List of cyanases from plants, fungi and bacteria.

<p>List of cyanases from plants, fungi and bacteria.</p

Coimmunoprecipitation assay demonstrating self-interaction of AtCYN (A) and OsCYN (B).

<p>Although an extremely low amount of OsCYN proteins was detected in the input samples, an identical pattern was shown by OsCYN in the assay. Lane 1: Mock; Lane 2: HA:CYN and FLAG:CYN were detected in the input samples; Lane 3: when anti-HA antibody was added, FLAG:CYN immunoprecipitated with HA:CYN (Lanes 6&8 were controls); Lane 4: when anti-FLAG antibody was added, HA:CYN immunoprecipitated with FLAG:CYN (Lanes 7&9 were controls); Lane 5: Native mouse IgG was used as negative control of antibodies. Bands of HA:CYN and Flag:CYN are indicated with arrows and bands of mouse IgG are indicated with stars.</p

List of primers used in this study.

<p>List of primers used in this study.</p

Quantitative RT-PCR analysis of <i>AtCYN</i> transcription.

<p>(A) The <i>AtCYN</i> transcript levels in different plant organs. RL (rosette leaves), CL (cauline leaves); (B) and (C) Samples are the 7-day-old seedlings which grew in the MS medium containing KCNO or NaCl. The <i>AtCYN</i> transcript levels in response to treatment with three KCNO concentrations (B) and in response to treatment with 150 mM NaCl (C); (D) The 7-day-old seedlings from the standard MS medium were transferred to the MS medium containing 1 mM KCNO or 150 mM NaCl, and the samples were harvested at different time points. Error bars represent the standard deviation of three biological replicates.</p

Homology modelling of AtCYN and OsCYN.

<p>(A) The predicted structures of AtCYN (blue) and OsCYN (magentas) were similar to the crystal structure of the EcCYN monomer (green). Ball-and-stick figures represent the conserved catalytic residues Arg96, Glu99 (B) and Ser122 of the EcCYN (C). Red dots indicate chloride ions.</p

Gel filtration and cyanase activities of His-tagged AtCYN, OsCYN and AtCYN mutants (E94L and S117A).

<p>(A) Gel filtration. High Molecular Weight (HMW) Standard: Thyroglobulin, 669 kDa; Ferritin, 440 kDa; Aldolase, 158 kDa; Conalbumin, 75 kDa and Ovalbumin, 43 kDa. And calculated molecular weight: AtCYN, 210.74 kDa; OsCYN, 211.72 kDa; AtCYN-E94L, 71.64 kDa and AtCYN-S117A, 62.76 kDa. (B) Cyanase activities of His-tagged AtCYN, OsCYN and AtCYN mutants (E94L and S117A) at pH 7.7 and 27°C.</p

Decomposition of cyanate by AtCYN and OsCYN <i>in vivo</i>.

<p>Seeds were plated on MS medium containing either 0 mM, 0.5 mM, 1 mM or 2 mM KCNO. Plates were incubated at 4°C for 3 days and then transferred to a growth chamber for 7 days.</p

Alignment of catalytic regions of cyanases from fungus, plant and bacterial species.

<p>Accession numbers for each cyanase are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018300#pone-0018300-t001" target="_blank">Table 1</a>. Residues conserved in all sequences are indicated in white type on a black background, the consensus residue or similar residues in all sequences are indicated in white type on a dark grey background and the consensus residue or similar residues in the sequences of cyanases from Dicotyledoneae and Monocotyledoneae are indicated in black type on a light grey background. The predicted catalytic residues in <i>E. coli</i> are indicated above the alignment.</p

Influence of pH and temperature on cyanase activity.

<p>The His:AtCYN and His:OsCYN enzymes were purified and their activities assayed <i>in vitro</i>. (A) Effect of pH on cyanase activity at 27°C. BSA was used as a control. (B) Effect of temperature on cyanase activity at pH 7.7.</p

Biochemical characteristics of heterologously expressed AtCYN and OsCYN.

<p>The apparent <i>Km</i> and <i>Vmax</i> values were calculated by double-reciprocal plots.</p>a<p>The cyanase reaction was assayed at 27°C, pH 7.7, in the presence of 5 mM KCNO,</p>b<p>The cyanase reaction was assayed at 27°C, pH 7.7, in the presence of 5 mM NaHCO₃.</p