title: Mutational analysis of conserved regions harboring catalytic triad residues of the levansucrase protein encoded by the lsc-3 gene (lsc3) of Pseudomonas syringae pv. tomato DC3000.
abstract: Levansucrase encoded by the lsc-3 (lsc3) gene at genomic locus PSPTOA0032 of Pseudomonas syringae pv. tomato DC3000 was mutationally analyzed. Altogether, 18 single-amino-acid mutants of 13 positions of Lsc3 were studied for catalytic properties, including production of fructooligosaccharides (FOS). Asp62, Asp219, and Glu303 were proved as members of the catalytic triad. Respective alanine replacement mutants were practically inactive with their kcat values reduced up to ∼130,000 times. Additionally, the requirements of Trp61, Gln301, and Arg304, located in conserved sequence blocks around the catalytic triad positions for the catalysis were shown. The catalytic significance of the position equivalent to Arg304 was shown for levansucrases for the first time. Replacement of Gln301 specifically affected the polymerizing ability of Lsc3. The Gln301Ala mutant was largely hydrolytic and produced 31 times less FOS than the wild type. Despite high conservation grades, Leu66, Pro220, Asp225, and His306 tolerated replacement well. Quantification of produced FOS showed a high biotechnological potential of Lsc3. Using 1 mg of Lsc3 protein, 15.4 g of FOS with a degree of polymerization from 3 to 7 can be synthesized in a 20 H reaction with 1,200 mM sucrose. Our expression system allowed us to produce up to 30 mg of Lsc3 protein from 1 L of induced culture of recombinant Escherichia coli.
authors.affiliation: Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia.
authors.forename: Karin
authors.initials: K
authors.lastname: Mardo
authors.forename: Triinu
authors.initials: T
authors.lastname: Visnapuu
authors.forename: Heiki
authors.initials: H
authors.lastname: Vija
authors.forename: Triin
authors.initials: T
authors.lastname: Elmi
authors.forename: Tiina
authors.initials: T
authors.lastname: Alamäe
mesh_terms: D000595:Amino Acid Sequence; D055162:Biocatalysis; D017124:Conserved Sequence / Q000235:genetics*; D004252:DNA Mutational Analysis; D006602:Hexosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009844:Oligosaccharides / Q000096:biosynthesis; D011487:Protein Conformation; D044224:Pseudomonas syringae / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D009844:Oligosaccharides; C116580:fructooligosaccharide; D006602:Hexosyltransferases; C021513:levansucrase
keywords: fructooligosaccharides; site-directed mutagenesis; structure-function analysis