Identification and characterization of DyP peroxidases from Rhodococcus jostii RHA1

• Main Author: Roberts, Joseph N.
• Language: English
• Published: University of British Columbia 2011
• Online Access: http://hdl.handle.net/2429/31830

The lignin-degrading soil bacterium Rhodococcus jostii RHA1 contains two genes encoding DyP-type peroxidases. Based on phylogenetic studies, the enzymes were classified as DypA, which carries a TAT sequence, and DypB, which carries a C-terminal sequence predicted to target it to an icosahedral protein nanocompartment. Consistent with other DyPs, DypA showed 6-fold greater apparent specificity for the anthraquinone dye Reactive Blue 4 (kcat/Km = 12,800 ± 600 M⁻¹1s⁻¹) than either ABTS or pyrogallol. By contrast, DypB showed greatest apparent specificity for ABTS (kcat/Km = 2000 ± 100 M⁻¹s⁻¹) and also oxidized Mn(II) (kcat/Km = 25.1 ± 0.1 M⁻¹s⁻¹). Herein the x-ray crystal structure of DypB is presented to 1.4 Å resolution, revealing a hexa-coordinated heme molecule and an additional Asn residue in the active site which is unique to DypB. Analysis of the DypB structural surface provides additional contrast to the structure of plant peroxidases, and identifies a potential substrate-binding pocket distal to the heme center. Assay of gene deletion mutants using a colorimetric lignin degradation assay reveals that a ΔdypB mutant shows greatly reduced lignin degradation activity, consistent with a role in lignin breakdown. Recombinant DypB protein also shows activity in the colorimetric assay, which is increased 5-fold in the presence of Mn(II). Overall, the different reactivities of the RHA1 DyPs with reducing substrates and Mn(II) enhanced ligninolytic activity of DypB have important implications for biotechnological applications.