PDBsum entry 1vfo

Hydrolase

PDB id: 1vfo

Contents

Protein chains

585 a.a. *

Ligands

GLC-GLC-GLC-GLC-GLC-GLC-GLC

GLC-GLC-GLC-GLC-GLC-BGC-GLC

Metals

_CA ×2

Waters ×228

* Residue conservation analysis

PDB id:

1vfo

Name:

Hydrolase

Title:

Crystal structure of thermoactinomyces vulgaris r-47 alpha-amylase 2/beta-cyclodextrin complex

Structure:

Neopullulanase 2. Chain: a, b. Synonym: alpha-amylase 2, alpha-amylase ii. 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:

2.81Å R-factor: 0.200 R-free: 0.256

Authors:

A.Ohtaki,M.Mizuno,T.Tonozuka,Y.Sakano,S.Kamitori

Key ref:

A.Ohtaki et al. (2004). Complex structures of Thermoactinomyces vulgaris R-47 alpha-amylase 2 with acarbose and cyclodextrins demonstrate the multiple substrate recognition mechanism. J Biol Chem, 279, 31033-31040. PubMed id: 15138257 DOI: 10.1074/jbc.M404311200

Date:

16-Apr-04 Release date: 08-Feb-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 2 residue positions (black crosses)

Enzyme reactions

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

DOI no: 10.1074/jbc.M404311200

J Biol Chem 279:31033-31040 (2004)

PubMed id: 15138257

Complex structures of Thermoactinomyces vulgaris R-47 alpha-amylase 2 with acarbose and cyclodextrins demonstrate the multiple substrate recognition mechanism.

A.Ohtaki, M.Mizuno, T.Tonozuka, Y.Sakano, S.Kamitori.

ABSTRACT

Thermoactinomyces vulgaris R-47 alpha-amylase 2 (TVAII) has the unique ability to hydrolyze cyclodextrins (CDs), with various sized cavities, as well as starch. To understand the relationship between structure and substrate specificity, x-ray structures of a TVAII-acarbose complex and inactive mutant TVAII (D325N/D421N)/alpha-, beta- and gamma-CDs complexes were determined at resolutions of 2.9, 2.9, 2.8, and 3.1 A, respectively. In all complexes, the interactions between ligands and enzymes at subsites -1, -2, and -3 were almost the same, but striking differences in the catalytic site structure were found at subsites +1 and +2, where Trp(356) and Tyr(374) changed the conformation of the side chain depending on the structure and size of the ligands. Trp(356) and Tyr(374) are thought to be responsible for the multiple substrate-recognition mechanism of TVAII, providing the unique substrate specificity. In the beta-CD complex, the beta-CD maintains a regular conical structure, making it difficult for Glu(354) to protonate the O-4 atom at the hydrolyzing site as a previously proposed hydrolyzing mechanism of alpha-amylase. From the x-ray structures, it is suggested that the protonation of the O-4 atom is possibly carried out via a hydrogen atom of the inter-glucose hydrogen bond at the hydrolyzing site.

Selected figure(s)

Figure 3.
FIG. 3. Stereo views of interactions between catalytic site residues (light gray) and acarbose (gray)(a), -CD (gray)(b), -CD (gray) (c), and -CD (gray)(d), illustrated by the program MOLSCRIPT (23). Trp356 and Tyr374 in the unliganded TVAII are superimposed (white). The selected hydrogen bonds are shown with dotted lines.

Figure 5.
FIG. 5. The proposed hydrolyzing mechanism for substrate with a linear structure (a) and the hypothetical -CD hydrolyzing mechanism of TVA II (this study) (b) are shown.

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2004, 279, 31033-31040) copyright 2004.

Figures were selected by an automated process.