Fig 1.
Glucosinolate breakdown pathways.
Pathways are illustrated using allylglucosinolate as an example. Myrosinase-catalyzed glucosinolate hydrolysis yields an unstable aglucone that undergoes a spontaneous Lossen-like rearrangement to the isothiocyanate. In the presence of specifier proteins, alternative breakdown products (simple nitrile, epithionitrile, thiocyanate) are formed. Boxes indicate hypothetic mechanisms supposively mediated by specifier proteins. Specifier proteins analyzed in the present study are assigned to the boxes. If the aglucone lacks the structural requirements for epithionitrile and/or thiocyanate formation, all types of specifier proteins promote simple nitrile formation (not shown). For more detailed explanations, see text. Boxed reaction schemes are redrawn from [20] and [21] with modifications.
Fig 2.
Ferene S quantification of iron in TaTFP wildtype and mutants.
Proteins (in 37.5 mM sodium acetate buffer, pH 5, with 6.25% (w/v) sucrose) were denatured and precipitated. Fe2+ in the supernatant was quantified colorimetrically using Ferene S reagent and calibration with (NH4)2Fe(SO4)2. OA, ovalbumin; Tf, holo-transferrin. Shown are means ± SD (N = 6 independent expression experiments).
Fig 3.
ICP-MS quantification of iron in TaTFP wildtype and mutants.
Proteins (in 37.5 mM sodium acetate buffer, pH 5, with 6.25% (w/v) sucrose) were denatured and precipitated. The supernatant was subjected to ICP-MS. Iron was quantified based on calibration with Iron ICP Standard. OA, ovalbumin; Tf, holo-transferrin. Shown are means ± SD (N = 3 independent expression experiments).
Fig 4.
Ferene S quantification of iron in AtESP and AtNSP3 wildtype and mutants.
Proteins (in 50 mM MES, pH 6.5, TaTFP in 6.25% (w/v) sucrose in 37.5 mM sodium acetate buffer, pH 5) were denatured and precipitated. Fe2+ in the supernatant was quantified colorimetrically using Ferene S reagent and calibration with (NH4)2Fe(SO4)2. OA, ovalbumin; Tf, holo-transferrin. Shown are means ± SD (N = 3 independent expression experiments). Different letters above bars indicate a significant difference (p<0.05; lower case letters: ANOVA with Tukey's test (when errors were normally distributed); upper case letter: Kruskal-Wallis non-parametric test (non-normal errors)).
Fig 5.
Structure of the final AtNSP3 homology model.
The β-propeller and JAL domains are shown in blue and orange, respectively. Fe2+ is represented by a purple sphere. (A) Top view. β-Sheets are labeled according to [34]. (B) Side view.
Fig 6.
Activity of AtNSP3 mutants with substitutions of proposed iron-binding residues.
Purified proteins were incubated with allylglucosinolate (A) or benzylglucosinolate (B) and myrosinase in 50 mM MES buffer, pH 6.0, supplemented with 0.01 mM Fe2+ for 40 min. Activity is expressed as the proportion of simple nitrile formed relative to the total amount (nmol) of detected breakdown products. Shown are means ± SD of N = 3 independent expression experiments.
Fig 7.
Docking arrangement of AtNSP3 with Fe2+ and allylglucosinolate aglucone.
Active site residues are shown as sticks with gray C-skeleton. The aglucone is shown with the C-skeleton in cyan. The color code for heteroatoms is as follows: yellow, sulfur; blue, nitrogen; red, oxygen. Fe2+ is represented by a purple sphere.
Fig 8.
Impact of Fe2+ and Fe3+ on specifier protein activity of TaTFP.
Purified TaTFP was incubated with allylglucosinolate and myrosinase (Myr) in 50 mM MES buffer, pH 6.0, with or without 0.01 mM Fe2+ (FeII, (NH4)2Fe(SO4)2) or 0.01 mM Fe3+ (FeIII, NH4Fe(SO4)2) for 40 min. Activity is expressed as the proportion of individual products ((A) epithionitrile, (B) organic thiocyanate) formed relative to the total amount (nmol) of detected breakdown products. Shown are means ± SD of N = 5 independent expression experiments. Different letters above bars indicate a significant difference (p<0.05; ANOVA with Tukey's test).