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

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

doi:10.3389/fbioe.2015.00074

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

Journal Article · Mon Jun 01 00:00:00 EDT 2015 · Frontiers in Bioengineering and Biotechnology

DOI:https://doi.org/10.3389/fbioe.2015.00074· OSTI ID:1630044

Zadvornyy, Oleg A. ^[1]; Boyd, Eric S. ^[2]; Posewitz, Matthew C. ^[3]; Zorin, Nikolay A. ^[4]; Peters, John W. ^[5]

Montana State Univ., Bozeman, MT (United States). Dept. of Chemistry and Biochemistry; Russian Academy of Sciences (RAS), Pushchino (Russian Federation). Inst. of Basic Biological Problems
Montana State Univ., Bozeman, MT (United States). Dept. of Microbiology and Immunology
Colorado School of Mines, Golden, CO (United States). Dept. of Chemistry and Geochemistry
Russian Academy of Sciences (RAS), Pushchino (Russian Federation). Inst. of Basic Biological Problems
Montana State Univ., Bozeman, MT (United States). Dept. of Chemistry and Biochemistry

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. K_m values for the 2-phosphoglycerate/phosphoenolpyruvate reaction determined at 25 and 80°C were 0.16 and 0.03 mM, respectively. The K_m 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

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Research Organization:: Montana State Univ., Bozeman, MT (United States); SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States). Stanford Synchrotron Radiation Lightsource (SSRL)

Sponsoring Organization:: US Air Force Office of Scientific Research (AFOSR); National Aeronautics and Space Administration (NASA); USDOE Office of Science (SC), Biological and Environmental Research (BER); National Institutes of Health (NIH); National Institute of General Medical Sciences (NIGMS); National Center for Research Resources (NCRR); USDOE Office of Science (SC), Basic Energy Sciences (BES)

Grant/Contract Number:: AC02-76SF00515; FA9550-14-110147; W911NF0510255; NNA 15BB02A

OSTI ID:: 1630044

Journal Information:: Frontiers in Bioengineering and Biotechnology, Vol. 3; ISSN 2296-4185

Publisher:: Frontiers Research FoundationCopyright Statement

Country of Publication:: United States

Language:: English

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37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
59 BASIC BIOLOGICAL SCIENCES
enolase
thermal stability
origin
evolution
hydropathy
green sulfur bacteria

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

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