Activity of hyperthermophilic glycosynthases is significantly enhanced at acidic pH.
We have previously shown that the hyperthermophilic glycosynthase from Sulfolobus solfataricus (Ssbeta-glyE387G) can promote the synthesis of branched oligosaccharides from activated beta-glycosides, at pH 6.5, in the presence of 2 M sodium formate as an external nucleophile. In an effort to increase the synthetic potential of hyperthermophilic glycosynthases, we report a new method to reactivate the Ssbeta-glyE387G glycosynthase and two novel mutants in the nucleophile of the beta-glycosidases from the hyperthermophilic Archaea Thermosphaera aggregans (Tabeta-gly) and Pyrococcus furiosus (CelB). We describe here that, at pH 3.0 and low concentrations of sodium formate buffer, the three hyperthermophilic glycosynthases show k(cat) values similar to those of the wild-type enzymes and 17-fold higher than those observed at the usual reactivation conditions in 2 M sodium formate at pH 6.5. Moreover, at acidic pH the three reactivated mutants have wide substrate specificity and improved efficiency in the synthetic reaction. The data reported suggest that the reactivation conditions modify the ionization state of the residue acting as an acid/base catalyst. This new reactivation method can be of general applicability on hyperthermophilic glycosynthases whose intrinsic stability allows their exploitation as synthetic tools at low pH.
Perugino
Giuseppe
G
Institute of Protein Biochemistry-Consiglio Nazionale delle Ricerche, Via P. Castellino 111, 80131, Naples Italy.
Trincone
Antonio
A
Giordano
Assunta
A
van der Oost
John
J
Kaper
Thijs
T
Rossi
Mosè
M
Moracci
Marco
M
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D002151:Calorimetry; D003001:Cloning, Molecular; D017931:DNA Primers; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction / Q000379:methods; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016944:Sulfolobus / Q000201:enzymology*
D017931:DNA Primers; D011994:Recombinant Proteins; D005964:Glucosyltransferases