The Role of Glu39 in Mn^II Binding and Oxidation by Manganese Peroxidase from Phanerochaete chrysoporium

Abstract

Manganese peroxidase (MnP) is a heme-containing enzyme produced by white-rot fungi and is part of the extracellular lignin degrading system in these organisms. MnP is unique among Mn binding enzymes in its ability to bind and oxidize Mn II and efficiently release Mn III . Initial site-directed mutagenesis studies identified the residues E35, E39, and D179 as the Mn binding ligands. However, an E39D variant was recently reported to display wild-type Mn binding and rate of oxidation, calling into question the role of E39 as an Mn ligand. To investigate this hypothesis, we performed computer modeling studies which indicated metal-ligand bond distances in the E39D variant and in an E35D-E39D-D179E triple variant which might allow Mn binding and oxidation. To test the model, we reconstructed the E35D and E39D variants used in the previous study, as well as an E39A single variant and the E35D-E39D-D179E triple variant of MnP isozyme 1 from Phanerochaete chrysosporium. We find that all of the variant proteins are impaired for Mn II binding (K m increases 20-30-fold) and Mn II oxidation (k cat decreases 50-400-fold) in both the steady state and the transient state. In particular, mutation of the E39 residue in MnP decreases both Mn binding and oxidation. The catalytic efficiency of the E39A variants decreased ∼10 4 -fold, while that of the E39D variant decreased ∼10 3 -fold. Contrary to initial modeling results, the triple variant performed only as well as any of the single Mn ligand variants. Interestingly, the catalytic efficiency of the triple variant decreased only 10 4 -fold, which is ∼10 2 -fold better than that reported for the E35Q-D179N double variant. These combined studies indicate that precise geometry of the Mn ligands within the Mn binding site of MnP is essential for the efficient binding, oxidation, and release of Mn by this enzyme. The results clearly indicate that E39 is a Mn ligand and that mutation of this ligand decreases both Mn binding and the rate of Mn oxidation.

Extracellular oxidative systems of the lignin-degrading Basidiomycete Phanerochaete chrysosporium

“…Site specific mutagenesis has implicated Asp179, Glu35, and Glu39 in Mn binding (Kusters-van Someren et al, 1995;Whitwam et al, 1997;Sollewijn Gelpke et al, 1999;Youngs et al, 2001).…”

High-Resolution Crystal Structure of Manganese Peroxidase: Substrate and Inhibitor Complexes,

“…Alteration of the proposed ligands in the Mn-binding site significantly affects Mn binding and oxidation (17)(18)(19)(20)(21)(22), and crystals of both the single variant, D179N, and the double variant, E35Q/D179N, lack electron density at the proposed Mn-binding site (23), suggesting that Mn II is not bound. MnP is unique among enzymes using manganese as a redox cofactor.…”

Ultrahigh (0.93Å) resolution structure of manganese peroxidase from Phanerochaete chrysosporium: Implications for the catalytic mechanism

“…Other ligands—the heme propionate and Asp179—do not move from their original positions whether a metal ion is bound or not, strongly suggesting that precise geometry is required for efficient Mn II -binding and oxidation. This is confirmed by steady-state and transient-state kinetic analyses of a MnP E39D single mutant and an E35D-E39D-D179E triple mutant [23]. The single and triple mutant variants exhibit 20- and 40-fold increase in K m , and 10 3 and 10 4 decrease in catalytic efficiency, respectively.…”