Hydroxylation Step Research Articles

Vitamin D Enhancement of Adipose Biology: Implications on Obesity-Associated Cardiometabolic Diseases

Vitamin D is activated into 1α,25(OH)2D through two hydroxylation steps that are primarily catalyzed by 25-hydroxylase in the liver and 1α-hydroxylase in the kidneys. The active form of vitamin D regulates myriads of cellular functions through its nuclear receptor, vitamin D receptor (VDR). Vitamin D metabolizing enzymes and VDR are expressed in adipose tissues and vitamin D regulates multiple aspects of adipose biology including the recruitment and differentiation of adipose stem cells into adipocytes and metabolic, endocrine, and immune properties. Obesity is associated with low vitamin D status, which is thought to be explained by its sequestration in large mass of adipose tissues as well as dysregulated vitamin D metabolism. Low vitamin D status in obesity may negatively impact adipose biology leading to adipose tissue dysfunctions, the major pathological factors for cardiometabolic diseases in obesity. In this review, the current understanding of vitamin D metabolism and its molecular mechanisms of actions, focusing on vitamin D–VDR regulation of adipose biology with their implications on obesity-associated diseases, is discussed. Whether improving vitamin D status leads to reductions in adiposity and risks for cardiometabolic diseases is also discussed.

Hydroxylase-oriented mining and functional characterization of camptothecin 10-hydroxylase from Camptotheca acuminata Decne

The regiospecific C-10 hydroxylation of camptothecin (CPT) is challenging from a chemical perspective. This step bridges the natural-sourced CPT to an important industrial material, 10-hydroxycamptothecin (10HCPT). 10HCPT is naturally generated in a medicinal plant, Camptotheca acuminata Decne. Functionally active hydroxylase responsible for regiospecific C-10 hydroxylation of CPT is undoubtedly existed in this plant. Therefore, the multi-omics database of C. acuminata was utilized to perform hydroxylase-oriented mining and screening. Three CYP81 genes were identified and two of them, including CYP81BQ18 and CYP81B256 catalyzed hydroxylation of CPT at the C-10 position. CYP81B256 and CYP81BN24 displayed quercetin C-5’ hydroxylase activity. CYP81B256 exhibited better catalytic performance for CPT than CYP81BQ18 and CPT10H in vitro. Furthermore, CYP81BQ18 and CYP81B256 displayed CPT 10-hydroxylase activity in tobacco. Functional verification in planta suggested that the newly observed CYP81BQ18 and CYP81B256 were involved in 10HCPT biosynthesis in C. acuminata. Subcellular localization analysis revealed that these CYP81 genes were located on the endoplasmic reticulum. Evolutionary analysis indicated that CYP81B256 and CYP81BN24 probably evolved from a same ancestral gene via gene duplication, while CYP81BQ18 evolved independently and acted as a specie-specific hydroxylase. CYP81B256 acted as an evolutionary intermediate gene bridging the alkaloid and flavonoid metabolism in C. acuminata. This research provides valuable insights into the function and evolutionary origin of CYP81s in the regiospecific hydroxylation step for 10HCPT biosynthesis.

How Ir–Rh Alloys Improve Electrochemical Ammonia Oxidation Activity Studied by Density Functional Theory

AbstractThe electrochemical ammonia oxidation reaction (AOR) has applications in hydrogen storage and ammonia waste remediation. Using density functional theory, we investigate the mechanism of AOR on Ir, Rh, and their alloys at varied atomic ratios (, , and ) toward (g), (aq), and (aq) formation. This work introduces a method for computational alloy design by considering both electronic energy and configurational entropy. The structures considered are selective to (g) formation and all favored *N–N coupling. An alloy was found to reduce the theoretical onset potential for (g) formation relative to pure Ir while not exhibiting a downhill coupling step corresponding to catalyst poisoning by *N as shown for pure Rh, consistent with previous experimental work. The formation of (aq) and (aq) demand significantly higher potentials, typically limited by the final hydroxylation step before desorption.

N-Alkane Assimilation by Pseudomonas aeruginosa and Its Interactions with Virulence and Antibiotic Resistance.

Pseudomonas aeruginosa strains with potential for degrading n-alkanes are frequently cultured from hydrocarbon-contaminated sites. The initial hydroxylation step of long-chain n-alkanes is mediated by the chromosomally encoded AlkB1 and AlkB2 alkane hydroxylases. The acquisition of an additional P. putida GPo1-like alkane hydroxylase gene cluster can extend the substrate range assimilated by P. aeruginosa to <C12 n-alkanes. Efficient niche colonization of hydrocarbon-contaminated sites is facilitated by avid iron-uptake systems, such as pyoverdine, and the production of several compounds with antimicrobial activities. A GPo1-like gene cluster can facilitate detoxification and solvent tolerance in P. aeruginosa. The overproduction of various multidrug efflux pumps, in particular, the MexAB-OprM system, can also contribute to solvent tolerance, which is often associated with reduced susceptibility or full resistance to certain clinically relevant antibiotics. These characteristics, together with the remarkable conservation of P. aeruginosa virulence determinants among human, animal, and environmental isolates, necessitate further studies from a One Health perspective into the acquired antibiotic resistance mechanisms of environmental P. aeruginosa strains and possible ways for their dissemination into the human population.

4-Hydroxy-1α,25-Dihydroxyvitamin D3: Synthesis and Structure-Function Study.

The active vitamin D metabolites, 25-hydroxyvitamin D3 (25D3) and 1,25-dihydroxyvitamin D3 (1,25D3), are produced by successive hydroxylation steps and play key roles in several cellular processes. However, alternative metabolic pathways exist, and among them, the 4-hydroxylation of 25D3 is a major one. This study aims to investigate the structure-activity relationships of 4-hydroxy derivatives of 1,25D3. Structural analysis indicates that 1,4α,25(OH)3D3 and 1,4β,25(OH)3D3 maintain the anchoring hydrogen bonds of 1,25D3 and form additional interactions, stabilizing the active conformation of VDR. In addition, 1,4α,25D3 and 1,4β,25D3 are as potent as 1,25D3 in regulating the expression of VDR target genes in rat intestinal epithelial cells and in the mouse kidney. Moreover, these two 4-hydroxy derivatives promote hypercalcemia in mice at a dose similar to that of the parent compound.

Anti-inflammatory and Neuroprotective α-Pyrones from a Marine-Derived Strain of the Fungus Arthrinium arundinis and Their Heterologous Expression.

Fungal linear polyketides, such as α-pyrones with a 6-alkenyl chain, have been a rich source of biologically active compounds. Two new (1 and 2) and four known (3-6) 6-alkenylpyrone polyketides were isolated from a marine-derived strain of the fungus Arthrinium arundinis. Their structures were determined based on extensive spectroscopic analysis. The biosynthetic gene cluster (alt) for alternapyrones was identified from A. arundinis ZSDS-F3 and validated by heterologous expression in Aspergillus nidulans A1145 ΔSTΔEM, which revealed that the cytochrome P450 monooxygenase Alt2' could convert the methyl group 26-CH3 to a carboxyl group to produce 4 from 3. Another cytochrome P450 monooxygenase, Alt3', catalyzed successive hydroxylation, epoxidation, and oxidation steps to produce 1, 2, 5, and 6 from 4. Alternapyrone G (1) not only suppressed M1 polarization in lipopolysaccharide (LPS)-stimulated BV2 microglia but also stimulated dendrite regeneration and neuronal survival after Aβ treatment, suggesting alternapyrone G may be utilized as a privileged scaffold for Alzheimer's disease drug discovery.

A Role in 15-Deacetylcalonectrin Acetylation in the Non-Enzymatic Cyclization of an Earlier Bicyclic Intermediate in Fusarium Trichothecene Biosynthesis.

The trichothecene biosynthesis in Fusarium begins with the cyclization of farnesyl pyrophosphate to trichodiene, followed by subsequent oxygenation to isotrichotriol. This initial bicyclic intermediate is further cyclized to isotrichodermol (ITDmol), a tricyclic precursor with a toxic trichothecene skeleton. Although the first cyclization and subsequent oxygenation are catalyzed by enzymes encoded by Tri5 and Tri4, the second cyclization occurs non-enzymatically. Following ITDmol formation, the enzymes encoded by Tri101, Tri11, Tri3, and Tri1 catalyze 3-O-acetylation, 15-hydroxylation, 15-O-acetylation, and A-ring oxygenation, respectively. In this study, we extensively analyzed the metabolites of the corresponding pathway-blocked mutants of Fusarium graminearum. The disruption of these Tri genes, except Tri3, led to the accumulation of tricyclic trichothecenes as the main products: ITDmol due to Tri101 disruption; a mixture of isotrichodermin (ITD), 7-hydroxyisotrichodermin (7-HIT), and 8-hydroxyisotrichodermin (8-HIT) due to Tri11 disruption; and a mixture of calonectrin and 3-deacetylcalonectrin due to Tri1 disruption. However, the ΔFgtri3 mutant accumulated substantial amounts of bicyclic metabolites, isotrichotriol and trichotriol, in addition to tricyclic 15-deacetylcalonectrin (15-deCAL). The ΔFgtri5ΔFgtri3 double gene disruptant transformed ITD into 7-HIT, 8-HIT, and 15-deCAL. The deletion of FgTri3 and overexpression of Tri6 and Tri10 trichothecene regulatory genes did not result in the accumulation of 15-deCAL in the transgenic strain. Thus, the absence of Tri3p and/or the presence of a small amount of 15-deCAL adversely affected the non-enzymatic second cyclization and C-15 hydroxylation steps.

An organic O donor for biological hydroxylation reactions

All biological hydroxylation reactions are thought to derive the oxygen atom from one of three inorganic oxygen donors, O2, H2O2, or H2O. Here, we have identified the organic compound prephenate as the oxygen donor for the three hydroxylation steps of the O2-independent biosynthetic pathway of ubiquinone, a widely distributed lipid coenzyme. Prephenate is an intermediate in the aromatic amino acid pathway and genetic experiments showed that it is essential for ubiquinone biosynthesis in Escherichia coli under anaerobic conditions. Metabolic labeling experiments with 18O-shikimate, a precursor of prephenate, demonstrated the incorporation of 18O atoms into ubiquinone. The role of specific iron-sulfur enzymes belonging to the widespread U32 protein family is discussed. Prephenate-dependent hydroxylation reactions represent a unique biochemical strategy for adaptation to anaerobic environments.

Biosynthesis of the highly oxygenated tetracyclic core skeleton of Taxol

Taxol is a widely-applied anticancer drug that inhibits microtubule dynamics in actively replicating cells. Although a minimum 19-step biosynthetic pathway has been proposed and 16 enzymes likely involved have been characterized, stepwise biosynthetic reactions from the well-characterized di-oxygenated taxoids to Taxol tetracyclic core skeleton are yet to be elucidated. Here, we uncover the biosynthetic pathways for a few tri-oxygenated taxoids via confirming the critical reaction order of the second and third hydroxylation steps, unearth a taxoid 9α-hydroxylase catalyzing the fourth hydroxylation, and identify CYP725A55 catalyzing the oxetane ester formation via a cascade oxidation-concerted acyl rearrangement mechanism. After identifying a acetyltransferase catalyzing the formation of C7-OAc, the pathway producing the highly-oxygenated 1β-dehydroxybaccatin VI with the Taxol tetracyclic core skeleton is elucidated and its complete biosynthesis from taxa-4(20),11(12)-diene-5α-ol is achieved in an engineered yeast. These systematic studies lay the foundation for the complete elucidation of the biosynthetic pathway of Taxol.

Metagenomic insights into phenanthrene biodegradation in electrical field-governed biofilms for groundwater bioremediation

Electrical fields (EFs)-assisted in-situ bioremediation of petroleum-contaminated groundwater, such as polycyclic aromatic hydrocarbons, has drawn increasing attention. However, the long-term stability, the EFs influence, and metabolic pathways are still poorly understood, hindering the further development of robust technology design. Herein, a series of EFs was applied to the phenanthrene-contaminated groundwater, and the corresponding system performance was investigated. The highest removal capacity of phenanthrene (phe) (7.63 g/(m3·d)) was achieved with EF_0.8 V biofilm at a hydrolytic retention time of 0.5 d. All the biofilms with four EFs exhibited a high removal efficiency of phe over 80% during a 100-d continuous-flow operation. Intermediates analysis revealed the main pathways of phe degradation: phthalate and salicylate via hydroxylation, methylation, carboxylation, and ring cleavage steps. Synergistic effects between phe-degraders (Dechloromonas), fermentative bacteria (Delftia), and electroactive microorganisms (Geobacter) were the main contributors to the complete phe mineralization. Genes encoding various proteins of methyl-accepting (mcp), response regulator (cheABDRY), and type IV pilus (pilABCMQV) were dominant, revealing the importance of cell motility and extracellular electron transfer. Metagenomics analysis unveiled phe-degrading genes, including ring reduction enzymes (bamBCDE), carboxylase of aromatics (ubiD), and methyltransferase protein (ubiE, pcm). These findings offered a molecular understanding of refractory organics’ decompositions in EFs-governed biotechnology.

Characterization of the Baeyer-Villiger monooxygenase in the pathway of the bacterial pyrrolizidine alkaloids, legonmycins.

The Baeyer-Villiger monooxygenase (BVMO), LgnC, plays a crucial role in the biosynthesis of bacterial pyrrolizidine alkaloids, legonmycins. It processes bicyclic indolizidine substrates generated from the coordinative action of two non-ribosomal peptide synthetases (LgnB and LgnD) and the standalone type II thioesterase-like enzyme (LgnA). It has been demonstrated that the enzyme selectively inserts molecular oxygen into the carbon-carbon bond adjacent to the carbonyl group in legonindolizidines to form bicyclic 1,3-oxazepine carbamate intermediates. After ring opening and contraction, the most advanced products, prelegonmycins, are formed. However, factors controlling the final hydroxylation step and how the enzyme handles the substrates have remained elusive. In this study, we show that the final hydroxylation at the activated carbon of the electron-rich pyrrole system is attributed to either spontaneous oxidation or the action of an endogenous redox reagent. Substrate docking on the structural model of LgnC combined with site-directed mutagenesis allows the identification of several key amino acids that are essential for substrate/intermediate binding and a mechanism of LgnC-catalysed transformation is proposed.

The multistep oxidation of cholesterol to pregnenolone by human cytochrome P450 11A1 is highly processive

Cytochrome P450 (P450, CYP) 11A1 is the classical cholesterol side chain cleavage enzyme (P450scc) that removes six carbons of the side chain, the first and rate-limiting step in the synthesis of all mammalian steroids. The reaction is a 3-step, 6-electron oxidation that proceeds via formation of 22R-hydroxy (OH) and 20R,22R-(OH)2 cholesterol, yielding pregnenolone. We expressed human P450 11A1 in bacteria, purified the enzyme in the absence of nonionic detergents, and assayed pregnenolone formation by HPLC-mass spectrometry of the dansyl hydrazone. The reaction was inhibited by the nonionic detergent Tween 20, and several lipids did not enhance enzymatic activity. The 22R-OH and 20R,22R-(OH)2 cholesterol intermediates were bound to P450 11A1 relatively tightly, as judged by steady-state optical titrations and koff rates. The electron donor adrenodoxin had little effect on binding; the substrate cholesterol showed a ∼5-fold stimulatory effect on the binding of adrenodoxin to P450 11A1. Presteady-state single-turnover kinetic analysis was consistent with a highly processive reaction with rates of intermediate oxidation steps far exceeding dissociation rates for products and substrates. The presteady-state kinetic analysis revealed a second di-OH cholesterol product, separable by HPLC, in addition to 20R,22R-(OH)2 cholesterol, which we characterized as a rotamer that was also converted to pregnenolone at a similar rate. The first oxidation step (at C-22) is the slowest, limiting the overall rate of cleavage. d3-Cholesterol showed no kinetic deuterium isotope effect on C-22, indicating that C-H bond cleavage is not rate-limiting in the first hydroxylation step.

Heterologous Production in the Synechocystis Chassis Suggests the Biosynthetic Pathway of Astaxanthin in Cyanobacteria.

Astaxanthin is a carotenoid species with the highest antioxidant capability. Its natural resource is very rare. The biosynthesis of astaxanthin from β-carotene includes a hydroxylation step and a ketolation step, for which the corresponding enzymes have been characterized in a few species. However, the sequence of these two reactions is unclear, and may vary with different organisms. In this study, we aimed to elucidate this sequence in Synechocystis, which is an ideal cyanobacterial synthetic biology chassis. We first silenced the endogenous carotene oxygenase gene SyneCrtO to avoid its possible interference in the carotenoid metabolic network. We then introduced the β-carotene ketolase gene from Haematococcus pluvialis (HpBKT) and the CrtZ-type carotene β-hydroxylase gene from Pantoea agglomerans (PaCrtZ) to this δCrtO strain. Our pigment analysis demonstrated that both the endogenous CrtR-type carotene hydroxylase SyneCrtR and HpBKT have the preference to use β-carotene as their substrate for hydroxylation and ketolation reactions to produce zeaxanthin and canthaxanthin, respectively. However, the endogenous SyneCrtR is not able to further catalyze the 3,3'-hydroxylation of canthaxanthin to generate astaxanthin. From our results, a higher accumulation of canthaxanthin and a much lower level of astaxanthin, as confirmed using liquid chromatography-tandem mass spectrometry analysis, were detected in our transgenic BKT+/CrtZ+/δCrtO cells. Therefore, we proposed that the bottleneck for the heterologous production of astaxanthin in Synechocystis might exist at the hydroxylation step, which requires a comprehensive screening or genetic engineering for the corresponding carotene hydroxylase to enable the industrial production of astaxanthin.

UbiN, a novel Rhodobacter capsulatus decarboxylative hydroxylase involved in aerobic ubiquinone biosynthesis.

Ubiquinone (UQ) is a lipophilic electron carrier that functions in the respiratory and photosynthetic electron transfer chains of proteobacteria and eukaryotes. Bacterial UQ biosynthesis is well studied in the gammaproteobacterium Escherichia coli, in which most bacterial UQ-biosynthetic enzymes have been identified. However, these enzymes are not always conserved among UQ-containing bacteria. In particular, the alphaproteobacterial UQ biosynthesis pathways contain many uncharacterized steps with unknown features. In this work, we identified in the alphaproteobacterium Rhodobacter capsulatus a new decarboxylative hydroxylase and named it UbiN. Remarkably, the UbiN sequence is more similar to a salicylate hydroxylase than the conventional flavin-containing UQ-biosynthetic monooxygenases. Under aerobic conditions, R. capsulatus ΔubiN mutant cells accumulate 3-decaprenylphenol, which is a UQ-biosynthetic intermediate. In addition, 3-decaprenyl-4-hydroxybenzoic acid, which is the substrate of UQ-biosynthetic decarboxylase UbiD, also accumulates in ΔubiN cells under aerobic conditions. Considering that the R. capsulatus ΔubiD-X double mutant strain (UbiX produces a prenylated FMN required for UbiD) grows as a wild-type strain under aerobic conditions, these results indicate that UbiN catalyzes the aerobic decarboxylative hydroxylation of 3-decaprenyl-4-hydroxybenzoic acid. This is the first example of the involvement of decarboxylative hydroxylation in ubiquinone biosynthesis. This finding suggests that the C1 hydroxylation reaction is, at least in R. capsulatus, the first step among the three hydroxylation steps involved in UQ biosynthesis. Although the C5 hydroxylation reaction is often considered to be the first hydroxylation step in bacterial UQ biosynthesis, it appears that the R. capsulatus pathway is more similar to that found in mammalians.

Lignin conversion to β-ketoadipic acid by Pseudomonas putida via metabolic engineering and bioprocess development.

Bioconversion of a heterogeneous mixture of lignin-related aromatic compounds (LRCs) to a single product via microbial biocatalysts is a promising approach to valorize lignin. Here, Pseudomonas putida KT2440 was engineered to convert mixed p-coumaroyl- and coniferyl-type LRCs to β-ketoadipic acid, a precursor for performance-advantaged polymers. Expression of enzymes mediating aromatic O-demethylation, hydroxylation, and ring-opening steps was tuned, and a global regulator was deleted. β-ketoadipate titers of 44.5 and 25 grams per liter and productivities of 1.15 and 0.66 grams per liter per hour were achieved from model LRCs and corn stover-derived LRCs, respectively, the latter representing an overall yield of 0.10 grams per gram corn stover-derived lignin. Technoeconomic analysis of the bioprocess and downstream processing predicted a β-ketoadipate minimum selling price of $2.01 per kilogram, which is cost competitive with fossil carbon-derived adipic acid ($1.10 to 1.80 per kilogram). Overall, this work achieved bioproduction metrics with economic relevance for conversion of lignin-derived streams into a performance-advantaged bioproduct.

Unveiling the hidden role of aquatic viruses in hydrocarbon pollution bioremediation

Hydrocarbon pollution poses substantial environmental risks to water and soil. Bioremediation, which utilizes microorganisms to manage pollutants, offers a cost-effective solution. However, the role of viruses, particularly bacteriophages (phages), in bioremediation remains unexplored. This study examines the diversity and activity of hydrocarbon-degradation genes encoded by environmental viruses, focusing on phages, within public databases. We identified 57 high-quality phage-encoded auxiliary metabolic genes (AMGs) related to hydrocarbon degradation, which we refer to as virus-encoded hydrocarbon degradation genes (vHYDEGs). These genes are encoded by taxonomically diverse aquatic phages and highlight the under-characterized global virosphere. Six protein families involved in the initial alkane hydroxylation steps were identified. Phylogenetic analyses revealed the diverse evolutionary trajectories of vHYDEGs across habitats, revealing previously unknown biodegraders linked evolutionarily with vHYDEGs. Our findings suggest phage AMGs may contribute to alkane and aromatic hydrocarbon degradation, participating in the initial, rate-limiting hydroxylation steps, thereby aiding hydrocarbon pollution bioremediation and promoting their propagation. To support future research, we developed vHyDeg, a database containing identified vHYDEGs with comprehensive annotations, facilitating the screening of hydrocarbon degradation AMGs and encouraging their bioremediation applications.

Insights into manganese(VII) enhanced oxidation of benzophenone-8 by ferrate(VI): Mechanism and transformation products

Benzophenones (BPs) are commonly used as UV filters in cosmetics and plastics products and are potentially toxic to the environment. This paper presents kinetics and products of BPs oxidation by ferrate(VI) (FeO42−, Fe(VI)) promoted by permanganate (Mn(VII)) . Degradation of 10.0 µM 2,2′-dihydroxy-4-methoxybenzophenone (BP-8)were determined under different experimental conditions ([Mn(VII)] = 0.5–1.5 µM, [Fe(VI)] = 50–150 µM, and pH = 7.0–10.0). The addition of Mn(VII) traces to Fe(VI)-BP-8 solution enhanced kinetics and efficiency of the removal. Similar enhanced removals were also seen for other BPs (BP-1, BP-3, and BP-4) under optimized conditions. The second-order rate constants (k, M−1s−1) of the degradation of BPs showed positive relationship with the energy of the highest occupied orbital (EHOMO). The possible interaction between Mn(VII) and BP-8 and the enhanced generation of Fe(V)/Fe(IV) and •OH was proposed to facilitate the oxidation of the target benzophenone, supported by in-situ electrochemical measurements, theoretical calculations and reactive species quenching experiments. Thirteen oxidation products of BP-8 suggested hydroxylation, bond breaking, polymerization and carboxylation steps in the oxidation. Toxicity assessments by ECOSAR program showed that the oxidized intermediate products posed a tapering ecological risk during the degradation process. Overall, the addition of Mn(VII) could improve the oxidation efficiency of Fe(VI).

Photoactivity of boron-or nitrogen-modified TiO2 for organic pollutants degradation: Unveiling the photocatalytic mechanisms and by-products

The degradation of fluoxetine (FLX) emerging pollutant and the formation of by-products, potentially present in environmental ecosystems, was investigated from photocatalytic processes. Applying different synthesis methods, nitrogen (TiN) or boron (TiB)-modified TiO2 presented oxygen vacancies (Vö) responsible for generating intermediate energy levels, which acted as shallow traps to minimize the recombination processes and maintain the efficient separation of charge carriers. All photocatalysts showed the potential to produce hydroxyl radicals (•OH) from the oxidation of water in h+(VB), achieving 100% of RhB degradation under UV or visible irradiation. For the degradation of FLX, an alternative mechanism mediated by one of the by-products was confirmed after LC-MS-Q-TOF analysis. The protonation, dehalogenation and hydroxylation steps of FLX (m/z[H+] =310.1419) produce the transformation products TP8 (m/z[H+] = 310.1430), TP6 (m/z[H+] = 286.1454), and TP9 (m/z = 161.0221 (-ESI)), respectively. Other by-products have been identified after photodegradation, and their presence in FLX-contaminated ecosystems cannot be ignored. Therefore, high-performance photocatalysts obtained in this study are real alternatives for the environmental remediation of organic pollutants in aquatic ecosystems.

De novo transcriptome assembly and functional analysis reveal a dihydrochalcone 3-hydroxylase(DHC3H) of wild Malus species that produces sieboldin in vivo.

Sieboldin is a specialised secondary metabolite of the group of dihydrochalcones (DHC), found in high concentrations only in some wild Malus species, closely related to the domesticated apple (Malus × domestica L.). To date, the first committed step towards the biosynthesis of sieboldin remains unknown. In this study, we combined transcriptomic analysis and a de novo transcriptome assembly to identify two putative 3-hydroxylases in two wild Malus species (Malus toringo (K. Koch) Carriere syn. sieboldii Rehder, Malus micromalus Makino) whose DHC profile is dominated by sieboldin. We assessed the in vivo activity of putative candidates to produce 3-hydroxyphloretin and sieboldin by de novo production in Saccharomyces cerevisiae. We found that CYP98A proteins of wild Malus accessions (CYP98A195, M. toringo and CYP98A196, M. micromalus) were able to produce 3-hydroxyphloretin, ultimately leading to sieboldin accumulation by co-expression with PGT2. CYP98A197-198 genes of M. × domestica, however, were unable to hydroxylate phloretin in vivo. CYP98A195-196 proteins exerting 3-hydroxylase activity co-localised with an endoplasmic reticulum marker. CYP98A protein model from wild accessions showed mutations in key residues close to the ligand pocket predicted using phloretin for protein docking modelling. These mutations are located within known substrate recognition sites of cytochrome P450s, which could explain the acceptance of phloretin in CYP98A protein of wild accessions. Screening a Malus germplasm collection by HRM marker analysis for CYP98A genes identified three clusters that correspond to the alleles of domesticated and wild species. Moreover, CYP98A isoforms identified in M. toringo and M. micromalus correlate with the accumulation of sieboldin in other wild and hybrid Malus genotypes. Taken together, we provide the first evidence of an enzyme producing sieboldin in vivo that could be involved in the key hydroxylation step towards the synthesis of sieboldin in Malus species.

Solar photoelectro-Fenton-like process with anodically-generated HClO in a flow reactor: Norfloxacin as a pollutant with a particular structure

The degradation and mineralization of the drug norfloxacin (NFX) has been assessed in a model solution, containing 15 mM NaCl +45 mM Na2SO4 at pH 3.0, using a flow plant with an FM01-LC filter-press reactor equipped with a Ti|Ir-Sn-Ru oxides anode to electrogenerate HClO from Cl− oxidation and a stainless-steel cathode. Unexpectedly, anodic oxidation with active chlorine (AO-HClO) outperformed electro-Fenton (EF-HClO), photoelectro-Fenton (PEF-HClO) and solar photoelectro-Fenton (SPEF-HClO) due to: (i) the formation of refractory complexes between iron ions and carboxyl group of NFX, and (ii) the conversion of HClO into less effective •OH upon its Fenton-like reaction with added Fe2+ catalyst. SPEF-HClO was superior among Fenton-based treatments because the •OH concentration was largely increased by the photolysis of Fe(III) species. At an initial NFX concentration in the range 0.103–0.146 mM, the optimum conditions for SPEF-HClO were 0.40 mM Fe2+ and 15 mA cm−2. A BDD anode allowed a higher production of •OH, accelerating the degradation and mineralization, with similar energy requirements as compared to trials with Ti|Ir-Sn-Ru oxides anode. The SPEF-HClO process in urban wastewater was less powerful because of the parallel oxidation of natural organic matter. HClO, Fe2+ and Fetotal were quantified in both water matrices. The initial degradation sequence for NFX, proposed from 10 primary by-products identified by LC-MS/MS, revealed the occurrence of hydroxylation, chlorination and defluorination steps. Additionally, 5 stable by-products were detected by GC–MS.