title: Identification of critical residues for the activity and thermostability of Streptomyces sp. SK glucose isomerase.
abstract: 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 Å, 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.
authors: Laboratoire de Micro-organismes et de Biomolécules, Centre de Biotechnologie de Sfax, Université de Sfax, BP "1177", 3018, Sfax, Tunisia.
authors: Hajer
authors: H
authors: Ben Hlima
authors: Samir
authors: S
authors: Bejar
authors: Jonas
authors: J
authors: Riguet
authors: Richard
authors: R
authors: Haser
authors: Nushin
authors: N
authors: Aghajari
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019943:Amino Acid Substitution; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004252:DNA Mutational Analysis; D004795:Enzyme Stability; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013696:Temperature
chemical_list: D050505:Mutant Proteins; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase