3.1. Determination of TdCAT1 activity
After recovery from the inclusion bodies, the recombinant His_TdCAT1 was purified using Ni-sepharose column chromatography (Fig. 1a, b). It is known that the activity of CATs towards their substrate is very low [41]. Thus, the optimum buffer concentration was also studied in presence of different buffer concentrations (25, 50, 75 and 100 mM phosphate buffer). These results showed that saturating buffer concentration is 75 mM. Moreover, the effect of pH upon TdCAT1 activity in the decomposition of H2O2 was studied. The pH selected ranged from 3.0 to 9.0. Below pH 5 the activity of TdCAT1 was very low (Fig. 1c). The reaction of CAT toward H2O2exhibits remarkable pH stability; negligible change is measured from pH 3 to 5. Moreover, this activity showed a sharp optimum at pH =7 in presence of 160 µg of recombinant His_TdCAT1 as a minimum quantity with which we register a significant activity. Thus, for the rest of the experiments, the pH was fixed at 7.
For the optimum temperature, we performed a series of CAT activity assays using different temperatures 10 to 80°C. Our results show that the catalase activity is important at 25°C and decreases gradually with temperature (Fig. 1d). This activity depends on the tertiary structure of the protein since protein denaturation by heat treatment completely abolishes the CAT activity (data not shown). Thus, in this study the optimum buffer concentration, pH, and temperature for TdCAT1 were determined as 75 mM, 7 and 25°C, respectively.
In a second step, we determined the initial reaction rate (Vo) by measuring enzyme's kinetics of the purified recombinant proteins His_TdCAT1 during the first min. As it is known that CAT activities can be modulated by bivalent cations [42], and the registered activity in our experimental assays was relatively low (96.27 µmol/min/mg of protein), we investigated whether His_TdCAT1 needs divalent cations to enhance its activity. For this purpose, different enzyme assays were performed with TdCAT1 in the presence of 2 mM of Mn2+, Mg2+, Ca2+, Fe2+, Zn2+, Cd2+ or Cu2+. Experimental results showed that the catalytic activity is significantly stimulated in the presence of 2 mM Mn2+ and Fe2+ and with a lesser extent by Zn2+, Cu2+ and Ca2+ and slightly by Mg2+ (Fig. 1e). In contrast, this activity was not significantly modified by Cd2+ (Fig. 1e). Thus, a dose response assay was performed with these cations separately and the results showed that the activity of TdCAT1 is enhanced by increasing Mn2+, Ca2+, Fe2+, Zn2+, Cu2+ or Mg2+ concentrations. In fact, the maximal activity of TdCAT1 (about 16-fold higher than in control conditions) was reached using 1 mM Fe2+ (Fig. 2a) or Mn2+ (Fig. 2b). In the presence of those cations, the catalytic activity of TdCAT1 started to increase with 0.5 mM of both cations (about 3-fold; Fig. 2a, b). Interestingly, the same result was observed in presence of Ca2+ cations. In fact, the catalase activity of TdCAT1 was stimulated 5 times than in absence of calcium (Fig. 2c). This stimulation started with 1 mM Ca2+ and reached its maximum in presence of 2 mM Ca2+. In another hand, in presence of Zn2+ and Cu2+, the catalytic activity of TdCAT1 increases with 0.5 mM of both cations (̴ about 8-fold increase) and reached to the maximum in presence of 3 mM (Suppl. Fig. 1a, b). In the presence of Mg2+, the activity of TdCAT1 also increases in a dose-dependent manner, but to a lesser extent than with Mn2+, Fe2+, Zn2+, Cu2+, and Ca2+ (Suppl. Fig. 1c). The stimulation started to increase in presence of 0.5 mM Mg2+ and reached its maximum of 3.2-fold increase using 3 mM Mg2+ compared to the basal activity (Suppl. Fig. 1c). The effects of all those cations were exerted gradually with no fold induction. Therefore, Mn2+ and Fe2+ appear more efficient than other cations (Mg2+, Zn2+, Cu2+, and Ca2+) on the TdCAT1 activity in vitro. Thus, those cations were used to perform the rest of experiments. Altogether, these results showed that the catalase activity of TdCAT1 increase in a gradual manner by increasing cations concentration in the medium. It is worth to note that cation stimulation is specific and not artifactual since no activity could be detected when similar assays are performed in absence of enzyme or when we used a heat denatured form of His_TdCAT1 incubated at 100°C for 10 minutes (data not shown).
3.2. TdCAT1 harbors conserved ion binding motifs required for its activations by divalent cations at different parts of the protein
The observed stimulatory effects of Fe2+, Cu2+, Zn2+, Mn2+ and Mg2+ on TdCAT1 activity suggest that durum wheat CAT may harbor cation binding motifs. Thus, we analyzed the structure of TdCAT1 to identify potential cations binding domains. Alignment with well-known Mn2+ or Mg2+ binding proteins (http://www.uniprot.org) revealed the presence of putative Mn2+(position 44-55) and Mg2+ (position 439-449) binding sites on the N-terminal and the C-terminal region of TdCAT1 sequence respectively (Fig. 3a). These domains are highly conserved among CATs proteins from different plant species (Suppl. Fig. 2a, b). Moreover, we identified putative Copper/Zinc binding domain, Calcium binding domain and Iron binding domain in the sequence of TdCAT1. In fact, the analysis of TdCAT1 amino acids sequence revealed that this protein contains H-(X)12 –H type motif, known as a cupper binding domain, which is localized at 165-177aa position in the TdCAT1 sequence (HIQENWRILDLFSH, Fig. 3a). In another hand, sequence investigation of zinc binding domains shows that TdCAT1 contains a domain homolog to the domain identified in protein AN1. These motifs bind a single zinc atom (the European Bioinformatic Institute: https://www.ebi.ac.uk/interpro/potm/2007_3/) and is localized at the amino acids 163-191 position. This motif is related to AtSAP10 protein (Q9STJ9). Thus, TdCAT1 harbors a Zn/Cu binding domain located at its N-terminal region (163-191aa; Suppl. Fig. 3). Moreover, using the Swiss model database, (https://swissmodel.expasy.org/interactive/T5XR77/models/), we found a degenerate domain for iron binding (HDV domain) located at 76-85aa position with conservation of Histidine residue implicated in iron binding. This domain is also well conserved in all studied CATs (data not shown). Finally, using the Supfam databases (http://supfam.org/SUPERFAMILY/cgi-bin/align.cgi), a Calcium binding domain called EF-hand was identified in the sequence of TdCAT1. The consensus pattern for calcium-binding is D-x-[DNS]-{ILVFYW}-[DENSTG]-[DNQGHRK]-{GP}-[LIVMC]-[DENQSTAGC]-x(2)-[DE]. In TdCAT1, this domain is localized at amino acids position 266-293 (Suppl. Fig. 4).
To confirm the presence of those putative cation binding domains, we generated three different deleted forms which are TdCAT200 (containing the first 200 aa), TdCAT295 (containing the first 295 aa), and TdCAT460 (containing the first 460 aa) (Fig. 3b). After production and purification of those forms, we measured their catalytic activities in absence of divalent cations. Interestingly, TdCAT200 has a very week basal activity (4.011 µmol/min/mg of protein) whereas TdCAT295 has a better catalytic activity (29.41 µmol/min/mg of protein) while TdCAT460 and the non-truncated protein TdCAT1 have the same catalytic activity (96.27 µmol/min/mg of protein) (Fig. 3c). This result could be explained by the fact that TdCAT1 protein contains one catalase domain (Pfam Id PF00199, 18-399 aa) and catalase related immune-responsive (Pfam Id PF06628.11, 421- 486; data not shown) as revealed by HMMER database. Those results were also shown for some OsCATs demonstrating that the presence of whole catalase domain is essential for protein activity [41]. As a result, the entire catalytic domain is important for the catalytic activity of TdCAT1.
To confirm the presence of these cation binding domains, the different deleted forms were used to perform CAT activity tests. We measured the catalytic activities of those forms in presence of divalent cations. We first used the TdCAT200 form that contains the Mn2+, Cu2+/Zn2+ and Fe2+ binding domains (Fig. 3b). As expected, TdCAT200 activity was stimulated by Mn2+, Fe2+, Cu2+ andZn2+ but not with Ca2+ and Mg2+ (Fig. 4a) confirming that the Ca2+ and Mg2+ binding domains are not located in the first 200 aa while Mn2+, Fe2+, Cu2+ and Zn2+ binding domains could be present in those first 200 aa. Similarly, TdCAT295 was stimulated by Mn2+, Fe2+, Cu2+, Ca2+ and Zn2+ but not with Mg2+ (Fig. 4b) while TdCAT460 was stimulated also by Mg2+ (Fig. 4c). Those results suggested strongly that TdCAT1 contains 5 different putative cations binding domains that are localized at different parts of the protein (Fig. 3a).
3.6. Effects of TdCaM1.3 on TdCAT1 activity
It has been demonstrated that CaM proteins interact with various target proteins and modulate their activities [22, 26, 27, 43]. Consequently, we investigated the effect of wheat calmodulin binding on the catalase activity of TdCAT1 using in vitro assays. As shown in Fig. 7a, in the absence of Ca2+, TdCaM1.3 alone did not modify TdCAT1 activity. Thus TdCaM1.3 alone had no effect on catalase activity of TdCAT1. In the presence of calcium, both His-TdCaM1.3 (Fig. 7b) and GST-TdCaM1.3 (data not shown) stimulated the catalytic activity of TdCAT1 with the same fold and it reached the maximum in the presence of 2 mM of Ca2+. Thus, for the rest of the catalase assays, we used His-TdCaM1.3. TdCAT1 activity started to increase with Ca2+ concentrations as low as 0.5 mM and stimulation reaches its maximum level using 2 mM of Ca2+ corresponding to 8 times increase of the Vo in presence of TdCaM1.3 (Fig. 7b). Moreover, the addition of EGTA, a well-known Ca2+ chelator, restores the activity of TdCAT1 in presence of TdCaM1.3/Ca2+ (Fig. 7b), whereas EGTA alone does not alter the TdCAT1 activity. This result indicated that Ca2+ is necessary for the activation of TdCAT1 by the calmodulin TdCaM1.3. As Mn2+ and Fe2+ were shown to enhance the TdCAT1 catalytic activity (Fig. 2), we also evaluated the effects of TdCaM1.3/Ca2+ on the TdCAT1 activity in the presence of those cations. Remarkably, in a buffer containing 2 mM of Mn2+ or Fe2+, addition of Ca2+ slightly increases the catalase activity of TdCAT1 (Fig. 8a). In the presence of TdCaM1.3/Ca2+ complex, addition of Mn2+stimulates the activity of TdCAT1 (Fig. 8b). TdCaM1.3 and calcium rather increase TdCAT1 activity by about 2-fold comparatively to the activity measured only in the presence of CaM/Ca2+ alone (Fig. 8b). This stimulatory effect of TdCaM1.3 occurs albeit with lower efficiency even with concentrations of Mn2+, Fe2+ and Ca2+ as low as 0.5 mM (Fig. 8b). In presence of Mn2+, the increase reached it’s maximum with 0.5 mM, while in presence of Fe2+, the activity started to increase gradually and reached it’s maximum in presence of 2 mM Fe2+ (Fig. 8c). This increase is calcium-dependent because addition of EGTA is sufficient to return the TdCAT1 activity to its initial level (Fig. 8b). Finally, to confirm the observed regulatory effects of TdCaMs/Ca2+ on TdCAT1 activity, we performed a new series of phosphatase assays using the truncated form His_TdCAT200. As expected, the activity of His_TdCAT200remains unchanged in the presence of TdCaMs/Ca2+. There is neither a negative (in the absence of Ca2+; Fig. 9a) nor a positive effect (in the presence of TdCAT1/ TdCaM1.3 ratio molar of 1:4 and Ca2+; Fig. 9b) of TdCaM1.3 on the catalytic activity of this truncated TdCAT200 protein mainly in presence of increasing quantities of CaMs in the medium (data not shown). The same effect is observed in presence ofMn2+ and Fe2+ cations (Fig. 9c). All together, these data confirm that the catalytic activity of TdCAT1 can be specifically activated by CaM/Ca2+ in the presence of Mn2+ and Fe2+.