PDBsum entry 1v3h

Hydrolase

PDB id: 1v3h

Contents

Protein chain

491 a.a. *

Ligands

GLC-GLC-GLC-GLC-GLC

SO4 ×6

Waters ×787

* Residue conservation analysis

PDB id:

1v3h

Name:

Hydrolase

Title:

The roles of glu186 and glu380 in the catalytic reaction of soybean beta-amylase

Structure:

Beta-amylase. Chain: a. Synonym: 1,4-alpha-d-glucan maltohydrolase. Engineered: yes. Mutation: yes

Source:

Glycine max. Soybean. Organism_taxid: 3847. Gene: bmy1. Expressed in: escherichia coli. Expression_system_taxid: 562.

Resolution:

1.60Å R-factor: 0.163 R-free: 0.181

Authors:

Y.N.Kang,M.Adachi,S.Utsumi,B.Mikami

Key ref:

Y.N.Kang et al. (2004). The roles of Glu186 and Glu380 in the catalytic reaction of soybean beta-amylase. J Mol Biol, 339, 1129-1140. PubMed id: 15178253 DOI: 10.1016/j.jmb.2004.04.029

Date:

02-Nov-03 Release date: 22-Jun-04

Protein chain

P10538  (AMYB_SOYBN) -  Beta-amylase from Glycine max

Seq: 496 a.a.

Struc: 491 a.a.*

* PDB and UniProt seqs differ at 4 residue positions (black crosses)

Enzyme reactions

• Enzyme class: E.C.3.2.1.2 - beta-amylase.
• Reaction: Hydrolysis of 1,4-alpha-glucosidic linkages in polysaccharides so as to remove successive maltose units from the non-reducing ends of the chains.

DOI no: 10.1016/j.jmb.2004.04.029

J Mol Biol 339:1129-1140 (2004)

PubMed id: 15178253

The roles of Glu186 and Glu380 in the catalytic reaction of soybean beta-amylase.

Y.N.Kang, M.Adachi, S.Utsumi, B.Mikami.

ABSTRACT

It has previously been suggested that the glutamic acid residues Glu186 and Glu380 of soybean beta-amylase play critical roles as a general acid and a general base catalyst, respectively. In order to confirm the roles of Glu186 and Glu380, each residue was mutated to a glutamine residue and the crystal structures of the substrate (E186Q/maltopentaose) and product (E380Q/maltose) complexes were determined at resolutions of 1.6 Angstrom and 1.9 Angstrom, respectively. Both mutant enzymes exhibited 16,000- and 37,000-fold decreased activity relative to that of the wild-type enzyme. The crystal structure of the E186Q/maltopentaose complex revealed an unambiguous five-glucose unit at subsites -2 to +3. Two maltose molecules bind on subsites -2 to -1 and +2 to +3 in the E380Q/maltose complex, whereas they bind in tandem to -2 to -1 and +1 to +2 in the wild-type/maltose complex. The conformation of the glucose residue at subsite -1 was identified as a stable (4)C(1) alpha-anomer in the E380Q/maltose complex, whereas a distorted ring conformation was observed in the wild-type/maltose complex. The side-chain movement of Gln380 to the position of a putative attacking water molecule seen in the wild-type enzyme caused the inactivation of the E380Q mutant and an altered binding pattern of maltose molecules. These results confirm the critical roles played by Glu186 in the donation of a proton to the glycosidic oxygen of the substrate, and by Glu380 in the activation of an attacking water molecule. The observed difference between the backbones of E186Q/maltopentaose and E380Q/maltose in terms of Thr342 suggests that the side-chain of Thr342 may stabilize the deprotonated form of Glu186 after the cleavage of the glycosidic bond.

Selected figure(s)

Figure 2.
Figure 2. (a) Stereo view of the E186Q/maltopentaose (shown in magenta) superimposed on the wild-type/maltose (shown in yellow) structure at the active site. The protein residues of each complex were superimposed using the RIGID program of TURBO-FRODO. Comparison of the two structures revealed almost the same conformation at the active site, except for in the main-chain region around the Thr342 residue. (b) Comparison of the hydrogen bonding pattern between the structures of E186Q/maltopentaose (magenta) and wild-type/maltose (yellow) around subsites -1 and +1 in stereo. The dotted lines indicate the hydrogen bond interactions. A putative attacking water molecule (H[2]O 712) was observed at the position corresponding to the O1 atom of Glc( -1) in the wild-type/maltose structure. The intramolecular hydrogen bonds between Glu186, Arg188, and Tyr192 were not greatly altered, but there was no interaction between Gln186 and Thr342 due to the main-chain conformational change in the 340-343 residues. The NE2 atom of Gln186 formed a hydrogen bond with the O4 atom of Glc(+1) at a distance of 2.9 Å.

Figure 4.
Figure 4. A schematic representation of the hydrogen bonding networks of (a) the wild-type enzyme in the attack by a catalytic water molecule on the C1 atom of Glc( -1) from above, and (b) the wild-type enzyme after the inversion at the O1 atom. Note that the Thr342 residue assumes different conformations in (a) and (b). (c) The hydrogen bonding network rearranged in the E380Q/maltose structure. The catalytic water molecule was eliminated and a new water molecule was introduced between Gln380 and Asn340. No interaction between Gln380 and Lys295 was observed.

The above figures are reprinted by permission from Elsevier: J Mol Biol (2004, 339, 1129-1140) copyright 2004.

Figures were selected by an automated process.