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Copper complexes supported by benzimidazole‐based tetradentate neutral ligands featuring thio‐ and selenoether donors, resemble the coordination environment of the active site in copper dependent monooxygenases Dopamine‐β‐monooxygenase (DβM) and peptidylglycine‐α‐hydroxylating monooxygenase (PHM). The cuprous complexes react with O2 at low temperature generating a reactive copper‐oxygen species assigned to a side‐on cupric–superoxo complex, which activates the C─H bond of dihydroanthracene. Based on structural, 1H and 77Se NMR, and EPR spectroscopic characterization, together with DFT computations, the selenoether moiety likely acts as a hemilabile ligand in these scaffolds, which results in an electrophilic cupric–superoxide intermediate poised for H‐atom transfer, as has been proposed for related bis(benzimidazole)‐thioether analogues and selenoether‐modified enzymatic systems. [ABSTRACT FROM AUTHOR]

Abstract Flavin-dependent monooxygenases (FMOs) are a valuable group of biocatalysts that can regioselectively introduce a hydroxy group for the targeted modification of biologically active compounds. Here, we present the fdeE, the FMO from Herbaspirillum seropedicae SmR1 that is a part of the naringenin degradation pathway and is active towards a wide range of flavonoids—flavanones, flavones, isoflavones, and flavonols. Bioinformatics and biochemical analysis revealed a high similarity between the analyzed enzyme and other F8H FMOs what might indicate convergent evolutionary mechanism of flavonoid degradation pathway emergence by microorganism. A simple approach with the manipulation of the reaction environment allowed the stable formation of hydroxylation products, which showed very high reactivity in both in vivo and in vitro assays. This approach resulted in an 8-hydroxyquercetin—gossypetin titer of 0.16 g/L and additionally it is a first report of production of this compound.

Polyacrylamide (PAM) is a high-molecular-weight polymer with extensive applications. However, the inefficient natural degradation of PAM results in environmental accumulation of the polymer. Biodegradation is an environmentally friendly approach in the field of PAM treatment. The first phase of PAM biodegradation is the deamination of PAM, forming the product poly(acrylic acid) (PAA). The second phase of PAM biodegradation involves the cleavage of PAA into small molecules, which is a crucial step in the degradation pathway of PAM. However, the enzyme that catalyzes the degradation of PAA and the molecular mechanism remain unclear. Here, a novel monooxygenase PCX02514 is identified as the key enzyme for PAA degradation. Through biochemical experiments, the monooxygenase PCX02514 oxidizes PAA with the participation of NADPH, causing the cleavage of carbon chains and a decrease in the molecular weight of PAA. In addition, the crystal structure of the monooxygenase PCX02514 is solved at a resolution of 1.97 Å. The active pocket is in a long cavity that extends from the C-terminus of the TIM barrel to the protein surface and exhibits positive electrostatic potential, thereby causing the migration of oxygen-negative ions into the active pocket and facilitating the reaction between the substrates and monooxygenase PCX02514. Moreover, Arg10-Arg125-Ser186-Arg187-His253 are proposed as potential active sites in monooxygenase PCX02514. Our research characterizes the molecular mechanism of this monooxygenase, providing a theoretical basis and valuable tools for PAM bioremediation. [ABSTRACT FROM AUTHOR]

Shiraia-like fungi, which are rare parasitic fungi found around bamboo, play an important role in traditional medicine. Their main active component, hypocrellin, is widely used in medicine, food, and cosmetics. By comparing strains with different hypocrellin yields, we identified a transcription factor (SbTF) in the hypocrellin biosynthesis pathway. SbTF from high-yielding zzz816 and low-yielding CNUCC C72 differed in its protein structure. Subsequently, SbTF from high-yielding zzz816 was overexpressed in several strains. This stabilised the yield in zzz816 and significantly increased the yield in low-yielding CNUCC C72. Comparing downstream non-essential genes between wild type and SbTF-overexpressing CNUCC C72 showed that SbMNF was significantly up-regulated. Therefore, it was selected for further study. SbMNF overexpression increased the hypocrellin yield in low-yielding CNUCC C72 and altered the composition of compounds in high-yielding CNUCC 1353PR and zzz816. This involved an increased elsinochrome C yield in CNUCC 1353PR and an increased hypocrellin B yield in zzz816 (by 2 and 70.3 times that in the corresponding wild type, respectively). This study is the first to alter hypocrellin synthesis to alter the levels of one bioactive agent compared to another. The results provide new insights regarding genetic modification and will help to optimise fungal fermentation. [ABSTRACT FROM AUTHOR]

ABSTRACTShiraia-like fungi, which are rare parasitic fungi found around bamboo, play an important role in traditional medicine. Their main active component, hypocrellin, is widely used in medicine, food, and cosmetics. By comparing strains with different hypocrellin yields, we identified a transcription factor (SbTF) in the hypocrellin biosynthesis pathway. SbTF from high-yielding zzz816 and low-yielding CNUCC C72 differed in its protein structure. Subsequently, SbTF from high-yielding zzz816 was overexpressed in several strains. This stabilised the yield in zzz816 and significantly increased the yield in low-yielding CNUCC C72. Comparing downstream non-essential genes between wild type and SbTF-overexpressing CNUCC C72 showed that SbMNF was significantly up-regulated. Therefore, it was selected for further study. SbMNF overexpression increased the hypocrellin yield in low-yielding CNUCC C72 and altered the composition of compounds in high-yielding CNUCC 1353PR and zzz816. This involved an increased elsinochrome C yield in CNUCC 1353PR and an increased hypocrellin B yield in zzz816 (by 2 and 70.3 times that in the corresponding wild type, respectively). This study is the first to alter hypocrellin synthesis to alter the levels of one bioactive agent compared to another. The results provide new insights regarding genetic modification and will help to optimise fungal fermentation.

Aim: We tested the hypothesis that low testosterone alters the effects of intermittent hypoxia (IH) on glucose homeostasis, hepatic oxidative stress, and transcriptomic profile in male mice. Methods: We used sham‐operated or orchiectomized (ORX) mice exposed to normoxia (Nx) or IH for 2 weeks. We performed fasting insulin and glucose tolerance tests and assessed fasting and postprandial insulin resistance with the HOMA‐IR. The activity of hepatic prooxidant (NADPH oxidase—NOX), antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase—SOD, Cat, GPx), lipid peroxidation (MDA concentration), and the total concentration of glutathione (GSH) were measured under postprandial conditions. mRNA sequencing and pathway enrichment analyses were used to identify hepatic genes underlying the interactions between IH and testosterone. Results: In Sham mice, IH improves fasting insulin sensitivity and glucose tolerance, while there are no effects of IH in ORX mice. In ORX mice, IH induces postprandial hyperinsulinemia, insulin resistance, and a prooxidant profile of enzyme activity (low SOD activity) without altering hepatic MDA and GSH content. ORX and IH altered the expression of genes involved in oxidoreductase activities, cytochromes‐dependent pathways, and glutathione metabolism. Among the genes upregulated in ORX‐IH mice, the flavin‐containing monooxygenases (FMO) are particularly relevant since these are potent hepatic antioxidants that could help prevent overt oxidative stress in ORX‐IH mice. Conclusion: Low levels of testosterone in male mice exposed to IH induce post‐prandial hyperinsulinemia and insulin resistance and determine the mechanisms by which the liver handles IH‐induced oxidative stress. [ABSTRACT FROM AUTHOR]

CYP2C19 is a member of the human microsomal cytochrome P450 (CYP). Significant variation in CYP2C19 levels and activity can be attributed to polymorphisms in this gene. Wildtype CYP2C19 and 13 mutants (CYP2C19.1B, CYP2C19.5A, CYP2C19.5B, CYP2C19.6, CYP2C19.8, CYP2C19.9, CYP2C19.10, CYP2C19.11, CYP2C19.13, CYP2C19.16, CYP2C19.19, CYP2C19.23, CYP2C19.30, and CYP2C19.33) were coexpressed with NADPH‐cytochrome P450 reductase in Escherichia coli. Hydroxylase activity toward testosterone and progesterone was also examined. Ten CYP2C19 variants showed Soret peaks (450 nm) typical of P450 in the reduced CO‐difference spectra. CYP2C19.11 and CYP2C19.23 showed higher testosterone 11α, 16α‐/17‐ and progesterone 6β‐,21‐,16α‐/17α‐hydroxylase activities than CYP2C19.1B. CYP2C19.6, CYP2C19.16, CYP2C19.19, and CYP2C19.30 showed lower activity than CYP2C19.1B. CYP2C19.9, CYP2C19.10. CYP2C19.13, and CYP2C19.33 showed different hydroxylation activities than CYP2C19.1B. These results indicated that CYP2C19 variants have very different substrate specificities for testosterone and progesterone. [ABSTRACT FROM AUTHOR]

Insect ommochrome biosynthesis pathways metabolize tryptophan to generate eye-color pigments and wild-type alleles of pathway genes are useful phenotypic markers in transgenesis studies. Pleiotropic effects of mutations in some genes exert a load on both survival and reproductive success in blood-feeding species. Here, we investigated the challenges imposed on mosquitoes by the increase of tryptophan metabolites resulting from blood meal digestion and the impact of disruptions of the ommochrome biosynthesis pathway. Female mosquitoes with spontaneous and induced mutations in the orthologs of the genes encoding kynurenine hydroxylase in Aedes aegypti, Anopheles stephensi and Culex quinquefasciatus exhibited impaired survival and reproductive phenotypes that varied in type and severity among the species. A compromised midgut permeability barrier function was also observed in An. stephensi. Surprisingly, mutant mosquitoes displayed an increase in microbiota compared to controls that was not accompanied by a general induction of immune genes. Antibiotic treatment rescued some deleterious traits implicating a role for the kynurenine pathway (KP) in midgut homeostasis. Supplemental xanthurenic acid, a KP end-product, rescued lethality and limited microbiota proliferation in Ae. aegypti. These data implicate the KP in the regulation of the host/microbiota interface. These pleiotropic effects on mosquito physiology are important in the development of genetic strategies targeting vector mosquitoes.

Oxygenases are important biocatalysts to produce many industrially important biomolecules. Here, a novel oxygenase, named MoxA, was identified through screening of a deep-sea sediment metagenomic library. Sequence analysis showed MoxA contains 424 amino acid residues with a predicated molecular mass of 46.9 kDa. Multiple sequence alignment and phylogenetic analysis indicated the sequence might be a new member of monooxygenase subfamily. A recombinant MoxA was obtained through the functional expression of moxA gene in Escherichia coli. Characterization of the purified MoxA indicated that it is an alkaline oxygenase showing maximal activity at pH 8.0. The optimal temperature of MoxA was 37 ℃, and it retained more than 70% of its initial activity after 1 h at 20–50 ℃ exhibiting good thermostability. Furthermore, effect of metal ions and organic solvents on enzymatic activity was investigated, and the results showed that the activity of MoxA was enhanced by Cu2+, Zn2+, Co2+ and Mg2+ at 1 mM, and by Co2+, Ca2+ and Mg2+ at 5 mM. Moreover, the recombinant strain harboring MoxA was used as a whole-cell biocatalyst for the efficient biosynthesis of indigo showing promising conversion efficiency. The biochemical properties of MoxA indicated that it would provide great contribution for the indigo bioproduction. Key points: • A novel monooxygenase from a metagenomic library was characterized. • The activity of MoxA was enhanced by metal ions at 1 mM and 5 mM. • MoxA has an optimal temperature of 37 ℃ and exhibited high conversion capacity. [ABSTRACT FROM AUTHOR]

Multi element compound-specific stable isotope analysis (ME-CSIA) is a tool to assess (bio)chemical reactions of molecules in the environment based on their isotopic fingerprints. To that effect, ME-CSIA concepts are initially developed with laboratory model experiments to determine the isotope fractionation factors specific for distinct (bio)chemical reactions. Here, we determined for the first time the carbon and hydrogen isotope fractionation factors for the monooxygenation of the short-chain alkanes ethane, propane, and butane. As model organism we used Thauera butanivorans strain Bu-B1211 which employs a non-haem iron monooxygenase (butane monooxygenase) to activate alkanes. Monooxygenation of alkanes was associated with strong carbon and hydrogen isotope effects: εbulkC = -2.95 ± 0.5 ‰ for ethane, -2.68 ± 0.1 ‰ for propane, -1.19 ± 0.18 ‰ for butane; εbulkH = -56.3 ± 15 ‰ for ethane, -40.5 ± 2.3 ‰ for propane, -14.6 ± 3.6 ‰ for butane. This resulted in lambda (Λ ≈ εHbulk/εCbulk) values of 16.2 ± 3.7 for ethane, 13.2 ± 0.7 for propane, and 11.4 ± 2.8 for butane. The results show that ME-CSIA can be used to track the occurrence and impact of monooxygenase-dependent aerobic processes converting short-chain alkanes in natural settings like marine and terrestrial seeps, gas reservoirs, and other geological formations impacted by natural gas. [ABSTRACT FROM AUTHOR]

Multi element compound-specific stable isotope analysis (ME-CSIA) is a tool to assess (bio)chemical reactions of molecules in the environment based on their isotopic fingerprints. To that effect, ME-CSIA concepts are initially developed with laboratory model experiments to determine the isotope fractionation factors specific for distinct (bio)chemical reactions. Here, we determined for the first time the carbon and hydrogen isotope fractionation factors for the monooxygenation of the short-chain alkanes ethane, propane, and butane. As model organism we used Thauera butanivorans strain Bu-B1211 which employs a non-haem iron monooxygenase (butane monooxygenase) to activate alkanes. Monooxygenation of alkanes was associated with strong carbon and hydrogen isotope effects: εbulkC = −2.95 ± 0.5 ‰ for ethane, −2.68 ± 0.1 ‰ for propane, −1.19 ± 0.18 ‰ for butane; εbulkH = −56.3 ± 15 ‰ for ethane, −40.5 ± 2.3 ‰ for propane, −14.6 ± 3.6 ‰ for butane. This resulted in lambda (Λ ≈ εHbulk/εCbulk) values of 16.2 ± 3.7 for ethane, 13.2 ± 0.7 for propane, and 11.4 ± 2.8 for butane. The results show that ME-CSIA can be used to track the occurrence and impact of monooxygenase-dependent aerobic processes converting short-chain alkanes in natural settings like marine and terrestrial seeps, gas reservoirs, and other geological formations impacted by natural gas.

Cytochromes P450 (CYPs) are heme-thiolate monooxygenases that prototypically catalyze the insertion of oxygen into unactivated C-H bonds but are capable of mediating more complex reactions. One of the most remarked-upon alternative reactions occurs during biosynthesis of the gibberellin A (GA) phytohormones, involving hydrocarbon ring contraction with coupled aldehyde extrusion of ent-kaurenoic acid to form the first gibberellin intermediate. While the unusual nature of this reaction has long been noted, its mechanistic basis has remained opaque. Building on identification of the relevant CYP114 from bacterial GA biosynthesis, detailed structure-function studies are reported here, including development of in vitro assays as well as crystallographic analyses both in the absence and presence of substrate. These structures provided insight into enzymatic catalysis of this unusual reaction, as exemplified by identification of a key role for the "missing" acid from an otherwise highly conserved acid-alcohol pair of residues. Notably, the results demonstrate that ring contraction requires dual factors, both the use of a dedicated ferredoxin and absence of the otherwise conserved acidic residue, with exclusion of either limiting turnover to just the initiating and more straightforward hydroxylation. The results provide detailed insight into the enzymatic structure-function relationships underlying this fascinating reaction and support the use of a semipinacol mechanism for the unusual ring contraction reaction. [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]

Though reactive flavin-N5/C4α-oxide intermediates can be spectroscopically profiled for some flavin-assisted enzymatic reactions, their exact chemical configurations are hardly visualized. Structural systems biology and stable isotopic labelling techniques were exploited to correct this stereotypical view. Three transition-like complexes, the α-ketoacid…N5-FMNox complex (I), the FMNox -N5-aloxyl-C'α- -C4α+ zwitterion (II), and the FMN-N5-ethenol-N5-C4α-epoxide (III), were determined from mandelate oxidase (Hmo) or its mutant Y128F (monooxygenase) crystals soaked with monofluoropyruvate (a product mimic), establishing that N5 of FMNox an alternative reaction center can polarize to an ylide-like mesomer in the active site. In contrast, four distinct flavin-C4α-oxide adducts (IV-VII) from Y128F crystals soaked with selected substrates materialize C4α of FMN an intrinsic reaction center, witnessing oxidation, Baeyer-Villiger/peroxide-assisted decarboxylation, and epoxidation reactions. In conjunction with stopped-flow kinetics, the multifaceted flavin-dependent reaction continuum is physically dissected at molecular level for the first time.

Background & objectives: Mosquito-borne diseases are major threats to human health worldwide. Successful control of vector mosquitoes requires periodic updates on their response to the insecticides that are in use. Different classes of neurotoxic insecticides have been used in vector control programs. Ae. aegypti and Ae. albopictus are the primary vectors of dengue and have developed resistance to organophosphates and synthetic pyrethroids that are used in vector control programs. Monitoring insecticide pressure and studying the underlying mechanisms of resistance in the field populations of Aedes aegypti are important to formulate resistant management strategies for their control programs. Methods: Aedes aegypti were collected from study sites Lawspet and Abishegapakkam and F1 progeny was subject to biochemical assays to determine the enzyme activity. Insecticide susceptibility tests were conducted to determine vector susceptibility/resistance to malathion and deltamethrin. Adult dried mosquitoes were subjected to multiplex PCR to detect point mutation in the VGSC gene. Results: Insecticide susceptibility test results revealed that Aedes aegypti is resistant to malathion and incipient resistance to deltamethrin has emerged. It was observed that β-esterase and monoxygense activity were significantly higher in Lawspet sample than the laboratory strain, whereas it was comparatively lower in Abishegapakkam sample than laboratory strain. Multiplex PCR assays showed no kdr mutation in all Ae. aegypti strains. Interpretation & conclusion: Monitoring insecticide resistance in Ae. aegypti would help the local health authorities to implement a rationalized approach for insecticide use in vector control.

Flavoprotein monooxygenases are a versatile group of enzymes for biocatalytic transformations. Among these, group E monooxygenases (GEMs) catalyze enantioselective epoxidation and sulfoxidation reactions. Here, we describe the crystal structure of an indole monooxygenase from the bacterium Variovorax paradoxus EPS, a GEM designated as VpIndA1. Complex structures with substrates reveal productive binding modes that, in conjunction with force‐field calculations and rapid mixing kinetics, reveal the structural basis of substrate and stereoselectivity. Structure‐based redesign of the substrate cavity yielded variants with new substrate selectivity (for sulfoxidation of benzyl phenyl sulfide) or with greatly enhanced stereoselectivity (from 35.1 % to 99.8 % ee for production of (1S,2R)‐indene oxide). This first determination of the substrate binding mode of GEMs combined with structure‐function relationships opens the door for structure‐based design of these powerful biocatalysts. [ABSTRACT FROM AUTHOR]

Flavoprotein‐Monooxygenasen sind eine vielseitige Gruppe von Enzymen für biokatalytische Reaktionen. Die dazugehörigen Monooxygenasen der Gruppe E (GEMs) katalysieren enantioselektive Epoxidierungs‐ und Sulfoxidierungsreaktionen. In dieser Arbeit beschreiben wir die Kristallstruktur einer Indol‐Monooxygenase aus dem Bakterium Variovorax paradoxus EPS, einer GEM mit der Bezeichnung VpIndA1. Basierend auf Substratkomplexstrukturen produktiver Bindungsmodi und in Verbindung mit Kraftfeldberechnungen sowie durch schnelle Mischungskinetik zeigen wir die strukturelle Grundlage der Substrat‐ und Stereoselektivität auf. Die strukturbasierte Umgestaltung der Substrattasche führte zu Varianten mit neuer Substratselektivität (für die Sulfoxidation von Benzylphenylsulfid) oder mit stark erhöhter Stereoselektivität (von 35.1 % auf 99.8 % ee für die Herstellung von (1S,2R)‐Indenoxid). Diese erste Bestimmung des Substratbindungsmodus von GEMs in Verbindung mit der Untersuchung von Struktur‐Funktions‐Beziehungen öffnet die Tür für ein strukturbasiertes Design dieser leistungsstarken Biokatalysatoren. [ABSTRACT FROM AUTHOR]

Monooxygenases, an important class of enzymes, have been the subject of enzyme engineering due to their high activity and versatile substrate scope. Reactions performed by these biocatalysts have long been monitored by a colorimetric method involving the coupling of a dye precursor to naphthalene hydroxylation products generated by the enzyme. Despite the popularity of this method, we found the dye product to be unstable, preventing quantitative readout. By incorporating an extraction step to solubilize the dye produced, we have improved this assay to the point where quantitation of enzyme activity is possible. Further, by incorporating spectral deconvolution, we have, for the first time, enabled independent quantification of the two possible regioisomeric products: 1‐naphthol and 2‐naphthol. Previously, such analysis was only possible with chromatographic separation, increasing the cost and complexity of analysis. The efficacy of our improved workflow was evaluated by monitoring the activity of a toluene‐4‐monooxygenase enzyme from Pseudomonas mendocina KR‐1. Our colorimetric regioisomer quantification was found to be consistent with chromatographic analysis by HPLC. The development and validation of a quantitative colorimetric assay for monooxygenase activity that enables regioisomeric distinction and quantification represents a significant advance in analytical methods to monitor enzyme activity. By maintaining facile, low‐cost, high‐throughput readout while incorporating quantification, this assay represents an important alternative to more expensive chromatographic quantification techniques. [ABSTRACT FROM AUTHOR]

Oxidative stress is associated with several acute and chronic disorders, including hematological malignancies such as acute myeloid leukemia, the most prevalent acute leukemia in adults. Xenobiotics are usually harmless compounds that may be detrimental, such as pharmaceuticals, environmental pollutants, cosmetics, and even food additives. The storage of xenobiotics can serve as a defense mechanism or a means of bioaccumulation, leading to adverse effects. During the absorption, metabolism, and cellular excretion of xenobiotics, three steps may be distinguished: (i) inflow by transporter enzymes, (ii) phases I and II, and (iii) phase III. Phase I enzymes, such as those in the cytochrome P450 superfamily, catalyze the conversion of xenobiotics into more polar compounds, contributing to an elevated acute myeloid leukemia risk. Furthermore, genetic polymorphism influences the variability and susceptibility of related myeloid neoplasms, infant leukemias associated with mixed-lineage leukemia (MLL) gene rearrangements, and a subset of de novo acute myeloid leukemia. Recent research has shown a sustained interest in determining the regulators of cytochrome P450, family 2, subfamily E, member 1 (CYP2E1) expression and activity as an emerging field that requires further investigation in acute myeloid leukemia evolution. Therefore, this review suggests that CYP2E1 and its mutations can be a therapeutic or diagnostic target in acute myeloid leukemia. [ABSTRACT FROM AUTHOR]

Peptidylglycine monooxygenase (PHM) is essential for the biosynthesis of many neuroendocrine peptides via a copper‐dependent hydroxylation of a glycine‐extended pro‐peptide. The "canonical" mechanism requires the transfer of two electrons from one mononuclear copper (CuH, H‐site) to a second mononuclear copper (CuM, M‐site) which is the site of oxygen binding and catalysis. In most crystal structures the copper centers are separated by 11 Å of disordered solvent, but recent work has established that a PHM variant H108A forms a closed conformer in the presence of citrate with a reduced Cu–Cu site separation of ~4 Å. Here we report three new PHM structures where the H and M sites are separated by a longer distance of ~14 Å. Variation in Cu–Cu distance is the result of a rotation of the M subdomain about a hinge point centered on the pro199‐leu200‐ile201 triad which forms the linker between subdomains. The energetic cost of domain dynamics is likely small enough to allow free rotation of the subdomains relative to each other, adding credence to recent suggestions that an open‐to‐closed transition to form a binuclear oxygen binding intermediate is an essential element of catalysis. This inference would explain many experimental observations that are inconsistent with the current canonical mechanism including substrate‐induced oxygen activation and isotope scrambling during the peroxide shunt. PDB Code(s): 8DSJ, 8DSL and 8DSN. [ABSTRACT FROM AUTHOR]

The Y128F single mutant of p-hydroxymandelate oxidase (Hmo) is capable of oxidizing mandelate to benzoate via a four-electron oxidative decarboxylation reaction. When benzoylformate (the product of the first two-electron oxidation) and hydrogen peroxide (an oxidant) were used as substrates the reaction did not proceed, suggesting that free hydrogen peroxide is not the committed oxidant in the second two-electron oxidation. How the flavin mononucleotide (FMN)-dependent four-electron oxidation reaction takes place remains elusive. Structural and biochemical explorations have shed new light on this issue. 15 high-resolution crystal structures of Hmo and its mutants liganded with or without a substrate reveal that oxidized FMN (FMNox) possesses a previously unknown electrophilic/nucleophilic duality. In the Y128F mutant the active-site perturbation ensemble facilitates the polarization of FMNox to a nucleophilic ylide, which is in a position to act on an α-ketoacid, forming an N5-acyl-FMNred dead-end adduct. In four-electron oxidation, an intramolecular disproportionation reaction via an N5-alkanol-FMNred C'α carbanion intermediate may account for the ThDP/PLP/NADPH-independent oxidative decarboxylation reaction. A synthetic 5-deaza-FMNox cofactor in combination with an α-hydroxyamide or α-ketoamide biochemically and structurally supports the proposed mechanism.

Abstract Background The regioselective hydroxylation of phenolic compounds, especially flavonoids, is still a bottleneck of classical organic chemistry that could be solved using enzymes with high activity and specificity. Yeast Rhodotorula glutinis KCh735 in known to catalyze the C-8 hydroxylation of flavones and flavanones. The enzyme F8H (flavonoid C8-hydroxylase) is involved in the reaction, but the specific gene has not yet been identified. In this work, we present identification, heterologous expression and characterization of the first F8H ortho-hydroxylase from yeast. Results Differential transcriptome analysis and homology to bacterial monooxygenases, including also a FAD-dependent motif and a GD motif characteristic for flavin-dependent monooxygenases, provided a set of coding sequences among which RgF8H was identified. Phylogenetic analysis suggests that RgF8H is a member of the flavin monooxygenase group active on flavonoid substrates. Analysis of recombinant protein showed that the enzyme catalyzes the C8-hydroxylation of naringenin, hesperetin, eriodyctiol, pinocembrin, apigenin, luteolin, chrysin, diosmetin and 7,4ʹ-dihydroxyflavone. The presence of the C7-OH group is necessary for enzymatic activity indicating ortho-hydroxylation mechanism. The enzyme requires the NADPH coenzyme for regeneration prosthetic group, displays very low hydroxyperoxyflavin decupling rate, and addition of FAD significantly increases its activity. Conclusions This study presents identification of the first yeast hydroxylase responsible for regioselective C8-hydroxylation of flavonoids (F8H). The enzyme was biochemically characterized and applied in in vitro cascade with Bacillus megaterium glucose dehydrogenase reactions. High in vivo activity in Escherichia coli enable further synthetic biology application towards production of rare highly antioxidant compounds.

Tyrosinase is the most predominant member of the coupled binuclear copper (CBC) protein family. The recent trapping and spectroscopic definition of the elusive catalytic ternary intermediate (enzyme/O2/monophenol) of tyrosinase dictates a monooxygenation mechanism that revises previous proposals and involves cleavage of the μ‐η2:η2‐peroxide dicopper(II) O–O bond to accept the phenolic proton, followed by monophenolate coordination to copper concomitant with aromatic hydroxylation by the non‐protonated μ‐oxo. Here, we compare and contrast previously proposed and current mechanistic models for monophenol monooxygenation of tyrosinase. Next, we discuss how these recent insights provide new opportunities towards uncovering structure–function relationships in CBC enzymes, as well as understanding fundamental principles for O2 activation and reactivity by bioinorganic active sites. [ABSTRACT FROM AUTHOR]

Fungi produce a plethora of natural products exhibiting a fascinating diversity of chemical structures with an enormous potential formedical applications.Despite the importance of understanding the scope of natural products and their biosynthetic pathways, a systematic analysis of the involved enzymes has not been undertaken. In our previous studies, we examined the flavoprotein encoding gene pool in archaea, eubacteria, the yeast Saccharomyces cerevisiae, Arabidopsis thaliana, and Homo sapiens. In the present survey, we have selected the model fungus Neurospora crassa as a starting point to investigate the flavoproteomes in the fungal kingdom. Our analysis showed that N. crassa harbors 201 flavoprotein-encoding genes amounting to 2% of the total protein-encoding genome. The majority of these flavoproteins (133) could be assigned to primary metabolism, termed the "core flavoproteome", with the remainder of flavoproteins (68) serving in, as yet unidentified, reactions. The latter group of "accessory flavoproteins" is dominated by monooxygenases, berberine bridge enzyme-like enzymes, and glucosemethanol- choline-oxidoreductases. Although the exact biochemical role of most of these enzymes remains undetermined, we propose that they are involved in activities closely associated with fungi, such as the degradation of lignocellulose, the biosynthesis of natural products, and the detoxification of harmful compounds in the environment. Based on this assumption, we have analyzed the accessory flavoproteomes in the fungal kingdom using the MycoCosm database. This revealed large differences among fungal divisions, with Ascomycota, Basidiomycota, and Mucoromycota featuring the highest average number of genes encoding accessory flavoproteins. Moreover, a more detailed analysis showed a massive accumulation of accessory flavoproteins in Sordariomycetes, Agaricomycetes, and Glomeromycotina. In our view, this indicates that these fungal classes are proliferative producers of natural products and also interesting sources for flavoproteins with potentially useful catalytic properties in biocatalytic applications. [ABSTRACT FROM AUTHOR]

Myzus persicae(Sulzer) (Hemiptera: Aphididae) is a polyphagous pest that causes significant losses in many crops. In the present study, the biochemical and molecular mechanism of acetamiprid resistance in a laboratory-selected M.persicae population of which the resistance ratios reached 57.5-fold were investigated. This study was conducted in the Isparta University of Applied Sciences, Agriculture Faculty, Department of Plant Protection in 2018 and 2020. Synergism, biochemical and molecular assays showed the absence of increased P450 activity in selected population. In addition, no point mutation in nicotinic acetylcholine receptor (nAChR), the target-site of neonicotinoids including acetamiprid, was detected in the selected population. These results suggests that high level of acetamiprid resistance might be developed via the mechanisms other than well-known mechanisms, such as increased P450 activity and target-site mutations. The population selected with acetamiprid showed decreased susceptibility to imidacloprid, sulfoxaflor, beta-cyfluthrin, and tau-fluvanite ranging from 1.54 to 4.76. Nonetheless, more studies are needed to support cross-resistance by M. persicae populations having different genetic backgrounds. [ABSTRACT FROM AUTHOR]

Abstract Background Non-ribosomal peptide synthetase-like (NRPS-like) enzymes are highly enriched in fungal genomes and can be discriminated into reducing and non-reducing enzymes. Non-reducing NRPS-like enzymes possess a C-terminal thioesterase domain that catalyses the condensation of two identical aromatic α-keto acids under the formation of enzyme-specific substrate-interconnecting core structures such as terphenylquinones, furanones, butyrolactones or dioxolanones. Ascocoryne sarcoides produces large quantities of ascocorynin, which structurally resembles a terphenylquinone produced from the condensation of p-hydroxyphenylpyruvate and phenylpyruvate. Since the parallel use of two different substrates by a non-reducing NRPS-like enzyme appeared as highly unusual, we investigated the biosynthesis of ascocorynin in A. sarcoides. Results Here, we searched the genome of A. sarcoides for genes coding for non-reducing NRPS-like enzymes. A single candidate gene was identified that was termed acyN. Heterologous gene expression confirmed that AcyN is involved in ascocorynin production but only produces the non-hydroxylated precursor polyporic acid. Although acyN is embedded in an ascocorynin biosynthesis gene cluster, a gene encoding a monooxygenase required for the hydroxylation of polyporic acid was not present. Expression analyses of all monooxygenase-encoding genes from A. sarcoides identified a single candidate that showed the same expression pattern as acyN. Accordingly, heterologous co-expression of acyN and the monooxygenase gene resulted in the production of ascocorynin. Structural modelling of the monooxygenase suggests that the hydrophobic substrate polyporic acid enters the monooxygenase from a membrane facing entry site and is converted into the more hydrophilic product ascocorynin, which prevents its re-entry for a second round of hydroxylation. Conclusion This study characterises the first naturally occurring polyporic acid synthetase from an ascomycete. It confirms the high substrate and product specificity of this non-reducing NRPS-like enzyme and highlights the requirement of a monooxygenase to produce the terphenylquinone ascocorynin.

The crosstalk between endoplasmic reticulum and mitochondria is of significance in apoptosis, in which cytochrome b 5 (Cyt b 5) is thought to be a major target for cytochrome c (Cyt c) upon its release from the mitochondria. In the absence of Cyt b 5 , the role of interactions of Cyt c with CYP-dependent monooxygenase system in apoptotic regulation was explored in this study. NADPH-dependent and Cyt c -induced formation of reactive oxygen species (ROS) and NADPH-independent Cyt c unfolding were revealed. With the aid of a CPR inhibitor and CYP antibodies, the interactions among Cyt c , cytochrome P450 reductase (CPR) and cytochrome P450 (CYP) are evidenced, which are found crucial for monooxygenase-derived ROS formation. The underlying structural basis of Cyt c −CYP complex was unveiled by molecular dynamics simulations. This study provides novel insights into how Cyt c regulates ROS formation through the interactions with CPR and CYP, and is implicated for a deeper understanding of the regulation mechanism in the mitochondria–endoplasmic reticulum apoptotic pathway. [Display omitted] [ABSTRACT FROM AUTHOR]

Tyrian purple, mainly composed of 6, 6′-dibromoindigo, is a precious dye extracted from sea snails. In this study, we found Tyrian purple can be selectively produced by a bacterial strain GS-2 when fed with 6-bromotryptophan in the presence of tryptophan. This GS-2 strain was then identified as Providencia rettgeri based on bacterial genome sequencing analysis. An indole degradation gene cluster for indole metabolism was identified from this GS-2 strain. The heterologous expression of the indole degradation gene cluster in Escherichia coli BL21 (DE3) and in vitro enzymatic reaction demonstrated that the indole biodegradation gene cluster may contribute to selectively biosynthesizing Tyrian purple. To further explore the underlying mechanism of the selectivity, we explored the intermediates in this indole biodegradation pathway using liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-MS/MS), which indicated that the indole biodegradation pathway in Providencia rettgeri is the catechol pathway. Interestingly, the monooxygenase GS-C co-expressed with its corresponding reductase GS-D in the cluster has better activity for the biosynthesis of Tyrian purple compared with the previously reported monooxygenase from Methylophaga aminisulfidivorans (MaFMO) or Streptomyces cattleya cytochrome P450 enzyme (CYP102G4). This is the first study to show the existence of an indole biodegradation pathway in Providencia rettgeri, and the indole biodegradation gene cluster can contribute to the selective production of Tyrian purple.

The reactions of enzymes and cofactors with gaseous molecules such as dioxygen (O2) are challenging to study and remain among the most contentious subjects in biochemistry. To date, it is largely enigmatic how enzymes control and fine-tune their reactions with O2, as exemplified by the ubiquitous flavin-dependent enzymes that commonly facilitate redox chemistry such as the oxygenation of organic substrates. Here we employ O2-pressurized X-ray crystallography and quantum mechanical calculations to reveal how the precise positioning of O2 within a flavoenzyme's active site enables the regiospecific formation of a covalent flavin-oxygen adduct and oxygenating species (i.e., the flavin-N5-oxide) by mimicking a critical transition state. This study unambiguously demonstrates how enzymes may control the O2 functionalization of an organic cofactor as prerequisite for oxidative catalysis. Our work thus illustrates how O2 reactivity can be harnessed in an enzymatic environment and provides crucial knowledge for future rational design of O2-reactive enzymes.

Enzymatic conversion of polysaccharides into lower-molecular-weight, soluble oligosaccharides is dependent on the action of hydrolytic and oxidative enzymes. Polysaccharide monooxygenases (PMOs) use an oxidative mechanism to break the glycosidic bond of polymeric carbohydrates, thereby disrupting the crystalline packing and creating new chain ends for hydrolases to depolymerize and degrade recalcitrant polysaccharides. PMOs contain a mononuclear Cu(II) center that is directly involved in C-H bond hydroxylation. Molecular oxygen was the accepted cosubstrate utilized by this family of enzymes until a recent report indicated reactivity was dependent on H2O2 Reported here is a detailed analysis of PMO reactivity with H2O2 and O2, in conjunction with high-resolution MS measurements. The cosubstrate utilized by the enzyme is dependent on the assay conditions. PMOs will directly reduce O2 in the coupled hydroxylation of substrate (monooxygenase activity) and will also utilize H2O2 (peroxygenase activity) produced from the uncoupled reduction of O2 Both cosubstrates require Cu reduction to Cu(I), but the reaction with H2O2 leads to nonspecific oxidation of the polysaccharide that is consistent with the generation of a hydroxyl radical-based mechanism in Fenton-like chemistry, while the O2 reaction leads to regioselective substrate oxidation using an enzyme-bound Cu/O2 reactive intermediate. Moreover, H2O2 does not influence the ability of secretome from Neurospora crassa to degrade Avicel, providing evidence that molecular oxygen is a physiologically relevant cosubstrate for PMOs.

The activities of critical metabolic and regulatory proteins can be altered by exposure to natural or synthetic redox-cycling compounds. Many bacteria, therefore, possess mechanisms to transport or transform these small molecules. The opportunistic pathogen Pseudomonas aeruginosa PA14 synthesizes phenazines, redox-active antibiotics that are toxic to other organisms but have beneficial effects for their producer. Phenazines activate the redox-sensing transcription factor SoxR and thereby induce the transcription of a small regulon, including the operon mexGHI-opmD, which encodes an efflux pump that transports phenazines, and PA14_35160 (pumA), which encodes a putative monooxygenase. Here, we provide evidence that PumA contributes to phenazine resistance and normal biofilm development, particularly during exposure to or production of strongly oxidizing N-methylated phenazines. We show that phenazine resistance depends on the presence of residues that are conserved in the active sites of other putative and characterized monooxygenases found in the antibiotic producer Streptomyces coelicolor. We also show that during biofilm growth, PumA is required for the conversion of phenazine methosulfate to unique phenazine metabolites. Finally, we compare ∆mexGHI-opmD and ∆pumA strains in assays for colony biofilm morphogenesis and SoxR activation, and find that these deletions have opposing phenotypic effects. Our results suggest that, while MexGHI-OpmD-mediated efflux has the effect of making the cellular phenazine pool more reducing, PumA acts on cellular phenazines to make the pool more oxidizing. We present a model in which these two SoxR targets function simultaneously to control the biological activity of the P. aeruginosa phenazine pool.

Fungi produce a plethora of natural products exhibiting a fascinating diversity of chemical structures with an enormous potential for medical applications. Despite the importance of understanding the scope of natural products and their biosynthetic pathways, a systematic analysis of the involved enzymes has not been undertaken. In our previous studies, we examined the flavoprotein encoding gene pool in archaea, eubacteria, the yeast Saccharomyces cerevisiae, Arabidopsis thaliana, and Homo sapiens. In the present survey, we have selected the model fungus Neurospora crassa as a starting point to investigate the flavoproteomes in the fungal kingdom. Our analysis showed that N. crassa harbors 201 flavoprotein-encoding genes amounting to 2% of the total protein-encoding genome. The majority of these flavoproteins (133) could be assigned to primary metabolism, termed the “core flavoproteome”, with the remainder of flavoproteins (68) serving in, as yet unidentified, reactions. The latter group of “accessory flavoproteins” is dominated by monooxygenases, berberine bridge enzyme-like enzymes, and glucose-methanol-choline-oxidoreductases. Although the exact biochemical role of most of these enzymes remains undetermined, we propose that they are involved in activities closely associated with fungi, such as the degradation of lignocellulose, the biosynthesis of natural products, and the detoxification of harmful compounds in the environment. Based on this assumption, we have analyzed the accessory flavoproteomes in the fungal kingdom using the MycoCosm database. This revealed large differences among fungal divisions, with Ascomycota, Basidiomycota, and Mucoromycota featuring the highest average number of genes encoding accessory flavoproteins. Moreover, a more detailed analysis showed a massive accumulation of accessory flavoproteins in Sordariomycetes, Agaricomycetes, and Glomeromycotina. In our view, this indicates that these fungal classes are proliferative producers of natural products and also interesting sources for flavoproteins with potentially useful catalytic properties in biocatalytic applications.

Most methanotrophs produce Mb to facilitate copper uptake, maintain Cu homeostasis, and protect against the toxic effects of high Cu concentration ( I M. trichosporium i OB3b on 10 µM Cu) [[98]]. The chelation of copper forms the Cu-Mb complex, which is a copper uptaking strategy of MOB in copper-limited conditions [[97]]. In the culture of I Methylococcus capsulatus i Bath, pMMO facilitates CH SB 4 sb oxidation in the cytoplasm when Cu/cell ratios are high (increased from 0 to 55 µM of Cu), while sMMO is activated in the periplasmic membrane space when Cu/cell ratios are low (decreased to 0 µM of Cu) because of different expression patterns (see above). Keywords: ammonia and methane oxidation; monooxygenase; copper; iron; methanobactin; siderophore EN ammonia and methane oxidation monooxygenase copper iron methanobactin siderophore 1806 18 11/17/22 20221101 NES 221101 1. On the other hand, Cu-chelating Mb also played a critical role in maintaining Cu homeostasis and protection against the potentially toxic effects of a high Cu concentration ( I M. trichosporium i OB3b on 10 µM copper) (see Figure 2) [[98]]. [Extracted from the article]

6‐Bromoindirubin (6BrIR), found in Murex sea snails, is a precursor of indirubin‐derivatives anticancer drugs. However, its synthesis remains limited due to uncharacterized biosynthetic pathways and difficulties in site‐specific bromination and oxidation at the indole ring. Here, we present an efficient 6BrIR production strategy in Escherichia coli by using four enzymes, that is, tryptophan 6‐halogenase fused with flavin reductase Fre (Fre‐L3‐SttH), tryptophanase (TnaA), toluene 4‐monooxygenase (PmT4MO), and flavin‐containing monooxygenase (MaFMO). Although most indole oxygenases preferentially oxygenate the electronically active C3 position of indole, PmT4MO was newly characterized to perform C2 oxygenation of 6‐bromoindole with 45% yield to produce 6‐bromo‐2‐oxindole. In addition, 6BrIR was selectively generated without indigo and indirubin byproducts by controlling the reducing power of cysteine and oxygen supply during the MaFMO reaction. These approaches led to 34.1 mg/L 6BrIR productions, making it possible to produce the critical precursor of the anticancer drugs only from natural ingredients such as tryptophan, NaBr, and oxygen. [ABSTRACT FROM AUTHOR]

The aim of this study was to apply biochemical parameters to Onychostoma barbatulum to understand whether Wuling Farm activities have caused organic pollution from xenoestrogens to flow into surrounding streams and thereby affected area aquatic ecosystems. Individuals of the Taiwan shovel-jaw carp (O. barbatulum) were collected by fyke netting in the Cijiawan, Kaoshan, and Yusheng Rivers in the protected area of Shei-Pa National Park and in the Ikawan River outside the protected areas from 4–5 June 2015 to 20–21 September 2015. The collected male individuals of O. barbatulum were divided into two groups. In the first group monooxygenase (Mon) activity, glutathione S-transferase (GST) activity, and vitellogenin (VTG) concentration in the liver were measured right after they were captured. Those in the second group were acclimated for 14 days and then exposed to 0 (control) or 10 ng/L 17β-estradiol for 10 days and then measured for changes in Mon, GST, and VTG in their livers. The value of VTG concentrations in O. barbatulum individuals collected from the Ikawan River was 2.12 ± 1.62 μg PO42+/g protein, which was the highest among all samples, followed by those collected from the Yusheng River (0.78 ± 1.00 μg PO42+/g protein). Individuals collected from the Yusheng River had the highest liver Mon activity (4.16 ± 1.08△A650/30 min/g protein) and the highest GST activity (1.58 ± 1.13 △A340/20 min/g protein), followed by those from the Ikawan River (Mon: 3.63 ± 1.13 △A650/30 min/g protein; GST: 1.24 ± 0.73 △A340/20 min/g protein). Comprehensive analyses showed that Mon and GST activities and VTG concentrations in livers measured right after being collected were lower than individuals exposed to 10 ng 17β-estradiol/L in the laboratory. Induced VTG concentrations were 9.87 ± 0.89 μg PO42+/g protein, Mon activity 8.02 ± 1.74 △A650/30 min/g protein, and GST activity 3.24 ± 0.62 △A340/20 min/g protein, indicating that farming activities have not significantly affected these aquatic organisms. However, pollution sources are still releasing pollutants containing xenoestrogens into the Yusheng River and Ikawan River. The impact of xenoestrogens is worthy of continuous long-term follow-up monitoring. [ABSTRACT FROM AUTHOR]

4-Hydroxyphenylacetate 3-hydroxylase (HpaB) has high potential for use in polyphenol synthesis via ortho-hydroxylation. Although the HpaB enzymes from Pseudomonas aeruginosa (PaHpaB) and Escherichia coli (EcHpaB) have been well studied, few studies have compared their activity and substrate selectivity. Thus, which HpaB is optimal for use in the biotechnological production of polyphenols is unclear. In this study, we performed a comparative analysis of the substrate- and regio-selectivity of PaHpaB, EcHpaB, and the recently discovered enzyme from Rhodococcus opacus (RoHpaB). The activity of these enzymes was first compared toward representative aromatic substrates. PaHpaB and EcHpaB exhibited very similar catalytic activity toward p-coumaric acid and tyrosol with one benzene ring, whereas PaHpaB exhibited greater activity than EcHpaB toward resveratrol and naringenin with two benzene rings. These results suggest that PaHpaB is superior to EcHpaB in converting bulky compounds. Furthermore, PaHpaB also exhibited catalytic activity toward a flavonoid, daidzein (7,4'-dihydroxyisoflavone), whereas EcHpaB did not. RoHpaB also exhibited strong activity toward daidzein in addition to other aromatic substrates. Interestingly, PaHpaB hydroxylated the 6-position of daidzein, whereas RoHpaB hydroxylated the 3'-position. PaHpaB and RoHpaB enabled the facile synthesis of not only 6-hydroxydaidzein and 3'-hydroxydaidzein but also 6,3'-dihydroxydaidzein via the cascade reaction. This study is the first to demonstrate synthesis of hydroxydaidzeins using HpaB enzymes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

As a typical polycyclic aromatic hydrocarbon (PAH), phenanthrene is often present in diverse environments, leading to severe environmental contamination. However, bacterial degradation plays a crucial role in remediating phenanthrene contamination and has been widely adopted. The widely distributed marine Roseobacter-clade bacteria are frequently found in phenanthrene-contaminated environments, but their catalyzing ability and related molecular mechanism have been rarely elucidated. Our previous work showed Ruegeria sp. PrR005 isolated from the Pearl River Estuary sediment could degrade phenanthrene and other PAHs. Integrated approaches including multi-omics and biochemical analysis were applied here to explore its catabolism mechanism. The genomic and transcriptomic analysis indicated that six new P450 monooxygenase proteins could be closely associated with phenanthrene degradation. Heterologous expression of P450 monooxygenase candidates revealed that PrR005_00615, PrR005_04282, PrR005_04577 have considerable activity in phenanthrene removal, with PrR005_00615 being the primary contributor. Further, the biochemical and metabolic analysis revealed that PrR005_00615 could catalyze phenanthrene to phenanthrene-9,10-epoxide by introducing an oxygen atom at 9,10-carbon positions, which functioned as a monooxygenase. The present study provides compelling evidences of a novel enzyme responsible for catalyzing the initial step of phenanthrene transformation in PrR005. These findings hold significant importance in unraveling the mechanism behind phenanthrene degradation by Roseobacter-clade bacteria., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Abstract Background Medium-chain-length α,ω-diols (mcl-diols) are important building blocks in polymer production. Recently, microbial mcl-diol production from alkanes was achieved in E. coli (albeit at low rates) using the alkane monooxygenase system AlkBGTL and esterification module Atf1. Owing to its remarkable versatility and conversion capabilities and hence potential for enabling an economically viable process, we assessed whether the industrially robust P. putida can be a suitable production organism of mcl-diols. Results AlkBGTL and Atf1 were successfully expressed as was shown by oxidation of alkanes to alkanols, and esterification to alkyl acetates. However, the conversion rate was lower than that by E. coli, and not fully to diols. The conversion was improved by using citrate instead of glucose as energy source, indicating that carbon catabolite repression plays a role. By overexpressing the activator of AlkBGTL-Atf1, AlkS and deleting Crc or CyoB, key genes in carbon catabolite repression of P. putida increased diacetoxyhexane production by 76% and 65%, respectively. Removing Crc/Hfq attachment sites of mRNAs resulted in the highest diacetoxyhexane production. When the intermediate hexyl acetate was used as substrate, hexanol was detected. This indicated that P. putida expressed esterases, hampering accumulation of the corresponding esters and diesters. Sixteen putative esterase genes present in P. putida were screened and tested. Among them, Est12/K was proven to be the dominant one. Deletion of Est12/K halted hydrolysis of hexyl acetate and diacetoxyhexane. As a result of relieving catabolite repression and preventing the hydrolysis of ester, the optimal strain produced 3.7 mM hexyl acetate from hexane and 6.9 mM 6-hydroxy hexyl acetate and diacetoxyhexane from hexyl acetate, increased by 12.7- and 4.2-fold, respectively, as compared to the starting strain. Conclusions This study shows that the metabolic versatility of P. putida, and the associated carbon catabolite repression, can hinder production of diols and related esters. Growth on mcl-alcohol and diol esters could be prevented by deleting the dominant esterase. Carbon catabolite repression could be relieved by removing the Crc/Hfq attachment sites. This strategy can be used for efficient expression of other genes regulated by Crc/Hfq in Pseudomonas and related species to steer bioconversion processes.

Background: Culex pipiens has a significant public health importance since it is an important vector of West Nile virus and Rift Valley fever virus. We, therefore, aimed to determine the insecticide resistance level in Cx. pipiens populations in the Aegean and Mediterranean regions of Turkey. Methods: Bioassays have been carried out against Dichlorodiphenyltrichloroethane (DDT) (4%), Malathion (5%), Fenitrothion (1%), Propoxur (0.1%), Bendiocarb (0.1%), Permethrin (0.75%) and Deltamethrin (0.05%). Biochemical analyses have been performed to detect non-specific esterase, mixed function oxidase, glutathione-s-transferase and acetylcholinesterase levels. A knockdown resistance (kdr) (L1014F) and Acetylcholinesterase (Ace-1) (G119S) mutations have been detected by using allele-specific primers and a polymerase chain reaction (PCR) amplification of specific alleles (PASA) diagnostic test was performed for detection of F290V mutation. Results: Bioassay results showed that all Cx. pipiens populations were resistant to DDT, Malathion, Fenitrothion, Bendiocarb, Propoxur and some of the populations have started to gain Permethrin and Deltamethrin resistance. Biochemical analyses results revealed that altered glutathione-s-transferases, P450 monooxygenases, esterase levels might be responsible for DDT, organophosphate, carbamate and pyrethroid resistance in Cx. pipiens populations. Results showed mild to high frequency of L1014F, low frequency of F290V but no Ace-1 G119S mutation within the populations. Additionally, acetylcholinesterase insensitivity was not significantly high within the most of these populations. Conclusion: Overall results may help to fulfil the lacking information in the literature regarding insecticide resistance status and underlying mechanism of Culex pipiens populations of the Mediterranean and Aegean region of Turkey by using all bioassays, molecular tests and biochemical assays.

Background: The regioselective hydroxylation of phenolic compounds, especially flavonoids, is still a bottleneck of classical organic chemistry that could be solved using enzymes with high activity and specificity. Yeast Rhodotorula glutinis KCh735 in known to catalyze the C-8 hydroxylation of flavones and flavanones. The enzyme F8H (flavonoid C8-hydroxylase) is involved in the reaction, but the specific gene has not yet been identified. In this work, we present identification, heterologous expression and characterization of the first F8H ortho-hydroxylase from yeast. Results: Differential transcriptome analysis and homology to bacterial monooxygenases, including also a FAD-dependent motif and a GD motif characteristic for flavin-dependent monooxygenases, provided a set of coding sequences among which RgF8H was identified. Phylogenetic analysis suggests that RgF8H is a member of the flavin monooxygenase group active on flavonoid substrates. Analysis of recombinant protein showed that the enzyme catalyzes the C8-hydroxylation of naringenin, hesperetin, eriodyctiol, pinocembrin, apigenin, luteolin, chrysin, diosmetin and 7,4ʹ-dihydroxyflavone. The presence of the C7-OH group is necessary for enzymatic activity indicating ortho-hydroxylation mechanism. The enzyme requires the NADPH coenzyme for regeneration prosthetic group, displays very low hydroxyperoxyflavin decupling rate, and addition of FAD significantly increases its activity. Conclusions: This study presents identification of the first yeast hydroxylase responsible for regioselective C8-hydroxylation of flavonoids (F8H). The enzyme was biochemically characterized and applied in in vitro cascade with Bacillus megaterium glucose dehydrogenase reactions. High in vivo activity in Escherichia coli enable further synthetic biology application towards production of rare highly antioxidant compounds. [ABSTRACT FROM AUTHOR]

Melanins are highly conjugated biopolymer pigments that provide photoprotection in a wide array of organisms, from bacteria to humans. The rate-limiting step in melanin biosynthesis, which is the ortho-hydroxylation of the amino acid L-tyrosine to L-DOPA, is catalyzed by the ubiquitous enzyme tyrosinase (Ty). Ty contains a coupled binuclear copper active site that binds O2 to form a μ:η²:η²-peroxide dicopper(II) intermediate (oxy-Ty), capable of performing the regioselective monooxygenation of para-substituted monophenols to catechols. The mechanism of this critical monooxygenation reaction remains poorly understood despite extensive efforts. In this study, we have employed a combination of spectroscopic, kinetic, and computational methods to trap and characterize the elusive catalytic ternary intermediate (Ty/O2/monophenol) under single-turnover conditions and obtain molecular-level mechanistic insights into its monooxygenation reactivity. Our experimental results, coupled with quantummechanics/molecular-mechanics calculations, reveal that the monophenol substrate docks in the active-site pocket of oxy-Ty fully protonated, without coordination to a copper or cleavage of the μ:η²:η²-peroxide O-O bond. Formation of this ternary intermediate involves the displacement of active-site water molecules by the substrate and replacement of their H bonds to the μ:η²:η²-peroxide by a single H bond from the substrate hydroxyl group. This H-bonding interaction in the ternary intermediate enables the unprecedented monooxygenation mechanism, where the μ:η²:η²-peroxide O-O bond is cleaved to accept the phenolic proton, followed by substrate phenolate coordination to a copper site concomitant with its aromatic ortho-hydroxylation by the nonprotonated μ-oxo. This study provides insights into O2 activation and reactivity by coupled binuclear copper active sites with fundamental implications in biocatalysis. [ABSTRACT FROM AUTHOR]

Aedes-borne diseases are on the rampant rise despite continued application of chemical insecticide-based interventions. The appearance of high degree of insecticide resistance in Aedes species and noxious effects on environment and non-targets have raised further concerns. Among new chemical interventions, neonicotinoids are considered a safe and effective approach. The present study investigated the control potency of acetamiprid and development of resistance in Aedes aegypti larvae; and the involvement of CYP450 monooxygenases in inducing resistance. The early fourth instars of Ae. aegypti parent susceptible strain (PS) were selected with acetamiprid for 15 generations (ACSF strain) increasing the resistance to 19.74-fold in ACSF-10 and 36.71-fold in ACSF-15. The ACSF-10 larvae were assayed with acetamiprid combined with piperonyl butoxide (PBO) in three different ratios (1:1, 1:5 and 1:10) and selected for next five generations with 1:10 combination. Selection with synergized acetamiprid (APSF strains) reversed as well as reduced the rate of resistance development resulting in only 1.35-fold resistance in APSF-15. The APSF strains showed %monooxygenase dependency ranging from 86.71 to 96.72%. The estimation of the monooxygenases levels in parent and selected larvae showed increased monooxygenase level in the ACSF strains by 2.42–2.87-fold. The APSF-15 strains exhibited 57.95% lower enzyme production than ACSF-15 strain. The reduction and reversion of resistance by using PBO and the elevated levels of monooxygenases in ACSF and reduction in APSF strains recommend the involvement of CYP450-mediated mechanism in the development of acetamiprid resistance in Ae. aegypti. These studies could help in devising resistance management strategies in order to preserve the efficiency of pre-existing insecticides. [ABSTRACT FROM AUTHOR]

Indigo is an important pigment widely used in industries of food, cosmetics, and textile. In this work, the styrene monooxygenase StyAB from Pseudomonas putida was co-expressed with the tryptophanase TnaA and the chaperone groES-groEL in Escherichia coli for indigo production. Over-expression of the gene styAB endowed the recombinant E. coli AB with the capacity of indigo biosynthesis from indole and tryptophan. Tryptophan fermentation in E. coli AB generated about five times more indigo than that from indole, and the maximum 530 mg/L of indigo was obtained from 1.2 mg/mL of tryptophan. The gene TnaA was then co-expressed with styAB, and the tryptophanase activity significantly increased in the recombinant E. coli ABT. However, TnaA expression led to a decrease in the activity of StyAB and indigo yield in E. coli ABT. Furthermore, the plasmid pGro7 harboring groES-groEL was introduced into E. coli AB, which obviously promoted the activity of StyAB and accelerated indigo biosynthesis in the recombinant E. coli ABP. In addition, the maximum yield of indigo was further increased to 550 mg/L from 1.2 mg/mL of tryptophan in E. coli ABP. The genetic manipulation strategy proposed in this work could provide new insights into construction of indigo biosynthesis cell factory for industrial production. [ABSTRACT FROM AUTHOR]

Herbicides are key weed-control tools, but their repeated use across large areas has favored the evolution of herbicide resistance. Although target-site has been the most prevalent and studied type of resistance, non-target-site resistance (NTSR) is increasing. However, the genetic factors involved in NTSR are widely unknown. In this study, four gene groups encoding putative NTSR enzymes, namely, cytochrome-P450, glutathione-S-transferase (GST), uridine 5′-diphospho-glucuronosyltransferase (UDPGT), and nitronate monooxygenase (NMO) were analyzed. The monocot and dicot gene sequences were downloaded from publicly available databases. Phylogenetic trees revealed that most of the CYP450 resistance-related sequences belong to CYP81 (5), and in GST, most of the resistance sequences belonged to GSTU18 (9) and GSTF6 (8) groups. In addition, the study of upstream promoter sequences of these NTSR genes revealed stress-related cis-regulatory motifs, as well as eight transcription factor binding sites (TFBS) were identified. The discovered TFBS were commonly present in both monocots and dicots, and the identified motifs are known to play key roles in countering abiotic stress. Further, we predicted the 3D structure for the resistant CYP450 and GST protein and identified the substrate recognition site through the homology approach. Our description of putative NTSR enzymes may be used to develop innovative weed control techniques to delay the evolution of NTSR. [ABSTRACT FROM AUTHOR]

Lytic polysaccharide monooxygenases (LPMOs) are widely distributed in fungi, and catalyze the oxidative degradation of polysaccharides such as cellulose. Despite their name, LPMOs possess a dominant peroxygenase activity that is reflected in high turnover numbers but also causes deactivation. We report on the influence of small molecules and ions on the activity and stability of LPMO during catalysis. Turbidimetric and photometric assays were used to identify LPMO inhibitors and measure their inhibitory effect. Selected inhibitors were employed to study LPMO activity and stability during cellulose depolymerization by HPLC and turbidimetry. It was found that the fungal metabolic products oxalic acid and citric acid strongly reduce LPMO activity, but also protect the enzyme from deactivation. QM calculations showed that the copper atom in the catalytic site could be ligated by bi- or tridentate chelating compounds, which replace two water molecules. MD simulations and QM calculations show that the most likely inhibition pattern is the competition between the inhibitor and reducing agent in the oxidized Cu(II) state. A correlation between the complexation energy and the IC50 values demonstrates that small, bidentate molecules interact strongest with the catalytic site copper and could be used by the fungus as physiological effectors to regulate LPMO activity. [ABSTRACT FROM AUTHOR]