3U75

Structure of E230A-fructofuranosidase from Schwanniomyces occidentalis complexed with fructosylnystose

Summary for 3U75

• Entry DOI: 10.2210/pdb3u75/pdb
• Related: 3KF3 3KF5 3U14
• Descriptor: Fructofuranosidase, alpha-D-glucopyranose-(1-2)-beta-D-fructofuranose-(2-1)-beta-D-fructofuranose-(2-1)-beta-D-fructofuranose-(2-1)-beta-D-fructofuranose, 2-acetamido-2-deoxy-beta-D-glucopyranose, ... (4 entities in total)
• Functional Keywords: glycosidase gh32, beta-propeller, hydrolase, carbohydrate/sugar binding, glycosylations
• Biological source: Schwanniomyces occidentalis (Yeast)
• Total number of polymer chains: 4
• Total formula weight: 249162.80

Authors

Sainz-Polo, M.A.,Sanz-Aparicio, J. (deposition date: 2011-10-13, release date: 2012-04-25, Last modification date: 2024-10-30)

Primary citation

Sainz-Polo, M.A.,Gonzalez-Perez, D.,Gonzalez, B.,Plou, F.J.,Fernandez-Lobato, M.,Sanz-Aparicio, J.
Structural and kinetic insights reveal that the amino acid pair GLN228/ASN254 modulates the transfructosylating specificity of Schwanniomyces occidentalis beta-fructofuranosidase, an enzyme that produces prebiotics.
J.Biol.Chem., 287:19674-19686, 2012

PubMed 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.

PubMed: 22511773
DOI: 10.1074/jbc.M112.355503

Experimental method

X-RAY DIFFRACTION (2.68 Å)