Multifunctional P450 Monooxygenase CftA Diversifies the Clifednamide Pool through Tandem C–H Bond Activations

Molecular evolution of a cytochrome P450 for the synthesis of potential antidepressant (2R,6R)-hydroxynorketamine

Chemical Communications ◽

10.1039/d0cc06729f ◽

2021 ◽

Author(s):

Ansgar Bokel ◽

Michael C. Hutter ◽

Vlada B. Urlacher

Keyword(s):

Cytochrome P450 ◽

Molecular Evolution ◽

Cytochrome P450 Monooxygenase ◽

P450 Monooxygenase ◽

Effective Synthesis

Engineered cytochrome P450 monooxygenase CYP154E1 enables the effective synthesis of the potential antidepressant (2R,6R)-hydroxynorketamine via N-demethylation and regio- and stereoselective hydroxylation of (R)-ketamine.

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Genomic and transcriptomic insights into the cytochrome P450 monooxygenase gene repertoire in the rice pest brown planthopper,Nilaparvata lugens

10.1603/ice.2016.108969 ◽

2016 ◽

Author(s):

Yanyuan Bao

Keyword(s):

Cytochrome P450 ◽

Brown Planthopper ◽

Nilaparvata Lugens ◽

Cytochrome P450 Monooxygenase ◽

Gene Repertoire ◽

P450 Monooxygenase ◽

Monooxygenase Gene ◽

Rice Pest

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Molecular Basis of Iterative C–H Oxidation by TamI, a Multifunctional P450 Monooxygenase from the Tirandamycin Biosynthetic Pathway

10.26434/chemrxiv.12710159.v1 ◽

2020 ◽

Author(s):

Sean A. Newmister ◽

Kinshuk Raj Srivastava ◽

Rosa V. Espinoza ◽

Kersti Caddell Haatveit ◽

Yogan Khatri ◽

...

Keyword(s):

Molecular Dynamics ◽

Molecular Dynamics Simulations ◽

Molecular Basis ◽

Hydrophobic Interactions ◽

Cytochromes P450 ◽

Targeted Mutagenesis ◽

P450 Monooxygenase ◽

Bond Functionalization ◽

Dynamics Simulations

Biocatalysis offers an expanding and powerful strategy to construct and diversify complex molecules by C-H bond functionalization. Due to their high selectivity, enzymes have become an essential tool for C-H bond functionalization and offer complementary reactivity to small-molecule catalysts. Hemoproteins, particularly cytochromes P450, have proven effective for selective oxidation of unactivated C-H bonds. Previously, we reported the in vitro characterization of an oxidative tailoring cascade in which TamI, a multifunctional P450 functions co-dependently with the TamL flavoprotein to catalyze regio- and stereoselective hydroxylations and epoxidation to yield tirandamycin A and tirandamycin B. TamI follows a defined order including 1) C10 hydroxylation, 2) C11/C12 epoxidation, and 3) C18 hydroxylation. Here we present a structural, biochemical, and computational investigation of TamI to understand the molecular basis of its substrate binding, diverse reactivity, and specific reaction sequence. The crystal structure of TamI in complex with tirandamycin C together with molecular dynamics simulations and targeted mutagenesis suggest that hydrophobic interactions with the polyene chain of its natural substrate are critical for molecular recognition. QM/MM calculations and molecular dynamics simulations of TamI with variant substrates provided detailed information on the molecular basis of sequential reactivity, and pattern of regio- and stereo-selectivity in catalyzing the three-step oxidative cascade.<br>

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Cytochrome P450 Monooxygenase-Mediated Metabolic Utilization of Benzo[a]Pyrene by Aspergillus Species

mBio ◽

10.1128/mbio.00558-19 ◽

2019 ◽

Vol 10 (3) ◽

Cited By ~ 3

Author(s):

Erin M. Ostrem Loss ◽

Mi-Kyung Lee ◽

Ming-Yueh Wu ◽

Julia Martien ◽

Wanping Chen ◽

...

Keyword(s):

Cytochrome P450 ◽

Environmental Pollutants ◽

Energy Generation ◽

Fungal Species ◽

Metabolic Changes ◽

Cytochrome P450 Monooxygenase ◽

Cellular Growth ◽

P450 Monooxygenase ◽

Content Type ◽

Fundamental Knowledge

ABSTRACT Soil-dwelling fungal species possess the versatile metabolic capability to degrade complex organic compounds that are toxic to humans, yet the mechanisms they employ remain largely unknown. Benzo[a]pyrene (BaP) is a pervasive carcinogenic contaminant, posing a significant concern for human health. Here, we report that several Aspergillus species are capable of degrading BaP. Exposing Aspergillus nidulans cells to BaP results in transcriptomic and metabolic changes associated with cellular growth and energy generation, implying that the fungus utilizes BaP as a growth substrate. Importantly, we identify and characterize the conserved bapA gene encoding a cytochrome P450 monooxygenase that is necessary for the metabolic utilization of BaP in Aspergillus. We further demonstrate that the fungal NF-κB-type velvet regulators VeA and VelB are required for proper expression of bapA in response to nutrient limitation and BaP degradation in A. nidulans. Our study illuminates fundamental knowledge of fungal BaP metabolism and provides novel insights into enhancing bioremediation potential. IMPORTANCE We are increasingly exposed to environmental pollutants, including the carcinogen benzo[a]pyrene (BaP), which has prompted extensive research into human metabolism of toxicants. However, little is known about metabolic mechanisms employed by fungi that are able to use some toxic pollutants as the substrates for growth, leaving innocuous by-products. This study systemically demonstrates that a common soil-dwelling fungus is able to use benzo[a]pyrene as food, which results in expression and metabolic changes associated with growth and energy generation. Importantly, this study reveals key components of the metabolic utilization of BaP, notably a cytochrome P450 monooxygenase and the fungal NF-κB-type transcriptional regulators. Our study advances fundamental knowledge of fungal BaP metabolism and provides novel insight into designing and implementing enhanced bioremediation strategies.

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Naturally Occurring Ecdysteroids in Triticum aestivum L. and Evaluation of Fenarimol as a Potential Inhibitor of Their Biosynthesis in Plants

International Journal of Molecular Sciences ◽

10.3390/ijms22062855 ◽

2021 ◽

Vol 22 (6) ◽

pp. 2855

Author(s):

Anna Janeczko ◽

Jana Oklestkova ◽

Danuše Tarkowská ◽

Barbara Drygaś

Keyword(s):

Triticum Aestivum ◽

Winter Wheat ◽

Triticum Aestivum L ◽

Growth Conditions ◽

Animal Kingdom ◽

Experimental Conditions ◽

P450 Monooxygenase ◽

Naturally Occurring ◽

Regulatory Effects ◽

Potential Use

Ecdysteroids (ECs) are steroid hormones originally found in the animal kingdom where they function as insect molting hormones. Interestingly, a relatively high number of these substances can also be formed in plant cells. Moreover, ECs have certain regulatory effects on plant physiology, but their role in plants still requires further study. One of the main aims of the present study was to verify a hypothesis that fenarimol, an inhibitor of the biosynthesis of ECs in the animal kingdom, also affects the content of endogenous ECs in plants using winter wheat Triticum aestivum L. as a model plant. The levels of endogenous ECs in winter wheat, including the estimation of their changes during a course of different temperature treatments, have been determined using a sensitive analytical method based on UHPLC-MS/MS. Under our experimental conditions, four substances of EC character were detected in the tissue of interest in amounts ranging from less than 1 to over 200 pg·g−1 FW: 20-hydroxyecdysone, polypodine B, turkesterone, and isovitexirone. Among them, turkesterone was observed to be the most abundant EC and accumulated mainly in the crowns and leaves of wheat. Importantly, the level of ECs was observed to be dependent on the age of the plants, as well as on growth conditions (especially temperature). Fenarimol, an inhibitor of a cytochrome P450 monooxygenase, was shown to significantly decrease the level of naturally occurring ECs in experimental plants, which may indicate its potential use in studies related to the biosynthesis and physiological function of these substances in plants.

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Exposure to low doses of pesticides induces an immune response and the production of nitric oxide in honeybees

Scientific Reports ◽

10.1038/s41598-021-86293-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Merle T. Bartling ◽

Susanne Thümecke ◽

José Herrera Russert ◽

Andreas Vilcinskas ◽

Kwang-Zin Lee

Keyword(s):

Nitric Oxide ◽

Immune Responses ◽

Bacterial Pathogen ◽

Wild Plants ◽

P450 Monooxygenase ◽

Abiotic Stressors ◽

Genes Encoding ◽

Pesticides Exposure ◽

Colony Survival ◽

Oxide Synthase

AbstractHoneybees are essential pollinators of many agricultural crops and wild plants. However, the number of managed bee colonies has declined in some regions of the world over the last few decades, probably caused by a combination of factors including parasites, pathogens and pesticides. Exposure to these diverse biotic and abiotic stressors is likely to trigger immune responses and stress pathways that affect the health of individual honeybees and hence their contribution to colony survival. We therefore investigated the effects of an orally administered bacterial pathogen (Pseudomonas entomophila) and low-dose xenobiotic pesticides on honeybee survival and intestinal immune responses. We observed stressor-dependent effects on the mean lifespan, along with the induction of genes encoding the antimicrobial peptide abaecin and the detoxification factor cytochrome P450 monooxygenase CYP9E2. The pesticides also triggered the immediate induction of a nitric oxide synthase gene followed by the delayed upregulation of catalase, which was not observed in response to the pathogen. Honeybees therefore appear to produce nitric oxide as a specific defense response when exposed to xenobiotic stimuli. The immunity-related and stress-response genes we tested may provide useful stressor-dependent markers for ecotoxicological assessment in honeybee colonies.

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Cooperative B–H and Si–H Bond Activations by κ2-N,S-Chelated Ruthenium Borate Complexes

Inorganic Chemistry ◽

10.1021/acs.inorgchem.0c03306 ◽

2021 ◽

Author(s):

Mohammad Zafar ◽

Rongala Ramalakshmi ◽

Asif Ahmad ◽

P. K. Sudhadevi Antharjanam ◽

Sébastien Bontemps ◽

...

Keyword(s):

Borate Complexes ◽

Bond Activations

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In Silico Structural Modeling and Analysis of Interactions of Tremellomycetes Cytochrome P450 Monooxygenases CYP51s with Substrates and Azoles

International Journal of Molecular Sciences ◽

10.3390/ijms22157811 ◽

2021 ◽

Vol 22 (15) ◽

pp. 7811

Author(s):

Olufunmilayo Olukemi Akapo ◽

Joanna M. Macnar ◽

Justyna D. Kryś ◽

Puleng Rosinah Syed ◽

Khajamohiddin Syed ◽

...

Keyword(s):

Cytochrome P450 ◽

Structural Analysis ◽

In Silico ◽

Web Application ◽

Cytochrome P450 Monooxygenases ◽

Agglomerative Clustering ◽

Ligand Complex ◽

P450 Monooxygenase ◽

Study Results

Cytochrome P450 monooxygenase CYP51 (sterol 14α-demethylase) is a well-known target of the azole drug fluconazole for treating cryptococcosis, a life-threatening fungal infection in immune-compromised patients in poor countries. Studies indicate that mutations in CYP51 confer fluconazole resistance on cryptococcal species. Despite the importance of CYP51 in these species, few studies on the structural analysis of CYP51 and its interactions with different azole drugs have been reported. We therefore performed in silico structural analysis of 11 CYP51s from cryptococcal species and other Tremellomycetes. Interactions of 11 CYP51s with nine ligands (three substrates and six azoles) performed by Rosetta docking using 10,000 combinations for each of the CYP51-ligand complex (11 CYP51s × 9 ligands = 99 complexes) and hierarchical agglomerative clustering were used for selecting the complexes. A web application for visualization of CYP51s’ interactions with ligands was developed (http://bioshell.pl/azoledocking/). The study results indicated that Tremellomycetes CYP51s have a high preference for itraconazole, corroborating the in vitro effectiveness of itraconazole compared to fluconazole. Amino acids interacting with different ligands were found to be conserved across CYP51s, indicating that the procedure employed in this study is accurate and can be automated for studying P450-ligand interactions to cater for the growing number of P450s.

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Pervasive cooperative mutational effects on multiple catalytic enzyme traits emerge via long-range conformational dynamics

Nature Communications ◽

10.1038/s41467-021-21833-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Carlos G. Acevedo-Rocha ◽

Aitao Li ◽

Lorenzo D’Amore ◽

Sabrina Hoebenreich ◽

Joaquin Sanchis ◽

...

Keyword(s):

Long Range ◽

Fitness Landscape ◽

Conformational Dynamics ◽

P450 Monooxygenase ◽

Access Channel ◽

Long Range Interactions ◽

Limited Protein ◽

Dynamics Simulations ◽

The Relationship ◽

Single Enzyme

AbstractMultidimensional 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.

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