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PDBsum entry 1b1y

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Hydrolase PDB id
1b1y
Jmol
Contents
Protein chain
500 a.a. *
Ligands
GLC-BGC
BGC
Waters ×140
* Residue conservation analysis

References listed in PDB file
Key reference
Title The crystal structure of the sevenfold mutant of barley beta-Amylase with increased thermostability at 2.5 a resolution.
Authors B.Mikami, H.J.Yoon, N.Yoshigi.
Ref. J Mol Biol, 1999, 285, 1235-1243. [DOI no: 10.1006/jmbi.1998.2379]
PubMed id 9918723
Abstract
The three-dimensional structure of the sevenfold mutant of barley beta-amylase (BBA-7s) with increased thermostability was determined by X-ray crystallography. The enzyme was purified as a single component and crystallized by a hanging drop method in the presence of 14 % PEG 6000. The crystals belong to space group P43212 with cell dimensions a=b=72.11 A, c=250.51 A. The diffraction data up to 2.5 A were collected after soaking the crystal in 100 mM maltose with Rsym of 8.6 %. The structure was determined by a molecular replacement method using soybean beta-amylase (SBA) as a search model and refined to an R-factor of 18.7 %. The final model included 500 amino acid residues, 141 water molecules and three glucose residues, which were located at subsites 1-2 and 4 in the active site. The r.m.s. distance of 485 Calpha atoms between BBA-7s and SBA was 0.62 A. Out of the seven mutated amino acids, four (Ser295Ala, Ile297Val, Ser351Pro and Ala376Ser) were substitutions from the common residues with SBA to the thermostable forms. A comparison of the structures of BBA-7s and SBA indicated that the side-chain of Ser376 makes new hydrogen bonds to the main-chain of an adjacent beta-strand, and that the side-chains of Val297 reduce an unfavorable interaction between the side-chains of Ala314. The mutation of Ser295Ala breaks the hydrogen bond between Ser295 OG and Tyr195 OH, which seems to be the reason for the unoccupied glucose residue at subsite 3. The tandem mutations at 350-352 including substitutions to two Pro residues suggested the reduction of main-chain entropy in the unfolded structure of this solvent-exposed protruded loop.
Figure 5.
Figure 5. Superposition of the mutated sites of Ser295 and Val297 and the bound maltose from BBA-7s on the corresponding sites from SBA (stereo view). BBA-7s and SBA protein models are colored purple and green, respectively. Maltose models in BBA-7s and SBA are colored red and cyan, respectively. Two molecular models are superimposed by a fitting program implemented in Turbo-Frodo.
Figure 6.
Figure 6. Superposition of the mutated site of Pro350- Asp352 from BBA-7s onto the corresponding site from SBA (stereo view). BBA-7s and SBA models are colored purple and green, respectively. Two molecular models are superimposed by a fitting program implemented in Turbo-Frodo.
The above figures are reprinted by permission from Elsevier: J Mol Biol (1999, 285, 1235-1243) copyright 1999.
Secondary reference #1
Title Construction of a plasmid used for the expression of a sevenfold-Mutant barley beta-Amylase with increased thermostability in escherichia coli and properties of the sevenfold-Mutant beta-Amylase.
Authors N.Yoshigi, Y.Okada, H.Maeba, H.Sahara, T.Tamaki.
Ref. J Biochem (tokyo), 1995, 118, 562-567.
PubMed id 8690718
Abstract
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