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Multidimensional fitness landscapes provide insights into the molecular basis of laboratory and natural evolution. To date, such efforts usually focus on limited protein families and a single enzyme trait, with little concern about the relationship between protein epistasis and conformational dynamics. Here, we report a multiparametric fitness landscape for a cytochrome P450 monooxygenase that was engineered for the regio- and stereoselective hydroxylation of a steroid. We develop a computational program to automatically quantify non-additive effects among all possible mutational pathways, finding pervasive cooperative signs and magnitude epistasis on multiple catalytic traits. By using quantum mechanics and molecular dynamics simulations, we show that these effects are modulated by long-range interactions in loops, helices and β-strands that gate the substrate access channel allowing for optimal catalysis. Our work highlights the importance of conformational dynamics on epistasis in an enzyme involved in secondary metabolism and offers insights for engineering P450s. Connecting conformational dynamics and epistasis has so far been limited to a few proteins and a single fitness trait. Here, the authors provide evidence of positive epistasis on multiple catalytic traits in the evolution and dynamics of engineered cytochrome P450 monooxygenase, offering insights for in silico protein design.

Candida albicans (C. albicans) is an opportunistic pathogen in immunocompromised individuals and a normal inhabitant of the oral cavity, throat, gastrointestinal tract, and genitourinary system among health populations. Our study focused on identifying new inhibitors capable of binding to the mutant cytochrome P450 family 51 (CYP-51) protein and intended to be effective against resistant C. albicans infections. The pharmacophore ligand-based model was used for the virtual screening of compound libraries. Molecular docking was performed on Maestro, Schrodinger. ADMET analysis was performed to check drug-likeness properties. Density function theory (DFT) calculations, molecular dynamic (MD) simulation, and free binding energy (MMPBSA) were also calculated. For docking, six compounds were selected from 11,022 hits from PubChem libraries, which showed the best interaction with mutant CYP-51 and were identified by pharmacophore mapping performed with the Pharma IT tool. Each of the six compounds was docked into the active site of the mutant CYP-51 protein. Overall, CP-3 exhibited significant binding affinity (−10.70 kcal/mol) as well as, showed good ADMET characteristics such as drug-likeness, absorption, distribution, metabolism, excretion, and toxicity. The lead compound, CP-3, was further used for MD simulation to observe the dynamic behavior of the complex in the active site of the mutant CYP-51 protein. Computational studies indicated that CP-3 could be a useful antagonist for the mutant protein, CYP-51. This study used computational approaches to identify potential inhibitors of C. albicans by targeting CYP-51 for antifungal drug development. Further invitro and in vivo studies are needed to evaluate its pharmacokinetic properties and efficacy as a novel antifungal drug.

Fruit growth and ripening are controlled by multiple phytohormones. How these hormones coordinate and interact with each other to control these processes at the molecular level is unclear. We found in the early stages of Fragaria vesca (woodland strawberry) fruit development, auxin increases both widths and lengths of fruits, while gibberellin [gibberellic acid (GA)] mainly promotes their longitudinal elongation. Auxin promoted GA biosynthesis and signaling by activating GA biosynthetic and signaling genes, suggesting auxin function is partially dependent on GA function. To prevent the repressive effect of abscisic acid (ABA) on fruit growth, auxin and GA suppressed ABA accumulation during early fruit development by activating the expression of FveCYP707A4a encoding cytochrome P450 monooxygenase that catalyzes ABA catabolism. At the onset of fruit ripening, both auxin and GA levels decreased, leading to a steep increase in the endogenous level of ABA that drives fruit ripening. ABA repressed the expression of FveCYP707A4a but promoted that of FveNCED, a rate-limiting step in ABA biosynthesis. Accordingly, altering FveCYP707A4a expression changed the endogenous ABA levels and affected FveNCED expression. Hence, ABA catabolism and biosynthesis are tightly linked by feedback and feedforward loops to limit ABA contents for fruit growth and to quickly increase ABA contents for the onset of fruit ripening. These results indicate that FveCYP707A4a not only regulates ABA accumulation but also provides a hub to coordinate fruit size and ripening times by relaying auxin, GA, and ABA signals.

Cytochromes P450 (P450 or CYP) are heme-containing enzymes that catalyze the introduction of one atom of molecular oxygen into nonactivated C–H bonds, often in a regio- and stereoselective manner. This ability, combined with a tremendous number of accepted substrates, makes P450s powerful biocatalysts. Sixty years after their discovery, P450 systems are recognized as essential bio-bricks in synthetic biology approaches to enable production of high-value complex molecules in recombinant hosts. Recent impressive results in protein engineering led to P450s with tailored properties that are even able to catalyze abiotic reactions. The introduction of P450s in artificial multi-enzymatic cascades reactions and chemo-enzymatic processes offers exciting future perspectives to access novel compounds that cannot be synthesized by nature or by chemical routes. Cytochromes P450 are ubiquitous enzymes accepting a tremendous number of substrates and catalyzing a broad range of reactions with potential applications in biotechnology and synthetic biology. P450s were engineered to catalyze abiotic reactions such as carbene or nitrene transfers, opening up completely new perspectives in synthetic chemistry. Lately, P450s have successfully been introduced into artificial multi-enzyme cascades, both in vitro and in vivo, providing alternative routes for retro-synthetic production of high-value oxyfunctionalized compounds. Harnessing the synthetic potential of P450s in chemo-enzymatic processes or as part of reconstituted biosynthetic pathways in microbial hosts provides promising strategies for de novo synthesis of synthons and complex natural products, even though there are still some obstacles to overcome.

Summary Suberin acts as stress‐induced antipathogen barrier in the root cell wall. CYP86A1 encodes cytochrome P450 fatty acid ω‐hydroxylase, which has been reported to be a key enzyme for suberin biosynthesis; however, its role in resistance to fungi and the mechanisms related to immune responses remain unknown. Here, we identified a disease resistance‐related gene, GbCYP86A1‐1, from Gossypium barbadense cv. Hai7124. There were three homologs of GbCYP86A1 in cotton, which are specifically expressed in roots and induced by Verticillium dahliae. Among them, GbCYP86A1‐1 contributed the most significantly to resistance. Silencing of GbCYP86A1‐1 in Hai7124 resulted in severely compromised resistance to V. dahliae, while heterologous overexpression of GbCYP86A1‐1 in Arabidopsis improved tolerance. Tissue sections showed that the roots of GbCYP86A1‐1 transgenic Arabidopsis had more suberin accumulation and significantly higher C16‐C18 fatty acid content than control. Transcriptome analysis revealed that overexpression of GbCYP86A1‐1 not only affected lipid biosynthesis in roots, but also activated the disease‐resistant immune pathway; genes encoding the receptor‐like kinases (RLKs), receptor‐like proteins (RLPs), hormone‐related transcription factors, and pathogenesis‐related protein genes (PRs) were more highly expressed in the GbCYP86A1‐1 transgenic line than control. Furthermore, we found that when comparing V. dahliae ‐inoculated and noninoculated plants, few differential genes related to disease immunity were detected in the GbCYP86A1‐1 transgenic line; however, a large number of resistance genes were activated in the control. This study highlights the role of GbCYP86A1‐1 in the defence against fungi and its underlying molecular immune mechanisms in this process.

Summary As one of the largest gene families in plants, the cytochrome P450 monooxygenase genes (CYPs) are involved in diverse biological processes including biotic and abiotic stress response. Moreover, P450 genes are prone to expanding due to gene tandem duplication during evolution, resulting in generations of novel alleles with the neo‐function or enhanced function. Here, the bread wheat (Triticum aestivum) gene TaCYP81D5 was found to lie within a cluster of five tandemly arranged CYP81D genes, although only a single such gene (BdCYP81D1) was present in the equivalent genomic region of the wheat relative Brachypodium distachyon. The imposition of salinity stress could up‐regulate TaCYP81D5, but the effect was abolished in plants treated with an inhibitor of reactive oxygen species synthesis. In SR3, a wheat cultivar with an elevated ROS content, the higher expression and the rapider response to salinity of TaCYP81D5 were related to the chromatin modification. Constitutively expressing TaCYP81D5 enhanced the salinity tolerance both at seedling and reproductive stages of wheat via accelerating ROS scavenging. Moreover, an important component of ROS signal transduction, Zat12, was proven crucial in this process. Though knockout of solely TaCYP81D5 showed no effect on salinity tolerance, knockdown of BdCYP81D1 or all TaCYP81D members in the cluster caused the sensitivity to salt stress. Our results provide the direct evidence that TaCYP81D5 confers salinity tolerance in bread wheat and this gene is prospective for crop improvement.

The regulatory process for assessing the risks of pesticides to bees relies heavily on the use of the honeybee, Apis mellifera, as a model for other bee species. However, the validity of using A. mellifera as a surrogate for other Apis and non-Apis bees in pesticide risk assessment has been questioned. Related to this line of research, recent work on A. mellifera has shown that specific P450 enzymes belonging to the CYP9Q subfamily act as critically important determinants of insecticide sensitivity in this species by efficiently detoxifying certain insecticide chemotypes. However, the extent to which the presence of functional orthologs of these enzymes is conserved across the diversity of bees is unclear. Here we used a phylogenomic approach to identify > 100 putative CYP9Q functional orthologs across 75 bee species encompassing all major bee families. Functional analysis of 26 P450s from 20 representative bee species revealed that P450-mediated detoxification of certain systemic insecticides, including the neonicotinoid thiacloprid and the butenolide flupyradifurone, is conserved across all major bee pollinator families. However, our analyses also reveal that CYP9Q-related genes are not universal to all bee species, with some Megachilidae species lacking such genes. Thus, our results reveal an evolutionary conserved capacity to metabolize certain insecticides across all major bee families while identifying a small number of bee species where this function may have been lost. Furthermore, they illustrate the potential of a toxicogenomic approach to inform pesticide risk assessment for nonmanaged bee species by predicting the capability of bee pollinator species to break down synthetic insecticides.

During cold stimulation, cholesterol is converted to bile acids in an alternative pathway. The bile acids then alter the microbiota, which in turn promotes more heat generation. Adaptive thermogenesis is an energy-demanding process that is mediated by cold-activated beige and brown adipocytes, and it entails increased uptake of carbohydrates, as well as lipoprotein-derived triglycerides and cholesterol, into these thermogenic cells. Here we report that cold exposure in mice triggers a metabolic program that orchestrates lipoprotein processing in brown adipose tissue (BAT) and hepatic conversion of cholesterol to bile acids via the alternative synthesis pathway. This process is dependent on hepatic induction of cytochrome P450, family 7, subfamily b, polypeptide 1 (CYP7B1) and results in increased plasma levels, as well as fecal excretion, of bile acids that is accompanied by distinct changes in gut microbiota and increased heat production. Genetic and pharmacological interventions that targeted the synthesis and biliary excretion of bile acids prevented the rise in fecal bile acid excretion, changed the bacterial composition of the gut and modulated thermogenic responses. These results identify bile acids as important metabolic effectors under conditions of sustained BAT activation and highlight the relevance of cholesterol metabolism by the host for diet-induced changes of the gut microbiota and energy metabolism.

Cytochrome P450 2U1 (CYP2U1) exhibits several distinctive characteristics among the 57 human CYPs, such as its presence in almost all living organisms with a highly conserved sequence, its particular gene organization with only five exons, its major location in thymus and brain, and its protein sequence involving an unusually long N-terminal region containing 8 proline residues and an insert of about 20 amino acids containing 5 arginine residues after the transmembrane helix. Few substrates, including fatty acids, N -arachidonoylserotonin (AS), and some drugs, have been reported so far. However, its biological roles remain largely unknown, even though CYP2U1 mutations have been involved in some pathological situations, such as complicated forms of hereditary spastic paraplegia. These data together with its ability to hydroxylate some fatty acids and AS suggest its possible role in lipid metabolism.

Cinnamate 4-hydroxylase (C4H) is the first key cytochrome P450 monooxygenase (P450) enzyme in the phenylpropanoid pathway. It belongs to the CYP73 family of P450 superfamily, and catalyzes the conversion of trans -cinnamic acid to p -coumaric acid. Since p -coumaric acid serves as the precursor for the synthesis of a wide variety of metabolites involved in plant development and stress resistance, alteration in the expression of soybean C4H genes is expected to affect the downstream metabolite levels, and its ability to respond to stress. In this study, we identified four C4H genes in the soybean genome that are distributed into both class I and class II CYP73 family. GmC4H2 , GmC4H14 and GmC4H20 displayed tissue- and developmental stage-specific gene expression patterns with their transcript accumulation at the highest level in root tissues. GmC4H10 appears to be a pseudogene as its transcript was not detected in any soybean tissues. Furthermore, protein homology modelling revealed substrate docking only for GmC4H2, GmC4H14 and GmC4H20. To demonstrate the function of GmC4Hs, we modified a cloning vector for the heterologous expression of P450s in yeast, and used it for microsomal protein production and enzyme assay. Our results confirmed that GmC4H2, GmC4H14 and GmC4H20 contain the ability to hydroxylate trans -cinnamic acid with varying efficiencies.