Effect of temperature on the structure of α- <scp>l</scp> -fucosidase from <i>Thermotoga maritima</i> : implications from molecular dynamics simulation

Human milk oligosaccharides, like 2'-fucosyllactose, have beneficial effects on newborn health, and they can be obtained by enzymatic synthesis with α-l-fucosidases. In this work, the impact of temperature on the α-l-fucosidase from <i>Thermotoga martima</i> (TmαFuc) structure was evaluated using molecular dynamics simulation (MD). The TmαFuc was found stable in a temperature range of 333-368 K since no differences in the RMSD, RMSF, H-bonds, solvent accessible surface area, radius of gyration, salt bridges and native contacts (<i>Q</i>) values were observed. Elevated temperature did not affect the protein secondary fold; nevertheless, increasing temperature to 473 K decreased the stabilizing structure, such as α-helices and β-sheets, and increased the presence of irregular structures. Eventually, these conformational changes caused the loss of enzymatic activity at high temperatures. Additionally, the MD results showed that the enzyme active site could adopt the following conformations: open, intermediate, or closed; these conformations are needed first to retain the substrates in the transglycosylation activity, such as the donor and the acceptor and then to release the transfructosylated product. Furthermore, Free Energy Landscape analysis showed that the increment in temperature facilitates the enzyme to fluctuate between conformational states, and that the system moves freely between states, suggesting frequent conformational transitions.