PDBsum entry 2owc

Transferase

PDB id: 2owc

Contents

Protein chain

499 a.a. *

Ligands

GLC-AC1

GOL

MLI

Waters ×411

* Residue conservation analysis

PDB id:

2owc

Name:

Transferase

Title:

Structure of a covalent intermediate in thermus thermophilus amylomaltase

Structure:

4-alpha-glucanotransferase. Chain: a. Fragment: amylomaltase. Engineered: yes

Source:

Thermus thermophilus. Organism_taxid: 274. Gene: malq. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Resolution:

2.05Å R-factor: 0.188 R-free: 0.216

Authors:

T.R.M.Barends,J.B.Bultema,T.Kaper,M.J.E.C.Van Der Maarel, L.Dijkhuizen,B.W.Dijkstra

Key ref:

T.R.Barends et al. (2007). Three-way stabilization of the covalent intermediate in amylomaltase, an alpha-amylase-like transglycosylase. J Biol Chem, 282, 17242-17249. PubMed id: 17420245 DOI: 10.1074/jbc.M701444200

Date:

16-Feb-07 Release date: 03-Apr-07

Protein chain

O87172  (MALQ_THETH) -  4-alpha-glucanotransferase from Thermus thermophilus

Seq: 500 a.a.

Struc: 499 a.a.*

* PDB and UniProt seqs differ at 2 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.2.4.1.25 - 4-alpha-glucanotransferase.
• Reaction: Transfers a segment of a (1,4)-alpha-D-glucan to a new 4-position in an acceptor, which may be glucose or (1,4)-alpha-D-glucan.

DOI no: 10.1074/jbc.M701444200

J Biol Chem 282:17242-17249 (2007)

PubMed id: 17420245

Three-way stabilization of the covalent intermediate in amylomaltase, an alpha-amylase-like transglycosylase.

T.R.Barends, J.B.Bultema, T.Kaper, M.J.van der Maarel, L.Dijkhuizen, B.W.Dijkstra.

ABSTRACT

Amylomaltases are glycosyl hydrolases belonging to glycoside hydrolase family 77 that are capable of the synthesis of large cyclic glucans and the disproportionation of oligosaccharides. Using protein crystallography, we have generated a flip-book movie of the amylomaltase catalytic cycle in atomic detail. The structures include a covalent glycosyl-enzyme intermediate, and a covalent intermediate in complex with an analogue of a co-substrate, and show how the structures of both enzyme and substrate respond to the changes required by the catalytic cycle as it proceeds. Notably, the catalytic nucleophile changes conformation dramatically during the reaction. Also, Gln256 on the 250s loop is involved in orienting the substrate in the +1 site. The absence of a suitable base in the covalent intermediate structure explains the low hydrolysis activity.

Selected figure(s)

Figure 1.
FIGURE 1. Crystallographic analysis of amylomaltase complexes. A, stereo figure of the F[o] - DF[c] electron density for acarbose bound to Asp-293, calculated prior to incorporation of acarbose in the model. The density was contoured at 2.5 and overlaid on the refined structure. B, F[o] - DF[c] electron density after refinement with a non-covalently bound acarbose. To check the density for the covalent bond and to correctly identify the various acarbose residues, the Asp-293 carboxylate group as well as the acarbose 6-OH groups were omitted from the calculations (see "Materials and Methods"). Positive difference density is shown in green (3.5 ), negative density in red (-3.5 ). 2mF[o] - DF[c] density (blue, 1.0 ) (C) F[o] - DF[c] electron density (green, 2.5 ) (D) for the covalent intermediate-4-deoxyglucose complex. To avoid model bias, both maps were calculated prior to the incorporation of 4-deoxyglucose in the model. All figures were produced using PyMol (37).

Figure 4.
FIGURE 4. Stereo images of structures representing different states during the reaction cycle of amylomaltase. A, empty active site at pH 5.6. The residues Asp-293, Glu-340, Asp-395, and Gln-256 are indicated. B, acarbose (ACR) complex as determined by Przylas et al. (22). The subsites +1 and -1 are indicated. C, acarbose covalent intermediate-4-deoxyglucose complex. D, covalent intermediate with acarbose. Possible hydrolytic water molecules are indicated as spheres.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2007, 282, 17242-17249) copyright 2007.

Figures were selected by the author.