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The heme enzymes of the cytochrome P450 superfamily (CYPs) catalyse the selective hydroxylation of unactivated C-H bonds in organic molecules. There is great interest in applying these enzymes as biocatalysts with a focus on self-sufficient CYP 'fusion' enzymes, comprising a single polypeptide chain with the electron transfer components joined to the heme domain. Here we elucidate the function of the self-sufficient CYP116B46 fusion enzyme, from the thermophilic bacterium Tepidiphilus thermophilus. We demonstrate that it efficiently hydroxylates aromatic organic acids, exemplified by oxidation of 2-hydroxyphenylacetic acid to homogentisic acid (2,5-dihydroxyphenylacetic acid), an important metabolite in bacterial catabolism. In line with the thermophilic nature of the source bacterium, activity increased at higher temperatures, (50 °C), with a catalytic preference for NADPH over NADH. While self-sufficient fusion enzymes simplify biocatalysis; engineered peroxygenase activity is also a key advance in the application of these enzymes as biocatalysts as it eliminates the need for electron transfer partner proteins and nicotinamide cofactors. We demonstrate that a T278E mutation in the heme domain of CYP116B46, confers peroxygenase activity. This engineered peroxygenase enzyme is stable to elevated temperatures and catalytic concentrations of hydrogen peroxide, with an observed optimal activity resulting in a total turnover number of ~650., (© 2024 Wiley-VCH GmbH.)

Thermostable protein folds of natural and synthetic origin are highly sought-after templates for biocatalyst generation due to their enhanced stability to elevated temperatures which overcomes one of the major limitations of applying enzymes for synthesis. Cytochrome P450 enzymes (CYPs) are a family of heme-thiolate monooxygenases that catalyse the oxidation of their substrates in a highly stereo- and regio-selective manner. The CYP enzyme (CYP107PQ1) from the thermophilic bacterium Meiothermus ruber binds the norisoprenoid β-ionone and was employed as a scaffold for catalyst design. The I-helix was modified to convert this enzyme from a monooxygenase into a peroxygenase (CYP107PQ1QE), enabling the enantioselective oxidation of β-ionone to (S)-4-hydroxy-β-ionone (94 % e.e.). The enzyme was resistant to 20 mM H 2 O 2 , 20 % (v/v) of organic solvent, supported over 1700 turnovers and was fully functional after incubation at 60 °C for 1 h and 30 °C for 365 days. The reaction was scaled-up to generate multi milligram quantities of the product for characterisation. Overall, we demonstrate that sourcing a CYP protein fold from an extremophile enabled the design of a highly stable enzyme for stereoselective C-H bond activation only using H 2 O 2 as the oxidant, providing a viable strategy for future biocatalyst design., (© 2024 The Author(s). ChemBioChem published by Wiley-VCH GmbH.)

Background: Opioid pain management in cancer survivorship is a complex and understudied topic., Methods: The authors conducted in-depth, qualitative interviews to understand clinician approaches to opioid pain management in chronic cancer pain and to generate ideas for improvement. They used a rigorous, inductive, qualitative, descriptive approach to examine clinician (n = 20) perspectives about opioid pain management in survivorship, including oncologists (n = 5), palliative care clinicians (n = 8), primary care clinicians (n = 5), and pain management specialists (n = 2)., Results: The findings indicated that no consistent medical home exists for chronic pain management in cancer survivors and that there are fundamental differences in how each subspecialty approaches chronic pain management in survivorship (e.g., "Do we think of this as noncancer pain or cancer pain?… This is in this limbo zone-this gray zone-because it's cancer-related pain, right?"). Simultaneously, clinicians are influenced by their peers' perceptions of their opioid prescribing decisions, sparking intraprofessional tension when disagreement occurs. In these instances, clinicians described overthinking and doubting their clinical decision-making as well as a sense of judgment, pressure, and/or shame. Finally, clinicians acknowledged a fear of consequences for opioid prescribing decisions. Specifically, participants cited conflict with patients, sometimes escalating to aggression and threats of violence, as well as potential disciplinary actions and/or legal consequences., Conclusions: Participants suggested that opportunities to improve chronic cancer pain care include developing clear, systematic guidance for chronic cancer pain management, facilitating clinician communication and consultation, creating tailored survivorship care plans in partnership with patients, and developing accessible, evidence-based, complementary pain treatments., (© 2024 The Authors. Cancer published by Wiley Periodicals LLC on behalf of American Cancer Society.)

Objectives: Minimally invasive surgery for treatment of gynecologic malignancies is associated with decreased pain, fewer complications, earlier return to activity, lower cost, and shorter hospital stays. Patients are often discharged the day of surgery, but occasionally stay overnight due to prolonged post-anesthesia care unit (PACU) stays. The objective of this study was to identify risk factors for prolonged PACU length of stay (LOS)., Methods: This is a single institution retrospective review of patients who underwent minimally invasive hysterectomy for gynecologic cancer from 2019 to 2022 and had a hospital stay <24-h. The primary outcome was PACU LOS. Demographics, pre-operative diagnoses, and surgical characteristics were recorded. After Box-Cox transformation, linear regression was used to determine significant predictors of PACU LOS., Results: For the 661 patients identified, median PACU LOS was 5.04 h (range 2.16-23.76 h). On univariate analysis, longer PACU LOS was associated with increased age (ρ = 0.106, p = 0.006), non-partnered status [mean difference (MD) = 0.019, p = 0.099], increased alcohol use (MD = 0.018, p = 0.102), increased Charlson Comorbidity Index (CCI) score (ρ = 0.065, p = 0.097), and ASA class ≥3 (MD = 0.033, p = 0.002). Using multivariate linear regression, increased age (R 2  = 0.0011, p = 0.043), non-partnered status (R 2  = 0.0389, p < 0.001), and ASA class ≥3 (R 2  = 0.0250, p = 0.023) were associated with increased PACU LOS., Conclusions: Identifying patients at risk for prolonged PACU LOS, including patients who are older, non-partnered, and have an ASA class ≥3, may allow for interventions to improve patient experience, better utilize hospital resources, decrease PACU overcrowding, and limit postoperative admissions and complications. The relationship between non-partnered status and PACU LOS is the most novel relationship identified in this study., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to disclose., (Copyright © 2024 Elsevier Inc. All rights reserved.)

This is the final column in a series of columns that began with the January/February 2021 issue of Neonatal Network , describing the evidence-based practice (EBP) project. The series has taken the reader through sparking the spirit of inquiry, asking a compelling question, and searching and critically appraising the literature. This column will briefly describe the final three steps: step 4, the integration of evidence with clinical expertise and patient/family preferences; step 5, the evaluation of outcomes of practice changes based on evidence; and step 6, the dissemination of the outcomes of the EBP change., (© Copyright 2024 Springer Publishing Company, LLC.)

Oxidised derivatives of cholesterol have been shown to inhibit the growth of Mycobacterium tuberculosis (Mtb). The bacteriostatic activity of these compounds has been attributed to their inhibition of CYP125A1 and CYP142A1, two metabolically critical cytochromes P450 that initiate degradation of the sterol side chain. Here, we synthesise and characterise an extensive library of 28 cholesterol derivatives to develop a structure-activity relationship for this class of inhibitors. The candidate compounds were evaluated for MIC with virulent Mtb and in binding studies with CYP125A1 and CYP142A1 from Mtb., (Copyright © 2024. Published by Elsevier Ltd.)

The cytochrome P450 family of heme metalloenzymes (CYPs) catalyse important biological monooxygenation reactions. Mycobacterium marinum contains a gene encoding a CYP105Q4 enzyme of unknown function. Other members of the CYP105 CYP family have key roles in bacterial metabolism including the synthesis of secondary metabolites. We produced and purified the cytochrome P450 enzyme CYP105Q4 to enable its characterization. Several nitrogen-donor atom-containing ligands were found to bind to CYP105Q4 generating type II changes in the UV-vis absorbance spectrum. Based on the UV-vis absorbance spectra none of the potential substrate ligands we tested with CYP105Q4 were able to displace the sixth distal aqua ligand from the heme, though there was evidence for binding of oleic acid and amphotericin B. The crystal structure of CYP105Q4 in the substrate-free form was determined in an open conformation. A computational structural similarity search (Dali) was used to find the most closely related characterized relatives within the CYP105 family. The structure of CYP105Q4 enzyme was compared to the GfsF CYP enzyme from Streptomyces graminofaciens which is involved in the biosynthesis of a macrolide polyketide. This structural comparison to GfsF revealed conformational changes in the helices and loops near the entrance to the substrate access channel. A disordered B/C loop region, usually involved in substrate recognition, was also observed., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Competing Interests: Declaration of Competing Interest BLM, KM, JM, NK, JAS, HC, MKH, RR, SGB, MR, CR, JKR, NK report no conflicts of interest with the current manuscript. NK reports receiving consulting fees from Eisana Corporation as a medical consultant and honoraria for lectures from AANEM. MKH reports receiving consulting fees from Argenx, Alexion, UCB Pharma, Janssen, and Immunovant and honoraria from Medscape, Springer Health, AANEM, Medlink Neurology, and Continuum Lifelong Learning in Neurology.

Introduction: Therapeutic alliance (TA), or the extent to which patients feel a sense of caring and trust with their physician, may have an impact on health care utilization. We sought to determine if TA is associated with: (1) emergency department (ED) visits within 30 days of death and (2) hospice enrollment. Methods and Materials: This is a secondary analysis of data from a randomized clinical trial. We used restricted cubic splines to assess the relationship between TA scores and health care utilization. Results: Six hundred seventy-two patients were enrolled in the study, with 331 (49.3%) dying within 12 months. Patients with higher TA were less likely to have an ED visit in the last 30 days of life, but there was no evidence of a relationship between TA and enrollment in hospice. Conclusions: Higher TA was associated with decreased ED visits within 30 days of death. There was no association between TA and rates of hospice enrollment. Clinical Registration Number: NCT02712229.

Mycotoxins are known environmental pollutants that may contaminate food and feed chains. Some mycotoxins are regulated in many countries to limit the trading of contaminated and harmful commodities. However, the so-called emerging mycotoxins are poorly understood and need to be investigated further. Fusaric acid is an emerging mycotoxin, noxious to plants and animals, but is known to be less toxic to plants when hydroxylated. The detoxification routes effective in animals have not been elucidated yet. In this context, this study integrated in silico and in vitro techniques to discover potential bioremediation routes to turn fusaric acid to its less toxic metabolites. The toxicodynamics of these forms in humans have also been addressed. An in silico screening process, followed by molecular docking and dynamics studies, identified CYP199A4 from the bacterium Rhodopseudomonas palustris HaA2 as a potential fusaric acid biotransforming enzyme. Its activity was confirmed in vitro. However, the effect of hydroxylation seemed to have a limited impact on the modelled toxicodynamics against human targets. This study represents a starting point to develop a hybrid in silico/in vitro pipeline to find bioremediation agents for other food, feed and environmental contaminants., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Daniel Zocchi Doherty reports financial support was provided by Australian Institute of Nuclear Science and Engineering (AINSE) Ltd. Lorenzo Pedroni reports financial support was provided by Emilia-Romagna Region. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The cytochrome P450 monooxygenases (CYPs) are a class of heme-thiolate enzymes that insert oxygen into unactivated C-H bonds. These enzymes can be converted into peroxygenases via protein engineering, which enables their activity to occur using hydrogen peroxide (H 2 O 2 ) without the requirement for additional nicotinamide co-factors or partner proteins. Here, we demonstrate that soaking crystals of an engineered P450 peroxygenase with H 2 O 2 enables the enzymatic reaction to occur within the crystal. Crystals of the designed P450 peroxygenase, the T252E mutant of CYP199A4, in complex with 4-methoxybenzoic acid were soaked with different concentrations of H 2 O 2 for varying times to initiate the in crystallo O-demethylation reaction. Crystal structures of T252E-CYP199A4 showed a distinct loss of electron density that was consistent with the O-demethylated metabolite, 4-hydroxybenzoic acid. A new X-ray crystal structure of this enzyme with the 4-hydroxybenzoic acid product was obtained to enable comparison alongside the existing substrate-bound structure. The visualisation of enzymatic catalysis in action is challenging in structural biology and the ability to initiate the reactions of P450 enzymes, in crystallo by simply soaking crystals with H 2 O 2 will enable new structural biology methods and techniques to be applied to study their mechanism of action., (© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Rhodococcus globerulus (R. globerulus) was isolated from the soil beneath a Eucalypt tree. Metabolic growth studies revealed that R. globerulus was capable of living on certain monoterpenes, including 1,8-cineole and p-cymene, as sole sources of carbon and energy. Multiple P450 genes were identified in the R. globerulus genome that shared homology to known bacterial, monoterpene hydroxylating P450s. To date, two of these P450s have been expressed and characterised as 1,8-cineole (CYP176A1) and p-cymene (CYP108N12) monooxygenases that are believed to initiate the biodegradation of these terpenes. In this work, another putative P450 gene (CYP108N14) was identified in R. globerulus genome. Given its amino acid sequence identity to other monoterpene hydroxylating P450s it was hypothesised to catalyse monoterpene hydroxylation. These include CYP108A1 from Pseudomonas sp. (47 % identity, 68 % similarity) which hydroxylates α-terpineol, and CYP108N12 also from R. globerulus (62 % identity, 77 % similarity). Also present in the operon containing CYP108N14 were putative ferredoxin and ferredoxin reductase genes, suggesting a typical Class I P450 system. CYP108N14 was successfully over-expressed heterologously and purified, resulting in a good yield of CYP108N14 holoprotein. However, neither the ferredoxin nor ferredoxin reductase could be produced heterologously. Binding studies with CYP108N14 revealed a preference for the monoterpenes p-cymene, (R)-limonene, (S)-limonene, (S)-α-terpineol and (S)-4-terpineol. An active catalytic system was reconstituted with the non-native redox partners cymredoxin (from the CYP108N12 system) and putidaredoxin reductase (from the CYP101A1 system). CYP108N14 when supported by these redox partners was able to catalyse the hydroxylation of the five aforementioned substrates selectively at the methyl benzylic/allylic positions., (Copyright © 2023. Published by Elsevier Inc.)

Critically appraising the literature is the third step in the evidence-based practice process. The lowest level of evidence includes expert opinion and related types of literature. This column describes the appraisal of this type of literature using a specific set of questions., (© Copyright 2024 Springer Publishing Company, LLC.)

Norcarane is a mechanistic probe of monooxygenase enzymes that is able to detect the presence of cationic or radical intermediates. The addition of substituents around the bicycloheptane ring of the norcarane scaffold can assist in improving enzyme binding affinity and thus improve the regioselectivity of oxidation. Here we prepare in three-step, diastereoselective syntheses, ten norcaranes monosubstituted α to the cyclopropane as advanced probes. Four of these compounds were examined in enzyme binding experiments to evaluate their potential as probe substrates. Additionally, 19 potential products of enzymatic oxidation were generated via two additional synthetic steps for use as product standards in future studies.

The cytochrome P450 enzymes (CYPs) are heme-thiolate monooxygenases that catalyse the insertion of an oxygen atom into the C-H bonds of organic molecules. In most CYPs, the activation of dioxygen by the heme is aided by an acid-alcohol pair of residues located in the I-helix of the enzyme. Mutation of the threonine residue of this acid-alcohol pair of CYP199A4, from the bacterium Rhodospeudomonas palustris HaA2, to a glutamate residue induces peroxygenase activity. In the X-ray crystal structures of this variant an interaction of the glutamate side chain and the distal aqua ligand of the heme was observed and this results in this ligand not being readily displaced in the peroxygenase mutant on the addition of substrate. Here we use a range of bulky hydrophobic and nitrogen donor containing ligands in an attempt to displace the distal aqua ligand of the T252E mutant of CYP199A4. Ligand binding was assessed by UV-visible absorbance spectroscopy, native mass spectrometry, electron paramagnetic resonance and X-ray crystallography. None of the ligands tested, even the nitrogen donor ligands which bind directly to the iron in the wild-type enzyme, resulted in displacement of the aqua ligand. Therefore, modification of the I-helix threonine residue to a glutamate residue results in a significant strengthening of the ferric distal aqua ligand. This ligand was not displaced using any of the ligands during this study and this provides a rationale as to why this mutant can shutdown the monooxygenase pathway of this enzyme and switch to peroxygenase activity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The members of the bacterial cytochrome P450 (CYP) monooxygenase family CYP125, catalyze the oxidation of steroid derivatives including cholesterol and phytosterols, as the initial activating step in their catabolism. However, several bacterial species contain multiple genes encoding CYP125 enzymes and other CYP enzymes which catalyze cholesterol/cholest-4-en-3-one hydroxylation. An important question is why these bacterium have more than one enzyme with overlapping substrate ranges capable of catalyzing the terminal oxidation of the alkyl chain of these sterols. To further understand the role of these enzymes we investigated CYP125A6 and CYP125A7 from Mycobacterium marinum with various cholesterol analogues. These have modifications on the A and B rings of the steroid and we assessed the substrate binding and catalytic activity of these with each enzyme. CYP125A7 gave similar results to those reported for the CYP125A1 enzyme from M. tuberculosis. Differences in the substrate binding and catalytic activity with the cholesterol analogues were observed with CYP125A6. For example, while cholesteryl sulfate could bind to both enzymes it was only oxidized by CYP125A6 and not by CYP125A7. CYP125A6 generated higher levels of metabolites with the majority of C-3 and C-7 substituted cholesterol analogues such 7-ketocholesterol. However, 5α-cholestan-3β-ol was only oxidized by CYP125A7 enzyme. The cholest-4-en-3-one and 7-ketocholesterol-bound forms of the CYP125A6 and CYP125A7 enzymes were modelled using AlphaFold. The structural models highlighted differences in the binding modes of the steroid derivatives within the same enzyme. Significant changes in the binding mode of the steroids between these CYP125 enzymes and other bacterial cholesterol oxidizing enzymes, CYP142A3 and CYP124A1, were also seen. Despite this, all these models predicted the selectivity for terminal methyl hydroxylation, in agreement with the experimental data., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

A highly sought after reaction in chemical synthesis is the activation of unactivated carbon-hydrogen bonds. We demonstrate the hydroxylation of fatty acids using an engineered thermostable archaeal cytochrome P450 enzyme. By replacing a seven amino acid section of the I-helix, the nicotinamide cofactor-dependent monooxygenase was converted into a hydrogen peroxide using peroxygenase, enabling the efficient biocatalytic oxidation of C-H bonds at room temperature to 90 °C.

The cytochrome P450 (CYP) superfamily of monooxygenase enzymes play important roles in the metabolism of molecules which contain heterocyclic, aromatic functional groups. Here we study how oxygen- and sulfur-containing heterocyclic groups interact with and are oxidized using the bacterial enzyme CYP199A4. This enzyme oxidized both 4-(thiophen-2-yl)benzoic acid and 4-(thiophen-3-yl)benzoic acid almost exclusively via sulfoxidation. The thiophene oxides produced were activated towards Diels-Alder dimerization after sulfoxidation, forming dimeric metabolites. Despite X-ray crystal structures demonstrating that the aromatic carbon atoms of the thiophene ring were located closer to the heme than the sulfur, sulfoxidation was still favoured with 4-(thiophen-3-yl)benzoic acid. These results highlight a preference of this cytochrome P450 enzyme for sulfoxidation over aromatic hydroxylation. Calculations predict a strong preference for homodimerization of the enantiomers of the thiophene oxides and the formation of a single major product, in broad agreement with the experimental data. 4-(Furan-2-yl)benzoic acid was oxidized to 4-(4'-hydroxybutanoyl)benzoic acid using a whole-cell system. This reaction proceeded via a γ-keto-α,β-unsaturated aldehyde species which could be trapped in vitro using semicarbazide to generate a pyridazine species. The combination of the enzyme structures, the biochemical data and theoretical calculations provides detailed insight into the formation of the metabolites formed from these heterocyclic compounds., (© 2023 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

Cholesterol catabolism is an important survival mechanism for the pathogenic Mycobacterium tuberculosis . Various other mycobacteria degrade not only cholesterol but plant sterols such as sitosterol and campesterol. In this work we demonstrate that the cytochrome P450 (CYP) CYP125 enzyme family is capable of sitosterol and campesterol side-chain oxidation and activation in these bacteria. We also show that the CYP142 and CYP124 cholesterol hydroxylating enzyme families are significantly less active for sitosterol hydroxylation compared to CYP125 enzymes.

Critical appraisal of the literature is the third step in the evidence-based practice process. There are 2 types of Level VI evidence, such as single qualitative studies and quality improvement (QI) projects. The process for critical appraisal of a single qualitative study is the same as that for an appraisal of a systematic review or metasynthesis of qualitative studies, as described in a previous evidence-based practice column. This column will describe the critical appraisal of QI projects., (© Copyright 2023 Springer Publishing Company, LLC.)

Cytochrome P450 (CYP) enzymes are heme-thiolate monooxygenases which catalyze the oxidation of aliphatic and aromatic C-H bonds and other reactions. The oxidation of halogens by cytochrome P450 enzymes has also been reported. Here we use CYP199A4, from the bacterium Rhodopseudomonas palustris strain HaA2, with a range of para-substituted benzoic acid ligands, which contain halogens, to assess if this enzyme can oxidize these species or if the presence of these electronegative atoms can alter the outcome of P450-catalyzed reactions. Despite binding to the enzyme, there was no detectable oxidation of any of the 4-halobenzoic acids. CYP199A4 was, however, able to efficiently catalyze the oxidation of both 4-chloromethyl- and 4-bromomethyl-benzoic acid to 4-formylbenzoic acid via hydroxylation of the α‑carbon. The 4-chloromethyl substrate bound in the enzyme active site in a similar manner to 4-ethylbenzoic acid. This places the benzylic α‑carbon hydrogens in an unfavorable position for abstraction indicating a degree of substrate mobility must be possible within the active site. CYP199A4 catalyzed oxidations of 4-(2'-haloethyl)benzoic acids yielding α-hydroxylation and desaturation metabolites. The α-hydroxylation product was the major metabolite. The desaturation pathway is significantly disfavored compared to 4-ethylbenzoic acid. This may be due to the electron-withdrawing halogen atom or a different positioning of the substrate within the active site. The latter was demonstrated by the X-ray crystal structures of CYP199A4 with these substrates. Overall, the presence of a halogen atom positioned close to the heme iron can alter the binding orientation and outcomes of enzyme-catalyzed oxidation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The CYP124 and CYP142 families of bacterial cytochrome P450 monooxygenases (CYPs), catalyze the oxidation of methyl branched lipids, including cholesterol, as one of the initial activating steps in their catabolism. Both enzymes are reported to supplement the CYP125 family of P450 enzymes. These CYP125 enzymes are found in the same bacteria, and are the primary cholesterol/cholest-4-en-3-one metabolizing enzymes. To further understand the role of the CYP124 and CYP142 cytochrome P450s we investigated the Mycobacterium marinum enzymes, MmarCYP124A1 and CYP142A3, with various cholesterol analogs with modifications on the A and B rings of the steroid. We assessed the substrate binding and catalytic activity of each enzyme. Neither enzyme could bind or oxidize cholesteryl acetate or 3,5-cholestadiene, which have modifications at the C3 hydroxyl moiety of cholesterol. The CYP142 enzyme was better able to accommodate and oxidize cholesterol analogs which have changes on the A/B rings including cholesterol-5α,6α-epoxide and diastereomers of 5-cholestan-3-ol. The CYP124 enzyme was more tolerant of changes at C7 of the cholesterol B ring, e.g., 7-ketocholesterol than in the A ring. The selectivity for oxidation at the ω-carbon of a branched chain was observed in all steroids that were oxidized. The 7-ketocholesterol-bound MmarCYP124A1 enzyme from M. marinum, was structurally characterized by X-ray crystallography to 1.81 Å resolution. The 7-ketocholesterol-bound X-ray crystal structure of the MmarCYP124A1 enzyme revealed that the substrate binding mode of this cholesterol derivative was altered compared to those observed with other non-steroidal ligands. The structure provided an explanation for the selectivity of the enzyme for terminal methyl hydroxylation., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

The cytochrome P450 enzyme CYP102A1 (P450BM3) is a versatile monooxygenase enzyme which has been adapted and engineered for multiple applications in chemical synthesis. Mutation of threonine 268 to glutamate (Thr268Glu) converted the heme domain of this enzyme into a H 2 O 2 utilizing peroxygenase. This variant displayed significantly increased peroxide driven hydroxylation activity towards the saturated linear fatty acids tested (undecanoic through to hexadecenoic acid) when compared to the wild-type heme domain. The product distributions arising from fatty acid oxidation using this peroxygenase variant were broadly similar to those obtained with the wild-type monooxygenase holoenzyme, with oxidation occurring predominantly at the ω-1 through to ω-3 positions. 10-Undecenoic acid was regioselectively hydroxylated at the allylic ω-2 carbon by the Thr268Glu peroxygenase. The effect of isotopic substitution were measured using [9,9,10,10-d 4 ]-dodecanoic acid. The kinetic isotope effect for both the monooxygenase and peroxygenase systems ranged between 7.9 and 9.5, with that of the peroxygenase enzyme being marginally lower. This highlights that carbon‑hydrogen bond abstraction is important in the mechanism of both the monooxygenase and peroxygenase systems. This would infer that the ferryl-oxo radical cation intermediate, compound I, is the likely reactive intermediate in both systems. The peroxygenase variant offers the possibility of simpler cytochrome P450 systems for selective oxidations. To demonstrate this we used this system to oxidize tetradecanoic acid using light driven generation of H 2 O 2 by a flavin., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The cytochrome P450 superfamily of heme-thiolate monooxygenase enzymes can catalyse various oxidation reactions. The addition of a substrate or an inhibitor ligand induces changes in the absorption spectrum of these enzymes and UV-visible (UV-vis) absorbance spectroscopy is the most common and readily available technique used to interrogate their heme and active site environment. Nitrogen-containing ligands can inhibit the catalytic cycle of heme enzymes by interacting with the heme. Here we evaluate the binding of imidazole and pyridine-based ligands to the ferric and ferrous forms of a selection of bacterial cytochrome P450 enzymes using UV-visible absorbance spectroscopy. The majority of these ligands interact with the heme as one would expect for type II nitrogen directly coordinated to a ferric heme-thiolate species. However, the spectroscopic changes observed in the ligand-bound ferrous forms indicated differences in the heme environment across these P450 enzyme/ligand combinations. Multiple species were observed in the UV-vis spectra of the ferrous ligand-bound P450s. None of the enzymes gave rise to the isolation of a single species with a Soret band at ∼442-447 nm, indicative of a 6-coordinate ferrous thiolate species with a nitrogen-donor ligand. A ferrous species with Soret band at ∼427 nm coupled with an α-band of increased intensity was observed with the imidazole ligands. With some enzyme-ligand combinations reduction resulted in breaking of the iron‑nitrogen bond yielding a 5-coordinate high-spin ferrous species. In other instances, the ferrous form was readily oxidised back to the ferric form on addition of the ligand., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

The cytochrome P450 (CYP) superfamily of heme monooxygenases has demonstrated ability to facilitate hydroxylation, desaturation, sulfoxidation, epoxidation, heteroatom dealkylation, and carbon-carbon bond formation and cleavage (lyase) reactions. Seeking to study the carbon-carbon cleavage reaction of α-hydroxy ketones in mechanistic detail using a microbial P450, we synthesized α-hydroxy ketone probes based on the physiological substrate for a well-characterized benzoic acid metabolizing P450, CYP199A4. After observing low activity with wild-type CYP199A4, subsequent assays with an F182L mutant demonstrated enzyme-dependent C-C bond cleavage toward one of the α-hydroxy ketones. This C-C cleavage reaction was subject to an inverse kinetic solvent isotope effect analogous to that observed in the lyase activity of the human P450 CYP17A1, suggesting the involvement of a species earlier than Compound I in the catalytic cycle. Co-crystallization of F182L-CYP199A4 with this α-hydroxy ketone showed that the substrate bound in the active site with a preference for the (S)-enantiomer in a position which could mimic the topology of the lyase reaction in CYP17A1. Molecular dynamics simulations with an oxy-ferrous model of CYP199A4 revealed a displacement of the substrate to allow for oxygen binding and the formation of the lyase transition state proposed for CYP17A1. This demonstration that a correctly positioned α-hydroxy ketone substrate can realize lyase activity with an unusual inverse solvent isotope effect in an engineered microbial system opens the door for further detailed biophysical and structural characterization of CYP catalytic intermediates.

The CYP124 family of cytochrome P450 enzymes, as exemplified by CYP124A1 from Mycobacterium tuberculosis, is involved in the metabolism of methyl branched lipids and cholesterol derivatives. The equivalent enzyme from Mycobacterium marinum was investigated to compare the degree of functional conservation between members of this CYP family from closely related bacteria. We compared substrate binding of each CYP124 enzyme using UV-vis spectroscopy and the catalytic oxidation of methyl branched lipids, terpenes and cholesterol derivatives was investigated. The CYP124 enzyme from M. tuberculosis displayed a larger shift to the ferric high-spin state on binding cholesterol derivatives compared to the equivalent enzyme from M. marinum. The biggest difference was observed with cholesteryl sulfate which induced distinct UV-vis spectra in each CYP124 enzyme. The selectivity for oxidation at the ω-carbon of a branched chain was maintained for all substrates, except cholesteryl sulfate which was not oxidized by either enzyme. The CYP124A1 enzyme from M. marinum, in combination with farnesol and farnesyl acetate, was structurally characterized by X-ray crystallography. These ligand-bound structures of the CYP124 enzyme revealed that the polar component of the substrates bound in a different manner to that of phytanic acid in the structure of CYP124A1 from M. tuberculosis. However, closer to the heme the structures were similar providing an explanation for the high selectivity of the enzyme for terminal methyl C-H bond oxidation. The work here demonstrates that there were differences in the biochemistry of the CYP124 enzymes from these closely related bacteria., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Rhodococcus globerulus is a metabolically active organism that has been shown to utilise eucalypt oil as its sole source of carbon and energy. This oil includes 1,8-cineole, p-cymene and limonene. Two identified and characterised cytochromes P450 (P450s) from this organism initiate the biodegradation of the monoterpenes 1,8-cineole (CYP176A1) and p-cymene (CYP108N12). Extensive characterisation has been completed for CYP176A1 and it has been successfully reconstituted with its immediate redox partner, cindoxin, and E. coli flavodoxin reductase. Two putative redox partner genes are encoded in the same operon as CYP108N12 and here the isolation, expression, purification, and characterisation of its specific [2Fe-2S] ferredoxin redox partner, cymredoxin is presented. Reconstitution of CYP108N12 with cymredoxin in place of putidaredoxin, a [2Fe-2S] redox partner of another P450, improves both the rate of electron transfer (from 13 ± 2 to 70 ± 1 μM NADH/min/μM CYP108N12) and the efficiency of NADH utilisation (the so-called coupling efficiency increases from 13% to 90%). Cymredoxin improves the catalytic ability of CYP108N12 in vitro. Aldehyde oxidation products of the previously identified substrates p-cymene (4-isopropylbenzaldehyde) and limonene (perillaldehyde) were observed in addition to major hydroxylation products 4-isopropylbenzyl alcohol and perillyl alcohol respectively. These further oxidation products had not previously been seen with putidaredoxin supported oxidation. Furthermore, when supported by cymredoxin CYP108N12 is able to oxidise a wider range of substrates than previously reported. These include o-xylene, α-terpineol, (-)-carveol and thymol yielding o-tolylmethanol, 7-hydroxyterpineol, (4R)-7-hydroxycarveol and 5-hydroxymethyl-2-isopropylphenol, respectively. Cymredoxin is also capable of supporting CYP108A1 (P450 terp ) and CYP176A1 activity, allowing them to catalyse the hydroxylation of their native substrates α-terpineol to 7-hydroxyterpineol and 1,8-cineole to 6β-hydroxycineole respectively. These results indicate that cymredoxin not only improves the catalytic capability of CYP108N12 but can also support the activity of other P450s and prove useful for their characterisation., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Critical appraisal of the evidence is the third step in the evidence-based practice (EBP) process. Many questions in nursing cannot be answered using quantitative methods. We often desire a better understanding of people's lived experiences. In the NICU, these questions may be related to the experiences of families or staff. Qualitative research can provide a deeper understanding of lived experiences. This column, the fifth in a multipart series describing the critical appraisal process focuses on the critical appraisal of a systematic review of qualitative studies., (© Copyright 2023 Springer Publishing Company, LLC.)

Cytochrome P450 (CYP) heme-thiolate monooxygenases catalyze the hydroxylation of the C-H bonds of organic molecules. This reaction is initiated by a ferryl-oxo heme radical cation (Cpd I). These enzymes can also catalyze sulfoxidation reactions and the ferric-hydroperoxy complex (Cpd 0) and the Fe(III)-H 2 O 2 complex have been proposed as alternative oxidants for this transformation. To investigate this, the oxidation of 4-alkylthiobenzoic acids and 4-methoxybenzoic acid by the CYP199A4 enzyme from Rhodopseudomonas palustris HaA2 was compared using both monooxygenase and peroxygenase pathways. By examining mutants at the mechanistically important, conserved acid alcohol-pair (D251N, T252A and T252E) the relative amounts of the reactive intermediates that would form in these reactions were disturbed. Substrate binding and X-ray crystal structures helped to understand changes in the activity and enabled an attempt to evaluate whether multiple oxidants can participate in these reactions. In peroxygenase reactions the T252E mutant had higher activity towards sulfoxidation than O-demethylation but in the monooxygenase reactions with the WT enzyme the activity of both reactions was similar. The peroxygenase activity of the T252A mutant was greater for sulfoxidation reactions than the WT enzyme, which is the reverse of the activity changes observed for O-demethylation. The monooxygenase activity and coupling efficiency of sulfoxidation and oxidative demethylation were reduced by similar degrees with the T252A mutant. These observations infer that while Cpd I is required for O-dealkylation, another oxidant may contribute to sulfoxidation. Based on the activity of the CYP199A4 mutants it is proposed that this is the Fe(III)-H 2 O 2 complex which would be more abundant in the peroxide-driven reactions., (© 2022 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

The cytochrome P450 family of monooxygenase enzymes have essential biological roles involving the selective oxidation of carbon-hydrogen bonds. They can also catalyze other important metabolic reactions including desaturation to form alkenes. Currently the factors that control the partition between P450 hydroxylation and desaturation pathways are poorly defined. The CYP199A4 enzyme from the bacterium Rhodopseudomonas palustris HaA2 catalyzes the oxidation of 4-ethyl- and 4-isopropyl- benzoic acids with hydroxylation and desaturation occurring in significant quantities. Here we demonstrate that 4-cyclopropylbenzoic acid is regioselectively hydroxylated by CYP199A4 at the benzylic carbon. In contrast, the oxidation of 4-n-propylbenzoic acid by CYP199A4 results in three major metabolites: an alkene from desaturation and two hydroxylation products at the benzylic (Cα) and Cβ carbons in similar quantities. Extending the length of the alkyl substituent resulted in 4-n-butylbenzoic acid being oxidized at the benzylic position (45%) and desaturated (55%). In contrast, 4-isobutylbenzoic generated very little alkene (5%) but was hydroxylated at the benzylic position (54%) and at the tertiary Cβ position (41%). The oxidation of 4-n-propylbenzoic acid by the F298 V mutant of CYP199A4 occurred with no hydroxylation at Cβ and a significant increase in metabolites arising from desaturation (73%). The X-ray crystal structures of CYP199A4 with each substrate revealed that they bind in the active site with the alkyl substituent positioned over the heme. However, the longer alkylbenzoic acids were bound in a different conformation as was 4-n-propylbenzoic acid in the F298 V mutant. Overall, the changes in metabolite distribution could be ascribed to bond strength differences and the position of the alkyl group relative to the heme., (© 2022 The Authors. Chemistry & An Asian Journal published by Wiley-VCH GmbH.)

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., (© 2022 The Authors. Chemistry - A European Journal published by Wiley-VCH GmbH.)

A crucial reaction in harnessing renewable carbon from lignin is O -demethylation. We demonstrate the selective O -demethylation of syringol and guaiacol using different cytochrome P450 enzymes. These can efficiently use hydrogen peroxide which, when compared to nicotinamide cofactor-dependent monooxygenases and synthetic methods, allows for cheap and clean O -demethylation of lignin-derived aromatics.

Critical appraisal of the evidence is the third step in the evidence-based practice process. This column, the third in a multipart series to describe the critical appraisal process, focuses on critical appraisal of quasi-experimental or nonrandomized experimental studies., (© Copyright 2022 Springer Publishing Company, LLC.)

Objective: Cervical cancer can be prevented by regular screening; however, screening rates are low in developing countries. We evaluated the proportion of women screened, modalities of screening utilized, and factors influencing uptake among Ghanaian women with access to free screening services., Methods: Participants were women aged 25-65 in Asokore-Mampong, Ghana. A structured questionnaire collected socio-demographic characteristics, risk factors, knowledge of, and utilization of cervical cancer screening. Adjusted logistic regression evaluated predictors of screening., Results: Of 710 participants, the majority had heard of cervical cancer (64.6%) and screening (57.7%). Screening utilization was 24.6%. Visual inspection with acetic acid was the most common screening method (97.1%). For those who had never been screened, common reasons were believing they were healthy (21.7%), fearing pain (12.9%), lacking awareness of screening (11.8%), and being too busy (11.6%). Participants who were aged 35-44 (aOR 1.82; 95% CI 1.09-3.03; p = 0.023), married (aOR 3.98; 95% CI 1.68-9.40; p = 0.002), formally employed (aOR 9.31; 95% CI 2.86-30.35; p <0.001), and had higher cervical cancer knowledge (aOR 3.98; 95% CI 2.64-6.02; p <0.001) were more likely to have been screened., Conclusion: Despite geographic proximity to a health center that provides free cervical cancer screening, screening uptake among Ghanaian women remains low., (© 2022 International Federation of Gynecology and Obstetrics.)