The Role of a Loop in the Non-catalytic Domain B on the Hydrolysis/Transglycosylation Specificity of the 4-& alpha;-Glucanotransferase from Thermotoga maritima
Por:
Llopiz, Alexey, Ramirez-Martinez, Marco A., Olvera, Leticia, Xolalpa-Villanueva, Wendy, Pastor, Nina, Saab-Rincon, Gloria
Publicada:
1 oct 2023
Ahead of Print:
1 jul 2023
Resumen:
The mechanism by which glycoside hydrolases control the reaction
specificity through hydrolysis or transglycosylation is a key element
embedded in their chemical structures. The determinants of reaction
specificity seem to be complex. We looked for structural differences in
domain B between the 4-& alpha;-glucanotransferase from Thermotoga
maritima (TmGTase) and the & alpha;-amylase from Thermotoga petrophila
(TpAmylase) and found a longer loop in the former that extends towards
the active site carrying a W residue at its tip. Based on these
differences we constructed the variants W131G and the partial deletion
of the loop at residues 120-124/128-131, which showed a 11.6 and
11.4-fold increased hydrolysis/transglycosylation (H/T) ratio relative
to WT protein, respectively. These variants had a reduction in the
maximum velocity of the transglycosylation reaction, while their
affinity for maltose as the acceptor was not substantially affected.
Molecular dynamics simulations allow us to rationalize the increase in
H/T ratio in terms of the flexibility near the active site and the
conformations of the catalytic acid residues and their associated pKas.
Filiaciones:
Llopiz, Alexey:
Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Morelos, Cuernavaca, 62209, Mexico
Ramirez-Martinez, Marco A.:
Centro de Investigación en Dinámica Celular, IICBA, Universidad Autónoma del Estado de Morelos, Morelos, Cuernavaca, 62209, Mexico
Olvera, Leticia:
Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Morelos, Cuernavaca, 62209, Mexico
Xolalpa-Villanueva, Wendy:
Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Morelos, Cuernavaca, 62209, Mexico
Pastor, Nina:
Centro de Investigación en Dinámica Celular, IICBA, Universidad Autónoma del Estado de Morelos, Morelos, Cuernavaca, 62209, Mexico
Saab-Rincon, Gloria:
Departamento de Ingeniería Celular y Biocatálisis, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Morelos, Cuernavaca, 62209, Mexico
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