Remote oxidative modifications induced by oxygen free radicals modify T/R allosteric equilibrium of a hyperthermophilic lactate dehydrogenase

Abstract

L-Lactate dehydrogenase (LDH) is a model protein allowing to shed light on the fundamental molecular mechanisms that drive the acquisition, evolution and regulation of enzyme properties. In this study, we test the hypothesis of a link between thermal stability of LDHs and their capacity against unfolding induced by reactive oxygen species (ROS) generated by γ-rays irradiation. By using circular dichroism spectroscopy, we analysed that high thermal stability of a thermophilic LDH favours strong resistance against ROS-induced unfolding, in contrast to its psychrophilic and mesophilic counterparts that are less resistant. We suggest that a protein's phenotype linking strong thermal stability and resistance against ROS damages would have been a selective evolutionary advantage. We also find that the enzymatic activity of the thermophilic LDH that is strongly resistant against ROS-unfolding is very sensitive to inactivation by irradiation. To address this counter-intuitive observation, we combined mass spectrometry analyses and enzymatic activity measurements. We demonstrate that the dramatic change on LDH activity was linked to remote chemical modifications away from the active site, that change the equilibrium between low-affinity tense (T-inactive) and high-affinity relaxed (R-active) forms. We found the T-inactive thermophilic enzyme obtained after irradiation can recover its LDH activity by addition of the allosteric effector 1, 6 fructose bis phosphate.We analyse our data within the general framework of allosteric regulation, which requires that an enzyme in solution populates a large diversity of dynamically-interchanging conformations. Our work demonstrates that the radiation-induced inactivation of an enzyme is controlled by its dynamical properties.

Protein Conformational Space at the Edge of Allostery: Turning a Nonallosteric Malate Dehydrogenase into an “Allosterized” Enzyme Using Evolution-Guided Punctual Mutations

“…Later, extensive analyses have shown that allosteric regulation is grounded in the protein dynamics, leading to the unifying and general “ensemble model” of allostery ( Hisler et al 2012 ; Motlagh et al 2014 ; Biddle et al 2021 ). In this model, the allosteric capacity of an enzyme depends on the reorganization of the protein conformational landscape, induced by events such as interactions with other protein partners or ligands, local unfolding, and physicochemical variations of the environment ( Schrank et al 2009 ; Arai et al 2010 ; Lisi et al 2018 ; Halgand et al 2020 ; Iorio et al 2021 ).…”

Biochemical, structural and dynamical studies reveal strong differences in the thermal-dependent allosteric behavior of two extremophilic lactate dehydrogenases

“…Since the introduction of this concept, extensive investigations have shown that allostery is rooted in the dynamical properties of proteins, and the general "ensemble model" of allostery has been proposed (Motlagh et al, 2014;Wei et al, 2016;Wodak et al, 2019). According to this model, the allosteric behavior of an enzyme depends on the reorganization of its conformational landscape induced by different events such as interactions with other protein partners and ligands, local unfolding, reactive oxygen species and physicochemical variations in the environment (Astl et al, 2020;Arai et al, 2010;Schrank et al, 2009;Halgand et al, 2020;Lisi et al, 2018). Temperature is a key environmental parameter with a major impact on conformational fluctuations and protein dynamics (Karshikoff et al, 2015;Kalimeri et al, 2013, Capdevila et al, 2017; therefore, understanding how temperature drives allosteric properties of an enzyme is of fundamental importance.…”