title: Structural and kinetic insights reveal that the amino acid pair Gln-228/Asn-254 modulates the transfructosylating specificity of Schwanniomyces occidentalis β-fructofuranosidase, an enzyme that produces prebiotics.
abstract: Schwanniomyces occidentalis β-fructofuranosidase (Ffase) is a GH32 dimeric enzyme that releases fructose from the nonreducing end of various oligosaccharides and essential storage fructans such as inulin. It also catalyzes the transfer of a fructosyl unit to an acceptor producing 6-kestose and 1-kestose, prebiotics that stimulate the growth of bacteria beneficial for human health. We report here the crystal structure of inactivated Ffase complexed with fructosylnystose and inulin, which shows the intricate net of interactions keeping the substrate tightly bound at the active site. Up to five subsites were observed, the sugar unit located at subsite +3 being recognized by interaction with the β-sandwich domain of the adjacent subunit within the dimer. This explains the high activity observed against long substrates, giving the first experimental evidence of the direct role of a GH32 β-sandwich domain in substrate binding. Crucial residues were mutated and their hydrolase/transferase (H/T) activities were fully characterized, showing the involvement of the Gln-228/Asn-254 pair in modulating the H/T ratio and the type β(2-1)/β(2-6) linkage formation. We generated Ffase mutants with new transferase activity; among them, Q228V gives almost specifically 6-kestose, whereas N254T produces a broader spectrum product including also neokestose. A model for the mechanism of the Ffase transfructosylation reaction is proposed. The results contribute to an understanding of the molecular basis regulating specificity among GH-J clan members, which represent an interesting target for rational design of enzymes, showing redesigned activities to produce tailor-made fructooligosaccharides.
authors.affiliation: Centro de Biología Molecular Severo Ochoa, Departamento de Biología Molecular, Universidad Autónoma de Madrid-Consejo Superior de Investigaciones, Cantoblanco, 28049 Madrid, Spain.
authors.forename: Miguel
authors.initials: M
authors.lastname: Álvaro-Benito
authors.forename: M Angela
authors.initials: MA
authors.lastname: Sainz-Polo
authors.forename: David
authors.initials: D
authors.lastname: González-Pérez
authors.forename: Beatriz
authors.initials: B
authors.lastname: González
authors.forename: Francisco J
authors.initials: FJ
authors.lastname: Plou
authors.forename: María
authors.initials: M
authors.lastname: Fernández-Lobato
authors.forename: Julia
authors.initials: J
authors.lastname: Sanz-Aparicio
mesh_terms: D019943:Amino Acid Substitution; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D005632:Fructose / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007444:Inulin / Q000378:metabolism; D020125:Mutation, Missense; D056692:Prebiotics; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D004718:Saccharomycetales / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D043324:beta-Fructofuranosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D056692:Prebiotics; D005632:Fructose; D007444:Inulin; D043324:beta-Fructofuranosidase