Alignment of sequences corresponding to Bmlp6.

<p>Amino acid sequence alignment of Bmlp6 (UniProt: A7LIK7; NCBI-Protein: NP_001095198), PBMHPC-23 (UniProt: P09338), PBMHPC-12 (UniPtot: P09335) and Bmlp6_SilkDB (SilkDB: BGIBMGA004457) calculated in ClustalW (<a href="http://www.ebi.ac.uk/Tools/msa/clustalw2/" target="_blank">http://www.ebi.ac.uk/Tools/msa/clustalw2/</a>). The alignment is colored according to identity (dark blue) and similarity (light blue) using Jalview (<a href="http://www.jalview.org/" target="_blank">http://www.jalview.org/</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108761#pone.0108761-Waterhouse1" target="_blank">[60]</a>. The evident sequencing error at the C-terminus of Bmlp6_SilkDB (highlighted in dark red, starting from position 194) has been disregarded in sequence similarity calculations. The only discrepancy between Bmlp6 (UniProt: A7LIK7) and the amino acid sequence determined by X-ray crystallography is indicated by white font, at position 217. The N-terminal sequence of Bmlp6 established by Edman degradation is boxed. The displayed sequences correspond to mature proteins without signal peptides.</p

Diffraction data collection and refinement statistics.

a<p>Values in parentheses are for the highest resolution shell.</p>b<p><i>R_merge</i> =  ∑_h∑_j | I_hj - h> |/∑_h∑_j I_hj, where I_hj is the intensity of observation j of reflection h.</p>c<p><i>R_work</i> =  ∑_h | | F_o| - | F_c| |/∑_h | F_o| for all reflections, where F_o and F_c are observed and calculated structure factors, respectively. <i>R_free</i> is calculated analogously for the test reflections, randomly selected and excluded from the refinement.</p><p>Diffraction data collection and refinement statistics.</p

Electrostatic surface potential of Bmlp6, Bmlp7 and Bmlp3 at pH 6.5.

<p>Electrostatic surface potential of Bmlp6, Bmlp7 and Bmlp3 was calculated using the <i>APBS</i> algorithm <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108761#pone.0108761-Baker1" target="_blank">[52]</a> and the <i>PDB2PQR</i> program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108761#pone.0108761-Dolinsky1" target="_blank">[53]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108761#pone.0108761-Dolinsky2" target="_blank">[54]</a> at pH 6.5, which is the physiological pH of silkworm hemolymph. The protein surfaces are shown in the same orientation, in two different views for each protein. The positive and negative charges are colored blue and red, respectively, according to the scale. The <i>APBS</i> writes out the electrostatic potential in dimensionless units of k_bTe_c⁻¹ where k_b is Boltzmann's constant, T is the temperature of calculation and e_c is the charge of an electron <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108761#pone.0108761-Baker1" target="_blank">[52]</a>.</p

Electron density maps of Bmlp6.

<p>(A, B) <i>Fo-Fc</i> and <i>2Fo-Fc</i> electron density maps (contour 5.0 σ and 1.0 σ, respectively), unequivocally demonstrate that residue 217 of Bmlp6 (green) is not Tyr but Asn. (C) <i>2Fo-Fc</i> electron density map (contour 1.0 σ) of a loop (residues 161–170) containing disulfide bridge is presented to illustrate that electron density maps were of a very good quality also at the loop regions. For both fragments the molecule A was arbitrary chosen.</p

Potential binding sites of Bmlp3 and Bmlp6.

<p>Potential binding sites of Bmlp3 (blue/pink) and of Bmlp6 (green/yellow) were marked as No. 1–4 and Po.1–2, respectively. Two different views of Bmlp6 are shown to present both cavities.</p

Crystal packing of Bmlp6.

<p>The asymmetric unit of Bmlp6 (shown with unit cell outline) is composed of five protein molecules, A–E, represented by different colors.</p

Potential binding sites of the Bmlp6 protein.

<p>Potential binding sites of the Bmlp6 protein.</p

DataSheet1_Biochemical characterization of L-asparaginase isoforms from Rhizobium etli—the boosting effect of zinc.pdf

L-Asparaginases, divided into three structural Classes, catalyze the hydrolysis of L-asparagine to L-aspartic acid and ammonia. The members of Class 3, ReAIV and ReAV, encoded in the genome of the nitrogen fixing Rhizobium etli, have the same fold, active site, and quaternary structure, despite low sequence identity. In the present work we examined the biochemical consequences of this difference. ReAIV is almost twice as efficient as ReAV in asparagine hydrolysis at 37°C, with the kinetic KM, kcat parameters (measured in optimal buffering agent) of 1.5 mM, 770 s-1 and 2.1 mM, 603 s-1, respectively. The activity of ReAIV has a temperature optimum at 45°C–55°C, whereas the activity of ReAV, after reaching its optimum at 37°C, decreases dramatically at 45°C. The activity of both isoforms is boosted by 32 or 56%, by low and optimal concentration of zinc, which is bound three times more strongly by ReAIV then by ReAV, as reflected by the KD values of 1.2 and 3.3 μM, respectively. We also demonstrate that perturbation of zinc binding by Lys→Ala point mutagenesis drastically decreases the enzyme activity but also changes the mode of response to zinc. We also examined the impact of different divalent cations on the activity, kinetics, and stability of both isoforms. It appeared that Ni2+, Cu2+, Hg2+, and Cd2+ have the potential to inhibit both isoforms in the following order (from the strongest to weakest inhibitors) Hg2+ > Cu2+ > Cd2+ > Ni2+. ReAIV is more sensitive to Cu2+ and Cd2+, while ReAV is more sensitive to Hg2+ and Ni2+, as revealed by IC50 values, melting scans, and influence on substrate specificity. Low concentration of Cd2+ improves substrate specificity of both isoforms, suggesting its role in substrate recognition. The same observation was made for Hg2+ in the case of ReAIV. The activity of the ReAV isoform is less sensitive to Cl− anions, as reflected by the IC50 value for NaCl, which is eightfold higher for ReAV relative to ReAIV. The uncovered complementary properties of the two isoforms help us better understand the inducibility of the ReAV enzyme.</p

Crystal packing of Bmlp3-p21 and Bmlp3-c2.

<p>Four protein molecules (shades of blue) are present in the asymmetric unit of Bmlp3-p21 (A), whereas the asymmetric unit of Bmlp3-c2 (B) contains a dimeric assembly (red and pink). Symmetry-related molecules are shown in gray.</p

Structural comparison of Bmlp3-p21, Bmlp3-c2 and Bmlp7.

<p>The structures of Bmlp3-p21 (chain A), Bmlp3-c2 (chains A and B) and Bmlp7 (chain A) were Cα-superposed using COOT <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061303#pone.0061303-Emsley1" target="_blank">[28]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061303#pone.0061303-Emsley2" target="_blank">[29]</a>. The main deviations are found in the CTD domain, in loop conformation. The most flexible loop and the N-terminus are encircled.</p