Metal-containing Enzymes Research Articles

Catalytic asymmetric fragmentation of cyclopropanes.

The stereoselective activation of alkanes constitutes a long-standing and grand challenge for chemistry. Although metal-containing enzymes oxidize alkanes with remarkable ease and selectivity, chemical approaches have largely been limited to transition metal-based catalytic carbon-hydrogen functionalizations. Alkanes can be protonated to form pentacoordinated carbonium ions and fragmented into smaller hydrocarbons in the presence of strong Brønsted acids. However, catalytic stereocontrol over such reactions has not previously been accomplished. We show here that strong and confined acids catalyze highly enantioselective fragmentations of a variety of cyclopropanes into the corresponding alkenes, expanding the boundaries of catalytic selective alkane activation. Computational studies suggest the involvement of the long-debated cycloproponium ions.

Tutorial Review on the Set-Up and Running of Quantum Mechanical Cluster Models for Enzymatic Reaction Mechanisms.

Enzymes turnover substrates into products with amazing efficiency and selectivity and as such have great potential for use in biotechnology and pharmaceutical applications. However, details of their catalytic cycles and the origins surrounding the regio- and chemoselectivity of enzymatic reaction processes remain unknown, which makes the engineering of enzymes and their use in biotechnology challenging. Computational modelling can assist experimental work in the field and establish the factors that influence the reaction rates and the product distributions. A popular approach in modelling is the use of quantum mechanical cluster models of enzymes that take the first- and second coordination sphere of the enzyme active site into consideration. These QM cluster models are widely applied but often the results obtained are dependent on model choice and model selection. Herein, we show that QM cluster models can give highly accurate results that reproduce experimental product distributions and free energies of activation within several kcal mol-1, regarded that large cluster models with >300 atoms are used that include key hydrogen bonding interactions and charged residues. In this tutorial review, we give general guidelines on the set-up and applications of the QM cluster method and discuss its accuracy and reproducibility. Finally, several representative QM cluster model examples on metal-containing enzymes are presented, which highlight the strength of the approach.

Mechanisms of muscle tissue adaptation in response to the influence of low-dose ionizing radiation

Mechanisms of interaction of ionizing radiation with biological objects are a chain of successive physical and physico-chemical changes, which manifest themselves in the form of excitation, primary and secondary ionization of molecules. Biosynthesis of ATP, which is carried out by a system of oxidation-reduction enzymes localized in the inner membrane of mitochondria - the respiratory chain, belongs to the vital processes that are directly disturbed under the action of ionizing radiation. The high degree of damage to this system is due to the significant radiosensitivity of metal-containing enzymes. The purpose of the work is to study the formation of the adaptive response of muscle tissue of sexually mature rats to the influence of ionizing radiation at a dose of 0.5 Gy. It was concluded that a day after irradiation with the dose of 0.5 Gy, the content of contractile proteins in skeletal muscle decreases slightly. On the 15th day, the content of contractile proteins began to decrease. By the 30th day, the content of contractile proteins decreased by 22.9% for myosin, by more than 11% for actin, and by 7 and 8% for troponin and tropomyosin, respectively, compared to the values of the intact group. A similar picture is observed in the cardiac muscle. Mg2+,Ca2+-ATP-ase activity of actomyosin, starting from the 1st day was shown to be increased in both the skeletal and cardiac muscles, reaching its peak in the cardiac muscle on the 15th day, in contrast to the skeletal muscle, where this indicator reached its peak on the 7th day. The authors conclude that irradiation of sexually mature animals with the dose of 0.5 Gy forms an adaptive response that is accompanied by an increase in Mg2+,Ca2+-ATP-ase activity due to the formation of a strong form of binding between F-actin and myosin, actin monomers go into the typical for actomyosin “turned on stage”, and the myosin heads acquire an ordered orientation in the muscle fiber. According to author’s idea, the data obtained indicated the benefit and reasonability of using in post-radiation dysfunctions complex pharmacological treatment drugs that are able to normalize intracellular homeostasis, eliminate probable acidic changes initiated by radiation exposure, activate the processes of intramuscle energy generation and which have protective properties in relation to the muscular system.

Electron-Transferring Metalloenzymes and their Potential Biotechnological Applications.

Modern societies rely heavily on centralized industrial processes to generate a multitude of products ranging from electrical energy to synthetic chemical building blocks to construction materials. To date, these processes have relied extensively on energy produced from fossil fuels, which has led to dramatically increased quantities of greenhouse gases (including carbon dioxide) being released into the atmosphere; the effects of the ensuing change to our climate are easily observed in day-to-day life. Some of the reactions catalyzed by these industrial processes can be catalyzed in nature by metal-containing enzymes (metalloenzymes) that have evolved over the course of up to 3.8 billion years to do so under mild physiological conditions using Earth-abundant metals. While such metalloenzymes could in principle facilitate the implementation of carbon-neutral processes around the globe, either in "bio-inspired" catalyst design or even by direct exploitation, many remaining questions surrounding their mechanisms often preclude both options. Here, our recent efforts in understanding and applying metalloenzymes that catalyze reactions such as dinitrogen reduction to ammonia or proton reduction to molecular hydrogen are discussed. In closing, an opinion on the question: "Can these types of enzymes really be used in new biotechnologies?" is offered.

Synthesis of some N-hydroxycinnamamide and N-hydroxybenzamide Derivatives Bearing Amide Bonds Targeting HDAC

Recently, hydroxamic acid has attracted considerable interest because of its ability to inhibit a variety of metal-containing enzymes such as metalloproteases, lipoxygenases, histone deacetylases, and cancer cells. For example, hydroxamic acid derivatives (vorinostat, belinostat, givinostat, panobinostat) have been approved by the US FDA for the treatment of various cancers. Based on the N-hydroxycinnamamide and N-hydroxybenzamide framework, the study described and synthesized N-hydroxycinnamamide and N-hydroxybenzamide derivatives bearing amide bonds as histone deacetylases enzyme inhibitors. The structures of the compounds were determined through modern NMR spectroscopy. The compounds were then molecularly docked to the active site of the HDAC6 enzyme, the results showed that the compounds strongly bound to the zinc ion and key amino acids. These results suggest that the synthesized N-hydroxycinnamamide and N-hydroxybenzamide derivatives are potential molecular targeted therapy for HDAC positive cancer in the future.

Superoxide dismutase gene family in cassava revealed their involvement in environmental stress via genome-wide analysis

Superoxide dismutase (SOD) is a crucial metal-containing enzyme that plays a vital role in catalyzing the dismutation of superoxide anions, converting them into molecular oxygen and hydrogen peroxide, essential for enhancing plant stress tolerance. We identified 8 SOD genes (4 CSODs, 2 FSODs, and 2 MSODs) in cassava. Bioinformatics analyses provided insights into chromosomal location, phylogenetic relationships, gene structure, conserved motifs, and gene ontology annotations. MeSOD genes were classified into two groups through phylogenetic analysis, revealing evolutionary connections. Promoters of these genes harbored stress-related cis-elements. Duplication analysis indicated the functional significance of MeCSOD2/MeCSOD4 and MeMSOD1/MeMSOD2. Through qRT-PCR, MeCSOD2 responded to salt stress, MeMSOD2 to drought, and cassava bacterial blight. Silencing MeMSOD2 increased XpmCHN11 virulence, indicating MeMSOD2 is essential for cassava's defense against XpmCHN11 infection. These findings enhance our understanding of the SOD gene family's role in cassava and contribute to strategies for stress tolerance improvement.

Discriminating Susceptibility of Xanthine Oxidoreductase Family to Metals.

The xanthine oxidoreductase (XOR) family are metal-containing enzymes that use the molybdenum cofactor (Moco), 2Fe-2S clusters, and flavin adenine dinucleotide (FAD) for their catalytic activity. This large molybdoenzyme family includes xanthine, aldehyde, and CO dehydrogenases. XORs are widely distributed from bacteria to humans due to their key roles in the catabolism of purines, aldehydes, drugs, and xenobiotics, as well as interconversions between CO and CO2. Assessing the effect of excess metals on the Rubrivivax gelatinosus bacterium, we found that exposure to copper (Cu) or cadmium (Cd) caused a dramatic decrease in the activity of a high-molecular-weight soluble complex exhibiting nitroblue tetrazolium reductase activity. Mass spectrometry and genetic analyses showed that the complex corresponds to a putative CO dehydrogenase (pCOD). Using mutants that accumulate either Cu+ or Cd2+ in the cytoplasm, we show that Cu+ or Cd2+ is a potent inhibitor of XORs (pCOD and the xanthine dehydrogenase [XDH]) in vivo. This is the first in vivo demonstration that Cu+ affects Moco-containing enzymes. The specific inhibitory effect of these compounds on the XOR activity is further supported in vitro by direct addition of competing metals to protein extracts. Moreover, emphasis is given on the inhibitory effect of Cu on bovine XOR, showing that the XOR family could be a common target of Cu. Given the conservation of XOR structure and function across the tree of life, we anticipate that our findings could be transferable to other XORs and organisms. IMPORTANCE The high toxicity of Cu, Cd, Pb, As, and other metals arises from their ability to cross membranes and target metalloenzymes in the cytoplasm. Identifying these targets provides insights into the toxicity mechanisms. The vulnerability of metalloenzymes arises from the accessibility of their cofactors to ions. Accordingly, many enzymes whose cofactors are solvent exposed are likely to be targets of competing metals. Here, we describe for the first time, with in vivo and in vitro experiments, a direct effect of excess Cu on the xanthine oxidoreductase family (XOR/XDH/pCOD). We show that toxic metal affects these Moco enzymes, and we suggest that access to the Moco center by Cu ions could explain the Cu inhibition of XORs in living organisms. Human XOR activity is associated with hyperuricemia, xanthinuria, gout arthritis, and other diseases. Our findings in vivo highlight XOR as a Cu target and thus support the potential use of Cu in metal-based therapeutics against these diseases.

The Co-Evolution Aspects of the Biogeochemical Role of Phytoplankton in Aquatic Ecosystems: A Review.

In freshwater and marine ecosystems, the phytoplankton community is based on microalgae and cyanobacteria, which include phylogenetically very diverse groups of oxygenic photoautotrophs. In the process of evolution, they developed a wide range of bio(geo)chemical adaptations that allow them to effectively use solar radiation, CO2, and nutrients, as well as major and trace elements, to form O2 and organic compounds with a high chemical bond energy. The inclusion of chemical elements in the key processes of energy and plastic metabolism in the cell is determined by redox conditions and the abundance and metabolic availability of elements in the paleoenvironment. Geochemical evolution, which proceeded simultaneously with the evolution of biosystems, contributed to an increase in the number of metals and trace elements acting as cofactors of enzymes involved in metabolism and maintaining homeostasis in the first photoautotrophs. The diversity of metal-containing enzymes and the adaptive ability to replace one element with another without losing the functional properties of enzymes ensured the high ecological plasticity of species and allowed microalgae and cyanobacteria to successfully colonize a wide variety of habitats. In this review, we consider the main aspects of the modern concepts of the biogeochemical evolution of aquatic ecosystems and the role of some metals in the main bioenergetic processes in photosynthetic prokaryotes and eukaryotes. We present generalized data on the efficiency of the assimilation of key nutrients by phytoplankton and their importance in the cycle of carbon, silicon, nitrogen, phosphorus, sulfur, and iron. This article presents modern views on the evolutionary prerequisites for the formation of elemental signatures in different systematic groups of microalgae, as well as the possibility of using the stoichiometric ratio in the study of biological and geochemical processes in aquatic ecosystems.

The Discovery of Tyrosinase Enzyme Inhibitors Activity from Polyphenolic Compounds in Red Grape Seeds through In Silico Study

Tyrosinases are essential metal-containing enzymes in the biosynthesis of melanin, therefore responsible for pigmentation of the skin. The upregulation of tyrosinase enzyme activities leads to hyperpigmentation that will become a health problems and interfere psychosocially. Inhibition of tyrosinase enzyme activity, both competitive and non-competitive become widely developed for most anti hyperpigmentation agent. Natural antioxidants are one of the potential compounds for this purpose. Red grape seeds contain high levels of antioxidant compounds, such as procyanidin, prodelphinidin, and propelargonidin. In this research in silico studies, including molecular docking, molecular dynamics simulations, and toxicity predictions, were used to assess the activity of the three molecules of polyphenolic compounds on macromolecules of the tyrosinase enzyme. Molecular docking studies show that the compound propelargonidin has the highest affinity against the macromolecule of the tyrosinase enzyme, with a binding free energy value of −32.87 kJ/mol. These results were confirmed in molecular dynamics simulations that show strong interactions at the macromolecular active site of the tyrosinase enzyme. Toxicity prediction results show that the three polyphenolic compound molecules were classified in the High-Class Category, which shows that safety is not guaranteed, but is likely, not carcinogenic and nongenotoxic. Therefore, the compound propelargonidin is predicted to be able to interact strongly with the tyrosinase enzyme. The results in this research are useful for further study in the development of tyrosinase enzyme inhibitors.

Potential inhibitors of methionine aminopeptidase type II identified via structure-based pharmacophore modeling.

Methionine aminopeptidase (MetAP2) is a metal-containing enzyme that removes initiator methionine from the N-terminus of a newly synthesized protein. Inhibition of the enzyme is crucial in diminishing cancer growth and metastasis. Fumagillin-a natural irreversible inhibitor of MetAP2-and its derivatives are used as potent MetAP2 inhibitors. However, because of their adverse effects, none of them has progressed to clinical studies. In search for potential reversible inhibitors, we built structure-based pharmacophore models using the crystal structure of MetAP2 complexed with fumagillin (PDB ID: 1BOA). The pharmacophore models were validated using Gunner-Henry scoring method. The best pharmacophore consisting of 1 H-bond donor, 1 H-bond acceptor, and 3 hydrophobic features was used to conduct pharmacophore-based virtual screening of ZINC15 database against MetAP2. The top 10 compounds with pharmacophore fit values > 3.00 were selected for further analysis. These compounds were subjected to absorption, distribution, metabolism, elimination, and toxicity (ADMET) prediction and found to have druglike properties. Furthermore, molecular docking calculations was performed on these hits using AutoDock4 to predict their binding mode and binding energy. Three diverse compounds: ZINC000014903160, ZINC000040174591, and ZINC000409110720 with respective binding energy/docking scores of - 9.22, - 9.21, and -817kcal/mol, were submitted to 100ns (MD) simulations using Nanoscale MD (NAMD) software. The compounds showed stable binding mode over time. Therefore, they may serve as a scaffold for further computational and experimental optimization toward the design of more potent and safer MetAP2 inhibitors.

A Cerium Vanadate Nanozyme with Specific Superoxide Dismutase Activity Regulates Mitochondrial Function and ATP Synthesis in Neuronal Cells.

Nanoparticles that functionally mimic the activity of metal-containing enzymes (metallo-nanozymes) are of therapeutic importance for treating various diseases. However, it is still not clear whether such nanozymes can completely substitute the function of natural enzymes in living cells. In this work, we show for the first time that a cerium vanadate (CeVO4 ) nanozyme can substitute the function of superoxide dismutase 1 and 2 (SOD1 and SOD2) in the neuronal cells even when the natural enzyme is down-regulated by specific gene silencing. The nanozyme prevents the mitochondrial damage in SOD1- and SOD2-depleted cells by regulating the superoxide levels and restores the physiological levels of the anti-apoptotic Bcl-2 family proteins. Furthermore, the nanozyme effectively prevents the mitochondrial depolarization, leading to a significant improvement in the cellular levels of ATP under oxidative stress.

Вплив іонів металів на функціонування антиоксидантної системи та ферменти міжклітинного матриксу пухлин за умов їх різної чутливості до доксорубіцину

The content of the essential macro- and microelements (MaE and ME) – copper, zinc, magnesium, iron and calcium was measured in blood plasma (BP) and tumor tissue (TT) of the animals engrafted with Walker 256 carcinosarcoma (tumor strains sensitive or resistant to doxorubicin). Also, the link between the content of these elements and the functional state of metal-containing enzymes of antioxidant system (metallothionein-1 – MT-1, ceruloplasmin – CP, ferritin – FR, myeloperoxidase – MPO, transferrin – TF) as well proteolytic enzymes (matrix metalloproteinases – MMP-2 and -9) was studied. In BP of animals engrafted with either sensitive or resistant to doxorubicin carcinosarcoma Cu/Zn and Ca/Mg ratios increase 2.8-fold and 1.4-fold, respectively as compared to the intact animals. The differences between MaE and ME content in the animals with tumors differed in doxorubicin sensitivity as compared to the intact animals affected the indices of prooxidant-antioxidant system: 2.7 and 1.6-fold increase of MT-1 (for sensitive and resistant tumors, correspondingly), 1.2 and 1.6-fold increase of CP, and 4.6- and 9-fold increase of FR against 5 and 7.5-fold decrease of MPO and 1.3 and 1.9-fold decrease in TF. In TT of the animals engrafted with the resistant strain 1.7-fold decrease of MT-1, 65-fold increase of FR, and 2.5-fold increase of TF was evident as compared with TT of animals engrafted with the sensitive strain. The change in prooxidant-antioxidant balance in tumorbearing animals was accompanied by the increased proteolytic activity of the enzymes involved in remodeling extracellular matrix, MMP-2 and MMP-8. Increased copper and iron content in BP and TT of the animals with doxorubicinresistant Walker 256 carcinosarcoma could initiate of freeradical compounds generation and enhancement of MMP-2 and MMP-9 activation. Therefore, the ionic misbalance might be the cause of changes of the antioxidant system functions contributing to the formation of the resistant phenotype of neoplasms. This provides the rationale for the use of chelating agents for increasing sensitivity of resistant neoplasms to cytostatics.

Carbon-fluorine bond cleavage mediated by metalloenzymes.

Fluorochemicals are a widely distributed class of compounds and have been utilized across a wide range of industries for decades. Given the environmental toxicity and adverse health threats of some fluorochemicals, the development of new methods for their decomposition is significant to public health. However, the carbon-fluorine (C-F) bond is among the most chemically robust bonds; consequently, the degradation of fluorinated hydrocarbons is exceptionally difficult. Here, metalloenzymes that catalyze the cleavage of this chemically challenging bond are reviewed. These enzymes include histidine-ligated heme-dependent dehaloperoxidase and tyrosine hydroxylase, thiolate-ligated heme-dependent cytochrome P450, and four nonheme oxygenases, namely, tetrahydrobiopterin-dependent aromatic amino acid hydroxylase, 2-oxoglutarate-dependent hydroxylase, Rieske dioxygenase, and thiol dioxygenase. While much of the literature regarding the aforementioned enzymes highlights their ability to catalyze C-H bond activation and functionalization, in many cases, the C-F bond cleavage has been shown to occur on fluorinated substrates. A copper-dependent laccase-mediated system representing an unnatural radical defluorination approach is also described. Detailed discussions on the structure-function relationships and catalytic mechanisms provide insights into biocatalytic defluorination, which may inspire drug design considerations and environmental remediation of halogenated contaminants.

Juglans regia and Ribes nigrum as potential nutraceuticals: Source of thermostable superoxide dismutase enzyme.

In the present study, superoxide dismutase (SOD) extracted from dry fruits; Juglans regia (Walnut; W) and Ribes nigrum (Munakka; M) was partially purified into 0%-40% and 40%-80% fractions based on ammonium sulfate saturation levels. The partially purified fractions (0%-40%) exhibited purification level of 3.09- (W) and 3.22- (M) fold with specific activity 79.32Umg-1 (W) and 125.23Umg-1 (M). SOD from both the sources was found to be thermally stable, that is, 80°C (W) and 70°C (M). Kinetic studies showed Km values to be 3.33mM (W) and 2.86mM (M), whereas the activation energy (Ea ) calculated as 24.52 KJmol-1 (W) and 26.25KJmol-1 (M). Na+ , Mn2+ , and Ba2+ ions acted as potential inhibitors, whereas Fe2+ stimulated SOD from both the sources. Among these metal ions, Na+ exhibited uncompetitive inhibition in both cases; with Ki values of 0.7mM (W) and 0.9mM (M), suggesting the more prominent binding affinity and effectiveness. PRACTICAL APPLICATIONS: Awareness need to be created among people for multifactorial health benefits of nutraceuticals in day-to-day life. Nutritional consumption from fruits, nuts, and vegetables safeguard against various maladies like cardiovascular diseases, diabetes, and cancers. Superoxide dismutase (EC 1.15.1.1) is a standout among the most critical metal-containing enzymes that act as a main line of defense against oxidative stress. Antioxidant-based drugs and formulations have been developed in the recent years and research is emphasized on its impact on oxidative stress levels. In this study, Juglans regia (W) and Ribes nigrum (M) were found to have thermostable SOD enzyme with excellent antioxidant properties. Thermal stability of an enzyme improves its significance making it industry friendly with therapeutically vital products, alongside their utilization as supplement in numerous therapeutic formulations.

The influence of lactoferrin on elemental homeostasis and activity of metal-containing enzymes in rats with Walker-256 carcinosarcoma

To investigate the content of essential elements (EE): copper, zinc, magnesium, iron and calcium and the evaluation of the activity of metal-containing enzymes- ceruloplasmin (CP), myeloperoxidase (MPO) and the content of transferrin (TF) in blood plasma(BP) and tumor tissue (TT) of animals with Walker-256 carcinosarcoma treated with lactoferrin (LF). The study of the EE content and the activity of the abovementioned enzymes was carried out on rats with Walker-256carcinosarcoma treated with LF at the doses of 1and 10mg/kg of body weight. The quantitative content of EE in BP and TT of animals was determined using the inductively coupled plasma atomic emission spectroscopy (ICP-AES). Determination of CP activity, content of TF and hemochromes was performed using the method of electron paramagnetic resonance (EPR), and MPO- by unified biochemical method. The introduction of LF at the doses of 1and 10mg/kg resulted in a decrease in the ratio ofCu/Zn in BP and even more expressed decrease of Ca/Mg ratio in TT. Administration of LF, especially at a dose of 10mg/kg, affected the increase in CP and MPO activity in BP. It has been shown that administration of LF at a dose of 10mg/kg led to an increase in oxidative products of destruction of the hemoglobin-hemochrom system in the TT, against the background of lowering the TF content. The administration of LF, especially at a dose of 10mg/kg, led to metabolic alterations associated with inhibition of the tumor process. The detected modulating effect of LF on the content of the EE and the activity of the CP and MPO may be a basis for correction of the elemental balance in carcinogenesis.

Cytochemical Localization of Polyphenol Oxidase Activity in K2-Bodies of Saprolegnia ferax Secondary Zoospores.

Zoospores of the oomycete Saprolegnia ferax release adhesive material from K-bodies at the onset of attachment to substrates. To understand more fully how K-bodies function in adhesion, enzyme activity was investigated cytochemically in secondary zoospores. Presence of catalase, a marker enzyme for microbodies, was explored in the diaminobenzidine (DAB) reaction. Although pH 9.2 DAB-staining characteristic of catalase activity was detected in the granular matrix regions of K-bodies, reaction controls indicated that the reaction was due to oxidative enzyme activity other than catalase. Because polyphenol oxidase (PPO) is another metal-containing enzyme capable of oxidizing DAB, activity of this enzyme was tested with a more specific substrate, dihydroxyphenylalanine (DOPA). In the DOPA procedure, reaction product was exclusively localized within K-bodies, indicating the presence of PPO. Results with three methods of reaction controls (elimination of substrate, addition of a PPO enzyme inhibitor, and heat-inactivation of enzymes) all supported the presence of PPO in K-bodies. This study highlights potential roles for K-body PPO in stabilization of adhesion bodies by: cross-linking matrix phenolic proteins or glycoproteins as K-bodies discharge adhesives onto substrates, or polymerizing phenolics protective against microbial attacks of the adhesion pad.

Stability of Benzotriazole Derivatives with Free Cu, Zn, Co and Metal-Containing Enzymes: Binding and Interaction of Methylbenzotriazoles with Superoxide Dismutase and Vitamin B12

Benzotriazole derivatives form very strong bonds with transition metals, and are the most widely used type of industrial corrosion inhibitor. Some benzotriazole derivatives have been implicated as hormone regulators which also carry the ability to induce uncoupling responses or otherwise inhibit respiration processes in some microorganisms. However, the mechanisms associated with benzotriazole toxicity and inhibition are unknown. Using Differential Pulse Polarography, the stability constants of commercially significant corrosion inhibitors, 4-and 5-methylbenzotriazole, coordinated with free Cu (II) and Co (III), were determined to be 1015 and 108, respectively. Polarographic analyses were extended to confirm that methylbenzotriazole also binds the copper center(s) in the ubiquitous enzyme superoxide dismutase, and the Corrin site in the coenzyme cobalamin (vitamin B12). These results suggest that the metal-chelating ability of this unique class of compounds may confer inhibition to certain enzyme systems.

Effects of Sulfate Reduction on Trichloroethene Dechlorination by Dehalococcoides-Containing Microbial Communities.

In order to elucidate interactions between sulfate reduction and dechlorination, we systematically evaluated the effects of different concentrations of sulfate and sulfide on reductive dechlorination by isolates, constructed consortia, and enrichments containing Dehalococcoides sp. Sulfate (up to 5 mM) did not inhibit the growth or metabolism of pure cultures of the dechlorinator Dehalococcoides mccartyi 195, the sulfate reducer Desulfovibrio vulgaris Hildenborough, or the syntroph Syntrophomonas wolfei In contrast, sulfide at 5 mM exhibited inhibitory effects on growth of the sulfate reducer and the syntroph, as well as on both dechlorination and growth rates of D. mccartyi Transcriptomic analysis of D. mccartyi 195 revealed that genes encoding ATP synthase, biosynthesis, and Hym hydrogenase were downregulated during sulfide inhibition, whereas genes encoding metal-containing enzymes involved in energy metabolism were upregulated even though the activity of those enzymes (hydrogenases) was inhibited. When the electron acceptor (trichloroethene) was limiting and an electron donor (lactate) was provided in excess to cocultures and enrichments, high sulfate concentrations (5 mM) inhibited reductive dechlorination due to the toxicity of generated sulfide. The initial cell ratio of sulfate reducers to D. mccartyi (1:3, 1:1, or 3:1) did not affect the dechlorination performance in the presence of sulfate (2 and 5 mM). In contrast, under electron donor limitation, dechlorination was not affected by sulfate amendments due to low sulfide production, demonstrating that D. mccartyi can function effectively in anaerobic microbial communities containing moderate sulfate concentrations (5 mM), likely due to its ability to outcompete other hydrogen-consuming bacteria and archaea.IMPORTANCE Sulfate is common in subsurface environments and has been reported as a cocontaminant with chlorinated solvents at various concentrations. Inconsistent results for the effects of sulfate inhibition on the performance of dechlorination enrichment cultures have been reported in the literature. These inconsistent findings make it difficult to understand potential mechanisms of sulfate inhibition and complicate the interpretation of bioremediation field data. In order to elucidate interactions between sulfate reduction and reductive dechlorination, this study systematically evaluated the effects of different concentrations of sulfate and sulfide on reductive dechlorination by isolates, constructed consortia, and enrichments containing Dehalococcoides sp. This study provides a more fundamental understanding of the competition mechanisms between reductive dechlorination by Dehalococcoides mccartyi and sulfate reduction during the bioremediation process. It also provides insights on the significance of sulfate concentrations on reductive dechlorination under electron donor/acceptor-limiting conditions during in situ bioremediation applications. For example, at a trichloroethene-contaminated site with a high sulfate concentration, proper slow-releasing electron donors can be selected to generate an electron donor-limiting environment that favors reductive dechlorination and minimizes the sulfide inhibition effect.

In Silico Analysis of Heavy Metal Assimilation Behaviors in the Genome of Methanosarcina barkeri str. Fusaro

A computational systems biology representation was utilized for understanding the molecular and metabolic behaviors of heavy metal assimilation system in the genome Methanosarcina barkeri str. Fusaro. A known functional protein of this system was identified by text mining. A combined functional annotation approach was employed to discover the missing proteins with unknown function. Herein, assimilation systems of cadmium, nickel and copper ions have been predicted by using bioinformatics resources. Metabolic flux balances of each pathway model have been optimized for its cellular behaviors in response to excessive heavy metal ions in the environment. Many heavy metals have been utilized by a typical assimilation system on producing the precursors required for methane biosynthesis, and other energy driven processes of this genome. Metal transporter and accessory proteins of methanogens were phylogenetically corresponded with similar proteins in the members of proteobacteria, but metal-containing enzymes are very exclusive to closely related methanogenic archaea. The evolutionary and metabolic relationships of heavy metal transporter system have been observed among archaea and some members in the proteobacteria. Thus, the metabolic models obtained from this study will optimistically be useful in understanding the heavy metal assimilation mechanism of this genome for producing methane, and applying in bioremediation process. Keywords: Cell behavior, heavy metal assimilation, metals detoxification, methanogens, molecular evolution, pathway model, protein function, transporters.

Identification of Novel Non-Metal Haloperoxidases from the Marine Metagenome

Haloperoxidase (HPO, E.C.1.11.1.7) is a metal-containing enzyme oxidizing halonium species, which can be used in the synthesis of halogenated organic compounds, for instance in the production of antimicrobial agents, cosmetics, etc., in the presence of halides and H2O2. To isolate and evaluate a novel non-metal HPO using a culture-independent method, a cassette PCR library was constructed from marine seawater in Japan. We first isolated a novel HPO gene from Pseudomonas putida ATCC11172 by PCR for constructing the chimeric HPO library (HPO11172). HPO11172 showed each single open-reading frame of 828 base pairs coding for 276 amino acids, respectively, and showed 87% similarity with P. putida IF-3 sequences. Approximately 600 transformants screened for chimeric genes between P. putida ATCC11173 and HPO central fragments were able to identify 113 active clones. Among them, we finally isolated 20 novel HPO genes. Sequence analyses of the obtained 20 clones showed higher homology genes with P. putida or Sinorhizobium or Streptomyces strains. Although the HPO A9 clone showed the lowest homology with HPO11172, clones in group B, including CS19, showed a relatively higher homology of 80%, with 70% identy. E. coli cells expressing these HPO chimeric genes were able to successfully bioconvert chlorodimedone with KBr or KCl as substrate.