PDBsum entry 3bcd

Hydrolase

PDB id: 3bcd

Contents

Protein chain

585 a.a. *

Ligands

BGC-GLC-GLC-GLC

GLC-GLC-GLC-GLC

GLC-GLC-GLC-GLC-GLC-GLC

Metals

_CA ×7

_NA

Waters ×259

* Residue conservation analysis

PDB id:

3bcd

Name:

Hydrolase

Title:

Alpha-amylase b in complex with maltotetraose and alpha-cyclodextrin

Structure:

Alpha amylase, catalytic region. Chain: a. Synonym: amyb. Engineered: yes

Source:

Halothermothrix orenii. Organism_taxid: 373903. Strain: h 168. Gene: amyb. Expressed in: escherichia coli bl21(de3). Expression_system_taxid: 469008.

Resolution:

2.20Å R-factor: 0.197 R-free: 0.240

Authors:

T.-C.Tan,B.N.Mijts,K.Swaminathan,B.K.C.Patel,C.Divne

Key ref:

T.C.Tan et al. (2008). Crystal structure of the polyextremophilic alpha-amylase AmyB from Halothermothrix orenii: details of a productive enzyme-substrate complex and an N domain with a role in binding raw starch. J Mol Biol, 378, 850-868. PubMed id: 18387632 DOI: 10.1016/j.jmb.2008.02.041

Date:

12-Nov-07 Release date: 22-Apr-08

Protein chain

B8CZ54  (B8CZ54_HALOH) -  Alpha amylase from Halothermothrix orenii (strain H 168 / OCM 544 / DSM 9562)

Seq: 623 a.a.

Struc: 585 a.a.

Enzyme reactions

• Enzyme class: E.C.3.2.1.98 - glucan 1,4-alpha-maltohexaosidase.
• Reaction: Hydrolysis of 1,4-alpha-D-glucosidic linkages in amylaceous polysaccharides so as to remove successive maltohexaose residues from the non-reducing chain ends.

DOI no: 10.1016/j.jmb.2008.02.041

J Mol Biol 378:850-868 (2008)

PubMed id: 18387632

Crystal structure of the polyextremophilic alpha-amylase AmyB from Halothermothrix orenii: details of a productive enzyme-substrate complex and an N domain with a role in binding raw starch.

T.C.Tan, B.N.Mijts, K.Swaminathan, B.K.Patel, C.Divne.

ABSTRACT

The gene for a membrane-bound, halophilic, and thermostable alpha-amylase, AmyB, from Halothermothrix orenii was cloned and sequenced. The crystal structure shows that, in addition to the typical domain organization of family 13 glycoside hydrolases, AmyB carries an additional N-terminal domain (N domain) that forms a large groove--the N-C groove--some 30 A away from the active site. The structure of AmyB with the inhibitor acarbose at 1.35 A resolution shows that a nonasaccharide has been synthesized through successive transglycosylation reactions of acarbose. Unexpectedly, in a complex of wild-type AmyB with alpha-cyclodextrin and maltoheptaose at 2.2 A resolution, a maltotetraose molecule is bound in subsites -1 to +3, spanning the cleavage point at -1/+1, with the -1 glucosyl residue present as a (2)S(o) skew boat. This wild-type AmyB complex was obtained in the presence of a large excess of substrate, a condition under which it is possible to capture Michaelis complexes, which may explain the observed binding across -1/+1 and ring distortion. We observe three methionine side chains that serve as "binding platforms" for glucosyl rings in AmyB, a seemingly rare occurrence in carbohydrate-binding proteins. The structures and results from the biochemical characterization of AmyB and AmyB lacking the N domain show that the N domain increases binding of the enzyme to raw starch. Furthermore, theoretical modeling suggests that the N-C groove can accommodate, spatially and chemically, large substrates such as A-starch.

Selected figure(s)

Figure 1.
Fig. 1. Overall structure of H. orenii AmyB and its N domain. (a) Ribbon drawing of AmyB and (b) its closest structural relative B. licheniformis α-amylase^23 (PDB code 1BLI), which lacks an N domain. (c) Structure of the AmyB N domain (amino acids 15–108). A tyrosine residue (Tyr86) forms a “tyrosine corner” with the Glu81 backbone. (d) Structural superposition of H. orenii AmyB-N (red) and P. syringae CopC^24 (blue; PDB code 2C9Q). The domains are colored as follows: A, red; B, green; C, orange; N, blue; the linker between N and A, magenta. Calcium and sodium ions are shown as blue and magenta spheres, respectively.

Figure 5.
Fig. 5. Comparison of the active site with related α-amylases. Superposition of the AmyB[ACR] active site around − 1 with that of B. licheniformis α-amylase^25 (PDB code 1VJS). Carbon atoms in AmyB are in beige, and those in the Bacillus enzyme are in green. (b) Superposition of the − 1 subsite in AmyB (beige) and AmyA^12 (green; PDB code 1WZA). In (a) and (b), the first amino-acid number given is for AmyB. The nonasaccharide in AmyB[ACR] is shown (yellow). (c) Surfaces of AmyB (left; beige) and AmyA (right; green). The nonasaccharide in the A–B groove and the acarbose molecule in the N–C groove of the AmyB[ACR] complex (left) are shown as blue and pink surfaces, respectively. To highlight the active-site loop that closes off the − 1 subsite in AmyA, the nonasaccharide (blue surface) was placed in AmyA.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2008, 378, 850-868) copyright 2008.

Figures were selected by an automated process.