polyphenol oxidases (ppos) comprise tyrosinases (tYRs) and catechol oxidases (cos), which catalyse the initial reactions in the biosynthesis of melanin. tYRs hydroxylate monophenolic (monophenolase activity) and oxidize diphenolic (diphenolase activity) substrates, whereas cos react only with diphenols. In order to elucidate the biochemical basis for the different reactions in PPOs, cDNA from walnut leaves was synthesized, the target gene encoding the latent walnut tyrosinase (jrPPO1) was cloned, and the enzyme was heterologously expressed in Escherichia coli. Mutations targeting the two activity controller residues (Asn240 and Leu244) as well as the gatekeeper residue (Phe260) were designed to impair monophenolase activity of jrPPO1. For the first time, monophenolase activity of jrPPO1 towards L-tyrosine was blocked in two double mutants (Asn240Lys/Leu244Arg and Asn240Thr/ Leu244Arg) while its diphenolase activity was partially preserved, thereby converting jrPPO1 into a CO. Kinetic data show that recombinant jrPPO1 resembles the natural enzyme, and spectrophotometric investigations proved that the copper content remains unaffected by the mutations. The results presented herein provide experimental evidence that a precisely tuned interplay between the amino acids located around the active center controls the substrate specificity and therewith the mono-versus diphenolase activity in the type-iii copper enzyme jrPPO1.Tyrosinases (TYRs), catechol oxidases (COs) and aurone synthases (AUSs) represent the polyphenol oxidase (PPO) family, which is an umbrella term for copper metalloenzymes 1-3 containing one type-III copper center. TYRs catalyse the hydroxylation of monophenols to o-diphenols (EC 1.14.18.1, monophenolase activity) as well as the subsequent oxidation of o-diphenols to their corresponding o-quinones (EC 1.10.3.1, diphenolase activity) 1,4 , whereas COs catalyse only the latter reaction, unable to react with monophenolic substrates (Fig. 1). AUSs participate in the formation of aurones from chalcone precursors and are involved in plant secondary metabolism 5,6 . Quinones produced by PPOs usually undergo non-enzymatic reactions, polymerize 7 and finally form melanin products 8,9 . PPOs occur in a broad spectrum of organisms, including archaea 10 , bacteria 11 , fungi 2 , plants 8 and animals 12,13 . In plants, they are believed to be involved in defence mechanisms associated with the formation of browning substances, which is triggered by mechanical damage or wounding 14 , while in animals their reaction products are responsible for coloring of skin, hair and eyes 13 .TYR from Juglans regia (walnut, jrPPO1) is expressed in vivo as a latent 66.8 kDa pro-enzyme consisting of three domains 15 : an N-terminal chloroplast transit peptide (~12 kDa) 15 , the catalytically active domain (~39 kDa) and the C-terminal domain (~16 kDa) that shields the entrance to the catalytic pocket and keeps the enzyme in a latent state. In vivo enzymatic activity is triggered by the removal of the C-terminal domain 16 . Alternati...
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.
Over the last millennia, wetlands have been sequestering carbon from the atmosphere via photosynthesis at a higher rate than releasing it and, therefore, have globally accumulated 550 × 10 15 g of carbon, which is equivalent to 73% of the atmospheric carbon pool. The accumulation of organic carbon in wetlands is effectuated by phenolic compounds, which suppress the degradation of soil organic matter by inhibiting the activity of organic-matter-degrading enzymes. The enzymatic removal of phenolic compounds by bacterial tyrosinases has historically been blocked by anoxic conditions in wetland soils, resulting from waterlogging. Bacterial tyrosinases are a subgroup of oxidoreductases that oxidatively remove phenolic compounds, coupled to the reduction of molecular oxygen to water. The biochemical properties of bacterial tyrosinases have been investigated thoroughly in vitro within recent decades, while investigations focused on carbon fluxes in wetlands on a macroscopic level have remained a thriving yet separated research area so far. In the wake of climate change, however, anoxic conditions in wetland soils are threatened by reduced rainfall and prolonged summer drought. This potentially allows tyrosinase enzymes to reduce the concentration of phenolic compounds, which in turn will increase the release of stored carbon back into the atmosphere. To offer compelling evidence for the novel concept that bacterial tyrosinases are among the key enzymes influencing carbon cycling in wetland ecosystems first, bacterial organisms indigenous to wetland ecosystems that harbor a TYR gene within their respective genome ( tyr + ) have been identified, which revealed a phylogenetically diverse community of tyr + bacteria indigenous to wetlands based on genomic sequencing data. Bacterial TYR host organisms covering seven phyla (Acidobacteria, Actinobacteria, Bacteroidetes, Firmicutes, Nitrospirae, Planctomycetes, and Proteobacteria) have been identified within various wetland ecosystems (peatlands, marshes, mangrove forests, bogs, and alkaline soda lakes) which cover a climatic continuum ranging from high arctic to tropic ecosystems. Second, it is demonstrated that (in vitro) bacterial TYR activity is commonly observed at pH values characteristic for wetland ecosystems (ranging from pH 3.5 in peatlands and freshwater swamps to pH 9.0 in soda lakes and freshwater marshes) and toward phenolic compounds naturally present within wetland environments ( p -coumaric acid, gallic acid, protocatechuic acid, p -hydroxybenzoic acid, caffeic acid, catechin, and epicatechin). Third, analyzing the available data confirmed that bacterial host organisms tend to exhibit in vitro growth optima at pH values similar to their respective wetland habitats. Based on these findings, it is concluded that, following increased aeration of previously anoxic wetland soils due to climate change, TYRs are among th...
Polyphenol oxidases (PPOs) are ubiquitously distributed among plants, bacteria, fungi and animals. They catalyze the hydroxylation of monophenols (monophenolase activity) and the oxidation of o-diphenols (diphenolase activity) to o-quinones. PPOs are commonly present as an isoenzyme family. In walnut (Juglans regia), two different genes (jrPPO1 and jrPPO2) encoding PPOs have been identified. In this study, jrPPO2 was, for the first time, heterologously expressed in E. coli and characterized as a tyrosinase (TYR) by substrate scope assays and kinetic investigations, as it accepted tyramine and L-tyrosine as substrates. Moreover, the substrate acceptance and kinetic parameters (kcat and Km values) towards 16 substrates naturally present in walnut were assessed for jrPPO2 (TYR) and its isoenzyme jrPPO1 (TYR). The two isoenzymes prefer different substrates, as jrPPO1 shows a higher activity towards monophenols, whereas jrPPO2 is more active towards o-diphenols. Molecular docking studies performed herein revealed that the amino acid residue in the position of the 1st activity controller (HisB1 + 1; in jrPPO1 Asn240 and jrPPO2 Gly240) is responsible for the different enzymatic activities. Additionally, interchanging the 1st activity controller residue of the two enzymes in two mutants (jrPPO1-Asn240Gly and jrPPO2-Gly240Asn) proved that the amino acid residue located in this position allows plants to selectively target or dismiss substrates naturally present in walnut.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.