## 3 Results and Discussion
### Catalytic Cleft and Sub Site Predictions  
The catalytic cleft is constituted by seven loops (\(\beta\)1-\(\alpha\)1, \(\beta\)2-\(\alpha\)2, \(\beta\)3-\(\alpha\)3, \(\beta\)4-A4, \(\beta\)5-\(\alpha\)5, \(\beta\)6-\(\alpha\)6, and \(\beta\)8-\(\alpha\)8) and the \(\alpha\)-helices 4 and 8, situated on opposite sides of the active site (Figure 2), at the C-terminal side of the \(\beta\)-strand with the same number (Figure 3). Superimposition of the modeled structures with crystal structures available from GH17 (Table 3), showed that the position of the catalytic residues was conserved in the structures and situated in loop \(\beta\)4-A4 (proton donor) and in the C-terminus of \(\beta\)7 (nucleophile) (Figures 3 and 5). The putative catalytic residues of the proteobacterial transglycoses are situated at positions corresponding to those of previously identified catalytic residues in crystallized enzymes in GH17 (including both the fungal _R. Thieli_ transglycoses (_Rm_Bgt17A) and several endoglucanas from plants). Identification of the residues was strengthened by mutagenesis in _Glt20_ where the single mutation in the respective putative catalytic residue: MalE-Glt20:E120Q and MalE-Glt20:E209Q (expressed to the same level as the _wt_ (MalE-Glt20) in _E. coli_) resulted in loss of detectable activity (Supplementary Figure S1), supporting their role in catalysis. Figure 4: Molecular dynamic simulations of Glt1, Glt3 and Glt20. (**A**) Root-mean-square deviation (RMSD) of alpha carbons. The peak observed the first 38 ns in Glt1 corresponds to a conformational change in a C-terminal loop. (**B**) Conformational change in the C-terminal loop of Glt1. (**C**) Root-mean-square fluctuations (RMSF). Peaks corresponding to loops surrounding the active site are indicated in gray, while the helixes in orange. With the exception of \(Rm\)Bgt17A, all crystallized enzymes in GH17 are endo-\(\beta\)-glucanases from plants. The endoglucanases have a higher number of subsites than the fungal transglycosylase and the currently investigated bacterial transglycosases. The fungal enzyme \(Rm\)Bgt17A has 5 subsites (\(-3\). \(-2\), \(-1\), \(+1\) and \(+2\)), in close accordance with the subsites in the bacterial transglycosases investigated here, for which the catalytic clefts include 6 putative subsites (\(-4\), \(-3\), \(-2\), \(-1\), \(+1\) and \(+2\)), while the endo-\(\beta\)-glucanases can have up to 8 subsites (\(-3\). \(-2\), \(-1\), \(+1\), \(+2\), \(+3\), \(+4\) and \(+5\)). An additional subdomain (Figure 5) around the \(\alpha\)6-helix at the end of the catalytic cleft could be the reason for the extended number of \"aglycone\" subsites in the endo-\(\beta\)-glucanases [14,32]. The lengths and conformation of all the loops involved in the catalytic cleft in the bacterial transglycosases were compared with the crystallographic structure of \(Rm\)Bgt17A (Figure 6 and Table 4). Subsite \(+2\) is surrounded by the loops \(\beta\)5-\(\alpha\)5 and \(\beta\)6-\(\alpha\)6, subsite +1 by \(\beta\)4-A4 and the A4 helix, the \(-2\) and \(-1\) subsites are surrounded by the \(\beta\)8-\(\alpha\)8 loop and the \(\alpha\)8 helix while subsite \(-3\) is influenced by the loops \(\beta\)1-\(\alpha\)1, \(\beta\)2-\(\alpha\)2 and \(\beta\)3-\(\alpha\)3. The bacterial transglycosases also host an additional \"glycone\" or donor subsite (\(-4\)), compared to their fungal counterpart. In Glt1, a putative subsite \(-4\) could be constituted by the loop \(\beta\)1-\(\alpha\)1, \(\beta\)2-\(\alpha\)2 and Arg101 in the \(\alpha\)3-helix. In Glt3, a putative subsite \(-4\) could be constituted by Arg94 and Arg97 in the \(\alpha\)3-helix, but in this case, the Arg-residues are  
Figure 5: Superimposition of the modeled Glt-structures and crystallographic structures available in GH17 (see Table 3). Plant endo-\(\beta\)-glucanases are in cyan, GT RmBglt1A in dark blue, Glt1 in ping, Glt3 in yellow and Glt20 in purple. Additional subdomains in endo-\(\beta\)-glucanases are depicted in grey. Catalytic amino acids are represented as red sticks.