## 3 Results and Discussion
### Catalytic Cleft and Sub Site Predictions
\begin{table}
\begin{tabular}{c c c c c c c c c} \hline \hline
**Loop** & \(\beta 1\)**-\(\alpha 1\)** & \(\beta 2\)**-\(\alpha 2\)** & \(\beta 3\)**-\(\alpha 3\)** & \(\beta 4\)**-\(\alpha 4\)** & \(\beta 5\)**-\(\alpha 5\)** & \(\beta 6\)**-\(\alpha 6\)** & \(\beta 8\)**-\(\alpha 8\)** & **At Helix** & **\(\alpha 8\) Helix** \\ \hline \multirow{2}{*}{\(Rm\)Bgt17A} & 9-17 & 38-41 & 65-68 & 97-99 & 134-139 & 153-161 & 230-235 & 100-103 & 236-243 \\  & GVNE-NSCPT & FALS & WIDR- & GSE & VYYFP- & AFPYWEGVT & & & VLVR & PYRG-GVEA \\ \hline \multirow{2}{*}{Glt1} & 26-41 (26-31)\({}^{\star}\) & 62-65 & 87-91 & 118-120 & 152-158 & 173-181 & 250-255 & 121-124 & 256-267 \\  & SFFRLNONPQGKRYS & Y5 VQ & WLFQPD & GNE & QWHVYWEATP & VLFQPOAETP & IEAFDQ & ALFR & PWKVAEGSVGA \\ \hline \multirow{2}{*}{Gt3} & 26-41 (26-32)\({}^{\star}\) & 62-65 & 87-91 & 118-120 & 152-158 & 173-181 & 250-255 & 121-124 & 256-267 \\  & SFFRLEGFSPQGCQYPT & YIVE & WISPD & GNE & QWHWKE & LPVWEFVP & IEAFDQ & ALFR & PWKADSEGSVGA \\ \hline \multirow{2}{*}{Gt20} & 26-41 & 61-64 & 87-91 & 118-120 & 152-158 & 173-181 & 250-255 & 121-124 & PWKFA- \\  & APFECSAHPDDINIPT & YSST & WIDKD & GNE & IWINWRD & VLPVWENFR & VEADID & IYR & FRGCGVGP \\ \hline \hline \end{tabular} \({}^{\star}\) Additional \(\alpha\)-helix in the middle of loop \(\beta 1\)-\(\alpha 1\) present only in Glt1 and Glt3.  
\end{table} 
Table 4: Alignment of amino acid sequences of the loops and helices located in the catalytic cleft. Figure 6: Comparison of conformations and lengths of the loops and \(\alpha\)-helixes involved in the catalytic clefts. Models: Glt1 in cyan, Glt3 in purple and Glt20 in brown. The crystallographic structure of \(Rm\)Bgt17A (PDB code: 4 WTR) is in orange (sequences are summarized in Table 4). Figure 7: Enzyme-substrate complexes modeled in this work, compared with the co-crystallized complexes \(Rm\)Bgt17A—laminartirose (4 WTS)—laminartiose (4 WTR) [14]. Glt1, Glt3 and Glt20 have a putative additional subsite \(-4\). However, in Glt3, the predicted interactions in \(-4\) subsite are less clear, Arg94 and Arg97 are the closest amino acids to the glucose in the putative \(-4\) subsite. In the other end, the \(+1\) and \(+2\) subsites are surrounded by loops more similar both in conformations and length (Figure 6 and Table 4), and some amino acids involved in the ligand interactions are conserved compared with \(Rm\)Bgt17A, including Trp157 (177 in Glt1, Glt3 and Glt20) and Glu158 (178 in Glt1, Glt3 and Glt20). The conserved residues Trp157, E158 of \(Rm\)Bgt17A, have been subjected to mutagenesis (along with the less conserved residue Tyr102, \(R\). _miehi_ numbering) [33]. This work showed that E158, which is also conserved in all the bacterial transglycos (Figure 7) was important and its removal increased hydrolytic activity [33]. The \(\alpha\)8-helix adopts different conformations in every protein studied (Figures 2 and 7). Molecular dynamic simulations have shown that this \(\alpha\)8-helix undergoes important conformational changes and as this secondary element is situated close to the \(-1\) and \(+1\) subsites, it could influence the catalytic mechanism (including the possibility of obtaining branched products in Glt20, see also below).