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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 DC 3000
Author(s) -
Mardo Karin,
Visnapuu Triinu,
Vija Heiki,
Elmi Triin,
Alamäe Tiina
Publication year - 2013
Publication title -
biotechnology and applied biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.468
H-Index - 70
eISSN - 1470-8744
pISSN - 0885-4513
DOI - 10.1002/bab.1129
Subject(s) - catalytic triad , mutant , pseudomonas syringae , levansucrase , escherichia coli , gene , biochemistry , alanine , biology , pseudomonas , locus (genetics) , chemistry , amino acid , peptide sequence , genetics , bacteria , bacillus subtilis
Levansucrase encoded by the lsc‐3 ( lsc3 ) gene at genomic locus PSPTOA 0032 of Pseudomonas syringae pv. tomato DC 3000 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 .