Identification of critical residues for the activity and thermostability of Streptomyces sp. SK glucose isomerase

• Published in: Applied microbiology and biotechnology Vol. 97; no. 22; pp. 9715 - 9726
• Main Authors: Ben Hlima, Hajer, Bejar, Samir, Riguet, Jonas, Haser, Richard, Aghajari, Nushin
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer-Verlag 01.11.2013; Springer Berlin Heidelberg; Springer; Springer Nature B.V; Springer Verlag
• Subjects: Aldose-Ketose Isomerases - chemistry; Aldose-Ketose Isomerases - genetics; Aldose-Ketose Isomerases - metabolism; Amino Acid Substitution; Analysis; Bacteria; Binding sites; Biomedical and Life Sciences; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Catalytic Domain; Chemical properties; Crystal structure; Crystallography, X-Ray; DNA Mutational Analysis; E coli; Enzyme Stability; Enzymes; Glucose; Isomerases; Life Sciences; metal ions; Microbial Genetics and Genomics; Microbiology; Models, Molecular; Mutagenesis, Site-Directed; Mutant Proteins - chemistry; Mutant Proteins - genetics; Mutant Proteins - metabolism; mutants; Mutation; Plasmids; Protein Conformation; Protein Folding; Protein Stability; Protein Structure, Tertiary; RNA polymerase; site-directed mutagenesis; Streptomyces; Streptomyces - enzymology; Streptomyces - genetics; Studies; Temperature; thermal stability; Yeast; Thermostability; Glucose isomerase; Crystal structure
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-013-4784-2

The role of residue 219 in the physicochemical properties of D-glucose isomerase from Streptomyces sp. SK strain (SKGI) was investigated by site-directed mutagenesis and structural studies. Mutants G219A, G219N, and G219F were generated and characterized. Comparative studies of their physicochemical properties with those of the wild-type enzyme highlighted that mutant G219A displayed increased specific activity and thermal stability compared to that of the wild-type enzyme, while for G219N and G219F, these properties were considerably decreased. A double mutant, SKGI F53L/G219A, displayed a higher optimal temperature and a higher catalytic efficiency than both the G219A mutant and the wild-type enzyme and showed a half-life time of about 150 min at 85 °C as compared to 50 min for wild-type SKGI. Crystal structures of SKGI wild-type and G219A enzymes were solved to 1.73 and 2.15 , respectively, and showed that the polypeptide chain folds into two structural domains. The larger domain consists of a (β/α)₈ unit, and the smaller domain forms a loop of α helices. Detailed analyses of the three-dimensional structures highlighted minor but important changes in the active site region as compared to that of the wild-type enzyme leading to a displacement of both metal ions, and in particular that in site M2. The structural analyses moreover revealed how the substitution of G219 by an alanine plays a crucial role in improving the thermostability of the mutant enzyme.