Table 1.
Data collection and refinement statistics.
Fig 1.
Structural and amino acid sequence alignment of VP (isoenzyme VPL2) from P. eryngii and MnP4 from P. ostreatus.
(A) Superimposition of VP (PDB 2BOQ) (grey) and MnP4 (PDB 4BM1) (orange) crystal structures (shown as cartoons) highlighting the VP amino acid residues mutated in this work (shown together with the heme group as CPK-colored sticks, and labeled according to the color code described below); and (B) alignment of their amino acid sequences (labeled using the same color code) (vertical lines denote conserved residues, and colons and periods indicate conservative and semi-conservative substitutions, respectively). Residues explored in the structural comparative analysis of VP and MnP4 searching for putative stabilizing motifs are shown in bold in the amino acid sequence alignment. VP amino acids subsequently substituted with those of MnP4 to generate the VPi variant appear on red background; those substituted by basic residues present in MnP4, introduced into VPi to form the VPi-br variant, are shown on blue background; and alanines substituted by cysteines to form an extra disulfide bridge in VPi resulting in the VPi-ss variant are highlighted on green background.
Fig 2.
Structural details of four solvent exposed regions (A, B, C and D) in MnP4 (left column), VP (middle column) and VPi variant (right column).
Residues mutated in VPi and their homologous in MnP4 and VP are highlighted in red color.
Table 2.
Steady-state kinetic constants [kcat (s-1), Km (μM), kcat /Km(s-1 mM-1)] of native VP and mutated variants for oxidation of Mn2+ (at the Mn-binding site), ABTS (at the main heme access channel), and VA and RB5 (at the catalytic Trp exposed to the solvent)a.
Fig 3.
Optimum pH for oxidation of Mn2+ (A), ABTS (B), VA (C) and RB5 (D) by native VP (●), VPi (○), VPi-br (■), VPi-ss (□) and VPi-br-ss (▲).
Reactions in 0.1 M B&R buffer for ABTS (7 mM), VA (20 mM) and RB5 (15 mM) oxidation; and 0.1 M sodium sodium tartrate for Mn2+ oxidation (pH 2.5–5.5), with 0.1 mM H2O2 at 25°C. Means and 95% confidence limits are shown.
Fig 4.
pH stability of native VP (A) and mutated variants VPi (B), VPi-br (C), VPi-ss (D) and VPi-br-ss (E).
The enzymes were incubated in 0.1 M B&R buffer (at pH 3, 3.5 and 7) at 25°C, and their residual activity was measured after 1 (), 4 (□), 24 (■) and 120 h (
), with 7 mM ABTS and 0.1 mM H2O2 in 0.1 M sodium tartrate pH 3.5, and referred to activity after 1 min incubation at pH 5. Means and 95% confidence limits are shown.
Fig 5.
Time course of the electronic absorption spectra of native VP (top) and VPi (bottom) at acidic and neutral pH.
UV-visible spectra of native VP and VPi after 0 (red line), 1 (green line) and 5 h (blue line) incubation at pH 3 (A and D), 3.5 (B and E) and 7 (C and F) in 0.1 M B&R buffer, at 25°C.
Fig 6.
Crystal structures of VPi, VPi-br and VPi-ss variants.
(A) Molecular structure of VPi (with 12 α-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 below the porphyrin plane; and mutated residues (all of them as CPK sticks) generating new H-bond and salt bridge interactions (yellow dashed lines) at four regions (named A to D) described in more detail in Fig 2. (B) Molecular structure of VPi-br, showing the same general elements described for VPi plus the seven solvent-exposed basic residues characterizing this variant (mutations described in VPi are also included in VPi-br but they have not been represented for simplifying purposes). (C) Structural detail of the VPi-ss variant showing the extra 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 one of the four disulfide bonds naturally existing in native VP between cysteine residues 34 and 114 that connects helices B and D (also depicted as cartoon) (heme and axial histidines are also shown).