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

Lionel Verdoucq,Mirjam Czjzek,Jeanne Moriniere,David R Bevan,Asim Esen

doi:10.1074/jbc.m301978200

Abstract

Plant beta-glucosidases display varying substrate specificities. The maize beta-glucosidase isozyme Glu1 (ZmGlu1) hydrolyzes a broad spectrum of substrates in addition to its natural substrate DIMBOA-Glc (2-O-beta-d-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxaxin-3-one), whereas the sorghum beta-glucosidase isozyme Dhr1 (SbDhr1) hydrolyzes exclusively its natural substrate dhurrin (p-hydroxy-(S)-mandelonitrile-beta-d-glucoside). Structural data from cocrystals of enzyme-substrate and enzyme-aglycone complexes have shown that five amino acid residues (Phe198, Phe205, 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 beta-glucosidases were generated by replacing Phe198, Phe205, Asp261, Met263, 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 beta-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.

Highlights

Introduction␤-Glucosidases (␤-D-glucoside glucohydrolase, EC 3.2.1.21) occur in all living organisms (bacteria, archaea, and eukarya) and play key roles in a variety of fundamental biological processes
␤-Glucosidases (␤-D-glucoside glucohydrolase, EC 3.2.1.21) occur in all living organisms and play key roles in a variety of fundamental biological processes
We have produced site-directed mutants of a maize (ZmGlu1) and a sorghum (SbDhr1) ␤-glucosidase and we have determined the structure of the most affected mutant of ZmGlu1 in complex with the natural substrate. These two enzymes share 70% sequence identity and represent extremes in substrate specificity [18] in that the former hydrolyzes a wide spectrum of substrates in addition to its natural substrate DIMBOA-Glc, whereas the latter hydrolyzes its natural substrate dhurrin only

Summary

Introduction

␤-Glucosidases (␤-D-glucoside glucohydrolase, EC 3.2.1.21) occur in all living organisms (bacteria, archaea, and eukarya) and play key roles in a variety of fundamental biological processes. The primary structures of maize and sorghum ␤-glucosidases contain the highly conserved peptide motifs TFNEP and ITENG, which contain the two glutamic acids (Glu191 and Glu406, numbering of ZmGlu, Fig. 1) involved in catalysis as the general acid/base catalyst and the nucleophile, respectively, in all family 1 ␤-glucosidases [12]. These residues form part of a slot-like active site [13] and are required in the two steps of the substrate hydrolysis [14]. The two catalytic glutamic acids (i.e. the nucleophile and the acid/base catalyst) of ZmGlu are positioned within the active site at expected distances (ϳ5.5 Å) for this mechanism [15]

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