## 3 Results and Discussion  
### Homology Modeling and Overall Structure  
The catalytic domains of the GH17 bacterial transglycoses from _P. aeruginosa_ (Glt1), _P. putida_ (Glt3) and _B. diazoefficients_ (Glt20) share relatively high sequence similarity with each other (especially the two Pseudomonas enzymes, Figure 1) but have low sequence homology with enzymes of known 3D-structure in GH17. The closest structure-determined homologue, and thus far only structure-determined transglycos in GH17 originates from the fungus _R. miehei_ (_Rm_Bgt17A) [14]. This enzyme shares 24-27% sequence identity (50-51% related residues) with the bacterial enzymes and has a query sequence coverage of 90-91% with any of the three bacterial sequences investigated here (Figure 1). To obtain structural data, despite the relatively low identity, hybrid modeling was used, including crystallographic structures of several proteins for modeling some fragments of the structures (detailed in Supplementary Table S2). This, together with molecular dynamics refinement, resulted in acceptable models according to several protein-structure validation approaches, including Ramachandran analysis (Table 1) and _Z_-scores (Table 2). Ramachandran diagram analysis of the respective model showed that most amino acids were in the preferred regions (more than 95%) and allowed regions (up to 3.9%), while less than 1.1% of the amino acids were outliers (Table 1). The _Z_-scores of the models were, for all of three transglycoses, in the acceptable distribution range and comparable with the Z-score for the crystal structure of _Rm_Bgt17A (Table 2), which was used as the main template. \begin{table}
\begin{tabular}{c c c c} \hline
**Protein** & **Preferred Regions** & **Allowed Regions** & **Outliers** \\ \hline Glt1 & 268 (95.04\%) & 11 (3.90\%) & 3 (1.06\%) Gln65, Arg217, Ala252 \\ Glt3 & 268 (95.01\%) & 11 (3.90\%) & 3 (1.06\%) Ala15, Ala252, Arg217 \\ Glt20 & 272 (95.77\%) & 11 (3.87\%) & 1 (0.35\%) Asp15 \\ \hline \end{tabular}
\end{table} 
Table 1: Ramachandran diagram analysis. All enzymes displayed the classical (\(\alpha/\beta\))s TIM-barrel fold typical for GH17-enzymes and related glycoside hydrolase families classified under clan A (Figure 2). The main differences between the three bacterial enzymes were found in loops surrounding the catalytic cleft. These loops differed in conformation and length (see below for more details). Additional secondary structure elements (short \(\beta\)-strands and/or short \(\alpha\)-helices) not present in the crystallographic structure of \(Rm\)Bgt17A, were also found close to the N- and/or C-termini of the amino acid sequences of the catalytic modules (Figure 3). The only non-conserved secondary structure element positioned in the catalytic cleft, was an additional \(\alpha\)-helix, proposed for Glt1 and Glt3 next to strand \(\beta\)1 (Figure 3). The molecular dynamic simulations at 298 \({}^{\circ}\)C with 0.9% NaCl ions for 500 ns, have shown overall conformational variations with an RMSD of alpha carbons between 4 and 6 A (Figure 4A). The trajectory for Glt1 has a peak from the beginning of the production phase until 38 ns, with a maximum RMSD of 7 A at 22 ns. This peak corresponds to a significant conformational change in the C-terminal tail, which includes a loop that connects the a8 helix with a short terminal a helix. This region changes from an extended to a contracted form (Figure 4B). After this change, the trajectory evolves without significant variations with an RMSD around 5 A, with an average structure keeping the C-terminal region in a contracted form. Glt3 and Glt20 do not show significant changes. These models have shown shorter loops in the C-terminal comparing with Glt1 (Figure 3). The RMSF analysis shows high peaks of fluctuations in the amino acids located in all the loops, including the A4 and \(\alpha\)8 helixes, that surround the active site (Figure 4C). The highest fluctuations correspond to the loops \(\beta\)1-\(\alpha\)1 and \(\beta\)8-\(\alpha\)8 and \(\alpha\)8 helix.