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The residue 179 is involved in product specificity of the Bacillus circulans DF 9R cyclodextrin glycosyltransferase

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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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Acknowledgments

We thank Drs. J. Delfino, L. Curto, and G. Gómez for performing circular dichroism experiments; Drs. O. Taboga and M.I. Gismondi for their supervision on cloning and sequencing; Lic. S. Linskens and Farm. C. Paván for their technical assistance on mass spectrometry, and Prof. Karen Todel for language supervision. This work was supported by grants from the Universidad Nacional de Luján, Universidad de Buenos Aires, and the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). AJD, CMB, and MBJB are career members of CONICET.

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Authors and Affiliations

  1. Departamento de Ciencias Básicas, Universidad Nacional de Luján, Rutas 5 y 7 (6700), Luján, Buenos Aires, Argentina

    Hernán Costa & Susana Alicia Ferrarotti

  2. Instituto de Biotecnología, CICVyA, INTA, Castelar, Los Reseros y Las Cabañas s/n (1686), Hurlingham, Buenos Aires, Argentina

    Ana Julia Distéfano

  3. Fundación Instituto Leloir, Avda. Patricias Argentinas 435 (C1405BWE), Autonomous City of Buenos Aires, Argentina

    Cristina Marino-Buslje

  4. Centro de Biología Molecular Severo Ochoa, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones Científicas, C/ Nicolás Cabrera 1(28049), Madrid, Spain

    Aurelio Hidalgo & José Berenguer

  5. Departamento de Química Biológica, Facultad de Farmacia y Bioquímica, Instituto de Química y Fisicoquímica Biológicas, Universidad de Buenos Aires, Junín 956 (1113), Autonomous City of Buenos Aires, Argentina

    Mirtha Biscoglio de Jiménez Bonino

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  1. Hernán Costa
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  2. Ana Julia Distéfano
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  3. Cristina Marino-Buslje
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Correspondence to Susana Alicia Ferrarotti.

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Costa, H., Distéfano, A.J., Marino-Buslje, C. et al. The residue 179 is involved in product specificity of the Bacillus circulans DF 9R cyclodextrin glycosyltransferase. Appl Microbiol Biotechnol 94, 123–130 (2012). https://doi.org/10.1007/s00253-011-3623-6

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  • DOI: https://doi.org/10.1007/s00253-011-3623-6

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