Mutational and Structural Analysis of Aglycone Specificity in Maize and Sorghum β-Glucosidases

Abstract

Plant β-glucosidases display varying substrate specificities. The maize β-glucosidase isozyme Glu1 (ZmGlu1) hydrolyzes a broad spectrum of substrates in addition to its natural substrate DIMBOA-Glc (2-O-β -D-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxaxin-3-one), whereas the sorghum β-glucosidase isozyme Dhr1 (SbDhr1) hydrolyzes exclusively its natural substrate dhurrin (p-hydroxy-(S)-mandelonitrile-β-D-glucoside). Structural data from cocrystals of enzyme-substrate and enzyme-aglycone complexes have shown that five amino acid residues (Phe198, Phe 205, Trp378, Phe466, and Ala467) are located in the aglycone-binding site of ZmGlu1 and form the basis of aglycone recognition and binding, hence substrate specificity. To study the mechanism of substrate specificity further, mutant β-glucosidases were generated by replacing Phe198, Phe205, Asp261, Phe377, Phe466 Ala467, and Phe473 of Glu1 by Dhr1 counterparts. The effects of mutations on enzyme activity and substrate specificity were studied using both natural and artificial substrates. The simple mutant replacing Phe198 by a valine had the most drastic effect on activity, because the capacity of this enzyme to hydrolyze β-glucosides was almost completely abolished. The analysis of this mutation was completed by a structural study of the double mutant ZmGlu1-E191D, F198V in complex with the natural substrate. The structure reveals that the single mutation F198V causes a cascade of conformational changes, which are unpredictable by standard molecular modeling techniques. Some other mutations led to drastic effects: replacing Asp261 by an asparagine decreases the catalytic efficiency of this simple mutant by 75% although replacing Tyr473 by a phenylalanine increase its efficiency by 300% and also provides a new substrate specificity by hydrolyzing dhurrin.