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Phytophthora infestans is a notorious oomycete pathogen that causes potato late blight. It secretes numerous effector proteins to manipulate host immunity. Understanding mechanisms underlying their host cell manipulation is crucial for developing disease resistance strategies. Here, we report that the conserved RXLR effector Pi05910 of P. infestans is a genotype-specific avirulence elicitor on potato variety Longshu 12 and contributes virulence by suppressing and destabilizing host glycolate oxidase StGOX4. By performing co-immunoprecipitation, yeast-two-hybrid assays, luciferase complementation imaging, bimolecular fluorescence complementation and isothermal titration calorimetry assays, we identified and confirmed potato StGOX4 as a target of Pi05910. Further analysis revealed that StGOX4 and its homologue NbGOX4 are positive immune regulators against P. infestans, as indicated by infection assays on potato and Nicotiana benthamiana overexpressing StGOX4 and TRV-NbGOX4 plants. StGOX4-mediated disease resistance involves enhanced reactive oxygen species accumulation and activated the salicylic acid signalling pathway. Pi05910 binding inhibited enzymatic activity and destabilized StGOX4. Furthermore, mutagenesis analyses indicated that the 25th residue (tyrosine, Y25) of StGOX4 mediates Pi05910 binding and is required for its immune function. Our results revealed that the core RXLR effector of P. infestans Pi05910 suppresses plant immunity by targeting StGOX4, which results in decreased enzymatic activity and protein accumulation, leading to enhanced plant susceptibility., (© 2024 The Author(s). Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

(2,3)-Butanediol dehydrogenases (BDHs) are widely utilized for the stereoselective interconversion between α-hydroxy ketones and vicinal diols to produce various functional building blocks. In this study, to enhance the specific activity towards (R)-phenyl-1,2-ethanediol (1a) for 2-hydroxyacetophenone (1b), the substrate-binding pocket of a Bacillus subtilis BDH (BsBDHA) was refined through site-directed mutagenesis. Based on molecular docking simulations, 14 residues were identified and subjected to alanine scanning mutagenesis. After screening, two residues, His 42 and Gly 292 , were singled out for partial site-saturation mutagenesis. The results revealed that BsBDHA H42A and BsBDHA G292A displayed high activities of 3.21 and 1.97 U/mg, respectively. Employing combinatorial mutagenesis, a superior mutant, BsBDHA I49L/V266L/G292A , was developed, exhibiting significantly enhanced specific activity and catalytic efficiency towards (R)-1a, achieving 14.81 U/mg and 4.47 mM -1  s -1 , respectively, which were 27.4- and 55.9-fold higher than those of BsBDHA. Further substrate spectrum analysis revealed that the superior mutant displayed increased specific activities for (R)-2a-6a by 1.4- to 10.3-fold. The integration of BsBDHA I49L/V266L/G292A into a three-enzymatic cascade for the synthesis of 1b effectively elevated the yield from 58.1 to 82.4%. Molecular mechanism analysis indicated that the mutation-induced changes in intermolecular forces resulted in a higher frequency of reactive conformations for (R)-1a in BsBDHA I49L/V266L/G292A compared to BsBDHA., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Nuclear Bcl-xL is found to promote cancer metastasis independently of its mitochondria-based anti-apoptotic activity. How Bcl-xL is translocated into the nucleus and how nuclear Bcl-xL regulates histone H3 trimethyl Lys4 (H3K4me3) modification have yet to be understood. Here, we report that C-terminal Binding Protein 2 (CtBP2) binds to Bcl-xL via its N-terminus and translocates Bcl-xL into the nucleus. Knockdown of CtBP2 by shRNA decreases the nuclear portion of Bcl-xL and reverses Bcl-xL-induced invasion and metastasis in mouse models. Furthermore, knockout of CtBP2 not only reduces the nuclear portion of Bcl-xL but also suppresses Bcl-xL transcription. The binding between Bcl-xL and CtBP2 is required for their interaction with MLL1, a histone H3K4 methyltransferase. Pharmacologic inhibition of the MLL1 enzymatic activity reverses Bcl-xL-induced H3K4me3 and TGFβ mRNA upregulation, as well as invasion. Moreover, the cleavage under targets and release using nuclease (CUT&RUN) assay coupled with next-generation sequencing reveals that H3K4me3 modifications are particularly enriched in the promotor regions of genes encoding TGFβ and its signaling pathway members in cancer cells overexpressing Bcl-xL. Altogether, the metastatic function of Bcl-xL is mediated by its interaction with CtBP2 and MLL1 and this study offers new therapeutic strategies to treat Bcl-xL-overexpressing cancer., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

l-2-Hydroxyglutarate (l-2-HG) has been regarded as a tumor metabolite, and it plays a crucial role in adaptation of tumor cells to hypoxic conditions. However, the role of l-2-HG in tumor radioresistance and the underlying mechanism have not yet been revealed. Here, we found that l-2-HG exhibited to have radioresistance effect on U87 human glioblastoma cells, which could reduce DNA damage and apoptosis caused by irradiation, promote cell proliferation and migration, and impair G2/M phase arrest. Mechanistically, l-2-HG upregulated the protein level of hypoxia-inducible factor-1α (HIF-1α) and the expression levels of HIF-1α downstream target genes. The knockdown of l-2-hydroxyglutarate dehydrogenase (L2HGDH) gene promoted the tumor growth and proliferation of U87 cells in nude mice by increasing HIF-1α expression level in vivo. In addition, the low expression level of L2HGDH gene was correlated with the short survival of patients with glioma or kidney cancer. In conclusion, our study revealed the role and mechanism of l-2-HG in tumor radioresistance and may provide a new perspective for overcoming tumor radioresistance and broaden our comprehension of the role of metabolites in tumor microenvironment., (© 2024 Wiley Periodicals LLC.)

Objectives: The mitochondrial enzyme L-2-hydroxyglutarate dehydrogenase (L2HGDH) regulates the abundance of L-2-hydroxyglutarate (L-2HG), a potent signaling metabolite capable of influencing chromatin architecture, mitochondrial metabolism, and cell fate decisions. Loss of L2hgdh activity in humans induces ectopic L-2HG accumulation, resulting in neurodevelopmental defects, altered immune cell function, and enhanced growth of clear cell renal cell carcinomas. To better understand the molecular mechanisms that underlie these disease pathologies, we used the fruit fly Drosophila melanogaster to investigate the endogenous functions of L2hgdh., Methods: L2hgdh mutant adult male flies were analyzed under normoxic and hypoxic conditions using a combination of semi-targeted metabolomics and RNA-seq. These multi-omic analyses were complemented by tissue-specific genetic studies that examined the effects of L2hgdh mutations on the Drosophila renal system (Malpighian tubules; MTs)., Results: Our studies revealed that while L2hgdh is not essential for growth or viability under standard culture conditions, L2hgdh mutants are hypersensitive to hypoxia and expire during the reoxygenation phase with severe disruptions of mitochondrial metabolism. Moreover, we find that the fly renal system is a key site of L2hgdh activity, as L2hgdh mutants that express a rescuing transgene within the MTs survive hypoxia treatment and exhibit normal levels of mitochondrial metabolites. We also demonstrate that even under normoxic conditions, L2hgdh mutant MTs experience significant metabolic stress and are sensitized to aberrant growth upon Egfr activation., Conclusions: These findings present a model in which renal L2hgdh activity limits systemic L-2HG accumulation, thus indirectly regulating the balance between glycolytic and mitochondrial metabolism, enabling successful recovery from hypoxia exposure, and ensuring renal tissue integrity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Cassava ( Manihot esculenta Crantz) is used as a biomass energy material and an effective supplement for food and feed. Cinnamyl alcohol dehydrogenase (CAD) catalyzes the final step of lignin biosynthesis and is responsible for various stresses. However, systematic investigations of the CAD gene family in cassava have been poorly understood. In this study, a genome-wide survey and bioinformatics analysis of CAD gene family was performed, transcriptomics, qRT-PCR, gene silencing and stress of yeast cell were used for excavate and validate the candidate MeCADs gene. 36 MeCADs genes unevenly distributed across 12 chromosomes were identified. Through phylogenetic analyses alongside their Arabidopsis counterparts, these MeCADs were divided into four groups, each containing a similar structure and conserved motifs. Interestingly, transcriptome data analysis revealed that 32 MeCAD genes were involved in the postharvest physiological deterioration (PPD) process, whereas 27 MeCAD genes showed significant changes. Additionally, the relative quantitative analysis of 6 MeCAD genes demonstrated that they were sensitive to PPD, suggesting that they may be involved in the regulation of PPD. Silencing MeCAD13 and MeCAD28 further showed that lignin content significantly decreased in the leaves. The wound-stress tolerance of transgenic yeast cells was enhanced after transformation with MeCAD13 and MeCAD28 . MeCAD13 and MeCAD28 may play positive roles in lignin biosynthesis and PPD response, respectively. These results provided a systematic functional analysis of MeCADs in cassava and paved a new way to genetically modify lignin biosynthesis and PPD tolerance.

The search for novel synthetic tools to prepare industrial chemicals in a safer and greener manner is a continuing challenge in synthetic chemistry. In this manuscript, we report the discovery, characterization, and synthetic potential of two novel aryl-alcohol oxidases from bacteria which are able to oxidize a variety of aliphatic and aromatic alcohols with efficiencies up to 4970 min -1  mM -1 . Both enzymes have shown a reasonable thermostability (thermal melting temperature values of 50.9 and 48.6 °C for ShAAO and SdAAO, respectively). Crystal structures revealed an unusual wide-open entrance to the active-site pockets compared to that previously described for traditional fungal aryl-alcohol oxidases, which could be associated with differences observed in substrate scope, catalytic efficiency, and other functional properties. Preparative-scale reactions and the ability to operate at high substrate loadings also demonstrate the potential of these enzymes in synthetic chemistry with total turnover numbers > 38000. Moreover, their availability as soluble and active recombinant proteins enabled their use as cell-free extracts which further highlights their potential for the large-scale production of carbonyl compounds. KEY POINTS: • Identification and characterization of two novel bacterial aryl-alcohol oxidases • Crystal structures reveal wide-open active-site pockets, impacting substrate scope • Total turnover numbers and cell-free extracts demonstrate the synthetic potential., (© 2024. The Author(s).)

Background: Breast cancer ranks among the most prevalent tumor types worldwide. Copy number amplification of chromosome 8q24 is frequently detected in breast cancer. ZNF623 is a relatively unexplored gene mapped to 8q24. Here, we explore the expression profile, prognostic significance, and biological action of ZNF623 in breast carcinogenesis., Methods: To evaluate the mRNA expression pattern and prognostic relevance of ZNF623 across different cancer types, we conducted bioinformatic analyses. The expression of the gene was suppressed using ZNF623 shRNAs/siRNAs and augmented through transfection with plasmids containing ZNF623 cDNA. Cell viability assay, clonogenic assay, and transwell migration assay were utilized to assess the proliferation, viability, and invasion capacity of breast cancer cell lines. Luciferase reporter assay served as a pivotal tool to ascertain the transcriptional activity of ZNF623. IP-MS and co-IP were employed to validate that ZNF623 interacted with CtBP1. ChIP analysis and ChIP-qPCR were conducted to assess the genes targeted by ZNF623/CtBP1 complex. Flow cytometry was conducted to evaluate the phosphorylation status of p65., Results: ZNF623 expression was notably elevated in breast cancer (BC). Prognostic analysis indicated higher expression of ZNF623 indicated worse survival. Functional experiments discovered that the upregulation of ZNF623 significantly enhanced both the proliferative and migratory capacities of breast cancer cells. Luciferase reporter assay indicated that ZNF623 was a transcription repressor. Immunoprecipitation coupled mass spectrometry analysis revealed a physical association between ZNF623 and CtBP1 in the interaction group. The conjoint analysis of ChIP-seq and TCGA DEG analysis revealed that the ZNF623/CtBP1 complex repressed a series of genes, such as negative regulation of the NF-kappaB signaling pathway. Flow cytometry analysis discovered that knockdown of ZNF623 decreased the phosphorylation level of p65, indicating that ZNF623 could regulate the activity of the NF-κB pathway., Conclusion: ZNF623 predicts poor prognosis of BC and enhances breast cancer growth and metastasis. By recruiting CtBP1, ZNF623 could suppress NF-κB inhibitors, including COMMD1, NFKBIL1, PYCARD, and BRMS1, expression from the transcription level., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

2-Hydroxyglutarate (2HG) is an oncometabolite that can contribute to tumor progression. Two enantiomer forms, L-2HG and D-2HG, arise from independent pathways starting from the precursor α-ketoglutarate (αKG). L-2HG production occurs through the promiscuous activities of malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) under acidic and/or hypoxic conditions. D-2HG frequently accumulates by gain-of-function mutations in the genes encoding two isoforms of isocitrate dehydrogenase (IDH1 and IDH2). Cognate metabolite repair enzymes, L- and D-2-hydroxyglutarate dehydrogenases, oxidize the enantiomers and cause abnormally high 2HG accumulation and disease when mutated. Elevated levels of either oncometabolite affect redox homeostasis, metabolism, and immune system functioning. Moreover, the oncometabolites inhibit several α-ketoglutarate-dependent dioxygenases resulting in epigenetic changes such as DNA and histone hypermethylation as well as deficiencies in DNA repair. L-2HG, and D-2HG in some cases, inhibit degradation of hypoxia-inducible factor (HIF1α), a transcription factor that alters gene expression to adapt to hypoxic conditions, favoring tumorigenesis. Patients with the rare disease 2-hydroxyglutaric aciduria (2HGA) have exceedingly high levels of 2HG, which is neurotoxic, causing developmental delays and brain abnormalities. D-2HG also has specific effects on collagen production and NADPH pools. Recently, D-2HG has been targeted in new chemotherapies aimed at disrupting the gain-of-function IDH1 and IDH2 mutants, resulting in successful clinical trials for several cancers., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Background: Noise-induced cochlear synaptopathy has recently emerged as a focus in hearing research., Purpose: This study aimed to examine the impact of repeated noise exposure on the quantification and mRNA expression levels of cochlear synapses., Methods: Measurements were conducted at baseline, 1 day, and 14 days post-exposure to 88 or 97 dB SPL noise (2 h/day for 7 days, frequency range 2-20 kHz). Auditory brainstem responses (ABRs), immunofluorescence and quantitative real-time PCR (qRT-PCR) were used to examine the results., Results: 1. Exposure to 88 dB SPL caused minimal changes in ABRs, ribbon morphology and medial olivocochlear (MOC) efferent synapses; elevation of synaptophysin(SYP) and α9α10 nAchR mRNA levels were observed. 2. Exposure to 97 dB SPL caused threshold shift and synaptopathy of ribbon and MOC; downregulation of α10nAchR, SYP and ctbp2 mRNA levels were observed., Conclusion: Noise-induced cochlear synaptic degeneration involves both afferent and efferent synaptopathy.

The advancement of acetylcholinesterase (AChE) activity and its inhibitor assays is crucial for clinical diagnosis, drug screening, and environmental monitoring. A nanozyme-mediated cascade reaction system could offer promising prospects for a wide range of applications in such biosensing; however, the creation of nanozyme catalysts with diverse functionalities remains a significant challenge. Herein, we have proposed a multifunctional iron-doped polymer dots (Fe-PDs) nanozyme possessing excellent fluorescence and peroxidase (POD)-mimicking activity. Notably, the Fe-PDs nanozyme is capable of catalyzing H 2 O 2 to produce a series of reactive oxygen species, which can simultaneously quench the fluorescence of Fe-PDs and induce a chromogenic reaction of 3,3',5,5'-tetramethylbenzidine (TMB), enabling the dual-mode detection of H 2 O 2 through both fluorescence turn-off and absorbance turn-on signals. Furthermore, by integrating acetylcholine (ACh) and choline oxidase (ChOx), we have developed a three-enzyme (AChE-ChOx-POD) cascade-based fluorometric and colorimetric dual-mode sensing platform for monitoring AChE activity and its inhibitors. The sensitive and convenient dual-mode sensor has achieved low limits of detection with 0.5 mU/mL (fluorometry) and 0.014 mU/mL (colorimetry) for AChE, respectively, which are superior to the traditional Ellman's assay. More significantly, this sensor can also be extended to detect the reversible and irreversible inhibitors of AChE, such as tacrine (IC 50 = 23.3 nM) and carbaryl (LOD = 0.8 nM). We firmly believe that this innovative dual-mode nanozyme-involved multienzyme cascade system-based sensing strategy will stimulate further exploration and serve as a versatile and practical tool for biochemical sensing applications.

Background: sepiapterine reductase deficiency (SRD) is a rare levodopa (L-dopa)-responsive disorder treated with a combination therapy of controlled-release L-dopa and carbidopa. The currently available formulation of controlled-release carbidopa/L-dopa does not entirely meet the requirements for the long-term therapy in pediatric patients. In fact, administration of a manufactured tablet at a dose intended for adults necessitates its adjustment to the child's needs, as the splitting of the tablet into smaller portions or its dilution in water. It's essential to emphasize that tablets must not be crushed, as this can compromise the controlled-release mechanism and affect the efficacy of the medication. At the moment, commercial liquid formulations are not available. Given these limitations, in house drug preparation in hospitals and community pharmacies is a valid option to ensure the proper therapeutic management of these patients., Materials and Methods: we described sample preparation, physical and microbiological analyses, taste testing, and tolerability of a 1:10 ratio carbidopa/L-dopa flavored (mint, raspberry, cacao, berries) and unflavored oral formulation (no sweetening agents were added). We also reported long-term follow-up of two pediatric patients with SRD., Results: we documented the stability for 28 days at 25 °C of the liquid solution. All formulations were well-tolerated, and no adverse events were observed during or after assessing taste and tolerability. The long-term follow up of two patients was characterized by effective symptom control and optimal treatment adherence and compliance., Conclusions: in-house liquid drug formulations can be a valid option for pediatric patients with SRD. Given the significant impact of taste on medication adherence, the use of flavoring agents in the development of liquid formulations of L-dopa/carbidopa results a very useful strategy to obtain optimal adherence in the pediatric population., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Anthocyanidins and anthocyanins are one subclass of flavonoids in plants with diverse biological functions and have health-promoting effects. Dihydroflavonol 4-reductase (DFR) is one of the important enzymes involved in the biosynthesis of anthocyanidins and other flavonoids. Here, a new MOF-based nano-immobilized DFR enzyme acting as a nano-biocatalyst for the production of anthocyanidins in vitro was designed. We prepared UiO-66-NH 2 MOF nano-carrier and recombinant DFR enzyme from genetic engineering. DFR@UiO-66-NH 2 nano-immobilized enzyme was constructed based on covalent bonding under the optimum immobilization conditions of the enzyme/carrier ratio of 250 mg/g, 37 °C, pH 6.5 and fixation time of 10 min. DFR@UiO-66-NH 2 was characterized and its catalytic function for the synthesis of anthocyanidins in vitro was testified using UPLC-QQQ-MS analysis. Compared with free DFR enzyme, the enzymatic reaction catalyzed by DFR@UiO-66-NH 2 was more easily for manipulation in a wide range of reaction temperatures and pH values. DFR@UiO-66-NH 2 had better thermal stability, enhanced adaptability, longer-term storage, outstanding tolerances to the influences of several organic reagents and Zn 2+ , Cu 2+ and Fe 2+ ions, and relatively good reusability. This work developed a new MOF-based nano-immobilized biocatalyst that had a good prospect of application in the green synthesis of anthocyanins in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Porcine carbonyl reductases (pCBR1 and pCBR-N1) and aldo-keto reductases (pAKR1C1 and pAKR1C4) exhibit hydroxysteroid dehydrogenase (HSD) activity. However, their roles in the metabolism of porcine-specific androgens (19-nortestosterone and epiandrosterone), 11-oxygenated androgens, neurosteroids, and corticosteroids remain unclear. Here, we compared the steroid specificity of the four recombinant enzymes by kinetic and product analyses. In C 18 /C 19 -steroids,11-keto- and 11β-hydroxy-5α-androstane-3,17-diones were reduced by all the enzymes, whereas 5α-dihydronandrolone (19-nortestosterone metabolite) and 11-ketodihydrotestosterone were reduced by pCBR1, pCBR-N1, and pAKR1C1, of which pCBR1 exhibited the lowest (submicromolar) K m values. Product analysis showed that pCBR1 and pCBR-N1 function as 3α/β-HSDs, in contrast to pAKR1C1 and pAKR1C4 (acting as 3β-HSD and 3α-HSD, respectively). Additionally, 17β-HSD activity was observed in pCBR1 and pCBR-N1 (toward epiandrosterone and its 11-oxygenated derivatives) and in pAKR1C1 (toward androsterone, 4-androstene-3,17-dione and their 11-oxygenated derivatives). The four enzymes also showed different substrate specificity for 3-keto-5α/β-dihydro-C 21 -steroids, including GABAergic neurosteroid precursors and corticosteroid metabolites. 5β-Dihydroprogesterone was reduced by all the enzymes, whereas 5α-dihydroprogesterone was reduced only by pCBR1, and 5α/β-dihydrodeoxycorticosterones by pCBR1 and pCBR-N1. The two pCBRs also reduced the 5α/β-dihydro-metabolites of cortisol, 11-deoxycortisol, cortisone, and corticosterone. pCBR1 exhibited lower K m values (0.3-2.9 μM) for the 3-keto-C 21 -steroids than pCBR-N1 (K m =10-36 μM). The reduced products of the 3-keto-C 21 -steroids by pCBR1 and pCBR-N1 were their 3α-hydroxy-metabolites. Finally, we found that human CBR1 has similar substrate specificity for the C 18 /C 19 /C 21 -steroids to pCBR-N1. Based on these results, it was concluded that porcine and human CBRs can be involved in the metabolism of the aforementioned steroids as 3α/β,17β-HSDs., Competing Interests: Declaration of Competing Interest The authors have no conflict of interest to declare., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Some patients with polycystic ovarian syndrome (PCOS) suffered from metabolic syndrome (MetS) including dyslipidemia, hyperinsulinism, but the underlying mechanism is unclear. Although C-terminal Binding Protein 1 (CTBP1) is a transcriptional co-repressor frequently involved in hormone secretion disorders and MetS-associated diseases, the role of CTBP1 in PCOS is rarely reported. In the present study, we found that CTBP1 expression was significantly elevated in primary granulosa cells (pGCs) derived from the PCOS with MetS patients and was positively associated with serum triglyceride, but negatively correlated with serum estradiol (E2) or high-density lipoprotein. Mechanistic study suggested that CTBP1 physically bound to the promoter II of cytochrome P450 family 19 subfamily A member 1 (CYP19A1) to inhibit the aromatase gene transcription and expression, resulting in the reduced E2 synthesis. Moreover, CTBP1 interacted with the phosphorylated SREBP1a at S396 in nuclei, leading to the FBXW7-dependent protein degradation, resulting in the reduced lipid droplets formation in pGCs. Therefore, we conclude that CTBP1 in GCs dysregulates the synthesis of steroid hormones and lipids through suppression of aromatase expression and promotion of SREBP1a protein degradation in PCOS patients, which may offer some fresh insights into the potential pathological mechanism for this tough disease., (© 2024. The Author(s).)

( R )-1, 3-Butanediol (1, 3-BDO) is an important intermediate in the synthesis of aromatics, pheromones, insecticides, and beta-lactam antibiotics. The Ch KRED20 is a robust NADH-dependent ketoreductase identified from Chryseobacterium sp. CA49 . We obtained a Ch KRED20 mutant (M12) through directed evolutionary screening of Ch KRED20, the mutant with significantly improved activity to asymmetrically reduce 4-hydroxy-2-butanone (4H2B) to ( R )-1, 3-BDO. So far, both Ch KRED20 and its mutants have been expressed in intracellular in E. coli , the process of purification after intracellular expression is complicated, which leads to high cost. Here, we expressed M12 by constructing multicopy expression strains in P. pastoris , and the target protein yield was 302 mg/L in shake-flask fermentation and approximately 3.5 g/L in high-density fermentation. The recombinant M12 showed optimal enzyme activity at 30 °C and had high activity within a broad pH range of 6.0-8.0, and also showed high thermal stability. The recombinant M12 was further used for the reduction of 4H2B to ( R )-1, 3-BDO, and 98.9% yield was achieved at 4540 mM 4H2B. The crude M12 enzyme extract was found to catalyze the bioreductive production of ( R )-1, 3-BDO with excellent stereoselectivity (ee > 99%) and meet the production requirements. Our research shows that the M12 mutant can be used for the synthesis of ( R )-1, 3-BDO, and the P. pastoris expression system is an ideal platform for the large-scale, low-cost preparation of Ch KRED20 or its mutants, which may have applications in industrial settings.

Dihydroflavonol 4-reductase (DFR) significantly influences the modification of flower color. To explore the role of DFR in the synthesis of strawberry anthocyanins, in this study, we downloaded the CDS sequences of the DFR gene family from the Arabidopsis genome database TAIR; the DFR family of forest strawberry was compared; then, a functional domain screen was performed using NCBI; the selected strawberry DFR genes were analyzed; and the expression characteristics of the family members were studied by qRT-PCR. The results showed that there are 57 members of the DFR gene family in strawberry, which are mainly expressed in the cytoplasm and chloroplast; most of them are hydrophilic proteins; and the secondary structure of the protein is mainly composed of α-helices and random coils. The analysis revealed that FvDFR genes mostly contain light, hormone, abiotic stress, and meristem response elements. From the results of the qRT-PCR analysis, the relative expression of each member of the FvDFR gene was significantly different, which was expressed throughout the process of fruit coloring. Most genes had the highest expression levels in the full coloring stage (S4). The expression of FvDFR30 , FvDFR54 , and FvDFR56 during the S4 period was 8, 2.4, and 2.4 times higher than during the S1 period, indicating that the DFR gene plays a key role in regulating the fruit coloration of strawberry. In the strawberry genome, 57 members of the strawberry DFR gene family were identified. The higher the DFR gene expression, the higher the anthocyanin content, and the DFR gene may be the key gene in anthocyanin synthesis. Collectively, the DFR gene is closely related to fruit coloring, which lays a foundation for further exploring the function of the DFR gene family.

Women in developing countries still face enormous challenges when accessing reproductive health care. Access to voluntary family planning empowers women allowing them to complete their education and join the paid workforce. This effectively helps to end poverty, hunger and promotes good health for all. According to the United Nations (UN) organization, in 2022, an estimated 257 million women still lacked access to safe and effective family planning methods globally. One of the main barriers is the associated cost of modern contraceptive methods. Funded by the Bill & Melinda Gates Foundation, Almac Group worked on the development of a novel biocatalytic route to etonogestrel and levonorgestrel, two modern contraceptive APIs, with the goal of substantially decreasing the cost of production and so enabling their use in developing nations. This present work combines the selection and engineering of a carbonyl reductase (CRED) enzyme from Almac's selectAZyme™ panel, with process development, to enable efficient and economically viable bioreduction of ethyl secodione to (13 R ,17 S )-secol, the key chirality introducing intermediate en route to etonogestrel and levonorgestrel API. CRED library screening returned a good hit with an Almac CRED from Bacillus weidmannii , which allowed for highly stereoselective bioreduction at low enzyme loading of less than 1% w/w under screening assay conditions. However, the only co-solvent tolerated was DMSO up to ∼30% v/v, and it was impossible to achieve reaction completion with any enzyme loading at substrate titres of 20 g L -1 and above, due to the insolubility of the secodione. This triggered a rapid enzyme engineering program fully based on computational mutant selection. A small panel of 93 CRED mutants was rationally designed to increase the catalytic activity as well as thermal and solvent stability. The best mutant, Mutant-75, enabled a reaction at 45 °C to go to completion at 90 g L -1 substrate titre in a buffer/DMSO/heptane reaction medium fed over 6 h with substrate DMSO stock solution, with a low enzyme loading of 3.5% w/w wrt substrate. In screening assay conditions, Mutant-75 also showed a 2.2-fold activity increase. Our paper shows which computations and rational decisions enabled this outcome.

α,ω-Dicarboxylic acids, ω-aminoalkanoic acids, and α,ω-diaminoalkanes are valuable building blocks for the production of biopolyesters and biopolyamides. One of the key steps in producing these chemicals is the oxidation of ω-hydroxycarboxylic acids using alcohol dehydrogenases (e.g., ChnD of Acinetobacter sp. NCIMB 9871). However, the reaction and structural features of these enzymes remain mostly undiscovered. Thereby, we have investigated characteristics of ChnD based on enzyme kinetics, substrate-docking simulations, and mutation studies. Kinetic analysis revealed a distinct preference of ChnD for medium chain ω-hydroxycarboxylic acids, with the highest catalytic efficiency of 18.0 mM -1 s -1 for 12-hydroxydodecanoic acid among C6 to C12 ω-hydroxycarboxylic acids. The high catalytic efficiency was attributed to the positive interactions between the carboxyl group of the substrates and the guanidino group of two arginine residues (i.e., Arg62 and Arg266) in the substrate binding site. The ChnD_R62L variant showed the increased efficiency and affinity, particularly for fatty alcohols (i.e., C6-C10) and branched-chain fatty alcohols, such as 3-methyl-2-buten-1-ol. Overall, this study contributes to the deeper understanding of medium-chain primary aliphatic alcohol dehydrogenases and their applications for the production of industrially relevant chemicals such as α,ω-dicarboxylic acids, ω-aminoalkanoic acids, and α,ω-diaminoalkanes from renewable biomass., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The discovery of new targets and lead compounds is the key to developing new pesticides. The herbicidal target of drupacine has been identified as shikimate dehydrogenase (SkDH). However, the mechanism of interaction between them remains unclear. This study found that drupacine specifically binds to SkDH with a dissociation equilibrium constant (K D ) of 8.88 μM and a K d value of 2.15 μM, as confirmed by surface plasmon resonance and microscale thermophoresis. Site-directed mutagenesis coupled with fluorescence quenching analysis indicated that residue THR431 was the key amino acid site for drupacine binding to SkDH. Nine compounds with the best binding ability to SkDH were identified by virtual screening from about 120,000 compounds. Among them, compound 8 showed the highest inhibition rate with values of 41.95% against SkDH, also exhibiting the strongest herbicidal activity. This research identifies a novel potential target SkDH and a candidate lead compound with high herbicidal activity for developing new herbicides., Competing Interests: Declaration of competing interest The authors declare no competing financial interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Climate warming poses a significant threat to global crop production and food security. However, our understanding of the molecular mechanisms governing thermoresponsive development in crops remains limited. Here we report that the auxiliary subunit of N-terminal acetyltransferase A (NatA) in rice OsNAA15 is a prerequisite for rice thermoresponsive growth. OsNAA15 produces two isoforms OsNAA15.1 and OsNAA15.2, via temperature-dependent alternative splicing. Among the two, OsNAA15.1 is more likely to form a stable and functional NatA complex with the potential catalytic subunit OsNAA10, leading to a thermoresponsive N-terminal acetylome. Intriguingly, while OsNAA15.1 promotes plant growth under elevated temperatures, OsNAA15.2 exhibits an inhibitory effect. We identified two glycolate oxidases (GLO1/5) as major substrates from the thermoresponsive acetylome. These enzymes are involved in hydrogen peroxide (H 2 O 2 ) biosynthesis via glycolate oxidation. N-terminally acetylated GLO1/5 undergo their degradation through the ubiquitin-proteasome system. This leads to reduced reactive oxygen species (ROS) production, thereby promoting plant growth, particularly under high ambient temperatures. Conclusively, our findings highlight the pivotal role of N-terminal acetylation in orchestrating the glycolate-mediated ROS homeostasis to facilitate thermoresponsive growth in rice., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

In this study, based on the self-assembly strategy, we fused CipA with carbonyl reductase LXCAR S154Y derived from Leifsonia xyli by gene coding, and successfully performed the carrier-free immobilization of LXCAR S154Y . The immobilized enzyme was then characterized using scanning electron microscope (SEM), dynamic light scattering (DLS) and fourier transform infrared spectroscopy (FTIR). Compared with the free enzyme, the immobilized LXCAR S154Y exhibited a 2.3-fold improvement in the catalytic efficiency k cat /k m for the synthesis of a chiral pharmaceutical intermediate (R)-3,5-bis(trifluoromethyl)phenyl ethanol ((R)-BTPE) by reducing 3,5-bis(trifluoromethyl)acetophenone (BTAP). Moreover, the immobilized enzyme showed the enhanced stability while maintaining over 61 % relative activity after 18 cycles of batch reaction. Further, when CipA-fused carbonyl reductase was employed for (R)-BTPE production in a continuous flow reaction, almost complete yield (97.0 %) was achieved within 7 h at 2 M (512.3 g/L) of BTAP concentration, with a space-time yield of 1717.1 g·L -1 ·d -1 . Notably, we observed the retention of cofactor NADH by CipA-based enzyme aggregates, resulting in a higher total turnover number (TTN) of 4815 to facilitate this bioreductive process. This research developed a concise strategy for efficient preparation of chiral intermediate with cofactor self-sufficiency via continuous flow biocatalysis, and the relevant mechanism was also explored., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Malonyl-CoA reductase utilizes two equivalents of NADPH to catalyze the reduction of malonyl-CoA to 3-hydroxypropionic acid (3HP). This reaction is part of the carbon fixation pathway in the phototrophic bacterium Chloroflexus aurantiacus. The enzyme is composed of two domains. The C-terminal domain catalyzes the reduction of malonyl-CoA to malonic semialdehyde, while the N-terminal domain catalyzes the reduction of the aldehyde to 3HP. The two domains can be produced independently and retain their enzymatic activity. This report focuses on the kinetic characterization of the C-terminal domain. Initial velocity patterns and inhibition studies showed the kinetic mechanism is ordered with NADPH binding first followed by malonyl-CoA. Malonic semialdehyde is released first, while CoA and NADP + are released randomly. Analogs of malonyl-CoA showed that the thioester carbon is reduced, while the carboxyl group is needed for proper positioning. The enzyme transfers the pro-S hydrogen of NADPH to malonyl-CoA and pH rate profiles revealed that a residue with a pK a value of about 8.8 must be protonated for activity. Kinetic isotope effects indicated that NADPH is not sticky (that is, NADPH dissociates from the enzyme faster than the rate of product formation) and product release is partially rate-limiting. Moreover, the mechanism is stepwise with the pH dependent step occurring before or after hydride transfer. The findings from this study will aid in the development of an eco-friendly biosynthesis of 3HP which is an industrial chemical used in the production of plastics and adhesives., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Decaprenylphosphoryl-β-D-ribose-oxidase (DprE1), a subunit of the essential decaprenylphosphoribose-2'-epimerase, plays a crucial role in the synthesis of cell wall arabinan components in mycobacteria, including the pathogen responsible for tuberculosis, Mycobacterium tuberculosis . In this study, we designed, synthesised, and evaluated 15 (BOK-1-BOK-10 and BOP-1-BOP-5) potential inhibitors of DprE1 from a series of 1,2,3-triazole ligands using a validated DprE1 inhibition assay. Two compounds, BOK-2 and BOK-3, demonstrated significant inhibition with IC 50 values of 2.2 ± 0.1 and 3.0 ± 0.6 μM, respectively, whereas the standard drug (TCA-1) showed inhibition at 3.0 ± 0.2 μM. Through molecular modelling and dynamic simulations, we explored the structural relationships between selected 1,2,3-triazole compounds and DprE1, revealing key features for effective drug-target interactions. This study introduces a novel approach for designing ligands against DprE1, offering a potential therapeutic strategy for tuberculosis treatment.

Introduction: Acute lymphoblastic leukaemia (ALL) is the most common type of childhood leukaemia with effective chemotherapeutic treatment. However, obesity has been associated with higher ALL chemoresistance rates and lower event-free survival rates. The molecular mechanism of how obesity promotes chemotherapy resistance is not well delineated., Objectives: This study evaluated the effect of adipocyte maturation on sequestration and metabolism of chemotherapeutic drug daunorubicin (DNR)., Methods: Using targeted LC-MS/MS multi-analyte assay, DNR sequestration and metabolism were studied in human preadipocyte and adipocyte cell lines, where expressions of DNR-metabolizing enzymes aldo-keto reductases (AKR) and carbonyl reductases (CBR) were also evaluated. In addition, to identify the most DNR-metabolizing AKR/CBR isoforms, recombinant human AKR and CBR enzymes were subject to DNR metabolism. The results were further validated by AKR-, CBR-specific inhibitors., Results: This report shows that adipocyte maturation upregulates expressions of AKR and CBR enzymes (by 4- to 60- folds, p < .05), which is positively associated with enhanced sequestration and metabolism of DNR in adipocytes compared to preadipocytes (by ~30%, p < .05). In particular, adipocyte maturation upregulates AKR1C3 and CBR1, which are the predominate metabolic enzyme isoforms responsible for DNR biotransformation to its metabolites., Conclusion: Fat is an expandable tissue that can sequester and detoxify DNR when stimulated by obesity, likely through the upregulation of DNR-metabolizing enzymes AKR1C3 and CBR1. Our data partially explains why obese ALL patients may be more likely to become chemoresistant towards DNR, and provides evidence for potential clinical investigation targeting obesity to reduce DNR chemoresistance., (© 2024 The Author(s). European Journal of Clinical Investigation published by John Wiley & Sons Ltd on behalf of Stichting European Society for Clinical Investigation Journal Foundation.)

Butanol dehydrogenase (BDH) plays a crucial role in butanol biosynthesis by catalyzing the conversion of butanal to butanol using the coenzyme NAD(P)H. In this study, we observed that BDH from Thermotoga maritima (TmBDH) exhibits dual coenzyme specificity and catalytic activity with NADPH as the coenzyme under highly alkaline conditions. Additionally, a thermal stability analysis on TmBDH demonstrated its excellent activity retention even at elevated temperatures of 80°C. These findings demonstrate the superior thermal stability of TmBDH and suggest that it is a promising candidate for large-scale industrial butanol production. Furthermore, we discovered that TmBDH effectively catalyzes the conversion of aldehydes to alcohols and exhibits a wide range of substrate specificities toward aldehydes, while excluding alcohols. The dimeric state of TmBDH was observed using rapid online buffer exchange native mass spectrometry. Additionally, we analyzed the coenzyme-binding sites and inferred the possible locations of the substrate-binding sites. These results provide insights that improve our understanding of BDHs., (© 2024 Wiley Periodicals LLC.)

Although the genome of Mycobacterium tuberculosis (Mtb) H37Rv, the causative agent of tuberculosis, has been repeatedly annotated and updated, a range of proteins from this human pathogen have unknown functions. Mtb Rv0439c, a member of the short-chain dehydrogenase/reductases superfamily, has yet to be cloned and characterized, and its function remains unclear. In this work, we present for the first time the optimized expression and purification of this enzyme, as well as bioinformatic analysis to unveil its potential coenzyme and substrate. Optimized expression in Escherichia coli yielded soluble Rv0439c, while certain tag fusions resulted in insolubility. Sequence and docking analyses strongly suggested that Rv0439c has a clear preference for NADP + , with Arg53 being a key residue that confers coenzyme specificity. Furthermore, functional prediction using CLEAN and DEEPre servers suggested that this protein is a potential NADP + -retinol dehydrogenase (EC No. 1.1.1.300) in retinol metabolism, and this was supported by a BLASTp search and docking studies. Collectively, our findings provide a solid basis for future functional characterization and structural studies of Rv0439c, which will contribute to enhanced understanding of Mtb biology., Competing Interests: Declarations Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: CINNAMYL ALCOHOL DEHYDROGENASE (CAD) catalyzes the NADPH-dependent reduction of cinnamaldehydes into cinnamyl alcohols and is a key enzyme found at the final step of the monolignol pathway. Cinnamyl alcohols and their conjugates are subsequently polymerized in the secondary cell wall to form lignin. CAD genes are typically encoded by multi-gene families and thus traditionally organized into general classifications of functional relevance., Results: In silico analysis of the hexaploid Triticum aestivum genome revealed 47 high confidence TaCAD copies, of which three were determined to be the most significant isoforms (class I) considered bone fide CADs. Class I CADs were expressed throughout development both in RNAseq data sets as well as via qRT-PCR analysis. Of the 37 class II TaCADs identified, two groups were observed to be significantly co-expressed with class I TaCADs in developing tissue and under chitin elicitation in RNAseq data sets. These co-expressed class II TaCADs were also found to be phylogenetically unrelated to a separate clade of class II TaCADs previously reported to be an influential resistance factor to pathogenic fungal infection. Lastly, two groups were phylogenetically identified as class III TaCADs, which possess distinct conserved gene structures. However, the lack of data supporting their catalytic activity for cinnamaldehydes and their bereft transcriptional presence in lignifying tissues challenges their designation and function as CADs., Conclusions: Taken together, our comprehensive transcriptomic analyses suggest that TaCAD genes contribute to overlapping but nonredundant functions during T. aestivum growth and development across a wide variety of agroecosystems and provide tolerance to various stressors., (© 2024. The Author(s).)

Background: Flavonoids, an important class of specialized metabolites, are synthesized from phenylalanine and present in almost all plant species. Different branches of flavonoid biosynthesis lead to products like flavones, flavonols, anthocyanins, and proanthocyanidins. Dihydroflavonols form the branching point towards the production of non-colored flavonols via flavonol synthase (FLS) and colored anthocyanins via dihydroflavonol 4-reductase (DFR). Despite the wealth of publicly accessible data, there remains a gap in understanding the mechanisms that mitigate competition between FLS and DFR for the shared substrate, dihydroflavonols., Results: An angiosperm-wide comparison of FLS and DFR sequences revealed the amino acids at positions associated with the substrate specificity in both enzymes. A global analysis of the phylogenetic distribution of these amino acid residues revealed that monocots generally possess FLS with Y132 (FLSY) and DFR with N133 (DFRN). In contrast, dicots generally possess FLSH and DFRN, DFRD, and DFRA. DFRA, which restricts substrate preference to dihydrokaempferol, previously believed to be unique to strawberry species, is found to be more widespread in angiosperms and has evolved independently multiple times. Generally, angiosperm FLS appears to prefer dihydrokaempferol, whereas DFR appears to favor dihydroquercetin or dihydromyricetin. Moreover, in the FLS-DFR competition, the dominance of one over the other is observed, with typically only one gene being expressed at any given time., Conclusion: This study illustrates how almost mutually exclusive gene expression and substrate-preference determining residues could mitigate competition between FLS and DFR, delineates the evolution of these enzymes, and provides insights into mechanisms directing the metabolic flux of the flavonoid biosynthesis, with potential implications for ornamental plants and molecular breeding strategies., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Choudhary, Pucker. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Gluconobacter oxydans succinic semialdehyde reductase (GoxSSAR) and Acetobacter aceti glyoxylate reductase (AacGR) represent a novel class in the β-hydroxyacid dehydrogenases superfamily. Kinetic analyses revealed GoxSSAR's activity with both glyoxylate and succinic semialdehyde, while AacGR is glyoxylate specific. GoxSSAR K167A lost activity with succinic semialdehyde but retained some with glyoxylate, whereas AacGR K175A lost activity. These findings elucidate differences between these homologous enzymes., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

2,3-butanediol (2,3-BD) is a versatile bio-based platform chemical. An artificial four-enzyme synthetic biosystem composed of ethanol dehydrogenase, NADH oxidase, formolase and 2,3-butanediol dehydrogenase was designed for upgrading ethanol to 2,3-BD in our previous study. However, a key challenge in developing in vitro enzymatic systems for 2,3-BD synthesis is the relatively sluggish catalytic efficiency of formolase, which catalyzes the rate-limiting step in such systems. Herein, this study reports how engineering the tunnel and substrate binding pocket of FLS improved its catalytic performance. A series of single-point and combinatorial variants were successfully obtained which displayed both higher catalytic efficiency and better substrate tolerance than wild-type FLS. Subsequently, a cell-free biosystem based on the FLS:I28V/L482E enzyme was implemented for upgrading ethanol to 2,3-BD. Ultimately, this system achieved efficient production of 2,3-BD from ethanol by the fed-batch method, reaching a concentration of 1.39 M (124.83 g/L) of the product and providing both excellent productivity and yield values of 5.94 g/L/h and 92.7%, respectively. Taken together, this modified enzymatic catalysis system provides a highly promising alternative approach for sustainable and cost-competitive production of 2,3-BD.

The hydroxyacid glycolate is a highly abundant carbon source in the environment. Glycolate is produced by unicellular photosynthetic organisms and excreted at petagram scales to the environment, where it serves as growth substrate for heterotrophic bacteria. In microbial metabolism, glycolate is first oxidized to glyoxylate by the enzyme glycolate oxidase. The recently described β-hydroxyaspartate cycle (BHAC) subsequently mediates the carbon-neutral assimilation of glyoxylate into central metabolism in ubiquitous Alpha- and Gammaproteobacteria. Although the reaction sequence of the BHAC was elucidated in Paracoccus denitrificans , little is known about the regulation of glycolate and glyoxylate assimilation in this relevant alphaproteobacterial model organism. Here, we show that regulation of glycolate metabolism in P. denitrificans is surprisingly complex, involving two regulators, the IclR-type transcription factor BhcR that acts as an activator for the BHAC gene cluster, and the GntR-type transcriptional regulator GlcR, a previously unidentified repressor that controls the production of glycolate oxidase. Furthermore, an additional layer of regulation is exerted at the global level, which involves the transcriptional regulator CceR that controls the switch between glycolysis and gluconeogenesis in P. denitrificans . Together, these regulators control glycolate metabolism in P. denitrificans , allowing the organism to assimilate glycolate together with other carbon substrates in a simultaneous fashion, rather than sequentially. Our results show that the metabolic network of Alphaproteobacteria shows a high degree of flexibility to react to the availability of multiple substrates in the environment.IMPORTANCEAlgae perform ca. 50% of the photosynthetic carbon dioxide fixation on our planet. In the process, they release the two-carbon molecule glycolate. Due to the abundance of algae, massive amounts of glycolate are released. Therefore, this molecule is available as a source of carbon for bacteria in the environment. Here, we describe the regulation of glycolate metabolism in the model organism Paracoccus denitrificans . This bacterium uses the recently characterized β-hydroxyaspartate cycle to assimilate glycolate in a carbon- and energy-efficient manner. We found that glycolate assimilation is dynamically controlled by three different transcriptional regulators: GlcR, BhcR, and CceR. This allows P. denitrificans to assimilate glycolate together with other carbon substrates in a simultaneous fashion. Overall, this flexible and multi-layered regulation of glycolate metabolism in P. denitrificans represents a resource-efficient strategy to make optimal use of this globally abundant molecule under fluctuating environmental conditions., Competing Interests: The authors declare no conflict of interest.

Capture of a photon by an opsin visual pigment isomerizes its 11-cis-retinaldehyde (11cRAL) chromophore to all-trans-retinaldehyde (atRAL), which subsequently dissociates. To restore light sensitivity, the unliganded apo-opsin combines with another 11cRAL to make a new visual pigment. Two enzyme pathways supply chromophore to photoreceptors. The canonical visual cycle in retinal pigment epithelial cells supplies 11cRAL at low rates. The photic visual cycle in Müller cells supplies cones with 11-cis-retinol (11cROL) chromophore precursor at high rates. Although rods can only use 11cRAL to regenerate rhodopsin, cones can use 11cRAL or 11cROL to regenerate cone visual pigments. We performed a screen in zebrafish retinas and identified ZCRDH as a candidate for the enzyme that converts 11cROL to 11cRAL in cone inner segments. Retinoid analysis of eyes from Zcrdh-mutant zebrafish showed reduced 11cRAL and increased 11cROL levels, suggesting impaired conversion of 11cROL to 11cRAL. By microspectrophotometry, isolated Zcrdh-mutant cones lost the capacity to regenerate visual pigments from 11cROL. ZCRDH therefore possesses all predicted properties of the cone 11cROL dehydrogenase. The human protein most similar to ZCRDH is RDH12. By immunocytochemistry, ZCRDH was abundantly present in cone inner segments, similar to the reported distribution of RDH12. Finally, RDH12 was the only mammalian candidate protein to exhibit 11cROL-oxidase catalytic activity. These observations suggest that RDH12 in mammals is the functional ortholog of ZCRDH, which allows cones, but not rods, to regenerate visual pigments from 11cROL provided by Müller cells. This capacity permits cones to escape competition from rods for visual chromophore in daylight-exposed retinas., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Engineering biocatalysts with enhanced stereoselectivity is highly desirable, and active-site loop dynamics play an important role in its regulation. However, knowledge of their precise roles in catalysis and evolution is limited. Here, we used the strategy of Rosetta enzyme design combined molecular dynamic simulations (MDs) to reprogram the landscapes of the key active-site loop dynamics of the carbonyl reductase LfSDR1 to improve stereoselectivity. The key flexible loop in the active site showed the potential to regulate the catalytic properties. A library of virtual variants was produced using the Rosetta design and assessed dynamic effect of the loop with the aid of MDs. A potential candidate was obtained with significant stereoselectivity (ee > 99 %) compared to the wild-type (ee = 42 %) without loss of catalytic activity or thermostability. The molecular basis of the catalytic property enhancement was flanked by MDs, which revealed the role of the G92L mutation in regulating loop dynamics to stabilize the environment of the active site. Finally, a series of the challenge bulky substrate derivatives were assessed using the G92L variant, and all showed improved stereoselectivity ee > 99 %. This study provides novel insights for improving stereoselectivity through rational engineering of the loop dynamics of biocatalysts., Competing Interests: Declaration of competing interest There are no conflicts to declare., (Copyright © 2024. Published by Elsevier B.V.)

l-threonine dehydrogenase (Tdh) is an enzyme that links threonine metabolism to epigenetic modifications and mitochondria biogenesis. In vitro studies show that it is critical for the regulation of trimethylation of histone H3 lysine 4 (H3K4me3) levels and cell fate determination of mouse embryonic stem cells (mESCs). However, whether Tdh regulates a developmental process in vivo and, if it does, whether it also primarily regulates H3K4me3 levels in this process as it does in mESCs, remains elusive. Here, we revealed that, in zebrafish hematopoiesis, tdh is preferentially expressed in neutrophils. Knockout of tdh causes a decrease in neutrophil number and slightly suppresses their acute injury-induced migration, but, unlike the mESCs, the level of H3K4me3 is not evidently reduced in neutrophils sorted from the kidney marrow of adult tdh-null zebrafish. These phenotypes are dependent on the enzymatic activity of Tdh. Importantly, a soluble supplement of nutrients that are able to fuel the acetyl-CoA pool, such as pyruvate, glucose and branched-chain amino acids, is sufficient to rescue the reduction in neutrophils caused by tdh deletion. In summary, our study presents evidence for the functional requirement of Tdh-mediated threonine metabolism in a developmental process in vivo. It also provides an animal model for investigating the nutritional regulation of myelopoiesis and immune response, as well as a useful tool for high-throughput drug/nutrition screening., (© 2024 Federation of European Biochemical Societies.)

Translational Relevance: A multi-stakeholder, patient centric approach will be critical to the design of future successful clinical trials with outcome measures relevant to the RDH12-IRD population.

Reactive carbonyl and oxygen species (RCS/ROS), often generated as metabolic byproducts, particularly under conditions of pathology, can cause direct damage to proteins, lipids, and nucleic acids. Glyoxal oxidases (Gloxs) oxidize aldehydes to carboxylic acids, generating hydrogen peroxide (H2O2). Although best characterized for their roles in lignin degradation, Glox in plant fungal pathogens are known to contribute to virulence, however, the mechanism underlying such effects are unclear. Here, we show that Glox in the insect pathogenic fungus, Metarhizium acridum, is highly expressed in mycelia and during formation of infection structures (appressoria), with the enzyme localizing to the cell membrane. MaGlox targeted gene disruption mutants showed RCS and ROS accumulation, resulting in cell toxicity, induction of apoptosis and increased autophagy, inhibiting normal fungal growth and development. The ability of the MaGlox mutant to scavenge RCS was significantly reduced, and the mutant exhibited increased susceptibility to aldehydes, oxidative and cell wall perturbing agents but not toward osmotic stress, with altered cell wall contents. The ΔMaGlox mutant was impaired in its ability to penetrate the host cuticle and evade host immune defense resulting in attenuated pathogenicity. Overexpression of MaGlox promoted fungal growth and conidial germination, increased tolerance to H2O2, but had little to other phenotypic effects. Transcriptomic analyses revealed downregulation of genes related to cell wall synthesis, conidiation, stress tolerance, and host cuticle penetration in the ΔMaGlox mutant. These findings demonstrate that MaGlox-mediated scavenging of RCS is required for virulence, and contributes to normal fungal growth and development, stress resistance., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Liu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Bacterial and fungal copper radical oxidases (CROs) from Auxiliary Activity Family 5 (AA5) are implicated in morphogenesis and pathogenesis. The unique catalytic properties of CROs also make these enzymes attractive biocatalysts for the transformation of small molecules and biopolymers. Despite a recent increase in the number of characterized AA5 members, especially from subfamily 2 (AA5&#95;2), the catalytic diversity of the family as a whole remains underexplored. In the present study, phylogenetic analysis guided the selection of six AA5&#95;2 members from diverse fungi for recombinant expression in Komagataella pfaffii (syn. Pichia pastoris ) and biochemical characterization in vitro . Five of the targets displayed predominant galactose 6-oxidase activity (EC 1.1.3.9), and one was a broad-specificity aryl alcohol oxidase (EC 1.1.3.7) with maximum activity on the platform chemical 5-hydroxymethyl furfural (EC 1.1.3.47). Sequence alignment comparing previously characterized AA5&#95;2 members to those from this study indicated various amino acid substitutions at active site positions implicated in the modulation of specificity.IMPORTANCEEnzyme discovery and characterization underpin advances in microbial biology and the application of biocatalysts in industrial processes. On one hand, oxidative processes are central to fungal saprotrophy and pathogenesis. On the other hand, controlled oxidation of small molecules and (bio)polymers valorizes these compounds and introduces versatile functional groups for further modification. The biochemical characterization of six new copper radical oxidases further illuminates the catalytic diversity of these enzymes, which will inform future biological studies and biotechnological applications., Competing Interests: The authors declare no conflict of interest.

Nitrogen heterocycles are commonly found in bioactive natural products and drugs. However, the biocatalytic tools for nitrogen heterocycle synthesis are limited. Herein, we report the discovery of vanillyl alcohol oxidases (VAOs) as efficient biocatalysts for the one-pot synthesis of 2-aryl thiazolines from various 4-hydroxybenzaldehydes and aminothiols. The wild-type biocatalyst features a broad scope of 4-hydroxybenzaldehydes. Though the scope of aminothiols is limited, it could be improved via semi-rational protein engineering, generating a variant to produce previously inaccessible cysteine-derived bioactive 2-aryl thiazolines using the wild-type VAO. Benefiting from the derivatizable functional groups in the enzymatic products, we further chemically modified these products to expand the chemical space, offering a new chemoenzymatic strategy for the green and efficient synthesis of structurally diverse 2-aryl-thiazoline derivatives to prompt their use in drug discovery and catalysis., (© 2024 Wiley-VCH GmbH.)

Background: Komagataella phaffii, a type of methanotrophic yeast, can use methanol, a favorable non-sugar substrate in eco-friendly bio-manufacturing. The dissimilation pathway in K. phaffii leads to the loss of carbon atoms in the form of CO 2 . However, the ΔFLD strain, engineered to lack formaldehyde dehydrogenase-an essential enzyme in the dissimilation pathway-displayed growth defects when exposed to a methanol-containing medium., Results: Inhibiting the dissimilation pathway triggers an excessive accumulation of formaldehyde and a decline in the intracellular NAD + /NADH ratio. Here, we designed dual-enzyme complex with the alcohol oxidase1/dihydroxyacetone synthase1 (Aox1/Das1), enhancing the regeneration of the formaldehyde receptor xylulose-5-phosphate (Xu5P). This strategy mitigated the harmful effects of formaldehyde accumulation and associated toxicity to cells. Concurrently, we elevated the NAD + /NADH ratio by overexpressing isocitrate dehydrogenase in the TCA cycle, promoting intracellular redox homeostasis. The OD 600 of the optimized combination of the above strategies, strain DF02-1, was 4.28 times higher than that of the control strain DF00 (ΔFLD, HIS4 + ) under 1% methanol. Subsequently, the heterologous expression of methanol oxidase Mox from Hansenula polymorpha in strain DF02-1 resulted in the recombinant strain DF02-4, which displayed a growth at an OD 600 4.08 times higher than that the control strain DF00 in medium containing 3% methanol., Conclusions: The reduction of formaldehyde accumulation, the increase of NAD + /NADH ratio, and the enhancement of methanol oxidation effectively improved the efficient utilization of a high methanol concentration by strain ΔFLD strain lacking formaldehyde dehydrogenase. The modification strategies implemented in this study collectively serve as a foundational framework for advancing the efficient utilization of methanol in K. phaffii., (© 2024. The Author(s).)

Human-borne acetone is a potent marker of lipid metabolism. Here, an enzyme immobilization method for secondary alcohol dehydrogenase (S-ADH), which is suitable for highly sensitive and selective biosensing of acetone, was developed, and then its applicability was demonstrated for spatiotemporal imaging of concentration distribution. After various investigations, S-ADH-immobilized meshes could be prepared with less than 5% variation by cross-linking S-ADH with glutaraldehyde on a cotton mesh at 40 °C for 15 min. Furthermore, high activity was obtained by adjusting the concentration of the coenzyme nicotinamide adenine dinucleotide (NADH) solution added to the S-ADH-immobilized mesh to 500 μM and the solvent to a potassium phosphate buffer solution at pH 6.5. The gas imaging system using the S-ADH-immobilized mesh was able to image the decrease in NADH fluorescence (ex 340 nm, fl 490 nm) caused by the catalytic reaction of S-ADH and the acetone distribution in the concentration range of 0.1-10 ppm-v, including the breath concentration of healthy people at rest. The exhaled breath of two healthy subjects at 6 h of fasting was quantified as 377 and 673 ppb-v, which were consistent with the values quantified by gas chromatography-mass spectrometry.

Theoretical concepts linking the structure, function, and evolution of a protein, while often intuitive, necessitate validation through investigations in real-world systems. Our study empirically explores the evolutionary implications of multiple gene copies in an organism by shedding light on the structure-function modulations observed in Pseudomonas aeruginosa 's second copy of ketopantoate reductase (PaKPR2). We demonstrated with two apo structures that the typical active site cleft of the protein transforms into a two-sided pocket where a molecular gate made up of two residues controls the substrate entry site, resulting in its inactivity toward the natural substrate ketopantoate. Strikingly, this structural modification made the protein active against several important α-keto-acid substrates with varied efficiency. Structural constraints at the binding site for this altered functional trait were analyzed with two binary complexes that show the conserved residue microenvironment faces restricted movements due to domain closure. Finally, its mechanistic highlights gathered from a ternary complex structure help in delineating the molecular perspectives behind its kinetic cooperativity toward these broad range of substrates. Detailed structural characteristics of the protein presented here also identified four key amino acid residues responsible for its versatile α-keto-acid reductase activity, which can be further modified to improve its functional properties through protein engineering.

We characterise a reversible bacterial zinc-containing benzyl alcohol dehydrogenase (BaDH) accepting either NAD + or NADP + as a redox cofactor. Remarkably, its redox cofactor specificity is pH-dependent with the phosphorylated cofactors favored at lower and the dephospho-forms at higher pH. BaDH also shows different steady-state kinetic behavior with the two cofactor forms. From a structural model, the pH-dependent shift may affect the charge of a histidine in the 2'-phosphate-binding pocket of the redox cofactor binding site. The enzyme is phylogenetically affiliated to a new subbranch of the Zn-containing alcohol dehydrogenases, which share this conserved residue. BaDH appears to have some specificity for its substrate, but also turns over many substituted benzyl alcohol and benzaldehyde variants, as well as compounds containing a conjugated C=C double bond with the aldehyde carbonyl group. However, compounds with an sp 3 -hybridised C next to the alcohol/aldehyde group are not or only weakly turned over. The enzyme appears to contain a Zn in its catalytic site and a mixture of Zn and Fe in its structural metal-binding site. Moreover, we demonstrate the use of BaDH in an enzyme cascade reaction with an acid-reducing tungsten enzyme to reduce benzoate to benzyl alcohol. KEY POINTS: •Zn-containing BaDH has activity with either NAD + or NADP + at different pH optima. •BaDH converts a broad range of substrates. •BaDH is used in a cascade reaction for the reduction of benzoate to benzyl alcohol., (© 2024. The Author(s).)

Synaptic ribbons are the eponymous specializations of continuously active ribbon synapses. They are primarily composed of the RIBEYE protein that consists of a unique amino-terminal A-domain and carboxy-terminal B-domain that is largely identical to the ubiquitously expressed transcriptional regulator protein CtBP2. Both RIBEYE A-domain and RIBEYE B-domain are essential for the assembly of the synaptic ribbon, as shown by previous analyses of RIBEYE knockout and knockin mice and related investigations. How exactly the synaptic ribbon is assembled from RIBEYE subunits is not yet clear. To achieve further insights into the architecture of the synaptic ribbon, we performed analytical post-embedding immunogold-electron microscopy with direct gold-labelled primary antibodies against RIBEYE A-domain and RIBEYE B-domain for improved ultrastructural resolution. With direct gold-labelled monoclonal antibodies against RIBEYE A-domain and RIBEYE B-domain, we found that both domains show a very similar localization within the synaptic ribbon of mouse photoreceptor synapses, with no obvious differential gradient between the centre and surface of the synaptic ribbon. These data favour a model of the architecture of the synaptic ribbon in which the RIBEYE A-domain and RIBEYE B-domain are located similar distances from the midline of the synaptic ribbon.

Derived from the denitrifying bacterium Aromatoleum aromaticum EbN1 ( Azoarcus sp.), the enzyme S -1-(4-hydroxyphenyl)-ethanol dehydrogenase (S-HPED) belongs to the short-chain dehydrogenase/reductase family. Using research techniques like UV-Vis spectroscopy, dynamic light scattering, thermal-shift assay and HPLC, we investigated the catalytic and structural stability of S-HPED over a wide temperature range and within the pH range of 5.5 to 9.0 under storage and reaction conditions. The relationship between aggregation and inactivation of the enzyme in various pH environments was also examined and interpreted. At pH 9.0, where the enzyme exhibited no aggregation, we characterized thermally induced enzyme inactivation. Through isothermal and multitemperature analysis of inactivation data, we identified and confirmed the first-order inactivation mechanism under these pH conditions and determined the kinetic parameters of the inactivation process. Additionally, we report the positive impact of glucose as an enzyme stabilizer, which slows down the dynamics of S-HPED inactivation over a wide range of pH and temperature and limits enzyme aggregation. Besides characterizing the stability of S-HPED, the enzyme's catalytic activity and high stereospecificity for 10 prochiral carbonyl compounds were positively verified, thus expanding the spectrum of substrates reduced by S-HPED. Our research contributes to advancing knowledge about the biocatalytic potential of this catalyst.

Recently, mounting evidence has highlighted the essential function of the C-terminal binding protein-1 divergent transcript (CTBP1-DT) in malignancies. However, its role in kidney renal clear cell carcinoma (KIRC) remains largely unknown. Our study aimed to identify the potential function of CTBP1-DT in KIRC. RT-qPCR, Kaplan-Meier survival analysis, Cox regression analysis, and nomogram analysis were utilized to determine the expression and effects of CTBP1-DT on survival. The subcellular localization of CTBP1-DT was determined using RNA fluorescence in situ hybridization (FISH). To investigate the functions of CTBP1-DT in regulating KIRC cell proliferation, migration, invasion, lipid synthesis, and apoptosis, we conducted CCK8, EdU, Transwell, and Oil Red O staining and cell apoptosis staining assays. The relationships between CTBP1-DT and the tumor microenvironment were investigated with multiple bioinformatics analysis algorithms and databases, including CYBERSORT, TIMER2, Spearman correlation test, tumor mutation burden (TMB), microsatellite instability (MSI), and immunophenoscore (IPS). According to our results, CTBP1-DT is a lncRNA located in the nucleus that is significantly upregulated in KIRC and is correlated with better clinical outcomes. Downregulating CTBP1-DT inhibited cell viability, migration, invasion, and lipid synthesis but triggered cell apoptosis. Additionally, we explored the potential effect of CTBP1-DT in regulating immune cell infiltration in KIRC and other malignancies. Furthermore, CTBP1-DT could be used to predict the effectiveness of targeted drugs and immune checkpoint inhibitors. In conclusion, we identified CTBP1-DT as a potential immunological biomarker and discovered the potential role of CTBP1-DT in regulating lipid synthesis and apoptosis resistance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Formaldehyde is a key metabolite in natural and synthetic one-carbon metabolism. To facilitate the engineering of formaldehyde-producing enzymes, the development of sensitive, user-friendly, and cost-effective detection methods is required. In this study, we engineered Escherichia coli to serve as a cellular biosensor capable of detecting a broad range of formaldehyde concentrations. Using both natural and promiscuous formaldehyde assimilation enzymes, we designed three distinct E. coli growth biosensor strains that depend on formaldehyde for cell growth. These strains were engineered to be auxotrophic for one or several essential metabolites that could be produced through formaldehyde assimilation. The respective assimilating enzyme was expressed from the genome to compensate the auxotrophy in the presence of formaldehyde. We first predicted the formaldehyde dependency of the biosensors by flux balance analysis and then analysed it experimentally. Subsequent to strain engineering, we enhanced the formaldehyde sensitivity of two biosensors either through adaptive laboratory evolution or modifications at metabolic branch points. The final set of biosensors demonstrated the ability to detect formaldehyde concentrations ranging approximately from 30 μM to 13 mM. We demonstrated the application of the biosensors by assaying the in vivo activity of different methanol dehydrogenases in the most sensitive strain. The fully genomic nature of the biosensors allows them to be deployed as "plug-and-play" devices for high-throughput screenings of extensive enzyme libraries. The formaldehyde growth biosensors developed in this study hold significant promise for advancing the field of enzyme engineering, thereby supporting the establishment of a sustainable one-carbon bioeconomy., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

The high infiltration of tumor-associated macrophages (TAMs) in the immunosuppressive tumor microenvironment prominently attenuates the efficacy of immune checkpoint blockade (ICB) therapies, yet the underlying mechanisms are not fully understood. Here, we investigate the metabolic profile of TAMs and identify S-2-hydroxyglutarate (S-2HG) as a potential immunometabolite that shapes macrophages into an antitumoral phenotype. Blockage of L-2-hydroxyglutarate dehydrogenase (L2HGDH)-mediated S-2HG catabolism in macrophages promotes tumor regression. Mechanistically, based on its structural similarity to α-ketoglutarate (α-KG), S-2HG has the potential to block the enzymatic activity of 2-oxoglutarate-dependent dioxygenases (2-OGDDs), consequently reshaping chromatin accessibility. Moreover, S-2HG-treated macrophages enhance CD8 + T cell-mediated antitumor activity and sensitivity to anti-PD-1 therapy. Overall, our study uncovers the role of blockage of L2HGDH-mediated S-2HG catabolism in orchestrating macrophage antitumoral polarization and, further, provides the potential of repolarizing macrophages by S-2HG to overcome resistance to anti-PD-1 therapy., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Key Message: DzMYB2 functions as an MYB activator, while DzMYB3 acts as an MYB repressor. They bind to promoters, interact with DzbHLH1, and influence phenolic contents, revealing their roles in phenylpropanoid regulation in durian pulps. Durian fruit has a high nutritional value attributed to its enriched bioactive compounds, including phenolics, carotenoids, and vitamins. While various transcription factors (TFs) regulate phenylpropanoid biosynthesis, MYB (v-myb avian myeloblastosis viral oncogene homolog) TFs have emerged as pivotal players in regulating key genes within this pathway. This study aimed to identify additional candidate MYB TFs from the transcriptome database of the Monthong cultivar at five developmental/postharvest ripening stages. Candidate transcriptional activators were discerned among MYBs upregulated during the ripe stage based on the positive correlation observed between flavonoid biosynthetic genes and flavonoid contents in ripe durian pulps. Conversely, MYBs downregulated during the ripe stage were considered candidate repressors. This study focused on a candidate MYB activator (DzMYB2) and a candidate MYB repressor (DzMYB3) for functional characterization. LC-MS/MS analysis using Nicotiana benthamiana leaves transiently expressing DzMYB2 revealed increased phenolic compound contents compared with those in leaves expressing green fluorescence protein controls, while those transiently expressing DzMYB3 showed decreased phenolic compound contents. Furthermore, it was demonstrated that DzMYB2 controls phenylpropanoid biosynthesis in durian by regulating the promoters of various biosynthetic genes, including phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), and dihydroflavonol reductase (DFR). Meanwhile, DzMYB3 regulates the promoters of PAL, 4-coumaroyl-CoA ligase (4CL), CHS, and CHI, resulting in the activation and repression of gene expression. Moreover, it was discovered that DzMYB2 and DzMYB3 could bind to another TF, DzbHLH1, in the regulation of flavonoid biosynthesis. These findings enhance our understanding of the pivotal role of MYB proteins in regulating the phenylpropanoid pathway in durian pulps., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Prior research has highlighted the involvement of a transcriptional complex comprising C-terminal binding protein 2 (CtBP2), histone acetyltransferase p300, and nuclear factor kappa B (NF-κB) in the transactivation of proinflammatory cytokine genes, contributing to inflammation in mice with acute respiratory distress syndrome (ARDS). Nonetheless, it remains uncertain whether the therapeutic targeting of the CtBP2-p300-NF-κB complex holds potential for ARDS suppression., Methods: An ARDS mouse model was established using lipopolysaccharide (LPS) exposure. RNA-Sequencing (RNA-Seq) was performed on ARDS mice and LPS-treated cells with CtBP2, p300, and p65 knockdown. Small molecules inhibiting the CtBP2-p300 interaction were identified through AlphaScreen. Gene and protein expression levels were quantified using RT-qPCR and immunoblots. Tissue damage was assessed via histological staining., Key Findings: We elucidated the specific role of the CtBP2-p300-NF-κB complex in proinflammatory gene regulation. RNA-seq analysis in LPS-challenged ARDS mice and LPS-treated CtBP2-knockdown (CtBP2 KD ), p300 KD , and p65 KD cells revealed its significant impact on proinflammatory genes with minimal effects on other NF-κB targets. Commercial inhibitors for CtBP2, p300, or NF-κB exhibited moderate cytotoxicity in vitro and in vivo, affecting both proinflammatory genes and other targets. We identified a potent inhibitor, PNSC928, for the CtBP2-p300 interaction using AlphaScreen. PNSC928 treatment hindered the assembly of the CtBP2-p300-NF-κB complex, substantially downregulating proinflammatory cytokine gene expression without observable cytotoxicity in normal cells. In vivo administration of PNSC928 significantly reduced CtBP2-driven proinflammatory gene expression in ARDS mice, alleviating inflammation and lung injury, ultimately improving ARDS prognosis., Conclusion: Our results position PNSC928 as a promising therapeutic candidate to specifically target the CtBP2-p300 interaction and mitigate inflammation in ARDS management., (© 2024. The Author(s).)