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Sutherland et al. recently reported the in vitro inhibition of Helicobacter hepaticus cytochrome c (cyt c) synthase, which may inactivate c-type cytochromes in this and in other pathogenic bacteria. Here, I discuss the impact of this work, which identifies this conserved synthase as a potential antibacterial target. [ABSTRACT FROM AUTHOR]

Cytochromes P450 (CYPs) catalyze a great number of metabolic reactions that have profound effects on the biological activities of xenobiotics and endobiotics. In this study, we aimed to characterize rhythmic expressions of drug-metabolizing CYPs using synchronized hepatoma cells, and to investigate the potential roles of cis-elements of circadian clock system (E-box, D-box and RevRE or RORE) in generating the rhythms. Serum was used to synchronize circadian cycles and to induce circadian gene expression in cultured hepatoma cells (HepRG and HepG2 cells). Regulation of CYP genes by circadian clock components was investigated by performing luciferase reporter, overexpression and knockdown experiments. mRNA and protein expression were determined by qPCR and Western blotting assays, respectively. Of ten major drug-metabolizing CYP genes, six are rhythmically expressed (CYP1A2, 2B6, 2C8, 2D6, 2E1 and 3A4), whereas other four are non-rhythmic (CYP1B1, 2A6, 2C9 and 2C19). The E-box binding protein BMAL1 directly controls the rhythmic expression of CYP1A2. Rhythmic expressions of CYP2E1 and CYP3A4 are generated via both E-box and D-box elements. The RevRE binding protein REV-ERBα contributes to rhythmic oscillations in CYP2B6 and CYP2C8. In conclusion, rhythmic expressions of five human CYPs (CYP1A2, 2B6, 2C8, 2E1 and 3A4) are generated and regulated by E-box-, D-box-, and/or RevRE-acting clock components. Our findings may have implications for understanding chronopharmacokinetic events in humans. [ABSTRACT FROM AUTHOR]

The 1,3‐enyne moiety is commonly found in cyclohexanoid natural products produced by endophytic and plant pathogenic fungi. Asperpentyn (1) is a 1,3‐enyne‐containing cyclohexanoid terpenoid isolated from Aspergillus and Pestalotiopsis. The genetic basis and biochemical mechanism of 1,3‐enyne biosynthesis in 1, and other natural products containing this motif, has remained enigmatic despite their potential ecological roles. Identified here is the biosynthetic gene cluster and characterization of two crucial enzymes in the biosynthesis of 1. A P450 monooxygenase that has a dual function, to first catalyze dehydrogenation of the prenyl chain to generate a cis‐diene intermediate and then serve as an acetylenase to yield an alkyne moiety, and thus the 1,3‐enyne, was discovered. A UbiA prenyltransferase was also characterized and it is unusual in that it favors transferring a five‐carbon prenyl chain, rather than a polyprenyl chain, to a p‐hydroxybenzoic acid acceptor. [ABSTRACT FROM AUTHOR]

The alkyne is a biologically significant moiety found in many natural products and a versatile functional group widely used in modern chemistry. Recent studies have revealed the biosynthesis of acetylenic bonds in fatty acids and amino acids. However, the molecular basis for the alkynyl moiety in acetylenic prenyl chains occurring in a number of meroterpenoids remains obscure. Here, we identify the biosynthetic gene cluster and characterize the biosynthetic pathway of an acetylenic meroterpenoid biscognienyne B based on heterologous expression, feeding experiments, and in vitro assay. This work shows that the alkyne moiety is constructed by an unprecedented cytochrome P450 enzyme BisI, which shows promiscuous activity towards C5 and C15 prenyl chains. This finding provides an opportunity for discovery of new compounds, featuring acetylenic prenyl chains, through genome mining, and it also expands the enzyme inventory for de novo biosynthesis of alkynes. [ABSTRACT FROM AUTHOR]

The 1,3‐enyne moiety is commonly found in cyclohexanoid natural products produced by endophytic and plant pathogenic fungi. Asperpentyn (1) is a 1,3‐enyne‐containing cyclohexanoid terpenoid isolated from Aspergillus and Pestalotiopsis. The genetic basis and biochemical mechanism of 1,3‐enyne biosynthesis in 1, and other natural products containing this motif, has remained enigmatic despite their potential ecological roles. Identified here is the biosynthetic gene cluster and characterization of two crucial enzymes in the biosynthesis of 1. A P450 monooxygenase that has a dual function, to first catalyze dehydrogenation of the prenyl chain to generate a cis‐diene intermediate and then serve as an acetylenase to yield an alkyne moiety, and thus the 1,3‐enyne, was discovered. A UbiA prenyltransferase was also characterized and it is unusual in that it favors transferring a five‐carbon prenyl chain, rather than a polyprenyl chain, to a p‐hydroxybenzoic acid acceptor. [ABSTRACT FROM AUTHOR]

The alkyne is a biologically significant moiety found in many natural products and a versatile functional group widely used in modern chemistry. Recent studies have revealed the biosynthesis of acetylenic bonds in fatty acids and amino acids. However, the molecular basis for the alkynyl moiety in acetylenic prenyl chains occurring in a number of meroterpenoids remains obscure. Here, we identify the biosynthetic gene cluster and characterize the biosynthetic pathway of an acetylenic meroterpenoid biscognienyne B based on heterologous expression, feeding experiments, and in vitro assay. This work shows that the alkyne moiety is constructed by an unprecedented cytochrome P450 enzyme BisI, which shows promiscuous activity towards C5 and C15 prenyl chains. This finding provides an opportunity for discovery of new compounds, featuring acetylenic prenyl chains, through genome mining, and it also expands the enzyme inventory for de novo biosynthesis of alkynes. [ABSTRACT FROM AUTHOR]

Cytochromes P450 are oxidizing enzymes; a few families of cytochromes P450 are implicated in drug metabolism. These enzymatic reactions involve many processes including (i) prodrug to drug conversion, (ii) easy excretion of drug, (iii) generation of reactive metabolites, many of which cause toxicity. In this review, the fundamental biochemical mechanisms associated with the conversion of drugs into the useful or toxic metabolites have been discussed. The mechanisms can be established with the help of many experimental methods like mass spectral analysis, NMR and in vitro analysis etc. Computational methods provide detailed atomic level information, which is generally not available from experimental studies. Thus, the in silico efforts in elucidating the molecular mechanisms are complementary to the known experimental methods and are often clearer (especially in providing 3D information about the metabolites and their reactions). Quantum chemical methods and molecular docking become especially very useful. This review includes five case studies, which explain how the atomic level details were obtained to explore the reaction mechanisms of drug metabolism by cytochromes P450. [ABSTRACT FROM AUTHOR]

Steroidal C7β alcohols and their respective esters have shown significant promise as neuroprotective and anti‐inflammatory agents to treat chronic neuronal damage like stroke, brain trauma, and cerebral ischemia. Since C7 is spatially far away from any functional groups that could direct C−H activation, these transformations are not readily accessible using modern synthetic organic techniques. Reported here are P450‐BM3 mutants that catalyze the oxidative hydroxylation of six different steroids with pronounced C7 regioselectivities and β stereoselectivities, as well as high activities. These challenging transformations were achieved by a focused mutagenesis strategy and application of a novel technology for protein library construction based on DNA assembly and USER (Uracil‐Specific Excision Reagent) cloning. Upscaling reactions enabled the purification of the respective steroidal alcohols in moderate to excellent yields. The high‐resolution X‐ray structure and molecular dynamics simulations of the best mutant unveil the origin of regio‐ and stereoselectivity. [ABSTRACT FROM AUTHOR]

Anaerobic microorganisms of the Geobacter genus are effective electron sources for the synthesis of nanoparticles, for bioremediation of polluted water, and for the production of electricity in fuel cells. In multistep reactions, electrons are transferred via iron/heme cofactors of c‐type cytochromes from the inner cell membrane to extracellular metal ions, which are bound to outer membrane cytochromes. We measured electron production and electron flux rates to 5×105 e s−1 per G. sulfurreducens. Remarkably, these rates are independent of the oxidants, and follow zero order kinetics. It turned out that the microorganisms regulate electron flux rates by increasing their Fe2+/Fe3+ ratios in the multiheme cytochromes whenever the activity of the extracellular metal oxidants is diminished. By this mechanism the respiration remains constant even when oxidizing conditions are changing. This homeostasis is a vital condition for living systems, and makes G. sulfurreducens a versatile electron source. [ABSTRACT FROM AUTHOR]

Philasterides dicentrarchi is a marine benthic microaerophilic scuticociliate and an opportunistic endoparasite that can infect and cause high mortalities in cultured turbot (Scophthalmus maximus). In addition to a cytochrome pathway (CP), the ciliate can use a cyanide-insensitive respiratory pathway, which indicates the existence of an alternative oxidase (AOX) in the mitochondrion. Although AOX activity has been described in P. dicentrarchi, based on functional assay results, genetic evidence of the presence of AOX in the ciliate has not previously been reported. In this study, we conducted genomic and transcriptomic analysis of the ciliate and identified the AOX gene and its corresponding mRNA. The AOX gene (size 1,106 bp) contains four exons and three introns that generate an open reading frame of 915 bp and a protein with a predicted molecular weight of 35.6 kDa. The amino acid (aa) sequence of the AOX includes an import signal peptide targeting the mitochondria and the protein is associated with the inner membrane of the mitochondria. Bioinformatic analysis predicted that the peptide is a homodimeric glycoprotein, although monomeric forms may also appear under native conditions, with EXXH motifs associated with the diiron active centers. The aa sequences of the AOX of different P. dicentrarchi isolates are highly conserved and phylogenetically closely related to AOXs of other ciliate species, especially scuticociliates. AOX expression increased significantly during infection in the host and after the addition of CP inhibitors. This confirms the important physiological roles of AOX in respiration under conditions of low levels of O2 and in protecting against oxidative stress generated during infection in the host. [ABSTRACT FROM AUTHOR]

The design of drugs from natural products is a re-emerging area due to the need for bioactive compounds. The exploitation of natural products and their derivatives obtained by biocatalysis is in line with the higher attention given today to new sustainable technologies that better preserve the environment (green chemistry). The research field of cytochromes P450 (CYPs) is continuously providing new enzymes and mutants that produce metabolites suitable for late-stage functionalization for new potential drugs. This review provides an overview of the exploitation of CYPs as biocatalysts in drug synthesis. Additionally, recent progress in protein and metabolic engineering is provided to show how these enzymes offer a toolbox that can be combined with other biocatalytic or chemical processes to build new platforms for the green production of new drugs. Cytochromes P450 (CYPs) are enzymes physiologically involved in the biosynthesis of thousands of bioactive natural compounds in bacteria, fungi, plants, and mammals. CYPs act on different molecular scaffolds and can be crucial for late-stage functionalization of bioactive compounds to produce new pharmaceuticals. Some CYPs are already used at industrial level for the production of nature-inspired pharmaceuticals, for example, for the production of pravastatin, a cholesterol-lowering drug, by bacterial P450sca-2. The use of CYPs in the synthesis of pharmaceuticals is expanding thanks to the progresses in protein, metabolic, and substrate engineering. [ABSTRACT FROM AUTHOR]

Background: The in vivo mechanistic static model (IMSM) is an effective method to predict the magnitude of drug-drug interactions (DDIs) mediated by cytochromes.Objective: The aim of this study was to extend the IMSM paradigm to DDIs mediated by organic anion transporting polypeptide (OATP) 1Bs, breast cancer resistance protein (BCRP) and cytochrome 2C8.Methods: First, a generic model for this kind of interaction was established, and a literature search was then conducted to retrieve the area under the concentration-time curve (AUC) ratio of a large set of DDIs involving OATP1B1, OATP1B3, BCRP and cytochromes 2C8 or 3A4. The model was fitted to the data to estimate the characteristic parameters (contribution ratios [CRs] and inhibition or induction potencies [IXs]) by nonlinear regression, and the model was qualified by external validation on a different dataset. Lastly, the model was used to identify the risks of overexposure by DDIs of this type.Results: A total of 27 substrates, 26 inhibitors, 3 inducers and 3 genetic variants were considered in the regression analysis. The number of observations (AUC ratios, denoted as Robs) was 101. Forty-six CRs and 47 IXs were estimated. The proportions of predictions within 0.67- to 1.5-fold and 0.5- to twofold Robs were 90% and 99%, respectively, for the internal validation, and 78% and 96%, respectively, for the external validation. The median fold-error was 1.03 (the ideal value is 1). The interquartile range of fold-error was 0.31, and the relative standard error of parameter estimates was, at most, 17%.Conclusions: The IMSM approach was successfully extended to DDIs mediated by OATP1Bs, BCRP and cytochromes 2C8 or 3A4. The method revealed good predictive performances by internal and external validation. [ABSTRACT FROM AUTHOR]

As a bacteriostatic agent, nitrite has been used in food preservation for centuries. When used in combination with antibiotics, nitrite is reported to work either cooperatively or antagonistically. However, the mechanism underlying these effects remains largely unknown. Here we show that nitrite mediates tolerance to aminoglycosides in both Gram-negative and Gram-positive bacteria, but has little interaction with other types of antibiotics. Nitrite directly and mainly inhibits cytochrome heme-copper oxidases (HCOs), and by doing so, the membrane potential is compromised, blocking uptake of aminoglycosides. In contrast, reduced respiration (oxygen consumption rate) resulting from nitrite inhibition is not critical for aminoglycoside tolerance. While our data indicate that nitrite is a promising antimicrobial agent targeting HCOs, cautions should be taken when used with other antibiotics, aminoglycosides in particular. Zhang et al. show that nitrite mediates bacterial tolerance to aminoglycosides without affecting other antibiotics. They find that nitrite inhibits cytochrome heme-copper oxidases, blocking bacterial uptake of aminoglycosides. This study suggests nitrite as a promising antimicrobial agent while discouraging its combined use with aminoglycosides. [ABSTRACT FROM AUTHOR]

Despite CYP102A1 (P450BM3) representing one of the most extensively researched metalloenzymes, crystallisation of its haem domain upon modification can be a challenge. Crystal structures are indispensable for the efficient structure‐based design of P450BM3 as a biocatalyst. The abietane diterpenoid derivative N‐abietoyl‐l‐tryptophan (AbiATrp) is an outstanding crystallisation accelerator for the wild‐type P450BM3 haem domain, with visible crystals forming within 2 hours and diffracting to a near‐atomic resolution of 1.22 Å. Using these crystals as seeds in a cross‐microseeding approach, an assortment of P450BM3 haem domain crystal structures, containing previously uncrystallisable decoy molecules and diverse artificial metalloporphyrins binding various ligand molecules, as well as heavily tagged haem‐domain variants, could be determined. Some of the structures reported herein could be used as models of different stages of the P450BM3 catalytic cycle. [ABSTRACT FROM AUTHOR]

Susceptibility and PBO synergist bioassays were done using 3–5 days old female Anopheles mosquito collected from Lagos State, Nigeria with WHO test papers DDT (4%), permethrin (0.75%), Bendiocarb (1%) and PBO (4%) according to standard procedures. The activities of cytochrome P450s, glutathione S-transferase and carboxylesterases were determined using biochemical assays. The presence of kdr-w, kdr-e and Ace-1R mutations were examined using molecular assays. Resistance to DDT and permethrin in An gambiae s.s from the four Local Government Areas (LGAs) was recorded while suspected resistance to bendiocarb was recorded in mosquitoes from Alimosho and Kosofe LGAs. PBO synergist reduced the knockdown time and also recorded significantly (P < 0.05) higher 24 hrs percentage mortality compared to non-synergized bioassays. Increased activities of detoxifying enzymes was recorded in wild mosquito compared to the insecticides susceptible laboratory strain and this was significant (P < 0.05) in P450s, esterase α and β. Kdr-w was detected in An. gambiae s.s from all the LGAs, kdr-e (L1014S) was detected in Alimosho, Kosofe and Ibeju-Lekki, while the Ace-1R gene was detected in Alimosho and Kosofe. Results from this study provide evidence for resistance of An. gambiae from Lagos State to multiple classes of neurotoxic insecticides with multiple resistance mechanisms to these insecticides. [ABSTRACT FROM AUTHOR]

Geobacter sulfurreducens is capable of reducing Pd(II) to Pd(0) using acetate as electron donor; however, the biochemical and genetic mechanisms involved in this process have not been described. In this work, we carried out transcriptome profiling analysis to identify the genes involved in Pd(II) reduction in this bacterium. Our results showed that 252 genes were upregulated while 141 were downregulated during Pd(II) reduction. Among the upregulated genes, 12 were related to energy metabolism and electron transport, 50 were classified as involved in protein synthesis, 42 were associated to regulatory functions and transcription, and 47 have no homologs with known function. RT-qPCR data confirmed upregulation of genes encoding PilA, the structural protein for electrically conductive pili, as well as c-type cytochromes GSU1062, GSU2513, GSU2808, GSU2934, GSU3107, OmcH, OmcM, PpcA, and PpcD under Pd(II)-reducing conditions. ΔpilA and ΔpilR mutant strains showed 20% and 40% decrease in the Pd(II)-reducing capacity, respectively, as compared to the wild type strain, indicating the central role of pili in this process. RT-qPCR data collected during Pd(II) reduction also confirmed downregulation of omcB, omcC, omcZ, and omcS genes, which have been shown to be involved in the reduction of Fe(III) and electrodes. The present study contributes to elucidate the mechanisms involved in Pd(II) reduction by G. sulfurreducens. Key points: • Transcriptome analysis provided evidence on Pd(II) reduction by G. sulfurreducens. • Results indicate that electrically conductive pili is involved in Pd(II) reduction. • G. sulfurreducens was not able to grow under Pd(II)-reducing conditions. • The study contributes to a better understanding of the mechanisms in Pd(II) reduction. [ABSTRACT FROM AUTHOR]

Background/Aims: The disruption of redox equilibrium by oxidative stress, which is characterized by an overproduction of reactive oxygen species (ROS), is considered to be associated with fibroblast death in severe lung diseases. Indole-6-carboxaldehyde (I6CA) is a natural indole derivative isolated from Sargassum thunbergii, which a type of brown algae. However, the antioxidative effects of I6CA, and their mechanisms, have not been identified. This study was conducted to investigate the potential protective effects of I6CA against oxidative stress in V79-4 Chinese hamster lung fibroblasts. Methods: Cell viability and mechanisms related to antioxidant activity of I6CA (ROS production, cell cycle, DNA damage, mitochondrial membrane potential (MMP) and apoptosis) were studied. Western blot analysis was carried out to understand the involvement of various genes at protein level. Results: Our results demonstrated that I6CA inhibited hydrogen peroxide (H2O2)-induced cytotoxicity by blocking abnormal ROS accumulation. H2O2 treatment of V79-4 fibroblasts caused cell cycle arrest at the G2/M phase, which was accompanied by increased expression of the cyclin-dependent kinase (Cdk) inhibitor p21WAF1/CIP1 and decreased expression of cyclin B1 and cyclin A. However, these effects were attenuated by treatment with I6CA. I6CA also effectively protected V79-4 cells against H2O2-induced apoptosis by increasing the Bcl-2/Bax ratio and suppressing the loss of MMP and the cytosolic release of cytochrome c. In addition, the activation of nuclear factor-erythroid-2-related factor 2 (Nrf2) was markedly promoted by I6CA, which was associated with enhanced expression and activity of heme oxygenase-1 (HO-1). However, inhibiting the activity of HO-1 by zinc protoporphyrin IX, a potent inhibitor of HO-1, eliminated the ROS scavenging and anti-apoptotic effects of I6CA, indicating that I6CA was able to protect V79-4 lung fibroblasts from H2O2-induced oxidative stress by activating the Nrf2 signaling pathway. Conclusion: We suggest that I6CA may be useful as a candidate therapeutic agent for the treatment of oxidative stress-related lung diseases. [ABSTRACT FROM AUTHOR]

The simulation of membrane proteins requires compatible protein and lipid force fields that reproduce the properties of both the protein and the lipid bilayer. Cytochrome P450 enzymes are bitopic membrane proteins with a transmembrane helical anchor and a large cytosolic globular domain that dips into the membrane. As such, they are representative and challenging examples of membrane proteins for simulations, displaying features of both peripheral and integral membrane proteins. We performed molecular dynamics simulations of three cytochrome P450 isoforms (2C9, 2C19 and 1A1) in a 2-oleoyl-1-palmitoyl-sn-glycerol-3-phosphocholine bilayer using two AMBER force field combinations: GAFF-LIPID with ff99SB for the protein, and LIPID14 with ff14SB for the protein. Comparison of the structural and dynamic properties of the proteins, the lipids and the protein-membrane interactions shows differing sensitivity of the cytochrome P450 isoforms to the choice of force field, with generally better agreement with experiment for the LIPID14 + ff14SB combination. [ABSTRACT FROM AUTHOR]

Introduction: Oxygenated water intake could increase cell death markers through increasing the free radicals. However, sport activities and antioxidant substances may prevent some of the symptoms caused by free radical production. The present study aimed to investigate the effects of endurance training (ET) and purslane (Portulaca oleracea; PO) seed consumption on cytochrome-C and malondialdehyde (MDA) in the heart tissues of rats poisoned with H2O2. Methods: In total, 45 male rats were randomly divided into nine groups of five, including control, 50 mg/kg of PO, 200 mg/kg of PO, 400 mg/kg of PO, ET, ET with 50 mg/kg of PO, ET with 200 mg/kg of PO, ET with 400 mg/kg of PO, and healthy control. During eight weeks, groups 1-8 received H2O2 (1 mmol/kg) intraperitoneally three times per week, and groups 5-8 ran on treadmill three days per week. Results: ET and PO significantly reduced cytochrome-C and MDA (P=0.001), while the interactive effects of ET and PO on the reduction of cytochrome-C (P=0.52) and MDA (P=0.08) were not considered significant. In addition, the administration of 200 mg/kg (P=0.01) and 400 mg/kg of PO (P=0.001) significantly decreased cytochrome-C, while 400 mg/kg of PO had more significant effects on the reduction of cytochrome-C compared to 200 mg/kg of the substance (P=0.01). Moreover, 400 mg/kg of PO significantly reduced MDA (P=0.001). Conclusion: According to the results, ET and PO could improve cytochrome-C and MDA in the heart tissues of the rats poisoned with H2O2. [ABSTRACT FROM AUTHOR]

Monoheme c‐type cytochromes are important electron transporters in all domains of life. They possess a common fold hallmarked by three α‐helices that surround a covalently attached heme. An intriguing feature of many monoheme c‐type cytochromes is their capacity to form oligomers by exchanging at least one of their α‐helices, which is often referred to as 3D domain swapping. Here, the crystal structure of NirC, a c‐type cytochrome co‐encoded with other proteins involved in nitrite reduction by the opportunistic pathogen Pseudomonas aeruginosa, has been determined. The crystals diffracted anisotropically to a maximum resolution of 2.12 Å (spherical resolution of 2.83 Å) and initial phases were obtained by Fe‐SAD phasing, revealing the presence of 11 NirC chains in the asymmetric unit. Surprisingly, these protomers arrange into one monomer and two different types of 3D domain‐swapped dimers, one of which shows pronounced asymmetry. While the simultaneous observation of monomers and dimers probably reflects the interplay between the high protein concentration required for crystallization and the structural plasticity of monoheme c‐type cytochromes, the identification of conserved structural motifs in the monomer together with a comparison with similar proteins may offer new leads to unravel the unknown function of NirC. [ABSTRACT FROM AUTHOR]

Milbemycins and their semisynthetic derivatives are recognized as effective and eco-friendly pesticides, whereas the high price limits their widespread applications in agriculture. One of the pivotal questions is the accumulation of milbemycin-like by-products, which not only reduces the yield of the target products milbemycin A3/A4, but also brings difficulty to the purification. With other analogous by-products abolished, α9/α10 and β-family milbemycins remain to be eliminated. Herein, we solved these issues by engineering of post-modification steps. First, Cyp41, a CYP268 family cytochrome P450, was identified to participate in α9/α10 biosynthesis. By deleting cyp41, milbemycin α9/α10 was eliminated with an increase of milbemycin A3/A4 titer from 2382.5 ± 55.7 mg/L to 2625.6 ± 64.5 mg/L. Then, MilE, a CYP171 family cytochrome P450, was determined to be responsible for the generation of the furan ring between C6 and C8a of milbemycins. By further overexpression of milE, the production of β-family milbemycins was reduced by 77.2%. Finally, the titer of milbemycin A3/A4 was increased by 53.1% to 3646.9 ± 69.9 mg/L. Interestingly, overexpression of milE resulted in increased transcriptional levels of milbemycin biosynthetic genes and production of total milbemycins, which implied that the insufficient function of MilE was a limiting factor to milbemycin biosynthesis. Our research not only provides an efficient engineering strategy to improve the production of a commercially important product milbemycins, but also offers the clues for future study about transcriptional regulation of milbemycin biosynthesis. [ABSTRACT FROM AUTHOR]

The evaluation of drug metabolites is compulsory during drug development. Since recently, bacterial cytochromes P450 and their mutated variants have attracted considerable interest as an alternative to hepatic P450s for the synthesis of human drug metabolites. Thus, straightforward screening approaches are required that enable rapid identification and evaluation of drug‐metabolizing bacterial P450s with different product selectivities. Herein, we report a two‐step screening method for discovery and characterization of new P450s from actinomycetes that enable oxidation of various drugs. In the first step, substrate profiling with three structurally different model drugs, ritonavir, testosterone, amitriptyline, allowed us to select CYP105D and CYP107Z from Streptomyces platensis DSM 40041 that accepted all model substrates and produced human‐like drug metabolites. In the second step, activity tests with an array of 25 structurally‐related molecules and derivatives of the three model compounds revealed a correlation between structural variations in the target drugs and the enzyme chemo‐ and regioselectivity. [ABSTRACT FROM AUTHOR]

Apicomplexan parasites such as Toxoplasma gondii possess an unusual heme biosynthesis pathway whose enzymes localize to the mitochondrion, cytosol, or apicoplast, a nonphotosynthetic plastid present in most apicomplexans. To characterize the involvement of the apicoplast in the T. gondii heme biosynthesis pathway, we investigated the role of the apicoplastlocalized enzyme uroporphyrinogen III decarboxylase (TgUroD). We found that TgUroD knockdown impaired parasite proliferation, decreased free heme levels in the parasite, and decreased the abundance of heme-containing c-type cytochrome proteins in the parasite mitochondrion. We validated the effects of heme loss on mitochondrial cytochromes by knocking down cytochrome c/c1 heme lyase 1 (TgCCHL1), a mitochondrial enzyme that catalyzes the covalent attachment of heme to c-type cytochromes. TgCCHL1 depletion reduced parasite proliferation and decreased the abundance of c-type cytochromes. We further sought to characterize the overall importance of TgUroD and TgCCHL1 for both mitochondrial and general parasite metabolism. TgUroD depletion decreased cellular ATP levels, mitochondrial oxygen consumption, and extracellular acidification rates. By contrast, depletion of TgCCHL1 neither diminished ATP levels in the parasite nor impaired extracellular acidification rate, but resulted in specific defects in mitochondrial oxygen consumption. Together, our results indicate that the apicoplast has a key role in heme biology in T. gondii and is important for both mitochondrial and general parasite metabolism. Our study highlights the importance of heme and its synthesis in these parasites. [ABSTRACT FROM AUTHOR]

Cytochrome c oxidase (CcO), the Cu A, heme a , heme a 3 , Cu B enzyme of respiratory chain, converts the free energy released by aerobic cytochrome c oxidation into a membrane electrochemical proton gradient (ΔμH+). ΔμH+ derives from the membrane anisotropic arrangement of dioxygen reduction to two water molecules and transmembrane proton pumping from a negative (N) space to a positive (P) space separated by the membrane. Spectroscopic, potentiometric, and X-ray crystallographic analyses characterize allosteric cooperativity of dioxygen binding and reduction with protonmotive conformational states of CcO. These studies show that allosteric cooperativity stabilizes the favorable conformational state for conversion of redox energy into a transmembrane ΔμH+. (i) Dioxygen binding to Fe a 3 at BNC selects a CcO state in which the conformation of the subunit I-helix X, next in sequence to the hemes a and a 3 histidine axial ligands, opens access of pumped protons from the N space to a Mg2+-storage site at the opposite (P) membrane side. (ii) Sequential electron transfer at Cu A and heme a is associated with additional CcO conformational change in which protons are translocated from the Mg2+-site to the propionate(s) of heme a. From there, protons are expelled in the P space on heme a oxidation by electrostatic repulsion along a hydrogen bond network of amino acid residues and/or structured water molecules. Image 1 • Cytochrome c oxidase converts the energy released in cytochrome c oxidation into a transmembrane DmH+. • The molecular mechanism of ΔμH+ generation in cytochrome c oxidase is a topic of advanced functional and structural studies. • Allosteric cooperativity in A1-type CcO plays a key role in proton energy conversion. • A scheme of the catalytic cycle in cytochrome c oxidase, which highlights the role of allosteric cooperativity, is proposed. [ABSTRACT FROM AUTHOR]

The co-occurrence of Geobacter and Methanosarcinales is often used as a proxy for the manifestation of direct interspecies electron transfer (DIET) in the environment. Here we tested eleven new co-culture combinations between methanogens and electrogens. Previously, only the most electrogenic Geobacter paired by DIET with Methanosarcinales methanogens, namely G. metallireducens and G. hydrogenophilus. Here we provide additional support, and show that five additional Methanosarcinales paired with G. metallireducens, while a strict hydrogenotroph could not. We also show that G. hydrogenophilus, which is incapable to grow with a strict hydrogenotrophic methanogen, could pair with a strict non-hydrogenotrophic Methanosarcinales. Likewise, an electrogen outside the Geobacter cluster (Rhodoferrax ferrireducens) paired with Methanosarcinales but not with strict hydrogenotrophic methanogens. The ability to interact with electrogens appears to be conserved among Methanosarcinales, the only methanogens with c-type cytochromes, including multihemes (MHC). Nonetheless, MHC, which are often linked to extracellular electron transfer, were neither unique nor universal to Methanosarcinales and only two of seven Methanosarcinales tested had MHC. Of these two, one strain had an MHC-deletion knockout available, which we hereby show is still capable to retrieve extracellular electrons from G. metallireducens or an electrode suggesting an MHC-independent strategy for extracellular electron uptake. [ABSTRACT FROM AUTHOR]

Gold compounds have a long history of use as immunosuppressants, but their precise mechanism of action is not completely understood. Using our recently developed liver-on-a-chip platform we now show that gold compounds containing planar N-heterocyclic carbene (NHC) ligands are potent ligands for the aryl hydrocarbon receptor (AHR). Further studies showed that the lead compound (MC3) activates TGFβ1 signaling and suppresses CD4+ T-cell activation in vitro, in human and mouse T cells. Conversely, genetic knockdown or chemical inhibition of AHR activity or of TGFβ1-SMAD-mediated signaling offsets the MC3-mediated immunosuppression. In scurfy mice, a mouse model of human immunodysregulation polyendocrinopathy enteropathy X-linked syndrome, MC3 treatment reduced autoimmune phenotypes and extended lifespan from 24 to 58 days. Our findings suggest that the immunosuppressive activity of gold compounds can be improved by introducing planar NHC ligands to activate the AHR-associated immunosuppressive pathway, thus expanding their potential clinical application for autoimmune diseases. Cheng, Haeberle et al. identify NHC–gold complexes as ligands of the aryl hydrocarbon receptor, which triggers TGFβ production for immune suppression. They find that the MC3 compound activates TGFβ signalling while reducing Il-2 expression and CD4+ T-cell activation and suppresses autoimmune phenotypes in a mouse model of IPEX syndrome. [ABSTRACT FROM AUTHOR]

Aphis gossypii Glover, 1877 (Hemiptera: Aphididae), which is a polyphagous species, is among the main pests of the cotton plants in the whole world. The Çukurova region is an area where polyculture agriculture activities are carried out. While insecticides in the neonicotinoid group are prevalently used in the cotton fields in this region for controlling A. gossypii, the problem of resistance is frequently encountered. This study determined the relative expression levels of the CYP6CY22 and CYP6CY13 genes, cytochrome P450, acetylcholine esterase (AChE) and glutathione S-transferase (GST) enzyme activities in six different populations of A. gossypii collected in 2018 from cotton fields in the province of Adana in Southern Turkey. In comparison to the reference culture, there was an increase in gene regulation by 81.9 fold in the CYP6CY22 gene in the Hamitbey population and by 6 fold in the CYP6CY13 gene in the Gazipaşa population. Additionally, the Hamitbey population showed 4.3 times higher (0.74 U/ml) cytochrome P450 enzyme activity. The Çiftlikler population showed 5.9 fold higher AChE (0.82 U/ml) and 4.3 fold higher GST (2.68 U/ml) activities. The analyses revealed that the A. gossypii populations were exposed to high amounts of insecticides. Consequently, overexpressed genes, high levels of enzyme activities and metabolic resistance in based on cytochrome P450 activity were observed in the study. [ABSTRACT FROM AUTHOR]

This study reveals that it is possible to secrete truncated versions of outer membrane cytochromes into the culture supernatant and that these proteins can provide a basis for the export of heterologously produced proteins. Different soluble and truncated versions of the outer membrane cytochrome MtrF were analyzed for their suitability to be secreted. A protein version with a very short truncation of the N-terminus to remove the recognition sequence for the addition of a lipid anchor is secreted efficiently to the culture supernatant, and moreover this protein could be further truncated by a deletion of 160 amino acid and still is detectable in the supernatant. By coupling a cellulase to this soluble outer membrane cytochrome, the export efficiency was measured by means of relative cellulase activity. We conclude that outer membrane cytochromes of S. oneidensis can be applied as transporters for the export of target proteins into the medium using the type II secretion pathway. [ABSTRACT FROM AUTHOR]

c-Type cytochromes (cyts c) are proteins that contain covalently bound heme and that thus require posttranslational modification for activity, a process carried out by the cytochrome c (cyt c) maturation system (referred to as the Ccm system) in many Gram-negative bacteria. It has been established that during cyt c maturation (CCM), two cysteine thiols of the heme binding motif (CXXCH) within apocytochromes c (apocyts c) are first oxidized largely by DsbA to form a disulfide bond, which is later reduced through a thio-reductive pathway involving DsbD. However, the physiological impacts of DsbA proteins on CCM in fact vary signifi- cantly among bacteria. In this work, we used the cyt c-rich Gram-negative bacterium Shewanella oneidensis as the research model to clarify the roles of DsbA proteins in CCM. We show that in terms of the oxidation of apocyts c, DsbA proteins are an important but not critical factor, and, strikingly, oxygen is not either. By exploiting the DsbD-independent pathway, we identify DsbA1, DsbA2, and DsbA3 as oxidants contributing to the oxidation of apocyts c and reductants, such as cysteine, to be an effective antagonist against DsbA-independent oxidation. We further show that DsbB proteins are partially responsible for the reoxidization of reduced DsbA proteins. Overall, our results indicate that the DsbA-DsbB redox pair has a limited role in CCM, challenging the established notion that it is the main oxidant for apocyts c. IMPORTANCE DsbA is a powerful oxidase that functions in the bacterial periplasm to introduce disulfide bonds in many proteins, including apocytochromes c. It has been well established that although DsbA is not essential, it plays a primary role in cytochrome c maturation, based on studies in bacteria hosting several cyts c. Here, with cyt c-rich S. oneidensis as a research model, we show that this is not always the case. Moreover, we demonstrate that DsbB is also not essential for cytochrome c maturation. These results underscore the need to identify oxidants other than DsbA/ DsbB that are crucial in the oxidation of apocyts c in bacteria. [ABSTRACT FROM AUTHOR]

Aconite alkaloids are the main bioactive ingredients existing in Aconitum, for instance aconitine (AC), which exhibit potent analgesic, antirheumatic and other pharmacological effects. In this study, effects of long-term treatment with liquorice on pharmacokinetics of AC in rats were investigated. Pharmacokinetics of AC after oral administration of AC at 1.5 mg/kg either with pre-treatment of liquorice water extracts at 0.433 or 1.299 g/kg (crude drug), respectively, for one week or not were studied. Additionally, LS-180 cells and human primary hepatocytes were utilized to explore the potential effects of bioactive ingredients of liquorice on P-glycoprotein (P-gp) and Cytochromes P450 (CYPs), respectively. The results revealed that exposure of AC after pre-treatment with liquorice was altered remarkably. Area under the concentration-time curve (AUC) decreased from 161 ± 37.8 to 58.8 ± 8.97 and 44.7 ± 8.20 ng/mL*h, respectively. Similarly, Cmax decreased from 26.2 ± 5.19 to 11.8 ± 1.15 and 6.86 ± 0.600 ng/mL, respectively. In addition, expressions of CYPs of human primary hepatocytes were enhanced to various contents after induction. Moreover, accumulation of AC and hypaconitine (HA), not mesaconitine (MA) inside of LS-180 cells were reduced after pre-treatment by comparison with control. In conclusion, the exposure of AC in vivo declined after pre-treatment with liquorice extract, which may be highly associated with upregulated expression and/or function of CYPs and P-gp. [ABSTRACT FROM AUTHOR]

Anaerobic ammonium-oxidizing (anammox) bacteria convert nitrite and ammonium via nitric oxide (NO) and hydrazine into dinitrogen gas by using a diverse array of proteins, including numerous c-type cytochromes. Many new catalytic and spectroscopic properties of c-type cytochromes have been unraveled by studies on the biochemical pathways underlying the anammox process. The unique anammox intermediate hydrazine is produced by a multiheme cytochrome c protein, hydrazine synthase, through the comproportionation of ammonium and NO and the input of three electrons. It is unclear how these electrons are delivered to hydrazine synthase. Here, we report the discovery of a functional tetraheme c-type cytochrome from the anammox bacterium Kuenenia stuttgartiensis with a naturally-occurring contracted Cys-Lys-Cys-His (CKCH) heme-binding motif, which is encoded in the hydrazine synthase gene cluster. The purified tetraheme protein (named KsTH) exchanged electrons with hydrazine synthase. Complementary spectroscopic techniques revealed that this protein harbors four low-spin hexa-coordinated hemes with His/Lys (heme 1), His/Cys (heme 2), and two His/His ligations (hemes 3 and 4). A genomic database search revealed that c-type cytochromes with a contracted CXCH heme-binding motif are present throughout the bacterial and archaeal domains in the tree of life, suggesting that this heme recognition site may be employed by many different groups of microorganisms. [ABSTRACT FROM AUTHOR]

Anaerobic ammonium oxidation (anammox) is a microbial process responsible for significant nitrogen loss from the oceans and other ecosystems. The redox reactions at the heart of anammox are catalyzed by large multiheme enzyme complexes that rely on small cytochrome c proteins for electron shuttling. Among the most highly abundant of these cytochromes is a unique heterodimeric complex composed of class I and class II c-type cytochromes called NaxLS, which has distinctive biochemical and spectroscopic properties. Here, we present the 1.7 Å resolution crystal structure of this complex from the anammox organism Kuenenia stuttgartiensis (KsNaxLS). The structure reveals that the heme irons in each subunit exhibit a rare His/Cys ligation, which, as we show by substitution, causes the observed unusual spectral properties. Unlike its individual subunits, the KsNaxLS complex binds nitric oxide (NO) only at the distal heme side, forming 6cNO adducts. This is likely due to steric immobilization of the proximal heme-binding motifs upon complex formation, a finding that may be of functional relevance, because NO is an intermediate in the central anammox metabolism. Pulldown experiments with K. stuttgartiensis cell-free extract showed that the KsNaxLS complex binds specifically to one of the central anammox enzyme complexes, hydrazine synthase, which uses NO as one of its substrates. It is therefore possible that the KsNaxLS complex plays a role in binding the volatile NO to retain it in the cell for transfer to hydrazine synthase. Alternatively, we propose that KsNaxLS may shuttle electrons to this enzyme complex. [ABSTRACT FROM AUTHOR]

Compound I from cytochrome P450 119 prepared by the photooxidation method involving peroxynitrite oxidation of the resting enzyme to Compound II followed by photooxidation to Compound I was compared to Compound I generated by m‐chloroperoxybenzoic acid (MCPBA) oxidation of the resting enzyme. The two methods gave the same UV/Visible spectra, the same products from oxidations of lauric acid and palmitic acid and their (ω‐2,ω‐2,ω‐3,ω‐3)‐tetradeuterated analogues, and the same kinetics for oxidations of lauric acid and caprylic acid. The experimental identities between the transients produced by the two methods leave no doubt that the same Compound I species is formed by the two methods. [ABSTRACT FROM AUTHOR]

Cytochromes P450 (P450s) are nature's catalysts of choice for performing demanding and physiologically vital oxidation reactions. Biochemical characterization of these enzymes over the past decades has provided detailed mechanistic insight and highlighted the diversity of substrates P450s accommodate and the spectrum of oxidative transformations they catalyze. Previously, we discovered that the bacterial P450 MycCI from the mycinamicin biosynthetic pathway in Micromonospora griseorubida possesses an unusually broad substrate scope, whereas the homologous P450 from tylosin-producing Streptomyces fradiae (TylHI) exhibits a high degree of specificity for its native substrate. Here, using biochemical, structural, and computational approaches, we aimed to understand the molecular basis for the disparate reactivity profiles of these two P450s. Turnover and equilibrium binding experiments with substrate analogs revealed that TylHI strictly prefers 16-membered ring macrolides bearing the deoxyamino sugar mycaminose. To help rationalize these results, we solved the X-ray crystal structure of TylHI in complex with its native substrate at 1.99-Å resolution and assayed several site-directed mutants. We also conducted molecular dynamics simulations of TylHI and MycCI and biochemically characterized a third P450 homolog from the chalcomycin biosynthetic pathway in Streptomyces bikiniensis. These studies provided a basis for constructing P450 chimeras to gain further insight into the features dictating the differences in reaction profile among these structurally and functionally related enzymes, ultimately unveiling the central roles of key loop regions in influencing substrate binding and turnover. Our work highlights the complex nature of P450/substrate interactions and raises interesting questions regarding the evolution of functional diversity among biosynthetic enzymes. [ABSTRACT FROM AUTHOR]

Geobacter sulfurreducens pili enable extracellular electron transfer and play a role in secretion of c-type cytochromes such as OmcZ. PilA-deficient mutants of G. sulfurreducens have previously been shown to accumulate cytochromes within their membranes. This cytochrome retaining phenotype allowed for enhanced growth of PilA-deficient mutants in electron donor and carbon-limited conditions where formate and fumarate are provided as the sole electron donor and acceptor with no supplementary carbon source. Conversely, wild-type G. sulfurreducens, which has normal secretion of cytochromes, has comparative limited growth in these conditions. This growth is further impeded for OmcZ-deficient and OmcS-deficient mutants. A PilB-deficient mutant which prevents pilin production but allows for secretion of OmcZ had moderate growth in these conditions, indicating a role for cytochrome localization to enabling survival in the electron donor and carbon-limited conditions. To determine which pathways enhanced growth using formate, Sequential Window Acquisition of all Theoretical Mass Spectra mass spectrometry (SWATH-MS) proteomics of formate adapted PilA-deficient mutants and acetate grown wild type was performed. PilA-deficient mutants had an overall decrease in tricarboxylic acid (TCA) cycle enzymes and significant upregulation of electron transport chain associated proteins including many c-type cytochromes and [NiFe]-hydrogenases. Whole genome sequencing of the mutants shows strong convergent evolution and emergence of genetic subpopulations during adaptation to growth on formate. The results described here suggest a role for membrane constrained c-type cytochromes to the enhancement of survival and growth in electron donor and carbon-limited conditions. [ABSTRACT FROM AUTHOR]

Summary: Hydroxylation of steroids has acquired special relevance for the pharmaceutical industries. Particularly, the 11β‐hydroxylation of steroids is a reaction of biotechnological importance currently carried out at industrial scale by the fungus Cochliobolus lunatus. In this work, we have identified the genes encoding the cytochrome CYP103168 and the reductase CPR64795 of C. lunatus responsible for the 11β‐hydroxylase activity in this fungus, which is the key step for the preparative synthesis of cortisol in industry. A recombinant Corynebacterium glutamicum strain harbouring a plasmid expressing both genes forming a synthetic bacterial operon was able to 11β‐hydroxylate several steroids as substrates. This is a new example to show that the industrial strain C. glutamicum can be used as a suitable chassis to perform steroid biotransformation expressing eukaryotic cytochromes. [ABSTRACT FROM AUTHOR]

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

The role of rs 4646421 polymorphism of the CYP1A1 gene (Cytochrome P450, family 1, member A1) in the development and clinical course of acne was analyzed. The study was carried out on samples of 165 patients with different clinical forms of acne and 161 conventionally healthy donors. Despite the significant contribution of CYP1A1 cytochrome enzyme level to the violation of sex hormone and vitamin A metabolism, the data obtained by us showed that there is no association of CYP1A1 gene polymorphism with acne development. The relative high prognostic efficacy of this locus as an independent marker of severe acne prognosis was revealed. The chance of detection of an unfavorable allelic variant of this genetic marker among patients with severe acne increased significantly in more than 2.1 times in comparison with the control group (χ²=6.8; P=0.01; OR=2.1; 95%CI1.186- 3.56). [ABSTRACT FROM AUTHOR]

One of the most important groups of metabolic enzymes is cytochrome P450 superfamily. These enzymes are important in terms of the catalytic diversity and the large number of xenobiotics that are detoxified or activated by converting to reactive metabolites. Flavonoids are xenobiotics to which humans are exposed through diet. Data on their oxidative metabolism mediated by cytochromes P450 are limited. The aim of this study was to determine the enzymatic kinetics of O-demethylation and aromatic hydroxylation of flavonoid aglycons on recombinant cytochrome P450 enzymes and human liver microsomes systems. The study was performed on ten flavonoids, namely 3,7- dihydroxyflavone, 7-hydroxyflavone, acacetin, apigenin, flavone, galangin, kaempferol, naringenin, sakuranetin, and tangeretin using liquid chromatography coupled with mass spectrometry and UV detector. Most relevant enzyme involved in metabolism of flavonoid aglycons is CYP1A2, and its catalytic effectiveness ranges from 0.5 to 2.9 × 106 M-1 min-1. Having in mind high expression and involvement of CYP1A2 in metabolism of xenobiotics including drugs, and its intraindividual differences in expression and activity, potential of drug-flavonoid competitive interactions/inhibitions should be considered when consuming dietary supplement and foods rich in flavonoids. [ABSTRACT FROM AUTHOR]

We have recently proposed a measure of the thermal stability of a protein: the water-entropy gain at 25 °C upon folding normalized by the number of residues, which is calculated using a hybrid of the angle-dependent integral equation theory combined with the multipolar water model and the morphometric approach. A protein with a larger value of the measure is thermally more stable. Here we extend the study to analyses on the effects of heme on the thermal stability of four cytochromes c (PA c551, PH c552, HT c552, and AA c555) whose denaturation temperatures are considerably different from one another despite that they share significantly high sequence homology and similar three-dimensional folds. The major conclusions are as follows. For all the four cytochromes c, the thermal stability is largely enhanced by the heme binding in terms of the water entropy. For the holo states, the measure is the largest for AA c555. However, AA c555 has the lowest packing efficiency of heme and the apo polypeptide with hololike structure, which is unfavorable for the water entropy. The highest stability of AA c555 is ascribed primarily to the highest efficiency of side-chain packing of the apo polypeptide itself. We argue for all the four cytochromes c that due to covalent heme linkages, the number of accessible conformations of the denatured state is decreased by the steric hindrance of heme, and the conformational-entropy loss upon folding becomes smaller, leading to an enhancement of the thermal stability. As for the apo state modeled as the native structure whose heme is removed, AA c555 has a much larger value of the measure than the other three. Overall, the theoretical results are quite consistent with the experimental observations (e.g., at 25 °C the α-helix content of the apo state of AA c555 is almost equal to that of the holo state while almost all helices are collapsed in the apo states of PA c551, PH c552, and HT c552). [ABSTRACT FROM AUTHOR]

The interaction of proteins with surfaces is a key phenomenon in many applications, but current understanding of the biophysics involved is lacking. At present, rational design of such emerging technologies is difficult as no methods or theories exist that correctly predict how surfaces influence protein behavior. Using molecular simulation and a coarse-grain model, this study illustrates for the first time that stability of proteins on surfaces can be correlated with tertiary structural elements for alpha-helical, orthogonal-bundle proteins. Results show that several factors contribute to stability on surfaces including the nature of the loop region where the tether is placed and the ability of the protein to freely rotate on the surface. A thermodynamic analysis demonstrates that surfaces stabilize proteins entropically and that any destabilization is an enthalpic effect. Moreover, the entropic effects are concentrated on the unfolded state of the protein while the ethalpic effects are focused on the folded state. [ABSTRACT FROM AUTHOR]

The time scales and pathways of vibrational energy relaxation (VER) of the ν4 and ν7 modes of three nickel porphyrin models, nickel porphine (NiP), nickel protoporphyrin IX (Ni-heme), and nickel octaethylporphyrin (NiOEP), were studied using a non-Markovian time-dependent perturbation theory at the B3LYP/6-31G(d) level. When NiP is calculated with D4h symmetry, it has the planar structure and the same VER properties as ferrous iron porphine (FeP). The porphine cores of both Ni-heme and NiOEP were distorted from a planar geometry, assuming a nonplanar structure, similar to that of the heme structure in cytochrome c. The VER time scales of Ni-heme are found to be similar to those predicted for a planar iron heme, but the derived pathways have distinctly different features. In particular, the strong coupling between the ν7 mode and the overtone of the ∼350 cm-1 γ7 mode, observed for planar porphyrins, is absent in both nonplanar nickel porphyrins. Direct energy exchange between the ν4 and ν7 modes is not observed in NiOEP, but is found to play an essential role in the VER of the ν4 mode in Ni-heme. The Ni-heme isopropionate groups are involved in the dominant VER pathways of both the ν4 and ν7 modes of Ni-heme. However, in contrast with VER pathways derived in planar iron heme, the isopropionate groups are not observed to play an essential role relative to other side chains in spatially directing the vibrational energy flow. [ABSTRACT FROM AUTHOR]

Density functional theory has been widely used to investigate the structural and electronic properties of heme-containing proteins such as cytochrome P450. Nevertheless, recent studies have shown that approximate exchange-correlation energy density functionals can incorrectly predict the stability order of spin states in, for instance, iron-containing pyridine and imidazole systems. This raises questions about the validity of earlier theoretical studies. In this work, we systematically investigate a few typical inorganic and organic iron-containing complexes and try to understand the performance difference of various density functionals. Two oxidation states of iron, Fe(II) and Fe(III), with different spin states and both adiabatic and vertical structures are considered. A different description of the outmost molecular orbital is found to play the crucial role. Local density and generalized gradient based functionals bias the lower spin state and produce a more localized frontier orbital that is higher in energy than the hybrid functionals. Energy component analysis has been performed, together with comparison of numerous structural and electronic properties. Implications of the present work to the theoretical study of heme-containing biological molecules and other spin-related systems are discussed. [ABSTRACT FROM AUTHOR]

• 58 screened cytochromes of five protein folds could wire enzymes to electrodes. • Electron uptake of eight cytochromes from a glucose dehydrogenase was studied. • c-Type cytochromes showed faster electron uptake than b-type cytochromes. • Complementary surface charges were not a predictor for fast electron shuttling. • Enzyme/cytochrome redox potential difference correlates to fast electron uptake. Cytochromes are a large and well-known group of electron transfer proteins, but only very few are used in bioelectrochemistry, e.g. to serve as electron shuttles between biocatalysts and electrodes. There, cytochromes generate an artificial electron transfer pathway if their structural and electrochemical properties are fitting. Cytochromes considered suitable are small and feature an exposed heme cofactor, a suitable redox potential, an electrochemically reversible redox process, sufficient protein production, and a fast electron transfer between other enzymes and hemoproteins. Out of 850 screened hemoproteins, 58 cytochromes met the requirements to function as an electron shuttle. They can be classified into five distinguished protein folds. Four representative b -type and three c -type cytochromes were selected for further analyses. The proteins were obtained in a yield of 0.075 to 375 mg L-1 in Escherichia coli or Komagataella phaffii. The electrochemical reversibility on 1-thioglycerol functionalized gold electrodes of all cytochromes was confirmed. The pH-dependent redox potentials range from -50 mV to +298 mV vs. SHE at pH 7.0. The electronic wiring ability was tested using the biocatalyst FAD-dependent glucose dehydrogenase (GDH). Cytochrome reduction by the GDH was monitored by spectrophotometry and amperometry. c -Type cytochromes showed a faster electron uptake from GDH than b -type cytochromes. Complementary surface charges at the cytochrome/GDH interface were not a predictor for a fast electron transfer but a higher difference in the redox potential of the cofactors was. This study supports the design of bioelectrocatalysts with high electron transfer rates, which are of great interest in 3rd generation biosensors and for direct bioelectrocatalysis. [Display omitted] [ABSTRACT FROM AUTHOR]

The marine archaeon Methanosarcina acetivorans contains a putative NAD + -independent d -lactate dehydrogenase (D-iLDH/glycolate oxidase) encoded by the MA4631 gene, belonging to the FAD-oxidase C superfamily. Nucleotide sequences similar to MA4631 gene, were identified in other methanogens and Firmicutes with >90 and 35–40% identity, respectively. Therefore, the lactate metabolism in M. acetivorans is reported here. Cells subjected to intermittent pulses of oxygen (air-adapted; AA-Ma cells) consumed lactate only in combination with acetate, increasing methane production and biomass yield. In AA-Ma cells incubated with d -lactate plus [14C]- l -lactate, the radioactive label was found in methane, CO 2 and glycogen, indicating that lactate metabolism fed both methanogenesis and gluconeogenesis. Moreover, d -lactate oxidation was coupled to O 2 -consumption which was sensitive to HQNO; also, AA-Ma cells showed high transcript levels of gene dld and those encoding subunits A (MA1006) and B (MA1007) of a putative cytochrome bd quinol oxidase, compared to anaerobic control cells. An E. coli mutant deficient in dld complemented with the MA4631 gene, grew with d -lactate as carbon source and showed membrane-bound d -lactate:quinone oxidoreductase activity. The product of the MA4631 gene is a FAD-containing monomer showing activity of iLDH with preference to d -lactate. The results suggested that air adapted M. acetivorans is able to co-metabolize lactate and acetate with associated oxygen consumption by triggering the transcription and synthesis of the D-iLDH and a putative cytochrome bd : methanophenazine (quinol) oxidoreductase. Biomass generation and O 2 consumption, suggest a potentially new oxygen detoxification mechanism coupled to energy conservation in this methanogen. [Display omitted] • Air-adapted Methanosarcina acetivorans can grow in presence of acetate + lactate. • Lactate was oxidized in the presence of O 2 , producing methane, glycogen and biomass. • D-iLDH and cyt bd oxidase transcripts increased in cells showing high O 2 -consumption. • Respiration linked to lactate oxidation was sensitive to HQNO and partially to azide. • Lactate oxidation potentially through cytochromes, consumed the otherwise toxic O 2. [ABSTRACT FROM AUTHOR]

Osilodrostat (LCI699) is a potent inhibitor of the human steroidogenic cytochromes P450 11β-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2). LCI699 is FDA-approved for the treatment of Cushing disease, which is characterized by chronic overproduction of cortisol. While phase II and III clinical studies have proven the clinical efficacy and tolerability of LCI699 for treating Cushing disease, few studies have attempted to fully assess the effects of LCI699 on adrenal steroidogenesis. To this end, we first comprehensively analyzed LCI699-mediated inhibition of steroid synthesis in the NCI-H295R human adrenocortical cancer cell line. We then studied LCI699 inhibition using HEK-293 or V79 cells stably expressing individual human steroidogenic P450 enzymes. Our studies using intact cells confirm the potent inhibition of CYP11B1 and CYP11B2 with negligible inhibition of 17-hydroxylase/17,20-lyase (CYP17A1) and 21-hydroxylase (CYP21A2). Furthermore, partial inhibition of the cholesterol side-chain cleavage enzyme (CYP11A1) was observed. To calculate the dissociation constant (K d) of LCI699 with the adrenal mitochondrial P450 enzymes, we successfully incorporated P450s into lipid nanodiscs and carried out spectrophotometric equilibrium and competition binding assays. Our binding experiments confirm the high affinity of LCI699 to CYP11B1 and CYP11B2 (K d ≈ 1 nM or less) and much weaker binding for CYP11A1 (K d = 18.8 μM). Our results confirm the selectivity of LCI699 for CYP11B1 and CYP11B2 and demonstrate partial inhibition of CYP11A1 but not CYP17A1 and CYP21A2. • LCI699 shows ∼1 nM affinity for CYP11B1 and CYP11B2 in 3 different systems. • LCI699 also weakly inhibits CYP11A1 (18 μM) but not CYP21A2 or CYP17A1. • Selectivity for CYP11B1∼CYP11B2 >CYP11A1 might explain clinical data using LCI699. [ABSTRACT FROM AUTHOR]

The strict requirement of cytochrome P450cam for its native ferredoxin redox partner, putidaredoxin (Pdx), is not exhibited by any other known cytochrome P450 (CYP) system and the molecular details of redox partner selectivity are still not completely understood. We therefore examined the selectivity of a related Pseudomonas cytochrome P450, P450lin, by testing its activity with non-native redox partners. We found that P450lin could utilize Arx, the native redox partner of CYP101D1, to enable turnover of its substrate, linalool, while Pdx showed limited activity. Arx exhibited a higher sequence similarity to P450lins native redox partner, linredoxin (Ldx) than Pdx, including several residues that are believed to be at the interface of the two proteins, based on the P450cam-Pdx complex structure. We therefore mutated Pdx to resemble Ldx and Arx and found that a double mutant, D38L/∆106, displayed higher activity than Arx. In addition, Pdx D38L/∆106 does not induce a low-spin shift in linalool bound P450lin but does destabilize the P450lin-oxycomplex. Together our results suggest that P450lin and its redox partners may form a similar interface to P450cam-Pdx, but the interactions that allow for productive turnover are different. Mutation of putidaredoxin (Pdx) at two critical residues to resemble the native redox partner of cytochromeP450lin, linredoxin, results in increased turnover rate. [Display omitted] • Selectivity of cytochromes P450cam and P450lin was tested with Pdx, Arx, and three Pdx variants. • Pdx was mutated to resemble Arx and Ldx at two key residues, D38L and Δ106. • P450lin exhibits turnover with Arx and Pdx variants. P450cam retains selectivity. • P450lin highest turnover rate and fastest oxycomplex decay rate with Pdx D38L/Δ106. [ABSTRACT FROM AUTHOR]

Simulations were performed for up to 150 simplified spherical horse heart cytochrome c molecules in the presence of a charged surface, which serves as an approximate model for a lipid membrane. Screened electrostatic and short-ranged attractive as well as repulsive van der Waals forces for interparticle and particle–membrane interactions are utilized in the simulations. At a distance from the membrane, where particle–membrane interactions are negligible, the simulation is coupled to a noninteraction continuum analogous to a heat bath [Geyer et al., J. Chem. Phys. 120, 4573 (2004)]. From the particles’ density profiles perpendicular to the planar surface binding isotherms are derived and compared to experimental results [Heimburg et al. (1999)]. Using a negatively charged structureless membrane surface a saturation effect was found for relatively large particle concentrations. Since biological membranes often contain membrane proteins, we also studied the influence of additional charges on our model membrane mimicking bacterial reaction centers. We find that the onset of the saturation occurs for much lower concentrations and is sensitive to the detailed implementation. Therefore we suggest that local distortion of membrane planarity (undulation), or lipid demixing, or the presence of charged integral membrane proteins create preferential binding sites on the membrane. Only then do we observe saturation at physiological concentrations. © 2004 American Institute of Physics. [ABSTRACT FROM AUTHOR]