3u75 | pdb_00003u75

Structure of E230A-fructofuranosidase from Schwanniomyces occidentalis complexed with fructosylnystose

spinqualitylabelsall models 3u75, resolution 2.68Å Show:Asymmetric Unit Biological Assembly Drag the structure with the mouse to rotate   Export Animated Image   Print3D Structural highlights 3u75 is a 4 chain structure with sequence from Atcc 2322. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance. Ligands: FRU, GLC, NAG Related: 3kf3, 3kf5, 3u14 Activity: Beta-fructofuranosidase, with EC number 3.2.1.26 Resources: FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT Publication Abstract from PubMed Schwanniomyces occidentalis beta-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 are observed, the sugar unit located at subsite +3 being recognized by interaction with the beta-sandwich domain of the adjacent subunit within the dimer. This explains the high activity observed against long substrates and gives the first experimental evidence of the direct role of a GH32 beta-sandwich domain in substrate binding. Crucial residues were mutated and their hydrolase/transferase (H/T) activities were fully characterized, showing the involvement of the Gln228/Asn254 pair in modulating the H/T ratio and the type beta(2-1)/beta(2-6) linkage formation. We have generated Ffase mutants with new transferase activity; among them, Q228V gives almost specifically 6-kestose while N254T produces a broader spectrum product including also neokestose. A model for the mechanism of the Ffase transfructosylation reaction has been proposed. The results contribute to understand 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 FOS. 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.,Alvaro-Benito M, Sainz-Polo MA, Gonzalez-Perez D, Gonzalez B, Plou FJ, Fernandez-Lobato M, Sanz Aparicio J J Biol Chem. 2012 Apr 18. PMID:22511773 From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

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