X-ray structure and enzymatic study of a bacterial NADPH oxidase highlight the activation mechanism of eukaryotic NOX.

NADPH oxidases (NOX) are transmembrane proteins, widely spread in eukaryotes and prokaryotes, that produce reactive oxygen species (ROS). Eukaryotes use the ROS products for innate immune defense and signaling in critical (patho)physiological processes. Despite the recent structures of human NOX isoforms, the activation of electron transfer remains incompletely understood. SpNOX, a homolog from Streptococcus pneumoniae, can serves as a robust model for exploring electron transfers in the NOX family thanks to its constitutive activity. Crystal structures of SpNOX full-length and dehydrogenase (DH) domain constructs are revealed here. The isolated DH domain acts as a flavin reductase, and both constructs use either NADPH or NADH as substrate. Our findings suggest that hydride transfer from NAD(P)H to FAD is the rate-limiting step in electron transfer. We identify significance of F397 in nicotinamide access to flavin isoalloxazine and confirm flavin binding contributions from both DH and Transmembrane (TM) domains. Comparison with related enzymes suggests that distal access to heme may influence the final electron acceptor, while the relative position of DH and TM does not necessarily correlate with activity, contrary to previous suggestions. It rather suggests requirement of an internal rearrangement, within the DH domain, to switch from a resting to an active state. Thus, SpNOX appears to be a good model of active NOX2, which allows us to propose an explanation for NOX2's requirement for activation.

A Redox-Regulated, Heterodimeric NADH:cinnamate Reductase in Vibrio ruber.

Genes of putative reductases of α,ß-unsaturated carboxylic acids are abundant among anaerobic and facultatively anaerobic microorganisms, yet substrate specificity has been experimentally verified for few encoded proteins. Here, we co-produced in Escherichia coli a heterodimeric protein of the facultatively anaerobic marine bacterium Vibrio ruber (GenBank SJN56019 and SJN56021; annotated as NADPH azoreductase and urocanate reductase, respectively) with Vibrio cholerae flavin transferase. The isolated protein (named Crd) consists of the sjn56021-encoded subunit CrdB (NADH:flavin, FAD binding 2, and FMN bind domains) and an additional subunit CrdA (SJN56019, a single NADH:flavin domain) that interact via their NADH:flavin domains (Alphafold2 prediction). Each domain contains a flavin group (three FMNs and one FAD in total), one of the FMN groups being linked covalently by the flavin transferase. Crd readily reduces cinnamate, p-coumarate, caffeate, and ferulate under anaerobic conditions with NADH or methyl viologen as the electron donor, is moderately active against acrylate and practically inactive against urocanate and fumarate. Cinnamates induced Crd synthesis in V. ruber cells grown aerobically or anaerobically. The Crd-catalyzed reduction started by NADH demonstrated a time lag of several minutes, suggesting a redox regulation of the enzyme activity. The oxidized enzyme is inactive, which apparently prevents production of reactive oxygen species under aerobic conditions. Our findings identify Crd as a regulated NADH-dependent cinnamate reductase, apparently protecting V. ruber from (hydroxy)cinnamate poisoning.

CmDOF18 positively regulates salinity tolerance in Chrysanthemum morifolium by activating the oxidoreductase system.

BACKGROUND: Chrysanthemum, one of the four major cut flowers all over the world, is very sensitive to salinity during cultivation. DNA binding with one finger (DOF) transcription factors play important roles in biological processes in plants. The response mechanism of CmDOF18 from chrysanthemum to salt stress remains unclear. RESULTS: In this study, CmDOF18 was cloned from Chrysanthemum morifolium, and its expression was induced by salinity stress. The gene encodes a 291-amino acid protein with a typical DOF domain. CmDOF18 was localized to the nucleus in onion epidermal cells and showed transcriptional activation in yeast. CmDOF18 transgenic plants were generated to identify the role of this gene in resistance to salinity treatment. Chrysanthemum plants overexpressing CmDOF18 were more resistant to salinity stress than wild-type plants. Under salinity stress, the malondialdehyde content and leaf electrolyte conductivity in CmDOF18-overexpressing transgenic plants were lower than those in wild-type plants, while the proline content, chlorophyll content, superoxide dismutase activity and peroxidase activity were higher than those in wild-type plants. The opposite findings were observed in gene-silenced plants compared with wild-type plants. The gene expression levels of oxidoreductase increased in CmDOF18-overexpressing transgenic plants but decreased in CmDOF18-SRDX gene-silenced transgenic plants. CONCLUSION: In summary, we analyzed the function of CmDOF18 from chrysanthemum, which may regulate salinity stress in plants, possibly due to its role in the regulation of oxidoreductase.

Tri-enzyme fusion of tryptophan halogenase achieves a concise strategy for coenzyme self-sufficiency and the continuous halogenation of L-tryptophan.

The halogenase-based catalysis is one of the most environmentally friendly methods for the synthesis of halogenated products, among which flavin-dependent halogenases (FDHs) have attracted great interest as one of the most promising biocatalysts due to the remarkable site-selectivity and wide substrate range. However, the complexity of constructing the NAD+-NADH-FAD-FADH2 bicoenzyme cycle system has affected the engineering applications of FDHs. In this work, a coenzyme self-sufficient tri-enzyme fusion was constructed and successfully applied to the continuous halogenation of L-tryptophan. SpFDH was firstly identified derived from Streptomyces pratensis, a highly selective halogenase capable of generating 6-chloro-tryptophan from tryptophan. Then, using gene fusion technology, SpFDH was fused with glucose dehydrogenase (GDH) and flavin reductase (FR) to form a tri-enzyme fusion, which increased the yield by 1.46-fold and making the coenzymes self-sufficient. For more efficient halogenation of L-tryptophan, a continuous halogenation bioprocess of L-tryptophan was developed by immobilizing the tri-enzyme fusion and attaching it to a continuous catalytic device, which resulted in a reaction yield of 97.6% after 12 h reaction. An FDH from S. pratensis was successfully applied in the halogenation and our study provides a concise strategy for the preparation of halogenated tryptophan mediated by multienzyme cascade catalysis.

Construction of a dual-signal readout platform for effective glutathione S-transferase sensing based on polyethyleneimine-capped silver nanoclusters and cobalt-manganese oxide nanosheets with oxidase-mimicking activity.

A novel dual-mode fluorometric and colorimetric sensing platform is reported for determining glutathione S-transferase (GST) by utilizing polyethyleneimine-capped silver nanoclusters (PEI-AgNCs) and cobalt-manganese oxide nanosheets (CoMn-ONSs) with oxidase-like activity. Abundant active oxygen species (O2•-) can be produced through the CoMn-ONSs interacting with dissolved oxygen. Afterward, the pink oxDPD was generated through the oxidation of colorless N,N-diethyl-p-phenylenediamine (DPD) by O2•-, and two absorption peaks at 510 and 551 nm could be observed. Simultaneously, oxDPD could quench the fluorescence of PEI-AgNCs at 504 nm via the inner filter effect (IFE). However, in the presence of glutathione (GSH), GSH prevents the oxidation of DPD due to the reducibility of GSH, leading to the absorbance decrease at 510 and 551 nm. Furthermore, the fluorescence at 504 nm was restored due to the quenching effect of oxDPD on decreased PEI-AgNCs. Under the catalysis of GST, GSH and1-chloro-2,4-dinitrobenzo (CDNB) conjugate to generate an adduct, initiating the occurrence of the oxidation of the chromogenic substrate DPD, thereby inducing a distinct colorimetric response again and the significant quenching of PEI-AgNCs. The detection limits for GST determination were 0.04 and 0.21 U/L for fluorometric and colorimetric modes, respectively. The sensing platform illustrated reliable applicability in detecting GST in real samples.

Temperature-resilient and sustainable Mn-MOF oxidase-like nanozyme (UoZ-4) for total antioxidant capacity sensing in some citrus fruits: Breaking the temperature barrier.

Current nanozyme applications rely heavily on peroxidase-like nanozymes and are limited to a specific temperature range, despite notable advancements in nanozyme development. In this work, we designed novel Mn-based metal organic frameworks (UoZ-4), with excellent oxidase mimic activity towards common substrates. UoZ-4 showed excellent oxidase-like activity (with Km 0.072 mM) in a wide range of temperature, from 10 °C to 100 °C with almost no activity loss, making it a very strong candidate for psychrophilic and thermophilic applications. Ascorbic acid, cysteine, and glutathione could quench the appearance of the blue color of oxTMB, led us to design a visual-based sensing platform for detection of total antioxidant capacity (TAC) in cold, mild and hot conditions. The visual mode successfully assessed TAC in citrus fruits with satisfactory recovery and precisions. Cold/hot adapted and magnetic property will broaden the horizon of nanozyme applications and breaks the notion of the temperature limitation of enzymes.

A gold speciation that adds a second layer to synergistic gold-copper toxicity in Cupriavidus metallidurans.

The metal-resistant bacterium Cupriavidus metallidurans occurs in metal-rich environments. In auriferous soils, the bacterium is challenged by a mixture of copper ions and gold complexes, which exert synergistic toxicity. The previously used, self-made Au(III) solution caused a synergistic toxicity of copper and gold that was based on the inhibition of the CupA-mediated efflux of cytoplasmic Cu(I) by Au(I) in this cellular compartment. In this publication, the response of the bacterium to gold and copper was investigated by using a commercially available Au(III) solution instead of the self-made solution. The new solution was five times more toxic than the previously used one. Increased toxicity was accompanied by greater accumulation of gold atoms by the cells. The contribution of copper resistance determinants to the commercially available Au(III) solution and synergistic gold-copper toxicity was studied using single- and multiple-deletion mutants. The commercially available Au(III) solution inhibited periplasmic Cu(I) homeostasis, which is required for the allocation of copper ions to copper-dependent proteins in this compartment. The presence of the gene for the periplasmic Cu(I) and Au(I) oxidase, CopA, decreased the cellular copper and gold content. Transcriptional reporter gene fusions showed that up-regulation of gig, encoding a minor contributor to copper resistance, was strictly glutathione dependent. Glutathione was also required to resist synergistic gold-copper toxicity. The new data indicated a second layer of synergistic copper-gold toxicity caused by the commercial Au(III) solution, inhibition of the periplasmic copper homeostasis in addition to the cytoplasmic one.IMPORTANCEWhen living in auriferous soils, Cupriavidus metallidurans is not only confronted with synergistic toxicity of copper ions and gold complexes but also by different gold species. A previously used gold solution made by using aqua regia resulted in the formation of periplasmic gold nanoparticles, and the cells were protected against gold toxicity by the periplasmic Cu(I) and Au(I) oxidase CopA. To understand the role of different gold species in the environment, another Au(III) solution was commercially acquired. This compound was more toxic due to a higher accumulation of gold atoms by the cells and inhibition of periplasmic Cu(I) homeostasis. Thus, the geo-biochemical conditions might influence Au(III) speciation. The resulting Au(III) species may subsequently interact in different ways with C. metallidurans and its copper homeostasis system in the cytoplasm and periplasm. This study reveals that the geochemical conditions may decide whether bacteria are able to form gold nanoparticles or not.

Contribution of Microorganisms with the Clade II Nitrous Oxide Reductase to Suppression of Surface Emissions of Nitrous Oxide.

The sources and sinks of nitrous oxide, as control emissions to the atmosphere, are generally poorly constrained for most environmental systems. Initial depth-resolved analysis of nitrous oxide flux from observation wells and the proximal surface within a nitrate contaminated aquifer system revealed high subsurface production but little escape from the surface. To better understand the environmental controls of production and emission at this site, we used a combination of isotopic, geochemical, and molecular analyses to show that chemodenitrification and bacterial denitrification are major sources of nitrous oxide in this subsurface, where low DO, low pH, and high nitrate are correlated with significant nitrous oxide production. Depth-resolved metagenomes showed that consumption of nitrous oxide near the surface was correlated with an enrichment of Clade II nitrous oxide reducers, consistent with a growing appreciation of their importance in controlling release of nitrous oxide to the atmosphere. Our work also provides evidence for the reduction of nitrous oxide at a pH of 4, well below the generally accepted limit of pH 5.

Radical fluorine transfer catalysed by an engineered nonheme iron enzyme.

Nonheme iron enzymes stand out as one of the most versatile biocatalysts for molecular functionalization. They facilitate a wide array of chemical transformations within biological processes, including hydroxylation, chlorination, epimerization, desaturation, cyclization, and more. Beyond their native biological functions, these enzymes possess substantial potential as powerful biocatalytic platforms for achieving abiological metal-catalyzed reactions, owing to their functional and structural diversity and high evolvability. To this end, our group has recently engineered a series of nonheme iron enzymes to employ non-natural radical-relay mechanisms for abiological radical transformations not previously known in biology. Notably, we have demonstrated that a nonheme iron enzyme, (S)-2-hydroxypropylphosphonate epoxidase from Streptomyces viridochromogenes (SvHppE), can be repurposed into an efficient and selective biocatalyst for radical fluorine transfer reactions. This marks the first known instance of a redox enzymatic process for C(sp3)F bond formation. This chapter outlines the detailed experimental protocol for engineering SvHPPE for fluorination reactions. Furthermore, the provided protocol could serve as a general guideline that might facilitate other engineering endeavors targeting nonheme iron enzymes for novel catalytic functions.

High- or Low-Yielding F2 Progeny of Wheat Is Result of Specific TaCKX Gene Coexpression Patterns in Association with Grain Yield in Paternal Parent.

Members of the TaCKX gene family (GFM) encode oxidase/dehydrogenase cytokinin degrading enzymes (CKX), which play an important role in the homeostasis of phytohormones, affecting wheat development and productivity. Therefore, the objective of this investigation was to test how the expression patterns of the yield-related TaCKX genes and TaNAC2-5A (NAC2) measured in 7 days after pollination (DAP) spikes and the seedling roots of parents are inherited to apply this knowledge in the breeding process. The expression patterns of these genes were compared between parents and their F2 progeny in crosses of one mother with different paterns of awnless cultivars and reciprocal crosses of awned and awnless lines. We showed that most of the genes tested in the 7 DAP spikes and seedling roots of the F2 progeny showed paternal expression patterns in crosses of awnless cultivars as well as reciprocal crosses of awned and awnless lines. Consequently, the values of grain yield in the F2 progeny were similar to the pater; however, the values of seedling root mass were similar to the mother or both parents. The correlation analysis of TaCKX GFMs and NAC2 in spikes and spikes per seedling roots reveals that the genes correlate with each other specifically with the pater and the F2 progeny or the mother and the F2 progeny, which shape phenotypic traits. The numbers of spikes and semi-empty spikes are mainly correlated with the specific coexpression of the TaCKX and NAC2 genes expressed in spikes or spikes per roots of the pater and F2 progeny. Variable regression analysis of grain yield and root mass with TaCKX GFMs and NAC2 expressed in the tested tissues of five crosses revealed a significant dependency of these parameters on the mother and F2 and/or the pater and F2 progeny. We showed that the inheritance of yield-related traits depends on the specific cooperative expression of some TaCKX GFMs, in some crosses coupled with NAC2, and is strongly dependent on the genotypes used for the crosses. Indications for parental selection in the breeding of high-yielding lines are discussed.

Characterization of a novel 3-quinuclidinone reductase possessing remarkable thermostability.

The 3-quinuclidinone reductase plays an irreplaceable role in the biopreparation of (R)-3-quinuclidinol, an intermediate vital for synthesis of various pharmaceuticals. Thermal robustness is a critical factor for enzymatic synthesis in industrial applications. This study characterized a new 3-quinuclidinone reductase, named SaQR, with significant thermal stability. The SaQR was overexpressed in a GST-fused state, and substrate and cofactor screening were conducted. Additionally, three-dimensional structure prediction using AlphaFold and analysis were performed, along with relevant thermostability tests, and the evaluation of factors influencing enzyme activity. The findings highlight the remarkable thermostability of SaQR, retaining over 90% of its activity after 72 h at 50°C, with an optimal operational temperature of 85°C. SaQR showed typical structural traits of the SDR superfamily, with its cofactor-determining residue being aspartic acid, conferring nicotinamide adenine dinucleotide (NAD(H)) preference. Moreover, K+ and Na+, at a concentration of 400 mM, could significantly enhance the activity, while Mg2+ and Mn2+ only display inhibitory effects within the tested concentration range. The findings of molecular dynamics simulations suggest that high temperatures may disrupt the binding of enzyme to substrate by increasing the flexibility of residues 205-215. In conclusion, this study reports a novel 3-quinuclidinone reductase with remarkable thermostability.

Engineering carboxylic acid reductases and unspecific peroxygenases for flavor and fragrance biosynthesis.

Emerging consumer demand for safer, more sustainable flavors and fragrances has created new challenges for the industry. Enzymatic syntheses represent a promising green production route, but the broad application requires engineering advancements for expanded diversity, improved selectivity, and enhanced stability to be cost-competitive with current methods. This review discusses recent advances and future outlooks for enzyme engineering in this field. We focus on carboxylic acid reductases (CARs) and unspecific peroxygenases (UPOs) that enable selective productions of complex flavor and fragrance molecules. Both enzyme types consist of natural variants with attractive characteristics for biocatalytic applications. Applying protein engineering methods, including rational design and directed evolution in concert with computational modeling, present excellent examples for property improvements to unleash the full potential of enzymes in the biosynthesis of value-added chemicals.

Multicopper oxidase-2 mediated cuticle formation: Its contribution to evolution and success of insects as terrestrial organisms.

The insect cuticle is a non-cellular matrix composed of polysaccharide chitins and proteins. The cuticle covers most of the body surface, including the trachea, foregut, and hindgut, and it is the body structure that separates the intraluminal environment from the external environment. The cuticle is essential to sustain their lives, both as a physical barrier to maintain homeostasis and as an exoskeleton that mechanically supports body shape and movement. Previously, we proposed a theory about the possibility that the cuticle-forming system contributes to the "evolution and success of insects." The main points of our theory are that 1) insects evolved an insect-specific system of cuticle formation and 2) the presence of this system may have provided insects with a competitive advantage in the early land ecosystems. The key to this theory is that insects utilize molecular oxygen abundant in the atmosphere, which differs from closely related crustaceans that form their cuticles with calcium ions. With newly obtained knowledge, this review revisits the significance of the insect-specific system for insects to adapt to terrestrial environments and also discusses the long-standing question in entomology as to why, despite their great success in terrestrial environments, they poorly adapt to marine environments.

Effect of pinoresinol-lariciresinol reductases on biosynthesis of lignans with substrate selectivity in Schisandra chinensis.

Schisandra lignans are the main bioactive compounds found in Schisandra chinensis fruits, such as schisandrol lignans and schisandrin lignans, which play important roles in organ protection or other clinical roles. Pinoresinol-lariciresinol reductase (PLR) plays a pivotal role in plant lignan biosynthesis, however, limited research has been conducted on S. chinensis PLR to date. This study identified five genes as ScPLR, successfully cloned their coding sequences, and elucidated their catalytic capabilities. ScPLR3-5 could recognize both pinoresinol and lariciresinol as substrates, and convert them into lariciresinol and secoisolariciresinol, respectively, while ScPLR2 exclusively catalyzed the conversion of (+)-pinoresinol into (+)-lariciresinol. Transcript-metabolite correlation analysis indicated that ScPLR2 exhibited unique properties that differed from the other members. Molecular docking and site-directed mutagenesis revealed that Phe271 and Leu40 in the substrate binding motif were crucial for the catalytic activity of ScPLR2. This study serves as a foundation for understanding the essential enzymes involved in schisandra lignan biosynthesis.

Structural enzymology studies with the substrate 3S-hydroxybutanoyl-CoA: bifunctional MFE1 is a less efficient dehydrogenase than monofunctional HAD.

Multifunctional enzyme, type-1 (MFE1) catalyzes the second and third step of the ß-oxidation cycle, being, respectively, the 2E-enoyl-CoA hydratase (ECH) reaction (N-terminal part, crotonase fold) and the NAD+-dependent, 3S-hydroxyacyl-CoA dehydrogenase (HAD) reaction (C-terminal part, HAD fold). Structural enzymological properties of rat MFE1 (RnMFE1) as well as of two of its variants, namely the E123A variant (a glutamate of the ECH active site is mutated into alanine) and the BCDE variant (without domain A of the ECH part), were studied, using as substrate 3S-hydroxybutanoyl-CoA. Protein crystallographic binding studies show the hydrogen bond interactions of 3S-hydroxybutanoyl-CoA as well as of its 3-keto, oxidized form, acetoacetyl-CoA, with the catalytic glutamates in the ECH active site. Pre-steady state binding experiments with NAD+ and NADH show that the kon and koff rate constants of the HAD active site of monomeric RnMFE1 and the homologous human, dimeric 3S-hydroxyacyl-CoA dehydrogenase (HsHAD) for NAD+ and NADH are very similar, being the same as those observed for the E123A and BCDE variants. However, steady state and pre-steady state kinetic data concerning the HAD-catalyzed dehydrogenation reaction of the substrate 3S-hydroxybutanoyl-CoA show that, respectively, the kcat and kchem rate constants for conversion into acetoacetyl-CoA by RnMFE1 (and its two variants) are about 10 fold lower as when catalyzed by HsHAD. The dynamical properties of dehydrogenases are known to be important for their catalytic efficiency, and it is discussed that the greater complexity of the RnMFE1 fold correlates with the observation that RnMFE1 is a slower dehydrogenase than HsHAD.

Low-molecular-weight thiol transferases in redox regulation and antioxidant defence.

Low-molecular-weight (LMW) thiols are produced in all living cells in different forms and concentrations. Glutathione (GSH), coenzyme A (CoA), bacillithiol (BSH), mycothiol (MSH), ergothioneine (ET) and trypanothione T(SH)2 are the main LMW thiols in eukaryotes and prokaryotes. LMW thiols serve as electron donors for thiol-dependent enzymes in redox-mediated metabolic and signaling processes, protect cellular macromolecules from oxidative and xenobiotic stress, and participate in the reduction of oxidative modifications. The level and function of LMW thiols, their oxidized disulfides and mixed disulfide conjugates in cells and tissues is tightly controlled by dedicated oxidoreductases, such as peroxiredoxins, glutaredoxins, disulfide reductases and LMW thiol transferases. This review provides the first summary of the current knowledge of structural and functional diversity of transferases for LMW thiols, including GSH, BSH, MSH and T(SH)2. Their role in maintaining redox homeostasis in single-cell and multicellular organisms is discussed, focusing in particular on the conjugation of specific thiols to exogenous and endogenous electrophiles, or oxidized protein substrates. Advances in the development of new research tools, analytical methodologies, and genetic models for the analysis of known LMW thiol transferases will expand our knowledge and understanding of their function in cell growth and survival under oxidative stress, nutrient deprivation, and during the detoxification of xenobiotics and harmful metabolites. The antioxidant function of CoA has been recently discovered and the breakthrough in defining the identity and functional characteristics of CoA S-transferase(s) is soon expected.

Identification and characterization of an ene-reductase from Corynebacterium casei.

The asymmetric reduction of α, ß-unsaturated compounds conjugated with electron-withdrawing group by ene-reductases (ERs) is a valuable method for the synthesis of enantiopure chiral compounds. This study introduced an ER from Corynebacterium casei (CcER) which was heterologously expressed in Escherichia coli BL21(DE3), and the purified recombinant CcER was characterized for its biocatalytic properties. CcER exhibited the highest specific activity at 40 °C and pH 6.5, and showcased appreciable stability below 40 °C over a pH range of 6.0-7.0. The enzyme displayed high resistance to methanol. CcER accepted NADH or NADPH as a cofactor and exhibited a broad substrate spectrum towards α, ß-unsaturated compounds. It achieved complete conversion of 2-cyclohexen-1-one and good performance for stereoselective reduction of (R)-carvone (conversion 98 %, diastereoselectivity 96 %). This study highlights the robustness and potential of CcER.

Dynamic Regulation of Intracellular Labile Cu(I)/Cu(II) Cycle in Microalgae Chlamydomonas reinhardtii: Disrupting the Balance by Cu Stress.

The labile metal pool involved in intracellular trafficking and homeostasis is the portion susceptible to environmental stress. Herein, we visualized the different intracellular distributions of labile Cu(I) and Cu(II) pools in the alga Chlamydomonas reinhardtii. We first demonstrated that labile Cu(I) predominantly accumulated in the granules within the cytoplasmic matrix, whereas the labile Cu(II) pool primarily localized in the pyrenoid and chloroplast. The cell cycle played an integral role in balancing the labile Cu(I)/Cu(II) pools. Specifically, the labile Cu(II) pool primarily accumulated during the SM phase following cell division, while the labile Cu(I) pool dynamically changed during the G phase as cell size increased. Notably, the labile Cu(II) pool in algae at the SM stage exhibited heightened sensitivity to environmental Cu stress. Exogenous Cu stress disrupted the intracellular labile Cu(I)/Cu(II) cycle and balance, causing a shift toward the labile Cu(II) pool. Our proteomic analysis further identified a putative cupric reductase, potentially capable of reducing Cu(II) to Cu(I), and four putative multicopper oxidases, potentially capable of oxidizing Cu(I) to Cu(II), which may be involved in the conversion between the labile Cu(I) pool and labile Cu(II) pool. Our study elucidated a dynamic cycle of the intracellular labile Cu(I)/Cu(II) pools, which were accessible and responsive to environmental changes.

Exploring functional genes' correlation with (S)-equol concentration and new daidzein racemase identification.

With its estrogenic activity, (S)-equol plays an important role in maintaining host health and preventing estrogen-related diseases. Exclusive production occurs through the transformation of soy isoflavones by intestinal bacteria, but the reasons for variations in (S)-equol production among different individuals and species remain unclear. Here, fecal samples from humans, pigs, chickens, mice, and rats were used as research objects. The concentrations of (S)-equol, along with the genetic homology and evolutionary relationships of (S)-equol production-related genes [daidzein reductase (DZNR), daidzein racemase (DDRC), dihydrodaidzein reductase (DHDR), tetrahydrodaidzein reductase (THDR)], were analyzed. Additionally, in vitro functional verification of the newly identified DDRC gene was conducted. It was found that approximately 40% of human samples contained (S)-equol, whereas 100% of samples from other species contained (S)-equol. However, there were significant variations in (S)-equol content among the different species: rats > pigs > chickens > mice > humans. The distributions of the four genes displayed species-specific patterns. High detection rates across various species were exhibited by DHDR, THDR, and DDRC. In contrast, substantial variations in detection rates among different species and individuals were observed with respect to DZNR. It appears that various types of DZNR may be associated with different concentrations of (S)-equol, which potentially correspond to the regulatory role during (S)-equol synthesis. This enhances our understanding of individual variations in (S)-equol production and their connection with functional genes in vitro. Moreover, the newly identified DDRC exhibits higher potential for (S)-equol synthesis compared to the known DDRC, providing valuable resources for advancing in vitro (S)-equol production. IMPORTANCE: (S)-equol ((S)-EQ) plays a crucial role in maintaining human health, along with its known capacity to prevent and treat various diseases, including cardiovascular diseases, metabolic syndromes, osteoporosis, diabetes, brain-related diseases, high blood pressure, hyperlipidemia, obesity, and inflammation. However, factors affecting individual variations in (S)-EQ production and the underlying regulatory mechanisms remain elusive. This study examines the association between functional genes and (S)-EQ production, highlighting a potential correlation between the DZNR gene and (S)-EQ content. Various types of DZNR may be linked to the regulation of (S)-EQ synthesis. Furthermore, the identification of a new DDRC gene offers promising prospects for enhancing in vitro (S)-EQ production.

Insights into the influence of polystyrene microplastics on the bio-degradation behavior of tetrabromobisphenol A in soil.

Soil contamination by emerging pollutants tetrabromobisphenol A (TBBPA) and microplastics has become a global environmental issue in recent years. However, little is known about the effect of microplastics on degradation of TBBPA in soil, especially aged microplastics. In this study, the effect of aged polystyrene (PS) microplastics on the degradation of TBBPA in soil and the mechanisms were investigated. The results suggested that the aged microplastics exhibited a stronger inhibitory effect on the degradation of TBBPA in soil than the pristine microplastics, and the degradation efficiency of TBBPA decreased by 21.57% at the aged microplastic content of 1%. This might be related to the higher TBBPA adsorption capacity of aged microplastics compared to pristine microplastics. Aged microplastics strongly altered TBBPA-contaminated soil properties, reduced oxidoreductase activity and affected microbial community composition. The decrease in soil oxidoreductase activity and relative abundance of functional microorganisms (e.g., Bacillus, Pseudarthrobacter and Sphingomonas) caused by aged microplastics interfered with metabolic pathways of TBBPA. This study indicated the importance the risk assessment and soil remediation for TBBPA-contaminated soil with aged microplastics.