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The work presented here investigates whether the TpMesFe+ moiety, which had been previously found to mediate the dioxygenation of cysteine and cysteamine, can do the same with simple thiols, which are not functionalized by an amine group. Accordingly, the complexes TpMesFeSR (R=Ph, iPr) were synthesized and after full characterization exposed to O2. They were found to react unselectively to yield sulfinates, disulfides, and thiosulfones as products. The sulfinate complex TpMesFeO2S‐p‐Tol was prepared independently and found to be air‐stable, suggesting that the additional products are not formed via overoxidation or subsequent reactions of coordinated sulfinate product. To investigate whether the larger reaction space, opened up due to the missing amine donor is responsible, a Tp‐ligand functionalized by a pyridyl donor was employed. A corresponding Tp‐iron(II) chlorido complex was prepared and converted through salt metathesis reactions into the respective thiolate analogues, which were fully characterized and structurally authenticated. However, subsequent O2 reactivity studies did not show an improved selectivity. The reason may lie in a more open reaction pocket in combination with an electronic situation which is less ideal than in case of TpMesFe(cysteamine). [ABSTRACT FROM AUTHOR]

The strain Achromobacter insolitus LCu2 isolated from the roots of alfalfa (Medicagosativa L.) utilized cinnamic acid and its methoxy derivatives (vanillic and ferulic acids) as the sole carbon source. Weak growth was observed with m-coumaric acid, but not on o- or p-coumaric acids. Growth on the cinnamic acid was slow and diauxic. The substrate decreased in the cultivation medium by 53%; the degradation efficiency during 14 days was 30 μg/mg wet weight. Despite the bactericidal effect of cinnamic acid, the culture A. insolitus LCu2 remained viable for a long time. Genomic analysis revealed two gene clusters (hca and mhp) responsible for the dihydroxylation of the phenyl ring (hcaA1A2CDB) and its subsequent cleavage to central metabolic products (mhpACDE), as well as a transcriptional regulator (hcaR) and a putative transporter (hcaT). A putative biochemical pathway for cinnamic acid degradation by the strain A. insolitus LCu2 was predicted using genomic data. [ABSTRACT FROM AUTHOR]

Alkylating modifications induced by either exogenous chemical agents or endogenous metabolites are some of the main types of damage to DNA, RNA, and proteins in the cell. Although research in recent decades has been almost entirely devoted to the repair of alkyl and in particular methyl DNA damage, more and more data lately suggest that the methylation of RNA bases plays an equally important role in normal functioning and in the development of diseases. Among the most prominent participants in the repair of methylation-induced DNA and RNA damage are human homologs of Escherichia coli AlkB, nonheme Fe(II)/α-ketoglutarate-dependent dioxygenases ABH1–8, and FTO. Moreover, some of these enzymes have been found to act on several protein targets. In this review, we present up-to-date data on specific features of protein structure, substrate specificity, known roles in the organism, and consequences of disfunction of each of the nine human homologs of AlkB. Special attention is given to reports about the effects of natural single-nucleotide polymorphisms on the activity of these enzymes and to potential consequences for carriers of such natural variants.

Vanillin is one of the world's most important flavor and fragrance compounds used in foods and cosmetics. In plants, vanillin is reportedly biosynthesized from ferulic acid via the hydratase/lyase-type enzyme VpVAN. However, in biotechnological and biocatalytic applications, the use of VpVAN limits the production of vanillin. Although microbial enzymes are helpful as substitutes for plant enzymes, synthesizing vanillin from ferulic acid in one step using microbial enzymes remains a challenge. Here, we developed a single enzyme that catalyzes vanillin production from ferulic acid in a coenzyme-independent manner via the rational design of a microbial dioxygenase in the carotenoid cleavage oxygenase family using computational simulations. This enzyme acquired catalytic activity toward ferulic acid by introducing mutations into the active center to increase its affinity for ferulic acid. We found that the single enzyme can catalyze not only the production of vanillin from ferulic acid but also the synthesis of other aldehydes from p-coumaric acid, sinapinic acid, and coniferyl alcohol. These results indicate that the approach used in this study can greatly expand the range of substrates available for the dioxygenase family of enzymes. The engineered enzyme enables efficient production of vanillin and other value-added aldehydes from renewable lignin-derived compounds. [ABSTRACT FROM AUTHOR]

Simple Summary: Ammonium (NH4+) is usually toxic to plant growth when used as the sole or dominant N source. Exploring the underlying molecular mechanisms of NH4+ toxicity and how to minimize NH4+ toxicity may greatly benefit crop productivity. In this study, the underlying mechanism of NH4+ toxicity and bicarbonate (HCO3−)-dependent alleviation in wheat was investigated. Comprehensive transcriptomic and physiological analyses suggested that NH4+ nutrition alone stimulated fermentation and glycolysis, promoted the activity of alternative respiratory pathways, suppressed TCA cycle pathways, and reduced ATP synthesis; adding HCO3− relieved the toxic effects of NH4+ nutrition. Our results reveal the importance of C and N interactions for alleviating NH4+ toxicity, likely by mitigating root hypoxic stress. As the first report on the hypoxic stress triggered by NH4+ treatment, this study provides novel insights into the mechanisms of NH4+ toxicity and its alleviation, which may present potential solutions for improving the nitrogen use efficiency in wheat. Ammonium (NH4+) toxicity is ubiquitous in plants. To investigate the underlying mechanisms of this toxicity and bicarbonate (HCO3−)-dependent alleviation, wheat plants were hydroponically cultivated in half-strength Hoagland nutrient solution containing 7.5 mM NO3− (CK), 7.5 mM NH4+ (SA), or 7.5 mM NH4+ + 3 mM HCO3− (AC). Transcriptomic analysis revealed that compared to CK, SA treatment at 48 h significantly upregulated the expression of genes encoding fermentation enzymes (pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH), and lactate dehydrogenase (LDH)) and oxygen consumption enzymes (respiratory burst oxidase homologs, dioxygenases, and alternative oxidases), downregulated the expression of genes encoding oxygen transporters (PIP-type aquaporins, non-symbiotic hemoglobins), and those involved in energy metabolism, including tricarboxylic acid (TCA) cycle enzymes and ATP synthases, but upregulated the glycolytic enzymes in the roots and downregulated the expression of genes involved in the cell cycle and elongation. The physiological assay showed that SA treatment significantly increased PDC, ADH, and LDH activity by 36.69%, 43.66%, and 61.60%, respectively; root ethanol concentration by 62.95%; and lactate efflux by 23.20%, and significantly decreased the concentrations of pyruvate and most TCA cycle intermediates, the complex V activity, ATP content, and ATP/ADP ratio. As a consequence, SA significantly inhibited root growth. AC treatment reversed the changes caused by SA and alleviated the inhibition of root growth. In conclusion, NH4+ treatment alone may cause hypoxic stress in the roots, inhibit energy generation, suppress cell division and elongation, and ultimately inhibit root growth, and adding HCO3− remarkably alleviates the NH4+-induced inhibitory effects on root growth largely by attenuating the hypoxic stress. [ABSTRACT FROM AUTHOR]

Bacterial degradation of natural rubber (NR) in an oxic environment is initiated by oxidative cleavage of double bonds in the NR-carbon backbone and is catalyzed by extracellular haem-containing rubber oxygenases. NR-cleavage products of sufficiently low molecular mass are taken up by the cells and metabolized for energy and biomass formation. Gram-negative and Gram-positive NR-degrading bacteria (usually) employ different types of rubber oxygenases such as RoxA and/or RoxB (most Gram-negative NR-degraders) or latex clearing protein Lcp (most Gram-positive NR-degraders). In order to find novel orthologues of Rox proteins, we have revisited databases and provide an update of Rox-like proteins. We describe the putative evolution of rubber oxygenases and confirm the presence of a third subgroup of Rox-related proteins (RoxCs), the biological function of which remains, however, unclear. We summarize the knowledge on the taxonomic position of Steroidobacter cummioxidans 35Y and related species. Comparison of genomic and biochemical features of strain 35Y with other species of the genus Steroidobacter suggests that strain 35Y represents a species of a novel genus for which the designation Aurantibaculum gen. nov. is proposed. A short summary on the capabilities of NR-degrading consortia, that could be superior in biotechnological applications compared to pure cultures, is also provided. Key points: • Three types of rubber oxygenases exist predominantly in Gram-negative microbes • S. cummioxidans 35Y contains RoxA and RoxB which are superior in activity • S. cummioxidans 35Y represents a species of a novel genus [ABSTRACT FROM AUTHOR]

Vanillin is a popular and versatile flavor compound, almost entirely produced from petroleum‐derived phenol by a multi‐step chemical synthesis. The process is hazardous to the environment and unsustainable for its fossil oil usage. Therefore, developing environmentally friendly, efficient, and sustainable routes to biobased vanillin is essential. Here, we report on vanillin production from 4‐n‐propylguaiacol (4PG), one of the main components in lignin oil obtained through reductive catalytic fractionation (RCF) of soft wood, by employing recombinant Escherichia coli cells. Conversion is based on the expression of two engineered oxidative enzymes: a 4‐n‐propylguaiacol oxidase and an isoeugenol dioxygenase. A high yield of vanillin, 66% from 4PG in RCF lignin oil was achieved through rounds of optimisation of the whole‐cell conversion process. This high‐performance strategy was readily scaled up to produce vanillin at an unprecedented 18% and 3% yield based on lignin oil and spruce wood respectively. The whole‐cell bioconversion process shows good tolerance even at high loadings of starting material, showcasing the robustness and applicability of the employed biocatalysts. This work paves the way for further development towards the efficient production of high‐titer biobased vanillin using depolymerised lignin as the feedstock. [ABSTRACT FROM AUTHOR]

Oxidative ring cleavage reactions have attracted great interest and various studies on the catechol ring-cleaving enzyme homoprotocatechuate dioxygenase (HPCD) have been reported in the literature. The available data on how the proton transfer takes place led us to design a potential HPCD model structure. A secondary sphere effect of utmost importance, the assistance of His200, which is critical for the catechol proton to migrate to dioxygen, was cautiously included on the first coordination shell. This was done mainly by modifying the axial ligands in the first coordination shell of HPCD such that the dual basic/acidic role in the proton transfer pathway of His200 was reproduced. Model systems with mono-, bi-, and tridentate ligands are reported. Energetically feasible reaction channels on synthetically promising ligand structures are identified. Key structural and electronic principles for obtaining viable proton transfer paths are outlined. [ABSTRACT FROM AUTHOR]

Background: Alkaptonuria is a rare metabolic autosomal recessive disorder. Its aetiology involves homogentisate 1,2 dioxygenase (HGD) deficiency resulting in homogentisic acid accumulation in the connective tissues (ochronosis). The classic triad of the disease is: i) homogentisic aciduria, ii) bluish-black pigmentation in tissues such as the sclera, cornea, cartilage and skin, and iii) degenerative arthropathy usually in the fourth decade of life. Case: In this case report, we present a 41-year-old man with diffuse musculoskeletal pain and additional clinical features in tissues such as the ear and urinary tract who was diagnosed late (>30 years). He was diagnosed with alkaptonuria based on clinical findings and elevated urinary homogentisic acid levels. Conclusion: This case report underscores the need for early diagnosis of alkaptonuria, which can help in managing symptoms and improving the quality of life of patients. Further research is needed to develop more targeted therapies for alkaptonuria, which can help slow down the progression of joint degeneration and improve overall patient. [ABSTRACT FROM AUTHOR]

A bacterial strain Citrobacter sp. HJS-1 was discovered from the sludge in a drainage canal of a coal mine. Firstly, its biodegradation capacity for benzo[a]pyrene (BaP) was detected under different concentrations. The results proved that the strain possessed excellent biodegradation capacity for BaP with high-efficiency degradation rates ranging from 78.9 to 86.8%. The highest degradation rate was observed in the low-concentration sample, and the high-concentration BaP had a slight influence on the biodegradation capacity due to the potential toxicity of BaP and its oxygen-containing derivatives. Meanwhile, the degradation test for the other five aromatic hydrocarbons (2- to 4-ring) proved that the strain had a comprehensive degradation potential. To clarify the biodegradation mechanism of BaP, a dioxygenase structure was constructed by homology modeling. Then, the interactions between dioxygenase and BaP were researched by molecular simulation. Combined with the identification of the vital BaP-cis-7,8-dihydrodiol intermediate and the interaction analysis, the initial oxidation mode and the binding site of BaP were revealed in the dioxygenase. Taken together, this study has offered a way to understand the biodegradation process of BaP and its interaction mechanism based on experimental and theoretical analysis. [ABSTRACT FROM AUTHOR]

Wild-type cells of Rhodococcuspyridinivorans 5Ap were found to be highly efficient naphthalene degraders, completely utilizing this compound (500 mg/L) after 3 days, and may be used for remediation of naphthalene-contaminated aquatic ecosystems. Inactivation of the biodegradation genes narAa (encoding the large subunit of naphthalene dioxygenase) and narB (encoding cis-naphthalene dihydrodiol dehydrogenase) resulted it the loss of ability to use naphthalene as the sole energy source, which indicated the absence in the genome of R.pyridinivorans 5Ap of the determinants responsible for alternative pathways of naphthalene oxidation. Moreover, narB inactivation resulted in accumulation of a polar colored compound (probably a product of primary naphthalene oxidation) in the medium. [ABSTRACT FROM AUTHOR]

Diphenyl ethers (DEs), which are widely used in the agricultural and chemical industries, have become hazardous contaminants in the environment. Although several DE-degrading bacteria have been reported, discovering new types of such microorganisms could enhance understanding of the degradation mechanism in the environment. In this study, we used a direct screening method based on detection of ether bond–cleaving activity to screen for microorganisms that degrade 4,4′-dihydroxydiphenyl ether (DHDE) as a model DE. Microorganisms isolated from soil samples were incubated with DHDE, and strains producing hydroquinone via ether bond cleavage were selected using hydroquinone-sensitive Rhodanine reagent. This screening procedure resulted in the isolation of 3 bacteria and 2 fungi that transform DHDE. Interestingly, all of the isolated bacteria belonged to one genus, Streptomyces. To our knowledge, these are the first microorganisms of the genus Streptomyces shown to degrade a DE. Streptomyces sp. TUS-ST3 exhibited high and stable DHDE-degrading activity. HPLC, LC-MS, and GC–MS analyses revealed that strain TUS-ST3 converts DHDE to its hydroxylated analogue and generates hydroquinone as an ether bond–cleavage product. Strain TUS-ST3 also transformed DEs other than DHDE. In addition, glucose-grown TUS-ST3 cells began to transform DHDE after incubation with this compound for 12 h, and produced 75 μM hydroquinone in 72 h. These activities of streptomycetes may play an important role in DE degradation in the environment. We also report the whole genome sequence of strain TUS-ST3. [Display omitted] • Streptomyces strains transforming DEs were discovered for the first time. • Streptomyces sp. TUS-ST3 generated hydroquinone as an ether bond cleavage product of DHDE. • Strain TUS-ST3 also exhibited transforming activity toward DEs other than DHDE. [ABSTRACT FROM AUTHOR]

The enzyme β-carotene oxygenase 2 (BCO2) converts carotenoids into more polar metabolites. Studies in mammals, fish, and birds revealed that BCO2 controls carotenoid homeostasis and is involved in the pathway for vitamin A production. However, it is controversial whether BCO2 function is conserved in humans, because of a 4-amino acid long insertion caused by a splice acceptor site polymorphism. We here show that human BCO2 splice variants, BCO2a and BCO2b, are expressed as pre-proteins with mitochondrial targeting sequence (MTS). The MTS of BCO2a directed a green fluorescent reporter protein to the mitochondria when expressed in ARPE-19 cells. Removal of the MTS increased solubility of BCO2a when expressed in Escherichia coli and rendered the recombinant protein enzymatically active. The expression of the enzymatically active recombinant human BCO2a was further improved by codon optimization and its fusion with maltose-binding protein. Introduction of the 4-amino acid insertion into mouse Bco2 did not impede the chimeric enzymes catalytic proficiency. We further showed that the chimeric BCO2 displayed broad substrate specificity and converted carotenoids into two ionones and a central C14-apocarotendial by oxidative cleavage reactions at C9,C10 and C9,C10. Thus, our study demonstrates that human BCO2 is a catalytically competent enzyme. Consequently, information on BCO2 becomes broadly applicable in human biology with important implications for the physiology of the eyes and other tissues.

Rhodococcus biphenylivorans GA1 was successfully isolated, which can efficiently degrade alkali lignin and a variety of lignin-derived aromatic compounds as the sole carbon source. Whole genome sequencing of strain GA1 showed that it possessed G + C content of 68% with the size of 6.0 Mb and 4319 putative open reading frames (ORFs). Four replicons consisting of one circular chromosome (ChrA1) and three circular plasmids (pGA1, pGA2, pGA3) were found. Among these annotated proteins, lignin depolymerizing peroxidases (Dyp) and two lignin-derived aromatic compounds cleavage dioxygenases, protocatechuate 3,4-dioxygenase(P34D) and catechol-1,2-dioxygenase (C12D) play key roles in the catabolism of lignin. [ABSTRACT FROM AUTHOR]

Protocatechuate 4,5-dioxygenase (LigAB) is a heterodimeric enzyme that catalyzes the dioxygenation of multiple lignin derived aromatic compounds. The active site of LigAB is at the heterodimeric interface, with specificity conferred by the alpha subunit and catalytic residues contributed by the beta subunit. Previous research has indicated that the phenylalanine at the 103 position of the alpha subunit (F103α) controls selectivity for the C5 position of the aromatic substrates, and mutations of this residue can enhance the rate of catalysis for substrates with larger functional groups at this position. While several of the mutations to this position (Valine, V; Threonine, T; Leucine, L; and Histidine, H) were catalytically active, other mutations (Alanine, A; and Serine, S) were found to have reduced dimer interface affinity, leading to challenges in copurifing the catalytically active enzyme complex under high salt conditions. In this study, we aimed to experimentally and computationally interrogate residues at the dimer interface to discern the importance of position 103α for maintaining the integrity of the heterodimer. Molecular dynamic simulations and electrophoretic mobility assays revealed a preference for nonpolar/aromatic amino acids in this position, suggesting that while substitutions to polar amino acids may produce a dioxygenase with a useful substrate utilization profile, those considerations may be off-set by potential destabilization of the catalytically active oligomer. Understanding the dimerization of LigAB provides insight into the multimeric proteins within the largely uncharacterized superfamily and characteristics to consider when engineering proteins that can degrade lignin efficiently. These results shed light on the challenges associated with engineering proteins for broader substrate specificity. [ABSTRACT FROM AUTHOR]

Fe(II)/2OG-dependent dioxygenases of the AlkB family catalyze a direct removal of alkylated damages in the course of DNA and RNA repair. A human homolog of the E. coli AlkB ALKBH3 protein is able to hydroxylate N1-methyladenine, N3-methylcytosine, and N1-methylguanine in single-stranded DNA and RNA. Due to its contribution to an antitumor drug resistance, this enzyme is considered a promising therapeutic target. The elucidation of ALKBH3's structural peculiarities is important to establish a detailed mechanism of damaged DNA recognition and processing, as well as to the development of specific inhibitors. This work presents new data on the wild type ALKBH3 protein and its four mutant forms (Y143F, Y143A, L177A, and H191A) obtained by circular dichroism (CD) spectroscopy. The dataset includes the CD spectra of proteins measured at different temperatures and a 3D visualization of the ALKBH3–DNA complex where the mutated amino acid residues are marked. These results show how substitution of the key amino acids influences a secondary structure content of the protein. Dataset: https://doi.org/10.17632/7bfsjtkgtb.1 Dataset License: CC BY 4.0 [ABSTRACT FROM AUTHOR]

Ammonium (NH4+) toxicity is ubiquitous in plants. To investigate the underlying mechanisms of this toxicity and bicarbonate (HCO3−)-dependent alleviation, wheat plants were hydroponically cultivated in half-strength Hoagland nutrient solution containing 7.5 mM NO3− (CK), 7.5 mM NH4+ (SA), or 7.5 mM NH4+ + 3 mM HCO3− (AC). Transcriptomic analysis revealed that compared to CK, SA treatment at 48 h significantly upregulated the expression of genes encoding fermentation enzymes (pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH), and lactate dehydrogenase (LDH)) and oxygen consumption enzymes (respiratory burst oxidase homologs, dioxygenases, and alternative oxidases), downregulated the expression of genes encoding oxygen transporters (PIP-type aquaporins, non-symbiotic hemoglobins), and those involved in energy metabolism, including tricarboxylic acid (TCA) cycle enzymes and ATP synthases, but upregulated the glycolytic enzymes in the roots and downregulated the expression of genes involved in the cell cycle and elongation. The physiological assay showed that SA treatment significantly increased PDC, ADH, and LDH activity by 36.69%, 43.66%, and 61.60%, respectively; root ethanol concentration by 62.95%; and lactate efflux by 23.20%, and significantly decreased the concentrations of pyruvate and most TCA cycle intermediates, the complex V activity, ATP content, and ATP/ADP ratio. As a consequence, SA significantly inhibited root growth. AC treatment reversed the changes caused by SA and alleviated the inhibition of root growth. In conclusion, NH4+ treatment alone may cause hypoxic stress in the roots, inhibit energy generation, suppress cell division and elongation, and ultimately inhibit root growth, and adding HCO3− remarkably alleviates the NH4+-induced inhibitory effects on root growth largely by attenuating the hypoxic stress.

Carotenoid cleavage dioxygenases (CCDs) use a nonheme Fe(II) cofactor to split alkene bonds of carotenoid and stilbenoid substrates. The iron centers of CCDs are typically five-coordinate in their resting states, with solvent occupying an exchangeable site. The involvement of this iron-bound solvent in CCD catalysis has not been experimentally addressed, but computational studies suggest two possible roles. 1) Solvent dissociation provides a coordination site for O2, or 2) solvent remains bound to iron but changes its equilibrium position to allow O2 binding and potentially acts as a proton source. To test these predictions, we investigated isotope effects (H2O versus D2O) on two stilbenoid-cleaving CCDs, Novosphingobium aromaticivorans oxygenase 2 (NOV2) and Neurospora crassa carotenoid oxygenase 1 (CAO1), using piceatannol as a substrate. NOV2 exhibited an inverse isotope effect (k H/k D ∼ 0.6) in an air-saturated buffer, suggesting that solvent dissociates from iron during the catalytic cycle. By contrast, CAO1 displayed a normal isotope effect (k H/k D ∼ 1.7), suggesting proton transfer in the rate-limiting step. X-ray absorption spectroscopy on NOV2 and CAO1 indicated that the protonation states of the iron ligands are unchanged within pH 6.5-8.5 and that the Fe(II)-aquo bond is minimally altered by substrate binding. We pinpointed the origin of the differential kinetic behaviors of NOV2 and CAO1 to a single amino acid difference near the solvent-binding site of iron, and X-ray crystallography revealed that the substitution alters binding of diffusible ligands to the iron center. We conclude that solvent-iron dissociation and proton transfer are both associated with the CCD catalytic mechanism.

Pipecolic acid (Pip) and its derivative hydroxypipecolic acids, such as (2S,3R)-3-hydroxypipecolic acid (cis-3-L-HyPip), are components of many natural and synthetic bioactive molecules. Fe(II)/α-ketoglutaric acid (Fe(II)/2-OG)-dependent dioxygenases can catalyze the hydroxylation of pipecolic acid. However, the available enzymes with desired activity and selectivity are limited. Herein, we compare the possible candidates in the Fe(II)/2-OG-dependent dioxygenase family, and cis-P3H is selected for potentially catalyzing selective hydroxylation of L-Pip. cis-P3H was further engineered to increase its catalytic efficiency toward L-Pip. By analyzing the structural confirmation and residue composition in substrate-binding pocket, a "handlebar" mode of molecular interactions is proposed. Using molecular docking, virtual mutation analysis, and dynamic simulations, R97, E112, L57, and G282 were identified as the key residues for subsequent site-directed saturation mutagenesis of cis-P3H. Consequently, the variant R97M showed an increased catalytic efficiency toward L-Pip. In this study, the kcat/Km value of the positive mutant R97M was about 1.83-fold that of the wild type. The mutation R97M would break the salt bridge between R97 and L-Pip and weaken the positive-positive interaction between R97 and R95. Therefore, the force on the amino and carboxyl groups of L-Pip was lightly balanced, allowing the molecule to be stabilized in the active pocket. These results provide a potential way of improving cis-P3H catalytic activity through rational protein engineering. [ABSTRACT FROM AUTHOR]

Nitrated polycyclic aromatic hydrocarbons (nitro-PAHs) enter the environment from natural sources and anthropogenic activities. To date, microorganisms able to mineralize nitro-PAHs have not been reported. Here, Sphingobium sp. strain JS3065 was isolated by selective enrichment for its ability to grow on 1-nitronaphthalene as the sole carbon, nitrogen, and energy source. Analysis of the complete genome of strain JS3065 indicated that the gene cluster encoding 1-nitronaphthalene catabolism (nin) is located on a plasmid. Based on the genetic and biochemical evidence, the nin genes share an origin with the nag-like genes encoding naphthalene degradation in Ralstonia sp. strain U2. The initial step in degradation of 1-nitronaphthalene is catalyzed by a three-component dioxygenase, NinAaAbAcAd, resulting in formation of 1,2-dihydroxynaphthalene which is also an early intermediate in the naphthalene degradation pathway. Introduction of the ninAaAbAcAd genes into strain U2 enabled its growth on 1-nitronaphthalene. Phylogenic analysis of NinAc suggested that an ancestral 1-nitronaphthalene dioxygenase was an early step in the evolution of nitroarene dioxygenases. Based on bioinformatic analysis and enzyme assays, the subsequent assimilation of 1,2-dihydroxynaphthalene seems to follow the well-established pathway for naphthalene degradation by Ralstonia sp. strain U2. This is the first report of catabolic pathway for 1-nitronaphthalene and is another example of how expanding the substrate range of Rieske type dioxygenase enables bacteria to grow on recalcitrant nitroaromatic compounds. [ABSTRACT FROM AUTHOR]

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The opportunistic pathogen Pseudomonas aeruginosa uses quorum sensing to control its virulence. One of its major signal molecules, the Pseudomonas quinolone signal PQS, has high affinity to membranes and is known to be trafficked mainly via outer membrane vesicles (OMVs). We previously reported that several 3-hydroxy-4(1H)-quinolone 2,4-dioxygenases (HQDs) catalyze the cleavage of PQS and thus act as quorum quenching enzymes. Further analysis showed that, in contrast to other HQDs, the activity of HQD from Streptomyces bingchenggensis (HQDS.b.) was unexpectedly stabilized by culture supernatants of P. aeruginosa. Interestingly, the stabilizing effect was higher with supernatants from the strain PA14 than with supernatants from the strain PAO1. Heat treatment and lyophilization hardly affected the stabilizing effect; however, fractionation of the supernatant excluded small molecules as stabilizing agents. In a pull-down assay, HQDS.b. appeared to interact with several P. aeruginosa proteins previously found in the OMV proteome. This prompted us to probe the physical interaction of HQDS.b. with prepared extracellular membrane vesicles. Homo-FRET of fluorescently labeled HQDS.b. indeed indicated a spatial clustering of the protein on the vesicles. Binding of a PQS-cleaving enzyme to the OMVs of P. aeruginosa may enhance PQS degradation and is highly reconcilable with its function as a quorum quenching enzyme. [ABSTRACT FROM AUTHOR]

To analyze the mechanisms involved in anthracene (ANT) degradation in the marine alga Ulva lactuca, total RNA was obtained from the alga cultivated without ANT and with 5μM of ANT for 24 h, and transcriptomic analyses were performed. A de novo transcriptome was assembled, transcripts di􀀀erentially expressed were selected, and those overexpressed were identified. Overexpressed transcripts potentially involved in ANT degradation were: one aromatic ring dioxygenase, three 2-oxoglutarate Fe (II) dioxygenases (2-OGDOs), and three dienelactone hydrolases that may account for anthraquinone, phthalic anhydride, salicylic acid, and phthalic acid production (pathway 1). In addition, two flavin adenine dinucleotide (FAD)-dependent monooxygenases, four cytP450 monooxygenases, two epoxide hydrolase, one hydroxyphenylpyruvic acid dioxygenase (HPPDO), and two homogentisic acid dioxygenases (HGDOs) were identified that may also participate in ANT degradation (pathway 2). Moreover, an alkane monooxygenase (alkB), two alcohol dehydrogenases, and three aldehyde dehydrogenases were identified, which may participate in linear hydrocarbon degradation (pathway 3). Furthermore, the level of transcripts encoding some of mentioned enzymes were quantified by qRT-PCR are in the alga cultivated with 5 μM of ANT for 0-48 h, and those more increased were 2-OGDO, HGDO, and alkB monooxygenase. Thus, at least three pathways for ANT and linear hydrocarbons degradation may be existed in U. lactuca. In addition, ANTmetabolites were analyzed by gas chromatography and mass spectrometry (GC-MS), allowing the identification of anthraquinone, phthalic anhydride, salicylic acid, and phthalic acid, thus validating the pathway 1. [ABSTRACT FROM AUTHOR]

Alkylating modifications induced by either exogenous chemical agents or endogenous metabolites are some of the main types of damage to DNA, RNA, and proteins in the cell. Although research in recent decades has been almost entirely devoted to the repair of alkyl and in particular methyl DNA damage, more and more data lately suggest that the methylation of RNA bases plays an equally important role in normal functioning and in the development of diseases. Among the most prominent participants in the repair of methylation-induced DNA and RNA damage are human homologs of Escherichia coli AlkB, nonheme Fe(II)/α-ketoglutarate-dependent dioxygenases ABH1-8, and FTO. Moreover, some of these enzymes have been found to act on several protein targets. In this review, we present up-to-date data on specific features of protein structure, substrate specificity, known roles in the organism, and consequences of disfunction of each of the nine human homologs of AlkB. Special attention is given to reports about the effects of natural single-nucleotide polymorphisms on the activity of these enzymes and to potential consequences for carriers of such natural variants.

To analyze the mechanisms involved in anthracene (ANT) degradation in the marine alga Ulva lactuca, total RNA was obtained from the alga cultivated without ANT and with 5 μM of ANT for 24 h, and transcriptomic analyses were performed. A de novo transcriptome was assembled, transcripts differentially expressed were selected, and those overexpressed were identified. Overexpressed transcripts potentially involved in ANT degradation were: one aromatic ring dioxygenase, three 2-oxoglutarate Fe (II) dioxygenases (2-OGDOs), and three dienelactone hydrolases that may account for anthraquinone, phthalic anhydride, salicylic acid, and phthalic acid production (pathway 1). In addition, two flavin adenine dinucleotide (FAD)-dependent monooxygenases, four cytP450 monooxygenases, two epoxide hydrolase, one hydroxyphenylpyruvic acid dioxygenase (HPPDO), and two homogentisic acid dioxygenases (HGDOs) were identified that may also participate in ANT degradation (pathway 2). Moreover, an alkane monooxygenase (alkB), two alcohol dehydrogenases, and three aldehyde dehydrogenases were identified, which may participate in linear hydrocarbon degradation (pathway 3). Furthermore, the level of transcripts encoding some of mentioned enzymes were quantified by qRT-PCR are in the alga cultivated with 5 μM of ANT for 0–48 h, and those more increased were 2-OGDO, HGDO, and alkB monooxygenase. Thus, at least three pathways for ANT and linear hydrocarbons degradation may be existed in U. lactuca. In addition, ANT metabolites were analyzed by gas chromatography and mass spectrometry (GC–MS), allowing the identification of anthraquinone, phthalic anhydride, salicylic acid, and phthalic acid, thus validating the pathway 1.

The columnar apple cultivar 'McIntosh Wijcik' was discovered as a spontaneous mutant from the top of a 'McIntosh' tree in the early 1960s. 'McIntosh Wijcik' exhibits the columnar growth phenotype: compact and sturdy growth, short internodes, and very few lateral shoots. Classical genetic analysis revealed that the columnar growth phenotype of 'McIntosh Wijcik' is controlled by a single dominant gene, Co. This review focuses on the advances made toward understanding the molecular mechanisms of columnar growth in the last decade. Molecular studies have shown that an 8.2 kb insertion in the intergenic region of the Co locus is responsible for the columnar growth phenotype of 'McIntosh Wijcik', implying that the insertion affects the expression patterns of adjacent genes. Among the candidate genes in the Co region, the expression pattern of MdDOX-Co, putatively encoding 2-oxoglutarate-dependent dioxygenase (DOX), was found to vary between columnar and non-columnar apples. Recent studies have found three functions of MdDOX-Co: facilitating bioactive gibberellin deficiency, increasing strigolactone levels, and positively regulating abscisic acid levels. Consequently, changes in these plant hormone levels caused by the ectopic expression of MdDOX-Co in the aerial organs of 'McIntosh Wijcik' can lead to dwarf trees with fewer lateral branches. These findings will contribute to the breeding and cultivation of new columnar apple cultivars with improved fruit quality. [ABSTRACT FROM AUTHOR]

The vicinal oxygen chelate (VOC) metalloenzyme superfamily catalyzes a highly diverse set of reactions with the mechanism characterized by the bidentate coordination of vicinal oxygen atoms to metal ion centers, but there remains a lack of a platform to steer the reaction trajectories, especially for o‐quinone metabolizing pathways. Herein, we present the directed‐evolution‐enabled bifunctional turnover of ChaP, which is a homotetramer and represents an unprecedented VOC enzyme class. Unlike the ChaP catalysis of extradiol‐like o‐quinone cleavage and concomitant α‐keto acid decarboxylation, a group of ChaP variants (CVs) catalyze intradiol‐like o‐quinone deconstruction and CO2 liberation from the resulting o‐hydroxybenzoic acid scaffolds with high regioselectivity. Enzyme crystal structures, labeling experiments and computational simulations corroborated that the D49L mutation allows the metal ion to change its coordination with the tyrosine phenoxy atoms in different monomers, thereby altering the reaction trajectory with the regiospecificity further improved by the follow‐up replacement of the Y92 residue with any of alanine, glycine, threonine, and serine. The study highlights the unpredicted catalytic versatility and enzymatic plasticity of VOC enzymes with biotechnological significance. [ABSTRACT FROM AUTHOR]

Stilbenes are diphenyl ethene compounds produced naturally in a wide variety of plant species and some bacteria. Stilbenes are also derived from lignin during kraft pulping. Stilbene cleavage oxygenases (SCOs) cleave the central double bond of stilbenes, forming two phenolic aldehydes. Here, we report the structure of an SCO. The X-ray structure of NOV1 from Novosphingobium aromaticivorans was determined in complex with its substrate resveratrol (1.89 Å), its product vanillin (1.75 Å), and without any bound ligand (1.61 Å). The enzyme is a seven-bladed β-propeller with an iron cofactor coordinated by four histidines. In all three structures, dioxygen is observed bound to the iron in a side-on fashion. These structures, along with EPR analysis, allow us to propose a mechanism in which a ferric-superoxide reacts with substrate activated by deprotonation of a phenol group at position 4 of the substrate, which allows movement of electron density toward the central double bond and thus facilitates reaction with the ferric superoxide electrophile. Correspondingly, NOV1 cleaves a wide range of other stilbene-like compounds with a 4'-OH group, offering potential in processing some solubilized fragments of lignin into monomer aromatic compounds.

Pharmaceuticals are relatively new to nature and often not completely removed in wastewater treatment plants (WWTPs). Consequently, these micropollutants end up in water bodies all around the world posing a great environmental risk. One exception to this recalcitrant conversion is paracetamol, whose full degradation has been linked to several microorganisms. However, the genes and corresponding proteins involved in microbial paracetamol degradation are still elusive. In order to improve our knowledge of the microbial paracetamol degradation pathway, we inoculated a bioreactor with sludge of a hospital WWTP (Pharmafilter, Delft, NL) and fed it with paracetamol as the sole carbon source. Paracetamol was fully degraded without any lag phase and the enriched microbial community was investigated by metagenomic and metatranscriptomic analyses, which demonstrated that the microbial community was very diverse. Dilution and plating on paracetamol-amended agar plates yielded two Pseudomonas sp. isolates: a fast-growing Pseudomonas sp. that degraded 200 mg/L of paracetamol in approximately 10 h while excreting 4-aminophenol, and a slow-growing Pseudomonas sp. that degraded paracetamol without obvious intermediates in more than 90 days. Each Pseudomonas sp. contained a different highly-expressed amidase (31% identity to each other). These amidase genes were not detected in the bioreactor metagenome suggesting that other as-yet uncharacterized amidases may be responsible for the first biodegradation step of paracetamol. Uncharacterized deaminase genes and genes encoding dioxygenase enzymes involved in the catabolism of aromatic compounds and amino acids were the most likely candidates responsible for the degradation of paracetamol intermediates based on their high expression levels in the bioreactor metagenome and the Pseudomonas spp. genomes. Furthermore, cross-feeding between different community members might have occurred to efficiently degrade paracetamol and its intermediates in the bioreactor. This study increases our knowledge about the ongoing microbial evolution towards biodegradation of pharmaceuticals and points to a large diversity of (amidase) enzymes that are likely involved in paracetamol metabolism in WWTPs.

Isonitrile lipopeptides discovered from Actinobacteria have attracted wide attention due to their fascinating biosynthetic pathways and relevance to the virulence of many human pathogens including Mycobacterium tuberculosis. Specifically, the identification of the new class of isonitrile-forming enzymes that belong to non-heme iron (II) and α-ketoglutarate dependent dioxygenases has intrigued several research groups to investigate their catalytic mechanism. Here we summarize the recent studies on the biosynthesis of isonitrile lipopeptides from Streptomyces and Mycobacterium. The latest research on the core and tailoring enzymes involved in the pathway as well as the isonitrile metabolic enzymes are discussed in this review. [ABSTRACT FROM AUTHOR]

Protocatechuate 4,5-dioxygenase (LigAB) is a heterodimeric enzyme that catalyzes the dioxygenation of multiple lignin derived aromatic compounds. The active site of LigAB is at the heterodimeric interface, with specificity conferred by the alpha subunit and catalytic residues contributed by the beta subunit. Previous research has indicated that the phenylalanine at the 103 position of the alpha subunit (F103α) controls selectivity for the C5 position of the aromatic substrates, and mutations of this residue can enhance the rate of catalysis for substrates with larger functional groups at this position. While several of the mutations to this position (Valine, V; Threonine, T; Leucine, L; and Histidine, H) were catalytically active, other mutations (Alanine, A; and Serine, S) were found to have reduced dimer interface affinity, leading to challenges in copurifing the catalytically active enzyme complex under high salt conditions. In this study, we aimed to experimentally and computationally interrogate residues at the dimer interface to discern the importance of position 103α for maintaining the integrity of the heterodimer. Molecular dynamic simulations and electrophoretic mobility assays revealed a preference for nonpolar/aromatic amino acids in this position, suggesting that while substitutions to polar amino acids may produce a dioxygenase with a useful substrate utilization profile, those considerations may be off-set by potential destabilization of the catalytically active oligomer. Understanding the dimerization of LigAB provides insight into the multimeric proteins within the largely uncharacterized superfamily and characteristics to consider when engineering proteins that can degrade lignin efficiently. These results shed light on the challenges associated with engineering proteins for broader substrate specificity.

Fe(II)/2OG-dependent dioxygenases of the AlkB family catalyze a direct removal of alkylated damages in the course of DNA and RNA repair. A human homolog of the E. coli AlkB ALKBH3 protein is able to hydroxylate N1-methyladenine, N3-methylcytosine, and N1-methylguanine in single-stranded DNA and RNA. Due to its contribution to an antitumor drug resistance, this enzyme is considered a promising therapeutic target. The elucidation of ALKBH3’s structural peculiarities is important to establish a detailed mechanism of damaged DNA recognition and processing, as well as to the development of specific inhibitors. This work presents new data on the wild type ALKBH3 protein and its four mutant forms (Y143F, Y143A, L177A, and H191A) obtained by circular dichroism (CD) spectroscopy. The dataset includes the CD spectra of proteins measured at different temperatures and a 3D visualization of the ALKBH3–DNA complex where the mutated amino acid residues are marked. These results show how substitution of the key amino acids influences a secondary structure content of the protein.

The present article will not attempt to deal with sulfide per se as a signaling molecule but will aim to examine the consequences of sulfide oxidation by mitochondrial sulfide quinone reductase in mammalian cells. This oxidation appears first as a priority to avoid self-poisoning by endogenous sulfide and second to occur with the lowest ATP/O2 ratio when compared to other mitochondrial substrates. This is explained by the injection of electrons in the respiratory chain after complex I (as for succinate) and by a sulfur oxidation step implying a dioxygenase that consumes oxygen but does not contribute to mitochondrial bioenergetics. Both contribute to increase cellular oxygen consumption if sulfide is provided below its toxic level (low µM). Accordingly, if oxygen supply or respiratory chain activity becomes a limiting factor, small variations in sulfide release impact the cellular ATP/ADP ratio, a major metabolic sensor. [ABSTRACT FROM AUTHOR]

Polycyclic aromatic hydrocarbons (PAHs), products from the incomplete combustion of crude oil, are pollutants present in nature. Ring hydroxylating dioxygenase enzymes are able to catalyze polycyclic aromatic hydrocarbons in the biodegradation process with a high degree of stereo-, regio-, and enantiospecificity. In this work, we present the first approximation of the binding modes of 9 PAHs with high aromaticity in the catalytic sites of biphenyl or naphthalene dioxygenases from four microorganisms usually used in bio-remediation processes: Sphingobium yanoikuyae, Rhodococcus jostii RHA1, Pseudomonas sp. C18, and Paraburkholderia xenovorans. Molecular modeling studies of two biphenyl dioxygenases from Sphingobium yanoikuyae and Paraburkholderia xenovorans showed good binding affinity for PAHs with 2–4 benzene rings (fluoranthene, pyrene, and chrysene), and both enzymes had a similar amount of substrate binding. Molecular docking studies using naphthalene dioxygenase from Pseudomonas sp. C18 showed that the enzyme is able to accommodate PAHs with high aromaticity (benzo(a)pyrene, indeno(1,2,3-cd)pyrene), with good docking scores. This study provides important insight into the utility of naphthalene dioxygenases in the degradation of HAPs with high aromaticity. [ABSTRACT FROM AUTHOR]

Carotenoid metabolism is critical for diverse physiological processes. The nematode Caenorhabditis elegans has two genes that are annotated as β-carotene 15,15′-monooxygenase (BCMO) and are 17 centimorgan apart on chromosome II, but the function of BCMO-1 and BCMO-2 remains uncharacterized. Sequence homology indicates that the two enzymes belong to the carotenoid cleavage dioxygenase family that share a seven-bladed β-propeller fold with a nonheme iron center. Here we determined crystal structures of BCMO-1 and BCMO-2 at resolutions of 1.8 and 1.9 Å, respectively. Structural analysis reveals that BCMO-1 and BCMO-2 are strikingly similar to each other. We also characterized their β-carotene cleavage activity, but the results suggest that they may not act as β-carotene 15,15′-oxygenases.

Pipecolic acid (Pip) and its derivative hydroxypipecolic acids, such as (2S,3R)-3-hydroxypipecolic acid (cis-3-L-HyPip), are components of many natural and synthetic bioactive molecules. Fe(II)/α-ketoglutaric acid (Fe(II)/2-OG)-dependent dioxygenases can catalyze the hydroxylation of pipecolic acid. However, the available enzymes with desired activity and selectivity are limited. Herein, we compare the possible candidates in the Fe(II)/2-OG-dependent dioxygenase family, and cis-P3H is selected for potentially catalyzing selective hydroxylation of L-Pip. cis-P3H was further engineered to increase its catalytic efficiency toward L-Pip. By analyzing the structural confirmation and residue composition in substrate-binding pocket, a “handlebar” mode of molecular interactions is proposed. Using molecular docking, virtual mutation analysis, and dynamic simulations, R97, E112, L57, and G282 were identified as the key residues for subsequent site-directed saturation mutagenesis of cis-P3H. Consequently, the variant R97M showed an increased catalytic efficiency toward L-Pip. In this study, the kcat/Km value of the positive mutant R97M was about 1.83-fold that of the wild type. The mutation R97M would break the salt bridge between R97 and L-Pip and weaken the positive-positive interaction between R97 and R95. Therefore, the force on the amino and carboxyl groups of L-Pip was lightly balanced, allowing the molecule to be stabilized in the active pocket. These results provide a potential way of improving cis-P3H catalytic activity through rational protein engineering.

ABSTRACT Perfluorinated carbon atoms in a diether linkage are common in commercial anesthetics, drugs, fungicides, and insecticides. An important chemical group comprising perfluorodiethers is the 2,2-fluoro-1,3-benzodioxole (DFBD) moiety. The fluorine atoms stabilize the molecule by mitigating against metabolism by humans and microbes, as used in drugs and pesticides, respectively. Pseudomonas putida F1 catalyzed defluorination of DFBD at an initial rate of 2,100 nmol/h per mg cellular protein. This is orders of magnitude higher than previously reported microbial defluorination rates with multiply fluorinated carbon atoms. Defluorination rates declined after several hours, and the medium darkened. Significant defluorination activity was observed with cells grown on toluene but not l-arginine. Defluorination required only toluene dioxygenase. Pseudomonas and recombinant Escherichia coli cells expressing toluene dioxygenase oxidized DFBD to DFBD-4,5-dihydrodiol. The dihydrodiol could be oxidized to 4,5-dihydroxy-DFBD via the dihydrodiol dehydrogenase from P. putida F1. The dihydrodiol dehydrated with acid to yield a mixture of 4-hydroxy-DFBD and 5-hydroxy-DFBD. All those metabolites retained the difluoromethylene group; no fluoride or dark color was observed. The major route of DFBD-4,5-dihydrodiol decomposition produced fluoride and 1,2,3-trihydroxybenzene, or pyrogallol, and that was shown to be the source of the dark colors in the medium. A mechanism for DFBD-4,5-dihydrodiol transformation to two fluoride ions and pyrogallol is proposed. The Pseudomonas genome database and other databases revealed hundreds of bacteria with enzymes sharing high amino acid sequence identity to toluene dioxygenase from P. putida F1, suggesting the mechanism revealed here may apply to the defluorination of DFBD-containing compounds in the environment. IMPORTANCE There are more than 9,000 polyfluorinated compounds developed for commercial use, some negatively impacting human health, and they are generally considered to be resistant to biodegradation. Only a limited number of studies have identified microbes with enzymes sufficiently reactive to defluorinate difluoromethylene carbon groups. The present study examined one important group of commercial fluorinated chemicals and showed its rapid defluorination by a bacterium and its key enzyme, a Rieske dioxygenase. Rieske dioxygenases are common in environmental bacteria, and those closely resembling toluene dioxygenase from Pseudomonas putida F1 are candidates for biodegradative defluorination of the common 2,2-fluoro-1,3-benzodioxole (DFBD) moiety.

Vanillin is one of the world's most important flavor and fragrance compounds used in foods and cosmetics. In plants, vanillin is reportedly biosynthesized from ferulic acid via the hydratase/lyase-type enzyme VpVAN. However, in biotechnological and biocatalytic applications, the use of VpVAN limits the production of vanillin. Although microbial enzymes are helpful as substitutes for plant enzymes, synthesizing vanillin from ferulic acid in one step using microbial enzymes remains a challenge. Here, we developed a single enzyme that catalyzes vanillin production from ferulic acid in a coenzyme-independent manner via the rational design of a microbial dioxygenase in the carotenoid cleavage oxygenase family using computational simulations. This enzyme acquired catalytic activity toward ferulic acid by introducing mutations into the active center to increase its affinity for ferulic acid. We found that the single enzyme can catalyze not only the production of vanillin from ferulic acid but also the synthesis of other aldehydes from p -coumaric acid, sinapinic acid, and coniferyl alcohol. These results indicate that the approach used in this study can greatly expand the range of substrates available for the dioxygenase family of enzymes. The engineered enzyme enables efficient production of vanillin and other value-added aldehydes from renewable lignin-derived compounds., Importance: The final step of vanillin biosynthesis in plants is reportedly catalyzed by the enzyme VpVAN. Prior to our study, VpVAN was the only reported enzyme that directly converts ferulic acid to vanillin. However, as many characteristics of VpVAN remain unknown, this enzyme is not yet suitable for biocatalytic applications. We show that an enzyme that converts ferulic acid to vanillin in one step could be constructed by modifying a microbial dioxygenase-type enzyme. The engineered enzyme is of biotechnological importance as a tool for the production of vanillin and related compounds via biocatalytic processes and metabolic engineering. The results of this study may also provide useful insights for understanding vanillin biosynthesis in plants., Competing Interests: Tokyo University of Science has filed a patent application related to vanillin production on behalf of T.F., S.S., and S.F.

The human indoleamine-2,3-dioxygenase 2 (hIDO2) protein is growing of interest as it is increasingly implicated in multiple diseases (cancer, autoimmune diseases, COVID-19). However, it is only poorly reported in the literature. Its mode of action remains unknown because it does not seem to catalyze the reaction for which it is attributed: the degradation of the L-Tryptophan into N-formyl-kynurenine. This contrasts with its paralog, the human indoleamine-2,3-dioxygenase 1 (hIDO1), which has been extensively studied in the literature and for which several inhibitors are already in clinical trials. Yet, the recent failure of one of the most advanced hIDO1 inhibitors, the Epacadostat, could be caused by a still unknown interaction between hIDO1 and hIDO2. In order to better understand the mechanism of hIDO2, and in the absence of experimental structural data, a computational study mixing homology modeling, Molecular Dynamics, and molecular docking was conducted. The present article highlights an exacerbated lability of the cofactor as well as an inadequate positioning of the substrate in the active site of hIDO2, which might bring part of an answer to its lack of activity.Communicated by Ramaswamy H. Sarma.

Two histidine-ligated heme-dependent monooxygenase proteins, TyrH and SfmD, have recently been found to resemble enzymes from the dioxygenase superfamily currently named after tryptophan 2,3-dioxygenase (TDO), that is, the TDO superfamily. These latest findings prompted us to revisit the structure and function of the superfamily. The enzymes in this superfamily share a similar core architecture and a histidine-ligated heme. Their primary functions are to promote O-atom transfer to an aromatic metabolite. TDO and indoleamine 2,3-dioxygenase (IDO), the foundingmembers, promote dioxygenation through a two-step monooxygenation pathway. However, the new members of the superfamily, including PrnB, SfmD, TyrH, and MarE, expand its boundaries and mediate monooxygenation on a broader set of aromatic substrates. We found that the enlarged superfamily contains eight clades of proteins. Overall, this protein group is a more sizeable, structure-based, histidine-ligated heme-dependent, and functionally diverse superfamily for aromatics oxidation. The concept of TDO superfamily or heme-dependent dioxygenase superfamily is no longer appropriate for defining this growing superfamily. Hence, there is a pressing need to redefine it as a hemedependent aromatic oxygenase (HDAO) superfamily. The revised concept puts HDAO in the context of thiol-ligated heme-based enzymes alongside cytochrome P450 and peroxygenase. It will update what we understand about the choice of heme axial ligand. Hemoproteins may not be as stringent about the type of axial ligand for oxygenation, although thiolate-ligated hemes (P450s and peroxygenases) more frequently catalyze oxygenation reactions. Histidine-ligated hemes found in HDAO enzymes can likewise mediate oxygenation when confronted with a proper substrate. [ABSTRACT FROM AUTHOR]

3-(2-Hydroxyethyl)catechol (HEC) is a polyphenol reported to exhibit skin-lightning and antioxidative effects, and hence is expected to be used as cosmetic and food additives and chemical products such as electronic materials. In this study, we established biocatalytic HEC production from 2-phenylethanol using the dioxygenase whose expression was induced by toluene, CumA, and its flanking dehydrogenase, CumB, from an isolated strain, Pseudomonas sp. K17. Escherichia coli cells coexpressing CumA and CumB were stained blue during cultivation in Luria–Bertani medium, and HEC was not produced upon using the cell-free extracts as biocatalysts, likely resulting from the inhibitory effects of the blue dyes. The disruption of the tryptophanase gene of E. coli was found to repress the generation of the blue dyes, and enhanced HEC production. The blue dyes were extracted from the cell-free extracts, and their molecular formula was C 16 H 10 N 2 O 3 , suggesting they were monooxygenated indigo or its isomers. Although repression of blue dye formation and enhancement of HEC production were observed when cells were cultivated with glucose, the percent yield of HEC was 84% at 20 h, whereas that with tryptophanase disruption strain was 84% at 4 h. It was suggested that tryptophanase gene disruption could contribute to more efficient HEC production. [ABSTRACT FROM AUTHOR]

NADH-dependent reductase enzyme catalyzes the phenolic aldehyde conversion and correspondingly improves the ethanol fermentability of the ethanologenic Zymomonas mobilis. This study constructed the transcriptional landscape of mono/dioxygenase genes in Z. mobilis ZM4 under the stress of the toxic phenolic aldehyde inhibitors of 4-hydroxybenzaldehyde, syringaldehyde, and vanillin. One specific dioxygenase encoding gene ZMO1721 was differentially expressed by 3.07-folds under the stress of 4-hydroxybenzaldehyde among the eleven mono/dioxygenase genes. The purified ZMO1721 shared 99.9% confidence and 48.0% identity with the oxidoreductase in Rhodoferax ferrireducens T118 was assayed and the NADH-dependent reduction activity was confirmed for phenolic aldehyde vanillin conversion. The ZMO1721 gene was then overexpressed in Z. mobilis ZM4 and the 4-hydroxybenzaldehyde conversion rate was accelerated. The cell growth, glucose consumption, and ethanol productivity of Z. mobilis ZM4 were also improved by ZMO1721 overexpression. The genes identified on improving phenolic aldehyde tolerance and ethanol fermentability in this study could be used as the synthetic biology tools for modification of ethanologenic strains. [ABSTRACT FROM AUTHOR]

The opportunistic pathogen Pseudomonas aeruginosa uses quorum sensing to control its virulence. One of its major signal molecules, the Pseudomonas quinolone signal PQS, has high affinity to membranes and is known to be trafficked mainly via outer membrane vesicles (OMVs). We previously reported that several 3-hydroxy-4(1H)-quinolone 2,4-dioxygenases (HQDs) catalyze the cleavage of PQS and thus act as quorum quenching enzymes. Further analysis showed that, in contrast to other HQDs, the activity of HQD from Streptomyces bingchenggensis (HQDS.b.) was unexpectedly stabilized by culture supernatants of P. aeruginosa. Interestingly, the stabilizing effect was higher with supernatants from the strain PA14 than with supernatants from the strain PAO1. Heat treatment and lyophilization hardly affected the stabilizing effect; however, fractionation of the supernatant excluded small molecules as stabilizing agents. In a pull-down assay, HQDS.b. appeared to interact with several P. aeruginosa proteins previously found in the OMV proteome. This prompted us to probe the physical interaction of HQDS.b. with prepared extracellular membrane vesicles. Homo-FRET of fluorescently labeled HQDS.b. indeed indicated a spatial clustering of the protein on the vesicles. Binding of a PQS-cleaving enzyme to the OMVs of P. aeruginosa may enhance PQS degradation and is highly reconcilable with its function as a quorum quenching enzyme.

Plants synthesize many diverse small molecules that affect function of the mammalian central nervous system, making them crucial sources of therapeutics for neurological disorders. A notable portion of neuroactive phytochemicals are lysine-derived alkaloids, but the mechanisms by which plants produce these compounds have remained largely unexplored. To better understand how plants synthesize these metabolites, we focused on biosynthesis of the Lycopodium alkaloids that are produced by club mosses, a clade of plants used traditionally as herbal medicines. Hundreds of Lycopodium alkaloids have been described, including huperzine A (HupA), an acetylcholine esterase inhibitor that has generated interest as a treatment for the symptoms of Alzheimer’s disease. Through combined metabolomic profiling and transcriptomics, we have identified a developmentally controlled set of biosynthetic genes, or potential regulon, for the Lycopodium alkaloids. The discovery of this putative regulon facilitated the biosynthetic reconstitution and functional characterization of six enzymes that act in the initiation and conclusion of HupA biosynthesis. This includes a type III polyketide synthase that catalyzes a crucial imine-polyketide condensation, as well as three Fe(II)/2-oxoglutarate–dependent dioxygenase (2OGD) enzymes that catalyze transformations (pyridone ring-forming desaturation, piperidine ring cleavage, and redox-neutral isomerization) within downstream HupA biosynthesis. Our results expand the diversity of known chemical transformations catalyzed by 2OGDs and provide mechanistic insight into the function of noncanonical type III PKS enzymes that generate plant alkaloid scaffolds. These data offer insight into the chemical logic of Lys-derived alkaloid biosynthesis and demonstrate the tightly coordinated coexpression of secondary metabolic genes for the biosynthesis of medicinal alkaloids. [ABSTRACT FROM AUTHOR]