Modulating Glycoside Hydrolase Activity between Hydrolysis and Transfer Reactions Using an Evolutionary Approach

Abstract

The proteins within the CAZy glycoside hydrolase family GH13 catalyze the hydrolysis of polysaccharides such as glycogen and starch. Many of these enzymes also perform transglycosylation in various degrees, ranging from secondary to predominant reactions. Identifying structural determinants associated with GH13 family reaction specificity is key to modifying and designing enzymes with increased specificity towards individual reactions for further applications in industrial, chemical, or biomedical fields. This work proposes a computational approach for decoding the determinant structural composition defining the reaction specificity. This method is based on the conservation of coevolving residues in spatial contacts associated with reaction specificity. To evaluate the algorithm, mutants of α-amylase (TmAmyA) and glucanotransferase (TmGTase) from Thermotoga maritima were constructed to modify the reaction specificity. The K98P/D99A/H222Q variant from TmAmyA doubled the transglycosydation/hydrolysis (T/H) ratio while the M279N variant from TmGTase increased the hydrolysis/transglycosidation ratio five-fold. Molecular dynamic simulations of the variants indicated changes in flexibility that can account for the modified T/H ratio. An essential contribution of the presented computational approach is its capacity to identify residues outside of the active center that affect the reaction specificity.

Keywords: amylase; coevolution; contact-residues; enrichment-factor; glucanotransferase; hydrolysis; specificity; transglycosidation.

Figure 1

Function classification of enzymes in the GH13 family based on the amino acid preference in their residue-residue contacts. (a) Dataset 1 (training group). (b) Dataset 2 (validation group). (c) Dataset 4 (updated dataset). In both cases, enzymes defined as transglycosidic or hydrolytic are shown with green and red dots, respectively.

Figure 2

Correlation of the residue-residue contact conservation between all enzymes and those of TmAmya, one of the enzymes modified in this study. The preservation of each contact in a protein is plotted against its conservation in TmAmyA. This parameter seems to correlate with phylogenetic relationships. A poor correlation is evident for the enzymes not belonging to the GH13 family (e.g., PDB ID: 2ZQ0, family GH97, and enzyme with the PDB ID: 3W37, family GH31).

Figure 3

Parameters of similarity distance between evaluated enzymes and TmAmyA based on their correlation between contact conservation (R-squared) and (a) structure (Dali) and (b) sequence (Clustal) alignments.

Figure 4

Schematic representation of substitutions in glycosidases based on enrichment factors. (a) K98P/D99A in TmAmyA (b) F72L/V86I for TmGTase. The residues more frequently found in hydrolases and transglycosylases are shown in the lower part of the figure for each pair in red and green, respectively.

Figure 5

Transglycosylation/Hydrolysis (T/H) ratio of glycosidases. (a) TmAmyA and its variants; (b) TmGTase and its variants. Transglycosidation and hydrolysis values were normalized to each wild-type enzyme, which has the coordinates (1,1). The dashed lines indicate equivalent T/H ratios. The central diagonal corresponds to the wild-type enzyme. Enzymes with a better T/H ratio are over the central diagonal line, and enzymes with a better H/T ratio are under the central diagonal line.

Figure 6

RMSF difference (in nanometers) along the structure with high and low transglycosidic variants of glycosidases. (a) TmAmyA K98P/D99A/H222Q triple mutant (less hydrolytic than the wild-type enzyme). (b) wild-type TmGTase (high transglycosidic) vs. T274V/M279N. The active center is delimited by acarbose (yellow stick) and is located away from the zone with the modified fluctuations. No differences in RMSF are shown in white, while positive or negative RMSF changes are red and green, respectively.

Figure 7

Distances in Angstrom between the mutation sites (green and cyan) and the catalytic residues (red) (a) TmAmyA. (b) TmGTase. The active center is delimited by the competitive inhibitor acarbose (yellow). In pink is shown the D310 residue that acts as a transition state stabilizer.