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. 2020 Sep 23;10:15493. doi: 10.1038/s41598-020-72418-4

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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Specific activities of the ancestral and extant IPMDHs. (a) Plot of specific activity as a function of temperature. The assay solution was composed of 50 mM HEPES (pH 8.0), 100 mM KCl, 5 mM MgCl₂, 0.2 mM D-3-IPM, 5 mM NAD⁺, and 0.1–1.0 μM protein. Each value is the average of three measurements. The specific activities of both ancIPMDH-IQ and ancIPMDH-ML were greater than that of T. thermophilus enzymes by a factor of more than three and two, respectively, below 30 °C (inset). (b) Plot of relative activity as a function of temperature. Shown are the relative values of specific activity at various temperatures compared with the activity at the optimal temperature for each enzyme. (c) Arrhenius plot of the specific activities of the ancestral and extant IPMDHs. Activation energy (E_a) was calculated from the slope of each plot. E_a: ancIPMDH-IQ, 50 kJ/mol; ancIPMDH-ML, 59 kJ/mol; T. thermophilus PMDH, 93 kJ/mol; B. subtilis IPMDH, 66 kJ/mol; S. cerevisiae IPMDH, 64 kJ/mol. Green, ancIPMDH-IQ; orange, ancIPMDH-ML; magenta, T. thermophilus PMDH; blue, B. subtilis IPMDH; cyan, S. cerevisiae IPMDH.