 27, 2015
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    • "Sequence similarity network analysis is a powerful and computation-economic method to depict the relationship among different protein sequences (Atkinson et al. 2009; Lukk et al. 2012; Zhao et al. 2014). In a network, each node represents a protein sequence, and each edge (line) indicates a pair of nodes (protein sequence) that have a BlastP e-value more stringent than a certain cutoff value. "
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    ABSTRACT: Threonine aldolases (TAs) catalyze the inter-conversion of threonine and glycine plus acetaldehyde in a pyridoxal phosphate-dependent manner. This class of enzymes complements the primary glycine biosynthetic pathway catalyzed by serine hydroxymethyltransferase (SHMT), and was shown to be necessary for yeast glycine auxotrophy. Because the reverse reaction of TA involves carbon–carbon bond formation, resulting in a b-hydroxyl-a-amino acid with two adjacent chiral centers, TAs are of high interests in synthetic chemistry and bioengineering studies. Here, we report systematic phylogenetic analysis of TAs. Our results demonstrated that L-TAs and D-TAs that are specific for L-and D-threonine, respectively, are two phylogenetically unique families, and both enzymes are different from their closely related enzymes SHMTs and bacterial alanine racemases (ARs). Interestingly, L-TAs can be further grouped into two evolutionarily distinct families, which share low sequence similarity with each other but likely possess the same structural fold, suggesting a convergent evolution of these enzymes. The first L-TA family contains enzymes of both prokaryotic and eukary-otic origins, and is related to fungal ARs, whereas the second contains only prokaryotic L-TAs. Furthermore, we show that horizontal gene transfer may occur frequently during the evolution of both L-TA families. Our results indicate the complex, dynamic, and convergent evolution process of TAs and suggest an updated classification scheme for L-TAs.
    Journal of Molecular Evolution 02/2015; 80(2). DOI:10.1007/s00239-015-9667-y · 1.68 Impact Factor
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    • "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) ( 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. "
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    ABSTRACT: Sensory modalities are essential for navigating through an ever-changing environment. From insects to mammals, transient receptor potential (TRP) channels are known mediators for cellular sensing. Chlamydomonas reinhardtii is a motile single-celled freshwater green alga that is guided by photosensory, mechanosensory, and chemosensory cues. In this type of alga, sensory input is first detected by membrane receptors located in the cell body and then transduced to the beating cilia by membrane depolarization. Although TRP channels seem to be absent in plants, C. reinhardtii possesses genomic sequences encoding TRP proteins. Here, we describe the cloning and characterization of a C. reinhardtii version of a TRP channel sharing key features present in mammalian TRP channels associated with sensory transduction. In silico sequence-structure analysis unveiled the modular design of TRP channels, and electrophysiological experiments conducted on Human Embryonic Kidney-293T cells expressing the Cr-TRP1 clone showed that many of the core functional features of metazoan TRP channels are present in Cr-TRP1, suggesting that basic TRP channel gating characteristics evolved early in the history of eukaryotes. © 2015 American Society of Plant Biologists. All rights reserved.
    The Plant Cell 01/2015; 27(1). DOI:10.1105/tpc.114.131862 · 9.34 Impact Factor
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    • "Historically these have been particularly susceptible to mis-and over-annotation (e.g. [16]) and are the targets of genome-scale projects, both bioinformatic [17] [18] and crystallographic [19]. One such superfamily is the 2-oxoglutarate, Fe 2+ -dependent oxygenases (here abbreviated as 2OG oxygenases) [20] [21] [22], themselves part of a much larger group called the cupins [23]. "
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    ABSTRACT: Different bioinformatics methods illuminate different aspects of protein function, from specific catalytic activities to broad functional categories. Here, a triple-pronged approach to predict function for a domain of unknown function, DUF2086, is applied. Distant homology to characterised enzymes and conservation of key residues suggest an oxygenase function. Modelling indicates that the substrate is most likely a nucleic acid. Finally, genomic context analysis linking DUF2086 to DNA repair, leads to a predicted activity of oxidative demethylation of damaged bases in DNA. The newly assigned activity is sporadically present in phyla not containing near relatives of the similarly active repair protein AlkB.
    FEBS letters 09/2012; 586(21). DOI:10.1016/j.febslet.2012.09.023 · 3.17 Impact Factor
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