Identification and Characterization of a Multifunctional Dye Peroxidase from a Lignin-Reactive Bacterium

Abstract

Plant biomass represents a renewable feedstock that has not yet been fully tapped because of the difficulty in accessing the carbon in its structural biopolymers. Lignin is an especially challenging substrate, but select microbes have evolved complex systems of enzymes for its breakdown through a radical-mediated oxidation process. Fungal systems are well-characterized for their ability to depolymerize lignin, but the ability of bacteria to react with this substrate remains elusive. We have therefore focused on elucidating strategies used by lignin-reactive soil bacteria and describing their oxidative enzyme systems. We now report the identification and characterization of an unusual C-type dye-decolorizing peroxidase from Amycolatopsis sp. 75iv2 (DyP2), which belongs to a family of heme peroxidases reported to be involved in bacterial lignin degradation. Biochemical studies indicate that DyP2 has novel function for this family, with versatile and high activity both as a peroxidase and Mn peroxidase (k(cat)/K(M) ≈ 10(5)-10(6) M(-1) s(-1)). It also has a Mn-dependent oxidase mode of action that expands its substrate scope. Crystallographic studies of DyP2 at 2.25 Å resolution show the existence of a Mn binding pocket and support its key role in catalysis.

Structure of Thermobifida fusca DyP-type peroxidase and activity towards Kraft lignin and lignin model compounds

“…The structure of TfuDyP was solved at 1.8 Å, and shows a ferredoxin-type fold that is similar to structures of other DyPs from bacteria [7,10] and fungi [3,29]. Similar to other DyPs [3], TfuDyP contains Asp-203 and Arg-315 on the distal face of the heme cofactor, which are both important for catalytic activity, and the catalytic Asp residue is found in a GxxDG sequence motif, as noted in other DyPs [3].…”

“…Dimeric -aryl ether lignin model compounds have been frequently used to study the action of fungal lignin-oxidising enzymes [23], and bacterial DyPs [9,10] and laccases [24]. TfuDyP was incubated with guaiacylglycerol--guaiacyl ether (1) at 1.6 mg/ml concentration in 100 mM sodium acetate buffer pH 6.0 in the presence of 1 mM hydrogen peroxide.…”

“…Reactive Black 5, a dye substrate with a high redox potential [10]. D-type DyPs, found in fungi such as Bjerkandera adusta [11] and Auricularia auricular-judae [12], show high activity towards phenolic substrates and ABTS, and also show activity towards high-redox potential dyes.…”

Enzymatic Degradation of Lignin in Soil: A Review

“…The DyPs are phylogenetically distinct [97] from other peroxidases as they possess an α + β ferredoxin-like fold [98]. However, their oxidation mechanism is similar to VP and MnP [99]. They are widely found in microorganisms [100] and classified into four types: A, B, C, and D. Bacterial enzymes are predominantly found in type A to C, while type D is mostly clustered to fungal DyPs.…”

Identification of Manganese Superoxide Dismutase from Sphingobacterium sp. T2 as a Novel Bacterial Enzyme for Lignin Oxidation

“…75iv2 that shows higher activity for Mn(II) oxidation. 6 Bacterial laccase enzymes have also been identified in Streptomyces coelicolor A3(2), S. viridosporus T7A, and Amycolatopsis sp. 75iv2, which catalyse C  oxidation of a lignin model compound, and whose genetic knockout significantly reduces the ability of the host to metabolise lignin.…”