Sequence and structure analyses of lytic polysaccharide monooxygenases mined from metagenomic DNA of humus samples around white-rot fungi in Cuc Phuong tropical forest, Vietnam.

Background: White-rot fungi and bacteria communities are unique ecosystems with different types of symbiotic interactions occurring during wood decomposition, such as cooperation, mutualism, nutritional competition, and antagonism. The role of chitin-active lytic polysaccharide monooxygenases (LPMOs) in these symbiotic interactions is the subject of this study. Method: In this study, bioinformatics tools were used to analyze the sequence and structure of putative LPMOs mined by hidden Markov model (HMM) profiles from the bacterial metagenomic DNA database of collected humus samples around white-rot fungi in Cuc Phuong primary forest, Vietnam. Two genes encoding putative LPMOs were expressed in E. coli and purified for enzyme activity assay. Result: Thirty-one full-length proteins annotated as putative LPMOs according to HMM profiles were confirmed by amino acid sequence comparison. The comparison results showed that although the amino acid sequences of the proteins were very different, they shared nine conserved amino acids, including two histidine and one phenylalanine that characterize the H1-Hx-Yz motif of the active site of bacterial LPMOs. Structural analysis of these proteins revealed that they are multidomain proteins with different functions. Prediction of the catalytic domain 3-D structure of these putative LPMOs using Alphafold2 showed that their spatial structures were very similar in shape, although their protein sequences were very different. The results of testing the activity of proteins GL0247266 and GL0183513 show that they are chitin-active LPMOs. Prediction of the 3-D structures of these two LPMOs using Alphafold2 showed that GL0247266 had five functional domains, while GL0183513 had four functional domains, two of which that were similar to the GbpA_2 and GbpA_3 domains of protein GbpA of Vibrio cholerae bacteria. The GbpA_2 - GbpA_3 complex was also detected in 11 other proteins. Based on the structural characteristics of functional domains, it is possible to hypothesize the role of chitin-active GbpA-like LPMOs in the relationship between fungal and bacterial communities coexisting on decomposing trees in primary forests.

TET1-Lipid Nanoparticle Encapsulating Morphine for Specific Targeting of Peripheral Nerve for Pain Alleviation.

Background: Opioids are irreplaceable analgesics owing to the lack of alternative analgesics that offer opioid-like pain relief. However, opioids have many undesirable central side effects. Restricting opioids to peripheral opioid receptors could reduce those effects while maintaining analgesia. Methods: To achieve this goal, we developed Tet1-LNP (morphine), a neural-targeting lipid nanoparticle encapsulating morphine that could specifically activate the peripheral opioid receptor in the dorsal root ganglion (DRG) and significantly reduce the side effects caused by the activation of opioid receptors in the brain. Tet1-LNP (morphine) were successfully prepared using the thin-film hydration method. In vitro, Tet1-LNP (morphine) uptake was assessed in differentiated neuron-like PC-12 cells and dorsal root ganglion (DRG) primary cells. The uptake of Tet1-LNP (morphine) in the DRGs and the brain was assessed in vivo. Von Frey filament and Hargreaves tests were used to assess the antinociception of Tet1-LNP (morphine) in the chronic constriction injury (CCI) neuropathic pain model. Morphine concentration in blood and brain were evaluated using ELISA. Results: Tet1-LNP (morphine) had an average size of 131 nm. Tet1-LNP (morphine) showed high cellular uptake and targeted DRG in vitro. CCI mice treated with Tet1-LNP (morphine) experienced prolonged analgesia for nearly 32 h compared with 3 h with free morphine (p < 0.0001). Notably, the brain morphine concentration in the Tet1-LNP (morphine) group was eight-fold lower than that in the morphine group (p < 0.0001). Conclusion: Our study presents a targeted lipid nanoparticle system for peripheral neural delivery of morphine. We anticipate Tet1-LNP (morphine) will offer a safe formulation for chronic neuropathic pain treatment, and promise further development for clinical applications.

Product Selectivity in Baeyer-Villiger Monooxygenase-Catalyzed Bacterial Alkaloid Core Structure Maturation.

Baeyer-Villiger monooxygenases (BVMOs) play crucial roles in the core-structure modification of natural products. They catalyze lactone formation by selective oxygen insertion into a carbon-carbon bond adjacent to a carbonyl group (Baeyer-Villiger oxidation, BVO). The homologous bacterial BVMOs, BraC and PxaB, thereby process bicyclic dihydroindolizinone substrates originating from a bimodular nonribosomal peptide synthetase (BraB or PxaA). While both enzymes initially catalyze the formation of oxazepine-dione intermediates following the identical mechanism, the final natural product spectrum diverges. For the pathway involving BraC, the exclusive formation of lipocyclocarbamates, the brabantamides, was reported. The pathway utilizing PxaB solely produces pyrrolizidine alkaloids, the pyrrolizixenamides. Surprisingly, replacing pxaB within the pyrrolizixenamide biosynthetic pathway by braC does not change the product spectrum to brabantamides. Factors controlling this product selectivity have remained elusive. In this study, we set out to solve this puzzle by combining the total synthesis of crucial pathway intermediates and anticipated products with in-depth functional in vitro studies on both recombinant BVMOs. This work shows that the joint oxazepine-dione intermediate initially formed by both BVMOs leads to pyrrolizixenamides upon nonenzymatic hydrolysis, decarboxylative ring contraction, and dehydration. Brabantamide biosynthesis is enzyme-controlled, with BraC efficiently transforming all the accepted substrates into its cognate final product scaffold. PxaB, in contrast, shows only considerable activity toward brabantamide formation for the substrate analog with a natural brabantamide-type side chain structure, revealing substrate-controlled product selectivity.

The Fluorescent Detection of Glucose and Lactic Acid Based on Fluorescent Iron Nanoclusters.

In this paper, a novel fluorescent detection method for glucose and lactic acid was developed based on fluorescent iron nanoclusters (Fe NCs). The Fe NCs prepared using hemin as the main raw material exhibited excellent water solubility, bright red fluorescence, and super sensitive response to hydrogen peroxide (H2O2). This paper demonstrates that Fe NCs exhibit excellent peroxide-like activity, catalyzing H2O2 to produce hydroxyl radicals (•OH) that can quench the red fluorescence of Fe NCs. In this paper, a new type of glucose sensor was established by combining Fe NCs with glucose oxidase (GluOx). With the increase in glucose content, the fluorescence of Fe NCs decreases correspondingly, and the glucose content can be detected in the scope of 0-200 µmol·L-1 (µM). Similarly, the lactic acid sensor can also be established by combining Fe NCs with lactate oxidase (LacOx). With the increase in lactic acid concentration, the fluorescence of Fe NCs decreases correspondingly, and the lactic acid content can be detected in the range of 0-100 µM. Furthermore, Fe NCs were used in the preparation of gel test strip, which can be used to detect H2O2, glucose and lactic acid successfully by the changes of fluorescent intensity.

Bi-directionalized promoter systems allow methanol-free production of hard-to-express peroxygenases with Komagataella Phaffii.

BACKGROUND: Heme-incorporating peroxygenases are responsible for electron transport in a multitude of organisms. Yet their application in biocatalysis is hindered due to their challenging recombinant production. Previous studies suggest Komagataella phaffi to be a suitable production host for heme-containing enzymes. In addition, co-expression of helper proteins has been shown to aid protein folding in yeast. In order to facilitate recombinant protein expression for an unspecific peroxygenase (AnoUPO), we aimed to apply a bi-directionalized expression strategy with Komagataella phaffii. RESULTS: In initial screenings, co-expression of protein disulfide isomerase was found to aid the correct folding of the expressed unspecific peroxygenase in K. phaffi. A multitude of different bi-directionalized promoter combinations was screened. The clone with the most promising promoter combination was scaled up to bioreactor cultivations and compared to a mono-directional construct (expressing only the peroxygenase). The strains were screened for the target enzyme productivity in a dynamic matter, investigating both derepression and mixed feeding (methanol-glycerol) for induction. Set-points from bioreactor screenings, resulting in the highest peroxygenase productivity, for derepressed and methanol-based induction were chosen to conduct dedicated peroxygenase production runs and were analyzed with RT-qPCR. Results demonstrated that methanol-free cultivation is superior over mixed feeding in regard to cell-specific enzyme productivity. RT-qPCR analysis confirmed that mixed feeding resulted in high stress for the host cells, impeding high productivity. Moreover, the bi-directionalized construct resulted in a much higher specific enzymatic activity over the mono-directional expression system. CONCLUSIONS: In this study, we demonstrate a methanol-free bioreactor production strategy for an unspecific peroxygenase, yet not shown in literature. Hence, bi-directionalized assisted protein expression in K. phaffii, cultivated under derepressed conditions, is indicated to be an effective production strategy for heme-containing oxidoreductases. This very production strategy might be opening up further opportunities for biocatalysis.

Mechanism of Nitrone Formation by a Flavin-Dependent Monooxygenase.

OxaD is a flavin-dependent monooxygenase (FMO) responsible for catalyzing the oxidation of an indole nitrogen atom, resulting in the formation of a nitrone. Nitrones serve as versatile intermediates in complex syntheses, including challenging reactions like cycloadditions. Traditional organic synthesis methods often yield limited results and involve environmentally harmful chemicals. Therefore, the enzymatic synthesis of nitrone-containing compounds holds promise for more sustainable industrial processes. In this study, we explored the catalytic mechanism of OxaD using a combination of steady-state and rapid-reaction kinetics, site-directed mutagenesis, spectroscopy, and structural modeling. Our investigations showed that OxaD catalyzes two oxidations of the indole nitrogen of roquefortine C, ultimately yielding roquefortine L. The reductive-half reaction analysis indicated that OxaD rapidly undergoes reduction and follows a "cautious" flavin reduction mechanism by requiring substrate binding before reduction can take place. This characteristic places OxaD in class A of the FMO family, a classification supported by a structural model featuring a single Rossmann nucleotide binding domain and a glutathione reductase fold. Furthermore, our spectroscopic analysis unveiled both enzyme-substrate and enzyme-intermediate complexes. Our analysis of the oxidative-half reaction suggests that the flavin dehydration step is the slow step in the catalytic cycle. Finally, through mutagenesis of the conserved D63 residue, we demonstrated its role in flavin motion and product oxygenation. Based on our findings, we propose a catalytic mechanism for OxaD and provide insights into the active site architecture within class A FMOs.

Integrated Experimental and Theoretical Investigation of Copper Active Site Properties of a Lytic Polysaccharide Monooxygenase from Serratia marcescens.

In this paper, we employed a multidisciplinary approach, combining experimental techniques and density functional theory (DFT) calculations to elucidate key features of the copper coordination environment of the bacterial lytic polysaccharide monooxygenase (LPMO) from Serratia marcescens (SmAA10). The structure of the holo-enzyme was successfully obtained by X-ray crystallography. We then determined the copper(II) binding affinity using competing ligands and observed that the affinity of the histidine brace ligands for copper is significantly higher than previously described. UV-vis, advanced electron paramagnetic resonance (EPR), and X-ray absorption spectroscopy (XAS) techniques, including high-energy resolution fluorescence detected (HERFD) XAS, were further used to gain insight into the copper environment in both the Cu(II) and Cu(I) redox states. The experimental data were successfully rationalized by DFT models, offering valuable information on the electronic structure and coordination geometry of the copper center. Finally, the Cu(II)/Cu(I) redox potential was determined using two different methods at ca. 350 mV vs NHE and rationalized by DFT calculations. This integrated approach not only advances our knowledge of the active site properties of SmAA10 but also establishes a robust framework for future studies of similar enzymatic systems.

Crape myrtle LiGAoxs displaying activities of gibberellin oxidases respond to branching architecture.

In the realm of ornamental horticulture, crape myrtle (Lagerstroemia indica) stands out for its aesthetic appeal, attributed largely to its vibrant flowers and distinctive branching architecture. This study embarked on a comprehensive exploration of the gibberellin oxidase (GAox) gene family in crape myrtle, illuminating its pivotal role in regulating GA levels, a key determinant of plant developmental processes. We identified and characterized 36 LiGAox genes, subdivided into GA2ox, GA3ox, GA20ox, and GAox-like subgroups, through genomic analyses. These genes' evolutionary trajectories were delineated, revealing significant gene expansions attributed to segmental duplication events. Functional analyses highlighted the divergent expression patterns of LiGAox genes across different crape myrtle varieties, associating them with variations in flower color and branching architecture. Enzymatic activity assays on selected LiGA2ox enzymes exhibited pronounced GA2 oxidase activity, suggesting a potential regulatory role in GA biosynthesis. Our findings offered a novel insight into the molecular underpinnings of GA-mediated growth and development in L. indica, providing a foundational framework for future genetic enhancements aimed at optimizing ornamental traits.

Establishing an Artificial Pathway for the Biosynthesis of Octopamine and Synephrine.

In this study, we designed an artificial pathway composed of tyramine ß-hydroxylase (TBH) and phenylethanolamine N-methyltransferase (PNMT) for the biosynthesis of both octopamine and synephrine. As most TBH and PNMT originate from eukaryotic animals and plants, the heterologous expression and identification of functional TBH and PNMT are critical for establishing the pathway in mode microorganisms like Escherichia coli. Here, three TBHs were evaluated, and only TBH from Drosophila melanogaster was successfully expressed in the soluble form in E. coli. Its expression was promoted by evaluating the effects of different expression strategies. The specific enzyme activity of TBH was optimized up to 229.50 U·g-1, and the first step in the biosynthetic pathway was successfully established and converted tyramine to synthesize 0.10 g/L of octopamine. Furthermore, the second step to produce synephrine from octopamine was developed by screening PNMT, enhancing enzyme activity, and optimizing reaction conditions, with a maximum synephrine production of 2.02 g/L. Finally, based on the optimization of the reaction conditions for each individual reaction, the one-pot cascade reaction for synthesizing synephrine from tyramine was constructed by combining the TBH and PNMT. The synthetic synephrine reached 30.05 mg/L with tyramine as substrate in the two-step enzyme cascade system. With further optimization and amplification, the titers of octopamine and synephrine were increased to 0.45 and 0.20 g/L, respectively, with tyramine as substrate. This work was the first achievement of the biosynthesis of octopamine and synephrine to date.

TET1 mediated m5C modification of RelB aggravates cerebral ischemia/reperfusion-induced neuroinflammation through regulating microglia polarization.

Microglia mediated neuroinflammation is one of the major contributors to brain damage in cerebral ischemia reperfusion injury (CI/RI). Recently, RNA modification was found to contribute to the regulation of microglia polarization and the subsequent development of cerebral I/R neuroinflammation. Herein, we investigated the effect and mechanism of m5C RNA modification in the microglia induced CI/RI neuroinflammation. We found that the m5C RNA modification levels decreased in the primary microglia isolated from a mouse model of intraluminal middle cerebral artery occlusion/reperfusion (MCAO/R) and the BV2 microglial cells subjected to oxygen-glucose deprivation and reoxygenation (OGD/R), and this change was accompanied by an increase in the M1/M2 polarization ratio. Furthermore, the expression of m5C demethylase TET1 in microglia increased, which promoted M1 polarization but impeded M2 polarization. Mechanistically, the higher TET1 expression decreased the m5C modification level of RelB and enhanced its mRNA stability, which subsequently increased the M1/M2 polarization ratio. In conclusion, this study provides insight into the role of m5C RNA modification in the pathogenesis of cerebral I/R neuroinflammation and may deepen our understanding on clinical therapy targeting the TET1-RelB axis.

Quantum chemical modeling of enantioselective sulfoxidation and epoxidation reactions by indole monooxygenase VpIndA1.

Indole monooxygenases (IMOs) are enzymes from the family of Group E monooxygenases, requiring flavin adenine dinucleotide (FAD) for their activities. IMOs play important roles in both sulfoxidation and epoxidation reactions. The broad substrate range and high selectivity of IMOs make them promising biocatalytic tools for synthesizing chiral compounds. In the present study, quantum chemical calculations using the cluster approach were performed to investigate the reaction mechanism and the enantioselectivity of the IMO from Variovorax paradoxus EPS (VpIndA1). The sulfoxidation of methyl phenyl sulfide (MPS) and the epoxidation of indene were chosen as the representative reactions. The calculations confirmed that the FADOOH intermediate is the catalytic species in the VpIndA1 reactions. The oxidation of MPS adopts a one-step mechanism involving the direct oxygen-transfer from FADOOH to the substrate and the proton transfer from the -OH group back to FAD, while the oxidation of indene follows a stepwise mechanism involving a carbocation intermediate. It was computationally predicted that VpIndA1 prefers the formation of (S)-product for the MPS sulfoxidation and (1S,2R)-product for the indene epoxidation, consistent with the experimental observations. Importantly, the factors controlling the stereo-preference of the two reactions are identified. The findings in the present study provide valuable insights into the VpIndA1-catalyzed reactions, which are essential for the rational design of this enzyme and other IMOs for industrial applications. It is also worth emphasizing that the quantum chemical cluster approach is again demonstrated to be powerful in studying the enantioselectivity of enzymatic reactions.

Rational engineering of Halomonas salifodinae to enhance hydroxyectoine production under lower-salt conditions.

Hydroxyectoine is an important compatible solute that holds potential for development into a high-value chemical with broad applications. However, the traditional high-salt fermentation for hydroxyectoine production presents challenges in treating the high-salt wastewater. Here, we report the rational engineering of Halomonas salifodinae to improve the bioproduction of hydroxyectoine under lower-salt conditions. The comparative transcriptomic analysis suggested that the increased expression of ectD gene encoding ectoine hydroxylase (EctD) and the decreased expressions of genes responsible for tricarboxylic acid (TCA) cycle contributed to the increased hydroxyectoine production in H. salifodinae IM328 grown under high-salt conditions. By blocking the degradation pathway of ectoine and hydroxyectoine, enhancing the expression of ectD, and increasing the supply of 2-oxoglutarate, the engineered H. salifodinae strain HS328-YNP15 (ΔdoeA::PUP119-ectD p-gdh) produced 8.3-fold higher hydroxyectoine production than the wild-type strain and finally achieved a hydroxyectoine titer of 4.9 g/L in fed-batch fermentation without any detailed process optimization. This study shows the potential to integrate hydroxyectoine production into open unsterile fermentation process that operates under low-salinity and high-alkalinity conditions, paving the way for next-generation industrial biotechnology. KEY POINTS: • Hydroxyectoine production in H. salifodinae correlates with the salinity of medium • Transcriptomic analysis reveals the limiting factors for hydroxyectoine production • The engineered strain produced 8.3-fold more hydroxyectoine than the wild type.

CYP116B5-SOX: An artificial peroxygenase for drug metabolites production and bioremediation.

CYP116B5 is a class VII P450 in which the heme domain is linked to a FMN and 2Fe2S-binding reductase. Our laboratory has proved that the CYP116B5 heme domain (CYP116B5-hd) is capable of catalyzing the oxidation of substrates using H2O2. Recently, the Molecular Lego approach was applied to join the heme domain of CYP116B5 to sarcosine oxidase (SOX), which provides H2O2 in-situ by the sarcosine oxidation. In this work, the chimeric self-sufficient fusion enzyme CYP116B5-SOX was heterologously expressed, purified, and characterized for its functionality by absorbance and fluorescence spectroscopy. Differential scanning calorimetry (DSC) experiments revealed a TM of 48.4 ± 0.04 and 58.3 ± 0.02°C and a enthalpy value of 175,500 ± 1850 and 120,500 ± 1350 cal mol-1 for the CYP116B5 and SOX domains respectively. The fusion enzyme showed an outstanding chemical stability in presence of up to 200 mM sarcosine or 5 mM H2O2 (4.4 ± 0.8 and 11.0 ± 2.6% heme leakage respectively). Thanks to the in-situ H2O2 generation, an improved kcat/KM for the p-nitrophenol conversion was observed (kcat of 20.1 ± 0.6 min-1 and KM of 0.23 ± 0.03 mM), corresponding to 4 times the kcat/KM of the CYP116B5-hd. The aim of this work is the development of an engineered biocatalyst to be exploited in bioremediation. In order to tackle this challenge, an E. coli strain expressing CYP116B5-SOX was employed to exploit this biocatalyst for the oxidation of the wastewater contaminating-drug tamoxifen. Data show a 12-fold increase in tamoxifen N-oxide production-herein detected for the first time as CYP116B5 metabolite-compared to the direct H2O2 supply, equal to the 25% of the total drug conversion.

Carotenoid biosynthesis genes LcLCYB, LcLCYE, and LcBCH from wolfberry confer increased carotenoid content and improved salt tolerance in tobacco.

Carotenoids play essential roles in plant growth and development and provide plants with a tolerance to a series of abiotic stresses. In this study, the function and biological significance of lycopene ß-cyclase, lycopene ε-cyclase, and ß-carotene hydroxylase, which are responsible for the modification of the tetraterpene skeleton procedure, were isolated from Lycium chinense and analyzed. The overexpression of lycopene ß-cyclase, lycopene ε-cyclase, and ß-carotene hydroxylase promoted the accumulation of total carotenoids and photosynthesis enhancement, reactive oxygen species scavenging activity, and proline content of tobacco seedlings after exposure to the salt stress. Furthermore, the expression of the carotenoid biosynthesis genes and stress-related genes (ascorbate peroxidase, catalase, peroxidase, superoxide dismutase, and pyrroline-5-carboxylate reductase) were detected and showed increased gene expression level, which were strongly associated with the carotenoid content and reactive oxygen species scavenging activity. After exposure to salt stress, the endogenous abscisic acid content was significantly increased and much higher than those in control plants. This research contributes to the development of new breeding aimed at obtaining stronger salt tolerance plants with increased total carotenoids and vitamin A content.

Stacked mutations in wheat homologues of rice SEMI-DWARF1 confer a novel semi-dwarf phenotype.

BACKGROUND: Semi-dwarfing alleles are used widely in cereals to confer improved lodging resistance and assimilate partitioning. The most widely deployed semi-dwarfing alleles in rice and barley encode the gibberellin (GA)-biosynthetic enzyme GA 20-OXIDASE2 (GA20OX2). The hexaploid wheat genome carries three homoeologous copies of GA20OX2, and because of functional redundancy, loss-of-function alleles of a single homoeologue would not be selected in wheat breeding programmes. Instead, approximately 70% of wheat cultivars carry gain-of-function mutations in REDUCED HEIGHT 1 (RHT1) genes that encode negative growth regulators and are degraded in response to GA. Semi-dwarf Rht-B1b or Rht-D1b alleles encode proteins that are insensitive to GA-mediated degradation. However, because RHT1 is expressed ubiquitously these alleles have pleiotropic effects that confer undesirable traits in some environments. RESULTS: We have applied reverse genetics to combine loss-of-function alleles in all three homoeologues of wheat GA20OX2 and its paralogue GA20OX1 and evaluated their performance in three years of field trials. ga20ox1 mutants exhibited a mild height reduction (approximately 3%) suggesting GA20OX1 plays a minor role in stem elongation in wheat. ga20ox2 mutants have reduced GA1 content and are 12-32% shorter than their wild-type segregants, comparable to the effect of the Rht-D1b 'Green Revolution' allele. The ga20ox2 mutants showed no significant negative effects on yield components in the spring wheat variety 'Cadenza'. CONCLUSIONS: Our study demonstrates that chemical mutagenesis can expand genetic variation in polyploid crops to uncover novel alleles despite the difficulty in identifying appropriate mutations for some target genes and the negative effects of background mutations. Field experiments demonstrate that mutations in GA20OX2 reduce height in wheat, but it will be necessary to evaluate the effect of these alleles in different genetic backgrounds and environments to determine their value in wheat breeding as alternative semi-dwarfing alleles.

Aspartate ß-Hydroxylase Is Upregulated in Head and Neck Squamous Cell Carcinoma and Regulates Invasiveness in Cancer Cell Models.

Aspartate ß-hydroxylase (ASPH) is a protein associated with malignancy in a wide range of tumors. We hypothesize that inhibition of ASPH activity could have anti-tumor properties in patients with head and neck cancer. In this study, we screened tumor tissues of 155 head and neck squamous cell carcinoma (HNSCC) patients for the expression of ASPH using immunohistochemistry. We used an ASPH inhibitor, MO-I-1151, known to inhibit the catalytic activity of ASPH in the endoplasmic reticulum, to show its inhibitory effect on the migration of SCC35 head and neck cancer cells in cell monolayers and in matrix-embedded spheroid co-cultures with primary cancer-associated fibroblast (CAF) CAF 61137 of head and neck origin. We also studied a combined effect of MO-I-1151 and HfFucCS, an inhibitor of invasion-blocking heparan 6-O-endosulfatase activity. We found ASPH was upregulated in HNSCC tumors compared to the adjacent normal tissues. ASPH was uniformly high in expression, irrespective of tumor stage. High expression of ASPH in tumors led us to consider it as a therapeutic target in cell line models. ASPH inhibitor MO-I-1151 had significant effects on reducing migration and invasion of head and neck cancer cells, both in monolayers and matrix-embedded spheroids. The combination of the two enzyme inhibitors showed an additive effect on restricting invasion in the HNSCC cell monolayers and in the CAF-containing co-culture spheroids. We identify ASPH as an abundant protein in HNSCC tumors. Targeting ASPH with inhibitor MO-I-1151 effectively reduces CAF-mediated cellular invasion in cancer cell models. We propose that the additive effect of MO-I-1151 with HfFucCS, an inhibitor of heparan 6-O-endosulfatases, on HNSCC cells could improve interventions and needs to be further explored.

Simultaneous production of biosurfactant and extracellular unspecific peroxygenases by Moesziomyces aphidis XM01 enables an efficient strategy for crude oil degradation.

Crude oil is a hazardous pollutant that poses significant and lasting harm to human health and ecosystems. In this study, Moesziomyces aphidis XM01, a biosurfactant mannosylerythritol lipids (MELs)-producing yeast, was utilized for crude oil degradation. Unlike most microorganisms relying on cytochrome P450, XM01 employed two extracellular unspecific peroxygenases, MaUPO.1 and MaUPO.2, with preference for polycyclic aromatic hydrocarbons (PAHs) and n-alkanes respectively, thus facilitating efficient crude oil degradation. The MELs produced by XM01 exhibited a significant emulsification activity of 65.9% for crude oil and were consequently supplemented in an "exogenous MELs addition" strategy to boost crude oil degradation, resulting in an optimal degradation ratio of 72.3%. Furthermore, a new and simple "pre-MELs production" strategy was implemented, achieving a maximum degradation ratio of 95.9%. During this process, the synergistic up-regulation of MaUPO.1, MaUPO.1 and the key MELs synthesis genes contributed to the efficient degradation of crude oil. Additionally, the phylogenetic and geographic distribution analysis of MaUPO.1 and MaUPO.1 revealed their wide occurrence among fungi in Basidiomycota and Ascomycota, with high transcription levels across global ocean, highlighting their important role in biodegradation of crude oil. In conclusion, M. aphidis XM01 emerges as a novel yeast for efficient and eco-friendly crude oil degradation.

Mutational dissection of a hole hopping route in a lytic polysaccharide monooxygenase (LPMO).

Oxidoreductases have evolved tyrosine/tryptophan pathways that channel highly oxidizing holes away from the active site to avoid damage. Here we dissect such a pathway in a bacterial LPMO, member of a widespread family of C-H bond activating enzymes with outstanding industrial potential. We show that a strictly conserved tryptophan is critical for radical formation and hole transference and that holes traverse the protein to reach a tyrosine-histidine pair in the protein's surface. Real-time monitoring of radical formation reveals a clear correlation between the efficiency of hole transference and enzyme performance under oxidative stress. Residues involved in this pathway vary considerably between natural LPMOs, which could reflect adaptation to different ecological niches. Importantly, we show that enzyme activity is increased in a variant with slower radical transference, providing experimental evidence for a previously postulated trade-off between activity and redox robustness.

Oxygen-transfer reactions by enzymatic flavin-N5 oxygen adducts-Oxidation is not a must.

Flavoenzymes catalyze numerous redox reactions including the transfer of an O2-derived oxygen atom to organic substrates, while the other one is reduced to water. Investigation of some of these monooxygenases led to a detailed understanding of their catalytic cycle, which involves the flavin-C4α-(hydro)peroxide as hallmark oxygenating species, and newly discovered flavoprotein monooxygenases were generally assumed to operate similarly. However, discoveries in recent years revealed a broader mechanistic versatility, including enzymes that utilize flavin-N5 oxygen adducts for catalysis in the form of the flavin-N5-(hydro)peroxide and the flavin-N5-oxide species. In this review, I will highlight recent developments in that area, including noncanonical flavoenzymes from natural product biosynthesis and sulfur metabolism that provide first insights into the chemical properties of these species. Remarkably, some enzymes may even combine the flavin-N5-peroxide and the flavin-N5-oxide species for consecutive oxygen-transfers to the same substrate and thereby in essence operate as dioxygenases.

SlCYP94B18 and SlCYP94B19 monooxygenases for the catabolic turnover of jasmonates in tomato leaves.

(3R,7S)-Jasmonoyl-L-isoleucine (JA-Ile) is a plant hormone that regulates plant defense responses and other physiological functions. The mechanism of attenuation of JA-Ile signaling in the plant body is essential because prolonged JA-Ile signaling can be detrimental to plant survival. In Arabidopsis thaliana, the cytochrome P450 monooxygenases, CYP94B1/B3/C1, inactivate JA-Ile by converting it into 12-hydroxy-jasmonoyl-L-isoleucine (12-OH-JA-Ile), and CYP94C1 converts 12-OH-JA-Ile into 12-carboxy-jasmonoyl-L-isoleucine (12-COOH-JA-Ile). In the present study, we aimed to identify the cytochrome P450 monooxygenases involved in the catabolic pathway of JA-Ile in tomato leaves. Based on a gene expression screening of SlCYP94 subfamily monooxygenases using qPCR and the time-course of JA-Ile catabolism, we identified SlCYP94B18 and SlCYP94B19 expressed in tomato leaves as candidate monooxygenases catalyzing the two-step catabolism of JA-Ile. An in vitro enzymatic assay using a yeast expression system revealed that these enzymes efficiently converted JA-Ile to 12-OH-JA-Ile, and then to 12-COOH-JA-Ile. SlCYP94B18 and SlCYP94B19 also catalyzed the oxidative catabolism of several JA-amino acid conjugates (JA-AAs), JA-Leu and JA-Val, in tomatoes. These results suggest that SlCYP94B18 and SlCYP94B19 plays a role in the two-step oxidation of JA-AAs, suggesting their broad involvement in regulating jasmonate signaling in tomatoes. Our results contribute to a deeper understanding of jasmonate signaling in tomatoes and may help to improve tomato cultivation and quality.