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 k cat 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. C 2013

Levansucrases of a Pseudomonas syringae pathovar as catalysts for the synthesis of potentially prebiotic oligo- and polysaccharides

“…A small size of the protein can be beneficial in case of heterologous expression -it should reduce the burden for the host and increase the expression level. In an agreement with that, a high yield, 30 mg of Lsc3 protein per 1 ml of recombinant Escherichia coli culture, was achieved at its overexpression [74].…”

“…Levansucrases of Pst (Lsc2, Lsc3) and Pca (LscA) produce from sucrose aside of levan also FOS with DP 3-7 [74,84,85,87,89]. Polymerizing properties of two studied levansucrases of Pst, Lsc2 and Lsc3 were reported to be similar that agrees with their very high (95%) sequence identity [85,89].…”

“…These bacteria have mostly been isolated from plant phyllosphere, some of them are endophytic, some have biocontrol abilities and several cause diseases in plants ( [70][71][72][73][74] and references therein). P. syringae pathovars cause diseases in many plants [75] and they are exceptional among levansucrase-possessing bacteria for having multiple (two or three) copies of respective genes [70,76,77].…”

“…The Lsc2 and Lsc3 of Pst have been produced as heterologous proteins in E coli and after the purification thoroughly biochemically characterized [74,77,84,85,[87][88][89][90]. Table 1 reviews some biochemical parameters of above-mentioned levansucrases.…”

“…A high sucrose concentration also promoted FOS synthesis by the Lsc3. From 410 g/L of sucrose approximately 100 g/L of FOS (15.4 g per mg of protein) was synthesized [74]. The FOS mixture produced by the Lsc3 contained 1-and 6-kestoses, nystose and longer FOS with DP up to 7.…”