Expression, Purification, and Characterization of a Well-Adapted Tyrosinase from Peatlands Identified by Partial Community Analysis

Abstract

In peatlands, bacterial tyrosinases (TYRs) are proposed to act as key regulators of carbon storage by removing phenolic compounds, which inhibit the degradation of organic carbon. Historically, TYR activity has been blocked by anoxia resulting from persistent waterlogging; however, recent events of prolonged summer drought have boosted TYR activity and, consequently, the release of carbon stored in the form of organic compounds from peatlands. Since 30% of the global soil carbon stock is stored in peatlands, a profound understanding of the production and activity of TYRs is essential to assess the impact of carbon dioxide emitted from peatlands on climate change. TYR partial sequences identified by degenerated primers suggest a versatile TYR enzyme community naturally present in peatlands, which is produced by a phylogenetically diverse spectrum of bacteria, including Proteobacteria and Actinobacteria. One full-length sequence of an extracellular TYR (SzTYR) identified from a soda-rich inland salt marsh has been heterologously expressed and purified. SzTYR exhibits a molecular mass of 30 891.8 Da and shows a pH optimum of 9.0. Spectroscopic studies and kinetic investigations characterized SzTYR as a tyrosinase and proved its activity toward monophenols (coumaric acid), diphenols (caffeic acid, protocatechuic acid), and triphenols (gallic acid) naturally present in peatlands.

The Novel Role of Tyrosinase Enzymes in the Storage of Globally Significant Amounts of Carbon in Wetland Ecosystems

“…ZL-24, Sz TYR) was tested in vitro toward phenolic compounds present in its natural environment, and activity toward monophenolic, diphenolic, and triphenolic substrates was detected. 16 Taken together, these data demonstrate that a broad spectrum of phenolic compounds naturally present in wetlands is accepted by TYRs as substrates, which substantiates a possible involvement of TYRs in the “latch mechanism” ( Figure 1 ) as well as the formation of humic substances and SAHLs in intact peatlands. Comparing the in vitro determined kinetic parameters ( k cat and K m values) of phenolic compounds with different functionalities toward bacterial TYRs revealed substantial variations in activity ( k cat ) and affinity ( K m ) values ( Table S4 ).…”

“…Additionally, with increasing sequence information on TYR enzymes present in wetlands, PCR-based methods, such as single-strand conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) provide fast and cheap tools for the community analysis of TYRs present in soil samples, using TYR specific primers. 16 Combined with microbial ecology techniques such as nucleotide analog labeling 144 or stable isotope probing 145 , 146 the effects of environmental stimuli on the subcommunity of TYR-producing bacteria can be assessed effectively. As an additional benefit, DNA-based investigations allow to selectively investigate TYRs, laccases, and peroxidases, due to specific DNA motives present in the nucleotide sequences of each of these enzyme classes.…”

“… 91 , 131 , 132 Accordingly, previous studies have linked Streptomyces species with the storage of organic carbon in peatlands. 16 , 156 …”

“…1 However, depending on the flow rate and chemistry of the groundwater basic pH values have been reported for peatlands (e.g., soda lakes or rich fens), as well. 1 , 16 Similar to peatlands, mangrove soils are often tilted toward mild acidity; 17 , 18 however, the presence of carbonate can cause neutral or basic pH values in these ecosystems. 1 , 19 − 21 The pH values of freshwater marshes generally range from pH 6.0 to pH 9.0, 1 whereas freshwater swamps often show water pH values between pH 6.0 and pH 7.0.…”

“… 64 So far, research investigating the “latch mechanism” in wetlands has been focused on carbon fluxes on a macroscopic scale. 65 , 66 Consequently, little information is available on the specific organisms involved in the “latch mechanism”, 16 the corresponding TYR enzymes (or other enzyme classes), their phylogenetic diversity, their biochemical properties, and their enzymatic characteristics. Here, we propose the key role of TYRs in carbon cycling in wetland ecosystems in the wake of climate change.…”