PDBsum entry 3a3v

Hydrolase

PDB id: 3a3v

Contents

Protein chain

376 a.a.

Ligands

GOL ×4

Metals

_NI

Waters ×516

References listed in PDB file

Key reference

Title

Structural explanation for the acquisition of glycosynthase activity.

Authors

M.Hidaka, S.Fushinobu, Y.Honda, T.Wakagi, H.Shoun, M.Kitaoka.

Ref.

J Biochem (tokyo), 2010, 147, 237-244.

PubMed id

19819900

Abstract

Glycosynthases are engineered glycoside hydrolases (GHs) that catalyse the synthesis of glycoside from glycosyl-fluoride donors and suitable acceptors. We have determined five crystal structures of the glycosynthase mutants reducing-end xylose-releasing exo-oligoxylanase, an inverting GH, that exhibit various levels of glycosynthetic activities. At the active site of the Y198F mutant, the most efficient glycosynthase, a water molecule is observed at the same position as nucleophilic water (NW) in the parent enzyme, and the loss of the fixation of the direction of the lone pair of water molecules in the mutant drastically decreases hydrolytic activity. Water molecules were also observed at each active site of the general base mutant, but they were shifted 1.0-3.0 A from the NW in the wild type. Their positions exhibited a strong correlation with the strength of glycosynthase activity. Here, we propose that a structural prerequisite for the sufficient glycosynthase reaction is the presence of a water molecule at the NW position, and mutation at the NW holder provides a general strategy for inverting GHs. The idea on the position of a water molecule may also be applicable to the design of efficient glycosynthases from retaining GHs.

Secondary reference #1

Title

Alternative strategy for converting an inverting glycoside hydrolase into a glycosynthase.

Authors

Y.Honda, S.Fushinobu, M.Hidaka, T.Wakagi, H.Shoun, H.Taniguchi, M.Kitaoka.

Ref.

Glycobiology, 2008, 18, 325-330.

PubMed id

18263897

Secondary reference #2

Title

The first glycosynthase derived from an inverting glycoside hydrolase.

Authors

Y.Honda, M.Kitaoka.

Ref.

J Biol Chem, 2006, 281, 1426-1431. [DOI no: 10.1074/jbc.M511202200]

PubMed id

16301312

Figure 4.
FIGURE 4. Hehre resynthesis-hydrolysis mechanism for hydrolysis of the wrong glycosylfluoride by inverting enzymes. The protonations of the catalytic acidic residues are reversed at the initial stage, because the initial reaction in the mechanism mimics the reverse hydrolysis of the inverting enzyme shown in Fig. 1B.

Figure 5.
FIGURE 5. Reaction mechanism of glycosynthase derived from inverting and retaining GHs. A, Rex. B, Agrobacterium sp. -glucosidase.

The above figures are reproduced from the cited reference with permission from the ASBMB

Secondary reference #3

Title

Structural basis for the specificity of the reducing end xylose-Releasing exo-Oligoxylanase from bacillus halodurans c-125.

Authors

S.Fushinobu, M.Hidaka, Y.Honda, T.Wakagi, H.Shoun, M.Kitaoka.

Ref.

J Biol Chem, 2005, 280, 17180-17186. [DOI no: 10.1074/jbc.M413693200]

PubMed id

15718242

Figure 2.
FIG. 2. Ribbon diagrams of the GH-8a enzymes. The catalytic residues, ligand molecules, and metal ions are shown as black sticks, a ball-and-stick model, and spheres, respectively. a, side view of the ( / )[6] barrel of Rex. b, top view of the barrel in a. The position of the [10] helix is indicated. c, top view of the barrel of the wild-type pXyl complexed with a xylose at subsite +4. The side chain of the catalytic proton donor (Glu78) is positioned differently from in the other two enzymes. d, top view of the barrel of CelA. A part of the cellopentaose molecule (subsites -3 to -1 out of -3 to +2) and the cellotriose molecule (subsites +1 to +3) are shown.

Figure 5.
FIG. 5. Schematic drawing of the active sites in the WT-xylose (a) and E70A-xylobiose (b) structures.

The above figures are reproduced from the cited reference with permission from the ASBMB

Secondary reference #4

Title

Crystallization and preliminary X-Ray analysis of reducing-End xylose-Releasing exo-Oligoxylanase from bacillus halodurans c-125.

Authors

Y.Honda, S.Fushinobu, M.Hidaka, T.Wakagi, H.Shoun, M.Kitaoka.

Ref.

Acta Crystallograph Sect F Struct Biol Cryst Commun, 2005, 61, 291-292. [DOI no: 10.1107/S1744309105003635]

PubMed id

16511021

Figure 1.
Figure 1 Crystals of Rex obtained under the optimized conditions. See text for experimental details.

The above figure is reproduced from the cited reference which is an Open Access publication published by the IUCr

Secondary reference #5

Title

A family 8 glycoside hydrolase from bacillus halodurans c-125 (bh2105) is a reducing end xylose-Releasing exo-Oligoxylanase.

Authors

Y.Honda, M.Kitaoka.

Ref.

J Biol Chem, 2004, 279, 55097-55103. [DOI no: 10.1074/jbc.M409832200]

PubMed id

15491996

Figure 2.
FIG. 2. Schematic drawing of BH2105 subsite structure. Numbers represent the subsites.

Figure 4.
FIG. 4. Reaction mechanism of X3 and X4 hydrolysis by the BH2105 protein.

The above figures are reproduced from the cited reference with permission from the ASBMB