Manganese Oxidation Site in Pleurotus eryngii Versatile Peroxidase:  A Site-Directed Mutagenesis, Kinetic, and Crystallographic Study^,

Abstract

The molecular architecture of versatile peroxidase (VP) includes an exposed tryptophan responsible for aromatic substrate oxidation and a putative Mn2+ oxidation site. The crystal structures (solved up to 1.3 A) of wild-type and recombinant Pleurotus eryngii VP, before and after exposure to Mn2+, showed a variable orientation of the Glu36 and Glu40 side chains that, together with Asp175, contribute to Mn2+ coordination. To evaluate the involvement of these residues, site-directed mutagenesis was performed. The E36A, E40A, and D175A mutations caused a 60-85-fold decrease in Mn2+ affinity and a decrease in the Mn2+ oxidation activity. Transient-state kinetic constants showed that reduction of both compounds I and II was affected (80-325-fold lower k2app and 103-104-fold lower k3app, respectively). The single mutants retained partial Mn2+ oxidation activity, and a triple mutation (E36A/E40A/D175A) was required to completely suppress the activity (<1% kcat). The affinity for Mn2+ also decreased ( approximately 25-fold) with the shorter carboxylate side chain in the E36D and E40D variants, which nevertheless retained 30-50% of the maximal activity, whereas similar mutations caused a 50-100-fold decrease in kcat in the case of the Phanerochaete chrysosporium manganese peroxidase (MnP). Additional mutations showed that introduction of a basic residue near Asp175 did not improve Mn2+ oxidation as found for MnP and ruled out an involvement of the C-terminal tail of the protein in low-efficiency oxidation of Mn2+. The structural and kinetic data obtained highlighted significant differences in the Mn2+ oxidation site of the new versatile enzyme compared to P. chrysosporium MnP.

Two Oxidation Sites for Low Redox Potential Substrates

“…By contrast, phenols at millimolar concentrations are oxidized in direct contact with the heme cofactor by VP-I A and VP-II A . According to our studies, phenols gain access to the ␦-heme edge through the main channel, whereas Mn 2ϩ , which is also oxidized directly by the heme, accesses to the internal propionate through a small secondary channel formed by the three acidic residues participating in its coordination (13).…”

“…The catalytic sites responsible for oxidation of Mn 2ϩ and high redox potential substrates have been exhaustively investigated (12,13,15,18). However, not much attention has been paid to clarify how low redox potential substrates are oxidized by this enzyme.…”

“…ϩ ) (VP-I A ). VP-I A catalyzes one-electron substrate oxidation in direct contact with heme, as in Mn 2ϩ oxidation (13), or through the formation of a tryptophanyl radical (resulting in VP-I B ) (14) responsible for the oxidation of high redox potential substrates (e.g. veratryl alcohol) (12).…”

“…The Mn 2ϩ oxidation site is formed by three acidic residues at a small channel giving access to the internal heme propionate. This catalytic site has higher plasticity than in MnP because VP is able to efficiently oxidize Mn 2ϩ in the absence of one of the three acidic residues (13). On the other hand, high redox-potential substrates are oxidized by both VP and LiP at an exposed tryptophanyl radical, followed by long-range electron transfer to heme (pyrrolic ring-C) (12,17).…”

Delivery of a transforming growth factor &beta;-1 plasmid to mesenchymal stem cells via cationized Pleurotus eryngii polysaccharide nanoparticles

“…45 So far, most of the research regarding P. eryngii has focused on its enzymes. [46][47][48] One of the major components of P. eryngii, the polysaccharide of P. eryngii (PEPS), has been studied because of its positive health effects, which include cholesterol-lowering, antioxidant, and anticancer activities; however, little investigation into its application as a gene vector has been carried out. 49 Previous studies have indicated that the polysaccharides isolated from P. eryngii were mostly β-glucans, which exhibited potential prebiotic activities.…”