This work provides spectroscopic, catalytic, and stability fingerprints of two new bacterial dye-decolorizing peroxidases (DyPs) from Bacillus subtilis (BsDyP) and Pseudomonas putida MET94 (PpDyP). DyPs are a family of microbial heme-containing peroxidases with wide substrate specificity, including high redox potential aromatic compounds such as synthetic dyes or phenolic and nonphenolic lignin units. The genes encoding BsDyP and PpDyP, belonging to subfamilies A and B, respectively, were cloned and heterologously expressed in Escherichia coli. The recombinant PpDyP is a 120-kDa homotetramer while BsDyP enzyme consists of a single 48-kDa monomer. The optimal pH of both enzymes is in the acidic range (pH 4-5). BsDyP has a bell-shape profile with optimum between 20 and 30 °C whereas PpDyP shows a peculiar flat and broad (10-30 °C) temperature profile. Anthraquinonic or azo dyes, phenolics, methoxylated aromatics, and also manganese and ferrous ions are substrates used by the enzymes. In general, PpDyP exhibits higher activities and accepts a wider scope of substrates than BsDyP; the spectroscopic data suggest distinct heme microenvironments in the two enzymes that might account for the distinctive catalytic behavior. However, the Bs enzyme with activity lasting for up to 53 h at 40 °C is more stable towards temperature or chemical denaturation than the PpDyP. The results of this work will guide future optimization of the biocatalytis towards their utilization in the fields of environmental or industrial biotechnology.

Dye-decolorizing peroxidases (DyPs) are a family of microbial heme-containing peroxidases that show important properties for lignocellulose biorefineries due to their ability to oxidize lignin-related compounds. Directed evolution was used to improve the efficiency of the bacterial PpDyP from Pseudomonas putida MET94 for phenolic compounds. Three rounds of random mutagenesis by errorprone PCR of the ppDyP gene followed by high-throughput screening allow identification of the 6E10 variant showing a 100-fold enhanced catalytic efficiency (k cat /K m ) for 2,6dimethoxyphenol (DMP), similar to that exhibited by fungal lignin peroxidases (∼10 5 M −1 s −1 ). The evolved variant showed additional improved efficiency for a number of syringyl-type phenolics, guaiacol, aromatic amines, Kraft lignin, and the lignin phenolic model dimer guaiacylglycerol-β-guaiacyl ether. Importantly, variant 6E10 displayed optimal pH at 8.5, an upshift of 4 units in comparison to the wild type, showed resistance to hydrogen peroxide inactivation, and was produced at 2-fold higher yields. The acquired mutations in the course of the evolution affected three amino acid residues (E188K, A142V, and H125Y) situated at the surface of the enzyme, in the second shell of the heme cavity. Biochemical analysis of hit variants from the laboratory evolution, and single variants constructed using site-directed mutagenesis, unveiled the critical role of acquired mutations from the catalytic, stability, and structural viewpoints. We show that epistasis between A142V and E188K mutations is crucial to determine the substrate specificity of 6E10. Evidence suggests that ABTS and DMP oxidation occurs at the heme access channel. Details of the catalytic cycle of 6E10 were elucidated through transient kinetics, providing evidence for the formation of a reversible enzyme−hydrogen peroxide complex (Compound 0) barely detected in the majority of heme peroxidases studied to date.

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The ubiquitous members of the multicopper oxidase family of enzymes oxidize a range of aromatic substrates such as polyphenols, methoxy-substituted phenols, amines and inorganic compounds, concomitantly with the reduction of molecular dioxygen to water. This family of enzymes can be broadly divided into two functional classes: metalloxidases and laccases. Several prokaryotic metalloxidases have been described in the last decade showing a robust activity towards metals, such as Cu(I), Fe(II) or Mn(II) and have been implicated in the metal metabolism of the corresponding microorganisms. Many laccases, with a superior efficiency for oxidation of organic compounds when compared with metals, have also been identified and characterized from prokaryotes, playing roles that more closely conform to those of intermediary metabolism. This review aims to present an update of current knowledge on prokaryotic multicopper oxidases, with a special emphasis on laccases, anticipating their enormous potential for industrial and environmental applications.

Multicopper oxidases oxidize a vast range of aromatic substrates coupled to the reduction of molecular oxygen to water. A vast broad spectrum of applications reflects their high biotechnological importance. The crystal structure of McoA from the hyperthermophilic bacteria Aquifex aeolicus has the most tightly compact and hydrophobic core among its prokaryotic counterparts. A 29-residue long loop enriched in glycines and methionines (Met-loop) close to the active T1 Cu center is not detected in the electron density maps. Accurate prediction of loop structures remains challenging, especially for long segments with sizable conformational space. Therefore, a combination of Rosetta and molecular dynamics simulations with ensemble-based small-angle X-ray scattering analysis was used to probe the conformational landscape of the Met-loop. The results indicate a highly flexible omega-loop, which is nevertheless not random but preferentially follows open-to-close transitions, exposing or occluding the T1 Cu site. Loop-truncated variants maintain wild-type stability and consistently lower and higher catalytic efficiencies (k cat /K m ) for organic and metal substrates, respectively. Our results suggest that the loop transient dynamic equilibrium can exert important switch-like regulatory function, defining a role for Met-rich motifs as dynamic gate-gappers. This work provides insights into the dynamics of Met-rich loops essential to understand the molecular determinants of substrate promiscuity and catalytic rates within multicopper oxidases. We anticipate that engineering the Met-loop structural dynamics will unleash important changes in enzyme function and specificity with impact on their applications.