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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.

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.

Saccharomyces cerevisiae maltases use maltose, maltulose, turanose and maltotriose as substrates, isomaltases use isomaltose, α‐methylglucoside and palatinose and both use sucrose. These enzymes are hypothesized to have evolved from a promiscuous α‐glucosidase ancMALS through duplication and mutation of the genes. We studied substrate specificity of the maltase protein MAL1 from an earlier diverged yeast, Ogataea polymorpha (Op), in the light of this hypothesis. MAL1 has extended substrate specificity and its properties are strikingly similar to those of resurrected ancMALS. Moreover, amino acids considered to determine selective substrate binding are highly conserved between Op MAL1 and ancMALS. Op MAL1 represents an α‐glucosidase in which both maltase and isomaltase activities are well optimized in a single enzyme. Substitution of Thr200 (corresponds to Val216 in S. cerevisiae isomaltase IMA1) with Val in MAL1 drastically reduced the hydrolysis of maltose‐like substrates (α‐1,4‐glucosides), confirming the requirement of Thr at the respective position for this function. Differential scanning fluorimetry (DSF) of the catalytically inactive mutant Asp199Ala of MAL1 in the presence of its substrates and selected monosaccharides suggested that the substrate‐binding pocket of MAL1 has three subsites (–1, +1 and +2) and that binding is strongest at the –1 subsite. The DSF assay results were in good accordance with affinity (K m) and inhibition (K i) data of the enzyme for tested substrates, indicating the power of the method to predict substrate binding. Deletion of either the maltase (MAL1) or α‐glucoside permease (MAL2) gene in Op abolished the growth of yeast on MAL1 substrates, confirming the requirement of both proteins for usage of these sugars. © 2016 The Authors. Yeast published by John Wiley & Sons, Ltd.

Gut microbiota influences more physiological and developmental processes of humans and animals than earlier expected. Therefore, the possibility to shape the composition and activity of this bacterial population by prebiotics becomes especially important. Inulin, a β-2,1 linked fructan polymer, from plants and fructooligosaccharides (FOS) derived from it are recognized and already widely used as prebiotics while β-2,6 linked fructans have received much less attention from scientific community. In this mini-review, we will address β-2,6 linked fructans: levan and levan-type FOS as novel potential prebiotics and summarize the literature data on levansucrases of Pseudomonas bacteria which are producing these fructans. The major attention is drawn to stable and highly efficient levansucrases of Pseudomonas syringae pv. tomato, among which the Lsc3 protein has been most thoroughly studied using biochemical methods as well as extensive mutagenesis of the protein.

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.

Levansucrases are bacterial extracellular enzymes that act on sucrose producing b-2,6-linked fructans of varied chain length. We summarize here our up-to-date results on (i) novel methods for the production of heterologous levansucrase proteins in Escherichia coli, (ii) biochemical characterization of the levansucrases encoded in the genome of a tomato and Arabidopsis pathogen Pseudomonas syringae pv. tomato DC3000, (iii) innovative chipbased mass spectrometric analysis of oligosaccharidic reaction products, (iv) isolation and characterization of the first mutants and structure-function analysis of a levansucrase of P. syringae pv. tomato. Data on the levansucrase LscA of P. chlororaphis subsp. aurantiaca will be presented for comparison. As most important features of the levansucrases from Pseudomonas bacteria we emphasize their ability to synthesize potentially prebiotic fructooligosaccharides from sucrose or raffinose and to transfructosylate a variety of alternative acceptor sugars with production of heterooligofructans.