In Silico insights on enhancing thermostability and activity of a plant Fructosyltransferase from Pachysandra terminalis via introduction of disulfide bridges

Abstract

Drawbacks of industrially-used fructosyltransferases (FTs) such as low optimum temperature and low fructooligosaccharides (FOS) yield necessitates the search for engineered FTs that are highly thermostable and active. With the availability of the first plant FT crystal structure from Pachysandra terminalis (PDB ID: 3UGH), computer-aided protein engineering of plant FT is now feasible. To obtain insights on the effect of specific mutations i.e. disulfide bridge introduction, wild-type and mutant FTs were subjected to a 15 μs Martini Coarse-grained Molecular Dynamics (CGMD) simulations at 303 K and 334 K. We report here the five mutants, M31C-Q49C, L144C-S193C, P34C-W300C, S219C-L226C and V470C-S498C with enhanced thermostabilities and/or activities relative to the wild type. Interestingly, M31C-Q49C, which is located within the catalytic-carrying blade of the catalytic domain, has an activity enhancement at both temperatures. At 334 K, three mutations, L144C-S193C, P34C-W300C and V470C-S498C, achieved thermostability relative to the wild type. Intriguingly, both activity and stability enhancement exhibited only at 334 K can be achieved provided that the mutation is located either on the catalytic-carrying residue blade of the catalytic domain or on the non-catalytic domain. Our results suggest that V470C-S498C and L144C-S193C are promising mutants and that domain-specific approach may be exploited to customize enzyme properties.

Keywords: Disulfide bridge; Fructosyltransferase; HOLE; Protein engineering; Thermostability.