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

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 thermop...

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Autores principales: Zadvornyy, Oleg A., Boyd, Eric S., Posewitz, Matthew C., Zorin, Nikolay A., Peters, John W.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4450660/
https://www.ncbi.nlm.nih.gov/pubmed/26082925
http://dx.doi.org/10.3389/fbioe.2015.00074
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author Zadvornyy, Oleg A.
Boyd, Eric S.
Posewitz, Matthew C.
Zorin, Nikolay A.
Peters, John W.
author_facet Zadvornyy, Oleg A.
Boyd, Eric S.
Posewitz, Matthew C.
Zorin, Nikolay A.
Peters, John W.
author_sort Zadvornyy, Oleg A.
collection PubMed
description 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 Mg(2+) 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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spelling pubmed-44506602015-06-16 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. Front Bioeng Biotechnol Bioengineering and Biotechnology 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 Mg(2+) 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. Frontiers Media S.A. 2015-06-01 /pmc/articles/PMC4450660/ /pubmed/26082925 http://dx.doi.org/10.3389/fbioe.2015.00074 Text en Copyright © 2015 Zadvornyy, Boyd, Posewitz, Zorin and Peters. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Bioengineering and Biotechnology
Zadvornyy, Oleg A.
Boyd, Eric S.
Posewitz, Matthew C.
Zorin, Nikolay A.
Peters, John W.
Biochemical and Structural Characterization of Enolase from Chloroflexus aurantiacus: Evidence for a Thermophilic Origin
title Biochemical and Structural Characterization of Enolase from Chloroflexus aurantiacus: Evidence for a Thermophilic Origin
title_full Biochemical and Structural Characterization of Enolase from Chloroflexus aurantiacus: Evidence for a Thermophilic Origin
title_fullStr Biochemical and Structural Characterization of Enolase from Chloroflexus aurantiacus: Evidence for a Thermophilic Origin
title_full_unstemmed Biochemical and Structural Characterization of Enolase from Chloroflexus aurantiacus: Evidence for a Thermophilic Origin
title_short Biochemical and Structural Characterization of Enolase from Chloroflexus aurantiacus: Evidence for a Thermophilic Origin
title_sort biochemical and structural characterization of enolase from chloroflexus aurantiacus: evidence for a thermophilic origin
topic Bioengineering and Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4450660/
https://www.ncbi.nlm.nih.gov/pubmed/26082925
http://dx.doi.org/10.3389/fbioe.2015.00074
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