Lactobacillus plantarum WCFS1 β-Fructosidase: Evidence for an Open Funnel-Like Channel Through the Catalytic Domain with Importance for the Substrate Selectivity.
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.
Facultad de Ciencias Químicas, Universidad de Colima, Carr. Colima-Coquimatlán, km 9, Coquimatlán, Colima, México, C.P. 28400.
Edgar Omar
EO
Mendoza-Llerenas
Facultad de Ciencias Químicas, Universidad de Colima, Carr. Colima-Coquimatlán, km 9, Coquimatlán, Colima, México, C.P. 28400.
David Javier
DJ
Pérez
Facultad de Ciencias Químicas, Universidad de Colima, Carr. Colima-Coquimatlán, km 9, Coquimatlán, Colima, México, C.P. 28400.
Zeferino
Z
Gómez-Sandoval
Laboratorio de Bioingeniería, Universidad de Colima, Carr. Colima-Coquimatlán, km 9, Coquimatlán, Colima, México, C.P. 28400.
Pilar
P
Escalante-Minakata
Laboratorio de Bioingeniería, Universidad de Colima, Carr. Colima-Coquimatlán, km 9, Coquimatlán, Colima, México, C.P. 28400.
Vrani
V
Ibarra-Junquera
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
RS
Razo-Hernández
Dipartimento di Scienze degli Alimenti, Facoltà di Agraria, Università degli Studi di Foggia, via Napoli 25, Foggia, Italy, C.P. 71122.
Vittorio
V
Capozzi
Dipartimento di Scienze degli Alimenti, Facoltà di Agraria, Università degli Studi di Foggia, via Napoli 25, Foggia, Italy, C.P. 71122.
Pasquale
P
Russo
Dipartimento di Scienze degli Alimenti, Facoltà di Agraria, Università degli Studi di Foggia, via Napoli 25, Foggia, Italy, C.P. 71122.
Giuseppe
G
Spano
Dipartimento di Medicina Clinica e Sperimentale, Università degli Studi di Foggia, viale Luigi Pinto 1, Foggia, Italy, C.P. 71100.
Daniela
D
Fiocco
Facultad de Ciencias Biológicas y Agropecuarias, Universidad de Colima, Autopista Colima-Manzanillo, km 40, Tecomán, Colima, México, C.P. 28100. osuna_juan@hotmail.com.
Juan Alberto
JA
Osuna-Castro
Instituto de Química, Universidad Nacional Autónoma de México, Circuito exterior Ciudad Universitaria, Ciudad de Mexico, 04510, México. carcamo@unam.mx.
Abel
A
Moreno
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D048191:Lactiplantibacillus plantarum / Q000201:enzymology* / Q000235:genetics; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D008970:Molecular Weight; D010455:Peptides / Q000737:chemistry; D011994:Recombinant Proteins / Q000378:metabolism; D012542:Scattering, Radiation; D020539:Sequence Analysis, Protein; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013395:Sucrose / Q000378:metabolism; D013696:Temperature; D043324:beta-Fructofuranosidase / Q000737:chemistry* / Q000378:metabolism*
D010455:Peptides; D011994:Recombinant Proteins; D013395:Sucrose; D043324:beta-Fructofuranosidase
Computer simulations; Invertase; Lactobacillus plantarum; Selectivity; β-Fructosidase; β-Propeller domain