Additional file 2: Table S1. of Crown-of-thorns starfish have true image forming vision

Starfish usage data. (DOC 23 kb

Additional file 1: Figure S1. of Crown-of-thorns starfish have true image forming vision

Spectral transmittance curve blue filter. The relative transmittance of the filter is drawn in blue and the spectral sensitivity of the crown-of-thorn starfish photoreceptors in black (taken from [34]). Grey shading indicating the standard deviation. (PDF 7 kb

Schematic representation of recombinant His-FeCh, FeCh, His-FeChΔ347 and FeChΔ347 of <i>Synechocystis</i> 6803.

<p>The C-terminal CAB domain is exclusive to plastidic ferrochelatases of photosynthetic organisms, it is connected via a linker region to the catalytical domain (amino acids 1-324), where chelating of divalent metal ions into protoporphyrin IX takes place. N-terminal His₆-tags have been added with the amino acid sequence MGSSHHHHHHSSGLVPRGSH (for His-FeCh, cleavable by a thrombin protease) or MAHHHHHHVDDDDK (for His-FeChΔ347, cleavable by an enterokinase), respectively.</p

Activity progress curves in dependency of the presence of pigments.

<p>Enzyme activity was tested without (closed square) and with (closed circle) pigment mix from <i>Synechocystis</i> 6803. Samples in assay buffer at pH 8 were preincubated 30 min at 30°C before the start of the assay. (A) 50 nM co-expressed His-FeCh with 1 µM Zn²⁺, 0.5 µM Proto9 and pigment mix consisting of 0.5 µM Chl and 0.23 µM carotenoids. (B) 30 nM FeChΔ347, 5 µM Zn, 0.5 µM Proto9 and pigment mix consisting of 1.1 µM Chl and 0.5 µM carotenoids.</p

Activity of refolded His-FeCh is dependent on buffer composition.

<p>Zn-Proto9 formation was measured at 30°C in assay buffer in the presence of 37 nM His-FeCh, 1 µM Zn²⁺ and 0.5 µM Proto9 (closed circle) using a continuous assay. (Open circle) addition of 0.5 µM Mn²⁺, (open triangle) the detergent β-DM was replaced by 10 mM Chaps, (open inverted triangles) KCl was removed from the buffer. Invoked graph: control activity of standard buffer depleted of His-FeCh, Zn²⁺ or Proto9.</p

Enzyme kinetic plots for FeCh and FeChΔ347 <i>lacking</i> His₆-tags.

<p>30 nM enzyme was analyzed in a continuous assay at 30°C. Hill equation fit relating initial rate (nM Zn-Proto9 s⁻¹) to Zn²⁺ concentration for FeChΔ347 (A), co-expressed FeCh (B) or refolded FeCh (C) to Zn²⁺ concentration. Michaelis-Menten equation fit was used when the variable substrate was Proto9 for FeChΔ347 (D), co-expressed FeCh (E) or refolded FeCh (F). Error bars represent standard deviation (n = 3). “Refolded FeCh” corresponds to <i>in vitro</i> folded enzyme, while “co-expressed FeCh” was co-expressed with chaperones assisting folding in <i>E. coli</i> cytosol.</p

Enzyme kinetic parameters for ferrochelatases from other species.

<p>Enzyme kinetic parameters for ferrochelatases from other species.</p

Enzyme characterization using a discontinuous enzyme assay.

<p>Effect of pH (A) and temperature (B) were tested, error bars represent standard deviation (n = 3).</p

Kinetic parameters K_M and k_cat and the Hill coefficient <i>n</i> of refolded His-FeCh and His-FeChΔ347. Data obtained from Fig. 5.

<p>Kinetic parameters K_M and k_cat and the Hill coefficient <i>n</i> of refolded His-FeCh and His-FeChΔ347. Data obtained from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055569#pone-0055569-g005" target="_blank">Fig. 5</a>.</p

Kinetic parameters K_M and k_cat and the Hill coefficient <i>n</i> of <i>in vitro</i> refolded FeCh, co-expressed FeCh folded in the presence of chaperones in <i>E. coli</i> cytosol, and truncated FeChΔ347, after removal of the N-terminal His₆-tag. Data were obtained from Fig. 6.

<p>Kinetic parameters K_M and k_cat and the Hill coefficient <i>n</i> of <i>in vitro</i> refolded FeCh, co-expressed FeCh folded in the presence of chaperones in <i>E. coli</i> cytosol, and truncated FeChΔ347, after removal of the N-terminal His₆-tag. Data were obtained from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055569#pone-0055569-g006" target="_blank">Fig. 6</a>.</p