The Activity of the Durum Wheat (Triticum durum L.) Catalase 1 (TdCAT1) Is Modulated by Calmodulin

Abstract

Plant catalases (CAT) are involved in the cellular scavenging of the reactive oxygen species during developmental processes and in response to abiotic and biotic stresses. However, little is known about the regulation of the CAT activity to ensure efficient antioxidant function. Using bioinformatic analyses, we showed that durum wheat catalase 1 (TdCAT1) harbors highly conserved cation-binding and calmodulin binding (CaMBD) domains which are localized at different positions of the protein. As a result, the catalytic activity of TdCAT1 is enhanced in vitro by the divalent cations Mn2+ and Fe2+ and to a lesser extent by Cu2+, Zn2+, and Mg2+. Moreover, the GST-pull down assays performed here revealed that TdCAT1 bind to the wheat CaM (TdCaM1.3) in a Ca2+-independent manner. Furthermore, the TdCaM1.3/Ca2+ complex is stimulated in a CaM-dose-dependent manner by the catalytic activity of TdCAT1, which is further increased in the presence of Mn2+ cations. The catalase activity of TdCAT1 is enhanced by various divalent cations and TdCaM1.3 in a Ca-dependent manner. Such effects are not reported so far and raise a possible role of CaM and cations in the function of CATs during cellular response to oxidative stress.

The Putative Auto-Inhibitory Domain of Durum Wheat Catalase (TdCAT1) Positively Regulates Bacteria Cells in Response to Different Stress Conditions

“…Thus, we determined the initial reaction rate (Vo) of TdCAT1 by measuring enzyme kinetics of the purified recombinant deleted proteins during the first minute. Our results confirmed TdCAT 200 to have a very low catalase activity, as previously shown [ 23 ]. Moreover, the catalase activity of TdCAT295 and TdCAT340 forms was lower than that of the complete TdCAT1 ( Figure 3 c), suggesting that the presence of the whole catalase domain of TdCAT1 is essential for the protein’s activity.…”

“…Treatment of TdCAT1 with λ-phosphatase causes the decrease of the catalytic activity of about 35%. Moreover, addition of divalent cations (Ca 2+ , Mg 2+ , Zn 2+ , Cu 2+ , Fe 2+ and Mn 2+ ) stimulates the catalytic activity of native TdCAT1, as previously shown [ 23 ], but not the dephosphorylated protein dpTdCAT1. This could reveal the importance of phosphorylation in regulating TdCAT1 in planta.…”

“…Furthermore, we verified that all mutated forms produced the same amount of proteins, as revealed by Western blot analysis ( Figure 3 b). The optimum buffer concentration, pH and temperature for TdCAT1 proved to be 75 mM, 7 and 25 °C, respectively [ 23 ]. Thus, we determined the initial reaction rate (Vo) of TdCAT1 by measuring enzyme kinetics of the purified recombinant deleted proteins during the first minute.…”

“…Moreover, TdCAT1 confers abiotic stress tolerance to yeast [ 22 ]. Recently, it has been demonstrated that TdCAT1 harbors a conserved Calmodulin- binding domain located at its C-terminal portion [ 23 ]. This domain interacts with Calmodulins (CaM) in a calcium-independent manner but stimulates the catalytic activity of the protein in a calcium-dependent manner.…”

“…This domain interacts with Calmodulins (CaM) in a calcium-independent manner but stimulates the catalytic activity of the protein in a calcium-dependent manner. Moreover, TdCAT1 harbors several conserved cation-binding domains located in different parts of the protein that stimulate its activity [ 23 ]. In this work, we performed bioinformatic analysis of TdCAT1.…”