Recognition of microbial viability via TLR8 drives TFH cell differentiation and vaccine responses.

Live attenuated vaccines are generally highly efficacious and often superior to inactivated vaccines, yet the underlying mechanisms of this remain largely unclear. Here we identify recognition of microbial viability as a potent stimulus for follicular helper T cell (TFH cell) differentiation and vaccine responses. Antigen-presenting cells (APCs) distinguished viable bacteria from dead bacteria through Toll-like receptor 8 (TLR8)-dependent detection of bacterial RNA. In contrast to dead bacteria and other TLR ligands, live bacteria, bacterial RNA and synthetic TLR8 agonists induced a specific cytokine profile in human and porcine APCs, thereby promoting TFH cell differentiation. In domestic pigs, immunization with a live bacterial vaccine induced robust TFH cell and antibody responses, but immunization with its heat-killed counterpart did not. Finally, a hypermorphic TLR8 polymorphism was associated with protective immunity elicited by vaccination with bacillus Calmette-Guérin (BCG) in a human cohort. We have thus identified TLR8 as an important driver of TFH cell differentiation and a promising target for TFH cell-skewing vaccine adjuvants.

Bacterial stigmasterol degradation involving radical flavin delta-24 desaturase and molybdenum-dependent C26 hydroxylase.

Sterols are ubiquitous membrane constituents that persist to a large extent in the environment due to their water insolubility and chemical inertness. Recently, an oxygenase-independent sterol degradation pathway was discovered in a cholesterol-grown denitrifying bacterium Sterolibacterium (S.) denitrificans. It achieves hydroxylation of the unactivated primary C26 of the isoprenoid side chain to an allylic alcohol via a phosphorylated intermediate in a four-step ATP-dependent enzyme cascade. However, this pathway is incompatible with the degradation of widely distributed steroids containing a double bond at C22 in the isoprenoid side chain such as the plant sterol stigmasterol. Here, we have enriched a prototypical delta-24 desaturase from S. denitrificans, which catalyzes the electron acceptor-dependent oxidation of the intermediate stigmast-1,4-diene-3-one to a conjugated (22,24)-diene. We suggest an α4ß4 architecture of the 440 kDa enzyme, with each subunit covalently binding an flavin mononucleotide cofactor to a histidyl residue. As isolated, both flavins are present as red semiquinone radicals, which can be reduced by stigmast-1,4-diene-3-one but cannot be oxidized even with strong oxidizing agents. We propose a mechanism involving an allylic radical intermediate in which two flavin semiquinones each abstract one hydrogen atom from the substrate. The conjugated delta-22,24 moiety formed allows for the subsequent hydroxylation of the terminal C26 with water by a heterologously produced molybdenum-dependent steroid C26 dehydrogenase 2. In conclusion, the pathway elucidated for delta-22 steroids achieves oxygen-independent hydroxylation of the isoprenoid side chain by bypassing the ATP-dependent formation of a phosphorylated intermediate.

An Enzymatic Cofactor Regeneration System for the in-Vitro Reduction of Isolated C=C Bonds by Geranylgeranyl Reductases.

Cofactor regeneration systems are of major importance for the applicability of oxidoreductases in biocatalysis. Previously, geranylgeranyl reductases have been investigated for the enzymatic reduction of isolated C=C bonds. However, an enzymatic cofactor-regeneration system for in vitro use is lacking. In this work, we report a ferredoxin from the archaea Archaeoglobus fulgidus that regenerates the flavin of the corresponding geranylgeranyl reductase. The proteins were heterologously produced, and the regeneration was coupled to a ferredoxin reductase from Escherichia coli and a glucose dehydrogenase from Bacillus subtilis, thereby enabling the reduction of isolated C=C bonds by purified enzymes. The system was applied in crude, cell-free extracts and gave conversions comparable to those of a previous method using sodium dithionite for cofactor regeneration. Hence, an enzymatic approach to the reduction of isolated C=C bonds can be coupled with common systems for the regeneration of nicotinamide cofactors, thereby opening new perspectives for the application of geranylgeranyl reductases in biocatalysis.

Structures and Protein Engineering of the α-Keto Acid C-Methyltransferases SgvM and MrsA for Rational Substrate Transfer.

S-adenosyl-l-methionine-dependent methyltransferases (MTs) are involved in the C-methylation of a variety of natural products. The MTs SgvM from Streptomyces griseoviridis and MrsA from Pseudomonas syringae pv. syringae catalyze the methylation of the ß-carbon atom of α-keto acids in the biosynthesis of the antibiotic natural products viridogrisein and 3-methylarginine, respectively. MrsA shows high substrate selectivity for 5-guanidino-2-oxovalerate, while other α-keto acids, such as the SgvM substrates 4-methyl-2-oxovalerate, 2-oxovalerate, and phenylpyruvate, are not accepted. Here we report the crystal structures of SgvM and MrsA in the apo form and bound with substrate or S-adenosyl-l-methionine. By investigating key residues for substrate recognition in the active sites of both enzymes and engineering MrsA by site-directed mutagenesis, the substrate range of MrsA was extended to accept α-keto acid substrates of SgvM with uncharged and lipophilic ß-residues. Our results showcase the transfer of the substrate scope of α-keto acid MTs from different biosynthetic pathways by rational design.

Intra-oscillator high harmonic source reaching 100-eV photon energy.

Resonant enhancement inside an optical cavity has been a wide-spread approach to increase efficiency of nonlinear optical conversion processes while reducing the demands on the driving laser power. This concept has been particularly important for high harmonic generation XUV sources, where passive femtosecond enhancement cavities allowed significant increase in repetition rates required for applications in photoelectron spectroscopy, XUV frequency comb spectroscopy, including the recent endeavor of thorium nuclear clock development. In addition to passive cavities, it has been shown that comparable driving conditions can be achieved inside mode-locked thin-disk laser oscillators, offering a simplified single-stage alternative. This approach is less sensitive to losses thanks to the presence of gain inside the cavity and should thus allow higher conversion efficiencies through tolerating higher intensity in the gas target. Here, we show that the intra-oscillator approach can indeed surpass the much more mature technology of passive enhancement cavities in terms of XUV flux, even reaching comparable values to single-pass sources based on chirped-pulse fiber amplifier lasers. Our system operates at 17 MHz repetition rate generating photon energies between 60 eV and 100 eV. Importantly, this covers the highly attractive wavelength for the silicon industry of 13.5 nm at which our source delivers 60 nW of outcoupled average power per harmonic order.

Enzymatic defluorination of a terminally monofluorinated pentyl moiety: oxidative or hydrolytic mechanism?

Fluorination of organic compounds plays an important role in the chemical and pharmaceutical industry and is often applied in order to improve physicochemical parameters or modify pharmacological properties. While oxidative and reductive defluorination have been shown to be responsible for the metabolic degradation of organofluorine compounds, the involvement of hydrolytic mechanisms catalyzed by human enzymes has not been reported so far. Here, we investigated the enzymatic defluorination of terminally monofluorinated aliphates with [1-(5-fluoropentyl)-1H-indol-3-yl]-1-naphthalenyl-methanone (AM-2201) as a model substance. We performed in vitro biotransformation using pooled human liver microsomes (pHLM) and human recombinant cytochrome P450 (CYP) assays. In order to elucidate the underlying mechanisms, modified incubation conditions were applied including the use of deuterium labeled AM-2201 (d2 -AM-2201). Identification of the main metabolites and analysis of their isotopic composition was performed by liquid-chromatography coupled to time-of-flight-mass-spectrometry (LC-QToF-MS). Quantification of the metabolites was achieved with a validated method based on liquid-chromatography-tandem-mass-spectrometry (LC-MS/MS). CYP 1A2 mediated defluorination of d2 -AM-2201 revealed an isotopic pattern of the defluorinated 5-hydroxypentyl metabolite (5-HPM) indicating a redox mechanism with an aldehyde as a plausible intermediate. In contrast, formation of 5-HPM by pHLM was observed independently of the presence of atmospheric oxygen or co-factors regenerating the redox system. pHLM incubation of d2 -AM-2201 confirmed the hypothesis of a non-oxidative mechanism involved in the defluorination of the 5-fluoropentyl moiety. So far, enzymatically catalyzed, hydrolytic defluorination was only described in bacteria and other prokaryotes. The presented data prove the involvement of a hydrolytic mechanism catalyzed by human microsomal enzymes other than CYP. Significance Statement Elucidating the mechanisms involved in the enzymatic detoxification of organofluorine compounds is crucial for enhancing our understanding and facilitating the design and development of drugs with improved pharmacokinetic profiles. The carbon-fluorine bond possesses a high binding energy, which suggests that non-activated fluoroalkanes would not undergo hydrolytic cleavage. However, our study provides evidence for the involvement of a non-oxidative mechanism catalyzed by human liver enzymes. It is important to consider CYP-independent, hydrolytic defluorination, when investigating the pharmacokinetic properties of fluorinated xenobiotics.

Differences between women and men in prolonged weaning.

BACKGROUND: In recent years, the importance of sex as a factor influencing medical care has received increasing attention in the field of intensive care medicine. The objective of this study was to examine the influence of sex in prolonged weaning. METHODS: A retrospective analysis of patients undergoing prolonged weaning at Thoraxklinik, University Hospital Heidelberg between 12/08 and 12/23 was conducted. Patients with neuromuscular diseases were excluded from the analyses. The risk factors for weaning failure in men and women were identified through stepwise cox-regression analyses. RESULTS: A total of 785 patients were included, of whom 313 (39.9%) were women. 77.9% of the women and 75.4% of the men were successfully weaned from invasive ventilation. In group comparisons and multivariable analyses, sex was not found to be a risk factor for weaning failure. Cox regression analyses were performed separately for both sexes on the outcome of weaning failure, adjusting for relevant covariates. The results indicated that age ≥ 65 years (HR 2.38, p < 0.001) and the duration of IMV before transfer to the weaning centre (HR 1.01/day, p < 0.001) were independent risk factors in men. In women, however, the duration of IMV before transfer (HR 1.01, p < 0.001), previous non-invasive ventilation (HR 2.9, p 0.005), the presence of critical illness polyneuropathy (HR 1.82; p = 0.040) and delirium (HR 2.50, p = 0.017) were identified as relevant risk factors. In contrast delirium was associated with a favourable weaning outcome in men (HR 0.38, p = 0.020) and nosocomial pneumonia as a reason for prolonged weaning in women (HR 0.43; p = 0.032). CONCLUSION: The analyses indicate that there are sex-based differences in the risk factors associated with weaning failure. Further studies, ideally prospective, should confirm these findings to assess whether sex is a factor that should be taken into account to improve weaning outcomes.

Role of multidrug-resistant bacteria in weaning from invasive mechanical ventilation.

BACKGROUND: Although multidrug-resistant bacteria (MDR) are common in patients undergoing prolonged weaning, there is little data on their impact on weaning and patient outcomes. METHODS: This is a retrospective analysis of consecutive patients who underwent prolonged weaning and were at a university weaning centre from January 2018 to December 2020. The influence of MDR colonisation and infection on weaning success (category 3a and 3b), successful prolonged weaning from invasive mechanical ventilation (IMV) with or without the need for non-invasive ventilation (NIV) compared with category 3c (weaning failure 3cI or death 3cII) was investigated. The pathogen groups considered were: multidrug-resistant gram-negative bacteria (MDRGN), methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus spp. (VRE). RESULTS: A total of 206 patients were studied, of whom 91 (44.2%) showed evidence of MDR bacteria (32% VRE, 1.5% MRSA and 16% MDRGN), with 25 patients also meeting the criteria for MDR infection. 70.9% of the 206 patients were successfully weaned from IMV, 8.7% died. In 72.2% of cases, nosocomial pneumonia and other infections were the main cause of death. Patients with evidence of MDR (infection and colonisation) had a higher incidence of weaning failure than those without evidence of MDR (48% vs. 34.8% vs. 21.7%). In multivariate analyses, MDR infection (OR 4.9, p = 0.004) was an independent risk factor for weaning failure, along with male sex (OR 2.3, p = 0.025), Charlson Comorbidity Index (OR 1.2, p = 0.027), pH (OR 2.7, p < 0.001) and duration of IMV before admission (OR 1.01, p < 0.001). In addition, MDR infection was the only independent risk factor for death (category 3cII), (OR 6.66, p = 0.007). CONCLUSION: Patients with MDR infection are significantly more likely to die during the weaning process. There is an urgent need to develop non-antibiotic approaches for the prevention and treatment of MDR infections as well as clinical research on antibiotic stewardship in prolonged weaning as well as in ICUs.

Risk factors for long-term invasive mechanical ventilation: a longitudinal study using German health claims data.

BACKGROUND: Long-term invasive mechanical ventilation (IMV) is a major burden for those affected and causes high costs for the health care system. Early risk assessment is a prerequisite for the best possible support of high-risk patients during the weaning process. We aimed to identify risk factors for long-term IMV within 96 h (h) after the onset of IMV. METHODS: The analysis was based on data from one of Germany's largest statutory health insurance funds; patients who received IMV ≥ 96 h and were admitted in January 2015 at the earliest and discharged in December 2017 at the latest were analysed. OPS and ICD codes of IMV patients were considered, including the 365 days before intubation and 30 days after discharge. Long-term IMV was defined as evidence of invasive home mechanical ventilation (HMV), IMV ≥ 500 h, or readmission with (re)prolonged ventilation. RESULTS: In the analysis of 7758 hospitalisations, criteria for long-term IMV were met in 38.3% of cases, of which 13.9% had evidence of HMV, 73.1% received IMV ≥ 500 h and/or 40.3% were re-hospitalised with IMV. Several independent risk factors were identified (p < 0.005 each), including pre-diagnoses such as pneumothorax (OR 2.10), acute pancreatitis (OR 2.64), eating disorders (OR 1.99) or rheumatic mitral valve disease (OR 1.89). Among ICU admissions, previous dependence on an aspirator or respirator (OR 5.13), and previous tracheostomy (OR 2.17) were particularly important, while neurosurgery (OR 2.61), early tracheostomy (OR 3.97) and treatment for severe respiratory failure such as positioning treatment (OR 2.31) and extracorporeal lung support (OR 1.80) were relevant procedures in the first 96 h after intubation. CONCLUSION: This comprehensive analysis of health claims has identified several risk factors for the risk of long-term ventilation. In addition to the known clinical risks, the information obtained may help to identify patients at risk at an early stage. Trial registration The PRiVENT study was retrospectively registered at ClinicalTrials.gov (NCT05260853). Registered at March 2, 2022.

Impact of seizure outcome and extent of neocortical resection on emotional well-being in patients with epilepsy one year after temporal lobe surgery.

BACKGROUND: Seizure frequency and cognitive function are common parameters in assessing epilepsy surgery outcomes. However, psychobehavioral outcomes, such as symptoms of depression and quality of life (QOL), have not found equal attention yet. OBJECTIVE: To assess the effect of seizure frequency, the extent of resection, and cognitive function on the psychobehavioral outcome of patients after temporal lobe surgery for pharmacoresistant epilepsy. METHODS: We retrospectively reviewed all consecutive patients who underwent surgery for intractable temporal lobe epilepsy between 09/2015 and 07/2019. We examined seizure outcome, surgical plan, resection volume, cognitive functions, and psychobehavioral outcome. RESULTS: This study included 77 patients (31 males, 46 females) who underwent temporal lobe surgery. One year after surgery, 53 patients (68.8 %) were completely seizure-free (Engel IA) and 92.2 % of patients showed a worthwhile improvement in seizure frequency (Engel I-III). Resection volume was significantly negatively correlated with QOL (r =  - 0.284, p = 0.041). However, after controlling for the effect of seizure outcome, no significant correlation remained. Patients with a worthwhile improvement in seizure frequency showed significantly fewer symptoms of depression (p = 0.024) and a significantly higher QOL (p = 0.012) one year after surgery. The differences in symptoms of depression (p = 0.044) and QOL (p = 0.030) between patients with and without improvements in seizure frequency remained significant after controlling for the effect of resection volume. After procedures sparing the amygdala and hippocampus (neocortical resection), patients presented significantly fewer symptoms of depression (p = 0.044) and significantly better QOL (p = 0.008) than patients after procedures involving mesial-temporal structures, independent of the resection volume, and after controlling for the side of the procedure (dominant vs. non-dominant). After also controlling for seizure outcome, the difference remained for QOL (p = 0.014) but not for symptoms of depression. CONCLUSIONS: A patient's emotional well-being one year after surgery for pharmacoresistant temporal lobe epilepsy strongly depends on their seizure outcome. As an individual factor, the extent of neocortical resection negatively affects postsurgical emotional well-being, but a favorable seizure outcome outweighs this effect, independent of the resection volume. A favorable seizure outcome even outweighs the negative effects of procedures involving mesial-temporal structures on symptoms of depression.

Counterion effects on the mesomorphic and electrochemical properties of guanidinium salts.

Ionic liquid crystals (ILCs) combine the ion mobility of ionic liquids with the order and self-assembly of thermotropic mesophases. To understand the role of the anion in ILCs, wedge-shaped arylguanidinium salts with tetradecyloxy side chains were chosen as benchmark systems and their liquid crystalline self-assembly in the bulk phase as well as their electrochemical behavior in solution were studied depending on the anion. Differential scanning calorimetry (DSC), polarizing optical microscopy (POM) and X-ray diffraction (WAXS, SAXS) experiments revealed that for spherical anions, the phase width of the hexagonal columnar mesophase increased with the anion size, while for non-spherical anions, the trends were less clear cut. Depending on the anion, the ILCs showed different stability towards electrochemical oxidation and reduction with the most stable being the PF6 based compound. Cyclic voltammetry (CV) and density functional theory (DFT) calculations suggest a possible contribution of the guanidinium cation to the oxidation processes.

Global patterns of tropical forest fragmentation.

Remote sensing enables the quantification of tropical deforestation with high spatial resolution. This in-depth mapping has led to substantial advances in the analysis of continent-wide fragmentation of tropical forests. Here we identified approximately 130 million forest fragments in three continents that show surprisingly similar power-law size and perimeter distributions as well as fractal dimensions. Power-law distributions have been observed in many natural phenomena such as wildfires, landslides and earthquakes. The principles of percolation theory provide one explanation for the observed patterns, and suggest that forest fragmentation is close to the critical point of percolation; simulation modelling also supports this hypothesis. The observed patterns emerge not only from random deforestation, which can be described by percolation theory, but also from a wide range of deforestation and forest-recovery regimes. Our models predict that additional forest loss will result in a large increase in the total number of forest fragments-at maximum by a factor of 33 over 50 years-as well as a decrease in their size, and that these consequences could be partly mitigated by reforestation and forest protection.

Refined diet consumption increases neuroinflammatory signalling through bile acid dysmetabolism.

Over recent decades, dietary patterns have changed significantly due to the increasing availability of convenient, ultra-processed refined foods. Refined foods are commonly depleted of key bioactive compounds, which have been associated with several deleterious health conditions. As the gut microbiome can influence the brain through a bidirectional communication system known as the 'microbiota-gut-brain axis', the consumption of refined foods has the potential to affect cognitive health. In this study, multi-omics approaches were employed to assess the effect of a refined diet on the microbiota-gut-brain axis, with a particular focus on bile acid metabolism. Mice maintained on a refined low-fat diet (rLFD), consisting of high sucrose, processed carbohydrates and low fibre content, for eight weeks displayed significant gut microbial dysbiosis, as indicated by diminished alpha diversity metrics (p < 0.05) and altered beta diversity (p < 0.05) when compared to mice receiving a chow diet. Changes in gut microbiota composition paralleled modulation of the metabolome, including a significant reduction in short-chain fatty acids (acetate, propionate and n-butyrate; p < 0.001) and alterations in bile acid concentrations. Interestingly, the rLFD led to dysregulated bile acid concentrations across both the colon (p < 0.05) and the brain (p < 0.05) which coincided with altered neuroinflammatory gene expression. In particular, the concentration of TCA, TDCA and T-α-MCA was inversely correlated with the expression of NF-κB1, a key transcription factor in neuroinflammation. Overall, our results suggest a novel link between a refined low-fat diet and detrimental neuronal processes, likely in part through modulation of the microbiota-gut-brain axis and bile acid dysmetabolism.

Acremonium sp. diglycosidase-aid chemical diversification: valorization of industry by-products.

The fungal diglycosidase α-rhamnosyl-ß-glucosidase I (αRßG I) from Acremonium sp. DSM 24697 catalyzes the glycosylation of various OH-acceptors using the citrus flavanone hesperidin. We successfully applied a one-pot biocatalysis process to synthesize 4-methylumbellipheryl rutinoside (4-MUR) and glyceryl rutinoside using a citrus peel residue as sugar donor. This residue, which contained 3.5 % [w/w] hesperidin, is the remaining of citrus processing after producing orange juice, essential oil, and peel-juice. The low-cost compound glycerol was utilized in the synthesis of glyceryl rutinoside. We implemented a simple method for the obtention of glyceryl rutinoside with 99 % yield, and its purification involving activated charcoal, which also facilitated the recovery of the by-product hesperetin through liquid-liquid extraction. This process presents a promising alternative for biorefinery operations, highlighting the valuable role of αRßG I in valorizing glycerol and agricultural by-products. KEYPOINTS: • αRßG I catalyzed the synthesis of rutinosides using a suspension of OPW as sugar donor. • The glycosylation of aliphatic polyalcohols by the αRßG I resulted in products bearing a single rutinose moiety. • αRßG I catalyzed the synthesis of glyceryl rutinoside with high glycosylation/hydrolysis selectivity (99 % yield).

The role of sarcopenia in fragility fractures of the pelvis - is sarcopenia an underestimated risk factor?

BACKGROUND: Fragility fractures of the pelvis (FFPs) represent a significant health burden, particularly for the elderly. The role of sarcopenia, an age-related loss of muscle mass and function, in the development and impact of these fractures is not well understood. This study aims to investigate the prevalence and impact of osteoporosis and sarcopenia in patients presenting with FFPs. METHODS: This retrospective study evaluated 140 elderly patients with FFPs. The diagnosis of sarcopenia was assessed by psoas muscle area (PMA) and the height-adjusted psoas muscle index (PMI) measured on computed tomography (CT) scans. Clinical data, radiological findings and functional outcomes were recorded and compared with the presence or absence of sarcopenia and osteoporosis. RESULTS: Our study cohort comprised 119 female (85.0%) and 21 (15.0%) male patients. The mean age at the time of injury or onset of symptoms was 82.26 ± 8.50 years. Sarcopenia was diagnosed in 68.6% (n = 96) patients using PMA and 68.8% (n = 88) using PMI. 73.6% (n = 103) of our study population had osteoporosis and 20.0% (n = 28) presented with osteopenia. Patients with sarcopenia and osteoporosis had longer hospital stays (p < 0.04), a higher rate of complications (p < 0.048) and functional recovery was significantly impaired, as evidenced by a greater need for assistance in daily living (p < 0.03). However, they were less likely to undergo surgery (p < 0.03) and the type of FFP differed significantly (p < 0.04). There was no significant difference in mortality rate, pre-hospital health status, age or gender. CONCLUSION: Our study highlights the important role of sarcopenia in FFPs in terms of the serious impact on health and quality of life in elderly patients especially when osteoporosis and sarcopenia occur together. Identifying and targeting sarcopenia in older patients may be an important strategy to reduce pelvic fractures and improve recovery. Further research is needed to develop effective prevention and treatment approaches that target muscle health in the elderly.

E-learning-an interventional element of the PRiVENT project to improve weaning expertise.

BACKGROUND: PRiVENT (PRevention of invasive VENTilation) is an evaluation of a bundle of interventions aimed at the prevention of long-term invasive mechanical ventilation. One of these elements is an e-learning course for healthcare professionals to improve weaning expertise. The aim of our analysis is to examine the implementation of the course in cooperating intensive care units. METHODS: The course has been developed through a peer review process by pulmonary and critical care physicians in collaboration with respiratory therapists, supported by health services researchers and a professional e-learning agency. The e-learning platform "weLearn" was made available online to participating healthcare professionals. Feedback on the e-learning programme was obtained and discussed in quality circles (QCs). We measured the acceptance and use of the programme through access statistics. RESULTS: The e-learning course "Joint Prevention of Long-Term Ventilation" consists of 7 separate modules with practice-oriented training units as well as a cross-module area and corresponding interactive case studies. Users can receive 23 CME (continuing medical education) credits. The platform was released on July 1, 2021. By June 28, 2023, 214 users from 33 clinics had registered. Most users (77-98%) completed the modules, thus performing well in the test, where 90-100% passed. In the QCs, the users commended the structure and practical relevance of the programme, as well as the opportunity to earn CME credits. CONCLUSION: Especially for medical staff in intensive care units, where continuous training is often a challenge during shift work, e-learning is a useful supplement to existing medical training. TRIAL REGISTRATION: The PRiVENT study is registered at ClinicalTrials.gov (NCT05260853) on 02/03/2022.

Redox Out of the Box: Catalytic Versatility Across NAD(P)H-Dependent Oxidoreductases.

The asymmetric reduction of double bonds using NAD(P)H-dependent oxidoreductases has proven to be an efficient tool for the synthesis of important chiral molecules in research and on industrial scale. These enzymes are commercially available in screening kits for the reduction of C=O (ketones), C=C (activated alkenes), or C=N bonds (imines). Recent reports, however, indicate that the ability to accommodate multiple reductase activities on distinct C=X bonds occurs in different enzyme classes, either natively or after mutagenesis. This challenges the common perception of highly selective oxidoreductases for one type of electrophilic substrate. Consideration of this underexplored potential in enzyme screenings and protein engineering campaigns may contribute to the identification of complementary biocatalytic processes for the synthesis of chiral compounds. This review will contribute to a global understanding of the promiscuous behavior of NAD(P)H-dependent oxidoreductases on C=X bond reduction and inspire future discoveries with respect to unconventional biocatalytic routes in asymmetric synthesis.

Identification of Kibdelomycin and Related Biosynthetic Gene Clusters and Characterization of the C-Branching of Amycolose.

Many bacterial natural products contain C-branched sugars, including components from the outer cell wall or antibiotically active metabolites. The enzymatic C-branching of keto sugars leading to longer side chains (≥C2) is catalyzed by thiamine diphosphate (ThDP)-dependent enzymes. Chiral tertiary α-hydroxy ketones are formed in this process. The ThDP-dependent enzymes that catalyze C-branching reactions belong to one of three enzymatic superfamilies: decarboxylases, transketolases, and α-ketoacid dehydrogenases 2, but branching of keto sugars has only been demonstrated for decarboxylases. In this study, we showed that an α-ketoacid dehydrogenase is responsible for C-branching of the deoxyketo sugar amycolose in the biosynthesis of kibdelomycin in Kibdelosporangium sp. MA7385. In addition, we characterized an amino transferase in the same biosynthetic gene cluster (BGC) that accepts a sterically demanding tertiary α-hydroxy ketone in a downstream reaction. Subsequently, we identified approximately 400 similar BGCs in silico, suggesting that there is a large diversity of possible ThDP-dependent enzymes catalyzing the C-branching of keto sugars and subsequent modifications.

Identification and Characterization of Thiamine Diphosphate-Dependent Lyases with an Unusual CDG Motif.

The thiamine diphosphate (ThDP)-binding motif, characterized by the canonical GDG(X)24-27N sequence, is highly conserved among ThDP-dependent enzymes. We investigated a ThDP-dependent lyase (JanthE from Janthinobacterium sp. HH01) with an unusual cysteine (C458) replacing the first glycine of this motif. JanthE exhibits a high substrate promiscuity and accepts long aliphatic α-keto acids as donors. Sterically hindered aromatic aldehydes or non-activated ketones are acceptor substrates, giving access to a variety of secondary and tertiary alcohols as carboligation products. The crystal structure solved at a resolution of 1.9 Šreveals that C458 is not primarily involved in cofactor binding as previously thought for the canonical glycine. Instead, it coordinates methionine 406, thus ensuring the integrity of the active site and the enzyme activity. In addition, we have determined the long-sought genuine tetrahedral intermediates formed with pyruvate and 2-oxobutyrate in the pre-decarboxylation states and deciphered the atomic details for their stabilization in the active site. Collectively, we unravel an unexpected role for the first residue of the ThDP-binding motif and unlock a family of lyases that can perform valuable carboligation reactions.

Regioselective Oxidative Phenol Coupling by a Mushroom Unspecific Peroxygenase.

Bioactive dimeric (pre-)anthraquinones are ubiquitous in nature and are found in bacteria, fungi, insects, and plants. Their biosynthesis via oxidative phenol coupling (OPC) is catalyzed by cytochrome P450 enzymes, peroxidases, or laccases. While the biocatalysis of OPC in molds (Ascomycota) is well-known, the respective enzymes in mushroom-forming fungi (Basidiomycota) are unknown. Here, we report on the biosynthesis of the atropisomers phlegmacin A1 and B1 of the mushroom Cortinarius odorifer. The biosynthesis of these unsymmetrically 7,10'-homo-coupled dihydroanthracenones was heterologously reconstituted in the mold Aspergillus niger. Methylation of the parental monomer atrochrysone to its 6-O-methyl ether torosachrysone by the O-methyltransferase CoOMT1 precedes the regioselective homocoupling to phlegmacin, catalyzed by the enzyme CoUPO1 annotated as an "unspecific peroxygenase" (UPO). Our results reveal an unprecedented UPO reaction, thereby expanding the biocatalytic portfolio of oxidative phenol coupling beyond the commonly reported enzymes. The results show that Basidiomycota use peroxygenases to selectively couple aryls independently of and convergently to any other group of organisms, emphasizing the central role of OPC in natural processes.