Chelation of Serine 39 to Mg2+ Latches a Gate at the Active Site of Enolase: Structure of the Bis(Mg2+) Complex of Yeast Enolase and the Intermediate Analog Phosphonoacetohydroxamate at 2.1-.ANG. Resolution

Wedekind, Joseph E.; Poyner, Russell R.; Reed, George H.; Rayment, Ivan

doi:10.1021/bi00197a038

Cited by 110 publications

(189 citation statements)

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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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“…We note that the C-terminal 17 amino acid residues of RecA protein also include three serine and threonine residues. There is ample precedent that these could also be involved in magnesium ion coordination (35,36). One of these is removed in each of the 6-, 13-, and 17-amino acid residue deletions.…”

Section: Mgmentioning

confidence: 99%

Magnesium Ion-dependent Activation of the RecA Protein Involves the C Terminus

Lusetti

Shaw

Cox

2003

Journal of Biological Chemistry

122

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Optimal conditions for RecA protein-mediated DNA strand exchange include 6 -8 mM Mg 2؉ in excess of that required to form complexes with ATP. We provide evidence that the free magnesium ion is required to mediate a conformational change in the RecA protein C terminus that activates RecA-mediated DNA strand exchange. In particular, a "closed" (low Mg 2؉ ) conformation of a RecA nucleoprotein filament restricts DNA pairing by incoming duplex DNA, although singlestranded overhangs at the ends of a duplex allow limited DNA pairing to occur. The addition of excess Mg 2؉ results in an "open" conformation, which can promote efficient DNA pairing and strand exchange regardless of DNA end structure. The removal of 17 amino acid residues at the Escherichia coli RecA C terminus eliminates a measurable requirement for excess Mg 2؉ and permits efficient DNA pairing and exchange similar to that seen with the wild-type protein at high Mg 2؉ levels. Thus, the RecA C terminus imposes the need for the high magnesium ion concentrations requisite in RecA reactions in vitro. We propose that the C terminus acts as a regulatory switch, modulating the access of double-stranded DNA to the presynaptic filament and thereby inhibiting homologous DNA pairing and strand exchange at low magnesium ion concentrations.The RecA protein of Escherichia coli plays a central role in the processes of homologous DNA recombination and DNA repair. RecA is a DNA-dependent ATPase that catalyzes an in vitro DNA strand exchange reaction between single-stranded (ssDNA) 1 and homologous double-stranded DNA (dsDNA) molecules. The DNA strand exchange reaction takes place in several stages (Fig. 1). The RecA protein forms a nucleoprotein filament that completely encompasses the circular ssDNA. This filament then aligns the bound single strand with a homologous duplex DNA to form a DNA pairing intermediate often referred to as a joint molecule. 1000 base pairs of DNA can be aligned and exchanged in a joint molecule under the empirically defined optimal reaction conditions, which typically include 1-3 mM ATP and about 10 mM magnesium ion. All steps to this point, including the formation of joint molecules, require ATP but not ATP hydrolysis. ATP hydrolysis is needed only to complete the late stages of strand exchange of long DNA substrates, often derived from bacteriophage DNAs. Whereas DNA pairing, leading to joint molecule formation, can occur at either end of a linear duplex, the subsequent and ATP hydrolysisdependent extension of the nascently paired regions is unidirectional, proceeding 5Ј to 3Ј relative to ssDNA initially bound in the filament. Thus, exchange proceeds in one direction along the linear duplex, and joints formed at the "wrong" end in the pairing phase are eliminated. In a DNA strand exchange involving quite long DNAs that leads to nicked circular product formation, the ends of the duplex where the exchange begins and ends are referred to as proximal and distal, respectively ( Fig. 1) (1, 2).Examination of the conditions for an optimal RecA prot...

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“…This density is in the same position as the second magnesium ion found in the yeast enolase complex with G2P and phosphoenolpyruvate 17 and with an intermediate analogue. 2 Also at the closed-state D subunit of the E. coli enzyme, electron density was observed at the position corresponding to the substrate's phospho group in the yeast enolase complex ( Figure 6). We placed a sulfate ion here, because the height of the peak was comparable to the electron density of the sulfur atoms of the methionine and cysteine side-chains.…”

Section: The Active Sitementioning

confidence: 99%

“…2 Two dimers were placed into the asymmetric unit. Strict non-crystallographic symmetry (NCS) was applied and iterative cycles of rigid body re®nement, simulated annealing, individual B-factor re®nement with CNS 39 and 4-fold non-crystallographic symmetry averaging and solvent attening with DM 40 were carried out.…”

Section: Structure Determinationmentioning

confidence: 99%

Crystal structure of the Escherichia coli RNA degradosome component enolase

Kühnel

Luisi

2001

Journal of Molecular Biology

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The crystal structure of Escherichia coli enolase (EC 4.2.1.11, phosphopyruvate hydratase), which is a component of the RNA degradosome, has been determined at 2.5 A Ê . There are four molecules in the asymmetric unit of the C2 cell, and in one of the molecules,¯exible loops close onto the active site. This closure mimics the conformation of the substratebound intermediate. A comparison of the structure of the E. coli enolase with the eukaryotic enolase structures available (lobster and yeast) indicates a high degree of conservation of the hydrophobic core and the subunit interface of this homodimeric enzyme. The dimer interface is enriched in charged residues compared with other protein homodimers, which may explain our observations from analytical ultracentrifugation that dimerisation is affected by ionic strength. The putative role of enolase in the RNA degradosome is discussed; although it was not possible to ascribe a speci®c role to it, a structural role is possible.

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Chelation of Serine 39 to Mg2+ Latches a Gate at the Active Site of Enolase: Structure of the Bis(Mg2+) Complex of Yeast Enolase and the Intermediate Analog Phosphonoacetohydroxamate at 2.1-.ANG. Resolution

Cited by 110 publications

References 50 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

Magnesium Ion-dependent Activation of the RecA Protein Involves the C Terminus

Crystal structure of the Escherichia coli RNA degradosome component enolase

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