Fig 1.
Enzymatic production of DFAs from inulin by IFTases.
Table 1.
Primers for site-directed mutagenesis.
The underlined sequences represented mutated codons.
Fig 2.
A phylogenetic tree of the reported DFA IIIases and IFTases.
The dendrogram was constructed by the neighbor-joining method from the amino acid sequences in Mega5.2 program. Bootstrap values of each branch were obtained as 1,000 by 1,000-repeated bootstrapping. The scale bar indicated the amino acid substitution per site. GenBank accession numbers of the enzymes were showed in the parentheses after each enzyme name.
Table 2.
Identity of amino acid sequences of the reported DFA IIIases and IFTases from various microorganisms.
Fig 3.
Estimation of the molecular mass of AaDFA IIIase by SDS-PAGE and gel filtration.
(A) SDS-PAGE analysis of AaDFA IIIase. The protein and markers were stained with Coomassie Blue. (B) Gel filtration analysis of AaDFA IIIase. The marker proteins include thyroglobulin (bovine, Mw: 670 kDa), γ-globulin (bovine, Mw: 158 kDa), ovalbumin (chicken, Mw: 44 kDa), myoglobin (horse, Mw: 17 kDa), and vitamin B12 (Mw: 1.35 kDa), respectively. The retention times of above corresponding markers are 7.0, 8.3, 9.7, 11.1 and 13.9 min, respectively, and 8.5 min for AaDFA IIIase.
Table 3.
13C-NMR chemical shifts of enzymatic product and standard inulobiose.
Fig 4.
Effect of pH (A) and temperature (B) on the activity of AaDFA IIIase.
(A) The relative activity was investigated at 55°C and different pH values. (B) The relative activity was investigated at temperatures varying from 30–60°C at pH 5.5. Data were plotted as ln (relative activity, %) versus T-1. Relative activity was expressed as a percentage of the maximal enzyme activity. Values were the means of three replications ± standard deviation.
Fig 5.
Three-dimensional model of AaDFA IIIase predicted by SWISS-MODEL.
(A) A model structure of AaDFA IIIase which shows a typical right-handed parallel β-helix. The models of mutants (D207A, D207N, E218A, and E218N) have similar three-dimensional structure with the wild-type enzyme. (B) Superimposition of the monomer of AaDFA IIIase (green) and IFTase from Bacillus sp. snu-7 (pink).
Fig 6.
Putative active site of AaDFA IIIase and molecular docking of DFA III into this putative catalytic pocket.
(A) The putative reactive pocket and active site residues of AaDFA IIIase based on superimposition of AaDFA IIIase model (gray) and crystal structure of Bacillus sp. IFTase (pink). All of active residues of IFTase were showed as pink and putative active residues of AaDFA IIIase in parenthesis as gray. (B) Molecular docking of DFA III into the putative active pocket of AaDFA IIIase model. DFA III was bound into the putative active site through hydrogen bonds (green dotted lines) with R285 (1.76 Å), D207 (2.01 Å), E218 (2.00 Å), S84 (1.89 Å), R266 (2.1 Å), and Y171 (1.74 Å). (C) SDS-PAGE analysis of the mutants. M represents standard protein markers and Arabic numbers 1–4 represented D207A, D207N, E218A, and E218N mutants, respectively.
Fig 7.
Molecular docking of DFA III to the putative active pocket of AaDFA IIIase mutants.
A, B, C, and D represented the molecular docking of D207A, D207N, E218A, and E218N mutants, respectively. The dotted and solid pink lines represented strong and relatively weak hydrogen bonds, respectively. The distances of H bonds were labeled with pink Arabic numbers.