Crystal structure of circular permuted RoCBM21 (CP90): dimerisation and proximity of binding sites

Glucoamylases, containing starch-binding domains (SBD), have a wide range of scientific and industrial applications. Random mutagenesis and DNA shuffling of the gene encoding a starch-binding domain have resulted in only minor improvements in the affinities of the corresponding protein to their liga...

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Bibliographic Details
Published inPloS one Vol. 7; no. 11; p. e50488
Main Authors Stephen, Preyesh, Cheng, Kuo-Chang, Lyu, Ping-Chiang
Format Journal Article
LanguageEnglish
Published United States Public Library of Science 30.11.2012
Public Library of Science (PLoS)
Subjects
Affinity
Amylose
Amylose - metabolism
Analysis
Aspergillus awamori
Binding Sites
Biochemistry
Bioinformatics
Biology
Carbohydrates
Chemistry
Circularity
Crystal structure
Crystallography, X-Ray
Deoxyribonucleic acid
Dihydrofolate reductase
DNA
DNA shuffling
Enzymes
Glucan 1,4-alpha-Glucosidase - chemistry
Glucan 1,4-alpha-Glucosidase - genetics
Glucan 1,4-alpha-Glucosidase - metabolism
Industrial applications
Ligands
Models, Molecular
Mutagenesis, Site-Directed
Mutation
Permutations
Protein Multimerization
Protein Structure, Quaternary
Proteins
Random mutagenesis
Rhizopus - enzymology
Rhizopus oryzae
Selectivity
Site-directed mutagenesis
Spectrum analysis
Starch
Structural analysis
Structure-function relationships
Taiwan
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Summary:Glucoamylases, containing starch-binding domains (SBD), have a wide range of scientific and industrial applications. Random mutagenesis and DNA shuffling of the gene encoding a starch-binding domain have resulted in only minor improvements in the affinities of the corresponding protein to their ligands, whereas circular permutation of the RoCBM21 substantially improved its binding affinity and selectivity towards longer-chain carbohydrates. For the study reported herein, we used a standard soluble ligand (amylose EX-I) to characterize the functional and structural aspects of circularly permuted RoCBM21 (CP90). Site-directed mutagenesis and the analysis of crystal structure reveal the dimerisation and an altered binding path, which may be responsible for improved affinity and altered selectivity of this newly created starch-binding domain. The functional and structural characterization of CP90 suggests that it has significant potential in industrial applications.
Bibliography:Conceived and designed the experiments: PS PCL. Performed the experiments: PS KCC. Analyzed the data: PS PCL. Wrote the paper: PS PCL.
Competing Interests: The authors have declared that no competing interests exist.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0050488