Efficient bio-remediation of multiple aromatic hydrocarbons using different types of thermotolerant, ring-cleaving dioxygenases derived from Aeribacillus pallidus HB-1.

As toxic contaminants, aromatic compounds are widespread in most environmental matrices, and bioenzymatic catalysis plays a critical role in the degradation of xenobiotics. Here, a thermophillic aromatic hydrocarbon degrader Aeribacillus pallidus HB-1 was found. Bioinformatic analysis of the HB-1 genome revealed two ring-cleaving extradiol dioxygenases (EDOs), among which, EDO-0418 was assigned to a new subfamily of type I.1 EDOs and exhibited a broad substrate specificity, particularly towards biarylic substrate. Both EDOs exhibited optimal activities at elevated temperatures (55 and 65 °C, respectively) and showed remarkable thermostability, pH stability, metal ion resistance and tolerance to chemical reagents. Most importantly, simulated wastewater bioreactor experiments demonstrated efficient and uniform degradation performance of mixed aromatic substrates under harsh environments by the two enzymes combined for potential industrial applications. The unveiling of two thermostable dioxygenases with broad substrate specificities and stress tolerance provides a novel approach for highly efficient environmental bioremediation using composite enzyme systems.

Black Carbon Impacts on Paraburkholderia xenovorans Strain LB400 Cell Enrichment and Activity: Implications toward Lower-Chlorinated Polychlorinated Biphenyls Biodegradation Potential.

Volatilization of lower-chlorinated polychlorinated biphenyls (LC-PCBs) from sediment poses health threats to nearby communities and ecosystems. Biodegradation combined with black carbon (BC) materials is an emerging bioaugmentation approach to remove PCBs from sediment, but development of aerobic biofilms on BC for long-term, sustained LC-PCBs remediation is poorly understood. This work aimed to characterize the cell enrichment and activity of biphenyl- and benzoate-grown Paraburkholderia xenovorans strain LB400 on various BCs. Biphenyl dioxygenase gene (bphA) abundance on four BC types demonstrated corn kernel biochar hosted at least 4 orders of magnitude more attached cells per gram than other feedstocks, and microscopic imaging revealed the attached live cell fraction was >1.5× more on corn kernel biochar than GAC. BC characteristics (i.e., sorption potential, pore size, pH) appear to contribute to cell attachment differences. Reverse transcription qPCR indicated that BC feedstocks significantly influenced bphA expression in attached cells. The bphA transcript-per-gene ratio of attached cells was >10-fold more than suspended cells, confirmed by transcriptomics. RNA-seq also demonstrated significant upregulation of biphenyl and benzoate degradation pathways on attached cells, as well as revealing biofilm formation potential/cell-cell communication pathways. These novel findings demonstrate aerobic PCB-degrading cell abundance and activity could be tuned by adjusting BC feedstocks/attributes to improve LC-PCBs biodegradation potential.

Oxygen Activation in Aromatic Ring Cleaving Salicylate Dioxygenase: Detection of Reaction Intermediates with a Nitro-substituted Substrate Analog.

Cupin dioxygenases such as salicylate 1,2-dioxygense (SDO) perform aromatic C-C bond scission via a 3-His motif tethered iron cofactor. Here, transient kinetics measurements are used to monitor the catalytic cycle of SDO by using a nitro-substituted substrate analog, 3-nitrogentisate. Compared to the natural substrate, the nitro group reduces the enzymatic kcat by 500-fold, thereby facilitating the detection and kinetic characterization of reaction intermediates. Sums and products of reciprocal relaxation times derived from kinetic measurements were found to be linearly dependent on O2 concentration, suggesting reversible formation of two distinct intermediates. Dioxygen binding to the metal cofactor takes place with a forward rate of 5.9×103 M-1 s-1: two orders of magnitude slower than other comparable ring-cleaving dioxygenses. Optical chromophore of the first intermediate is distinct from the in situ generated SDO Fe(III)-O2⋅- complex but closer to the enzyme-substrate precursor.

Reply to Correspondence on "Structural Insight into the Catalytic Mechanism of the Endoperoxide Synthase FtmOx1''.

The high-resolution X-ray crystal structure of the ternary complex FtmOx1 ⋅ 2OG ⋅ fumitremorgin B and the catalytic mechanism were recently reported by us (DOI 10.1002/anie.202112063). In their Correspondence, Zhang, Costello, Liu et al. criticize our work in several aspects. Herein, we address these questions one by one. These structural clarifications and new computational results further support the CarC-like mechanistic model.

The case for an oxidopyrylium intermediate in the mechanism of quercetin dioxygenases.

The quercetin dioxygenases (QDOs) are unusual metalloenzymes in that they display ring-opening dioxygenase activity with several different first-row transition metal ions which do not undergo redox changes during turnover. The QDOs are also unique in that the substrate binds as an η1-flavonolate rather than the η2 -bidentate mode seen in all reported model complexes. The flavonol substrates were early examples of excited state intramolecular proton transfer (ESIPT) phenomena, in which photoexcitation causes an H-atom exchange between the adjacent hydroxyl and ketone, generating an oxidopyrylium emissive state. These oxidopyryliums undergo ring-opening dioxygenations analogous to the enzymatic reactions. Our hypothesis is that lability of the divalent metal ion may allow access to a reactive oxidopyrylium intermediate via coordination switching from the oxy to ketone position, which allows reaction with O2. In this report, we use a straight-forward methylation strategy to generate a panel of flavonol and thioflavonol derivatives modeling several η1- and η2-coordination modes. Methylation of 3-hydroxythioflavone generates an air stable η1 hydroxopyrylium salt, which undergoes rapid ring-opening dioxygenation by deprotonation or photoexcitation. By comparison, the η1-methoxyflavonol does not react with O2 under any condition. We find that any of the studied flavonol derivatives, η1 or η2, which demonstrates ESIPT-like oxidopyrylium emissions undergo QDO-like ring-opening reactions with dioxygen. The implications of these results concerning the mechanism of QDOs and related dioxygenases is discussed.

Nuclear Resonance Vibrational Spectroscopy Definition of Peroxy Intermediates in Catechol Dioxygenases: Factors that Determine Extra- versus Intradiol Cleavage.

The extradiol dioxygenases (EDOs) and intradiol dioxygenases (IDOs) are nonheme iron enzymes that catalyze the oxidative aromatic ring cleavage of catechol substrates, playing an essential role in the carbon cycle. The EDOs and IDOs utilize very different FeII and FeIII active sites to catalyze the regiospecificity in their catechol ring cleavage products. The factors governing this difference in cleavage have remained undefined. The EDO homoprotocatechuate 2,3-dioxygenase (HPCD) and IDO protocatechuate 3,4-dioxygenase (PCD) provide an opportunity to understand this selectivity, as key O2 intermediates have been trapped for both enzymes. Nuclear resonance vibrational spectroscopy (in conjunction with density functional theory calculations) is used to define the geometric and electronic structures of these intermediates as FeII-alkylhydroperoxo (HPCD) and FeIII-alkylperoxo (PCD) species. Critically, in both intermediates, the initial peroxo bond orientation is directed toward extradiol product formation. Reaction coordinate calculations were thus performed to evaluate both the extra- and intradiol O-O cleavage for the simple organic alkylhydroperoxo and for the FeII and FeIII metal catalyzed reactions. These results show the FeII-alkylhydroperoxo (EDO) intermediate undergoes facile extradiol O-O bond homolysis due to its extra e-, while for the FeIII-alkylperoxo (IDO) intermediate the extradiol cleavage involves a large barrier and would yield the incorrect extradiol product. This prompted our evaluation of a viable mechanism to rearrange the FeIII-alkylperoxo IDO intermediate for intradiol cleavage, revealing a key role in the rebinding of the displaced Tyr447 ligand in this rearrangement, driven by the proton delivery necessary for O-O bond cleavage.

Crystal structure of the monocupin ring-cleaving dioxygenase 5-nitrosalicylate 1,2-dioxygenase from Bradyrhizobium sp.

5-Nitrosalicylate 1,2-dioxygenase (5NSDO) is an iron(II)-dependent dioxygenase involved in the aerobic degradation of 5-nitroanthranilic acid by the bacterium Bradyrhizobium sp. It catalyzes the opening of the 5-nitrosalicylate aromatic ring, a key step in the degradation pathway. Besides 5-nitrosalicylate, the enzyme is also active towards 5-chlorosalicylate. The X-ray crystallographic structure of the enzyme was solved at 2.1 Šresolution by molecular replacement using a model from the AI program AlphaFold. The enzyme crystallized in the monoclinic space group P21, with unit-cell parameters a = 50.42, b = 143.17, c = 60.07 Å, ß = 107.3°. 5NSDO belongs to the third class of ring-cleaving dioxygenases. Members of this family convert para-diols or hydroxylated aromatic carboxylic acids and belong to the cupin superfamily, which is one of the most functionally diverse protein classes and is named on the basis of a conserved ß-barrel fold. 5NSDO is a tetramer composed of four identical subunits, each folded as a monocupin domain. The iron(II) ion in the enzyme active site is coordinated by His96, His98 and His136 and three water molecules with a distorted octahedral geometry. The residues in the active site are poorly conserved compared with other dioxygenases of the third class, such as gentisate 1,2-dioxygenase and salicylate 1,2-dioxygenase. Comparison with these other representatives of the same class and docking of the substrate into the active site of 5NSDO allowed the identification of residues which are crucial for the catalytic mechanism and enzyme selectivity.

Computational Study of the Fe(II) and α-Ketoglutarate-Dependent Aryloxyalkanoate Dioxygenase (AAD-1) in the Degradation of the Herbicide 2,4-Dichlorophenoxyacetic Acid.

The AAD-1 enzyme belongs to the Fe(II) and α-ketoglutarate (Fe/αKG)-dependent nonheme aryloxyalkanoate dioxygenase family (AADs), which catalyzes the breakdown of 2,4-dichlorophenoxyacetic acid (2,4-D, an active ingredient of thousands of commercial herbicides) by using the highly active Fe(IV)═O complex. Multiple species of bacteria degrade 2,4-D via a pathway initiated by AADs; however, the detail of how they promote the cleavage of the ether C-O bond to generate 2,4-dichlorophenol (2,4-DCP) and glyoxylate is still unclear, which is the prerequisite for the further degradation of these halogenated aromatics. In this work, based on the crystal structure of AAD-1, the computational models were constructed, and a series of QM/MM and QM-only calculations were performed to explore the cleavage of the ether bond in 2,4-D with the catalysis of AAD-1. Our calculations reveal that AAD-1 may be only responsible for the hydroxylation of the substrate to generate the intermediate hemiacetal, which corresponds to an overall energy barrier of 14.2 kcal/mol on the quintet state surface, and the decomposition of the hemiacetal in the active site center of AAD-1 was calculated to be rather slow, corresponding to an energy barrier of 24.5 kcal/mol. In contrast, the decomposition of the free hemiacetal molecule in a solvent was calculated to be quite easy. Whether the decomposition of the hemiacetal occurs inside or outside the activation site is still worthy of experimental verification.

Underlying Role of Hydrophobic Environments in Tuning Metal Elements for Efficient Enzyme Catalysis.

The catalytic functions of metalloenzymes are often strongly correlated with metal elements in the active sites. However, dioxygen-activating nonheme quercetin dioxygenases (QueD) are found with various first-row transition-metal ions when metal swapping inactivates their innate catalytic activity. To unveil the molecular basis of this seemingly promiscuous yet metal-specific enzyme, we transformed manganese-dependent QueD into a nickel-dependent enzyme by sequence- and structure-based directed evolution. Although the net effect of acquired mutations was primarily to rearrange hydrophobic residues in the active site pocket, biochemical, kinetic, X-ray crystallographic, spectroscopic, and computational studies suggest that these modifications in the secondary coordination spheres can adjust the electronic structure of the enzyme-substrate complex to counteract the effects induced by the metal substitution. These results explicitly demonstrate that such noncovalent interactions encrypt metal specificity in a finely modulated manner, revealing the underestimated chemical power of the hydrophobic sequence network in enzyme catalysis.

Cofactor-Free Dioxygenases-Catalyzed Reaction Pathway via Proton-Coupled Electron Transfer.

Understanding the general mechanism of the metal-free and cofactor-free oxidases and oxygenases catalyzed activation of triplet O2 is one of the most challenging questions in the field of enzymatic catalysis. Herein, we have performed Quantum Mechanics/Molecular Mechanics (QM/MM) multiscale simulations to reveal the detailed mechanism of the HOD catalyzed (i.e., 1-H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase from Arthrobacter nitroguajacolicus Rü61a) decomposition of N-heteroaromatic compounds. The complete catalytic mechanism includes four steps: (1) proton transfer from 1-H-3-hydroxy-4-oxoquinaldine (QND) substrate to His251 residue coupled with an electron transfer from QND to triplet O2 (i.e., PCET), (2) formation of C-O bond via an open-shell singlet diradical recombination pathway, (3) ring-closure to form a bicyclic ring, and (4) dissociation of CO. The dissociation of CO is determined as the rate-limiting step, and its calculated energy barrier of 14.9 kcal/mol is consistent with the 15.5 kcal/mol barrier derived from experimental kinetic data. The mechanistic profile is not only valuable for understanding the fundamental pathway of cofactor-free oxidases and oxygenases-catalyzed reactions involving the triplet O2 activation but also discloses a new pathway that undergoes the processes of PCET and open-shell singlet transition state.

Flavonol dioxygenase chemistry mediated by a synthetic nickel superoxide.

Nature exploits transition metal centers to enhance and tune the oxidizing power of natural oxidants such as O2 and H2O2. The design and interrogation of synthetic metallocomplexes with similar reactivity to metalloproteins provides one strategy for gaining insight into the mechanistic underpinnings of oxygen-activating enzymes such as oxidases, oxygenases, and dioxygenases like Ni-quercetinase (Ni-QueD). Ni-QueD catalyzes the oxidative ring opening of the polyphenol quercetin, a natural product with antioxidant properties. Herein, we report the synthesis and characterization of Ni(13-DOB), a Ni(II) species complexed by an N4-macrocycle that has been characterized by single crystal X-ray crystallography. Ni(13-DOB) forms a Ni-superoxide intermediate (Ni(13-DOB)O2•-) upon treatment with H2O2 and Et3N, as verified by resonance Raman spectroscopy. We demonstrate through UV/vis and LCMS that Ni(13-DOB)O2•- is capable of the 1-electron oxidation of flavonols, including both 3-hydroxyflavone (3-HF, the simplest flavonol) and quercetin itself. Incorporation of two O-atoms into the flavonol radical via superoxide from Ni(13-DOB)O2•- precedes oxidative cleavage of the flavonol scaffold in each case, consistent with quercetinase ring cleavage by Ni-QueD in Streptomyces sp. FLA. Conversion of 3-HF into 2-hydroxybenzoylbenzoic acid was accomplished with catalytic turnover of Ni(13-DOB) at ambient temperature, as confirmed by HPLC timecourses and GCMS analysis of isotopic labeling studies. The Ni(13-DOB)-mediated oxidative cleavage of quercetin to the corresponding biomimetic phenolic ester was also verified through 18O-isotopic labeling studies. Through the HPLC characterization of both on- and off-pathway products of flavonol dioxygenation by Ni(13-DOB)O2•-, the stringent reaction pathway control provided by enzyme active sites is highlighted.

The effects of metal cofactors on the reactivity of quercetin 2,4-dioxygenase: synthetic model studies with M(II)-complexes (M = Mn, Co, Ni, Cu, Zn) and assessment of the regulatory factors in catalytic efficacy.

This paper demonstrates the metal ion effects on the quercetin 2,4-dioxygenase (2,4-QD)-like reactivity. For this purpose, a series of five metal(II)-acetato complexes [MII(L)(OAc)] {M = Mn (1OAc), Co (2OAc), Ni (3OAc), Cu (4OAc), Zn (5OAc); OAc = acetate} supported with a newly designed N3O-donor carboxylato ligand L- {L- = 2-((benzyl((6'-methyl-[2,2'-bipyridin]-6-yl)methyl)amino)methyl)benzoate} has been synthesised as models for the active sites of MII-substituted 2,4-QDs. The enzyme-substrate (ES) model complexes [MII(L)(fla)] {M = Mn (1fla), Co (2fla), Ni (3fla), Cu (4fla), Zn (5fla); flaH = flavonol} have been synthesised by reacting flaH with their corresponding acetate-bound complexes in basic conditions. Detailed physicochemical properties of all the compounds are reported. Furthermore, single-crystal X-ray diffractions have been done to determine the structures of the compounds 2OAc·2H2O, 3OAc, 4OAc·CH2Cl2·2H2O, 5OAc·2H2O and 2fla·MeOH. The enzymatic reactivities of complexes 1OAc-5OAc towards the dioxygenation of flavonol have been explored in detail. All the complexes effectively catalyse the oxygenative degradation of flavonol in N,N-dimethylformamide (DMF) medium at 70 °C under multiple-turnover conditions and produce enzyme-type products. Kinetic investigations were performed to see the metal ions' effects on reactivity. The reaction rates vary with the metal ions, showing the order Co > Ni > Zn > Mn > Cu. The studies reveal that the reactivities of the [MII(L)(OAc)] complexes are governed primarily by three factors viz the ES adduct formation constant (Kf), the redox potential (Epa) of the bound fla-/fla˙ couple, and the degree of delocalisation of the fla˙ radical with the metal electrons, which are drastically influenced by the M2+ ions. In the mechanistic interpretation, a single-electron transfer (SET) from the bound-flavonolate to dioxygen has been proposed to generate the catalytically important "M(II)-fla˙" radical and superoxide ion, which react further to bring about the dioxygenation reaction. The identification of the metal(II)-bound flavonoxy radical intermediate for the case of cobalt using EPR spectroscopy and the detection of superoxide ion by NBT2+ test and EPR spin-trapping experiment (DMPO test) are remarkable in envisaging the reaction pathway.

Peroxy Intermediate Drives Carbon Bond Activation in the Dioxygenase AsqJ.

Dioxygenases catalyze stereoselective oxygen atom transfer in metabolic pathways of biological, industrial, and pharmaceutical importance, but their precise chemical principles remain controversial. The α-ketoglutarate (αKG)-dependent dioxygenase AsqJ synthesizes biomedically active quinolone alkaloids via desaturation and subsequent epoxidation of a carbon-carbon bond in the cyclopeptin substrate. Here, we combine high-resolution X-ray crystallography with enzyme engineering, quantum-classical (QM/MM) simulations, and biochemical assays to describe a peroxidic intermediate that bridges the substrate and active site metal ion in AsqJ. Homolytic cleavage of this moiety during substrate epoxidation generates an activated high-valent ferryl (FeIV = O) species that mediates the next catalytic cycle, possibly without the consumption of the metabolically valuable αKG cosubstrate. Our combined findings provide an important understanding of chemical bond activation principles in complex enzymatic reaction networks and molecular mechanisms of dioxygenases.

Differences in the Second Coordination Sphere Tailor the Substrate Specificity and Reactivity of Thiol Dioxygenases.

In recent years, considerable progress has been made toward elucidating the geometric and electronic structures of thiol dioxygenases (TDOs). TDOs catalyze the conversion of substrates with a sulfhydryl group to their sulfinic acid derivatives via the addition of both oxygen atoms from molecular oxygen. All TDOs discovered to date belong to the family of cupin-type mononuclear nonheme Fe(II)-dependent metalloenzymes. While most members of this enzyme family bind the Fe cofactor by two histidines and one carboxylate side chain (2-His-1-carboxylate) to provide a monoanionic binding motif, TDOs feature a neutral three histidine (3-His) facial triad. In this Account, we present a bioinformatics analysis and multiple sequence alignment that highlight the significance of the secondary coordination sphere in tailoring the substrate specificity and reactivity among the different TDOs. These insights provide the framework within which important structural and functional features of the distinct TDOs are discussed.The best studied TDO is cysteine dioxygenase (CDO), which catalyzes the conversion of cysteine to cysteine sulfinic acid in both eukaryotes and prokaryotes. Crystal structures of resting and substrate-bound mammalian CDOs revealed two surprising structural motifs in the first- and second coordination spheres of the Fe center. The first is the presence of the abovementioned neutral 3-His facial triad that coordinates the Fe ion. The second is the existence of a covalent cross-link between the sulfur of Cys93 and an ortho carbon of Tyr157 (mouse CDO numbering scheme). While the exact role of this cross-link remains incompletely understood, various studies established that it is needed for proper substrate Cys positioning and gating solvent access to the active site. Intriguingly, bacterial CDOs lack the Cys-Tyr cross-link; yet, they are as active as cross-linked eukaryotic CDOs.The other known mammalian TDO is cysteamine dioxygenase (ADO). Initially, it was believed that ADO solely catalyzes the oxidation of cysteamine to hypotaurine. However, it has recently been shown that ADO additionally oxidizes N-terminal cysteine (Nt-Cys) peptides, which indicates that ADO may play a much more significant role in mammalian physiology than was originally anticipated. Though predicted on the basis of sequence alignment, site-directed mutagenesis, and spectroscopic studies, it was not until last year that two crystal structures, one of wild-type mouse ADO (solved by us) and the other of a variant of nickel-substituted human ADO, finally provided direct evidence that this enzyme also features a 3-His facial triad. These structures additionally revealed several features that are unique to ADO, including a putative cosubstrate O2 access tunnel that is lined by two Cys residues. Disulfide formation under conditions of high O2 levels may serve as a gating mechanism to prevent ADO from depleting organisms of Nt-Cys-containing molecules.The combination of kinetic and spectroscopic studies in conjunction with structural characterizations of TDOs has furthered our understanding of enzymatic sulfhydryl substrate regulation. In this article, we take advantage of the fact that the ADO X-ray crystal structures provided the final piece needed to compare and contrast key features of TDOs, an essential family of metalloenzymes found across all kingdoms of life.

Preparation of carotenoid cleavage dioxygenases for X-ray crystallography.

Carotenoid cleavage dioxygenases (CCDs) constitute a superfamily of enzymes that are found in all domains of life where they play key roles in the metabolism of carotenoids and apocarotenoids as well as certain phenylpropanoids such as resveratrol. Interest in these enzymes stems not only from their biological importance but also from their remarkable catalytic properties including their regioselectivity, their ability to accommodate diverse substrates, and the additional activities (e.g., isomerase) that some of these enzyme possess. X-ray crystallography is a key experimental approach that has allowed detailed investigation into the structural basis behind the interesting biochemical features of these enzymes. Here, we describe approaches used by our lab that have proven successful in generating single crystals of these enzymes in resting or ligand-bound states for high-resolution X-ray diffraction analysis.

An artificial metalloprotein with metal-adaptive coordination sites and Ni-dependent quercetinase activity.

Engineering non-native metal active sites into proteins using canonical amino acids offers many advantages but is hampered by significant challenges. The TIM barrel protein, imidazole glycerol phosphate synthase from the hyperthermophilic organism Thermotoga maritima (tHisF), is well-suited for the construction of artificial metalloenzymes by this approach. To this end, we have generated a tHisF variant (tHisFEHH) with a Glu/His/His motif for metal ion coordination. Crystal structures of ZnII:tHisFEHH and NiII:tHisFEHH reveal that both metal ions bind to the engineered histidines. However, the two metals bind at distinct sites with different geometries, demonstrating the adaptability of tHisF. Only ZnII additionally ligates the Glu residue and adopts a tetrahedral geometry. The pseudo-octahedral NiII site comprises the two His and a native Ser residue. NiII:tHisFEHH catalyzes the oxidative cleavage of the flavanols quercetin and myricetin, providing an unprecedented example of an artificial metalloprotein with quercetinase activity.

Ten-Eleven Translocation-3 CXXC domain is critical for postfertilization demethylation and expression of pluripotency genes in pig embryos†.

Enzymes of the ten-eleven translocation family are considered to play an important role in the regulation of DNA methylation patterns by converting 5-methylcytosine to 5-hydroxymethylcytosine. Known as a maternal transcript enriched in mature oocytes, ten-eleven translocation-3 (TET3) has been suggested to initiate DNA demethylation of the paternal genome in zygotes. Previous studies in mouse cells indicate that the N-terminal CXXC domain of TET3 is important in catalyzing the oxidation of 5-methylcytosine through its potential DNA binding ability; however, it is not clear whether the DNA binding capacity of CXXC domain is required for the 5-hydroxymethylcytosine conversion in mammalian embryos. Here, we identified TET3 isoforms in porcine oocytes and investigated the role of the oocyte specific TET3 isoform (pTET3L) in controlling postfertilization demethylation in porcine embryos. The pTET3L possessed sequences representing a known DNA binding domain, the CXXC, and injection of the TET3 CXXC fused with GFP into mature porcine oocytes resulted in exclusive localization of the GFP-CXXC in the pronuclei. The CXXC overexpression reduced the 5-methylcytosine level in zygotes and enhanced the DNA demethylation of the NANOG promoter in 2-cell stage embryos. Furthermore, there was an increase in the transcript abundance of NANOG and ESRRB in blastocysts developed from GFP-CXXC injected oocytes. Targeted knockdown of pTET3L resulted in the downregulation of pluripotency genes in subsequently developed blastocysts. The findings indicate that the CXXC domain of TET3 serves as a critical component for the postfertilization demethylation of porcine embryos and coordinates proper expression of pluripotency related genes in blastocysts.

The ArsI C-As lyase: Elucidating the catalytic mechanism of degradation of organoarsenicals.

Organoarsenicals such as monosodium methylarsenate (MSMA or MAs(V)) and roxarsone (4-hydroxyl-3-nitrophenylarsenate or Rox(V)) have been extensively used as herbicides and growth enhancers for poultry, respectively. Degradation of organoarsenicals to inorganic arsenite (As(III)) contaminates crops and drinking water. One such process is catalyzed by the bacterial enzyme ArsI, whose gene is found in many soil bacteria. ArsI is a non-heme ferrous iron (Fe(II))-dependent dioxygenase that catalyzes oxygen-dependent cleavage of the carbon­arsenic (C-As) bond in trivalent organoarsenicals, degrading them to inorganic As(III). From previous crystal structures of ArsI, we predicted that a loop-gating mechanism controls the catalytic reaction. Understanding the catalytic mechanism of ArsI requires knowledge of the mechanisms of substrate binding and activation of dioxygen. Here we report new ArsI structures with bound Rox(III) and mutant enzymes with alteration of active site residues. Our results elucidate steps in the catalytic cycle of this novel dioxygenase and enhance understanding of the recycling of environmental organoarsenicals.

One-pot synthesis of dihydro-ß-ionone from carotenoids using carotenoid cleavage dioxygenase and enoate reductase.

Dihydro-ß-ionone is a characteristic aroma compound of Osmanthus fragrans and is widely applied in the flavor & fragrance industry. However, the main focus is on chemical synthesis due to the metabolic pathways of dihydro-ß-ionone is still unclear. Here, we explored the one-pot synthesis system for dihydro-ß-ionone production using carotenoid cleavage dioxygenase (CCD) and enoate reductase. After screening the CCD enzyme, PhCCD1 from the Petunia hybrid was identified as the suitable enzyme for the first step of dihydro-ß-ionone synthesis due to the high enzyme activity for carotenoid. The PhCCD1 was expressed in Escherichia coli and further characterized. The optimal activity of PhCCD1 was observed at pH 6.8 and 45 °C. The enzyme was stable over the pH range of 6.0-8.0 and had good thermal stability below 40 °C. Then, we optimized the coupled reaction conditions for dihydro-ß-ionone production by PhCCD1 and enoate reductase AaDBR1 from Artemisia annua. Furthermore, we introduced the NADPH regeneration system with a 1.5-fold enhancement for dihydro-ß-ionone production. Collectively, approximately 13.34 mg/L dihydro-ß-ionone was obtained by the one-pot biosystem with a corresponding molar conversion of 85.8%. For the first time, we successfully designed and constructed a new synthesis pathway for dihydro-ß-ionone production in vitro. The coupled catalysis reported herein illustrates the feasibility of producing dihydro-ß-ionone from carotenoids and guides further engineering in the food industry.

Oxygenolysis of a series of copper(II)-flavonolate adducts varying the electronic factors on supporting ligands as a mimic of quercetin 2,4-dioxygenase-like activity.

Four copper(II)-flavonolate compounds of type [Cu(LR)(fla)] {where LR = 2-(p-R-benzyl(dipyridin-2-ylmethyl)amino)acetate; R = -OMe (1), -H (2), -Cl (3) and -NO2 (4)} have been developed as a structural and functional enzyme-substrate (ES) model of the Cu2+-containing quercetin 2,4-dioxygenase enzyme. The ES model complexes 1-4 are synthesized by reacting 3-hydroxyflavone in the presence of a base with the respective acetate-bound copper(II) complexes, [Cu(LR)(OAc)]. In the presence of dioxygen the ES model complexes undergo enzyme-type oxygenolysis of flavonolate (dioxygenase type bond cleavage reaction) at 80 °C in DMF. The reactivity shows a substituent group dependent order as -OMe (1) > -H (2) > -Cl (3) > -NO2 (4). Experimental and theoretical studies suggest a single-electron transfer (SET) from flavonolate to dioxygen, rather than valence tautomerism {[CuII(fla-)] ↔ [CuI(fla˙)]}, to generate the reactive flavonoxy radical (fla˙) that reacts further with the superoxide radical to bring about the oxygenative ring opening reaction. The SET pathway has been further verified by studying the dioxygenation reaction with a redox-inactive Zn2+ complex, [Zn(LOMe)(fla)] (5).