Engineering disulfide bonds in Selenomonas ruminantium β-xylosidase by experimental and computational methods

Homotetrameric β-xylosidase from Selenomonas ruminantium (SXA) is one of the most efficient enzymes known for the hydrolysis of cell wall hemicellulose. SXA shows a rapid rate of activity loss at temperatures above 50 °C. In this study, we have introduced two inter-subunit disulfide bridges with one point mutation. Lys237 was chosen to be replaced with cysteine since it interacts with the same residue in the opposite subunit. While pH optimum, temperature profile and catalytic efficiency of the mutated variant were similar to the native enzyme, the mutated enzyme showed about 40% increase in thermal stability at 55 °C. Our results showed that introduction of a single residue mutation in structure of SXA results in appearance of two disulfide bonds at dimer-dimer interface of the enzyme. Coarse-grained molecular dynamics (CG-MD) simulations also proved lower amounts of root mean square fluctuation (RMSF) for position 237 and potential energy for mutated SXA. Based these results, we suggest that choosing a correct residue for mutation in multi subunit proteins results in multiple site conversions which equals to several simultaneous mutations. Furthermore, CG-MD simulation in agreement with experimental methods showed higher thermostability of mutated SXA which proved applicability of this simulation for thermostability analysis.