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

Abstract

Manganese peroxidase (MnP) is an extracellular heme enzyme that catalyzes the peroxidedependent oxidation of Mn II to Mn III . The Mn III is released from the enzyme in complex with oxalate. One heme propionate and the side chains of Glu35, Glu39, and Asp179 were identified as Mn II ligands in the 2.0 Å resolution crystal structure. The new 1.45 Å crystal structure of MnP complexed with Mn II provides a more accurate view of the Mn-binding site. New features include possible partial protonation of Glu39 in the Mn-binding site and glycosylation at Ser336. This is also the first report of MnPinhibitor complex structures. At the Mn-binding site, divalent Cd II exhibits octahedral, hexacoordinate ligation geometry similar to that of Mn II . Cd II also binds to a putative second weak metal-binding site with tetrahedral geometry at the C-terminus of the protein. Unlike that for Mn II and Cd II , coordination of trivalent Sm III at the Mn-binding site is octacoordinate. Sm III was removed from a MnP-Sm III crystal by soaking the crystal in oxalate and then reintroduced into the binding site. Thus, direct comparisons of Sm III -bound and metal-free structures were made using the same crystal. No ternary complex was observed upon incubation with oxalate. The reversible binding of Sm III may be a useful model for the reversible binding of Mn III to the enzyme, which is too unstable to allow similar examination.White-rot basidiomycetous fungi are the only organisms capable of degrading the phenylpropanoid, plant cell wall polymer, lignin (1-4). The lignin-degrading system of these fungi also can oxidize a variety of economically and environmentally important aromatic pollutants (5-9). Under ligninolytic conditions, the best-studied lignin-degrading fungus, Phanerochaete chrysosporium, secretes two families of extracellular heme peroxidases, lignin peroxidase (LiP) and manganese peroxidase (MnP) 1 (2-4, 10), and a hydrogen peroxide-generating system (2, 4, 6).MnP from P. chrysosporium has been studied extensively by a variety of biochemical and biophysical methods (11)(12)(13)(14)(15). The crystal structure illustrates that the heme environment of MnP is similar to that of other plant and fungal peroxidases (16). However, MnP is the only heme peroxidase capable of the one-electron oxidation of Mn II in a typical peroxidase reaction cycle:where MnPI and MnPII are the oxidized intermediates MnP compounds I and II, respectively. The 2.0 Å resolution crystal structure of MnP, determined at room temperature, shows that the substrate, Mn II , binds to one heme propionate and the side chains of three amino acids, Glu35, Glu39, and Asp179, as well as two solvent ligands (16) (Figure 1). This site was confirmed by kinetic and biophysical studies of wild-type MnP and of proteins containing point mutations in the putative binding site. † This research was supported by Grants GM42614 (to T.L.P.) and DK62524 (to M.S.) from the National Institutes of Health and Grants MCB-9808420 from the National Science Foundation and DE-03-9...

Structure and Action Mechanism of Ligninolytic Enzymes

“…The Mn(II) is located in a cation-binding site at the surface of the protein and coordinates to the carboxylate oxygens of Glu35, Glu39, and Asp179, the heme propionate oxygen, and two water oxygens. The site has considerable flexibility to accommodate the binding of a wide variety of metal ions [119,120]. Two heptacoordinate structural calcium ions, one tightly bound on the proximal side and the other bound on the distal side of the heme, are important for thermal stabilization of the active site of the enzyme [119].…”

Recent developments in biodegradation of industrial pollutants by white rot fungi and their enzyme system

“…Mn 2+ performs the role of mediator for MnP. High resolution crystal structure has revealed that MnP catalyzes the peroxide dependent oxidation of Mn 2+ to Mn 3+ and Mn 3+ is released from the enzyme in complex with oxalate or with other chelators (Sundramoorthy et al 2005). Along with pyrophosphate, the various Mn 2+ chelators that enhance the activity are malonate, oxalate, L-tartrate, oxaloacetate, Lmalate, and methylmalonate (Mäkelä et al 2005).…”

Multifrequency pulsed EPR studies of biologically relevant manganese(II) complexes

“…(1a) (1b) Other examples of such mononuclear redox-active enzymes include the manganese peroxidase responsible for lignin degradation by white-rot fungus [10][11][12]; a unique Mndependent form of lipoxygenase [13][14][15][16]; oxalate decarboxylase [17,18]; as well as an extradiol catechol dioxygenase [19][20][21]. …”

“…Many Mn-dependent enzymes take advantage o f the rich redox chemistry available to the metal, accessing the +2, +3, +4, and perhaps even the +5 oxidation states during their turnover. For example, Mn-superoxide dismutase (MnSOD), which detoxifies the cell of the superoxide radical , cycles between the Mn(II) and Mn(III) oxidation states via the ping-pong type mechanism shown below [5][6][7][8][9].(1a) (1b) Other examples of such mononuclear redox-active enzymes include the manganese peroxidase responsible for lignin degradation by white-rot fungus [10][11][12]; a unique Mndependent form of lipoxygenase [13][14][15][16]; oxalate decarboxylase [17,18]; as well as an extradiol catechol dioxygenase [19][20][21]. …”