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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.

Applied biochemistry and biotechnology (2016-11-05)
Edgar Omar Mendoza-Llerenas, David Javier Pérez, Zeferino Gómez-Sandoval, Pilar Escalante-Minakata, Vrani Ibarra-Junquera, Rodrigo Said Razo-Hernández, Vittorio Capozzi, Pasquale Russo, Giuseppe Spano, Daniela Fiocco, Juan Alberto Osuna-Castro, Abel Moreno
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.

MATERIALS
Product Number
Brand
Product Description

Supelco
1-Kestose, analytical standard
Supelco
Nystose, analytical standard