PDBsum entry 1vb9

Hydrolase

PDB id: 1vb9

Contents

Protein chains

585 a.a. *

Ligands

GLC-GLC-GLC-GLC-GLC-GLC ×2

Metals

_CA ×2

Waters ×508

* Residue conservation analysis

PDB id:

1vb9

Name:

Hydrolase

Title:

Crystal structure of thermoactinomyces vulgaris r-47 alpha-amylase ii (tva ii) complexed with transglycosylated product

Structure:

Alpha-amylase ii. Chain: a, b. Synonym: neopullulanase 2. Engineered: yes. Mutation: yes

Source:

Thermoactinomyces vulgaris. Organism_taxid: 2026. Strain: r-47. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Dimer (from PQS)

Resolution:

2.20Å R-factor: 0.194 R-free: 0.233

Authors:

M.Mizuno,T.Tonozuka,A.Uechi,A.Ohtaki,K.Ichikawa,S.Kamitori, A.Nishikawa,Y.Sakano

Key ref:

M.Mizuno et al. (2004). The crystal structure of Thermoactinomyces vulgaris R-47 alpha-amylase II (TVA II) complexed with transglycosylated product. Eur J Biochem, 271, 2530-2538. PubMed id: 15182368 DOI: 10.1111/j.1432-1033.2004.04183.x

Date:

25-Feb-04 Release date: 08-Mar-05

Protein chains

Q08751  (NEPU2_THEVU) -  Neopullulanase 2 from Thermoactinomyces vulgaris

Seq: 585 a.a.

Struc: 585 a.a.*

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

Enzyme reactions

• Enzyme class: E.C.3.2.1.135 - neopullulanase.
• Reaction: Hydrolysis of pullulan to panose (6-alpha-D-glucosylmaltose).

DOI no: 10.1111/j.1432-1033.2004.04183.x

Eur J Biochem 271:2530-2538 (2004)

PubMed id: 15182368

The crystal structure of Thermoactinomyces vulgaris R-47 alpha-amylase II (TVA II) complexed with transglycosylated product.

M.Mizuno, T.Tonozuka, A.Uechi, A.Ohtaki, K.Ichikawa, S.Kamitori, A.Nishikawa, Y.Sakano.

ABSTRACT

Alphan alpha-amylase (TVA II) from Thermoactinomyces vulgaris R-47 efficiently hydrolyzes alpha-1,4-glucosidic linkages of pullulan to produce panose in addition to hydrolyzing starch. TVA II also hydrolyzes alpha-1,4-glucosidic linkages of cyclodextrins and alpha-1,6-glucosidic linkages of isopanose. To clarify the basis for this wide substrate specificity of TVA II, we soaked 4(3)-alpha-panosylpanose (4(3)-P2) (a pullulan hydrolysate composed of two panosyl units) into crystals of D325N inactive mutated TVA II. We then determined the crystal structure of TVA II complexed with 4(2)-alpha-panosylpanose (4(2)-P2), which was produced by transglycosylation from 4(3)-P2, at 2.2-A resolution. The shape of the active cleft of TVA II is unique among those of alpha-amylase family enzymes due to a loop (residues 193-218) that is located at the end of the cleft around the nonreducing region and forms a 'dam'-like bank. Because this loop is short in TVA II, the active cleft is wide and shallow around the nonreducing region. It is assumed that this short loop is one of the reasons for the wide substrate specificity of TVA II. While Trp356 is involved in the binding of Glc +2 of the substrate, it appears that Tyr374 in proximity to Trp356 plays two roles: one is fixing the orientation of Trp356 in the substrate-liganded state and the other is supplying the water that is necessary for substrate hydrolysis.

Selected figure(s)

Figure 3.
Fig. 3. Stereo-view of the active site with 4^2-P2.(A) The whole shape of the active cleft formed collaboratively with domain N of MOL-2 (green surface model) is shown in the molecular surface model. The surface model was produced using PYMOL (http://www.pymol.org). (B) Unliganded TVA II (green) superimposed into the complex structure (magenta) around the nonreducing region. 4^2-P2, separated between –1 and +1, is displayed as dark gray sticks. The residues with asterisks are located in domain N of the MOL-2 molecule. (C) Reducing region. The explanation is the same as for (B).

Figure 4.
Fig. 4. Schematic drawing of the interactions of 4^2-P2 bound to the active site. Hydrogen bonds of less than 3.5 Å are shown as dashed lines. Water molecules are shown as spheres. The residues with asterisks are located in domain N of the MOL-2 molecule. Three catalytic residues, except for Asn325, which is aspartic acid in native TVA II, are surrounded by an elliptical box.

The above figures are reprinted by permission from the Federation of European Biochemical Societies: Eur J Biochem (2004, 271, 2530-2538) copyright 2004.

Figures were selected by an automated process.