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Lactobacillus plantarum WCFS1 β-Fructosidase: Evidence for an Open Funnel-Like Channel Through the Catalytic Domain with Importance for the Substrate Selectivity

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Abstract

β-Fructosidase, a glycoside hydrolase of a biotechnologically important strain, was studied for its biochemical, physicochemical, and three-dimensional structure characteristics. This enzyme was heterologously expressed in Escherichia coli as a C-terminal His-tagged protein (SacB). β-Fructosidase catalyzes the cleavage of glycoside bonds toward certain carbohydrates with β-fructofuranosyl linkages; however, SacB exhibited selectivity toward sucrose and an optimum activity at pH 6.0–6.5 and 37 °C. In such optimum enzymatic activity conditions, the SacB was commonly observed as a monodisperse protein by dynamic light scattering (DLS). As β-fructosidase belongs to glycoside hydrolase family 32 (GH32), a β-sandwich and a five-bladed β-propeller domain are typical predicted folds in its structure. Docking and molecular dynamic simulations revealed for the first time a funnel-like channel perfectly exposed in the β-propeller domain of the Lactobacillus plantarum β-fructosidase (this allows the interaction between its entire catalytic triad and substrates that are larger than sucrose). In contrast, SacB showed a closed central tunnel collaterally induced by its His-tag.

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Acknowledgments

This work was funded by FRABA 14-2013 grant of University of Colima, Mexico, and by CONACyT PhD scholarship (registration number 270848). We thank Dr. Jean Jakoncic (BNL, NSLS-II) for his support on X-ray data collection using synchrotron radiation in order to go further on the crystallographic work for this investigation.

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Authors and Affiliations

  1. Facultad de Ciencias Químicas, Universidad de Colima, Carr. Colima-Coquimatlán, km 9, Coquimatlán, Colima, México, C.P. 28400

    Edgar Omar Mendoza-Llerenas, David Javier Pérez & Zeferino Gómez-Sandoval

  2. Laboratorio de Bioingeniería, Universidad de Colima, Carr. Colima-Coquimatlán, km 9, Coquimatlán, Colima, México, C.P. 28400

    Pilar Escalante-Minakata & Vrani Ibarra-Junquera

  3. Laboratorio de Dinámica de Proteínas y Ácidos Nucleicos, Centro de Investigación en Dinámica Celular, Instituto de Investigación en Ciencias Básicas y Aplicadas, Universidad Autónoma del Estado de Morelos, Cuernavaca, Morelos, México, C.P. 62209

    Rodrigo Said Razo-Hernández

  4. Dipartimento di Scienze degli Alimenti, Facoltà di Agraria, Università degli Studi di Foggia, via Napoli 25, Foggia, Italy, C.P. 71122

    Vittorio Capozzi, Pasquale Russo & Giuseppe Spano

  5. Dipartimento di Medicina Clinica e Sperimentale, Università degli Studi di Foggia, viale Luigi Pinto 1, Foggia, Italy, C.P. 71100

    Daniela Fiocco

  6. Facultad de Ciencias Biológicas y Agropecuarias, Universidad de Colima, Autopista Colima-Manzanillo, km 40, Tecomán, Colima, México, C.P. 28100

    Juan Alberto Osuna-Castro

  7. Instituto de Química, Universidad Nacional Autónoma de México, Circuito exterior Ciudad Universitaria, Ciudad de Mexico, 04510, México

    Abel Moreno

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  1. Edgar Omar Mendoza-Llerenas
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  2. David Javier Pérez
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Mendoza-Llerenas, E.O., Pérez, D.J., Gómez-Sandoval, Z. et al. Lactobacillus plantarum WCFS1 β-Fructosidase: Evidence for an Open Funnel-Like Channel Through the Catalytic Domain with Importance for the Substrate Selectivity. Appl Biochem Biotechnol 180, 1056–1075 (2016). https://doi.org/10.1007/s12010-016-2152-2

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