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Chiral methyl mandelates are useful synthons in organic transformation and pharmaceutical synthesis. Green synthesis of these valuable compounds by direct C–H activating oxidative hydroxylation has attracted keen interest. Described herein is achieving the stereoselective and efficient bio-hydroxylation of methyl 2-phenylacetates to the chiral methyl mandelates by directed evolution of the cytochrome P450DA hydroxylase. In the present study, a new colorimetric high-throughput screening assay was successfully developed based on a dualenzyme cascade for the engineering of the P450DA's hydroxylation activity. Several beneficial variants with enhanced bio-hydroxylation activity were created by combining random mutagenesis and site-saturated/directed mutagenesis strategies. Whole-cell bio-hydroxylation of various methyl 2-phenylacetates using the best septupletmutant P450DA-11 yielded the corresponding chiral methyl mandelates in up to 92% isolated yields and >99% ee. [ABSTRACT FROM AUTHOR]

As compounds of natural origin enter human body, it is necessary to investigate their possible interactions with the metabolism of drugs and xenobiotics in general, namely with the cytochrome P450 (CYP) system. Phytic acid (myo-inositol hexaphosphoric acid, IP6) is mainly present in plants but is also an endogenous compound present in mammalian cells and tissues. It has been shown to exhibit protective effect in many pathological conditions. For this paper, its interaction with CYPs was studied using human liver microsomes, primary human hepatocytes, the HepG2 cell line, and molecular docking. Docking experiments and absorption spectra demonstrated the weak ability of IP6 to interact in the heme active site of CYP1A. Molecular docking suggested that IP6 preferentially binds to the protein surface, whereas binding to the active site of CYP1A2 was found to be less probable. Subsequently, we investigated the ability of IP6 to modulate the metabolism of xenobiotics for both the mRNA expression and enzymatic activity of CYP1A enzymes. Our findings revealed that IP6 can slightly modulate the mRNA levels and enzyme activity of CYP1A. However, thanks to the relatively weak interactions of IP6 with CYPs, the chances of the mechanisms of clinically important drug–drug interactions involving IP6 are low. [ABSTRACT FROM AUTHOR]

Human cytochrome P450 2F1, as well as its ortholog 2F3 in domestic goat, is considered to be a rather unusual enzyme. The only type of reaction it catalyzes has been described: the conversion of skatole, a product of anaerobic tryptophan metabolism, into a pulmonary toxin. Endogenous substrates of CYP2F are unknown, and although more than 30 years have passed since the discovery of the enzyme, its biological role remains unclear. We hypothesized that the physiological functions of CYP2F can be specifically implemented in the brain, remaining previously unnoticed owing to the high compartmentalization of the organ. Using open data, we studied the covariation of the expression of CYP2F1 and genes for behavioral traits: in the human brain, as well as the polymorphism of their orthologs and CYP2F3 in 180 populations of domestic goats (Capra hircus). Two SNPs were found in the CYP2F3 gene, one of which had pronounced traces of selection, and the frequency of homozygotes increased with geographic distance from the center of domestication. Expression of CYP2F1 mRNA in the human brain also had regional specificity. In both species, factor analysis revealed the relationship between CYP2F1/3 and a number of genes regulating behavior: the serotonin transporter SLC6A4 and its receptor HTR2A3, the ABCB1 transporter, the purine receptor P2RX7, the GABA receptor GABRA4, the circadian rhythm regulator PER3, and T-cadherin CDH13. Thus, analysis of the genomic data of the domestic goat and human transcriptomic data revealed the evolutionary and functional relationships of CYP2F cytochromes and neurochemical systems for regulating behavior. This evidence of the cerebral function of the enzyme is indirect, since it is based on correlation analysis, but indicates the promise of further search in this direction. [ABSTRACT FROM AUTHOR]

This article explores the potential of cytochromes P450 (P450s) as biocatalysts for oxidation reactions. Despite their potential, P450s have not been extensively used in research and industry due to challenges. The authors present a case study on the iron-catalyzed Wacker-type oxidation of olefins using ambient air as the sole oxidant, which has shown promising results for the functionalization of complex molecules. The text also discusses other strategies for the oxidation of organic compounds to produce aryl aldehydes, highlighting their selectivity and compatibility with complex molecules. The article concludes by discussing recent advances in catalysis inspired by P450 enzymes, which have the potential to accelerate the development of versatile synthetic catalysts with applications in drug synthesis and bioremediation. [Extracted from the article]

This study assessed the effect of tubeimoside I on CYP1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 to reveal the potential of tubeimoside I to induce drug-drug interaction. The evaluation of cytochromes P450 enzyme (CYP) activity was performed in pooled human liver microsomes with probing substrates of CYP1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4. Typical inhibitors were employed as positive controls and the effect of 0, 2.5, 5, 10, 25, 50, and 100 μM tubeimoside I was investigated. The activity of CYP2D6, 2E1, and 3A4 was significantly inhibited by tubeimoside I with the IC50 values of 10.34, 11.58, and 9.74 μM, respectively. The inhibition of CYP2D6 and 2E1 was competitive with the Ki value of 5.66 and 5.29 μM, respectively. While the inhibition of CYP3A4 was non-competitive with the Ki value of 4.87 μM. Moreover, the inhibition of CYP3A4 was time-dependent with the KI and Kinact values of 0.635 μM−1 and 0.0373 min−1, respectively. Tubeimoside I served as a competitive inhibitor of CYP2D6 and 2E1 exerting weak inhibition and a non-competitive inhibitor of CYP3A4 exerting moderate inhibition. [ABSTRACT FROM AUTHOR]

Aim: Cytochrome P450 is a hepatic enzyme system responsible for drug metabolism that comprises different iso-enzymes. The CYP2D6 enzyme, which composes 2-4% of all cytochrome enzymes, is responsible for 25% of the metabolisms of the drugs used in clinics. The genetic polymorphisms of this enzyme can change the efficiency and toxicity of the drugs used. In this study, the retrospective evaluation of CYP2D6 gene polymorphisms that influence enzyme activity in the Turkish population is targeted. Materials and Methods: From the 192 patients sent from psychiatry, medical genetics, and neurology polyclinics to our laboratory, we determined CYP2D6 enzyme gene polymorphisms by multiplex polymerase chain reaction (PCR) and multiplex allele-specific primer extension (ASPE). Results: Of all the cases, 82.29% were determined to be extensive, 12.5% intermediate, 2.08% poor, and 3.13% ultra-rapid metabolizers. In our population, the most frequent alleles were *1 (37.63%), *2 (24.75%), *41 (15.15%), *4 (9.85%), and *10 (4.29%), respectively. Conclusion: Compared to previous studies conducted on Turkish society, the percentage of extensive metabolizers was higher in the current examined population. In contrast, the percentages of poor and ultra-rapid metabolizers were lower. In addition, the *41 allele, which has not been studied before in our population, that follows the decrease in enzyme function was detected as the most frequent allele in this study. The fact that the percentage of the cases in which changes were observed was 17.1% will help determine the CYP2D6 gene mutations in the pre-treatment cases. [ABSTRACT FROM AUTHOR]

Cytochrome P450 3A4 and 2D6 (EC 1.14.13.97 and 1.14.14.1; CYP3A4 and 2D6) are heme-containing enzymes that catalyze the oxidation of a wide number of xenobiotic and drug substrates and thus broadly impact human biology and pharmacologic therapies. Although their activities are directly proportional to their heme contents, little is known about the cellular heme delivery and insertion processes that enable their maturation to functional form. We investigated the potential involvement of GAPDH and chaperone Hsp90, based on our previous studies linking these proteins to intracellular heme allocation. We studied heme delivery and insertion into CYP3A4 and 2D6 after they were transiently expressed in HEK293T and GlyA CHO cells or when naturally expressed in HEPG2 cells in response to rifampicin, and also investigated their associations with GAPDH and Hsp90 in cells. The results indicate that GAPDH and its heme binding function is involved in delivery of mitochondria-generated heme to apo-CYP3A4 and 2D6, and that cell chaperone Hsp90 is additionally involved in driving their heme insertions. Uncovering how cells allocate heme to CYP3A4 and 2D6 provides new insight on their maturation processes and how this may help to regulate their functions in health and disease. [ABSTRACT FROM AUTHOR]

The fossil record demonstrates the preservation of porphyrins (e.g., heme) in organic sediments and the fossilized remains of animals. These molecules are essential components in modern metabolic processes, such as electron transport (cytochromes) and oxygen transport (hemoglobin), and likely originated before the emergence of life. The integration and adaptation of porphyrins and structurally similar molecules (e.g., chlorophylls) are key aspects in the evolution of energy production (i.e., aerobic respiration and photosynthesis) and complex life (i.e., eukaryotes and multicellularity). Here, we discuss the evolution and functional diversity of heme-bound hemoglobin proteins in vertebrates, along with the preservation of these molecules in the fossil record. By elucidating the pivotal role of these molecules in the evolution of life, this review lays the groundwork necessary to explore hemoglobin as a means to investigate the paleobiology of extinct taxa, including non-avian dinosaurs. [ABSTRACT FROM AUTHOR]

Cytochromes P450 (CYPs) are a group of monooxygenases that can be found in almost all kinds of organisms. For CYPs to receive electrons from co-substrate NADPH, the activity of NADPH-Cytochrome-P450-oxidoreductase (CPR) is required as well. In humans, CYPs are an integral part of liver-based phase-1 biotransformation, which is essential for the metabolization of multiple xenobiotics and drugs. Consequently, CYPs are important players during drug development and therefore these enzymes are implemented in diverse screening applications. For these applications it is usually advantageous to use mono CYP microsomes containing only the CYP of interest. The generation of mono-CYP containing mammalian cells and vesicles is difficult since endogenous CYPs are present in many cell types that contain the necessary co-factors. By obtaining translationally active lysates from a modified CHO-CPR cell line, it is now possible to generate mono CYPs in a cell-free protein synthesis process in a straightforward manner. As a proof of principle, the synthesis of active human CYPs from three different CYP450 gene families (CYP1A2, CYP2B6 and CYP3A4), which are of outstanding interest in industry and academia was demonstrated. Luciferase based activity assays confirm the activity of the produced CYPs and enable the individual adaptation of the synthesis process for efficient cell-free enzyme production. Furthermore, they allow for substrate and inhibitor screenings not only for wild-type CYPs but also for mutants and further CYP isoforms and variants. As an example, the turnover of selected CYP substrates by cell-free synthesized CYPs was demonstrated via an indirect luciferase assay-based screening setup. [ABSTRACT FROM AUTHOR]

Benthodytes occidentpalauta sp. nov. was collected from the Kyushu-Palau Ridge at a depth of 5 481 m in 2021. This new species is characterized by a gelatinous body wall, violet skin, six pairs of dorsal papillae, and a rough mid-ventral surface without tube feet. The dorsal deposits are rod-shaped and tripartite. Two types of papillae deposits as crosses with four arms with central bipartite apophyses. Ventral deposits are rods. Tentacle ossicles are rod-shaped with end protrusions. Gonad deposits are rod-shaped, tripartite, and cross-shaped. The phylogenetic analyses based on cytochrome oxidase subunit 1 (COI) and 16S individually and a concatenated dataset of COI and 16S genes of this species support that B. occidentpalauta sp. nov. belongs to Benthodytes. [ABSTRACT FROM AUTHOR]

Electroactive microorganisms (EAMs) could utilize extracellular electron transfer (EET) pathways to exchange electrons and energy with their external surroundings. Conductive cytochrome proteins and nanowires play crucial roles in controlling electron transfer rate from cytosol to extracellular electrode. Many previous studies elucidated how the c‐type cytochrome proteins and conductive nanowires are synthesized, assembled, and engineered to manipulate the EET rate, and quantified the kinetic processes of electron generation and EET. Here, we firstly overview the electron transfer pathways of EAMs and quantify the kinetic parameters that dictating intracellular electron production and EET. Secondly, we systematically review the structure, conductivity mechanisms, and engineering strategies to manipulate conductive cytochromes and nanowire in EAMs. Lastly, we outlook potential directions for future research in cytochromes and conductive nanowires for enhanced electron transfer. This article reviews the quantitative kinetics of intracellular electron production and EET, and the contribution of engineered c‐type cytochromes and conductive nanowire in enhancing the EET rate, which lay the foundation for enhancing electron transfer capacity of EAMs. [ABSTRACT FROM AUTHOR]

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a large class of secondary metabolites that have garnered scientific attention due to their complex scaffolds with potential roles in medicine, agriculture, and chemical ecology. RiPPs derive from the cleavage of ribosomally synthesized proteins and additional modifications, catalyzed by various enzymes to alter the peptide backbone or side chains. Of these enzymes, cytochromes P450 (P450s) are a superfamily of heme-thiolate proteins involved in many metabolic pathways, including RiPP biosyntheses. In this review, we focus our discussion on P450 involved in RiPP pathways and the unique chemical transformations they mediate. Previous studies have revealed a wealth of P450s distributed across all domains of life. While the number of characterized P450s involved in RiPP biosyntheses is relatively small, they catalyze various enzymatic reactions such as C–C or C–N bond formation. Formation of some RiPPs is catalyzed by more than one P450, enabling structural diversity. With the continuous improvement of the bioinformatic tools for RiPP prediction and advancement in synthetic biology techniques, it is expected that further cytochrome P450-mediated RiPP biosynthetic pathways will be discovered. Summary The presence of genes encoding P450s in gene clusters for ribosomally synthesized and post-translationally modified peptides expand structural and functional diversity of these secondary metabolites, and here, we review the current state of this knowledge. [ABSTRACT FROM AUTHOR]

Cytochrome c552 from Thermus thermophilus is one of the hot topics for creating smart biomaterials as it possesses remarkable stability, is tolerant to multiple mutations and has therefore been recently reported for a number of functionalizations upon substitution of the original prosthetic group with an artificial prosthetic group. However, all of the substitutions were driven by the coordination through the axial ligands followed by complete reconstitution with a metal–porphyrin complex. This limits the scope of the cytochrome c for incorporating a metal-less non-natural heme species that could improve the versatility of cytochrome c for a new generation of engineered cytochrome proteins for further enhancement in their functionalities such as biocatalysts. In this connection, a new variant of Cytochrome c (rC552 C14A) from Thermus thermophilus was reported, where an easy approach to remove the original prosthetic group was achieved, followed by the incorporation of a number of metal-PPIX derivatives that ultimately led to the formation of artificial c-type cytochromes through covalent bonding. The apo-cytochrome was found to be thermally tolerant and to possess a distinctive overall structure as that of the wild type, as was evident from the corresponding CD spectra, which ultimately encouraged reconstitution with a metal-less protoporphyrin derivative for better understanding the role of axial ligands in the reconstitution process. Successful reconstitution was achieved, resulting in a new type of Cytochrome b-type artificial protein without the metal in its active site, indicating the non-involvement of the axial ligand. In order to prove the non-involvement of the axial ligand, a subsequent double mutant (C14A/M69A) was constructed, replacing the methionine at 69 position with non-coordinating alanine residue. Accordingly, the apo-C14A/M69A was prepared and found to be extremely stable as the earlier mutants and the WT showed no signs of denaturation, even at the elevated temperature of 98°C. Subsequently, heme b was successfully incorporated into the apo-C14A/M69A, which demonstrated itself as a highly thermally tolerant protein scaffold for incorporating a metal-less artificial prosthetic group in the absence of the axial ligand. Further improvement in the reconstitution process is achieved by replacing the methionine at 69 position with phenyl alanine (C14A/M69F mutant), resulting in further stabilization of heme species, possibly through non-covalent π–interactions, as corroborated by molecular docking. [ABSTRACT FROM AUTHOR]

Background: Vitamins have a great impact on metabolis. Aims: To determine the role of 1,25(OH)2D On Cytochromes CYP27A1 and CYP27B1 in Periodontitis. Material and Method: The investigation was carried out on 45 participants of ages within the range of (30-45 years) who were attending the private dental clinics. Diagnosis of chronic periodontitis was established depending on dental history, clinical examinations (periodontal indices). All participants were examined by the same dentist. They were classified into three groups: Group 1 (control negative): (15) participants with normal serum vitamin D3 level and with pocket depth =3 mm, good oral health and normal periodontal tissues and no previous history of periodontal diseases. Group 2 (control positive): (15) participants with normal serum vitamin D3 level and periodontitis with pocket depth =5 mm, they received placebo medication orally, Group3(treatment): (15) participants with vitamin D3 deficiency (below 30 IU), and periodontitis with pocket depth ≥5 mm, they received oral Vitamin D3 fast acting liquid soft gel capsule 2000 IU/day for 3 months. 3 blood samples were taken from each participant at 0,45,90 days, for research examinations. CYP27A1, CYP27B1 serum levels was measured for each sample in three groups by ELISA kit. Result: there was a highly significant reduction in CYP27A1 serum level in the treatment group at the ninety days of the study while there was no significant elevation CYP27B1 serum level in all groups during 45,90 days of the study. Conclusion: The present study suggested that the 1,25(OH)2D has effects on serum levels of both Cytochromes CYP27A1 and CYP27B1 and this was associated with periodontitis. [ABSTRACT FROM AUTHOR]

We evaluate cryoEM and crystal structures of two molecular machines that traffick heme and attach it to cytochrome c (cyt c), the second activity performed by a cyt c synthase. These integral membrane proteins, CcsBA and CcmF/H, both covalently attach heme to cyt c, but carry it out via different mechanisms. A CcsB-CcsA complex transports heme through a channel to its external active site, where it forms two thioethers between reduced (Fe+2) heme and CysXxxXxxCysHis in cyt c. The active site is formed by a periplasmic WWD sequence and two histidines (P-His1 and P-His2). We evaluate each proposed functional domain in CcsBA cryoEM densities, exploring their presence in other CcsB-CcsA proteins from a wide distribution of organisms (e.g., from Gram positive to Gram negative bacteria to chloroplasts.) Two conserved pockets, for the first and second cysteines of CXXCH, explain stereochemical heme attachment. In addition to other universal features, a conserved periplasmic beta stranded structure, called the beta cap, protects the active site when external heme is not present. Analysis of CcmF/H, here called an oxidoreductase and cyt c synthase, addresses mechanisms of heme access and attachment. We provide evidence that CcmF/H receives Fe+3 heme from holoCcmE via a periplasmic entry point in CcmF, whereby heme is inserted directly into a conserved WWD/P-His domain from above. Evidence suggests that CcmF acts as a heme reductase, reducing holoCcmE (to Fe+2) through a transmembrane electron transfer conduit, which initiates a complicated series of events at the active site. [ABSTRACT FROM AUTHOR]

Electroactive microbial cells have evolved unique extracellular electron transfer to conduct the reactions via redox outer-membrane (OM) proteins. However, the electron transfer mechanism at the interface of OM proteins and nanomaterial remains unclear. In this study, the mechanism for the electron transfer at biological/inorganic interface is investigated by integrating molecular modeling with electrochemical and spectroscopic measurements. For this purpose, a model system composed of OmcA, a typical OM protein, and the hexagonal tungsten trioxide (h-WO3) with good biocompatibility is selected. The interfacial electron transfer is dependent mainly on the special molecular configuration of OmcA and the microenvironment of the solvent exposed active center. Also, the apparent electron transfer rate can be tuned by site-directed mutagenesis at the axial ligand of the active center. Furthermore, the equilibrium state of the OmcA/h-WO3 systems suggests that their attachment is attributed to the limited number of residues. The electrochemical analysis of OmcA and its variants reveals that the wild type exhibits the fastest electron transfer rate, and the transient absorption spectroscopy further shows that the axial histidine plays an important role in the interfacial electron transfer process. This study provides a useful approach to promote the site-directed mutagenesis and nanomaterial design for bioelectrocatalytic applications. [ABSTRACT FROM AUTHOR]

c-type Cytochromes (c-Cyts), primarily as electron carriers and oxidoreductases, play a key role in energy transduction processes in virtually all living organisms. Many bacteria, such as Shewanella oneidensis, are particularly rich in c-Cyts, supporting respiratory versatility not seen in eukaryotes. Unfortunately, a large number of c-Cyts are underexplored, and their biological functions remain unknown. In this study, we identify SorCABD of S. oneidensis as a novel sulfite dehydrogenase (SDH), which catalyzes the oxidation of sulfite to sulfate. In addition to catalytic subunit SorA, this enzymatic complex includes three c-Cyt subunits, which all together carry out electron transfer. The electrons extracted from sulfite oxidation are ultimately delivered to oxygen, leading to oxygen reduction, a process relying on terminal oxidase cyt cbb3. Genomic analysis suggests that the homologs of this SDH are present in a small number of bacterial genera, Shewanella and Vibrio in particular. Because these bacteria are generally capable of reducing sulfite under anaerobic conditions, the co-existence of a sulfite oxidation system implies that they may play especially important roles in the transformation of sulfur species in natural environments. [ABSTRACT FROM AUTHOR]

N-nitrosamine impurities have been increasingly detected in human drugs. This is a safety concern as many nitrosamines are mutagenic in bacteria and carcinogenic in rodent models. Typically, the mutagenic and carcinogenic activity of nitrosamines requires metabolic activation by cytochromes P450 enzymes (CYPs), which in many in vitro models are supplied exogenously using rodent liver homogenates. There are only limited data on the genotoxicity of nitrosamines in human cell systems. In this study, we used metabolically competent human HepaRG cells, whose metabolic capability is comparable to that of primary human hepatocytes, to evaluate the genotoxicity of eight nitrosamines [N-cyclopentyl-4-nitrosopiperazine (CPNP), N-nitrosodibutylamine (NDBA), N-nitrosodiethylamine (NDEA), N-nitrosodimethylamine (NDMA), N-nitrosodiisopropylamine (NDIPA), N-nitrosoethylisopropylamine (NEIPA), N-nitroso-N-methyl-4-aminobutyric acid (NMBA), and N-nitrosomethylphenylamine (NMPA)]. Under the conditions we used to culture HepaRG cells, three-dimensional (3D) spheroids possessed higher levels of CYP activity compared to 2D monolayer cells; thus the genotoxicity of the eight nitrosamines was investigated using 3D HepaRG spheroids in addition to more conventional 2D cultures. Genotoxicity was assessed as DNA damage using the high-throughput CometChip assay and as aneugenicity/clastogenicity in the flow-cytometry-based micronucleus (MN) assay. Following a 24-h treatment, all the nitrosamines induced DNA damage in 3D spheroids, while only three nitrosamines, NDBA, NDEA, and NDMA, produced positive responses in 2D HepaRG cells. In addition, these three nitrosamines also caused significant increases in MN frequency in both 2D and 3D HepaRG models, while NMBA and NMPA were positive only in the 3D HepaRG MN assay. Overall, our results indicate that HepaRG spheroids may provide a sensitive, human-based cell system for evaluating the genotoxicity of nitrosamines. [ABSTRACT FROM AUTHOR]

Nivasorexant, a selective orexin‐1‐receptor antagonist, has recently been assessed in the treatment of humans with binge‐eating disorder. Herein, the inhibitory potential of nivasorexant on cytochromes P450 (CYPs) 2C9, 2C19, and 3A4 was evaluated. Human liver microsomes/recombinant CYP enzymes were evaluated in vitro. In vivo, a single‐center, open‐label, fixed‐sequence study was performed in healthy adults to explore the effect of 100 mg nivasorexant administered twice daily (b.i.d.) on the pharmacokinetics (PK) of flurbiprofen (50 mg, CYP2C9), omeprazole (20 mg, CYP2C19), midazolam (2 mg, CYP3A4) making use of a cocktail approach. Plasma PK sampling was performed over 24 h on Day 1 (Cocktail alone), 8 (Cocktail + nivasorexant), and 15 (Cocktail + nivasorexant at steady state). Genotyping of subjects' CYPs was performed while safety and tolerability were also assessed. In vitro, nivasorexant inhibited CYP2C9, 2C19, and 3A4 in competitive inhibition assays with IC50 values of 8.6, 1.6, and 19–44 μM, respectively, while showing a significant time‐dependent CYP2C19 inhibition. In 22 subjects, exposure to flurbiprofen, omeprazole, and midazolam was generally higher during concomitant single‐ (i.e., area under the plasma concentration–time curve [AUC] ratio increased by 1.04‐, 2.05‐, and 1.56‐fold, respectively) and repeated‐dose (i.e., AUC ratio increased by 1.47‐, 6.84‐, and 3.71‐fold, respectively) nivasorexant administration compared with the cocktail substrates administered alone. The most frequently reported adverse event was somnolence. According to regulatory guidance, nivasorexant is classified as a moderate CYP2C19 and weak CYP3A4 inhibitor after 1 day and as a weak CYP2C9, strong CYP2C19, and moderate CYP3A4 inhibitor after 8 days of 100 mg b.i.d. administration. Clinicaltrials.gov ID: NCT05254548. [ABSTRACT FROM AUTHOR]

Cytochromes P450 are a unique family of enzymes found in all Kingdoms of living organisms (animals, bacteria, plants, fungi, and archaea), whose main function is biotransformation of exogenous and endogenous compounds. The review discusses approaches to enhancing the efficiency of electrocatalysis by cytochromes P450 for their use in biotechnology and design of biosensors and describes main methods in the development of reconstituted and electrochemical catalytic systems based on the biochemical mechanism of cytochromes P450, as well as and modern trends for their practical application. [ABSTRACT FROM AUTHOR]

Microbial extracellular reduction of insoluble compounds requires soluble electron shuttles that diffuse in the environment, freely diffusing cytochromes, or direct contact with cellular conductive appendages that release or harvest electrons to assure a continuous balance between cellular requirements and environmental conditions. In this work, we produced and characterized the three cytochrome domains of PgcA, an extracellular triheme cytochrome that contributes to Fe(III) and Mn(IV) oxides reduction in Geobacter sulfurreducens. The three monoheme domains are structurally homologous, but their heme groups show variable axial coordination and reduction potential values. Electron transfer experiments monitored by NMR and visible spectroscopy show the variable extent to which the domains promiscuously exchange electrons while reducing different electron acceptors. The results suggest that PgcA is part of a new class of cytochromes - microbial heme-tethered redox strings - that use low-complexity protein stretches to bind metals and promote intra- and intermolecular electron transfer events through its cytochrome domains. [ABSTRACT FROM AUTHOR]

Understanding the effects of inflammation and cirrhosis on the regulation of drug metabolism during the progression of hepatocellular carcinoma (HCC) is critical for developing patient‐specific treatment strategies. Herein, novel 3D vascularized HCC on chips (HCCoCs), composed of HCC, endothelial, stellate, and Kupffer cells tuned to mimic normal or cirrhotic liver stiffness, are created. HCC inflammation is controlled by tuning Kupffer macrophage numbers, and the impact of cytochrome P450‐3A4 (CYP3A4) is investigated by culturing HepG2 HCC cells transfected with CYP3A4 to upregulate expression from baseline. This model allows for the simulation of chemotherapeutic delivery methods such as intravenous injection and transcatheter arterial chemoembolization (TACE). It is shown that upregulation of metabolic activity, incorporation of cirrhosis and inflammation, increases vascular permeability due to upregulated inflammatory cytokines leading to significant variability in chemotherapeutic treatment efficacy. Specifically, it is shown that further modulation of CYP3A4 activity of HCC cells by TACE delivery of doxorubicin provides an additional improvement to treatment response and reduces chemotherapy‐associated endothelial porosity increase. The HCCoCs are shown to have utility in uncovering the impact of the tumor microenvironment during cancer progression on vascular properties, tumor response to therapeutics, and drug delivery strategies. [ABSTRACT FROM AUTHOR]

Distances between Fe ions in multiheme cytochromes are sufficiently short to make the intramolecular dipole-dipole interaction between hemes probable. In the analysis of EPR data from cytochromes, this interaction has thus far been ignored under the assumption that spectra are the simple sum of non-interacting components. Here, we use a recently developed low-frequency broadband EPR spectrometer to establish the extent of dipolar interaction in the example cytochromes, characterize its spectral signatures, and identify present limitations in the analysis. Broadband EPR spectra of Shewanella oneidensis MR-1 small tetraheme cytochrome (STC) have been collected over the frequency range of 0.45 to 13.11 GHz, and they have been compared to similar data from Desulfovibrio vulgaris Hildenborough cytochrome c3. The two cases are representative examples of two very different heme topologies and corresponding electron-transfer properties in tetraheme proteins. While in cytochrome c3, the six Fe-Fe distances can be sorted into two well-separated groups, those in STC are diffuse. Since the onset of dipolar interaction between Fe-Fe pairs is already observed in the X-band, the g values are determined in the simulation of the 13.11 GHz spectrum. Low-frequency spectra are analyzed with the inclusion of dipolar interaction based on available structural data on mutual distances and orientations between all hemes. In this procedure, all 24 possible assignments of individual heme spectra to heme topologies are sampled. The 24 configurations can be reduced to a few, but inspection falls short of a unique assignment, due to a remaining lack of understanding of the fine details of these complex spectra. In general, the EPR analysis suggests the four-heme system in c3 to be more rigid than that in STC, which is proposed to be related to different physiological roles in electron transfer. [ABSTRACT FROM AUTHOR]

Improper drug prescription is a main cause of both drug-related harms (inefficacy and toxicity) and ineffective spending and waste of the healthcare system's resources. Nowadays, strategies to support an improved, informed prescription process may benefit from the adequate use of pharmacogenomic testing. Using next-generation sequencing, we analyzed the genomic profile for three major cytochromes P450 (CYP2C9, CYP2C19, CYP2D6) and studied the frequencies of dysfunctional isozymes (e.g., poor, intermediate, or rapid/ultra-rapid metabolizers) in a cohort of 298 Italian subjects. We found just 14.8% of subjects with a fully normal set of cytochromes, whereas 26.5% of subjects had combined cytochrome dysfunction (more than one isozyme involved). As improper drug prescription is more frequent, and more burdening, in polytreated patients, since drug–drug interactions also cause patient harm, we discuss the potential benefits of a more comprehensive PGX testing approach to support informed drug selection in such patients. [ABSTRACT FROM AUTHOR]

Cytochromes P450 (CYPs) are heme-thiolate monooxygenases that prototypically catalyze the insertion of oxygen into unactivated C-H bonds but are capable of mediating more complex reactions. One of the most remarked-upon alternative reactions occurs during biosynthesis of the gibberellin A (GA) phytohormones, involving hydrocarbon ring contraction with coupled aldehyde extrusion of ent-kaurenoic acid to form the first gibberellin intermediate. While the unusual nature of this reaction has long been noted, its mechanistic basis has remained opaque. Building on identification of the relevant CYP114 from bacterial GA biosynthesis, detailed structure-function studies are reported here, including development of in vitro assays as well as crystallographic analyses both in the absence and presence of substrate. These structures provided insight into enzymatic catalysis of this unusual reaction, as exemplified by identification of a key role for the "missing" acid from an otherwise highly conserved acid-alcohol pair of residues. Notably, the results demonstrate that ring contraction requires dual factors, both the use of a dedicated ferredoxin and absence of the otherwise conserved acidic residue, with exclusion of either limiting turnover to just the initiating and more straightforward hydroxylation. The results provide detailed insight into the enzymatic structure-function relationships underlying this fascinating reaction and support the use of a semipinacol mechanism for the unusual ring contraction reaction. [ABSTRACT FROM AUTHOR]

The structural basis by which gas-binding heme proteins control their interactions with NO, CO, andO2 is fundamental to enzymology, biotechnology, and human health. Cytochromes c0 (cyts c0) are a group of putative NO-binding heme proteins that fall into two families: the well-characterized four alpha helix bundle fold (cyts c0-a) and an unrelated family with a large betasheet fold (cyts c0-ß) resembling that of cytochromes P460. A recent structure of cyt c0-ß from Methylococcus capsulatus Bath revealed two heme pocket phenylalanine residues (Phe 32 and Phe 61) positioned near the distal gas-binding site. This feature, dubbed the "Phe cap," is highly conserved within the sequences of other cyts c0-ß but is absent in their close homologs, the hydroxylamine-oxidizing cytochromes P460, although some do contain a single Phe residue. Here, we report an integrated structural, spectroscopic, and kinetic characterization of cyt c0-ß from Methylococcus capsulatus Bath complexes with diatomic gases, focusing on the interaction of the Phe cap with NO and CO. Significantly, crystallographic and resonance Raman data show that orientation of the electron-rich aromatic ring face of Phe 32 toward distally bound NO or CO is associated with weakened backbonding and higher off rates. Moreover, we propose that an aromatic quadrupole also contributes to the unusually weak backbonding reported for some heme-based gas sensors, including the mammalian NO sensor, soluble guanylate cyclase. Collectively, this study sheds light on the influence of highly conserved distal Phe residues on heme-gas complexes of cytochrome c'-ß, including the potential for aromatic quadrupoles to modulate NO and CO binding in other heme proteins. [ABSTRACT FROM AUTHOR]

In the biosynthesis of the tryptophan‐linked dimeric diketopiperazines (DKPs), cytochromes P450 selectively couple DKP monomers to generate a variety of intricate and isomeric frameworks. To determine the molecular basis for selectivity of these biocatalysts we obtained a high‐resolution crystal structure of selective Csp2−N bond forming dimerase, AspB. Overlay of the AspB structure onto C−C and C−N bond forming homolog NzeB revealed no significant structural variance to explain their divergent chemoselectivities. Molecular dynamics (MD) simulations identified a region of NzeB with increased conformational flexibility relative to AspB, and interchange of this region along with a single active site mutation led to a variant that catalyzes exclusive C−N bond formation. MD simulations also suggest that intermolecular C−C or C−N bond formation results from a change in mechanism, supported experimentally through use of a substrate mimic. [ABSTRACT FROM AUTHOR]

In the biosynthesis of the tryptophan‐linked dimeric diketopiperazines (DKPs), cytochromes P450 selectively couple DKP monomers to generate a variety of intricate and isomeric frameworks. To determine the molecular basis for selectivity of these biocatalysts we obtained a high‐resolution crystal structure of selective Csp2−N bond forming dimerase, AspB. Overlay of the AspB structure onto C−C and C−N bond forming homolog NzeB revealed no significant structural variance to explain their divergent chemoselectivities. Molecular dynamics (MD) simulations identified a region of NzeB with increased conformational flexibility relative to AspB, and interchange of this region along with a single active site mutation led to a variant that catalyzes exclusive C−N bond formation. MD simulations also suggest that intermolecular C−C or C−N bond formation results from a change in mechanism, supported experimentally through use of a substrate mimic. [ABSTRACT FROM AUTHOR]

The recent development of tailored cytochrome enzymes has enabled "new‐to‐nature" reactivities, such as the biocatalytic formation of carbon‐silicon bonds using the cytochrome c from Rhodothermus marinus. To maximise the potential of this remarkable biocatalyst by increasing its turnover numbers (TON) and to enable its reusability in continuous processes, we report the use of the SpyTag/SpyCatcher bioconjugation system to immobilise this enzyme. We successfully modified the enzyme with a SpyTag without significant effects on its catalytic activity. Even after immobilization on microparticles the enzyme retained 60 % activity. When the immobilized enzyme was used in sequential batch or continuous flow to produce an organosilicon, we observed up to 6‐fold higher turnover numbers over a total period of 10 days compared to the free enzyme reaction, however we observed a drop in stereoselectivity under these conditions. This is the first report on the successful immobilisation of a carbon‐silicon bond forming enzyme for the continuous, biocatalytic production of organosilicons. [ABSTRACT FROM AUTHOR]

To study magnetoreception of Arabidopsis thaliana, we analysed several developmental responses including cryptochrome-independent seed germination and the phytochrome- and cryptochrome-dependent hypocotyl elongation and photo-accumulation of anthocyanins and chlorophylls in weak static magnetic fields ranging from near null to 122 μT. A field of 50 μT accelerated seed germination by about 20 h relative to samples maintained in a near-null field. The double mutant, cry1cry2, lacking cryptochromes 1 and 2 displayed the same magnetic field–induced germination acceleration under blue light as the wild-type strain. Magnetic field–induced germination acceleration was masked in the presence of exogenous sucrose. Stimulus–response curves for hypocotyl elongation in a range between near-null to 122 μT indicated maxima near 9 and 60 μT for the wild-type strain as well as mutant cry1cry2. The photo-accumulation of anthocyanins and chlorophylls could be effectively modulated by magnetic fields in the presence of low-irradiance red and blue light, respectively. The findings indicate that Arabidopsis thaliana possesses light-independent mechanisms of magnetic field reception, which remain presently unidentified. Our results are in better agreement with predictions of the level crossing mechanism (LCM) of magnetoreception rather than those of the cryptochrome-associated radical-pair mechanism (RPM). [ABSTRACT FROM AUTHOR]

Periplasmic nanowires and electric conductive filaments made of the polymeric assembly of c-type cytochromes from Geobacter sulfurreducens bacterium are crucial for electron storage and/or extracellular electron transfer. The elucidation of the redox properties of each heme is fundamental to the understanding of the electron transfer mechanisms in these systems, which first requires the specific assignment of the heme NMR signals. The high number of hemes and the molecular weight of the nanowires dramatically decrease the spectral resolution and make this assignment extremely complex or unattainable. The nanowire cytochrome GSU1996 (~42 kDa) is composed of four domains (A to D) each containing three c-type heme groups. In this work, the individual domains (A to D), bi-domains (AB, CD) and full-length nanowire were separately produced at natural abundance. Sufficient protein expression was obtained for domains C (~11 kDa/three hemes) and D (~10 kDa/three hemes), as well as for bi-domain CD (~21 kDa/six hemes). Using 2D-NMR experiments, the assignment of the heme proton NMR signals for domains C and D was obtained and then used to guide the assignment of the corresponding signals in the hexaheme bi-domain CD. This new biochemical deconstruction-based procedure, using nanowire GSU1996 as a model, establishes a new strategy to functionally characterize large multiheme cytochromes. [ABSTRACT FROM AUTHOR]

The development of environment‐friendly, step economic couplings to generate structurally diverse macrocyclic compounds is highly desirable but poses a marked challenge. Inspired by the C−H oxidation mechanism of cytochromes P450, an unprecedented and practical RhIII‐catalyzed acylmethylation macrocyclization via C−H/O2 dual activation has been developed by us. The process of macrocyclization is facilitated by a synergic coordination from pyridine and ester group. Interestingly, the reaction mode derives from a three‐component coupling which differs from established olefination and alkylation paths. Density functional theory (DFT) calculations and control experiments revealed the mechanism of this unique C−H/O2 dual activation. The newly achieved acylmethylation macrocyclic products and their derivatives showed a potent anti‐H1N1 bioactivity, which may provide an opportunity for the discovery of novel anti‐H1N1 macrocyclic leading compounds. [ABSTRACT FROM AUTHOR]

The development of environment‐friendly, step economic couplings to generate structurally diverse macrocyclic compounds is highly desirable but poses a marked challenge. Inspired by the C−H oxidation mechanism of cytochromes P450, an unprecedented and practical RhIII‐catalyzed acylmethylation macrocyclization via C−H/O2 dual activation has been developed by us. The process of macrocyclization is facilitated by a synergic coordination from pyridine and ester group. Interestingly, the reaction mode derives from a three‐component coupling which differs from established olefination and alkylation paths. Density functional theory (DFT) calculations and control experiments revealed the mechanism of this unique C−H/O2 dual activation. The newly achieved acylmethylation macrocyclic products and their derivatives showed a potent anti‐H1N1 bioactivity, which may provide an opportunity for the discovery of novel anti‐H1N1 macrocyclic leading compounds. [ABSTRACT FROM AUTHOR]

The effects of two anticancer active copper(II) mixed-ligand complexes of the type [Cu(qui)(mphen)]Y·H2O, where Hqui = 2-phenyl-3-hydroxy- 1H-quinolin-4-one, mphen = bathophenanthroline, and Y = NO3 (complex 1) or BF4 (complex 2) on the activities of different isoenzymes of cytochrome P450 (CYP) have been evaluated. The screening revealed significant inhibitory effects of the complexes on CYP3A4/5 (IC50 values were 2.46 and 4.88 μM), CYP2C9 (IC50 values were 16.34 and 37.25 μM), and CYP2C19 (IC50 values were 61.21 and 77.07 μM). Further, the analysis of mechanisms of action uncovered a non-competitive type of inhibition for both the studied compounds. Consequent studies of pharmacokinetic properties proved good stability of both the complexes in phosphate buffer saline (>96% stability) and human plasma (>91% stability) after 2 h of incubation. Both compounds are moderately metabolised by human liver microsomes (<30% after 1 h of incubation), and over 90% of the complexes bind to plasma proteins. The obtained results showed the potential of complexes 1 and 2 to interact with major metabolic pathways of drugs and, as a consequence of this finding, their apparent incompatibility in combination therapy with most chemotherapeutic agents. [ABSTRACT FROM AUTHOR]

Hemoproteins include several heme-binding proteins with distinct structure and function. The presence of the heme group confers specific reactivity and spectroscopic properties to hemoproteins. In this review, we provide an overview of five families of hemoproteins in terms of dynamics and reactivity. First, we describe how ligands modulate cooperativity and reactivity in globins, such as myoglobin and hemoglobin. Second, we move on to another family of hemoproteins devoted to electron transport, such as cytochromes. Later, we consider heme-based reactivity in hemopexin, the main heme-scavenging protein. Then, we focus on heme–albumin, a chronosteric hemoprotein with peculiar spectroscopic and enzymatic properties. Eventually, we analyze the reactivity and dynamics of the most recently discovered family of hemoproteins, i.e., nitrobindins. [ABSTRACT FROM AUTHOR]

Methanogenic archaea belonging to the Order Methanosarcinales conserve energy using an electron transport chain (ETC). In the genetically tractable strain Methanosarcina acetivorans, ferredoxin donates electrons to the ETC via the Rnf (Rhodobacter nitrogen fixation) complex. The Rnf complex in M. acetivorans, unlike its counterpart in Bacteria, contains a multiheme c‐type cytochrome (MHC) subunit called MmcA. Early studies hypothesized MmcA is a critical component of Rnf, however recent work posits that the primary role of MmcA is facilitating extracellular electron transport. To explore the physiological role of MmcA, we characterized M. acetivorans mutants lacking either the entire Rnf complex (∆mmcA‐rnf) or just the MmcA subunit (∆mmcA). Our data show that MmcA is essential for growth during acetoclastic methanogenesis but neither Rnf nor MmcA is required for methanogenic growth on methylated compounds. On methylated compounds, the absence of MmcA alone leads to a more severe growth defect compared to a Rnf deletion likely due to different strategies for ferredoxin oxidation that arise in each strain. Transcriptomic data suggest that the ∆mmcA mutant might oxidize ferredoxin by upregulating the cytosolic Wood‐Ljundahl pathway for acetyl‐CoA synthesis, whereas the ∆mmcA‐rnf mutant may repurpose the F420 dehydrogenase complex (Fpo) to oxidize ferredoxin coupled to proton translocation. Beyond energy conservation, the deletion of rnf or mmcA leads to global transcriptional changes of genes involved in methanogenesis, carbon assimilation and regulation. Overall, our study provides systems‐level insights into the non‐overlapping roles of the Rnf bioenergetic complex and the associated MHC, MmcA. [ABSTRACT FROM AUTHOR]

Metallic nanoparticles have been extensively used to adsorb biomolecules and improve their stability under broader conditions. This study focuses on the interaction of Cytochrome C (CytC) and gold nanoparticles (AuNP), CytC@AuNP. Circular dichroism (CD) and Raman show significant differences in secondary structure, mainly in α‐helix. CD ramp temperatures revealed enhanced thermal stability of CytC@AuNP, as evidenced by a refolding of the α‐helix conformation of CytC and a delay in denaturation temperature by approximately 15 °C. Catalytic oxidation performed with CytC@AuNP showed a hyperbolic profile up to 104 % higher yields compared to CytC, which exhibited a linear profile and 30 % loss of activity after thermal treatment. The same reaction performed upon plasmon stimulus comparatively increased the catalytic yield of CytC@AuNP in 12 %, although it had no effect on free CytC. This study highlights the advantages of adsorbing biomolecules on nanoparticles and is a glimpse at plasmon‐driven nanobiocatalysis. [ABSTRACT FROM AUTHOR]

I present here a personal perspective of the evolution of the two-light reaction two-pigment scheme for the electron transport in oxygenic photosynthesis – as I have lived through it – first as a graduate student of Robert Emerson, from September 1956–January 1959, and then of Eugene Rabinowitch from February 1959–September 1960. I have provided here some of the key published work in my way and have also provided a few photographs. It is essential to remind ourselves upfront that different individuals may have different recollections of the same event (see e.g., the Japanese movie ‘Rashomon’ https://en.wikipedia.org/wiki/Rashomon). Thus, I encourage others to write about their perspective. Further, I recognize, upfront, the support and encouragement I have received from Robert Blankenship for writing this perspective, and from Győző Garab for submission of this story to Photosynthetica. I begin my perspective with a dedication to Robert Emerson, whose pioneering work led to the concept of the two-light reaction two-pigment system in oxygenic photosynthesis. [ABSTRACT FROM AUTHOR]

The 2-oxidation, 3-methyl hydroxylation, and 6-hydroxylation of skatole (a contributor to boar taint) mediated by minipig liver microsomes and recombinant P450 enzymes expressed in bacterial membranes were investigated. At low substrate concentrations of 10 µM, the formation rates of indole-3-carbinol, 6-hydroxyskatole, and the sum of 3-methyloxindole, indole-3-carbinol, and 6-hydroxyskatole in male minipig liver microsomes were significantly lower than those in female minipig liver microsomes. Compensatory 3-methyloxindole and indole-3-carbinol formation in minipig liver microsomes, which lack 6-hydroxyskatole formation in males, was mediated partly by liver microsomal P450 1A2 and P450 1A2/2E1, respectively. These enzymes were suppressed by typical P450 inhibitors in female minipig liver microsomes. Among the 14 pig P450 forms evaluated, P450 2A19 was the dominant form mediating 3-methyloxindole, indole-3-carbinol, and 6-hydroxyskatole formation from skatole at substrate concentrations of 100 µM. Positive cooperativity was observed in 3-methyloxindole formation from skatole mediated by male minipig liver microsomes and by pig P450 3A22 with Hill coefficients of 1.2–1.5. These results suggest high skatole 2-oxidation, 3-methyl hydroxylation, and 6-hydroxylation activities of pig P450 2A19 and compensatory skatole oxidations mediated by pig P450 1A2, 2E1, or 3A22 in male minipig liver microsomes. [ABSTRACT FROM AUTHOR]

Cytochromes are enzymes of the dehydrogenase class with a hemoprotein structure in which the iron in these compounds undergoes oxidation and reduction reactions upon receiving or losing electrons. Quantitatively speaking, CYP3A4 is the most important isoenzyme of cytochrome P450, oxidizing foreign organic molecules such as drugs or toxins to cause them to leave the body. Many drugs and antibiotics can induce or inhibit the activity of cytochrome P450, including dexamethasone. Dexamethasone is a steroidal anti-inflammatory drug used to treat of inflammatory diseases and chronic autoimmunity. This study aimed to investigate the induction effect of dexamethasone in biotransformation pathways by in silico tools. Molegro Virtual Docker software was used to investigate the molecular docking of the enzyme and dexamethasone, which indicated the binding of the drug to the enzyme. The molecular simulation was performed in Linux with the GROMACS program. Root-mean-square distance (RMSD), and radius of gyration (Rg), were evaluated. The results were analyzed with Pymol and VMD software, and the obtained curve was plotted with GRACE software. Docking results show that a cluster with a bond energy of -60.81 was the best cluster, and the bond size between ligand and internal atoms was -23.191in the complex. In addition, the amount of bond between the ligand and water for this pose was zero. The stability of the enzyme-ligand complex and the induction effect of dexamethasone on CYP3A4 were indicated by RMSD and RG results. Results of RMSD and RG of CYP3A4 glucocorticoid obtained from the simulation showed the stability of binding of the drug to the enzyme. Also, RMSD results showed the stability of glucocorticoid and dexamethasone complex during molecular dynamics simulation. It reached relative stability at 0.8 nm after 80,000 ps until the end of the simulation. [ABSTRACT FROM AUTHOR]

Mycolicibacterium smegmatis mc2155 has been genetically modified to be used as a platform for the expression of heterologous cytochrome P450 monooxygenases by introducing deletions in the kshB and kstD genes that encode key stages of the steroid nucleus enzymatic degradation. Three sets of genetic constructs have been created for heterologous expression of the genes encoding P450 cytochromes CYP106A1 from Bacillus megaterium DSM319 and CYP106A2 from B. megaterium ATCC13368 in M. smegmatis mc2155 (ΔkshBΔkstD) cells. The recombinant plasmids contained monocistronic expression cassettes of cytochrome genes (pNS31 and pNS32), tricistronic cassettes of cytochrome genes together with cDNA copies of adrenodoxin and adrenodoxin reductase genes of the bovine adrenal cortex (pNS33 and pNS34), or monocistronic gene cassettes of chimeric cytochromes fused with the DNA sequence encoding the CYP116B2 reductase domain from the soil bacterium Rhodococcus sp. NCIMB 9784 (pNS35 and pNS36). The recombinant strains of mycolicibacteria were shown to selectively monohydroxylate androstenedione (AD) under growth conditions. The product was identified as 15β-hydroxyandrostenedione (15β-OH-AD) by mass spectrometry and 1H and 13C NMR spectroscopy. The maximum level of 15β-OH-AD production (17.3 ± 1.5 mg/L) was observed when using the recombinant M. smegmatis mc2155 (ΔkshBΔkstD) (pNS32) strain, which expresses a single cyp106A2 gene from B. megaterium ATCC13368. Host proteins of M. smegmatis mc2155 were shown to be capable of supplying electrons to heterologous cytochromes to support their hydroxylation activity. The results we obtained are of a priority character, expand the understanding of the hydroxylation of steroid compounds by bacterial cytochromes CYP106A1/A2, and are important for the creation of microbial strains for producing valuable hydroxysteroids. [ABSTRACT FROM AUTHOR]

Theodore "Ted" Kuwana and his research group played a central role in the invention and early development of spectroelectrochemistry at optically transparent electrodes (OTEs). The history of the "idea" and the first ten years of research are reviewed. Topics include OTEs and spectroelectrochemical cells for transmission and internal reflection experiments, rapid scan spectrometer development, and applications to kinetics of homogeneous chemical reactions associated with electrode processes and redox active biological compounds such as cytochromes. Relevant work of other contemporary research groups is also presented. [ABSTRACT FROM AUTHOR]

The cytochrome P450 (CYP) family of heme monooxygenases catalyse the selective oxidation of C−H bonds under ambient conditions. The CYP199A4 enzyme from Rhodopseudomonas palustris catalyses aliphatic oxidation of 4‐cyclohexylbenzoic acid but not the aromatic oxidation of 4‐phenylbenzoic acid, due to the distinct mechanisms of aliphatic and aromatic oxidation. The aromatic substrates 4‐benzyl‐, 4‐phenoxy‐ and 4‐benzoyl‐benzoic acid and methoxy‐substituted phenylbenzoic acids were assessed to see if they could achieve an orientation more amenable to aromatic oxidation. CYP199A4 could catalyse the efficient benzylic oxidation of 4‐benzylbenzoic acid. The methoxy‐substituted phenylbenzoic acids were oxidatively demethylated with low activity. However, no aromatic oxidation was observed with any of these substrates. Crystal structures of CYP199A4 with 4‐(3′‐methoxyphenyl)benzoic acid demonstrated that the substrate binding mode was like that of 4‐phenylbenzoic acid. 4‐Phenoxy‐ and 4‐benzoyl‐benzoic acid bound with the ether or ketone oxygen atom hydrogen‐bonded to the heme aqua ligand. We also investigated whether the substitution of phenylalanine residues in the active site could permit aromatic hydroxylation. Mutagenesis of the F298 residue to a valine did not significantly alter the substrate binding position or enable the aromatic oxidation of 4‐phenylbenzoic acid; however the F182L mutant was able to catalyse 4‐phenylbenzoic acid oxidation generating 2′‐hydroxy‐, 3′‐hydroxy‐ and 4′‐hydroxy metabolites in a 83 : 9 : 8 ratio, respectively. Molecular dynamics simulations, in which the distance and angle of attack were considered, demonstrated that in the F182L variant, in contrast to the wild‐type enzyme, the phenyl ring of 4‐phenylbenzoic acid attained a productive geometry for aromatic oxidation to occur. [ABSTRACT FROM AUTHOR]

Cytochrome P450 is an enzyme involved in the metabolism of phase 1 xenobiotics, toxins, endogenous hormones, and drugs, including those used in COVID-19 treatment. Cytochrome p450 genes are linked to the pathogenesis of some multifactorial traits and diseases, such as cancer, particularly prostate cancer, colorectal cancer, breast cancer, and cervical cancer. Genotyping was performed on 540 supposedly healthy individuals of 5 Finno-Permic populations from the territories of the European part of the Russian Federation. There was a statistically significant difference between Veps and most of the studied populations in the rs4986774 locus of the CYP2D6 gene; data on the rs3892097 locus of the CYP2D6 gene shows that Izhemsky Komis are different from the Mordovian and Udmurt populations. [ABSTRACT FROM AUTHOR]

Heme is a vital cofactor of proteins with roles in oxygen transport (e.g. hemoglobin), storage (e.g. myoglobin), and activation (e.g. P450) as well as electron transfer (e.g. cytochromes) and many other functions. However, its structural and functional role in oxygen sensing proteins differs markedly from that in most other enzymes, where it serves as a catalytic or functional center. This minireview discusses the mechanism of signal transduction in two heme-based oxygen sensors: the histidine kinase AfGcHK and the diguanylate cyclase YddV (EcDosC), both of which feature a heme-binding domain containing a globin fold resembling that of hemoglobin and myoglobin. [ABSTRACT FROM AUTHOR]

The genomic architecture underlying the origins and maintenance of biodiversity is an increasingly accessible feature of species, due in large part to third‐generation sequencing and novel analytical toolsets. Applying these techniques to woodrats (Neotoma spp.) provides a unique opportunity to study how herbivores respond to environmental change. Neotoma bryanti and N. lepida independently achieved a major dietary feat in the aftermath of a natural climate change event: switching to the novel, toxic food source creosote bush (Larrea tridentata). To better understand the genetic mechanisms underlying this ability, we employed a trio binning sequencing approach with a N. bryanti × N. lepida F1 hybrid, allowing the simultaneous assembly of genomes representing each parental species. The resulting phased, chromosome‐level, highly complete haploid references enabled us to explore the genomic architecture of several gene families—cytochromes P450, UDP‐glucuronosyltransferases (UGTs), and ATP‐binding cassette (ABC) transporters—known to play key roles in the metabolism of naturally occurring toxic dietary compounds. In addition to duplication events in the ABCG and UGT2B subfamilies, we found expansions in three P450 gene families (2A, 2B, 3A), including the evolution of multiple novel gene islands within the 2B and 3A subfamilies, which may have provided the crucial substrate for dietary adaptation. Our assemblies demonstrate that trio binning from an F1 hybrid rodent effectively recovers parental genomes from species that diverged more than a million years ago. [ABSTRACT FROM AUTHOR]

Shewanella oneidensis is the best understood model microorganism for the study of diverse cytochromes (cytos) c that support its unparallel respiratory versatility. Although RNA chaperone Hfq has been implicated in regulation of cyto c production, little is known about the biological pathways that it affects in this bacterium. In this study, from a spontaneous mutant that secretes pyomelanin and has a lowered cyto c content, we identified Hfq to be the regulator that critically associates with both phenotypes in S. oneidensis. We found that expression of the key genes in biosynthesis and degradation of heme is differentially affected by Hfq at under- and overproduced levels, and through modulating heme levels, Hfq influences the cyto c content. Although Hfq in excess results in overproduction of the enzymes responsible for both generation and removal of homogentisic acid (HGA), the precursor of pyomelanin, it is compromised activity of HmgA that leads to excretion and polymerization of HGA to form pyomelanin. We further show that Hfq mediates HmgA activity by lowering intracellular iron content because HmgA is an iron-dependent enzyme. Overall, our work highlights the significance of Hfq-mediated posttranscriptional regulation in the physiology of S. oneidensis, unraveling unexpected mechanisms by which Hfq affects cyto c biosynthesis and pyomelanin production. [ABSTRACT FROM AUTHOR]

Cytochrome P450 oxidoreductase (POR) is the redox partner of steroid and drug-metabolising cytochromes P450 located in the endoplasmic reticulum. Mutations in POR cause a broad range of metabolic disorders. The POR variant rs17853284 (P228L), identified by genome sequencing, has been linked to lower testosterone levels and reduced P450 activities. We expressed the POR wild type and the P228L variant in bacteria, purified the proteins, and performed protein stability and catalytic functional studies. Variant P228L affected the stability of the protein as evidenced by lower unfolding temperatures and higher sensitivity to urea denaturation. A significant decline in the rate of electron transfer to cytochrome c and thiazolyl blue tetrazolium (MTT) was observed with POR P228L, while activities of CYP3A4 were reduced by 25% and activities of CYP3A5 and CYP2C9 were reduced by more than 40% compared with WT POR. The 17,20 lyase activity of CYP17A1, responsible for the production of the main androgen precursor dehydroepiandrosterone, was reduced to 27% of WT in the presence of the P228L variant of POR. Based on in silico and in vitro studies, we predict that the change of proline to leucine may change the rigidity of the protein, causing conformational changes in POR, leading to altered electron transfer to redox partners. A single amino acid change can affect protein stability and cause a severe reduction in POR activity. Molecular characterisation of individual POR mutations is crucial for a better understanding of the impact on different redox partners of POR. [ABSTRACT FROM AUTHOR]

The search of a putative physiological electron acceptor for thiocyanate dehydrogenase (TcDH) newly discovered in the thiocyanate-oxidizing bacteria Thioalkalivibrio paradoxus revealed an unusually large, single-heme cytochrome c (CytC552), which was co-purified with TcDH from the periplasm. Recombinant CytC552, produced in Escherichia coli as a mature protein without a signal peptide, has spectral properties similar to the endogenous protein and serves as an in vitro electron acceptor in the TcDH-catalyzed reaction. The CytC552 structure determined by NMR spectroscopy reveals significant differences compared to those of the typical class I bacterial cytochromes c: a high solvent accessible surface area for the heme group and so-called "intrinsically disordered" nature of the histidine-rich N- and C-terminal regions. Comparison of the signal splitting in the heteronuclear NMR spectra of oxidized, reduced, and TcDH-bound CytC552 reveals the heme axial methionine fluxionality. The TcDH binding site on the CytC552 surface was mapped using NMR chemical shift perturbations. Putative TcDH-CytC552 complexes were reconstructed by the information-driven docking approach and used for the analysis of effective electron transfer pathways. The best pathway includes the electron hopping through His528 and Tyr164 of TcDH, and His83 of CytC552 to the heme group in accordance with pH-dependence of TcDH activity with CytC552. [ABSTRACT FROM AUTHOR]

Light-induced oxidation of the reaction center dimer and periplasmic cytochromes was detected by fast kinetic difference absorption changes in intact cells of wild type and cytochrome mutants (cycA, cytC4 and pufC) of Rubrivivaxgelatinosus and Rhodobactersphaeroides. Constant illumination from a laser diode or trains of saturating flashes enabled the kinetic separation of acceptor and donor redox processes, and the electron contribution from the cyt bc1 complex via periplasmic cytochromes. Under continuous excitation, concentrations of oxidized cytochromes increased in three phases where light intensity, electron transfer rate and the number of reduced cytochromes were the rate liming steps, respectively. By choosing suitable flash timing, gradual steps of cytochrome oxidation in whole cells were observed; each successive flash resulted in a smaller, damped oxidation. We attribute this damping to lowered availability of reduced cytochromes resulting from both exchange (unbinding/binding) of the cytochromes and electron transfer at the reaction center interface since a similar effect is observed upon deletion of genes encoding periplasmic cytochromes. In addition, we present a simple model to calculate the damping effect; application of this method may contribute to understanding the function of the diverse range of c-type cytochromes in the electron transport chains of anaerobic phototrophic bacteria. [ABSTRACT FROM AUTHOR]