Non-enzymatic degradation of carbon nanotubes in the presence of bacterial enzymes

The enzymatic degradation of carbon nanotubes (CNTs) by several enzymes has been reported. However, because organisms that possess these enzymes have limited habitats and distribution areas, it is unclear whether CNTs can be degraded in the general environment. The investigation of CNTs degradation by enzymes derived from bacteria, which inhabit a wide range of environments and have diverse metabolic systems, is inevitable for predicting the environmental fate of CNTs. In this study, the degradation of oxidized (carboxylated) single-walled CNTs (O-SWCNTs) by mt2DyP, a dye-decolorizing peroxidase of Pseudomonas putida mt-2, a common soil bacterium, was investigated. Suspensions of O-SWCNTs gradually became transparent and their optical absorbance decreased during 30 d of incubation in the presence of mt2DyP produced by a recombinant Brevibacillus choshinensis strain and its substrate, H2O2. The degradation was enhanced by higher H2O2 concentrations. The measurement of Raman spectra revealed the complete degradation of O-SWCNTs after 30 d of incubation with 100 mM H2O2. However, surprisingly, this heme enzyme was inactivated within 60 min of the incubation with O-SWCNTs, which suggested that the degradation of O-SWCNTs was not catalyzed by the enzyme. The inactivation of mt2DyP was accompanied by the release of iron, which suggested that the degradation of the O-SWCNTs was owing to the Fenton reaction caused by the iron released from mt2DyP and the supplied H2O2. A chelating agent, diethylenetriaminepentaacetic acid, significantly inhibited the O-SWCNTs degradation, proving the degradation by the Fenton reaction. These phenomena were also observed with another heme enzyme, Cytochrome P450. These results are important for predicting the fate of CNTs in a wide range of environments, as heme enzymes are secreted by many bacteria in the environment. This study also shows that the effect of the Fenton reaction should be considered to validate the degradation of CNTs by heme enzymes.

Biotransformation of Lignin by an Artificial Heme Enzyme Designed in Myoglobin With a Covalently Linked Heme Group

The conversion of Kraft lignin in plant biomass into renewable chemicals, aiming at harvesting aromatic compounds, is a challenge process in biorefinery. Comparing to the traditional chemical methods, enzymatic catalysis provides a gentle way for the degradation of lignin. Alternative to natural enzymes, artificial enzymes have been received much attention for potential applications. We herein achieved the biodegradation of Kraft lignin using an artificial peroxidase rationally designed in myoglobin (Mb), F43Y/T67R Mb, with a covalently linked heme cofactor. The artificial enzyme of F43Y/T67R Mb has improved catalytic efficiencies at mild acidic pH for phenolic and aromatic amine substrates, including Kraft lignin and the model lignin dimer guaiacylglycerol-β-guaiacyl ether (GGE). We proposed a possible catalytic mechanism for the biotransformation of lignin catalyzed by the enzyme, based on the results of kinetic UV-Vis studies and UPLC-ESI-MS analysis, as well as molecular modeling studies. With the advantages of F43Y/T67R Mb, such as the high-yield by overexpression in E. coli cells and the enhanced protein stability, this study suggests that the artificial enzyme has potential applications in the biodegradation of lignin to provide sustainable bioresource.

Indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors in clinical trials for cancer immunotherapy

Indoleamine 2,3-dioxygenase 1 (IDO1) is a heme enzyme that catalyzes the oxidation of L-tryptophan. Functionally, IDO1 has played a pivotal role in cancer immune escape via catalyzing the initial step of the kynurenine pathway, and overexpression of IDO1 is also associated with poor prognosis in various cancers. Currently, several small-molecule candidates and peptide vaccines are currently being assessed in clinical trials. Furthermore, the “proteolysis targeting chimera” (PROTAC) technology has also been successfully used in the development of IDO1 degraders, providing novel therapeutics for cancers. Herein, we review the biological functions of IDO1, structural biology and also extensively summarize medicinal chemistry strategies for the development of IDO1 inhibitors in clinical trials. The emerging PROTAC-based IDO1 degraders are also highlighted. This review may provide a comprehensive and updated overview on IDO1 inhibitors and their therapeutic potentials.

Unexpected photosensitivity of the well-characterized heme enzyme chlorite dismutase

Chlorite dismutase is a heme enzyme that catalyzes the conversion of the toxic compound ClO2− (chlorite) to innocuous Cl− and O2. The reaction is a very rare case of enzymatic O–O bond formation, which has sparked the interest to elucidate the reaction mechanism using pre-steady-state kinetics. During stopped-flow experiments, spectroscopic and structural changes of the enzyme were observed in the absence of a substrate in the time range from milliseconds to minutes. These effects are a consequence of illumination with UV–visible light during the stopped-flow experiment. The changes in the UV–visible spectrum in the initial 200 s of the reaction indicate a possible involvement of a ferric superoxide/ferrous oxo or ferric hydroxide intermediate during the photochemical inactivation. Observed EPR spectral changes after 30 min reaction time indicate the loss of the heme and release of iron during the process. During prolonged illumination, the oligomeric state of the enzyme changes from homo-pentameric to monomeric with subsequent protein precipitation. Understanding the effects of UV–visible light illumination induced changes of chlorite dismutase will help us to understand the nature and mechanism of photosensitivity of heme enzymes in general. Furthermore, previously reported stopped-flow data of chlorite dismutase and potentially other heme enzymes will need to be re-evaluated in the context of the photosensitivity. Graphic abstract Illumination of recombinantly expressed Azospira oryzae Chlorite dismutase (AoCld) with a high-intensity light source, common in stopped-flow equipment, results in disruption of the bond between FeIII and the axial histidine. This leads to the enzyme losing its heme cofactor and changing its oligomeric state as shown by spectroscopic changes and loss of activity.

Use of an Artificial Miniaturized Enzyme in Hydrogen Peroxide Detection by Chemiluminescence

Advanced oxidation processes represent a viable alternative in water reclamation for potable reuse. Sensing methods of hydrogen peroxide are, therefore, needed to test both process progress and final quality of the produced water. Several bio-based assays have been developed so far, mainly relying on peroxidase enzymes, which have the advantage of being fast, efficient, reusable, and environmentally safe. However, their production/purification and, most of all, batch-to-batch consistency may inherently prevent their standardization. Here, we provide evidence that a synthetic de novo miniaturized designed heme-enzyme, namely Mimochrome VI*a, can be proficiently used in hydrogen peroxide assays. Furthermore, a fast and automated assay has been developed by using a lab-bench microplate reader. Under the best working conditions, the assay showed a linear response in the 10.0–120 μM range, together with a second linearity range between 120 and 500 μM for higher hydrogen peroxide concentrations. The detection limit was 4.6 μM and quantitation limits for the two datasets were 15.5 and 186 μM, respectively. In perspective, Mimochrome VI*a could be used as an active biological sensing unit in different sensor configurations.

Murburn concept explains the acutely lethal effect of cyanide

Cyanide (CN) toxicity is traditionally understood to result from its binding of hemeFe centers, thereby disrupting mitochondrial cytochrome oxidase function and oxygen utilization by other globin proteins. Recently, a diffusible reactive oxygen species (DROS) mediated reaction mechanism called murburn concept was proposed to explain mitochondrial ATP-synthesis and heat generation. Per this purview, it was theorized that CN ion-radical equilibrium dissipates the catalytically vital DROS into futile cycles, producing water. In the current study, a comparative quantitative assessment of the above two explanations is made for: (i) lethal dosage or concentrations of CN, (ii) thermodynamics and kinetics of the binding/reaction, and (iii) correlation of CN with the binding data and reaction chemistry of H2S/CO. The quantitative findings suggest that the hemeFe binding-based toxicity explanation is untenable. CN also inhibited the experimental in vitro DROS-mediated coupling of inorganic phosphate with ADP. Further, pH-dependent inhibition profiles of heme enzyme catalyzed oxidation of a phenolic (wherein an -OH group reacts with DROS to form water, quite akin to the murburn model of ATP synthesis) indicated that- (i) multiple competitive reactions in milieu controlled outcomes and (ii) low concentrations of CN cannot disrupt activity via a coordination (binding) of cyanide at the distal hemeFe. Therefore, the μM-level IC50 and the acutely lethal effect of CN on cellular respiration could be explained by the deleterious interaction of CN ion-radical equilibrium with DROS in matrix, disrupting mitochondrial ATP synthesis. This work supports the murburn explanation for cellular respiration.