The residue 179 is involved in product specificity of the <Emphasis Type="Italic">Bacillus circulans</Emphasis> DF 9R cyclodextrin glycosyltransferase |
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Authors: | Hernán Costa Ana Julia Distéfano Cristina Marino-Buslje Aurelio Hidalgo José Berenguer Mirtha Biscoglio de Jiménez Bonino Susana Alicia Ferrarotti |
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Institution: | 1.Departamento de Ciencias Básicas,Universidad Nacional de Luján,Buenos Aires,Argentina;2.Instituto de Biotecnología, CICVyA, INTA, Castelar,Buenos Aires,Argentina;3.Fundación Instituto Leloir,Autonomous City of Buenos Aires,Argentina;4.Centro de Biología Molecular Severo Ochoa,Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas,Madrid,Spain;5.Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Instituto de Química y Fisicoquímica Biológicas,Universidad de Buenos Aires,Autonomous City of Buenos Aires,Argentina |
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Abstract: | Cyclodextrin glycosyltransferases (CGTases) are important enzymes in biotechnology because of their ability to produce cyclodextrin
(CD) mixtures from starch whose relative composition depends on enzyme source. A multiple alignment of 46 CGTases and Shannon
entropy analysis allowed us to find differences and similarities that could be related to product specificity. Interestingly,
position 179 has Gly in all the CGTases except in that from Bacillus circulans DF 9R which possesses Gln. The absence of a side chain at that position has been considered as a strong requirement for substrate
binding and cyclization process. Therefore, we constructed two mutants of this enzyme, Q179L and Q179G. The activity and kinetic
parameters of Q179G remained unchanged while the Q179L mutant showed a different CDs ratio, a lower catalytic efficiency,
and a decreased ability to convert starch into CDs. We show that position 179 is involved in CGTase product specificity and
must be occupied by Gly—without a side chain—or by amino acid residues able to interact with the substrate through hydrogen
bonds in a way that the cyclization process occurs efficiently. These findings are also explained on the basis of a structural
model. |
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