Evolution of Enzymatic Activities in the Enolase Superfamily: <scp>l</scp>-Rhamnonate Dehydratase

Rakus, John F.; Fedorov, A.A.; Fedorov, E.V.; Glasner, Margaret E.; Hubbard, Brian K.; Delli, Joseph D.; Babbitt, Patricia C.; Almo, Steven C.; Gerlt, J.A.

doi:10.1021/bi800914r

Cited by 50 publications

(43 citation statements)

References 32 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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“…This approach has been successful for identifying substrates for dipeptide epimerases. However, for the putative dehydratases, this approach has been less successful (21)(22)(23)(24). We have assembled a library of all mono-and diacids derived from D-and L-hexoses, D-and L-pentoses, and D-and L-tetroses; a partial set of hexuronic acids; and the (very few) commercially available phosphorylated acid sugars (20).…”

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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144

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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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“…This substitution suggests the hypothesis that functional diversity has evolved in the DJ-1 superfamily through a subtle modulation of the active site environment of the conserved reactive cysteine in order to achieve different chemical outcomes. This hypothesis, which has precedent in other protein families (34), would help explain the varied functions of characterized DJ-1 superfamily proteins and also provides an example of natural selection capitalizing on the unique reactivity of the cysteine thiol(ate) to achieve functional diversity while maintaining structural parsimony.…”

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confidence: 99%

Evolution of New Enzymatic Function by Structural Modulation of Cysteine Reactivity in Pseudomonas fluorescens Isocyanide Hydratase

Lakshminarasimhan¹,

Madzelan²,

Nan³

et al. 2010

Journal of Biological Chemistry

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Isocyanide (formerly isonitrile) hydratase (EC 4.2.1.103) is an enzyme of the DJ-1 superfamily that hydrates isocyanides to yield the corresponding N-formamide. In order to understand the structural basis for isocyanide hydratase (ICH) catalysis, we determined the crystal structures of wild-type and several sitedirected mutants of Pseudomonas fluorescens ICH at resolutions ranging from 1.0 to 1.9 Å . We also developed a simple UV-visible spectrophotometric assay for ICH activity using 2-naphthyl isocyanide as a substrate. ICH contains a highly conserved cysteine residue (Cys 101 ) that is required for catalysis and interacts with Asp 17 , Thr 102 , and an ordered water molecule in the active site. Asp 17 has carboxylic acid bond lengths that are consistent with protonation, and we propose that it activates the ordered water molecule to hydrate organic isocyanides. In contrast to Cys 101 and Asp 17 , Thr 102 is tolerant of mutagenesis, and the T102V mutation results in a substrate-inhibited enzyme. Although ICH is similar to human DJ-1 (1.6 Å C-␣ root mean square deviation), structural differences in the vicinity of Cys 101 disfavor the facile oxidation of this residue that is functionally important in human DJ-1 but would be detrimental to ICH activity. The ICH active site region also exhibits surprising conformational plasticity and samples two distinct conformations in the crystal. ICH represents a previously uncharacterized clade of the DJ-1 superfamily that possesses a novel enzymatic activity, demonstrating that the DJ-1 core fold can evolve diverse functions by subtle modulation of the environment of a conserved, reactive cysteine residue.

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Evolution of Enzymatic Activities in the Enolase Superfamily: l-Rhamnonate Dehydratase

Cited by 50 publications

References 32 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

Divergent Evolution in Enolase Superfamily: Strategies for Assigning Functions

Evolution of New Enzymatic Function by Structural Modulation of Cysteine Reactivity in Pseudomonas fluorescens Isocyanide Hydratase

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