Heavy Metals, Halogenated Hydrocarbons, Phthalates, Glyphosate, Cordycepin, Alcohol, Drugs, and Herbs, Assessed for Liver Injury and Mechanistic Steps.

Aluminum, arsenic, cadmium, chromium, cobalt, copper, iron, lead, mercury, nickel, thallium, titanium, zinc, carbon tetrachloride, phthalates, glyphosate, alcohol, drugs, and herbs are under discussion having the potential to injure the human liver, but allocation of the injury to the hepatotoxicant as exact cause is difficult for physicians and requires basic clinical knowledge of toxicology details. Liver injury occurs at a variable extent depending on the dose, mostly reproducible in animal models that allow studies on molecular steps leading to the hepatocellular injury. These exogenous hepatotoxins may cause an overproduction of reactive oxidative species (ROS), which are generated during microsomal or mitochondrial oxidative stress from incomplete oxygen split and trigger the injury if protective antioxidant capacities are reduced. Primary subcelluar target organelles involved are liver mitochondria through lipid peroxidation of membrane structures and the action of free radicals such as singlet radical 1O2, superoxide radical HO•2, hydrogen peroxide H2O2, hydroxyl radical HO•, alkoxyl radical RO•, and peroxyl radical ROO•. They attempt covalent binding to macromolecular structural proteins. As opposed to inorganic chemicals, liver injury due to chemicals with an organic structure proceedes via the hepatic microsomal cytochrome P450 with its different isoforms. In sum, many exogenous chemicals may have the potential of liver injury triggerd by overproduced ROS leading primarily to impairment of mitochondial functions in the course of structural mitochondial membrane dearrangement. As clinical data were often incomplete, future clinical prototols should focus on meeting liver injury criteria, exclusion of alternative causes, a robust causality evaluation management, and obtaining liver histology if clinically indicated and of benefit for the patient.

Substrate Profiling of Anion Methyltransferases for Promiscuous Synthesis of S-Adenosylmethionine Analogs from Haloalkanes.

Biocatalytic alkylation reactions can be performed with high chemo-, regio- and stereoselectivity using S-adenosyl-l-methionine (SAM)-dependent methyltransferases (MTs) and SAM analogs. Currently, however, this methodology is limited in application due to the rather laborious protocols to access SAM analogs. It has recently been shown that halide methyltransferases (HMTs) enable synthesis and recycling of SAM analogs with readily available haloalkanes as starting material. Here we expand this work by using substrate profiling of the anion MT enzyme family to explore promiscuous SAM analog synthesis. Our study shows that anion MTs are in general very promiscuous with respect to the alkyl chain as well as the halide leaving group. Substrate profiling further suggests that promiscuous anion MTs cluster in sequence space. Next to iodoalkanes, cheaper, less toxic, and more available bromoalkanes have been converted and several haloalkanes bearing short alkyl groups, alkyl rings, and functional groups such as alkene, alkyne and aromatic moieties are accepted as substrates. Further, we applied the SAM analogs as electrophiles in enzyme-catalyzed regioselective pyrazole allylation with 3-bromopropene as starting material.

Biocatalytic Cross-Coupling of Aryl Halides with a Genetically Engineered Photosensitizer Artificial Dehalogenase.

Devising artificial photoenzymes for abiological bond-forming reactions is of high synthetic value but also a tremendous challenge. Disclosed herein is the first photobiocatalytic cross-coupling of aryl halides enabled by a designer artificial dehalogenase, which features a genetically encoded benzophenone chromophore and site-specifically modified synthetic NiII(bpy) cofactor with tunable proximity to streamline the dual catalysis. Transient absorption studies suggest the likelihood of energy transfer activation in the elementary organometallic event. This design strategy is viable to significantly expand the catalytic repertoire of artificial photoenzymes for useful organic transformations.

A Review: Halogenated Compounds from Marine Fungi.

Marine fungi produce many halogenated metabolites with a variety of structures, from acyclic entities with a simple linear chain to multifaceted polycyclic molecules. Over the past few decades, their pharmaceutical and medical application have been explored and still the door is kept open due to the need of new drugs from relatively underexplored sources. Biological properties of halogenated compounds such as anticancer, antiviral, antibacterial, anti-inflammatory, antifungal, antifouling, and insecticidal activity have been investigated. This review describes the chemical structures and biological activities of 217 halogenated compounds derived mainly from Penicillium and Aspergillus marine fungal strains reported from 1994 to 2019.

Polyhalogenated Compounds (Halogenated Natural Products and POPs) in Sardine (Sardinops sagax) from the South Atlantic and Indian Oceans.

Halogenated natural products (HNPs) and persistent organic pollutants (POPs) were quantified in South African sardines (Sardinops sagax) from one site in the South Atlantic Ocean and one in the Indian Ocean. At both sites, HNPs [2,3,3',4,4',5,5'-heptachloro-1'-methyl-1,2'-bipyrrole (Q1), mixed halogenated compound 1 (MHC-1), 2,4,6-tribromoanisole (2,4,6-TBA), 2'-MeO-BDE 68 (BC-2), and 6-MeO-BDE 47 (BC-3)] were 1 order of magnitude higher concentrated than anthropogenic POPs [mainly polychlorinated biphenyls (PCBs) and dichlorodiphenyltrichloroethane (DDT), ∼3 ng/g lipids]. MHC-1 and Q1 were the major HNPs in the samples from both sites, contributing with up to 49 and 52 ng/g lipids, respectively. The same 1,1-dichloro-2,2-bis(4-chlorophenyl)ethane (p,p'-DDE)/PCB ratio suggested that the major POPs were evenly distributed at both sites. Different ratios of Q1/MHC-1 in the samples from the Indian (∼2:1) and South Atlantic (∼1:1) Oceans indicated that the occurrence of HNPs in seafood is difficult to predict and should be investigated more in detail. The PCB levels in sardines were found to pose no risk to human consumers, whereas HNPs could not be evaluated because of the lack of toxicological data.

A competition between hydrophobic and electrostatic interactions in protein-ligand systems. Binding of heterogeneously halogenated benzotriazoles by the catalytic subunit of human protein kinase CK2.

A series of chlorine-substituted benzotriazole derivatives, representing all possible substitution patterns of halogen atoms attached to the benzotriazole benzene ring, were synthetized as potential inhibitors of human protein kinase CK2. Basic ADME parameters for the free solutes (hydrophobicity, electronic properties) together with their binding affinity to the catalytic subunit of protein kinase CK2 were determined with reverse-phase HPLC, spectrophotometric titration, and Thermal Shift Assay Method, respectively. The analysis of position-dependent thermodynamic contribution of a chlorine atom attached to the benzotriazole ring confirmed the previous observation for brominated benzotriazoles, in which substitution at positions 5 and 6 with bromine was found crucial for ligand binding. In all tested halogenated benzotriazoles the replacement of Br with Cl decreases the hydrophobicity, while the electronic properties remain virtually unaffected. Supramolecular architecture identified in the just resolved crystal structures of three of the four possible dichloro-benzotriazoles shows how substitution distant from the triazole ring affects the pattern of intermolecular interactions. Summarizing, the benzotriazole benzene ring substitution pattern has been identified as the main driver of ligand binding, predominating the non-specific hydrophobic effect.

Transcriptional regulation of organohalide pollutant utilisation in bacteria.

Organohalides are organic molecules formed biotically and abiotically, both naturally and through industrial production. They are usually toxic and represent a health risk for living organisms, including humans. Bacteria capable of degrading organohalides for growth express dehalogenase genes encoding enzymes that cleave carbon-halogen bonds. Such bacteria are of potential high interest for bioremediation of contaminated sites. Dehalogenase genes are often part of gene clusters that may include regulators, accessory genes and genes for transporters and other enzymes of organohalide degradation pathways. Organohalides and their degradation products affect the activity of regulatory factors, and extensive genome-wide modulation of gene expression helps dehalogenating bacteria to cope with stresses associated with dehalogenation, such as intracellular increase of halides, dehalogenase-dependent acid production, organohalide toxicity and misrouting and bottlenecks in metabolic fluxes. This review focuses on transcriptional regulation of gene clusters for dehalogenation in bacteria, as studied in laboratory experiments and in situ. The diversity in gene content, organization and regulation of such gene clusters is highlighted for representative organohalide-degrading bacteria. Selected examples illustrate a key, overlooked role of regulatory processes, often strain-specific, for efficient dehalogenation and productive growth in presence of organohalides.

Reduction of halocarbons to hydrocarbons by NADH models and NADH.

The reduction of halocarbons by NADH models and NADH under ambient conditions is reported as a new type of reactivity pointing towards a hitherto unknown disruptive pathway for NADH/NADPH-dependent processes. The reaction was studied with the omnipresent pesticide DDT, the inhalation anesthetic halothane, and several simple halocarbons. The halide-hydride exchange represents a biochemical equivalent for the reduction of halocarbons by traditional synthetic reagents like silanes (R3Si-H) and stannanes (R3Sn-H). High precision thermochemical calculations (CBS-QB3) reveal the carbon-hydrogen bond dissociation energy of NADH (70.8 kcal·mol-1) to be lower than that of stannane (SnH4: 78.1 kcal·mol-1), approaching that of the elusive plumbane (PbH4: 68.9 kcal·mol-1). The ready synthetic accessibility of NADH models, their low carbon-hydrogen bond dissociation energy, and their dehalogenation activity in the presence of air and moisture recommend these compounds as substitutes for the air-sensitive or toxic metal hydrides currently employed in synthesis.

Stabilization of microtubules by cevipabulin.

We report the utility of cevipabulin as a stabilizing agent for microtubules. Cevipabulin-stabilized microtubules were more flexible compared to the microtubules stabilized by paclitaxel, the most commonly used microtubule stabilizing agent. Similar to the paclitaxel-stabilized microtubules, cevipabulin-stabilized microtubules were driven by kinesins in an in vitro gliding assay. The velocity of cevipabulin-stabilized microtubules was significantly higher than that of paclitaxel-stabilized microtubules. These findings will enrich the variety of microtubules with difference in mechanical and dynamic properties and widen their applications in nanotechnology.

Regulation of organohalide respiration.

Organohalide respiration (OHR) is an anaerobic metabolism by which bacteria conserve energy with the use of halogenated compounds as terminal electron acceptors. Genes involved in OHR are organized in reductive dehalogenase (rdh) gene clusters and can be found in relatively high copy numbers in the genomes of organohalide-respiring bacteria (OHRB). The minimal rdh gene set is composed by rdhA and rdhB, encoding the catalytic enzyme involved in reductive dehalogenation and its putative membrane anchor, respectively. In this chapter, we present the major findings concerning the regulatory strategies developed by OHRB to control the expression of the rdh gene clusters. The first section focuses on the description of regulation patterns obtained from targeted transcriptional analyses, and from transcriptomic and proteomic studies, while the second section offers a detailed overview of the biochemically characterized OHR regulatory proteins identified so far. Depending on OHRB, transcriptional regulators belonging to three different protein families are found in the direct vicinity of rdh gene clusters, suggesting that they activate the transcription of their cognate gene cluster. In this chapter, strong emphasis was laid on the family of CRP/FNR-type RdhK regulators which belong to members of the genera Dehalobacter and Desulfitobacterium. Whereas only chlorophenols have been identified as effectors for RdhK regulators, the protein sequence diversity suggests a broader organohalide spectrum. Thus, effector identification of new regulators offers a promising alternative to elucidate the substrates of yet uncharacterized reductive dehalogenases. Future work investigating the possible cross-talk between OHR regulators and their possible use as biosensors is discussed.

Crystal structure of the cold-adapted haloalkane dehalogenase DpcA from Psychrobacter cryohalolentis K5.

Haloalkane dehalogenases (HLDs) convert halogenated aliphatic pollutants to less toxic compounds by a hydrolytic mechanism. Owing to their broad substrate specificity and high enantioselectivity, haloalkane dehalogenases can function as biosensors to detect toxic compounds in the environment or can be used for the production of optically pure compounds. Here, the structural analysis of the haloalkane dehalogenase DpcA isolated from the psychrophilic bacterium Psychrobacter cryohalolentis K5 is presented at the atomic resolution of 1.05 Å. This enzyme exhibits a low temperature optimum, making it attractive for environmental applications such as biosensing at the subsurface environment, where the temperature typically does not exceed 25°C. The structure revealed that DpcA possesses the shortest access tunnel and one of the most widely open main tunnels among structural homologs of the HLD-I subfamily. Comparative analysis revealed major differences in the region of the α4 helix of the cap domain, which is one of the key determinants of the anatomy of the tunnels. The crystal structure of DpcA will contribute to better understanding of the structure-function relationships of cold-adapted enzymes.

Microbial transformation of chiral organohalides: Distribution, microorganisms and mechanisms.

Chiral organohalides including dichlorodiphenyltrichloroethane (DDT), Hexabromocyclododecane (HBCD) and polychlorinated biphenyls (PCBs) raise a significant concern in the environmental occurrence, fate and ecotoxicology due to their enantioselective biological effects. This review provides a state-of-the-art overview on enantioselective microbial transformation of the chiral organohalides. We firstly summarized worldwide field assessments of chiral organohalides in a variety of environmental matrices, which suggested the pivotal role of microorganisms in enantioselective transformation of chiral organohalides. Then, laboratory studies provided experimental evidences to further link enantioselective attenuation of chiral organohalides to specific functional microorganisms and enzymes, revealing mechanistic insights into the enantioselective microbial transformation processes. Particularly, a few amino acid residues in the functional enzymes could play a key role in mediating the enantioselectivity at the molecular level. Finally, major challenges and further developments toward an in-depth understanding of the enantioselective microbial transformation of chiral organohalides are identified and discussed.

Halogenated and Nonhalogenated Metabolites from the Marine-Alga-Endophytic Fungus Trichoderma asperellum cf44-2.

One new bisabolane sesquiterpene, bisabolan-1,10,11-triol (1), one new norbisabolane sesquiterpene, 12-nor-11-acetoxybisabolen-3,6,7-triol (2), two new naturally occurring monoterpenes, (7S)- and (7R)-1-hydroxy-3-p-menthen-9-oic acids (3 and 4), one new naturally occurring trichodenone, dechlorotrichodenone C (5), one new chlorine-containing trichodenone, 3-hydroxytrichodenone C (6), one new diketopiperazine, methylcordysinin A (7), and one new naturally occurring oxazole derivative, 4-oxazolepropanoic acid (8), were isolated from the culture of a marine brown alga-endophytic strain (cf44-2) of Trichoderma asperellum. Their structures and relative configurations were determined by extensive 1D/2D NMR and mass spectrometric data, and the absolute configurations of 3⁻6 were assigned by analysis of the ECD spectra aided by quantum chemical computations. Compounds 1, 2, 5, and 6 showed growth inhibition of some marine phytoplankton species and pathogenic bacteria.

Dehalogenases: From Improved Performance to Potential Microbial Dehalogenation Applications.

The variety of halogenated substances and their derivatives widely used as pesticides, herbicides and other industrial products is of great concern due to the hazardous nature of these compounds owing to their toxicity, and persistent environmental pollution. Therefore, from the viewpoint of environmental technology, the need for environmentally relevant enzymes involved in biodegradation of these pollutants has received a great boost. One result of this great deal of attention has been the identification of environmentally relevant bacteria that produce hydrolytic dehalogenases­key enzymes which are considered cost-effective and eco-friendly in the removal and detoxification of these pollutants. These group of enzymes catalyzing the cleavage of the carbon-halogen bond of organohalogen compounds have potential applications in the chemical industry and bioremediation. The dehalogenases make use of fundamentally different strategies with a common mechanism to cleave carbon-halogen bonds whereby, an active-site carboxylate group attacks the substrate C atom bound to the halogen atom to form an ester intermediate and a halide ion with subsequent hydrolysis of the intermediate. Structurally, these dehalogenases have been characterized and shown to use substitution mechanisms that proceed via a covalent aspartyl intermediate. More so, the widest dehalogenation spectrum of electron acceptors tested with bacterial strains which could dehalogenate recalcitrant organohalides has further proven the versatility of bacterial dehalogenators to be considered when determining the fate of halogenated organics at contaminated sites. In this review, the general features of most widely studied bacterial dehalogenases, their structural properties, basis of the degradation of organohalides and their derivatives and how they have been improved for various applications is discussed.

Electron transport chains in organohalide-respiring bacteria and bioremediation implications.

In situ remediation employing organohalide-respiring bacteria represents a promising solution for cleanup of persistent organohalide pollutants. The organohalide-respiring bacteria conserve energy by utilizing H2 or organic compounds as electron donors and organohalides as electron acceptors. Reductive dehalogenase (RDase), a terminal reductase of the electron transport chain in organohalide-respiring bacteria, is the key enzyme that catalyzes halogen removal. Accumulating experimental evidence thus far suggests that there are distinct models for respiratory electron transfer in organohalide-respirers of different lineages, e.g., Dehalococcoides, Dehalobacter, Desulfitobacterium and Sulfurospirillum. In this review, to connect the knowledge in organohalide-respiratory electron transport chains to bioremediation applications, we first comprehensively review molecular components and their organization, together with energetics of the organohalide-respiratory electron transport chains, as well as recent elucidation of intramolecular electron shuttling and halogen elimination mechanisms of RDases. We then highlight the implications of organohalide-respiratory electron transport chains in stimulated bioremediation. In addition, major challenges and further developments toward understanding the organohalide-respiratory electron transport chains and their bioremediation applications are identified and discussed.

Complete biosynthesis of noscapine and halogenated alkaloids in yeast.

Microbial biosynthesis of plant natural products from simple building blocks is a promising approach toward scalable production and modification of high-value compounds. The pathway for biosynthesis of noscapine, a potential anticancer compound, from canadine was recently elucidated as a 10-gene cluster from opium poppy. Here we demonstrate the de novo production of noscapine in Saccharomyces cerevisiae, through the reconstruction of a biosynthetic pathway comprising over 30 enzymes from plants, bacteria, mammals, and yeast itself, including 7 plant endoplasmic reticulum (ER)-localized enzymes. Optimization directed to tuning expression of pathway enzymes, host endogenous metabolic pathways, and fermentation conditions led to an over 18,000-fold improvement from initial noscapine titers to ∼2.2 mg/L. By feeding modified tyrosine derivatives to the optimized noscapine-producing strain we further demonstrated microbial production of halogenated benzylisoquinoline alkaloids. This work highlights the potential for microbial biosynthetic platforms to support the synthesis of valuable and novel alkaloid compounds, which can advance alkaloid-based drug discovery and development.

Understanding and Improving the Activity of Flavin-Dependent Halogenases via Random and Targeted Mutagenesis.

Flavin-dependent halogenases (FDHs) catalyze the halogenation of organic substrates by coordinating reactions of reduced flavin, molecular oxygen, and chloride. Targeted and random mutagenesis of these enzymes have been used to both understand and alter their reactivity. These studies have led to insights into residues essential for catalysis and FDH variants with improved stability, expanded substrate scope, and altered site selectivity. Mutations throughout FDH structures have contributed to all of these advances. More recent studies have sought to rationalize the impact of these mutations on FDH function and to identify new FDHs to deepen our understanding of this enzyme class and to expand their utility for biocatalytic applications.

Emission of volatile halogenated compounds, speciation and localization of bromine and iodine in the brown algal genome model Ectocarpus siliculosus.

This study explores key features of bromine and iodine metabolism in the filamentous brown alga and genomics model Ectocarpus siliculosus. Both elements are accumulated in Ectocarpus, albeit at much lower concentration factors (2-3 orders of magnitude for iodine, and < 1 order of magnitude for bromine) than e.g. in the kelp Laminaria digitata. Iodide competitively reduces the accumulation of bromide. Both iodide and bromide are accumulated in the cell wall (apoplast) of Ectocarpus, with minor amounts of bromine also detectable in the cytosol. Ectocarpus emits a range of volatile halogenated compounds, the most prominent of which by far is methyl iodide. Interestingly, biosynthesis of this compound cannot be accounted for by vanadium haloperoxidase since the latter have not been found to catalyze direct halogenation of an unactivated methyl group or hydrocarbon so a methyl halide transferase-type production mechanism is proposed.

Impact of the access tunnel engineering on catalysis is strictly ligand-specific.

The traditional way of rationally engineering enzymes to change their biocatalytic properties utilizes the modifications of their active sites. Another emerging approach is the engineering of structural features involved in the exchange of ligands between buried active sites and the surrounding solvent. However, surprisingly little is known about the effects of mutations that alter the access tunnels on the enzymes' catalytic properties, and how these tunnels should be redesigned to allow fast passage of cognate substrates and products. Thus, we have systematically studied the effects of single-point mutations in a tunnel-lining residue of a haloalkane dehalogenase on the binding kinetics and catalytic conversion of both linear and branched haloalkanes. The hotspot residue Y176 was identified using computer simulations and randomized through saturation mutagenesis, and the resulting variants were screened for shifts in binding rates. Strikingly, opposite effects of the substituted residues on the catalytic efficiency toward linear and branched substrates were observed, which was found to be due to substrate-specific requirements in the critical steps of the respective catalytic cycles. We conclude that not only the catalytic sites, but also the access pathways must be tailored specifically for each individual ligand, which is a new paradigm in protein engineering and de novo protein design. A rational approach is proposed here to address more effectively the task of designing ligand-specific tunnels using computational tools.

Use of Phenoxyaniline Analogues To Generate Biochemical Insights into the Interactio n of Polybrominated Diphenyl Ether with CYP2B Enzymes.

Human hepatic cytochromes P450 (CYP) are integral to xenobiotic metabolism. CYP2B6 is a major catalyst of biotransformation of environmental toxicants, including polybrominated diphenyl ethers (PBDEs). CYP2B substrates tend to contain halogen atoms, but the biochemical basis for this selectivity and for species specific determinants of metabolism has not been identified. Spectral binding titrations and inhibition studies were performed to investigate interactions of rat CYP2B1, rabbit CYP2B4, and CYP2B6 with a series of phenoxyaniline (POA) congeners that are analogues of PBDEs. For most congeners, there was a <3-fold difference between the spectral binding constants (KS) and IC50 values. In contrast, large discrepancies between these values were observed for POA and 3-chloro-4-phenoxyaniline. CYP2B1 was the enzyme most sensitive to POA congeners, so the Val-363 residue from that enzyme was introduced into CYP2B4 or CYP2B6. This substitution partially altered the protein-ligand interaction profiles to make them more similar to that of CYP2B1. Addition of cytochrome P450 oxidoreductase (POR) to titrations of CYP2B6 with POA or 2'4'5'TCPOA decreased the affinity of both ligands for the enzyme. Addition of cytochrome b5 to a recombinant enzyme system containing POR and CYP2B6 increased the POA IC50 value and decreased the 2'4'5'TCPOA IC50 value. Overall, the inconsistency between KS and IC50 values for POA versus 2'4'5'TCPOA is largely due to the effects of redox partner binding. These results provide insight into the biochemical basis of binding of diphenyl ethers to human CYP2B6 and changes in CYP2B6-mediated metabolism that are dependent on POA congener and redox partner identity.