## 3 Results and Discussion
### Ligand Interactions after Docking of Laminaripentaose and Laminarilexaose  
Experimental biochemical studies of \(\beta\)1,3-glucooligosaccharides have shown that \(Rm\)Bgt17A [14] and Glt20 [13] cleave the donor substrate 2 glucose units from the reducing end, releasing a laminaribiose and transferring the rest of the substrate to an acceptor oligosaccharide. In contrast, Glt1 and Glt3 transfer laminaroligos of defined length (mainly 3-4 glucose moieties) from the non-reducing end of the substrate to the acceptor oligosaccharide [12]. Despite these differences in product profile, the enzymes are predicted to share most of the amino acids involved in the subsite interactions with the substrates (Figure 7), supporting substrate binding in the same orientation in all enzymes (the reducing end in subsite \(+2\) and non-reducing end in subsite \(-4\)). The major cleavage site of Glt1 is at the fourth (\(\beta\)1-3) linkage from the non-reducing end (in accordance with presence of subsite \(-4\) to \(-1\)). Glt3 has been shown to cleave the substrate at the third and fourth (\(\beta\)1-3) linkage from the non-reducing end (indicating lower affinity to the \(-4\) subsite). The length of the respective donor experimentally observed for Glt1 and Glt3 is thus in accordance with the number of glycone subsites \(-4\) to \(-1\) in Glt1 and Glt3) predicted in the respective enzyme (Figure 7). To further investigate interacting residues and based on the above subsite-comparison and conformations of laminaritriose (PDB code: 4 WTS) and laminaribiose (PDB code: 4 WTR) co-crystallized in \(Rm\)Bgt17A [14], the ligand laminaripentaose was placed in the catalytic cleft of the models orientated with the reducing end in the potential subsite \(+2\) and the non-reducing end in the subsite \(-3\). Laminaripentaose MD simulations resulted in the most stable conformer, a structure with an overall shape very close to the crystallographic ligands laminaritetraose and laminaribiose placed together. Only the dihedral angle of the glycosidic bond in the reducing end was rotated \(180^{\circ}\) to match with the conformation co-crystallized in \(Rm\)Bgt17A of the laminaribiose (PDB 4 WTR). After docking calculations, the conformation of the ligand did not change significantly. As the catalytic clefts of Glt1, Glt3 and Glt20 have space for an additional sugar, one glucose residue was added in a putative subsite \(-4\), with a proper conformation and linked to the laminaripentaose by a \(\beta\)-1,3-glycosidic bond. The resulting complexes with laminarihexaose were energetically minimized to determine the putative interactions in subsite \(-4\). It should be noted that the docking simulations performed in the present work do not consider structural water molecules in the catalytic cleft, which could increase the number of hydrogen bonds between ligand and enzyme residues as three water molecules are found in the crystal complexes of \(Rm\)Bgt17A. Due to the absence of these water molecules, some distances between ligand and potential hydrogen bonding amino acids may be relatively long, and these predictions are approximated. The Glt20 complex is most similar to the \(Rm\)Bgt17A crystallographic complex in terms of interactions with the ligand, since two additional residues, besides Trp65 (87 in Glt20) mentioned in the previous paragraph, Asp67 (Asp89 in Glt20) in subsite \(-3\) and Tyr102(Tyr123) are conserved and interact with the ligand in a similar way. Other potential interactions are shown in Figure 7.  
In Gltl, subsite \(-4\), Gln65 and Arg102 makes two potential hydrogen bonds, each one with the OH-2, OH-3, OH-4 and OH-6 of the non-reducing end glucose of the laminariahexaose. In addition, the hydroxyl group of Ser63 is in the proximity of the sugar residue.