Probing the role of helix α1 in the acid-tolerance and thermal stability of the Streptomyces sp. SK Glucose Isomerase by site-directed mutagenesis.
In order to investigate the role of helix α1 in the different biochemical properties between class I and class II Glucose Isomerases, a histidine and a phenylalanine residue were inserted at position 17 and 19 of Streptomyces sp. SK Glucose Isomerase (SKGI). In addition, W16 was substituted by a histidine. The H17/F19 insertion displaced the optimal pH of SKGI from 6.5 to 7-8 and slightly decreased the thermostability. As for the W16H mutant, a shift in optimal pH of SKGI from 6.5 to 6 was observed along with a decrease in the enzyme thermostability at 85°C with a half-life time reduced twice compared to the wild-type enzyme. Three-dimensional structure analysis suggested that the insertion of a histidine at position 17 results in the formation of new hydrogen bond with D287, thereby preventing it from deprotonating the O2 hydroxyl of the sugar at low pH, while the substitution W16H induced opposite effect by preventing hydrogen bond formation between D287 and W16 and thereby probably facilitating the hydrogen transfer during the isomerization reaction. The findings highlight the essential role of helix α1, which bears the three introduced mutations, in the acid-tolerance and the thermostability of SKGI and of glucose isomerases in general.
Laboratoire de Microorganismes et de Biomolécules, Centre de Biotechnologie de Sfax, Université de Sfax, BP "1177" 3018 Sfax, Tunisie. Electronic address: hajer_benhlima@yahoo.fr.
Ben Hlima
BH
Hajer
Laboratoire de Microorganismes et de Biomolécules, Centre de Biotechnologie de Sfax, Université de Sfax, BP "1177" 3018 Sfax, Tunisie.
Zouari Ayadi
ZA
Dorra
Laboratoire de Microorganismes et de Biomolécules, Centre de Biotechnologie de Sfax, Université de Sfax, BP "1177" 3018 Sfax, Tunisie.
Mezghani
M
Monia
Laboratoire de Microorganismes et de Biomolécules, Centre de Biotechnologie de Sfax, Université de Sfax, BP "1177" 3018 Sfax, Tunisie.
Bejar
B
Samir
Laboratoire de BioCristallographie et Biologie Structurale des Cibles Thérapeutiques, Bases Moléculaires et Structurales des Systèmes Infectieux UMR 5086-CNRS/Université Lyon 1, Institut de Biologie et Chimie des Protéines FR3302, 7 Passage du Vercors, 69367 Lyon cedex 7, France.
Aghajari
A
Nushin
D019747:Aldose-Ketose Isomerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain / Q000235:genetics*; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D006639:Histidine / Q000378:metabolism; D006860:Hydrogen Bonding; D008958:Models, Molecular; D016254:Mutagenesis, Insertional; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013302:Streptomyces / Q000737:chemistry / Q000145:classification / Q000201:enzymology* / Q000502:physiology; D013816:Thermodynamics
D001426:Bacterial Proteins; D010649:Phenylalanine; D006639:Histidine; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase
Acid-tolerance; Glucose Isomerase; Streptomyces; Thermostability