Search Results (415)

Plant growth-promoting (PGP) bacteria are beneficial microbes that can help plants mitigate various biotic and abiotic stresses through different PGP functions. Flavins (FLs) are involved in flavoprotein-mediated reactions essential for plant metabolism and could act as PGP molecules. The aim of this study was to isolate and characterize potential FLs secreting bacteria from apple (Malus domestica [Suckow] Borkh) roots based on their fluorescence and to evaluate their PGP properties, including FLs secretion. A total of 26 bacteria with increased fluorescence in liquid culture were isolated from the apple roots. Based on 16S rRNA sequencing analysis, 11 genetically different strains mostly from Burkholderia and Rhizobia spp. were identified. All isolates secreted considerable amounts of riboflavin. In vitro plant assays showed that under nitrogen (N) limitation, inoculated alfalfa (Medicago sativa) plants yielded at least 25% more dry mass than non-inoculated plants, and inoculation with AK7 and FL112 enriched plant tissue N content compared to non-inoculated plants. This improved N acquisition was not linked to symbiotic N fixation. Additionally, the isolates exhibited some other PGP properties. However, no specific PGP functions were linked to improved plant N acquisition but could potentially be linked to the FLs secretion. For future investigation, the mechanisms underlying improved plant N uptake should be assessed to gain a more in-depth understanding. Full article

Free flavin adenine dinucleotide (FAD) is metabolized to flavin mononucleotide (FMN) and adenine monophosphate (AMP) by hydrolases and to 4′,5′-cyclic phosphoriboflavin (cFMN) and AMP by the triose kinase FMN cyclase (TKFC). Yet, the lack of analytical standards for cFMN might have resulted in the incidence of cFMN in biological specimens being underreported. To address this shortcoming, cFMN was synthesized from either FMN or FAD. The optimization of the FAD to cFMN reaction conditions revealed that an equimolar ratio of ZnSO₄ and FAD yielded pure cFMN upon the precipitation of AMP-Zn salts. cFMN is stable to aqueous acidic and basic conditions and is readily extracted from biological samples for detection by liquid chromatography coupled with mass spectrometry. Although cFMN is hydrolyzed by liver tissue extracts to FMN and riboflavin, the mechanisms for this conversion remain elusive. Full article

Sodium-pumping NADH: ubiquinone oxidoreductase (Na⁺-NQR) is an important component of the aerobic respiratory chain of Vibrio cholerae. It oxidizes NADH, reduces ubiquinone, and uses the free energy of this redox reaction to move sodium across the cell membrane. The enzyme is a membrane complex of six subunits, two 2Fe−2S centers, and four flavins. Both the oxidized and reduced forms of Na⁺-NQR exhibit EPR signals due to flavin semiquinone radicals. It has been shown that in the oxidized form of the enzyme, the radical is a neutral flavin, while in the NADH-reduced form, the radical is an anionic flavin. Electron Spin Echo Envelope Modulation Spectroscopy (ESEEM) was used to probe the presence of the magnetic nucleus ²³Na in the immediate vicinity of the paramagnetic centers. The contribution of the ²³Na nucleus was observed only in the ESEEM spectra of the anionic flavin semiquinone previously assigned to FMN_NqrB. Analysis shows that the Na⁺ ion is within ~3–4 Å of the flavin radical. This distance is consistent with two models: (i) complexation of the Na⁺ ion with the carbonyl group of CO4; or alternatively, (ii) a “cation-π interaction,” between Na⁺ and the electron-rich π-system of the flavin aromatic rings. Full article

Electroactive biofilms (EABs) are essential for the performance of bioelectrochemical systems (BESs), but their formation in Geobacter, critically on conductive pili and exopolysaccharides, limits application under conditions where these components are deficient. Herein, we investigated the restorative effects of exogenous flavin mononucleotide (FMN) on EAB formation and extracellular electron transfer (EET) in two defective mutants of Geobacter sulfurreducens: the pili-deficient PCAΔ1496 and exopolysaccharides-deficient PCAΔ1501. Results show that FMN significantly promoted biofilm thickness in PCAΔ1496 (250%) and PCAΔ1501 (33%), while boosting maximum current outputs by 175-fold and 317.7%, respectively. Spectroscopic and electrochemical analyses revealed that FMN incorporates into biofilms, binds to outer membrane c-type cytochromes (c-Cyts), and enhances electron exchange capacity. Differential pulse voltammetry further confirmed that FMN did not exist independently in the biofilm but bound to outer membrane c-Cyts as a cofactor. Collectively, exogenous FMN plays dual roles (electron shuttle and cytochrome-bound cofactor) in defective Geobacter EABs, effectively restoring biofilm formation and enhancing EET efficiency. This study expands the understanding of the formation mechanism of Geobacter EABs and provides a novel strategy for optimizing BES performance. Full article

Stem mustard (Brassica juncea var. tumida Tsen et Lee) is an important economic vegetable in China. Premature bolting induced by temperature fluctuations has become a major cultivation constraint. Photoreceptors (PHRs) serve as critical photosensor proteins that interpret light signals and regulate physiological responses in plants. In this study, five core PHR families, namely F-box-containing flavin binding proteins (ZTL/FKF1/LKP2), phytochrome (PHY), cryptochrome (CRY), phototropin (PHOT) and UV RESISTANCE LOCUS 8 (UVR8) were identified in Brassica species. RNA-seq analysis revealed their expression patterns during organogenesis in B. juncea. Seven candidate PHRs were validated by qRT-PCR in B. juncea early-bolting (‘YA-1’) and late-bolting (‘ZT-1’) cultivars. Agrobacterium-mediated BjuFKF1_1 overexpression (OE) lines resulted in significantly earlier flowering under field conditions. Histochemical GUS staining indicated that BjuFKF1_1 was expressed in seedlings, leaves, flower buds and siliques. Transcript analysis revealed that the expression level of BjuFKF1_1 was up-regulated in all tissues at both the vegetative and reproductive stages, whereas the expression of BjuFKF1_1 interacting protein-encoding genes were down-regulated in flowers. Under blue light, genes encoding interacting proteins (BjuCOL5, BjuSKP1, BjuCOL3, BjuAP2, BjuAP2-1 and BjuLKP2) were up-regulated in flower buds, whereas BjuCOL and BjuPP2C52 were down-regulated in flowers. Developmental stage analysis revealed the up-regulation of five (BjuAP2, BjuCOL3, BjuCOL5, BjuAP2-1 and BjuLKP2) and four (BjuCOL, BjuCOL5, BjuAP2 and BjuLKP2) interaction protein-encoding genes during the reproductive stage under white and blue light, respectively. These findings elucidate the role of BjuFKF1_1 in flowering regulation and provide molecular targets for B. juncea bolting-resistant variety breeding. Full article

Background/Objectives: Caffeic acid is a hydroxycinnamic acid that has a wide range of applications in the medical field. The synthesis of caffeic acid using microbial fermentation technology is an environmentally friendly method. Methods: By engaging various enzymes, specifically 4-hydroxyphenylacetate 3-monooxygenase (HpaB), sourced from diverse bacterial strains, we successfully engineered a functional version of this enzyme within Escherichia coli, enabling the production of caffeic acid. In addition to the two common tyrosine ammonia lyases (TAL) and HpaC, different combinations of HpaB demonstrated varying abilities in converting the substrate L-tyrosine into the desired product, caffeic acid. Results: Under shake-flask culture conditions, the highest yield of caffeic acid was achieved with an enzyme mixture containing HpaB from Escherichia coli, reaching 75.88 mg/L. Enhancing the activity of the rate-limiting enzyme through engineering could potentially increase caffeic acid titer. This study aims to conduct a semi-rational design of HpaB through structure-based approaches to screen for mutants that can enhance the production of caffeic acid. Initially, the predicted three-dimensional structure of HpaB was generated using AlphaFold2, and subsequent analysis was conducted to pinpoint the critical mutation sites within the substrate-binding pocket. Five key amino acid residues (R113, Y117, H155, S210 and Y461) located in the vicinity of the flavin adenine dinucleotide binding domain in HpaB from Escherichia coli could be instrumental in modulating enzyme activity. Subsequently, the mutant S210G/Y117A was obtained by iterative saturation mutagenesis, which increased the titer of caffeic acid by 1.68-fold. The caffeic acid titer was further improved to 2335.48 mg/L in a 5 L fermenter. The findings show that the yield of caffeic acid was significantly enhanced through the integration of semi-rational design and fermentation process optimization. Full article

Solute carriers (SLCs) mediate cell- and organelle-specific import and export of nutrients and metabolites required for every biochemical process that occurs in a cell. Functional studies have ascribed activities to many human genes annotated as SLCs, but more than 100 SLCs remain orphans. Here, we applied a set of computational tools to characterize the orphan carriers SLC35F4 and SLC35F5. Phylogenetic analysis grouped SLC35F4 sister to SLC35F3, a suspected thiamine transporter, in a clade with SLC35F5, and distinct from an SLC35F6/2/1 clade. Transcriptome datasets revealed a restricted function for SLC35F4 in the cerebellum, in contrast to the more widespread distribution of SLC35F5. Gene ontology identified the Golgi apparatus as the likely residence of both transporters. Conceptual docking of 71 candidate substrates predicted high affinities of SLC35F4 (10–40 nM) and SLC35F5 (0.1–0.4 nM) for flavin adenine dinucleotide (FAD), straddling that of the known FAD transporter SLC25A32 (2–4 nM), while returning much lower affinities (by 30–fold or more) for all other tested substrates. Docking to SLC35F3 returned low affinity for both FAD and thiamine as candidate substrates. Thus, SLC35F4 and SLC35F5 but not closely related SLC35F3 likely import FAD into the Golgi apparatus, where the cofactor serves as the oxidant for disulfide-bond formation during tissue-specific, post-translational modification of secretory proteins. These findings provide strong direction for the definitive experiments yet needed to confirm the carriers’ subcellular localization, transport activities, and contributions to protein maturation and trafficking. Full article

Chlorobaculum tepidum ferredoxin: ${\mathrm{NADP}}^{+}$ oxidoreductase (CtFNR) is a dimeric thioredoxin reductase (TrxR)-type FNR, whose mechanism and redox properties are poorly characterized. In this work, we focused on the reoxidation mechanisms of its flavin adenine dinucleotide (FAD) cofactor using quinones (Q), nitroaromatics (${\mathrm{ArNO}}_2$), and other nonphysiological oxidants with different single-electron reduction midpoint potentials ($E_7^1$) and electrostatic charge. Like in other FNRs, the rate-limiting step of the reaction is the reoxidation of FAD semiquinone (${\mathrm{FADH}}^{•}$). However, only one FAD per dimer functions in CtFNR due to some nonequivalence of the NADP(H) binding domains in separate subunits. The reactivity of Q increases with increasing $E_7^1$, while ${\mathrm{ArNO}}_2$ form another analogous series of lower reactivity. The compounds are reduced in a dominant single-electron way. These data are consistent with an “outer sphere” electron transfer mechanism. On the basis of reactions with 3-acetylpyridine adenine dinucleotide phosphate, the two-electron reduction midpoint potential of FAD at pH 7.0 is −0.282 V. In CtFNR, 11% ${\mathrm{FADH}}^{•}$ was stabilized at equilibrium. Calculated electron transfer distances in reactions with Q and ${\mathrm{ArNO}}_2$ were in the range of 2.6–3.4 Å. Taken together with previous studies of Rhodopseudomonas palustris and Bacillus subtilis FNRs, this work allows us to generalize the information on the catalytic ant thermodynamic properties of TrxR-type FNRs. In addition, our data may be valuable from an applied perspective, e.g., the use of redox mediators in photobioelectrochemical systems or microbial cells based on anoxygenic phototrophic bacteria. Full article

Ene reductases, belonging to the Old Yellow Enzyme (OYE) family, are widely used for biocatalysis. The OYE from Thermus scotoductus SA-01 (TsOYE) gained great attention due to its broad substrate scope, high stereoselectivity, thermostability, and catalytic versatility. Recently, the otherwise noncovalently bound flavin cofactor (FMN) was covalently anchored in several TsOYE mutants using the “flavin-fixing” method. However, the biochemical properties of these mutants remained unexplored. A detailed comparative study of wild-type (WT) TsOYE and the flavin-fixing variant F1 (F1 TsOYE) revealed that F1 TsOYE has a lower stability and poorer catalytic activity. Interestingly, both WT and F1 TsOYE have comparable redox potential values. These results suggest that the decrease in activity and stability is primarily caused by changes in structure and structural dynamics induced by the mutations and the covalent flavin-protein linkage. Replacing residues in the flavinylation recognition site did not result in significant repair of enzyme activity. Our findings highlight the sensitivity of TsOYE activity to covalent FMN incorporation and its associated mutations and underscore the necessity of structural insights for further rational design. This study also provides critical groundwork for optimizing the flavin-fixing strategy. Full article

Mitochondrial energy metabolism is fundamental to sperm function, and fatty acid β-oxidation is an important pathway for adenosine triphosphate (ATP) production. Riboflavin, a precursor of key flavin cofactors, plays a critical role in regulating β-oxidation and supports multiple physiological processes. This study aimed to determine whether adding riboflavin to semen dilution media could enhance goat sperm motility and to elucidate the underlying metabolic mechanisms. Goat semen was diluted in tris-citrate-glucose (TCG) medium containing 0, 5, 10, 15, and 20 μM riboflavin and incubated at 37 °C, after which sperm motility, acrosome integrity, mitochondrial membrane potential, ATP levels, malate dehydrogenase (MDH) and succinate dehydrogenase (SDH) activities, and the NADH/NAD⁺ were evaluated. The localization and expression of the β-oxidation enzymes carnitine palmitoyltransferase 1 (CPT1) and extremely long chain acyl-CoA dehydrogenase (ACADVL) were examined, and CPT1 activity was quantified. The results showed that CPT1 and ACADVL were present in goat sperm, and that 10 μM riboflavin significantly increased sperm motility, acrosome integrity, mitochondrial activity, ATP levels, and the activities of MDH, SDH, and CPT1, while also elevating NADH/NAD⁺ levels (p < 0.05). Notably, these enhancements were suppressed by 100 μM etomoxir, a mitochondrial β-oxidation inhibitor, which reduced total motility, ATP Levels, and CPT1 activity after riboflavin supplementation (p < 0.05). These findings indicate that goat sperm at least partly rely on mitochondrial β-oxidation for ATP generation and that riboflavin supplementation enhances mitochondrial metabolism, thereby improving sperm quality. Full article

Leaf morphogenesis is governed by a tightly integrated regulatory network centered on auxin, which operates through a sequential axis of synthesis, transport, and signal transduction. This review elucidates how pivotal molecular hubs previously identified in this regulatory network, including biosynthetic enzymes, polar transporters, and auxin response factors, interconnect through dynamic feedback mechanisms to orchestrate leaf initiation, polarity establishment, and the determination of its final size and shape. Notably, recent breakthroughs are transforming the field: the re-evaluation of established pathways like indole-3-acetaldoxime (IAOx), whose direct contribution to auxin pools is under scrutiny, hinting at the existence of undiscovered enzymes or alternative metabolic branches and the paradigm-shifting discovery that cAMP functions as a second messenger produced by Transport Inhibitor Resistant 1/Auxin signaling F-box (TIR1/AFB) receptors, which directly activates Auxin Response Factor (ARF)-mediated transcription. These foundational mechanistic insights provide the critical groundwork for application. Key network nodes—such as PIN-FORMED (PIN) transporters and YUCCA (YUC) flavin-containing monooxygenases—are now validated targets for crop improvement. Consequently, the elucidated network serves as a blueprint for rationally designing crop architecture. Full article

Background: Flavin cofactors, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are indispensable for plant metabolism, supporting photosynthesis, photorespiration, mitochondrial electron transport, nitrogen assimilation, and cellular redox balance. Both cofactors derive from riboflavin (vitamin B₂), which plants synthesize de novo, unlike animals, which rely on dietary intake. While the riboflavin biosynthesis pathway has been biochemically well-characterized, its transcriptional regulation and cellular organization remain poorly understood. Methods: Here, using large-scale transcriptomic datasets as well as co-expression and cis-element analyses, we systematically investigated the expression dynamics of riboflavin metabolism genes in Arabidopsis thaliana. In addition, HPLC was employed to monitor flavin level fluctuations in plants under abiotic stresses. Results: Most genes displayed strong expression in photosynthetic and reproductive tissues, consistent with elevated metabolic demands for flavins in redox reactions and energy metabolism. Under osmotic stress, RIBA1, RIBA3, PYRD, PYRR, COS1/LS, and RS, genes encoding enzymes involved in the early and intermediate steps of riboflavin biosynthesis were transcriptionally downregulated. In contrast, RIBA2, FHY1/PYRP1 and FMN/FHY were upregulated, whereas FADS1 and NUDX23, genes encoding enzymes responsible for interconversion between FMN and FAD, were suppressed. Gene expression responses are consistent with the maintenance of flavin homeostasis, affecting flavin level changes under abiotic stress. Conclusions: This study establishes a comprehensive framework for the transcriptional regulation of flavin biosynthesis in plants. The findings reveal stress-responsive reprogramming of flavin metabolism and identify promising strategies for engineering crops for biofortification, metabolic efficiency, and stress resilience. Full article

Choline is a central metabolite that connects membrane turnover, neurotransmission, and one-carbon metabolism, and its reliable measurement across diverse biological matrices remains a significant analytical challenge. This review brings together biological context, electrochemical mechanisms, and device engineering to define realistic performance targets for choline sensors in blood, cerebrospinal fluid, extracellular space, and milk. We examine enzymatic sensor architectures ranging from peroxide-based detection to mediated electron transfer via ferrocene derivatives, quinones, and osmium redox polymers and assess how applied potential, oxygen availability, and film structure shape electron-transfer pathways. Evidence for direct electron transfer with choline oxidase is critically evaluated, with emphasis on the essential controls needed to distinguish true flavin-based communication from peroxide-related artefacts. We also examine bienzymatic formats that allow operation at low or negative bias and discuss strategies for matrix-matched validation, selectivity, drift control, and resistance to fouling. To support reliable translation, we outline reporting standards that include matrix-specific concentration ranges, reference electrode notation, mediator characteristics, selectivity panels, and access to raw electrochemical traces. By connecting biological requirements to mechanistic pathways and practical design considerations, this review provides a coherent framework for developing choline sensors that deliver stable, reproducible performance in real samples. Full article

Singlet oxygen is a type of reactive oxygen species that is typically generated via type II photosensitization reactions. Since 1,3-diphenylisobenzofuran (DPBF), a commonly used chromogenic probe, exhibits peak absorbance at 410 nm for singlet oxygen detection, it severely interferes with blue light irradiation and compounds that absorb in this wavelength region. This study investigated developing and validating a fluorescence-based method using DPBF to quantitatively analyze the type II photosensitizing property of riboflavin (RF) and its heterocyclic flavin derivatives. DPBF fluorescence-based analysis provided more sensitive and practical results than traditional colorimetric methods. It effectively overcomes spectral interference from colored photosensitizers, such as RF and its derivatives, under blue light irradiation (λ peak 447 nm). This method permitted more effective measurement of their activity without interference from their intrinsic color and maintained high linearity and low variation across different sample concentrations, even with short irradiation times. The type II photosensitizing potency of the tested compounds under blue light was consistently ranked as follows: RF > flavin mononucleotide > flavin adenine dinucleotide > lumiflavin > lumichrome. The results suggest that the DPBF fluorescence-based assay is a more effective approach than colorimetric analysis, making it a practical and reproducible tool for assessing the type II photosensitizing properties of diverse compounds. This study also provides a refinement of an existing probe-based assay for relative comparisons under visible light conditions. Full article

Escherichia coli (E. coli) is a ubiquitous opportunistic pathogen in nature and serves as an important reservoir for antibiotic resistance genes. The tet(X4) gene is a key determinant mediating tigecycline resistance. Although its core resistance mechanism, encoding a flavin-dependent monooxygenase, has been elucidated, the broader impact of the tet(X4) gene on the secondary regulatory networks of E. coli remains not fully understood. In recent years, multiple studies have indicated that the tet(X4) gene participates in pathways contributing to resistance to other antibiotics by regulating the expression of various genes. In this study, E. coli tet(X4) gene deletion and complementation strains were constructed to investigate the mechanisms by which the tet(X4) gene influences the growth characteristics and antibiotic resistance of E. coli. The minimum inhibitory concentrations (MICs) of 24 different antibiotics, as well as the degradation capacities of tetracycline and tigecycline, were determined for the wild-type, deletion, and complementation strains. In addition, a four-week starvation stress experiment was performed under both the presence and absence of sub-inhibitory concentrations of tigecycline, during which the bacterial growth curves, survival rates, and MIC variations were analyzed. Transcriptomic sequencing of the wild-type, deletion, and complementation strains identified 531 differentially expressed genes associated with ABC transporter activity, drug metabolism, and bacterial two-component systems. These findings provide reliable evidence for elucidating the mechanism by which the tet(X4) gene affects E. coli resistance, offering valuable insights into the prevention and control of tigecycline-resistant E. coli infections. Full article