Search Results (92)

Steroids are found in bacteria and eukaryotes, and genes potentially encoding steroid metabolic enzymes have also been identified in giant viruses. For decades, hydroxylated steroids have been utilized in medicine to treat various human diseases. The hydroxylation of steroids can be achieved using microbial enzymes, especially cytochrome P450 monooxygenases (CYPs/P450s) and is well documented. Understanding the structural determinants that govern the regio- and stereoselectivity of steroid hydroxylation by P450s is essential in order to fully exploit their potential. Herein, we present a comprehensive analysis of the steroid-hydroxylating CYP109 family across the domains of life and delineate the structural determinants that govern steroid hydroxylation. Data mining, annotation, and phylogenetic analysis revealed that CYP109 family members are highly populated in bacteria, and indeed, these members passed from bacteria to archaea by horizontal gene transfer, leading to the evolution of P450s in archaea. Analysis of twelve CYP109 crystal structures revealed large, flexible, and dynamic active site cavities that can accommodate multiple ligands. The correct positioning and orientation of the steroid in the active site cavity and the nature of the C17 substituent on the steroid molecule influence catalysis. In an analogous fashion to the CYP107 family, the amino acid residues within the CYP109 binding pocket involve hydrophilic and hydrophobic interactions, influencing substrate orientations and anchoring and determining the site of hydroxylation and catalytic activity. A handful of amino acids, such as Val84, Val292, and Ser387 in CYP109B4, have been found to play a role in determining the catalytic regiospecificity, and a single amino acid, such as Arg74 in CYP109A2, has been found to be essential for the enzymatic activity. This work serves as a reference for the precise understanding of CYP109 structure–function relationships and for P450 enzymes in general. The findings will guide the genetic engineering of CYP109 enzymes to produce valuable steroid molecules of medicinal and biotechnological importance. Full article

Symbiotic interactions between legumes and a group of soil bacteria, known as rhizobia, lead to the formation of a specialized organs called root nodules. Inside them, atmospheric nitrogen (N₂) is fixed by bacteria and reduced to forms available to plants, catalyzed by the nitrogenase enzyme complex. The development of a symbiotic relationship between legumes and nodule bacteria is a multi-stage, precisely regulated process, characterized by a high specificity of partner selection. Nodulation involves the enhanced expression of certain plant genes, referred to as early- and late-nodulin genes. Many nodulin genes encode hydroxyproline-rich glycoproteins (HRGPs) and proline-rich proteins (PRPs) which are involved in various processes, including infection thread formation, cell signaling, and defense responses, thereby affecting nodule formation and function. Cyclophilins (CyPs) belong to a family of proteins with peptidyl-prolyl cis–trans isomerase activity. Proteins with cyclophilin domain can be found in the cytoplasm, endoplasmic reticulum, nucleus, chloroplast, and mitochondrion. They are involved in various processes, such as protein folding, cellular signaling, mRNA maturation, and response to biotic and abiotic stress. In this review, we aim to summarize the molecular processes involved in the development of symbiosis and highlight the potential role of cyclophilins (peptidyl-prolyl cis-trans isomerases) in this process. Full article

The Chinese tongue sole (Cynoglossus semilaevis) is a commercially important mariculture species; however, its fertilization and hatching rates under artificial conditions remain relatively low. Zona pellucida proteins (ZPs), which mediate sperm–egg binding, were previously identified as differentially expressed genes between newly differentiated ovaries and testes in C. semilaevis. In this study, we identified 25 ZPs of C. semilaevis through genomic analysis and classified them into five subfamilies. All genes possessed a conserved ZP domain, characteristic of the gene family from mammals to teleosts. Among them, nine genes were highly expressed in ovary cells, with the expression levels increasing during ovarian development, while another three genes were predominantly expressed in liver cells. Protein–protein interaction analysis predicted that 12 ZPs interacted with key reproductive regulators such as Gdf9, Arid4a, Arid4b, and Rbl, which were involved in steroidogenesis, sperm–egg recognition, and folliculogenesis. Functional analyses using RNA interference revealed that Cszpc7-1 knockdown in ovarian cells led to the downregulation of cyp19a, esr2, bmp15, and adamts-1, while the expression of rbl, gnas, adgrl1, and adgrl2 was upregulated. In contrast, Cszpax1 knockdown resulted in decreased expression of cyp19a, foxl2, arid4a, and zeb1, along with upregulation of arid4b, ogg1, and gdf9. These results suggested that ZP genes might contribute to ovarian homeostasis by regulating steroid hormone synthesis, follicular development, and ovulation. This study contributed to a deeper understanding of the reproductive mechanisms of C. semilaevis and provided evolutionary insights into the functional divergence of the ZP gene family across teleosts. Full article

In this study, red–blue light (7R3B) was used as the control (CK), while far-red (FR) and ultraviolet-A (UVA) light were supplemented to evaluate their effects on basil growth. The results showed that the FR treatment promoted plant height, stem diameter, and biomass, but reduced chlorophyll and carotenoid content, while the UVA treatment increased stem diameter and chlorophyll b content. Meanwhile, transcriptomic and metabolomic analyses were employed to examine changes in gene expression and metabolite accumulation in basil. The FR treatment reduced the levels of differentially accumulated metabolites (DAMs) in the carotenoid biosynthesis pathway, potentially contributing to the observed decrease in chlorophyll. The FR treatment upregulated the levels of five DAMs (gibberellin, cytokinin, brassinosteroid, jasmonic acid, and salicylic acid) and altered the differentially expressed genes (DEGs) such as gibberellin receptor (GID1) and jasmonate ZIM domain-containing protein (JAZ) in the plant hormone signal transduction pathway, thereby promoting plant growth and shade avoidance responses. The UVA treatment upregulated the 9-cis-epoxycarotenoid dioxygenase (NCED) expression in the carotenoid biosynthesis pathway, possibly indirectly promoting flavonoid synthesis. In the flavonoid biosynthesis pathway, the UVA treatment also promoted flavonoid accumulation by upregulating DEGs including flavonol synthase (FLS), anthocyanidin synthase (ANS), 5-O-(4-coumaroyl)-D-quinate 3′-monooxygenase (CYP98A), and flavanone 7-O-glucoside 2″-O-beta-L-rhamnosyltransferase (C12RT1), as well as increasing the levels of DAMs such as kaempferol, luteolin, apigenin, and leucopelargonidin. The accumulation of flavonoids improved antioxidant capacity and nutritional value in basil. Through a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, this study provided valuable insights into the molecular and metabolic mechanisms of the FR and UVA regulation of basil growth, providing guidance for optimizing supplementary lighting strategies in plant factories. Full article

Aspergillus flavus and its secondary metabolites aflatoxins pose a significant threat to the health of humans, animals, and plants. Therefore, there is an urgent need to control A. flavus contamination. AfverB plays a key role in the aflatoxin gene cluster; however, its function and mechanism in fungal development and virulence remain poorly understood. In this study, we constructed afVerB gene deletion mutants (∆afVerB−1 and ∆afVerB−2) and two CYP domain mutants (afVerB^∆D1 and afVerB^∆D2) through homologous recombination. Phenotype analysis revealed that, via its two CYP domains, AfVerB is deeply involved in fungal morphogenesis and aflatoxin synthesis. Insect and crop colonization models revealed that AfVerB plays a key role in the fungus’s ability to infect hosts, and stress experiments discovered that AfVerB plays a significant role in the response to various environmental stresses, which explains why AfVerB is a key factor in fungal infection to some extent. RT-qPCR analysis demonstrated that AfVerB performs its bio-function through corresponding regulatory factors. We ultimately discovered that AfVerB is deeply involved in cell membrane stress stability, thereby participating in the regulation of fungal drug resistance (sensitive to AMB and resistant to VOR in this study). The CYP domain of AfVerB, particularly its second CYP domain, is crucial for the execution of its biological functions. This study elucidated the regulatory mechanisms by which AfVerB regulates fungal pathogenicity and aflatoxin biosynthesis, providing potential strategies for controlling A. flavus and its aflatoxin contamination. Full article

In the domain of swine production, body weight (BW) and average daily gain (ADG) are recognized as the primary performance indicators. Nevertheless, the genetic architecture of ADG and BW in Dongliao black (DLB) pigs remains to be fully elucidated. In this study, we performed a genome-wide association analysis of BW, ADG, and body mass index (BMI) in 358 DLB pigs of different days of age. The genome-wide association study (GWAS) showed the following: (1) The most significant single nucleotide polymorphism (SNP) detected for BW was on Sus scrofa chromosome (SSC) 11:100,808 (p-value = 1.16 × 10⁻⁶) that was also the most significant SNP for ADG. (2) The most significant SNP associated with BMI was SSC17:51,463,521 (p-value = 5.16 × 10⁻⁸). (3) SNPs SSC10:6,523,844 and SSC17:23,852,682 were identified in both BW and ADG. A meta-analysis was conducted on BW at different days and demonstrated SSC5:39,028,335 (p-value = 8.37 × 10⁻⁶) which was not identified in the results of each single trait. The regions of two SNPs (SSC11:100,808, SSC4:10,703,277) exhibited considerable influence on both BW and ADG and the related regions were selected for linkage disequilibrium (LD) analyses that exhibited a notable linkage. In addition, several genes were identified that are associated with obesity and play roles in lipid metabolism, including MACROD2, PHLPP2, CYP2E1, and STT3B. Full article

It is well known that the host response to different human and animal coronaviruses infection is regulated by the aryl hydrocarbon receptor, a ligand-activated transcription factor. The present study investigates the expression of the aryl hydrocarbon receptor during bovine coronavirus infection, through in vitro and in silico investigations. The in vitro studies demonstrate that the aryl hydrocarbon receptor and as well as its targets, CYP1A1 and CYP1B1, were significantly activated by bovine coronavirus infection in bovine cells (MDBK). During infection, the pretreatment of cells with non-cytotoxic doses of CH223191, a selective inhibitor of the aryl hydrocarbon receptor, resulted in a significant reduction in virus yield and a downregulation in the viral spike protein expression. These findings occurred in the presence of the inhibition of aryl hydrocarbon receptor signaling. Our results reveal that the bovine coronavirus acts on viral replication, upregulating the aryl hydrocarbon receptor and its downstream target proteins, CYP1A1 and CYP1B1. In addition, following the in silico studies, the three-dimensional structural model of the bovine aryl hydrocarbon receptor in complex with the antagonist CH223191 indicates that the molecular mechanism, by which the PASB and TAD domains of the receptor interact with the inhibitor, is mainly driven by an extensive network of hydrophobic interactions, with a series of hydrogen bonds contributing to stabilizing the complex. Interestingly, bioinformatic analyses revealed that the PASB and TAD domains in the human and bovine aryl hydrocarbon receptor present high similarity at the primary sequence and three-dimensional structure levels. Taken together, these findings represent a fundamental step for the development of innovative drugs targeting AhR as a potential object for CoVs therapy. Full article

The CYP1B1 gene encodes a cytochrome p450 monooxygenase enzyme, and over 150 variants have been associated with a spectrum of eye diseases, including primary congenital glaucoma, anterior segment dysgenesis, juvenile open-angle glaucoma, and primary open-angle glaucoma. Clinical genetics has yielded insights into the functions of the various CYP1B1 gene domains; however, animal studies are required to investigate the molecular role of CYP1B1 in the eye. While both zebrafish and mice express CYP1B1 in the developing eye, embryonic studies have shown disparate species-specific functions. In zebrafish, CYP1B1 regulates ocular fissure closure such that overexpression causes a remarkable phenotype consisting of the absence of the posterior eye wall. Adult CYP1B1 null zebrafish lack an ocular phenotype but show mild craniofacial abnormalities. In contrast, CYP1B1^−/− mice display post-natal mild to severe trabecular meshwork degeneration due to increased oxidative stress damage. Interestingly, the retinal ganglion cells in CYP1B1 null mice may be more susceptible to damage secondary to increased intraocular pressure. Future studies, including detailed genotype–phenotype information and animal work elucidating the regulation, substrates, and downstream effects of CYP1B1, will yield important insights for developing molecularly targeted therapies that will aim to prevent vision loss in CYP1B1-related eye diseases. Full article

Cytochromes P450 are a superfamily of heme-containing monooxygenases involved in a variety of oxidative metabolic reactions, primarily catalyzing the insertion of an oxygen atom into a C-H bond. CYP102 represents the first example of a bacterial P450 that can be classified as a type II (eukaryotic-like) P450 and functions as a catalytically self-sufficient enzyme. These unique features have made CYP102 an attractive system for studying P450 structure and function. However, an overall picture of the specific amino acid residues that are crucial to the functioning of CYP102 and the effect of mutations on the P450 structure and catalysis is yet to be reported. Such an approach will aid protein engineering approaches used to improve this enzyme. To address this research knowledge gap, we have investigated 105 CYP102 crystal structures in this study. We demonstrate that the CYP102 active site is highly dynamic and flexible. Amino acid residues that play critical roles in substrate binding, orientation, and anchoring were identified. Mutational studies highlighted the roles of amino acids and provided possible bioengineering improvement strategies for CYP102. Decoy molecules are a promising agent for deceiving CYP102 and permitting non-native substrates into the active site. Ru(II)-diimine photosensitizers and zinc/cobalt (III) sepulchrate (Co(III)Sep) could be used as alternative electron sources. The present study serves as a reference for understanding the structure–functional analysis of CYP102 family members precisely and of P450 enzymes in general. Significantly, this work contributes to the effort to develop an improved CYP102 enzyme, thereby advancing the field of P450 research and potentially leading to new industrial applications. Full article

The CYP450 enzyme is a superfamily enzyme ubiquitously found in nearly all organisms, playing a vital role in the metabolism of both endogenous and exogenous compounds, and in biosynthesis. Unfortunately, an understanding of its classification, functions, expression characteristics, and other biological traits in Hyalomma asiaticum, a vector for Crimean–Congo Hemorrhagic Fever, as well as of the genes implicated in its natural product metabolism, is lacking. Towards this end, this study has identified 120 H. asiaticum CYP450 genes via transcriptome data in the face of a joint genome threat from terpinolene. The proteins these genes encode are of higher molecular weight, devoid of a signal peptide, and composed of unstable hydrophobic proteins principally containing 1–3 variable transmembrane regions. Phylogenetic evolution classifies these H. asiaticum CYP450 genes into four subfamilies. These genes all encompass complete CYP450 conserved domains, and five specific conserved motifs, albeit with different expression levels. GO and KEGG annotation findings suggest a widespread distribution of these CYP450 genes in many physiological systems, predominantly facilitating lipid metabolism, terpenoid compound metabolism, and polyketone compound metabolism, as well as cofactor and vitamin metabolism at a cellular level. Molecular docking results reveal a hydrophobic interaction between the ARG-103, ARG-104, LEU-106, PHE-109, and ILE-119 amino acid residues in CYP3A8, which is primarily expressed in the fat body, and terpinolene, with a notably up-regulated expression, with affinity = −5.6 kcal/mol. The conservation of these five key amino acid residues varies across 12 tick species, implying differences in terpinolene metabolism efficacy among various tick species. This study thereby fills an existing knowledge gap regarding the biological characteristics of H. asiaticum CYP450 genes and paves the way for further research into the functions of these particular genes. Full article

Gametogenesis, the intricate developmental process responsible for the generation of germ cells (gametes), serves as a fundamental prerequisite for the perpetuation of the reproductive cycle across diverse organisms. The g2e3 enzyme is a putative ubiquitin E3 ligase implicated in the intricate regulatory mechanisms underlying cellular proliferation and division processes. The present study delves into the function of G2/M phase-specific E3 ubiquitin protein ligase (Cs-g2e3) in gametogenesis in Chinese Tongue Sole (Cynoglossus semilaevis). Sequence analysis shows that the Cs-g2e3 mRNA spans 6479 bp, encoding a 733 amino acid protein characterized by three conserved structural domains: PHD, RING, and HECT—typical of HECT E3 ubiquitin ligases. The predominant expression of Cs-g2e3 in the gonad tissues is further verified by qPCR. The expression profile of Cs-g2e3 in the gonads of the Chinese Tongue Sole is analyzed at different ages, and the results show that its expression peaks at 8 months of age and then begins to decline and stabilize. It is noteworthy that the expression level remains significantly elevated compared to that observed during the juvenile period. In situ hybridization shows that the mRNA of Cs-g2e3 is mainly localized in the germ cells of the ovary and the testis. RNA interference experiments show that the knockdown of Cs-g2e3 in ovarian and testicular germ cell lines significantly downregulates the expression of key genes involved in oogenesis (e.g., sox9 and cyp19a) and spermatogenesis (e.g., tesk1 and piwil2), respectively. Furthermore, the analysis of mutations in the transcription factor binding sites reveals that mutations within the Myogenin, YY1, and JunB binding sites significantly impact the transcriptional activity of the Cs-g2e3 gene, with the mutation in the YY1 binding site exhibiting the most pronounced effect (p < 0.001). This study contributes to a deeper understanding of the tissue-specific expression patterns of Cs-g2e3 across various tissues in Cynoglossus semilaevis, as well as the potential regulatory influences of transcription factors on its promoter activity. These findings may facilitate future research endeavors aimed at elucidating the expression and functional roles of the Cs-g2e3 gene. Full article

The versatility of cytochrome P450 reductase (CPR) in transferring electrons to P450s from other closely related species has been extensively exploited, e.g., by using An. gambiae CPR (AgCPR), as a homologous surrogate, to validate the role of An. funestus P450s in insecticide resistance. However, genomic variation between the AgCPR and An. funestus CPR (AfCPR) suggests that the full metabolism spectrum of An. funestus P450s might be missed when using AgCPR. To test this hypothesis, we expressed AgCPR and AfCPR side-by-side with CYP6P9a and CYP6P9b and functionally validated their role in the detoxification of insecticides from five different classes. Major variations were observed within the FAD- and NADP-binding domains of AgCPR and AfCPR, e.g., the coordinates of the second FAD stacking residue AfCPR-Y⁴⁵⁶ differ from that of AgCPR-His⁴⁵⁶. While no significant differences were observed in the cytochrome c reductase activities, when co-expressed with their endogenous AfCPR, the P450s significantly metabolized higher amounts of permethrin and deltamethrin, with CYP6P9b-AfCPR membrane metabolizing α-cypermethrin as well. Only the CYP6P9a-AfCPR membrane significantly metabolized DDT (producing dicofol), bendiocarb, clothianidin, and chlorfenapyr (bioactivation into tralopyril). This demonstrates the broad substrate specificity of An. funestus CYP6P9a/-b, capturing their role in conferring cross-resistance towards unrelated insecticide classes, which can complicate resistance management. Full article

Cuttage is the main propagation method of tea plant cultivars in China. However, some tea softwood cuttings just form an expanded and loose callus at the base, without adventitious root (AR) formation during the propagation period. Meanwhile, exogenous auxin could promote the AR formation of tea plant cuttings, but the regulation mechanism has not yet explained clearly. We conducted this study to elucidate the regulatory mechanism of exogenous auxin-induced adventitious root (AR) formation of such cuttings. The transcriptional expression profile of non-rooting tea calluses in response to exogenous IBA and NAA was analyzed using ONT RNA Seq technology. In total, 56,178 differentially expressed genes (DEGs) were detected, and most of genes were significantly differentially expressed after 12 h of exogenous auxin treatment. Among these DEGs, we further identified 80 DEGs involved in the auxin induction pathway and AR formation. Specifically, 14 auxin respective genes (ARFs, GH3s, and AUX/IAAs), 3 auxin transporters (AUX22), 19 auxin synthesis- and homeostasis-related genes (cytochrome P450 (CYP450) and calmodulin-like protein (CML) genes), and 44 transcription factors (LOB domain-containing protein (LBDs), SCARECROW-LIKE (SCL), zinc finger protein, WRKY, MYB, and NAC) were identified from these DEGs. Moreover, we found most of these DEGs were highly up-regulated at some stage before AR formation, suggesting that they may play a potential role in the AR formation of tea plant cuttings. In summary, this study will provide a theoretical foundation to deepen our understanding of the molecular mechanism of AR formation in tea cuttings induced by auxin during propagation time. Full article

Drought stress is one of the significant abiotic stresses that limit soybean (Glycine max [L.] Merr.) growth and production. Ankyrin repeat (ANK) proteins, being highly conserved, occupy a pivotal role in diverse biological processes. ANK genes were classified into nine subfamilies according to conserved domains in the soybean genome. However, the function of ANK-TM subfamily proteins (Ankyrin repeat proteins with a transmembrane domain) in the abiotic-stress response to soybean remains poorly understood. In this study, we first demonstrated the subcellular localization of GmANKTM21 in the cell membrane and nucleus. Drought stress-induced mRNA levels of GmANKTM21, which encodes proteins belonging to the ANK-TM subfamily, Transgenic 35S:GmANKTM21 soybean improved drought tolerance at the germination and seedling stages, with higher stomatal closure in soybean, lower water loss, lower malondialdehyde (MDA) content, and less reactive oxygen species (ROS) production compared with the wild-type soybean (Dongnong50). RNA-sequencing (RNA-seq) and RT-qPCR analysis of differentially expressed transcripts in overexpression of GmANKTM21 further identified potential downstream genes, including GmSPK2, GmSPK4, and GmCYP707A1, which showed higher expression in transgenic soybean, than those in wild-type soybean and KEGG enrichment analysis showed that MAPK signaling pathways were mostly enriched in GmANKTM21 overexpressing soybean plants under drought stress conditions. Therefore, we demonstrate that GmANKTM21 plays an important role in tolerance to drought stress in soybeans. Full article