Computation-Facilitated Assignment of the Function in the Enolase Superfamily: A Regiochemically Distinct Galactarate Dehydratase from <i>Oceanobacillus iheyensis</i>,

Rakus, John F.; Kalyanaraman, Chakrapani; Fedorov, A.A.; Fedorov, E.V.; Mills-Groninger, F.; Toro, R.; Bonanno, J.B.; Bain, K.T.; Sauder, J.M.; Burley, S.K.; Almo, Steven C.; Jacobson, Matthew P.; Gerlt, J.A.

doi:10.1021/bi901731c

Cited by 32 publications

(42 citation statements)

References 34 publications

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“…S1). Like most other members of the enolase superfamily, the five enzymes from the NSAR/OSBS subfamily are multimers (21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40). The three previously characterized NSAR/OSBS subfamily enzymes are (53).…”

Section: Resultsmentioning

confidence: 97%

Loss of quaternary structure is associated with rapid sequence divergence in the OSBS family

Odokonyero

Sakai

Patskovsky³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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The rate of protein evolution is determined by a combination of selective pressure on protein function and biophysical constraints on protein folding and structure. Determining the relative contributions of these properties is an unsolved problem in molecular evolution with broad implications for protein engineering and function prediction. As a case study, we examined the structural divergence of the rapidly evolving o-succinylbenzoate synthase (OSBS) family, which catalyzes a step in menaquinone synthesis in diverse microorganisms and plants. On average, the OSBS family is much more divergent than other protein families from the same set of species, with the most divergent family members sharing <15% sequence identity. Comparing 11 representative structures revealed that loss of quaternary structure and large deletions or insertions are associated with the family's rapid evolution. Neither of these properties has been investigated in previous studies to identify factors that affect the rate of protein evolution. Intriguingly, one subfamily retained a multimeric quaternary structure and has small insertions and deletions compared with related enzymes that catalyze diverse reactions. Many proteins in this subfamily catalyze both OSBS and N-succinylamino acid racemization (NSAR). Retention of ancestral structural characteristics in the NSAR/OSBS subfamily suggests that the rate of protein evolution is not proportional to the capacity to evolve new protein functions. Instead, structural features that are conserved among proteins with diverse functions might contribute to the evolution of new functions.enolase superfamily | protein structure | protein structure-function relationships I nvestigating the causes and effects of protein sequence divergence is the key to identifying properties that enable proteins to evolve new functions. Previous studies found that constraints imposed by biophysical properties such as protein folding and stability, translational accuracy, and interactions with other proteins make a large contribution to the rate of protein evolution (1-11). However, the relative contributions of biophysical properties versus functional constraints is an open question (2). Given that the rate of protein evolution varies over several orders of magnitude, the evolutionary rate of each protein is probably determined by a unique blend of biophysical and functional constraints (12-15). Thus, the evolutionary simulations and statistical analyses of large protein datasets that comprise the primary focus of this field need to be supplemented with case studies.Here, we present the extraordinarily diverse o-succinylbenzoate synthase (OSBS) family as such a case study. The OSBS family belongs to the enolase superfamily, a group of evolutionarily related protein families that have a common fold but catalyze diverse reactions (16). The rate of sequence divergence in the OSBS family is much faster than other families in the enolase superfamily. For example, the average pairwise amino acid sequence identity of OSBSs fr...

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Section: Resultsmentioning

confidence: 97%

Loss of quaternary structure is associated with rapid sequence divergence in the OSBS family

Odokonyero

Sakai

Patskovsky³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…Thus, the AEE substrate specificity can be assigned to three distinct groups, highlighting the ability of divergent evolution within the enolase superfamily to deliver the same function. Previous examples of the malleability of structure to enable conserved substrate specificity within the enolase superfamily are provided by N-succinylamino acid racemases (10, 13), cis,cis-muconate lactonizing enzymes (13), and galactarate dehydratases (producing enantiomeric products) (14).…”

Section: Resultsmentioning

confidence: 99%

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

Lukk

Sakai

Kalyanaraman

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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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

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“…The differences allow the superfamily to be partitioned into several subgroups that provide structure-based restrictions on the mechanism and therefore the overall reaction: enolase, MLE, MR, and D-glucarate dehydratase (31); D-mannonate dehydratase (23); ␤-methylaspartate ammonia lyase (32,33); and galactarate dehydratase (28). The structures of the barrel domains in the MLE and MR subgroups are compared in Fig.…”

Section: Sequence-structure-function Relationships In Enolase Superfamentioning

confidence: 99%

“…In the case of Protein Data Bank code 2OQY, a divergent member of the enolase superfamily, in silico ligand docking was used to focus in vitro enzymatic assays (28). Unlike other members, the structure of 2OQY revealed the presence of two Mg 2ϩ ions in the active site: one in the canonical position and a second 10.6 Å away in the capping domain.…”

Section: Assigning Functionmentioning

confidence: 99%

Divergent Evolution in Enolase Superfamily: Strategies for Assigning Functions

Gerlt

Babbitt

Jacobson

et al. 2012

Journal of Biological Chemistry

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Nature's strategies for evolving catalytic functions can be deciphered from the information contained in the rapidly expanding protein sequence databases. However, the functions of many proteins in the protein sequence and structure databases are either uncertain (too divergent to assign function based on homology) or unknown (no homologs), thereby limiting the utility of the databases. The mechanistically diverse enolase superfamily is a paradigm for understanding the structural bases for evolution of enzymatic function. We describe strategies for assigning functions to members of the enolase superfamily that should be applicable to other superfamilies. Nature continues to evolve an extraordinarily diverse collection of enzymes.2 The reactions they catalyze and the metabolic pathways in which they participate provide organisms with the ability to thrive in different metabolic niches; they also provide potential for biomedical and chemical applications. To fully understand the importance and implications of this diversity, the challenges are both to provide descriptions of the full range of functional diversity, i.e. molecular and biological functions, and to use those descriptions to understand and exploit natural design principles. However, the number of protein sequences 2 is growing much faster than the number of experimentally based annotations (ExPASy UniProtKB/Swiss-Prot Database). Therefore, novel approaches for reliable functional annotation are required to maximize the utility of the data.In genome sequencing projects, automated methods identify the sequences and then typically annotate their functions by transfer from a homolog using simple pairwise comparisons (1), despite the availability of more sophisticated and orthogonal tools (2). A recent analysis of the annotation error rate revealed that it can be very high, sometimes exceeding 80% for particular monofunctional families (3). Even now, this error rate prevents reliable use of annotations to infer in vitro molecular and in vivo biological functions; the problem will become even more critical as the errors in functional annotation are propagated.Assignment of correct functions to homologous enzymes is confounded because they need not catalyze the same reaction. From a survey of structurally characterized superfamilies, almost 40% are functionally diverse, i.e. different members catalyze reactions with different EC numbers (4). Thus, trivial annotation transfer by sequence homology is often not sufficient to assign function. In mechanistically diverse enzyme superfamilies, all of the homologous enzymes catalyze different reactions using a conserved partial reaction; in functionally distinct enzyme suprafamilies, homologous enzymes catalyze different reactions that do not share any mechanistic attribute (5).This minireview focuses on approaches for functional assignment in the mechanistically diverse enolase superfamily, a paradigm for understanding relationships among sequence, structure, and function in homologous enzymes. The current challenge...

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Computation-Facilitated Assignment of the Function in the Enolase Superfamily: A Regiochemically Distinct Galactarate Dehydratase from Oceanobacillus iheyensis,

Cited by 32 publications

References 34 publications

Loss of quaternary structure is associated with rapid sequence divergence in the OSBS family

Loss of quaternary structure is associated with rapid sequence divergence in the OSBS family

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

Divergent Evolution in Enolase Superfamily: Strategies for Assigning Functions

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