Uted into four solventexposed regions (named AD in Fig 6A). Region A (containing mutations D69S/T70D/S86E) is located at the heme distal side above the heme plane, whereas regions B, C and D (containing mutations D146T/Q239R, Q202L/H232E and S301K, respectively) are discovered in the proximal side under the heme plane. The 3 mutations introduced in region A fail to emulate the contacts identified in MnP4 (Fig 2A, left). Nonetheless, compared with all the native VP (Fig 2A, middle), they contribute to reinforce the Phytosphingosine Protocol interaction between helices B’b and C by escalating the Hbond network in this region, as shown in the crystal structure (Fig 2A, correct). Similarly, the two substitutions in area B strengthen the loop amongst helices H and I by interaction on the Arg239 guanidinium group with the Asp237 carboxylate (Fig 2B, right), mimicking that observed between Arg245 and N-Acetyl-DL-methionine Description Asp243 in MnP4 (Fig 2B, left). In addition, the two mutated residues in this area (Thr146 and Arg239) are capable to retain the Hbond that connects the loop involving helices H and I using the Nterminal finish of helix E established amongst Asp146 and Gln239 in the native VP (Fig 2B, middle). Regarding the region C, the introduction of a glutamate at position 232 in helix H promotes the formation of a salt bridge involving this amino acid and Arg227 (Fig 2C, proper) emulating that observed between Glu238 and Arg233 in MnP4 (Fig 2C, left). This interaction, not current in the native enzyme (Fig 2C, middle), reinforces an extensive Hbond networkPLOS One particular | DOI:10.1371/journal.pone.0140984 October 23,13 /pHStability Improvement of a PeroxidaseFig 6. Crystal structures of VPi, VPibr and VPiss variants. (A) Molecular structure of VPi (with 12 helices named from A to J, shown as cylinders) including general structural elements such as four disulfide bonds (cyan sticks) and two Ca2 ions (green spheres); heme cofactor; the two catalytic histidines above and under the porphyrin plane; and mutated residues (all of them as CPK sticks) producing new Hbond and salt bridge interactions (yellow dashed lines) at four regions (named A to D) described in far more detail in Fig two. (B) Molecular structure of VPibr, showing precisely the same general components described for VPi plus the seven solventexposed standard residues characterizing this variant (mutations described in VPi are also integrated in VPibr but they have not been represented for simplifying purposes). (C) Structural detail of the VPiss variant showing the additional disulfide bond (formed by Cys49 and Cys61) that connects helices B and B’a (shown as cartoons); the amino acid residues (CPK sticks) and water molecules (w) coordinating the distal Ca2 ion; and among the list of four disulfide bonds naturally current in native VP amongst cysteine residues 34 and 114 that connects helices B and D (also depicted as cartoon) (heme and axial histidines are also shown). doi:ten.1371/journal.pone.0140984.gthat anchors the helix H each for the Cterminal end of helix G and to Glu304 situated in the Cterminal area of the protein consisting of 66 residues without the need of clearly defined secondary structures (except for two 3amino acids strands plus a single turn 310 helix). Ultimately, unlike what was described for the other regions, the S301K substitution integrated in area D (Fig 2D, suitable) don’t have the expected effect. This must consist in formation of a new Hbond, as observed in MnP4 (Fig 2D, left). By contrast, the sidechain of Lys301 appears exposed to the solvent. Summarizing, three from the.
