المؤلفون: Kerschbaumer, Bianca¹ (AUTHOR), Totaro, Massimo G.¹ (AUTHOR), Friess, Michael² (AUTHOR), Breinbauer, Rolf² (AUTHOR), Bijelic, Aleksandar¹ (AUTHOR) aleksandar.bijelic@tugraz.at, Macheroux, Peter¹ (AUTHOR) peter.macheroux@tugraz.at

المصدر: FEBS Journal. Apr2024, Vol. 291 Issue 7, p1560-1574. 15p.

مصطلحات موضوعية: *COFACTORS (Biochemistry), *FLAVIN mononucleotide, *MOLECULAR dynamics, *CARBON-carbon bonds, *TOMATOES, *COENZYMES, *OXIDOREDUCTASES, *NICOTINAMIDE

مستخلص: Flavin mononucleotide (FMN)‐dependent ene‐reductases constitute a large family of oxidoreductases that catalyze the enantiospecific reduction of carbon–carbon double bonds. The reducing equivalents required for substrate reduction are obtained from reduced nicotinamide by hydride transfer. Most ene‐reductases significantly prefer, or exclusively accept, either NADPH or NADH. Despite their usefulness in biocatalytic applications, the structural determinants for cofactor preference remain elusive. We employed the NADPH‐preferring 12‐oxophytodienoic acid reductase 3 from Solanum lycopersicum (SlOPR3) as a model enzyme of the ene‐reductase family and applied computational and structural methods to investigate the binding specificity of the reducing coenzymes. Initial docking results indicated that the arginine triad R283, R343, and R366 residing on and close to a critical loop at the active site (loop 6) are the main contributors to NADPH binding. In contrast, NADH binds unfavorably in the opposite direction toward the β‐hairpin flap within a largely hydrophobic region. Notably, the crystal structures of SlOPR3 in complex with either NADPH4 or NADH4 corroborated these different binding modes. Molecular dynamics simulations confirmed NADH binding near the β‐hairpin flap and provided structural explanations for the low binding affinity of NADH to SlOPR3. We postulate that cofactor specificity is determined by the arginine triad/loop 6 and the residue(s) controlling access to a hydrophobic cleft formed by the β‐hairpin flap. Thus, NADPH preference depends on a properly positioned arginine triad, whereas granting access to the hydrophobic cleft at the β‐hairpin flap favors NADH binding. [ABSTRACT FROM AUTHOR]

المؤلفون: Li, Na¹ (AUTHOR), Wang, Yuan¹ (AUTHOR), Meng, Yinyin² (AUTHOR), Lv, Yangyong¹ (AUTHOR), Zhang, Shuaibing¹ (AUTHOR), Wei, Shan¹ (AUTHOR), Ma, Pingan (AUTHOR), Hu, Yuansen¹ (AUTHOR) hys308@126.com, Lin, Hui² (AUTHOR) huilin@henau.edu.cn

المصدر: Applied Microbiology & Biotechnology. 1/13/2024, Vol. 108 Issue 1, p1-11. 11p.

مصطلحات موضوعية: *ASPERGILLUS flavus, *FLAVIN mononucleotide, *THERMOPHILIC bacteria, *MANUFACTURING processes, *NITROALKENES, *AFLATOXINS, *CRYSTAL structure

مستخلص: Old yellow enzymes (OYEs) have been proven as powerful biocatalysts for the asymmetric reduction of activated alkenes. Fungi appear to be valuable sources of OYEs, but most of the fungal OYEs are unexplored. To expand the OYEs toolbox, a new thermophilic-like OYE (AfOYE1) was identified from Aspergillus flavus strain NRRL3357. The thermal stability analysis showed that the T1/2 of AfOYE1 was 60 °C, and it had the optimal temperature at 45 °C. Moreover, AfOYE1 exhibited high reduction activity in a wide pH range (pH 5.5–8.0). AfOYE1 could accept cyclic enones, acrylamide, nitroalkenes, and α, β-unsaturated aldehydes as substrates and had excellent enantioselectivity toward prochiral alkenes (> 99% ee). Interestingly, an unexpected (S)-stereoselectivity bioreduction toward 2-methylcyclohexenone was observed. The further crystal structure of AfOYE1 revealed that the "cap" region from Ala132 to Thr182, the loop of Ser316 to Gly325, α short helix of Arg371 to Gln375, and the C-terminal "finger" structure endow the catalytic cavity of AfOYE1 quite deep and narrow, and flavin mononucleotide (FMN) heavily buried at the bottom of the active site tunnel. Furthermore, the catalytic mechanism of AfOYE1 was also investigated, and the results confirmed that the residues His211, His214, and Tyr216 compose its catalytic triad. This newly identified thermophilic-like OYE would thus be valuable for asymmetric alkene hydrogenation in industrial processes. Key points: A new thermophilic-like OYE AfOYE1 was identified from Aspergillus flavus, and the T1/2of AfOYE1 was 60 °C AfOYE1 catalyzed the reduction of 2-methylcyclohexenone with (S)-stereoselectivity The crystal structure of AfOYE1 was revealedv [ABSTRACT FROM AUTHOR]

المؤلفون: Vinacour, Matias¹, Moiana, Mauro¹, Forné, Ignasi², Jung, Kirsten³, Bertea, Micaela¹, Calero Valdayo, Patricia M.⁴, Nikel, Pablo I.⁴, Imhof, Axel², Palumbo, Miranda C.⁵, Fernández Do Porto, Dario^5,6, Ruiz, Jimena A.^1,3,4 jruiz@agro.uba.ar

المصدر: Applied & Environmental Microbiology. Dec2023, Vol. 89 Issue 12, p1-22. 22p.

مصطلحات موضوعية: *BURKHOLDERIA, *FLAVIN mononucleotide, *MICROBIAL enzymes, *SOIL degradation, *WILT diseases, *QUERCETIN, *RHIZOBACTERIA

مستخلص: Fusaric acid (FA) is a mycotoxin produced by several Fusarium species. Burkholderia ambifaria T16 is a rhizosphere bacterium, able to use FA as sole nitrogen, carbon, and energy source. By screening a transposon insertional library, combined with proteomic analysis, genes and enzymes involved in the microbial degradation of FA were identified for the first time. A functional 2-methylcitrate cycle, an anaplerotic pathway where propionyl-coenzyme A (CoA) is converted to pyruvate and succinate, was shown to be essential for growth in the presence of FA. The proteomic profile of B. ambifaria T16 showed that more than 50 enzymes (including those belonging to the 2-methylcitrate cycle, fatty acid metabolism, valine catabolism, and flavin biosynthesis) were significantly more abundant when growing on FA than on citrate. Flavin mononucleotide (FMN)- dependent luciferases like monooxygenase (LLM) are shown to catalyze the pyridine-ring cleavage reaction of several N-heterocyclic compounds. Deletion of a gene encoding a predicted LLM enzyme that was highly upregulated during growth on FA, completely abolished the capability of B. ambifaria T16 to grow with this mycotoxin as sole nitrogen, carbon, and energy source. Re-introduction of the wild type gene was able to restore growth. The mentioned gene is part of a gene cluster of unknown function that we termed fua, due to its probable role in fusaric acid catabolism. Our results suggest that the LLM encoded in the fua cluster catalyzes the pyridine-ring opening reaction during FA degradation, and that propionyl-CoA is one of the intermediates of FA catabolism in B. ambifaria T16. IMPORTANCE Fusaric acid (FA) is an important virulence factor produced by several Fusarium species. These fungi are responsible for wilt and rot diseases in a diverse range of crops. FA is toxic for animals, humans and soil-borne microorganisms. This mycotoxin reduces the survival and competition abilities of bacterial species able to antagonize Fusarium spp., due to its negative effects on viability and the production of antibiotics effective against these fungi. FA biodegradation is not a common characteristic among bacteria, and the determinants of FA catabolism have not been identified so far in any microorganism. In this study, we identified genes, enzymes, and metabolic pathways involved in the degradation of FA in the soil bacterium Burkholderia ambifaria T16. Our results provide insights into the catabolism of a pyridine-derivative involved in plant pathogenesis by a rhizosphere bacterium. [ABSTRACT FROM AUTHOR]

المؤلفون: Moltó, Eduardo¹ (AUTHOR), Pintado, Cristina¹ (AUTHOR), Louzada, Ruy Andrade² (AUTHOR), Bernal-Mizrachi, Ernesto² (AUTHOR), Andrés, Antonio³ (AUTHOR), Gallardo, Nilda³ (AUTHOR), Bonzon-Kulichenko, Elena¹ (AUTHOR) elena.bonzon@uclm.es

المصدر: International Journal of Molecular Sciences. Nov2023, Vol. 24 Issue 22, p16172. 18p.

مصطلحات موضوعية: *FLAVIN mononucleotide, *POST-translational modification, *CELL physiology, *MITOCHONDRIAL proteins, *MASS spectrometry, *INSULIN

مستخلص: Post-translational modifications (PTMs) of proteins are paramount in health and disease. Phosphoproteome analysis by enrichment techniques is becoming increasingly attractive for biomedical research. Recent findings show co-enrichment of other phosphate-containing biologically relevant PTMs, but these results were obtained by closed searches focused on the modifications sought. Open searches are a breakthrough in high-throughput PTM analysis (OS-PTM), identifying practically all PTMs detectable by mass spectrometry, even unknown ones, with their modified sites, in a hypothesis-free and deep manner. Here we reanalyze liver phosphoproteome by OS-PTM, demonstrating its extremely complex nature. We found extensive Lys glycerophosphorylations (pgK), as well as modification with glycerylphosphorylethanolamine on Glu (gpetE) and flavin mononucleotide on His (fmnH). The functionality of these metabolite-derived PTMs is demonstrated during metabolic dysfunction-associated steatotic liver disease (MASLD) development in mice. MASLD elicits specific alterations in pgK, epgE and fmnH in the liver, mainly on glycolytic enzymes and mitochondrial proteins, suggesting an increase in glycolysis and mitochondrial ATP production from the early insulin-resistant stages. Thus, we show new possible mechanisms based on metabolite-derived PTMs leading to intrahepatic lipid accumulation during MASLD development and reinforce phosphoproteome enrichment as a valuable tool with which to study the functional implications of a variety of low-abundant phosphate-containing PTMs in cell physiology. [ABSTRACT FROM AUTHOR]

المؤلفون: Silvestri, Giuseppe¹ (AUTHOR), Arrigoni, Federica¹ (AUTHOR), Persico, Francesca¹ (AUTHOR), Bertini, Luca¹ (AUTHOR), Zampella, Giuseppe¹ (AUTHOR), De Gioia, Luca¹ (AUTHOR) luca.degioia@unimib.it, Vertemara, Jacopo¹ (AUTHOR) luca.degioia@unimib.it

المصدر: Molecules. Aug2023, Vol. 28 Issue 16, p6016. 13p.

مصطلحات موضوعية: *REDUCTION potential, *FLAVIN mononucleotide, *TECHNOLOGICAL innovations, *FLAVOPROTEINS, *CHARGE exchange, *NON-equilibrium reactions

مستخلص: Flavodoxins are enzymes that contain the redox-active flavin mononucleotide (FMN) cofactor and play a crucial role in numerous biological processes, including energy conversion and electron transfer. Since the redox characteristics of flavodoxins are significantly impacted by the molecular environment of the FMN cofactor, the evaluation of the interplay between the redox properties of the flavin cofactor and its molecular surroundings in flavoproteins is a critical area of investigation for both fundamental research and technological advancements, as the electrochemical tuning of flavoproteins is necessary for optimal interaction with redox acceptor or donor molecules. In order to facilitate the rational design of biomolecular devices, it is imperative to have access to computational tools that can accurately predict the redox potential of both natural and artificial flavoproteins. In this study, we have investigated the feasibility of using non-equilibrium thermodynamic integration protocols to reliably predict the redox potential of flavodoxins. Using as a test set the wild-type flavodoxin from Clostridium Beijerinckii and eight experimentally characterized single-point mutants, we have computed their redox potential. Our results show that 75% (6 out of 8) of the calculated reaction free energies are within 1 kcal/mol of the experimental values, and none exceed an error of 2 kcal/mol, confirming that non-equilibrium thermodynamic integration is a trustworthy tool for the quantitative estimation of the redox potential of this biologically and technologically significant class of enzymes. [ABSTRACT FROM AUTHOR]

المؤلفون: Huwyler, Florian^1,2,3 (AUTHOR), Eden, Janina² (AUTHOR), Binz, Jonas¹ (AUTHOR), Cunningham, Leslie^1,2,3 (AUTHOR), Sousa Da Silva, Richard X.^2,3 (AUTHOR), Clavien, Pierre‐Alain^2,3 (AUTHOR), Dutkowski, Philipp² (AUTHOR) philipp.dutkowski@usz.ch, Tibbitt, Mark W.^1,3 (AUTHOR) mtibbitt@ethz.ch, Hefti, Max³ (AUTHOR) max.hefti@proton.me

المصدر: Advanced Science. 8/15/2023, Vol. 10 Issue 23, p1-10. 10p.

مصطلحات موضوعية: *PATIENT monitoring, *FLAVIN mononucleotide, *TRANSPLANTATION of organs, tissues, etc., *OPERATING rooms, *HEMODIALYSIS patients, *POINT-of-care testing, *BIOLOGICAL laboratories

مستخلص: Biomarkers are powerful clinical diagnostics and predictors of patient outcome. However, robust measurements often require time and expensive laboratory equipment, which is insufficient to track rapid changes and limits direct use in the operating room. Here, this study presents a portable spectrophotometric device for continuous real‐time measurements of fluorescent and non‐fluorescent biomarkers at the point of care. This study measures the mitochondrial damage biomarker flavin mononucleotide (FMN) in 26 extended criteria human liver grafts undergoing hypothermic oxygenated perfusion to guide clinical graft assessment. Real‐time data identified seven organs unsuitable for transplant that are discarded. The remaining grafts are transplanted and FMN values correlated with post‐transplant indicators of liver function and patient recovery. Further, this study shows how this device can be used to monitor dialysis patients by measuring creatinine in real‐time. Our approach provides a simple method to monitor biomarkers directly within biological fluids to improve organ assessment, patient care, and biomarker discovery. [ABSTRACT FROM AUTHOR]

المؤلفون: Da'dara, Akram A., Nation, Catherine S., Skelly, Patrick J.

المصدر: BMC Infectious Diseases; 6/25/2024, Vol. 24 Issue 1, p1-14, 14p

مصطلحات موضوعية: SCHISTOSOMA mansoni, VITAMIN B2, FLAVIN adenine dinucleotide, FLAVIN mononucleotide, METABOLISM

مستخلص: Background: Schistosomiasis is a parasitic disease caused by trematodes of the genus Schistosoma. The intravascular worms acquire the nutrients necessary for their survival from host blood. Since all animals are auxotrophic for riboflavin (vitamin B2), schistosomes too must import it to survive. Riboflavin is an essential component of the coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD); these support key functions of dozens of flavoenzymes. Methods: Here, using a combination of metabolomics, enzyme kinetics and in silico molecular analysis, we focus on the biochemistry of riboflavin and its metabolites in Schistosoma mansoni (Sm). Results: We show that when schistosomes are incubated in murine plasma, levels of FAD decrease over time while levels of FMN increase. We show that live schistosomes cleave exogenous FAD to generate FMN and this ability is significantly blocked when expression of the surface nucleotide pyrophosphatase/phosphodiesterase ectoenzyme SmNPP5 is suppressed using RNAi. Recombinant SmNPP5 cleaves FAD with a Km of 178 ± 5.9 µM and Kcat/Km of 324,734 ± 36,347 M^{− 1}.S^{− 1}. The FAD-dependent enzyme IL-4I1 drives the oxidative deamination of phenylalanine to produce phenylpyruvate and H2O2. Since schistosomes are damaged by H2O2, we determined if SmNPP5 could impede H2O2 production by blocking IL-4I1 action in vitro. We found that this was not the case; covalently bound FAD on IL-4I1 appears inaccessible to SmNPP5. We also report that live schistosomes can cleave exogenous FMN to generate riboflavin and this ability is significantly impeded when expression of a second surface ectoenzyme (alkaline phosphatase, SmAP) is suppressed. Recombinant SmAP cleaves FMN with a Km of 3.82 ± 0.58 mM and Kcat/Km of 1393 ± 347 M^{− 1}.S^{− 1}. Conclusions: The sequential hydrolysis of FAD by tegumental ecto-enzymes SmNPP5 and SmAP can generate free vitamin B2 around the worms from where it can be conveniently imported by the recently described schistosome riboflavin transporter SmaRT. Finally, we identified in silico schistosome homologs of enzymes that are involved in intracellular vitamin B2 metabolism. These are riboflavin kinase (SmRFK) as well as FAD synthase (SmFADS); cDNAs encoding these two enzymes were cloned and sequenced. SmRFK is predicted to convert riboflavin to FMN while SmFADS could further act on FMN to regenerate FAD in order to facilitate robust vitamin B2-dependent metabolism in schistosomes. [ABSTRACT FROM AUTHOR]

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المؤلفون: van de Leemkolk, Fenna E. M.^1,2,3 (AUTHOR) f.e.m.van_de_leemkolk@lumc.nl, Lo Faro, M. Letizia^3,4 (AUTHOR), Shaheed, Sadr^3,4 (AUTHOR), Mulvey, John F.³ (AUTHOR), Huurman, Volkert A. L.^1,2 (AUTHOR), Alwayn, Ian P. J.^1,2 (AUTHOR), Putter, Hein^2,5 (AUTHOR), Jochmans, Ina^6,7 (AUTHOR), Lindeman, Jan H. N.^1,2 (AUTHOR), Ploeg, Rutger J.^2,3,4 (AUTHOR)

المصدر: PLoS ONE. 6/23/2023, Vol. 17 Issue 6, p1-15. 15p.

مصطلحات موضوعية: *FLAVIN mononucleotide, *PERFUSION, *KIDNEYS, *LIQUID chromatography-mass spectrometry, *BIOMARKERS, *FLUORESCENCE spectroscopy

مستخلص: Hypothermic machine perfusion (HMP) provides preservation superior to cold storage and may allow for organ assessment prior to transplantation. Since flavin mononucleotide (FMN) in perfusate has been proposed as a biomarker of organ quality during HMP of donor livers, the aim of this study was to validate FMN as a biomarker for organ quality in the context of HMP preserved kidneys. Perfusate samples (n = 422) from the paired randomised controlled COPE-COMPARE-trial, comparing HMP with oxygenation (HMPO2) versus standard HMP in kidneys, were used. Fluorescence intensity (FI) was assessed using fluorescence spectroscopy (excitation 450nm; emission 500-600nm) and validated by fluorospectrophotometer and targeted liquid chromatography mass spectrometry (LC-MS/MS). Fluorescence intensity (FI)(ex450;em500-600) increased over time during machine perfusion in both groups (p<0.0001). This increase was similar for both groups (p = 0.83). No correlation, however, was found between FI(ex450;em500-600) and post-transplant outcomes, including day 5 or 7 serum creatinine (p = 0.11; p = 0.16), immediate graft function (p = 0.91), creatinine clearance and biopsy-proven rejection at one year (p = 0.14; p = 0.59). LC-MS/MS validation experiments of samples detected FMN in only one perfusate sample, whilst the majority of samples with the highest fluorescence (n = 37/38, 97.4%) remained negative. In the context of clinical kidney HMP, fluorescence spectroscopy unfortunately appears to be not specific and probably unsuitable for FMN. This study shows that FMN does not classify as a clinically relevant predictive biomarker of kidney graft function after transplantation. [ABSTRACT FROM AUTHOR]

المؤلفون: Sakamoto, Sodai¹ (AUTHOR) soudai114@gmail.com, Bochimoto, Hiroki² (AUTHOR) botimoto@jikei.ac.jp, Shibata, Kengo³ (AUTHOR), Zin, Nur Khatijah Mohd² (AUTHOR), Fukai, Moto¹ (AUTHOR) db7m-fki@hotmail.co.jp, Nakamura, Kosei¹ (AUTHOR), Ishikawa, Takahisa¹ (AUTHOR), Fujiyoshi, Masato¹ (AUTHOR), Shimamura, Tsuyoshi⁴ (AUTHOR), Taketomi, Akinobu^1,3 (AUTHOR)

المصدر: Journal of Clinical Medicine. Jun2023, Vol. 12 Issue 11, p3845. 14p.

مصطلحات موضوعية: *PERFUSION, *FLAVIN mononucleotide, *LIVER, *LIVER enzymes, *SCANNING electron microscopy, *CHONDROITIN sulfates

مستخلص: Ex vivo hypothermic machine perfusion (HMP) is a strategy for controlling ischemia-reperfusion injury in donation after circulatory death (DCD) liver transplantation. The pH of blood increases with a decrease in temperature and water dissociation, leading to a decrease in [H+]. This study aimed to verify the optimal pH of HMP for DCD livers. Rat livers were retrieved 30 min post-cardiac arrest and subjected to 3-h cold storage (CS) in UW solution (CS group) or HMP with UW-gluconate solution (machine perfusion [MP] group) of pH 7.4 (original), 7.6, 7.8, and 8.0 (MP-pH 7.6, 7.8, 8.0 groups, respectively) at 7–10 °C. The livers were subjected to normothermic perfusion to simulate reperfusion after HMP. All HMP groups showed greater graft protection compared to the CS group due to the lower levels of liver enzymes in the former. The MP-pH 7.8 group showed significant protection, evidenced by bile production, diminished tissue injury, and reduced flavin mononucleotide leakage, and further analysis by scanning electron microscopy revealed a well-preserved structure of the mitochondrial cristae. Therefore, the optimum pH of 7.8 enhanced the protective effect of HMP by preserving the structure and function of the mitochondria, leading to reduced reperfusion injury in the DCD liver. [ABSTRACT FROM AUTHOR]

المؤلفون: Strotmann, Lisa¹ (AUTHOR), Harter, Caroline¹ (AUTHOR), Gerasimova, Tatjana¹ (AUTHOR), Ritter, Kevin² (AUTHOR), Jessen, Henning J.² (AUTHOR), Wohlwend, Daniel¹ (AUTHOR), Friedrich, Thorsten¹ (AUTHOR) Friedrich@bio.chemie.uni-freiburg.de

المصدر: Scientific Reports. 5/11/2023, Vol. 13 Issue 1, p1-9. 9p.

مصطلحات موضوعية: *FLAVIN mononucleotide, *CHARGE exchange, *IRON, *REACTIVE oxygen species, *NAD (Coenzyme), *ENERGY metabolism

مستخلص: NADH:ubiquinone oxidoreductase, respiratory complex I, plays a major role in cellular energy metabolism by coupling electron transfer with proton translocation. Electron transfer is catalyzed by a flavin mononucleotide and a series of iron-sulfur (Fe/S) clusters. As a by-product of the reaction, the reduced flavin generates reactive oxygen species (ROS). It was suggested that the ROS generated by the respiratory chain in general could damage the Fe/S clusters of the complex. Here, we show that the binuclear Fe/S cluster N1b is specifically damaged by H2O2, however, only at high concentrations. But under the same conditions, the activity of the complex is hardly affected, since N1b can be easily bypassed during electron transfer. [ABSTRACT FROM AUTHOR]