## 3 Results and Discussion
### Presence of Bound Donor Allows Acceptor Binding to Branching Subsites in Glt20  
A deep biochemical characterization of Glt20 showed the enzyme to be an efficient catalyst for transglycosylation when substrates longer than DP4 were used [34]. Studies using laminaripentaose showed that the donor substrate is transferred to the glucose situated in the third position from the non-reducing end of the acceptor substrate with the formation of a \(\beta\)-(1\(\rightarrow\)6) glycosidic bond. Moreover, a branching point was created, predominantly 2 residues from the formed \(\beta\)-(1\(\rightarrow\)6) glycosidic bond, between the reducing end of the donor and third moiety of the acceptor from the non-reducing end [34]. The structural implications of the experimental evidence were studied by molecular modeling of complexes between Glt20 using laminaripentaose (DP5) as ligand (occupying subsites \(-3\) to +2).  
The molecular model shows that amino acids in subsites \(-3\), \(-2\) and \(-1\) are mostly polar, while those in subsites +1 and +2 are mostly hydrophobic. Indeed, the hydrogen bonds predicted between the ligand and enzyme in the Glt20-laminaripentaose complex (Figure 7) are mainly in subsites \(-3\) and \(-2\). Weaker (hydrophobic) enzyme-ligand interactions in subsites +1 and +2 are consistent with release of the reducing end laminaribiose moiety. At the same time, the laminaritriose remains bound to subsites \(-1\), \(-2\) and \(-3\), stabilized by hydrogen bonds. In accordance with experimental data, smaller substrates are disadvantaged, which is consistent with experimental evidence, indicating that binding to sub site \(-3\) is crucial for activity.  
Nuclear magnetic resonance (NMR) analyses have also demonstrated that Glt20 retains the configuration of theomeric group [34]. Retaining glycosyl hydrolases form a covalent intermediate between the substrate and the catalytic nucleophile Thus, the covalent intermediate with laminaritriose (in subsite \(-3\), \(-2\), \(-1\)) to the catalytic nucleophile E120 (bound via the \(\alpha\)-carbon) was modeled for Glt20 (Figure 8B). Energy minimization of this intermediate conserved most of the predicted interactions in subsites \(-3\), \(-2\) and \(-1\), and no significant conformational changes happened, expect in the glucose moiety covalently bound to E120 (Figure 8B). In addition, Asp35 and Lys258 made additional interactions in subsite \(-3\).  
Formation of the covalent bond between the ligand and enzymes also created space for accommodation of the laminaripentaose acceptor substrate in the active site (Figure 8C). The third glucose moiety from the non-reducing end of the acceptor was situated perpendicular to the glucose of the covalently bound donor. The two glucose moieties in the non-reducing end were stabilized via interactions with amino acids of the \(\alpha\)8-helix and loop 258-265 (Figure 8C), of which the moiety in the non-reducing end is stabilized by Lys258, Arg124 and Asp124. The next glucose moiety is stabilized by the loop 258-265 backbone. These interactions could define the branching subsite \(-3\)* and \(-2\)*, while the \(-1\)* is surrounded by the catalytic residue E209 and the donor substrate. This conformation favors the formation of the \(\beta\)(1-6) bond between the donor and acceptor, giving a branched product, as characterized by NMR and mass spectrometry [34].