An acceptor-substrate binding site determining glycosyl transfer emerges from mutant analysis of a plant vacuolar invertase and a fructosyltransferase

Abstract

Glycoside hydrolase family 32 (GH32) harbors hydrolyzing and transglycosylating enzymes that are highly homologous in their primary structure. Eight amino acids dispersed along the sequence correlated with either hydrolase or glycosyltransferase activity. These were mutated in onion vacuolar invertase (acINV) according to the residue in festuca sucrose:sucrose 1-fructosyltransferase (saSST) and vice versa. acINV(W440Y) doubles transferase capacity. Reciprocally, saSST(C223N) and saSST(F362Y) double hydrolysis. SaSST(N425S) shows a hydrolyzing activity three to four times its transferase activity. Interestingly, modeling acINV and saSST according to the 3D structure of crystallized GH32 enzymes indicates that mutations saSST(N425S), acINV(W440Y), and the previously reported acINV(W161Y) reside very close together at the surface in the entrance of the active-site pocket. Residues in- and outside the sucrose-binding box determine hydrolase and transferase capabilities of GH32 enzymes. Modeling suggests that residues dispersed along the sequence identify a location for acceptor-substrate binding in the 3D structure of fructosyltransferases.

Fig. 1

Schematic drawing of the activities of invertase and the fructosyltransferase 1-SST. Sucrose is split into glucose and fructose, the glucose is released and fructose stays bound to the enzyme, subsequently fructose is released via hydrolysis in the case of invertase whereas fructose is coupled to another sucrose molecule in the case of 1-SST

Fig. 2

Saturation curves of acINV and saSST for sucrose. Production of glucose, fructose and kestose is determined at different sucrose concentrations with the recombinant enzyme preparations incubated at 28°C for 20 min. For AcINV sucrose concentrations up to 200 mM are used, whereas saSST was tested for up to 1 M of sucrose. AcINV is saturated at relatively low sucrose concentrations, whereas saSST shows increased product formation with higher sucrose concentrations

Fig. 3

Hydrolysis as a percentage of total hydrolysis plus transfer activity by the transferase saSST and its mutants at sites I–VIII (Table 2) incubated at 28°C for 20 min with different sucrose concentrations. Hydrolysis is measured through the fructose released and increases with increasing sucrose concentrations. Measurements from three independent protein expression experiments were analyzed, the error bars represent the standard error

Fig. 4

Saturation curves for sucrose of saSST mutated at position VIII. Production of glucose, fructose and kestose is determined after 20 min incubation at 28°C with sucrose concentrations up to 1 M. SST mutation VIII (N425S) shows high fructose production (i.e. sucrose hydrolysis) at all sucrose concentrations tested

Fig. 5

Transfer as a percentage of total hydrolysis plus transfer activity by the hydrolase acINV and its mutants at sites I–VIII (Table 2) incubated at 28°C for 20 min with different sucrose concentrations. Transfer is measured through the kestose produced and increases with increasing sucrose concentrations. Measurements from three independent protein expression experiments were analyzed, the error bars represent the standard error

Fig. 6

Solvent accessible surface for the homology models of (a) vacuolar invertase of onion (Allium cepa); residues Trp161 (blue), Trp440 (red) and Ser457 (green); and (b) 1-SST of festuca (Schedonorus arundinaceus); residues Tyr133 (blue), Tyr408 (red) and Asn425 (green). The active site is marked for reference with a sucrose molecule obtained by superposing the coordinates from β-fructosidase from Thermotoga maritima (PDB code 1W2T, light green). Colored residues were subtracted from each surface calculation in order to stress their spatial distribution. (c) Stereo-view of the substrate pocket of INV in yellow the three catalytic residues are indicated. The figure was prepared with CCP4 mg (Potterton et al. 2002)