Abstract 1087 Implications on ionic concentration and mutations for the dimerization of a GH1 β-Glycosidase

Many enzymes are presented in oligomeric forms, including the GH1 β Glucosidase from Spodoptera frugiperda (Sfβglu), which was shown to be more active in its homodimeric form. However, the interface of that homodimer and the interactions between the monomers remain to be better explored. Here, using size exclusion chromatography performed in a range of buffer concentrations and subsequently mutations directed to the interface of the homodimer, we showed that the hydrophobic effect is the main component that leads to dimerization. The dissociation constant (Kd) for the Sfbglu dimer decreases while the concentration of the buffer (Phosphate pH 6 ; P) increases, ranging from 135 μM in 5mM P to 7.8 mM in 100 mM P, an effect similar to that observed in a hydrophobic interaction chromatography (HIC). Also, site-directed mutagenesis of three residues, N112S, M210A and L214A, all of them having more than 70% of the surface area buried in the dimerization interface, resulted in Kd drastic changes, moving it from μM to mM. As an ongoing project the next steps are the Kd determination for the site-directed mutants containing replacement of residues involved in hydrogen bonds in the Sfbglu dimer interface and Small Angle X-ray Scattering (SAXS) experiments of the wild-type and mutant Sfbglu homodimers. In conclusion, the results obtained to date indicate that the hydrophobic effect is the main modulator of the dimerization of GH1 β Glucosidase Sfβglu. These results improve the knowledge about GH1 enzymes and are potentially useful for those seeking to optimize enzyme oligomerization. Supported by CNPq and FAPESP.