Biochemical and Structural Characterization of Enolase from Chloroflexus aurantiacus: Evidence for a Thermophilic Origin.

Oleg A Zadvornyy,Nikolay A Zorin,Matthew C Posewitz,John W Peters,Eric S Boyd

doi:10.3389/fbioe.2015.00074

Abstract

Enolase catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate during both glycolysis and gluconeogenesis, and is required by all three domains of life. Here, we report the purification and biochemical and structural characterization of enolase from Chloroflexus aurantiacus, a thermophilic anoxygenic phototroph affiliated with the green non-sulfur bacteria. The protein was purified as a homodimer with a subunit molecular weight of 46 kDa. The temperature optimum for enolase catalysis was 80°C, close to the measured thermal stability of the protein which was determined to be 75°C, while the pH optimum for enzyme activity was 6.5. The specific activities of purified enolase determined at 25 and 80°C were 147 and 300 U mg−1 of protein, respectively. Km values for the 2-phosphoglycerate/phosphoenolpyruvate reaction determined at 25 and 80°C were 0.16 and 0.03 mM, respectively. The Km values for Mg2+ binding at these temperatures were 2.5 and 1.9 mM, respectively. When compared to enolase from mesophiles, the biochemical and structural properties of enolase from C. aurantiacus are consistent with this being thermally adapted. These data are consistent with the results of our phylogenetic analysis of enolase, which reveal that enolase has a thermophilic origin.

Highlights

Enolase (2-phospho- -glycerate hydrolyase, EC 4.2.1.11) catalyzes the conversion of 2-phosphoglycerate (2-PGA) to phosphoenolpyruvate (PEP) during both glycolysis and gluconeogenesis in all three domains of life (Ballou and Wold, 1957; Wold, 1971)
Thermophilic Origin and Properties of Thermal Adaptation of Enolase from C. aurantiacus enolase-1 from Chloroflexus aurantiacus (EnoCa) shares significant sequence identity with enolases from other organisms distributed across Bacteria and Archaea
Evolutionary analysis of archaeal and bacterial enolase-1 protein representatives, when rooted with representative enolase-2 proteins, indicate that the earliest branching lineages derive from thermophilic taxa indicating that the enzyme likely has a thermophilic origin

Summary

Introduction

Enolase (2-phospho- -glycerate hydrolyase, EC 4.2.1.11) catalyzes the conversion of 2-phosphoglycerate (2-PGA) to phosphoenolpyruvate (PEP) during both glycolysis and gluconeogenesis in all three domains of life (Ballou and Wold, 1957; Wold, 1971). Enolases are highly conserved enzymes and commonly exist as homodimers. Abbrevations: EnoCa, enolase-1 from Chloroflexus aurantiacus; FIRST, floppy inclusion rigid substructure topography; 2-PGA, 2-phosphoglycerate; PEP, phosphoenolpyruvate; PAGE, polyacrylamide gel electrophoresis; PEG, polyethylene glycol; PDB, protein data bank; R.m.s.d.s, root-mean-square deviations; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis. Enolase from the anaerobic, hyperthermophilic archaeon Pyrococcus furiosus was reported as a homodimer (Peak et al, 1994)

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Conclusion