Levansucrases from Pseudomonas syringae pv. tomato and P. chlororaphis subsp. aurantiaca: substrate specificity, polymerizing properties and usage of different acceptors for fructosylation |
| |
Authors: | Visnapuu Triinu Mardo Karin Mosoarca Cristina Zamfir Alina D Vigants Armands Alamäe Tiina |
| |
Institution: | a Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010 Tartu, Estonia b Mass Spectrometry Laboratory, National Institute for Research and Development in Electrochemistry and Condensed Matter, Plautius Andronescu 1, 300224 Timisoara, Romania c Department of Chemistry and Biology, “Aurel Vlaicu” University of Arad, Revolutiei Blvd. 77, 310130 Arad, Romania d Institute of Microbiology and Biotechnology, University of Latvia, Kronvalda Blvd. 4, LV-1586 Riga, Latvia |
| |
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. |
| |
Keywords: | CID collision-induced dissociation DP degree of polymerization FOS fructooligosaccharides HOF heterooligofructans m/z mass to charge ratio nanoESI HCT MS nanoelectrospray ionization high-capacity ion trap mass spectrometry Mw molecular weight PDB Protein Data Bank TLC thin layer chromatography |
本文献已被 ScienceDirect PubMed 等数据库收录! |
|