Glucose isomerase of the Streptomyces sp. SK strain: purification, sequence analysis and implication of alanine 103 residue in the enzyme thermostability and acidotolerance.
The glucose isomerase gene (xylA) from the Streptomyces sp. SK strain encodes a 386-amino-acid protein (42.7 kDa) showing extensive identities with many other bacterial glucose isomerases. We have shown by gel filtration chromatography and SDS-PAGE analysis that the purified recombinant glucose isomerase (SKGI) is a 180 kDa tetramer of four 43 kDa subunits. Sequence inspection revealed that this protein, present some special characteristics like the abundance of hydrophobic residues and some original amino-acid substitutions, which distinguish SKGI from the other GIs previously reported. The presence of an Ala residue at position 103 in SKGI is especially remarkable, since the same amino-acid was found at the equivalent position in the extremely thermostable GIs from Thermus thermophilus and Thermotoga neapolitana; whereas a Gly was found in the majority of less thermostable GIs from Streptomyces. The Ala103Gly mutation, introduced in SKGI, significantly decreases the half-life time at 90 degrees C from 80 to 50 min and also shifts the optimum pH from 6.5 to 7.5. This confirms the implication of the Ala103 residue on SKGI thermostability and activity at low pH. A homology model of SKGI based on the SOGI (that of Streptomyces olivochromogenes) crystal structure has been constructed in order to understand the mutational effects on a molecular scale. Hence, the Ala103Gly mutation, affecting enzyme properties, is presumed to increase molecular flexibility and to destabilize, in particular at elevated temperature, the 91-109 loop that includes the important catalytic residue, Phe94.
Borgi
Mohamed Ali
MA
Laboratoire d'Enzymes et de Métabolites des Procaryotes, Centre de Biotechnologie de Sfax BP K 3038 Sfax, Tunisie.
Srih-Belguith
Karima
K
Ben Ali
Mamdouh
M
Mezghani
Monia
M
Tranier
Samuel
S
Haser
Richard
R
Bejar
Samir
S
D000143:Acids / Q000494:pharmacology; D000409:Alanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019747:Aldose-Ketose Isomerases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D004795:Enzyme Stability / Q000187:drug effects; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D016415:Sequence Alignment; D017421:Sequence Analysis; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics
D000143:Acids; D011994:Recombinant Proteins; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase; D000409:Alanine