title: Developing fructan-synthesizing capability in a plant invertase via mutations in the sucrose-binding box.
abstract: Fructans are fructose polymers that are synthesized from sucrose by fructosyltransferases. Fructosyltransferases are present in unrelated plant families suggesting a polyphyletic origin for their transglycosylation activity. Based on sequence comparisons and enzymatic properties, fructosyltransferases are proposed to have evolved from vacuolar invertases. Between 1% and 5% of the total activity of vacuolar invertase is transglycosylating activity. We investigated the nature of the changes that can convert a hydrolysing invertase into a transglycosylating enzyme. Remarkably, replacing 33 amino acids (amino acids 143-175) corresponding to the N-terminus of the mature onion vacuolar invertase with the corresponding region of onion fructan:fructan 6G-fructosyltransferase (6G-FFT) led to a shift in activity from hydrolysis of sucrose towards transglycosylation between two sucrose molecules. The substituted N-terminal region contains the sucrose-binding box that harbours the nucleophile involved in sucrose hydrolysis (Asp164). Subsequent research into the individual amino acids responsible for the enhanced transglycosylation activity revealed that mutations in amino acids Trp161 and Asn166, can give rise to a shift towards polymerase activity. Changing the amino acid at either of these positions in the sucrose-binding box increases the transglycosylation capacity of invertases two- to threefold compared to wild type. Combining the two mutations had an additive effect on transglycosylation ability, resulting in an approximately fourfold enhancement. The mutations generated correspond with natural variation present in the sucrose-binding boxes of vacuolar invertases and fructosyltransferases. These relatively small changes that increase the transglycosylation capacity of invertases might explain the polyphyletic origin of the fructan accumulation trait.
authors.affiliation: Zürich Basel Plant Science Center, Botanisches Institut der Universität Basel, Hebelstrasse 1, 4056 Basel, Switzerland. t.ritsema@bio.uu.nl
authors.forename: Tita
authors.initials: T
authors.lastname: Ritsema
authors.forename: Lázaro
authors.initials: L
authors.lastname: Hernández
authors.forename: Auke
authors.initials: A
authors.lastname: Verhaar
authors.forename: Denise
authors.initials: D
authors.lastname: Altenbach
authors.forename: Thomas
authors.initials: T
authors.lastname: Boller
authors.forename: Andres
authors.initials: A
authors.lastname: Wiemken
authors.forename: Sjef
authors.initials: S
authors.lastname: Smeekens
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D018389:Codon, Nonsense; D019143:Evolution, Molecular; D005630:Fructans / Q000096:biosynthesis*; D006031:Glycosylation; D006602:Hexosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019697:Onions / Q000378:metabolism; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment; D013395:Sucrose / Q000378:metabolism*; D043324:beta-Fructofuranosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins
chemical_list: D018389:Codon, Nonsense; D005630:Fructans; D006602:Hexosyltransferases; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D013395:Sucrose; D043324:beta-Fructofuranosidase; C021451:inulosucrase; D001426:Bacterial Proteins