**10. Discussion**

of secondary structure organization, it was possible to determine that about 33% of the sequence can form α‐helix/3‐10 helix and that about 23% can take part of β‐sheet conforma-

**Figure 3.** Sequence alignment of caspases and metacaspases, displaying the corresponding secondary structure. Blue: catalytic histidine; yellow: catalytic cysteine; brown: polar residues; grey: aliphatic residues; pink: contact aspartic acid and arginine residues; green: hydrophobic residues. Blue box: β conformation; red box: α‐helix/310helix. Star: catalytic residues.

The search for a protein that could be applied as a template using the individual subunits from *G. max* metacaspase, resulted in only one choice with a significant sequence similarity rate for the p20 subunit: the chain A of a protein complex from *Geobacter sulfureduccens* (PDB‐3BIJ). The identity and similarity rates between the template (from the 60th to the

**9.2. Molecular structure prediction of** *G. max* **metacaspase 4**

tion (**Figure 3**).

38 Enzyme Inhibitors and Activators

The sequence comparison of metacaspases and caspases domains p20 and p10 clearly shows differences on the amino acid composition and disposition. Nevertheless, the segments, once aligned, displayed conserved positions of catalytic residues and of other amino acid residues with conserved physical‐chemical properties, what is important to the arrangement on a similar secondary structure. Among the p20 secondary structure, six peptide sequences participate on α‐helixes and 3–10 helixes, and seven composes β‐conformations; for p10, three sequences form helix structures and two originate β‐conformations. Together, these structures are organized in a similar way to that observed for the protein from the CD clan of C14 family of the cysteine proteases, which includes caspases and metacaspases [86]. The constructed *G. max* metacaspase model shows that these sequences are organized in form to present a core of β conformations encircled by five helixes, with the amino acid residues which compose the active site localized in one of the enzyme central axis poles, out of the β‐sheets and α‐helixes region.

The used template here was the chain A of the protein not functionally uncharacterized 3BIJ protein of *G. sulfureduccens*. This is the same protein used by Dudkiewicz and Piszczek [87], for the prediction of a model for *Triticum aestivum* type II metacaspase. Interestingly, 3BIJ was seen to be a better template for the considered metacaspase than the *Homo sapiens* caspase 7, whose similarity with soybean metacaspase was also high.

Curiously, a number of recent reports have demonstrated a difference of cleavage specificity among caspases and metacaspases. The recombinant metacaspase McIIPa, from *P. abies,* was shown to be efficient on the cleavage of the peptide sequence EGR and GRR, but not of VEID and YVAD, which are processed by caspases [46]. In 2008, He et al. [88] demonstrated that the recombinant metacaspase 8 from *Arabidopsis thaliana* was efficient on the cleavage of the sequence GRR, being unable to process DEVD, VEID, IETD and YVAD. This difference on enzymatic activity nature of metacaspases and caspases are generating an open discussion on literature. The denomination "caspase" itself gives clues to these discussions concerning to the particularities presented by caspases and metacaspases. Carmona‐Gutierrez et al. [51] suggested that metacaspases and caspases present characteristics that fulfil the homology criteria, as they participate of a common program, share substrates and by the fact that metacaspase genes are present in all organisms, except superior animal taxa. In this scenario, the caspase genes could be derived from metacaspases.

In response, Enoksson and Salvesen [52] defended that yeasts and plants would employ PCD programmes other than apoptosis, what would be an innovation when compared to animals. Also they argue that, even if metacaspases and caspases share the tridimensional structure, the cleavage specificity displayed by them could show that they are derived from a common ancestor, which was neither caspase nor metacaspase.

This scenery is reinforced by data from the work of Koonin and Aravind [11], which showed that metacaspases have similarities with α‐proteobacteria homologues, the group of endosymbiotic mitochondria ancestors, being the metacaspases from prokaryotic origin. Also, it was demonstrated that bacterial homologues of caspase‐related proteins showed a greater diversity of phyletic distribution, domain architecture and sequence than their eukaryotic counterparts, suggesting that events of gene transference from prokaryote to eukaryotes could be an explanation for the distribution of caspase‐related genes, what could have been assured by multiple bacterial gene infusions [87].
