Structural basis for the intracellular regulation of ferritin degradation.

The interaction between nuclear receptor coactivator 4 (NCOA4) and the iron storage protein ferritin is a crucial component of cellular iron homeostasis. The binding of NCOA4 to the FTH1 subunits of ferritin initiates ferritinophagy-a ferritin-specific autophagic pathway leading to the release of the iron stored inside ferritin. The dysregulation of NCOA4 is associated with several diseases, including neurodegenerative disorders and cancer, highlighting the NCOA4-ferritin interface as a prime target for drug development. Here, we present the cryo-EM structure of the NCOA4-FTH1 interface, resolving 16 amino acids of NCOA4 that are crucial for the interaction. The characterization of mutants, designed to modulate the NCOA4-FTH1 interaction, is used to validate the significance of the different features of the binding site. Our results explain the role of the large solvent-exposed hydrophobic patch found on the surface of FTH1 and pave the way for the rational development of ferritinophagy modulators.

FTH1 overexpression using a dCasRx translation enhancement system protects the kidney from calcium oxalate crystal-induced injury.

The engineered clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system is currently widely applied in genetic editing and transcriptional regulation. The catalytically inactivated CasRx (dCasRx) has the ability to selectively focus on the mRNA coding region without disrupting transcription and translation, opening up new avenues for research on RNA modification and protein translation control. This research utilized dCasRx to create a translation-enhancement system for mammals called dCasRx-eIF4GI, which combined eukaryotic translation initiation factor 4G (eIF4GI) to boost translation levels of the target gene by recruiting ribosomes, without affecting mRNA levels, ultimately increasing translation levels of different endogenous proteins. Due to the small size of dCasRx, the dCasRx-eIF4GI translation enhancement system was integrated into a single viral vector, thus optimizing the delivery and transfection efficiency in subsequent applications. Previous studies reported that ferroptosis, mediated by calcium oxalate (CaOx) crystals, significantly promotes stone formation. In order to further validate its developmental potential, it was applied to a kidney stone model in vitro and in vivo. The manipulation of the ferroptosis regulatory gene FTH1 through single-guide RNA (sgRNA) resulted in a notable increase in FTH1 protein levels without affecting its mRNA levels. This ultimately prevented intracellular ferroptosis and protected against cell damage and renal impairment caused by CaOx crystals. Taken together, this study preliminarily validated the effectiveness and application prospects of the dCasRx-eIF4GI translation enhancement system in mammalian cell-based disease models, providing novel insights and a universal tool platform for protein translation research and future therapeutic approaches for nephrolithiasis.

An unexpected role of EasDaf: catalyzing the conversion of chanoclavine aldehyde to chanoclavine acid.

Ergot alkaloids (EAs) are a diverse group of indole alkaloids known for their complex structures, significant pharmacological effects, and toxicity to plants. The biosynthesis of these compounds begins with chanoclavine-I aldehyde (CC aldehyde, 2), an important intermediate produced by the enzyme EasDaf or its counterpart FgaDH from chanoclavine-I (CC, 1). However, how CC aldehyde 2 is converted to chanoclavine-I acid (CC acid, 3), first isolated from Ipomoea violacea several decades ago, is still unclear. In this study, we provide in vitro biochemical evidence showing that EasDaf not only converts CC 1 to CC aldehyde 2 but also directly transforms CC 1 into CC acid 3 through two sequential oxidations. Molecular docking and site-directed mutagenesis experiments confirmed the crucial role of two amino acids, Y166 and S153, within the active site, which suggests that Y166 acts as a general base for hydride transfer, while S153 facilitates proton transfer, thereby increasing the acidity of the reaction. KEY POINTS: • EAs possess complicated skeletons and are widely used in several clinical diseases • EasDaf belongs to the short-chain dehydrogenases/reductases (SDRs) and converted CC or CC aldehyde to CC acid • The catalytic mechanism of EasDaf for dehydrogenation was analyzed by molecular docking and site mutations.

A functional study reveals CsNAC086 regulated the biosynthesis of flavonols in Camellia sinensis.

MAIN CONCLUSION: CsNAC086 was found to promote the expression of CsFLS, thus promoting the accumulation of flavonols in Camellia sinensis. Flavonols, the main flavonoids in tea plants, play an important role in the taste and quality of tea. In this study, a NAC TF gene CsNAC086 was isolated from tea plants and confirmed its regulatory role in the expression of flavonol synthase which is a key gene involved in the biosynthesis of flavonols in tea plant. Yeast transcription-activity assays showed that CsNAC086 has self-activation activity. The transcriptional activator domain of CsNAC086 is located in the non-conserved C-terminal region (positions 171-550), while the conserved NAC domain (positions 1-170) does not have self-activation activity. Silencing the CsNAC086 gene using antisense oligonucleotides significantly decreased the expression of CsFLS. As a result, the concentration of flavonols decreased significantly. In overexpressing CsNAC086 tobacco leaves, the expression of NtFLS was significantly increased. Compared with wild-type tobacco, the flavonols concentration increased. Yeast one-hybrid assays showed CsNAC086 did not directly regulate the gene expression of CsFLS. These findings indicate that CsNAC086 plays a role in regulating flavonols biosynthesis in tea plants, which has important implications for selecting and breeding of high-flavonols-concentration containing tea-plant cultivars.

Zeaxanthin epoxidase 3 Knockout Mutants of the Model Diatom Phaeodactylum tricornutum Enable Commercial Production of the Bioactive Carotenoid Diatoxanthin.

Carotenoids are pigments that have a range of functions in human health. The carotenoid diatoxanthin is suggested to have antioxidant, anti-inflammatory and chemo-preventive properties. Diatoxanthin is only produced by a few groups of microalgae, where it functions in photoprotection. Its large-scale production in microalgae is currently not feasible. In fact, rapid conversion into the inactive pigment diadinoxanthin is triggered when cells are removed from a high-intensity light source, which is the case during large-scale harvesting of microalgae biomass. Zeaxanthin epoxidase (ZEP) 2 and/or ZEP3 have been suggested to be responsible for the back-conversion of high-light accumulated diatoxanthin to diadinoxanthin in low-light in diatoms. Using CRISPR/Cas9 gene editing technology, we knocked out the ZEP2 and ZEP3 genes in the marine diatom Phaeodactylum tricornutum to investigate their role in the diadinoxanthin-diatoxanthin cycle and determine if one of the mutant strains could function as a diatoxanthin production line. Light-shift experiments proved that ZEP3 encodes the enzyme converting diatoxanthin to diadinoxanthin in low light. Loss of ZEP3 caused the high-light-accumulated diatoxanthin to be stable for several hours after the cultures had been returned to low light, suggesting that zep3 mutant strains could be suitable as commercial production lines of diatoxanthin.

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.

Clinical application value of pre-pregnancy carrier screening in Chinese Han childbearing population.

BACKGROUND: To explore the clinical application value of pre-conception expanded carrier screening (PECS) in the Chinese Han ethnicity population of childbearing age. METHODS: The results of genetic testing of infertile parents who underwent PECS in the Reproductive Medicine Center of the Second Affiliated Hospital of Zhengzhou University, China, from September 2019 to December 2021, were retrospectively analyzed. The carrier rate of single gene disease, the detection rate of high-risk parents, and the clinical outcome of high-risk parents were statistically analyzed. RESULTS: A total of 1372 Chinese Han ethnicity patients underwent PECS, among which 458 patients underwent the extended 108-gene test, their overall carrier rate was 31.7%, and the detection rate of high-risk parents was 0.3%. The highest carrier rates were SLC22A (2.4%), ATP7B (2.4%), MMACHC (2.2%), PAH (1.8%), GALC (1.8%), MLC1 (1.3%), UNC13D (1.1%), CAPN3 (1.1%), and PKHD1 (1.1%). There were 488 women with fragile X syndrome-FMR1 gene detection, and 6 patients (1.2%) had FMR1 gene mutation. A total of 426 patients were screened for spinal muscular atrophy-SMN1, and the carrier rate was 3.5%, and the detection rate of parents' co-carrier was 0.5%. CONCLUSION: Monogenic recessive hereditary diseases had a high carrier rate in the population. Pre-pregnancy screening could provide good prenatal and postnatal care guidance for patients and preimplantation genetic testing for monogenic/single gene disorders (PGT-M) and prenatal diagnosis could provide more precise reproductive choices for high-risk parents.

Bradyrhizobium ontarionense sp. nov., a novel bacterial symbiont isolated from Aeschynomene indica (Indian jointvetch), harbours photosynthesis, nitrogen fixation and nitrous oxide (N2O) reductase genes.

A novel bacterial symbiont, strain A19T, was previously isolated from a root-nodule of Aeschynomene indica and assigned to a new lineage in the photosynthetic clade of the genus Bradyrhizobium. Here data are presented for the detailed genomic and taxonomic analyses of novel strain A19T. Emphasis is placed on the analysis of genes of practical or ecological significance (photosynthesis, nitrous oxide reductase and nitrogen fixation genes). Phylogenomic analysis of whole genome sequences as well as 50 single-copy core gene sequences placed A19T in a highly supported lineage distinct from described Bradyrhizobium species with B. oligotrophicum as the closest relative. The digital DNA-DNA hybridization and average nucleotide identity values for A19T in pair-wise comparisons with close relatives were far lower than the respective threshold values of 70% and ~ 96% for definition of species boundaries. The complete genome of A19T consists of a single 8.44 Mbp chromosome and contains a photosynthesis gene cluster, nitrogen-fixation genes and genes encoding a complete denitrifying enzyme system including nitrous oxide reductase implicated in the reduction of N2O, a potent greenhouse gas, to inert dinitrogen. Nodulation and type III secretion system genes, needed for nodulation by most rhizobia, were not detected. Data for multiple phenotypic tests complemented the sequence-based analyses. Strain A19T elicits nitrogen-fixing nodules on stems and roots of A. indica plants but not on soybeans or Macroptilium atropurpureum. Based on the data presented, a new species named Bradyrhizobium ontarionense sp. nov. is proposed with strain A19T (= LMG 32638T = HAMBI 3761T) as the type strain.

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.

Systems Metabolic Engineering for Efficient Violaxanthin Production in Yeast.

Violaxanthin is a plant-derived orange xanthophyll with remarkable antioxidant activity that has wide applications in various industries, such as food, agriculture, and cosmetics. In addition, it is the key precursor of important substances such as abscisic acid and fucoxanthin. Saccharomyces cerevisiae, as a GRAS (generally regarded as safe) chassis, provides a good platform for producing violaxanthin production with a yield of 7.3 mg/g DCW, which is far away from commercialization. Herein, an integrated strategy involving zeaxanthin epoxidase (ZEP) source screening, cytosol redox state engineering, and nicotinamide adenine dinucleotide phosphate (NADPH) regeneration was implemented to enhance violaxanthin production in S. cerevisiae. 58aa-truncated ZEP from Vitis vinifera exhibited optimal efficiency in an efficient zeaxanthin-producing strain. The titer of violaxanthin gradually increased by 17.9-fold (up to 119.2 mg/L, 15.19 mg/g DCW) via cytosol redox state engineering and NADPH supplementation. Furthermore, balancing redox homeostasis considerably improved the zeaxanthin concentration by 139.3% (up to 143.9 mg/L, 22.06 mg/g DCW). Thus, the highest reported titers of violaxanthin and zeaxanthin in S. cerevisiae were eventually achieved. This study not only builds an efficient platform for violaxanthin biosynthesis but also serves as a useful reference for the microbial production of xanthophylls.

Identification of novel molecules and pathways associated with fascin actin­bundling protein 1 in laryngeal squamous cell carcinoma through comprehensive transcriptome analysis.

Laryngeal squamous cell carcinoma (LSCC) is a common malignant tumor with a poor prognosis. Fascin actin­bundling protein 1 (FSCN1) has been reported to play a crucial role in the development and progression of LSCC; however, the underlying molecular mechanisms remain unknown. Herein, a whole transcriptome microarray analysis was performed to screen for differentially expressed genes (DEGs) in cells in which FSCN1 was knocked down. A total of 462 up and 601 downregulated mRNA transcripts were identified. Functional annotation analysis revealed that these DEGs were involved in multiple biological functions, such as transcriptional regulation, response to radiation, focal adhesion, extracellular matrix­receptor interaction, steroid biosynthesis and others. Through co­expression and protein­protein interaction analysis, FSCN1 was linked to novel functions, including defense response to virus and steroid biosynthesis. Furthermore, crosstalk analysis with FSCN1­interacting proteins revealed seven DEGs, identified as FSCN1­interacting partners, in LSCC cells, three of which were selected for further validation. Co­immunoprecipitation validation confirmed that FSCN1 interacted with prostaglandin reductase 1 and 24­dehydrocholesterol reductase (DHCR24). Of note, DHCR24 is a key enzyme involved in cholesterol biosynthesis, and its overexpression promotes the proliferation and migration of LSCC cells. These findings suggest that DHCR24 is a novel molecule associated with FSCN1 in LSCC, and that the FSCN1­DHCR24 interaction may promote LSCC progression by regulating cholesterol metabolism­related signaling pathways.

Identification of redox activators for continuous reactivation of glyoxal oxidase from Trametes versicolor in a two-enzyme reaction cascade.

Glyoxal oxidases, belonging to the group of copper radical oxidases (CROs), oxidize aldehydes to carboxylic acids, while reducing O2 to H2O2. Their activity on furan derivatives like 5-hydroxymethylfurfural (HMF) makes these enzymes promising biocatalysts for the environmentally friendly synthesis of the bioplastics precursor 2,5-furandicarboxylic acid (FDCA). However, glyoxal oxidases suffer from inactivation, which requires the identification of suitable redox activators for efficient substrate conversion. Furthermore, only a few glyoxal oxidases have been expressed and characterized so far. Here, we report on a new glyoxal oxidase from Trametes versicolor (TvGLOX) that was expressed at high levels in Pichia pastoris (reclassified as Komagataella phaffii). TvGLOX was found to catalyze the oxidation of aldehyde groups in glyoxylic acid, methyl glyoxal, HMF, 2,5-diformylfuran (DFF) and 5-formyl-2-furancarboxylic acid (FFCA), but barely accepted alcohol groups as in 5-hydroxymethyl-2-furancarboxylic acid (HMFCA), preventing formation of FDCA from HMF. Various redox activators were tested for TvGLOX reactivation during catalyzed reactions. Among them, a combination of horseradish peroxidase and its substrate 2,2'-azino-di-(3-ethylbenzthiazoline sulfonic acid) (ABTS) most efficiently reactivated TvGLOX. Through continuous reactivation of TvGLOX in a two-enzyme system employing a recombinant Moesziomyces antarcticus aryl-alcohol oxidase (MaAAO) almost complete conversion of 8 mM HMF to FDCA was achieved within 24 h.

Functional diversity of YbbN/CnoX proteins: Insights from a comparative analysis of three thioredoxin-like oxidoreductases from Pseudomonas aeruginosa, Xylella fastidiosa and Escherichia coli.

YbbN/CnoX are proteins that display a Thioredoxin (Trx) domain linked to a tetratricopeptide domain. YbbN from Escherichia coli (EcYbbN) displays a co-chaperone (holdase) activity that is induced by HOCl. Here, we compared EcYbbN with YbbN proteins from Xylella fastidiosa (XfYbbN) and from Pseudomonas aeruginosa (PaYbbN). EcYbbN presents a redox active Cys residue at Trx domain (Cys63), 24 residues away from SQHC motif (SQHC[N24]C) that can form mixed disulfides with target proteins. In contrast, XfYbbN and PaYbbN present two Cys residues in the CXXC (CAPC) motif, while only PaYbbN shows the Cys residue equivalent to Cys63 of EcYbbN. Our phylogenetic analysis revealed that most of the YbbN proteins are in the bacteria domain of life and that their members can be divided into four groups according to the conserved Cys residues. EcYbbN (SQHC[N24]C), XfYbbN (CAPC[N24]V) and PaYbbN (CAPC[N24]C) are representatives of three sub-families. In contrast to EcYbbN, both XfYbbN and PaYbbN: (1) reduced an artificial disulfide (DTNB) and (2) supported the peroxidase activity of Peroxiredoxin Q from X. fastidiosa, suggesting that these proteins might function similarly to the canonical Trx enzymes. Indeed, XfYbbN was reduced by XfTrx reductase with a high catalytic efficiency (kcat/Km = 1.27 x 107 M-1 s-1), similar to the canonical XfTrx (XfTsnC). Furthermore, EcYbbN and XfYbbN, but not PaYbbN displayed HOCl-induced holdase activity. Remarkably, EcYbbN gained disulfide reductase activity while lost the HOCl-activated chaperone function, when the SQHC was replaced by CQHC. In contrast, the XfYbbN CAPA mutant lost the disulfide reductase activity, while kept its HOCl-induced chaperone function. In all cases, the induction of the holdase activity was accompanied by YbbN oligomerization. Finally, we showed that deletion of ybbN gene did not render in P. aeruginosa more sensitive stressful treatments. Therefore, YbbN/CnoX proteins display distinct properties, depending on the presence of the three conserved Cys residues.

In Vitro Comparative Study on Antineoplastic Effects of Pinoresinol and Lariciresinol on Healthy Cells and Breast Cancer-Derived Human Cells.

Background: Herbal medicines are the preferred anticancer agents due to their lower cytotoxic effects on healthy cells. Plant lignans play an important role in treating various diseases, especially cancer. The present study aimed to evaluate the effect of podophyllotoxin, pinoresinol, and lariciresinol on cellular toxicity and inducing apoptosis in fibroblasts, HEK-293, and SkBr3 cell lines. Methods: An in vitro study was conducted from 2017 to 2019 at the Faculty of Biological Sciences, Tarbiat Modares University (Tehran, Iran). The cell lines were treated for 24 and 48 hours with different concentrations of lignans. Cell viability and apoptosis were examined using MTT and flow cytometry, respectively. Expression levels of cell cycle and apoptosis regulator genes were determined using quantitative real-time polymerase chain reaction. Data were analyzed using a two-way analysis of variance followed by Tukey's HSD test. P<0.05 was considered statistically significant. Results: Podophyllotoxin significantly increased apoptosis in fibroblast cells compared to pinoresinol and lariciresinol (P<0.001). The percentage of cell viability of fibroblast cells treated for 48 hours with pinoresinol, lariciresinol, and podophyllotoxin was reduced by 49%, 47%, and 36%, respectively. Treatment with pinoresinol and lariciresinol significantly overexpressed pro-apoptotic genes and underexpressed anti-apoptotic genes in SkBr3 cells (P<0.001). SkBr3 cells treated with lariciresinol significantly reduced gene expression (P<0.001). Conclusion: Pinoresinol and lariciresinol can potentially be used as new therapeutic agents for the treatment of breast cancer.

Identification of bacterial dissimilatory antimonate reductase AnrA: genes and proteins involved in antimonate respiration and resistance in Geobacter sp. strain SVR.

Geobacter sp. strain SVR uses antimonate [Sb(V)] as a terminal electron acceptor for anaerobic respiration. Here, we visualized a possible key enzyme, periplasmic Sb(V) reductase (Anr), via active staining and non-denaturing gel electrophoresis. Liquid chromatography-tandem mass spectrometry analysis revealed that a novel dimethyl sulfoxide (DMSO) reductase family protein, WP_173201954.1, is involved in Anr. This protein was closely related with AnrA, a protein suggested to be the catalytic subunit of a respiratory Sb(V) reductase in Desulfuribacillus stibiiarsenatis. The anr genes of strain SVR (anrXSRBAD) formed an operon-like structure, and their transcription was upregulated under Sb(V)-respiring conditions. The expression of anrA gene was induced by more than 1 µM of antimonite [Sb(III)]; however, arsenite [As(III)] did not induce the expression of anrA gene. Tandem mass tag-based proteomic analysis revealed that, in addition to Anr proteins, proteins in the following categories were upregulated under Sb(V)-respiring conditions: (i) Sb(III) efflux systems such as Ant and Ars; (ii) antioxidizing proteins such as ferritin, rubredoxin, and thioredoxin; (iii) protein quality control systems such as HspA, HslO, and DnaK; and (iv) DNA repair proteins such as UspA and UvrB. These results suggest that strain SVR copes with antimony stress by modulating pleiotropic processes to resist and actively metabolize antimony. To the best of our knowledge, this is the first report to demonstrate the involvement of AnrA in Sb(V) respiration at the protein level. Furthermore, this is the first example to show high expression of the Ant system proteins in the Sb(V)-respiring bacterium.IMPORTANCEAntimony (Sb) exists mainly as antimonite [Sb(III)] or antimonate [Sb(V)] in the environment, and Sb(III) is more toxic than Sb(V). Recently, microbial involvement in Sb redox reactions has received attention. Although more than 90 Sb(III)-oxidizing bacteria have been reported, information on Sb(V)-reducing bacteria is limited. Especially, the enzyme involved in dissimilatory Sb(V) reduction, or Sb(V) respiration, is unclear, despite this pathway being very important for the circulation of Sb in nature. In this study, we demonstrated that the Sb(V) reductase (Anr) of an Sb(V)-respiring bacterium (Geobacter sp. SVR) is a novel member of the dimethyl sulfoxide (DMSO) reductase family. In addition, we found that strain SVR copes with Sb stress by modulating pleiotropic processes, including the Ant and Ars systems, and upregulating the antioxidant and quality control protein levels. Considering the abundance and diversity of putative anr genes in the environment, Anr may play a significant role in global Sb cycling in both marine and terrestrial environments.

Mining and tailor-made engineering of a novel keto reductase for asymmetric synthesis of structurally hindered γ- and δ-lactones.

A novel carbonyl reductase from Hyphopichia burtoni (HbKR) was discovered by gene mining. HbKR is a NADPH-dependent dual function enzyme with reduction and oxidation activity belonging to SDR superfamily. HbKR strictly follows Prelog priority in the reduction of long-chain aliphatic keto acids/esters containing remote carbonyl groups, such as 4-oxodecanoic acid and 5-oxodecanoic acid, producing (S)-γ-decalactone and (S)-δ-decalactone in >99 % e.e. Tailor-made engineering of HbKR was conducted to improve its catalytic efficiency. Variant F207A/F86M was obtained with specific activity of 8.37 U/mg toward 5-oxodecanoic acid, which was 9.7-fold of its parent. Employing F207A/F86M, 100 mM 5-oxodecanoic acid could be reduced into optically pure (S)-δ-decalactone. Molecular docking analysis indicates that substitution of aromatic Phe with smaller residues renders sufficient space for accommodating substrates in a more stable conformation. This study offers an efficient biocatalyst for the biosynthesis of (S)-lactones, and provides guidance for engineering carbonyl reductases toward structurally hindered substrates.

Mitochondrial amidoxime-reducing component 1 p.Ala165Thr increases protein degradation mediated by the proteasome.

OBJECTIVE: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a global health concern with no effective and specific drug treatment available. The rs2642438 minor allele in mitochondrial amidoxime-reducing component 1 (MARC1) results in an aminoacidic substitution (p.Ala165Thr) and associates with protection against MASLD. However, the mechanisms behind this protective effect are unknown. In this study, we examined the consequences of this aminoacidic substitution on protein stability and subcellular localization. METHODS: We overexpressed the human MARC1 A165 (wild-type) or 165T (mutant) in vivo in mice and in vitro in human hepatoma cells (HepG2 and HuH-7), generated several mutants at position 165 by in situ mutagenesis and then examined protein levels. We also generated HepG2 cells stably overexpressing MARC1 A165 or 165T to test the effect of this substitution on MARC1 subcellular localization. RESULTS: MARC1 165T overexpression resulted in lower protein levels than A165 both in vivo and in vitro. Similarly, any mutant at position 165 showed lower protein levels compared to the wild-type protein. We showed that the 165T mutant protein is polyubiquitinated and its degradation is accelerated through lysine-48 ubiquitin-mediated proteasomal degradation. We also showed that the 165T substitution does not affect the MARC1 subcellular localization. CONCLUSIONS: This study shows that alanine at position 165 in MARC1 is crucial for protein stability, and the threonine substitution at this position leads to a hypomorphic protein variant due to lower protein levels. Our result supports the notion that lowering hepatic MARC1 protein level may be a successful therapeutic strategy for treating MASLD.

Genome-Wide Identification, Expression Analysis, and Subcellular Localization of DET2 Gene Family in Populus yunnanensis.

(1) Background: Brassinosteroids (BRs) are important hormones involved in almost all stages of plant growth and development, and sterol dehydrogenase is a key enzyme involved in BRs biosynthesis. However, the sterol dehydrogenase gene family of Populus yunnanensis Dode (P. yunnanensis) has not been studied. (2) Methods: The PyDET2 (DEETIOLATED2) gene family was identified and analyzed. Three genes were screened based on RNA-seq of the stem tips, and the PyDET2e was further investigated via qRT-PCR (quantitative real-time polymerase chain reaction) and subcellular localization. (3) Results: The 14 DET2 family genes in P. yunnanensis were categorized into four groups, and 10 conserved protein motifs were identified. The gene structure, chromosome distribution, collinearity, and codon preference of all PyDET2 genes in the P. yunnanensis genome were analyzed. The codon preference of this family is towards the A/U ending, which is strongly influenced by natural selection. The PyDET2e gene was expressed at a higher level in September than in July, and it was significantly expressed in stems, stem tips, and leaves. The PyDET2e protein was localized in chloroplasts. (4) Conclusions: The PyDET2e plays an important role in the rapid growth period of P. yunnanensis. This systematic analysis provides a basis for the genome-wide identification of genes related to the brassinolide biosynthesis process in P. yunnanensis, and lays a foundation for the study of the rapid growth mechanism of P. yunnanensis.

Anaerobic hexadecane degradation by a thermophilic Hadarchaeon from Guaymas Basin.

Hadarchaeota inhabit subsurface and hydrothermally heated environments, but previous to this study, they had not been cultured. Based on metagenome-assembled genomes, most Hadarchaeota are heterotrophs that grow on sugars and amino acids, or oxidize carbon monoxide or reduce nitrite to ammonium. A few other metagenome-assembled genomes encode alkyl-coenzyme M reductases (Acrs), ß-oxidation, and Wood-Ljungdahl pathways, pointing toward multicarbon alkane metabolism. To identify the organisms involved in thermophilic oil degradation, we established anaerobic sulfate-reducing hexadecane-degrading cultures from hydrothermally heated sediments of the Guaymas Basin. Cultures at 70°C were enriched in one Hadarchaeon that we propose as Candidatus Cerberiarchaeum oleivorans. Genomic and chemical analyses indicate that Ca. C. oleivorans uses an Acr to activate hexadecane to hexadecyl-coenzyme M. A ß-oxidation pathway and a tetrahydromethanopterin methyl branch Wood-Ljungdahl (mWL) pathway allow the complete oxidation of hexadecane to CO2. Our results suggest a syntrophic lifestyle with sulfate reducers, as Ca. C. oleivorans lacks a sulfate respiration pathway. Comparative genomics show that Acr, mWL, and ß-oxidation are restricted to one family of Hadarchaeota, which we propose as Ca. Cerberiarchaeaceae. Phylogenetic analyses further indicate that the mWL pathway is basal to all Hadarchaeota. By contrast, the carbon monoxide dehydrogenase/acetyl-coenzyme A synthase complex in Ca. Cerberiarchaeaceae was horizontally acquired from Bathyarchaeia. The Acr and ß-oxidation genes of Ca. Cerberiarchaeaceae are highly similar to those of other alkane-oxidizing archaea such as Ca. Methanoliparia and Ca. Helarchaeales. Our results support the use of Acrs in the degradation of petroleum alkanes and suggest a role of Hadarchaeota in oil-rich environments.

Mutation in BrFLS encoding flavonol synthase induced anthocyanin accumulation in Chinese cabbage.

KEY MESSAGE: BrFLS mutation promoted anthocyanin accumulation in Chinese cabbage, which was verified in four allelic mutants. Chinese cabbage is a major vegetable crop in Eastern Asia. Anthocyanin-rich vibrantly colored varieties are increasingly favored by consumers for their higher nutritional and aesthetic value compared to the typical green varieties of Chinese cabbage. Herein, we identified an anthocyanin accumulation mutant aam1 from a mutant library of EMS-mutagenized Chinese cabbage DH line 'FT', which appeared partial purple on leaves, bolting stems and floral buds. This anthocyanin accumulation trait was genetically controlled by a recessive nuclear gene, and through MutMap mapping and KASP genotyping, BraA10g030950.3C was identified as the candidate causal gene with a G202 to A202 non-synonymous SNP variation in exon 1. Three additional mutants allelic to aam1 were obtained via screening of similar-phenotype mutants from the mutant library, namely aam2/3/4, where the causal SNPs reside in the same gene as aam1, corroborating that the mutation of BraA10g030950.3C caused anthocyanin accumulation. BraA10g030950.3C encodes a flavonol synthase that catalyzes dihydroflavonols substrate into flavonols and is homologous to Arabidopsis FLS1 (AT5G08640), named BrFLS. Compared to wildtype, the expression level of BrFLS was significantly reduced in the mutants, while BrDFR, which is involved in the anthocyanin biosynthesis and competes with FLS for the common substrate dihydroflavonols, was increased. The flavonol synthase activity decreased, and dihydroflavonol 4-reductase activity was elevated. Differentially accumulated flavonoid metabolites were detected between wildtype and aam1, which were enriched primarily in flavonol and anthocyanin pathways. Our results revealed that mutations in the BrFLS gene could contribute to anthocyanin accumulation and provide a new target for Chinese cabbage color modification.