PDBsum entry 1e4l

Hydrolase

PDB id: 1e4l

Contents

Protein chains

490 a.a. *

Ligands

GOL ×2

Waters ×382

* Residue conservation analysis

PDB id:

1e4l

Name:

Hydrolase

Title:

Crystal structure of the inactive mutant monocot (maize zmglu1) beta- glucosidase zm glu191asp

Structure:

Beta-glucosidase, chloroplastic. Chain: a, b. Synonym: beta-d-glucoside glucohydrolase, cellobiase, gentiobiase. Engineered: yes. Mutation: yes

Source:

Zea mays. Maize. Organism_taxid: 4577. Strain: cv. Mutin. Tissue: coleoptile. Organelle: chloroplast. Expressed in: escherichia coli. Expression_system_taxid: 562. Expression_system_cell: plys s cells.

Resolution:

2.20Å R-factor: 0.207 R-free: 0.244

Authors:

M.Czjzek,M.Cicek,D.R.Bevan,B.Henrissat,A.Esen

Key ref:

M.Czjzek et al. (2000). The mechanism of substrate (aglycone) specificity in beta -glucosidases is revealed by crystal structures of mutant maize beta -glucosidase-DIMBOA, -DIMBOAGlc, and -dhurrin complexes. Proc Natl Acad Sci U S A, 97, 13555-13560. PubMed id: 11106394 DOI: 10.1073/pnas.97.25.13555

Date:

10-Jul-00 Release date: 11-Dec-00

Protein chains

P49235  (HGGL1_MAIZE) -  4-hydroxy-7-methoxy-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl glucoside beta-D-glucosidase 1, chloroplastic from Zea mays

Seq: 566 a.a.

Struc: 490 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class 2: E.C.3.2.1.182 - 4-hydroxy-7-methoxy-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl glucoside
• Reaction:
  1. DIMBOA beta-D-glucoside + H2O = DIMBOA + D-glucose
  2. DIBOA beta-D-glucoside + H2O = DIBOA + D-glucose

DIMBOA beta-D-glucoside + H2O = DIMBOA (Bound ligand (Het Group name = GOL) matches with 50.00% similarity) + D-glucose

DIBOA beta-D-glucoside + H2O = DIBOA (Bound ligand (Het Group name = GOL) matches with 50.00% similarity) + D-glucose

• Enzyme class 3: E.C.3.2.1.21 - beta-glucosidase.
• Reaction: Hydrolysis of terminal, non-reducing beta-D-glucose residues with release of beta-D-glucose.

Note, where more than one E.C. class is given (as above), each may correspond to a different protein domain or, in the case of polyprotein precursors, to a different mature protein.

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1073/pnas.97.25.13555

Proc Natl Acad Sci U S A 97:13555-13560 (2000)

PubMed id: 11106394

The mechanism of substrate (aglycone) specificity in beta -glucosidases is revealed by crystal structures of mutant maize beta -glucosidase-DIMBOA, -DIMBOAGlc, and -dhurrin complexes.

M.Czjzek, M.Cicek, V.Zamboni, D.R.Bevan, B.Henrissat, A.Esen.

ABSTRACT

The mechanism and the site of substrate (i.e., aglycone) recognition and specificity were investigated in maize beta-glucosidase (Glu1) by x-ray crystallography by using crystals of a catalytically inactive mutant (Glu1E191D) in complex with the natural substrate 2-O-beta-d-glucopyranosyl-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOAGlc), the free aglycone DIMBOA, and competitive inhibitor para-hydroxy-S-mandelonitrile beta-glucoside (dhurrin). The structures of these complexes and of the free enzyme were solved at 2.1-, 2.1-, 2.0-, and 2.2-A resolution, respectively. The structural data from the complexes allowed us to visualize an intact substrate, free aglycone, or a competitive inhibitor in the slot-like active site of a beta-glucosidase. These data show that the aglycone moiety of the substrate is sandwiched between W378 on one side and F198, F205, and F466 on the other. Thus, specific conformations of these four hydrophobic amino acids and the shape of the aglycone-binding site they form determine aglycone recognition and substrate specificity in Glu1. In addition to these four residues, A467 interacts with the 7-methoxy group of DIMBOA. All residues but W378 are variable among beta-glucosidases that differ in substrate specificity, supporting the conclusion that these sites are the basis of aglycone recognition and binding (i.e., substrate specificity) in beta-glucosidases. The data also provide a plausible explanation for the competitive binding of dhurrin to maize beta-glucosidases with high affinity without being hydrolyzed.

Selected figure(s)

Figure 2.
Fig. 2. Structure of the ligands and the active site of Glu1E191D. (A) The natural substrate DIMBOAGlc (Left), the aglycone DIMBOA (Center), and the competitive inhibitor dhurrin (Right). (B) Ribbon diagram of the structure of the maize -glucosidase Glu1 and its inactive Glu1E191D mutant, showing the catalytic residues E191 (D191 in the mutant) and E406 (red), four residues (F198, F205, W378, and F466) forming the aglycone-binding pocket (blue), and two other residues (A467 and Y473) that are probably important for aglycone recognition (yellow). Different colors and the color transitions in -helices and -strands trace the polypeptide backbone in the barrel-shaped three-dimensional structure from the N terminus (dark blue) to the C terminus (dark red) direction. The figure was produced with MOLSCRIPT (35) and RASTER3D (36). (C) Electrostatic surface representation of the active site region of Glu1E191D showing positively charged regions in blue, negatively charged regions in red, and neutral regions in white. The slot-like active site, measuring 23 Å × 7.1 Å at the entrance, contains the natural substrate DIMBOAGlc in compact representation with standard atom-type colors. In this view, only the aglycone moiety is visible in its binding site as glucose is hidden below aglycone. C was produced with GRASP (37).

Figure 3.
Fig. 3. Aglycone recognition and binding in -glycosidases as revealed by DIMBOAGlc-, DIMBOA-, and dhurrin-Glu1E191D inactive mutant complexes. (A) Closeup view of the active site of Glu1, showing the catalytic glutamates E191 and E406 (red), the four residues (F198, F205, W378, and F466) forming the aglycone-binding pocket (light blue), and the additional residues (A467 and Y473) that are probably important for aglycone recognition (light green). (B) Glu1E191D with bound DIMBOAGlc. The glycone moiety is in blue, whereas the aglycone is in atom-type colors. The bulky aryl group is sandwiched between W378 on one side and F198, F205, and F466 on the other. (C) Same as B but with bound DIMBOA, showing a slightly different orientation than DIMBOA in DIMBOAGlc, which is constrained by the glycosidic linkage. (D) Same as B but with bound dhurrin. The aglycone moiety of the inhibitor dhurrin is in the same position as the aglycone of the natural substrate. Figs. 3. and 4 were produced with TURBO-FRODO (38).

Figures were selected by an automated process.