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. 2022 Apr 1;78(Pt 4):177-184.
doi: 10.1107/S2053230X22003612. Epub 2022 Apr 3.

The structure of Synechococcus elongatus enolase reveals key aspects of phosphoenolpyruvate binding

Affiliations

The structure of Synechococcus elongatus enolase reveals key aspects of phosphoenolpyruvate binding

Javier M González et al. Acta Crystallogr F Struct Biol Commun. .

Abstract

A structure-function characterization of Synechococcus elongatus enolase (SeEN) is presented, representing the first structural report on a cyanobacterial enolase. X-ray crystal structures of SeEN in its apoenzyme form and in complex with phosphoenolpyruvate are reported at 2.05 and 2.30 Å resolution, respectively. SeEN displays the typical fold of enolases, with a conformationally flexible loop that closes the active site upon substrate binding, assisted by two metal ions that stabilize the negatively charged groups. The enzyme exhibits a catalytic efficiency of 1.2 × 105 M-1 s-1 for the dehydration of 2-phospho-D-glycerate, which is comparable to the kinetic parameters of related enzymes. These results expand the understanding of the biophysical features of these enzymes, broadening the toolbox for metabolic engineering applications.

Keywords: Synechococcus elongatus; cyanobacteria; enolases; phosphoenolpyruvate.

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Figures

Figure 1
Figure 1
Overall crystal structure of SeEN. The enzyme is likely to adopt an octameric quaternary structure comprising four heart-shaped dimers or pseudo-dimers (shaded yellow). In the resting state, SeEN shows one Ca2+ ion (Ca1 site) coordinated to Asp246, Glu287 and Asp314. The segment 43-PSGASTGTF-51 is conformationally flexible and closes on the active-site cleft upon sequential entrance of the substrate and a second Ca2+ ion (Ca2 site). In this way, the backbone carbonyl of Ala46 becomes a ligand of Ca2. Proton extraction from 2PGA is assumed to occur from behind by Lys339 acting as a strong base, whereas hydration of PEP is believed to take place from the opposite side by a water molecule hydrogen-bonded to Glu209 and Glu168.
Figure 2
Figure 2
Electron-density distribution around the ligand-binding sites of apo SeEN (PDB entry 4rop) (a) and PEP-bound SeEN (PDB entry 5j04) chains A and B (b, c). For simplicity, only those residues that directly interact (dotted lines) with the Ca2+ ions and PEP are displayed. Wireframe surfaces indicate 2F o − F c electron-density maps contoured at 1.5σ. Grey spheres indicate the Ca1 and Ca2 metal-binding sites. Red spheres indicate water molecules.
Figure 3
Figure 3
(a) Sequence-similarity network (SSN) of all enolase sequences currently available in the UniProt database (1231 rep nodes; 46 008 edges). Edges connecting nodes indicate at least 50% sequence similarity. Node size is proportional to the number of IDs per rep node, whereas square rep nodes indicate the presence of at least one experimentally determined enolase structure (i.e. associated PDB entries). The node containing the SeEN sequence is indicated by a black arrow. (b) The NC distribution indicates that all functionally and structurally characterized enolases belong to the largest cluster, accounting for 73% of the family, of which about 50% is composed of eukaryotic enolases. (c) Unrooted phylogenetic tree of 32 structurally characterized enolases (see Supplementary Table S1). Organism names highlighted in orange indicate enolase structures that are predicted to exhibit an octameric quaternary structure.
Figure 4
Figure 4
Binding of PEP in the active site of SeEN. The PEP molecule is shown in the two conformations found in chains A (green) and B (blue) of PDB entry 5j04. The water molecule ‘w’ is 3.5 Å from C3 of the PEP molecule (red dotted line) and is hydrogen-bonded to His367 and Glu168. Compare with Fig. 2 ▸. Presumably, during the dehydration of 2PGA or the hydration of PEP (bottom), a water molecule is exchanged from the Glu168 side, whereas proton extraction occurs from the opposite side, with Lys339 acting as a strong base.

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