title: Levansucrases from Pseudomonas syringae pv. tomato and P. chlororaphis subsp. aurantiaca: substrate specificity, polymerizing properties and usage of different acceptors for fructosylation.
abstract: Levansucrases of Pseudomonas syringae pv. tomato DC3000 (Lsc3) and Pseudomonas chlororaphis subsp. aurantiaca (also Pseudomonas aurantiaca) (LscA) have 73% identity of protein sequences, similar substrate specificity and kinetic properties. Both enzymes produce levan and fructooligosaccharides (FOS) of varied length from sucrose, raffinose and sugar beet molasses. A novel high-throughput chip-based nanoelectrospray mass spectrometric method was applied to screen alternative fructosyl acceptors for levansucrases. Lsc3 and LscA could both transfructosylate D-xylose, D-fucose, L- and D-arabinose, D-ribose, D-sorbitol, xylitol, xylobiose, D-mannitol, D-galacturonic acid and methyl-α-D-glucopyranoside and heterooligofructans with degree of polymerization up to 5 were detected. The ability of D-sorbitol, xylobiose, D-galacturonic acid, D-mannitol, xylitol and methyl-α-D-glucopyranoside to serve as fructosyl acceptors for levansucrases is shown for the first time. Expectedly, site-directed mutagenesis of His321 in Lsc3 to Arg, Lys, Leu and Ser resulted in proteins with decreased catalytic activity, affinity for sucrose and polymerizing ability. Random mutagenesis yielded a Lsc3 mutant Thr302Pro with reduced synthesis of levan and long-chain FOS. Thr302 is located in conserved DQTERP region of levansucrases adjacent to predicted acid-base catalyst Glu303. Thr302 and His321 are predicted to belong to +1 subsite of the substrate binding region of Lsc3.
authors.affiliation: Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010 Tartu, Estonia.
authors.forename: Triinu
authors.initials: T
authors.lastname: Visnapuu
authors.forename: Karin
authors.initials: K
authors.lastname: Mardo
authors.forename: Cristina
authors.initials: C
authors.lastname: Mosoarca
authors.forename: Alina D
authors.initials: AD
authors.lastname: Zamfir
authors.forename: Armands
authors.initials: A
authors.lastname: Vigants
authors.forename: Tiina
authors.initials: T
authors.lastname: Alamäe
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002855:Chromatography, Thin Layer; D005630:Fructans / Q000378:metabolism; D005632:Fructose / Q000378:metabolism*; D006602:Hexosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006639:Histidine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides; D011549:Pseudomonas / Q000201:enzymology* / Q000235:genetics; D044224:Pseudomonas syringae / Q000201:enzymology / Q000235:genetics; D011887:Raffinose / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D021241:Spectrometry, Mass, Electrospray Ionization; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000235:genetics; D013395:Sucrose / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005630:Fructans; C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D005632:Fructose; D006639:Histidine; D013395:Sucrose; D006602:Hexosyltransferases; C021513:levansucrase; D011887:Raffinose