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Three types of smectite with various crystallochemical compositions and interlayer cations as well as kaolinite and illite were fired at 800°C and 650°C and tested for their rehydroxylation's (RHX) potential and kinetics in the presence of water vapor at 200°C–350°C. A dehydroxylated structure of 2:1 Al‐rich mineral with the interlayer pillared by a large cation (like K+ or Cs+) is wide enough to allow H2O diffusion which results in advanced RHX, that is, restoring up to several tens of percent of the original OH content. Such 2:1 layer structures (beidellite, illite) do not follow the time‐to‐the‐quarter (TTTQ) kinetics during RHX and show a non‐Arrhenius behavior for isothermal RHX. Because TTTQ kinetics is assumed in an RHX dating of ceramic artifacts in archaeometry, fired‐clay ceramics prepared from material dominated by Al‐rich 2:1 minerals was found unfeasible for RHX dating. Kaolinite and Mg2+‐ or Ca2+‐exchanged smectites show the Arrhenius behavior and follow the TTTQ kinetics of RHX, resulting in apparent Ea increase with the progress of reaction (up to α ∼0.2) of ∼20–50 kJ/mol in beidellite and 15–35 kJ/mol in montmorillonite. We suggest using 'hydroxylation' rather than 'rehydroxylation' for structurally‐disordered fired clays gaining OH groups in an unknown position. [ABSTRACT FROM AUTHOR]

The Von Hippel–Lindau (VHL) protein, which is frequently mutated in clear-cell renal cell carcinoma (ccRCC), is a master regulator of hypoxia-inducible factor (HIF) that is involved in oxidative stresses. However, whether VHL possesses HIF-independent tumor-suppressing activity remains largely unclear. Here, we demonstrate that VHL suppresses nutrient stress-induced autophagy, and its deficiency in sporadic ccRCC specimens is linked to substantially elevated levels of autophagy and correlates with poorer patient prognosis. Mechanistically, VHL directly binds to the autophagy regulator Beclin1, after its PHD1-mediated hydroxylation on Pro54. This binding inhibits the association of Beclin1-VPS34 complexes with ATG14L, thereby inhibiting autophagy initiation in response to nutrient deficiency. Expression of non-hydroxylatable Beclin1 P54A abrogates VHL-mediated autophagy inhibition and significantly reduces the tumor-suppressing effect of VHL. In addition, Beclin1 P54-OH levels are inversely correlated with autophagy levels in wild-type VHL-expressing human ccRCC specimens, and with poor patient prognosis. Furthermore, combined treatment of VHL-deficient mouse tumors with autophagy inhibitors and HIF2α inhibitors suppresses tumor growth. These findings reveal an unexpected mechanism by which VHL suppresses tumor growth, and suggest a potential treatment for ccRCC through combined inhibition of both autophagy and HIF2α. Synopsis: PHD1-mediated prolyl hydroxylation targets HIF-1α for VHL-dependent ubiquitination and is a key step in the cell's oxygen sensing machinery. This work shows that PHD1 and VHL also inhibit Beclin1 and autophagy initiation, with dysregulation leading to clear-cell renal cell carcinoma progression. PHD1 mediates Beclin1 Pro54 hydroxylation on pre-autophagosomal structures in clear-cell renal cell carcinoma cells. VHL binds to Pro54-hydroxylated Beclin1 and prevents the association between ATG14L and Beclin1/VPS34. VHL inhibits VPS34-dependent PI(3)P production, autophagy initiation, and kidney tumor growth. The Von Hippel-Lindau (VHL) E3 ligase binds to proline-hydroxylated Beclin1, inhibiting the initiation of autophagy and blocking kidney tumor growth. [ABSTRACT FROM AUTHOR]

Electronic components made by inorganics (e.g., mica) play key roles in the function exertion of instruments working in outer‐layer space, enduring long‐term β‐ray irradiation. Its damage is vital while explored rarely. Herein, pure muscovite crystals were chosen and irradiated by an electron beam (EB) in air with a dose up to 1000 kGy. Then, micro‐geometry morphology variation in Z‐axis and microstructural transformation mechanism were explored by X‐ray diffraction, Fourier transform attenuated total reflection infrared spectrum, thermogravimetric analysis, X‐ray photoelectron spectroscopy, and CA analysis. Main results reveal that muscovite lattice is unstable to EB irradiation. With dose increases to 1000 kGy, interlayer space d of (0 0 2) lattice varied 2% near 0.2 Å. At low‐dose, irradiation lattice expansion readily occurs, whereas at higher dose, it is lattice shrinkage, showing a transformation from expansion to shrinkage. Concurrently, chemical structure varied, H2O amount increased, and metal element valance and surface wettability were reduced, so dehydroxylation occurred exceeding H2O radiolysis and evaporation, and framework cleavage could be severe. Binding energies of metals of 1000 kGy‐irradiated species decreased by 0.2 eV, and CA of 100 kGy‐irradiated species increased by 10°. Intrinsic mechanisms involve framework destruction, H‐atom migration, hydrogen bond formation/destruction, and H2O radiolysis. At low‐dose irradiation, H‐atom migration and hydrogen bond formation/destruction are predominant, inducing lattice expansion; at higher dose, framework cleavage is intensive and crucial, inducing shrinkage. During which, partial H2O occurred radiolysis, reducing valence. Generally, H‐atom migration and interface cleavage played key roles in microgeometry morphology a variation of muscovite crystal under EB irradiation at 0–1000 kGy. To enhance muscovite geometry‐morphology stability under a long‐term β‐ray irradiation condition, OH and hydrogen bond contents should be reduced and interface region should be strengthened (e.g., lessening vacancies or compacting stacking). This conclusion promotes the design of radiation‐resistant silicate material, guiding the manufacturing of electronic component used in aerospace industry significantly. [ABSTRACT FROM AUTHOR]

The P450 monooxygenase CYP109A2 from Bacillus megaterium DSM319 was previously found to convert vitamin D3 (VD3) to 25‐hydroxyvitamin D3. Here, we show that this enzyme is also able to convert testosterone in a highly regio‐ and stereoselective manner to 16β‐hydroxytestosterone. To reveal the structural determinants governing the regio‐ and stereoselective steroid hydroxylation reactions catalyzed by CYP109A2, two crystal structures of CYP109A2 were solved in similar closed conformations, one revealing a bound testosterone in the active site pocket, albeit at a nonproductive site away from the heme‐iron. To examine whether the closed crystal structures nevertheless correspond to a reactive conformation of CYP109A2, docking and molecular dynamics (MD) simulations were performed with testosterone and vitamin D3 (VD3) present in the active site. These MD simulations were analyzed for catalytically productive conformations, the relative occurrences of which were in agreement with the experimentally determined stereoselectivities if the predicted stability of each carbon‐hydrogen bond was taken into account. Overall, the first‐time determination and analysis of the catalytically relevant 3D conformation of CYP109A2 will allow for future small molecule ligand screening in silico, as well as enabling site‐directed mutagenesis toward improved enzymatic properties of this enzyme. [ABSTRACT FROM AUTHOR]

Electrochemical oxidation of the new psychoactive substance 3‐fluorophenmetrazine (FPM) was studied in phosphate buffers by cyclic voltammetry and differential pulse voltammetry (DPV) on a glassy carbon electrode. The redox potential of FPM in buffered solution strongly depends on pH. Cyclic voltammetry behavior shows the partial influence of adsorption on the electrode process not allowing detailed analysis of the individual steps of the reaction scheme, it means the involvement of electron transfer (E) and chemical reaction (C). Nevertheless, the irreversible shape of the cyclic voltammogram is explained by the participation of hydroxylation nucleophilic addition of water (hydroxylation) after two‐electron/two‐proton oxidation of molecule at the tetrahydro‐1,4‐oxazine ring. The suggested mechanism leading to hydroxylated derivative 2‐(3‐fluorophenyl)‐3‐methyl‐5‐hydroxymorfolin is supported by the calculated highest occupied molecular orbital spatial distribution and atomic charges calculations for electrochemically formed radical cation. Infrared spectroelectrochemistry performed during oxidation in acetonitrile/water also supported the formation of this product. The analytical method of FPM determination on glassy carbon electrode was developed using DPV with an attained limit of detection = 4.7 μmol/L in phosphate buffer of pH 9. The linear range of the calibration curve is from 7.0 to 107.00 μmol/L, correlation coefficient (r) = 0.9988. [ABSTRACT FROM AUTHOR]

Tyrosinase is the most predominant member of the coupled binuclear copper (CBC) protein family. The recent trapping and spectroscopic definition of the elusive catalytic ternary intermediate (enzyme/O2/monophenol) of tyrosinase dictates a monooxygenation mechanism that revises previous proposals and involves cleavage of the μ‐η2:η2‐peroxide dicopper(II) O–O bond to accept the phenolic proton, followed by monophenolate coordination to copper concomitant with aromatic hydroxylation by the non‐protonated μ‐oxo. Here, we compare and contrast previously proposed and current mechanistic models for monophenol monooxygenation of tyrosinase. Next, we discuss how these recent insights provide new opportunities towards uncovering structure–function relationships in CBC enzymes, as well as understanding fundamental principles for O2 activation and reactivity by bioinorganic active sites. [ABSTRACT FROM AUTHOR]

The electrocatalyzed ammonia oxidation reaction (AOR) is a potential pathway toward waste ammonia remediation, energy generation, and the synthesis of value‐added products. To date, mechanistic studies have focused on elucidating the progress of AOR on Pt‐based catalysts with an established pathway for N2$\mathrm{N_2}$ only. In this work, density functional theory was applied to determine the lowest energy intermediates toward nitrogen gas, nitrite, and nitrate formation on β$\beta$‐Ni(OH)2$\mathrm{Ni(OH)_2}$, a promising electrocatalyst material for AOR. It was found that dinitrogen formation progresses via NH‐NH coupling, whereas nitrite and nitrate formation occurs via deprotonation of ammonia to form adsorbed N and subsequent hydroxylation to form oxygenated intermediates. This work is the first to report a mechanism for nitrite and nitrate formation and will also serve as a benchmark for future studies on Ni‐based materials. [ABSTRACT FROM AUTHOR]

Many natural products comprise N‐O containing functional groups with crucial roles for biological activity. Their enzymatic formation is predominantly achieved by oxidation of an amine to form a hydroxylamine, which enables further functionalization. N‐hydroxylation by flavin‐dependent enzymes has so far been attributed to a distinct group of flavoprotein monooxygenases (FPMOs) containing two dinucleotide binding domains. Here, we present three flavoprotein N‐hydroxylases that exhibit a glutathione reductase 2 (GR2)‐type topology with only one nucleotide binding domain, which belong to a distinct phylogenetic branch within the GR2‐fold FPMOs. In addition to PqsL of Pseudomonas aeruginosa, which catalyses the N‐hydroxylation of a primary aromatic amine during biosynthesis of 2‐alkyl‐4‐hydroxyquinoline N‐oxide respiratory chain inhibitors, we analysed isofunctional orthologs from Burkholderia thailandensis (HmqL) and Chryseobacterium nematophagum (PqsLCn). Pre‐steady‐state kinetics revealed that the oxidative half‐reaction of all three enzymes is highly efficient despite the soft nucleophile substrate. Ligand binding studies indicated that HmqL and PqsLCn show displacement of the oxidized flavin cofactor from the active site by the organic substrate, which likely abolishes the substrate inhibition observed in PqsL. Despite mechanistic heterogeneity, the investigated monooxygenases in principle follow the catalytic mechanism of GR2‐fold FPMOs and thus differ from previously described N‐hydroxylating enzymes. The discovery of this yet unrecognized family of flavoprotein N‐hydroxylases expands the current knowledge on the catalytic repertoire of GR2‐type FPMOs and provides a basis for the discovery of other nitrogen functionalizing reactions. [ABSTRACT FROM AUTHOR]

SUMMARY: Diosgenin is an important compound in the pharmaceutical industry and it is biosynthesized in several eudicot and monocot species, herein represented by fenugreek (a eudicot), and Dioscorea zingiberensis (a monocot). Formation of diosgenin can be achieved by the early C22,16‐oxidations of cholesterol followed by a late C26‐oxidation. This study reveals that, in both fenugreek and D. zingiberensis, the early C22,16‐oxygenase(s) shows strict 22R‐stereospecificity for hydroxylation of the substrates. Evidence against the recently proposed intermediacy of 16S,22S‐dihydroxycholesterol in diosgenin biosynthesis was also found. Moreover, in contrast to the eudicot fenugreek, which utilizes a single multifunctional cytochrome P450 (TfCYP90B50) to perform the early C22,16‐oxidations, the monocot D. zingiberensis has evolved two separate cytochrome P450 enzymes, with DzCYP90B71 being specific for the 22R‐oxidation and DzCYP90G6 for the C16‐oxidation. We suggest that the DzCYP90B71/DzCYP90G6 pair represent more broadly conserved catalysts for diosgenin biosynthesis in monocots. [ABSTRACT FROM AUTHOR]

Salicylate is a typical aromatic compound widely distributed in nature. Microbial degradation of salicylate has been well studied and salicylate hydroxylases play essential roles in linking the peripheral and ring‐cleavage catabolic pathways. The direct hydroxylation of salicylate catalyzed by salicylate‐1‐hydroxylase or salicylate‐5‐hydroxylase has been well studied. However, the CoA mediated salicylate 5‐hydroxylation pathway has not been characterized in detail. Here, we elucidate the molecular mechanism of the reaction in the conversion of salicylate to gentisate in the carbaryl‐degrading strain Rhizobium sp. X9. Three enzymes (salicylyl‐CoA ligase CehG, salicylyl‐CoA hydroxylase CehH and gentisyl‐CoA thioesterase CehI) catalyzed the conversion of salicylate to gentisate via a route, including CoA thioester formation, hydroxylation and thioester hydrolysis. Further analysis indicated that genes cehGHI are also distributed in other bacteria from terrestrial environment and marine sediments. These genomic evidences highlight the role of this salicylate degradation pathway in the carbon cycle of soil organic compounds and marine sediments. Our findings of this three‐step strategy enhanced the current understanding of CoA mediated degradation of salicylate. [ABSTRACT FROM AUTHOR]

Oxidative functionalization of alkenes is a versatile strategy for the preparation of many oxygen-containing scaffolds, such as epoxides, diols, or carbonylcontaining compounds. In addition to conventional chemical methods, which rely on the utilization of stoichiometric amounts of oxidizing reagents, some electrochemical procedures have been developed to achieve these transformations. Typical electrochemical procedures employ tailored and often complex redox mediators to achieve the target transformation. Herein we present a modular approach for the synthesis of epoxides, diols, and aldehydes from a single set of reaction components. With sodium bromide as an inexpensive electrocatalyst and water as the oxygen donor, the outcome of the reaction (epoxide, 1,2-diol, or aldehyde) can be selected by simply tuning the electrolysis conditions. This convenient platform has been accomplished by developing a selective one-pot three-step bromide-electrocatalyzed epoxidation/ring opening/carboncarbon cleavage sequence. Its modularity permits the selection of the desired product, as the sequential reaction can be ended when the target compound has been formed. Themethod has been applied to a diversely functionalized terminal and non-terminal olefins. [ABSTRACT FROM AUTHOR]

Two‐step chemical vapor deposition (CVD) method is a promising technique for industrial fabrication of low‐hydroxyl silica glass ingot, in which a porous silica preform is first synthesized by flame hydrolysis deposition, which is subsequently sintered and vitrified to form silica glass. During sintering hydroxyls can be easily removed through the small pores in porous silica preform, which is called dehydroxylation. A deep understanding of the heat and mass transport characteristics involved in the dehydroxylation process is prerequisite to its controlling. In our previous work, a numerical model was developed to simulate the sintering and dehydroxylation of porous silica preform considering heat and mass transfer in porous media at high temperatures, chemical reaction of dehydroxylation, and volume shrinkage of preform. The results revealed that the dehydroxylation effect largely depends on the temperature fields in the furnace. In present work, the influences of thermal environments, including heating curve, heater position, and crucible structure on the dehydroxylation process of porous silica preform are systematically studied by numerical methods. Based on the results, increasing temperature at either stage is helpful for hydroxyl removal as well as increasing the hydroxyl distribution uniformity. However, long‐time exposure to 1500°C will instead worsen the dehydroxylation effect. Compared to top or bottom heating, side heating is more beneficial to hydroxyl removal. To improve the dehydroxylation condition at the bottom of porous silica preform, a novel crucible structure is designed which shows better performance than the ordinary crucible. At last, an optimized heating scheme is proposed in this study, which obtains much better dehydroxylation effect than other cases. [ABSTRACT FROM AUTHOR]

Summary: Sphingolipids are enriched in microdomains in the plant plasma membrane (PM). Hydroxyl groups in the characteristic long‐chain base (LCB) moiety might be essential for the interaction between sphingolipids and sterols during microdomain formation. Investigating LCB hydroxylase mutants in Physcomitrium patens might therefore reveal the role of certain plant sphingolipids in the formation of PM subdomains.Physcomitrium patens mutants for the LCB C‐4 hydroxylase S4H were generated by homologous recombination. Plants were characterised by analysing their sphingolipid and steryl glycoside (SG) profiles and by investigating different gametophyte stages.s4h mutants lost the hydroxyl group at the C‐4 position of their LCB moiety. Loss of this hydroxyl group caused global changes in the moss sphingolipidome and in SG composition. Changes in membrane lipid composition may trigger growth defects by interfering with the localisation of membrane‐associated proteins that are crucial for growth processes such as signalling receptors or callose‐modifying enzymes.Loss of LCB‐C4 hydroxylation substantially changes the P. patens sphingolipidome and reveals a key role for S4H during development of nonvascular plants. Physcomitrium patens is a valuable model for studying the diversification of plant sphingolipids. The simple anatomy of P. patens facilitates visualisation of physiological processes in biological membranes. [ABSTRACT FROM AUTHOR]

Hexagonal Boron nitride (BN) can be used as a lubricant additive, which can dramatically reduce friction energy. However, due to the accumulation of BN in the water‐based lubricants and the difficulty of entering the friction contact area, the tribological performance of BN becomes worse. To address this issue, the atomically thin hydroxylated boron nitride nanosheets (HO‐BNNS) were successfully prepared. The thickness of HO‐BNNS is only 0.6‐0.8 nm. Meanwhile, the HO‐BNNS has excellent dispersion stability in water‐based lubricants because of the hydroxyl group on the surface of HO‐BNNS. The as‐prepared HO‐BNNS exhibits unique friction properties as a water‐based dispersible lubricant additive. Additionally, the average wear scar diameter, average friction coefficient, and mean wear volume of HO‐BNNS/water‐based (0.05 wt%) drop by about 64.7%, 60%, and 95.73%. Finally, the introduction of HO‐BNNS can well control the temperature of water‐based lubricant. [ABSTRACT FROM AUTHOR]

The importance of oxygen for the survival of multicellular and aerobic organisms is well established and documented. Over the years, increased knowledge of its use for bioenergetics has placed oxygen at the centre of research on mitochondria and ATP‐generating processes. Understanding the molecular mechanisms governing cellular oxygen sensing and response has allowed for the discovery of novel pathways oxygen is involved in, culminating with the award of the Nobel Prize for Medicine and Physiology in 2019 to the pioneers of this field, Greg Semenza, Peter Ratcliffe and William Kaelin. However, it is now beginning to be appreciated that oxygen can be a signalling molecule involved in a vast array of molecular processes, most of which impinge on gene expression control. This review will focus on the knowns and unknowns of oxygen as a signalling molecule, highlighting the role of 2‐oxoglutarate‐dependent dioxygenases as central players in the cellular response to deviations in oxygen tension. [ABSTRACT FROM AUTHOR]

Background and Purpose: The 5-lipoxygenase product 5-oxo-6E,8Z,11Z,14Z-eicosatetraenoic acid (5-oxo-ETE), acting through the OXE receptor, is a potent eosinophil chemoattractant that may be an important proinflammatory mediator in eosinophilic diseases such as asthma. We previously identified a series of indole-based OXE receptor antagonists that rapidly appear in the blood following oral administration but have limited lifetimes. The objective of this study was to increase the potency and plasma half-lives of these compounds and thereby identify the optimal candidate for future preclinical studies in monkeys, as rodents do not have an OXE receptor orthologue.Experimental Approach: We synthesized a series of substituted phenylalkyl indoles and compared their antagonist potencies, pharmacokinetics, and metabolism to those of our earlier compounds. The potencies of some of their metabolites were also investigated.Key Results: Among the compounds tested, the S-enantiomer of the m-chlorophenyl compound (S-Y048) was the most potent, with an pIC50 of about 10.8 for inhibition of 5-oxo-ETE-induced calcium mobilization in human neutrophils. When administered orally to cynomolgus monkeys, S-Y048 rapidly appeared in the blood and had a half-life in plasma of over 7 hr, considerably longer than any of the other OXE analogues tested. A major hydroxylated metabolite, with a potency close to that of its precursor, was identified in plasma.Conclusion and Implications: Because of its highly potent antagonist activity and its long lifetime in vivo, S-Y048 may be a useful anti-inflammatory agent for the treatment of eosinophilic diseases such as asthma, allergic rhinitis, and atopic dermatitis. [ABSTRACT FROM AUTHOR]

The human cytochrome P450 (CYP) enzyme CYP4A22 is an orphan CYP with unknown function. Here, through functional expression in fission yeast, we show that CYP4A22 catalyzes fatty acid hydroxylation as well as aliphatic or aromatic hydroxylations of luciferin‐based probe substrates. Mechanistic molecular modeling of CYP4A22 suggests that its ω‐hydroxylation activity is hampered by a more spacious active site compared to CYP4B1. Substrate recognition via side‐chains R96 and R233 is indicated by dynamic three‐dimensional pharmacophores (dynophores) derived from molecular dynamics simulations. CYP4A22 activity is inhibited by three unspecific CYP inhibitors. A comparison of CYP4A22*1 (the reference standard sequence) with CYP4A22‐WT (the most common allele) revealed that for the four substrates tested the WT‐enzyme always had lower activity. [ABSTRACT FROM AUTHOR]

Here, a complete set of recombinant fission yeast strains that coexpress each of the 57 human cytochrome P450 (CYP) enzymes together with their natural human electron transfer partner(s) was cloned. This strain collection was tested with two luminogenic probe substrates, and 31 human CYPs (including the orphan enzymes CYP2A7, CYP4A22 and CYP20A1) were found to metabolize at least one of these. Since other substrates are known for the remaining enzymes, all human CYPs are now shown to be active. Interestingly, CYP5A1 was found for the first time to work on a substrate other than prostaglandin H2, and, moreover, to catalyze an aliphatic hydroxylation reaction that consumes molecular oxygen. Also, the ability of CYP11A1 to catalyze an aryl hydroxylation is another unexpected result. [ABSTRACT FROM AUTHOR]

Bacterial P450s have considerable potential for biotechnological applications. The P450 CYP106A2 from Bacillus megaterium ATCC 13368 converts progesterone to several hydroxylated products that are important precursors for pharmaceutical substances. As high yields of monohydroxylated products are required for biotechnological processes, improving this conversion is of considerable interest. It has previously been shown that the binding mode of the redox partner can affect the selectivity of the progesterone hydroxylation, being more stringent in case of the Etp1 compared with Adx(4–108). Therefore, in this study we aimed to improve hydroxylation selectivity by optimizing the binding of Adx(4–108) with CYP106A2 allowing for a shorter distance between both redox centers. To change the putative binding interface of Adx(4–108) with CYP106A2, molecular docking was used to choose mutation sites for alteration. Mutants at positions Y82 and P108 of Adx were produced and investigated, and confirmed our hypothesis. Protein–protein docking, as well as conversion studies, using the mutants demonstrated that the iron–sulfur(FeS) cluster/heme distance diminished significantly, which subsequently led to an approximately 2.5‐fold increase in 15β‐hydroxyprogesterone, the main product of progesterone conversion by CYP106A2. CYP106A2 can convert progesterone to several hydroxylated products (important pharmaceutical precursors). Here, we aim to improve hydroxylation selectivity by optimizing the binding of Adx (4–108) with CYP106A2. Adx(4–108) mutants were chosen via molecular docking and confirmed our hypothesis. The protein–protein docking showed a shorter distance between both redox centers and the conversion studies confirmed a considerable increase in the main progesterone product. [ABSTRACT FROM AUTHOR]

Background and Purpose: The aim of this study was to characterize the human cytochrome P450s (CYPs) involved in oxidative bioactivation of flucloxacillin to 5-hydroxymethyl flucloxacillin, a metabolite with high cytotoxicity towards biliary epithelial cells.Experimental Approach: The CYPs involved in hydroxylation of flucloxacillin were characterized using recombinant human CYPs, pooled liver microsomes in the presence of CYP-specific inhibitors and by correlation analysis using a panel of liver microsomes from 16 donors.Key Results: Recombinant CYPs showing the highest specific activity were CYP3A4, CYP3A7 and to lower extent CYP2C9 and CTP2C8. Michaelis-Menten enzyme kinetics were determined for pooled human liver microsomes, recombinant CYP3A4, CYP3A7 and CYP2C9. Surprisingly, sulfaphenazole appeared to be a potent inhibitor of 5'-hydroxylation of flucloxacillin by both recombinant CYP3A4 and CYP3A7.Conclusions and Implications: The combined results show that the 5'-hydroxylation of flucloxacillin is primarily catalysed by CYP3A4, CYP3A7 and CYP2C9. The large variability of the hepatic expression of these enzymes could affect the formation of 5'-hydroxymethyl flucloxacillin, which may determine the differences in susceptibility to flucloxacillin-induced liver injury. Additionally, the strong inhibition in CYP3A-catalysed flucloxacillin metabolism by sulfaphenazole suggests that unanticipated drug-drug interactions could occur with coadministered drugs. [ABSTRACT FROM AUTHOR]

Here, we report the first bacterial cytochrome P450, CYP154C8, that catalyzes the C–C bond cleavage reaction of steroids. A major change in product distribution is observed with CYP154C8, when the reactions are supported by NADPH and spinach redox partners ferredoxin and ferredoxin reductase, compared with previously reported reactions supported by NADH and redox partners containing putidaredoxin and putidaredoxin reductase. The NMR‐based structural elucidation of reaction products reveals 21‐hydroxyprednisone as the major product for prednisone, while the other product is identified as 1‐dehydroadrenosterone obtained due to C–C bond cleavage. A similar pattern of product formation is observed with cortisone, hydrocortisone, and prednisone. The reaction catalyzed by CYP154C8 in the presence of oxygen surrogates also prominently shows the formation of C–C bond cleavage products. [ABSTRACT FROM AUTHOR]

The achievement of an effective process of 9α‐hydroxylation of 4‐androstene‐3,17‐dione is of significant importance as it leads to the formation of the key intermediate 9α‐hydroxy‐4‐androstene‐3,17‐dione which is not possible by chemical means. In this study, the 9α‐hydroxylation of 4‐androstene‐3,17‐dione was carried out by resting Rhodococcus sp. cells. The ability of the naturally hydrophobic Rhodococcus to assimilate n‐alkanes was employed to obtain a cell depot with an intentionally increased cell surface hydrophobicity. The control Rhodococcus sp. cells were cultivated on medium containing glucose instead of n‐alkanes as a source of carbon and energy. Cells were harvested, washed from the cultivation media, and subjected to transformation of crystal androstenedione in buffer medium. The hydrophobicity of the n‐alkanes‐ and glucose‐grown cells, their total lipid content, and fatty acid composition were determined. The ultrastructure of the n‐alkanes‐ and glucose‐grown cells and their steroid hydroxylating activities were examined and compared. The results obtained in the present study showed that the intentionally achieved growth‐driven enhancement of the already hydrophobic Rhodococcus sp. cells made them even more compatible with the hydrophobic steroid substrate and enhanced its accessibility, which provided an increased steroid hydroxylating activity and lack of the accompanying product destruction. [ABSTRACT FROM AUTHOR]

Summary: l‐lysine catabolic routes in plants include the saccharopine pathway to α‐aminoadipate and decarboxylation of lysine to cadaverine. The current review will cover a third l‐lysine metabolic pathway having a major role in plant systemic acquired resistance (SAR) to pathogen infection that was recently discovered in Arabidopsis thaliana. In this pathway, the aminotransferase AGD2‐like defense response protein (ALD1) α‐transaminates l‐lysine and generates cyclic dehydropipecolic (DP) intermediates that are subsequently reduced to pipecolic acid (Pip) by the reductase SAR‐deficient 4 (SARD4). l‐pipecolic acid, which occurs ubiquitously in the plant kingdom, is further N‐hydroxylated to the systemic acquired resistance (SAR)‐activating metabolite N‐hydroxypipecolic acid (NHP) by flavin‐dependent monooxygenase1 (FMO1). N‐hydroxypipecolic acid induces the expression of a set of major plant immune genes to enhance defense readiness, amplifies resistance responses, acts synergistically with the defense hormone salicylic acid, promotes the hypersensitive cell death response and primes plants for effective immune mobilization in cases of future pathogen challenge. This pathogen‐inducible NHP biosynthetic pathway is activated at the transcriptional level and involves feedback amplification. Apart from FMO1, some cytochrome P450 monooxygenases involved in secondary metabolism catalyze N‐hydroxylation reactions in plants. In specific taxa, pipecolic acid might also serve as a precursor in the biosynthesis of specialized natural products, leading to C‐hydroxylated and otherwise modified piperidine derivatives, including indolizidine alkaloids. Finally, we show that NHP is glycosylated in Arabidopsis to form a hexose‐conjugate, and then discuss open questions in Pip/NHP‐related research. [ABSTRACT FROM AUTHOR]

Summary: The astounding structural and biological diversities of the large class of terpenoid natural products are imparted by both their complex hydrocarbon backbones and further elaboration by the addition of multiple hydroxyl groups, which provide both solubility and specific binding properties. While the role of terpene synthases (TPSs) in generating hydrocarbons with complex backbones is well known, these also are known to generate (singly) hydroxylated products by the addition of water prior to terminating deprotonation. Here a maize sesquiterpene synthase was unexpectedly found to generate dually hydroxylated products directly from (E,E)‐farnesyl diphosphate, primarily eudesmane‐2,11‐diol, along with two closely related structural isomers. The unprecedented formation of these diols was proposed to proceed via initial addition of water to a germacradienyl+ intermediate, followed by protonation of the internal carbon‐6,7‐double‐bond in the resulting hedycarol, with subsequent cyclization and further addition of water to an eudesmolyl+ intermediate. Evidence for the proposed mechanism was provided by labeling studies, as well as site‐directed mutagenesis, based on structural modeling, which identified an active site phenylalanine required for the protonation and further elaboration of hedycaryol. This dihydroxylated sesquiterpenoid synthase was specifically expressed in maize roots and induced by pathogen infection, with its major enzymatic product only detected in root exudates or infected roots, suggesting a role in defense. Regardless of the ultimate metabolic fate or physiological role of these diols, this report not only reveals an unanticipated extension of the catalytic prowess of TPSs, but also provides insight into the underlying enzymatic mechanism. [ABSTRACT FROM AUTHOR]

We recently described a complex multisystem syndrome in which mild‐moderate myopia segregated as an independent trait. A plethora of genes has been related to sporadic and familial myopia. More recently, in Chinese patients severe myopia (MYP25, OMIM:617238) has been linked to mutations in P4HA2 gene. Seven family members complaining of reduced distance vision especially at dusk underwent complete ophthalmological examination. Whole‐exome sequencing was performed to identify the gene responsible for myopia in the pedigree. Moderate myopia was diagnosed in the family which was associated to the novel missense variant c.1147A > G p.(Lys383Glu) in the prolyl 4‐hydroxylase,alpha‐polypeptide 2 (P4HA2) gene, which catalyzes the formation of 4‐hydroxyproline residues in the collagen strands. In vitro studies demonstrated P4HA2 mRNA and protein reduced expression level as well as decreased collagen hydroxylation and deposition in mutated fibroblast primary cultures compared to healthy cell lines. This study suggests that P4HA2 mutations may lead to myopic axial elongation of eyeball as a consequence of quantitative and structural alterations of collagen. This is the first confirmatory study which associates a novel dominant missense variant in P4HA2 with myopia in Caucasian patients. Further studies in larger cohorts are advisable to fully clarify genotype‐phenotype correlations. [ABSTRACT FROM AUTHOR]

Abstract: The effects of strain σ on the binding position preference of oxygen atoms and hydroxyl groups adsorbed on Pt(111) have been investigated using density functional theory. A transition between the bridge and FCC binding occurs under compressive strain of the O/Pt(111) surface. A significant reconstruction occurs under compressive strain of the OH/Pt(111) surface, and the surface OH groups preferentially occupy on‐top (bridge) positions at highly compressive (less compressive/tensile) strains. Changes to magnetisation of the O‐ and OH‐populated surfaces are discussed and for O/Pt(111) oxygenation reduces the surface magnetism via a delocalised mechanism. The origins of the surface magnetisation for both O‐ and OH‐bearing systems are discussed in terms of the state‐resolved electronic populations and of the surface charge density. [ABSTRACT FROM AUTHOR]

The mini‐M conotoxins are peptidic scaffolds found in the venom of cones snails. These scaffolds are tightly folded structures held together by three disulfide bonds with a CC‐C‐C‐CC arrangement (conotoxin framework III) and belong to the M Superfamily of conotoxins. Here, we describe mini‐M conotoxins from the venom of Conus regius, a Western Atlantic worm‐hunting cone snail species using transcriptomic and peptidomic analyses. These C. regius conotoxins belong to three different subtypes: M1, M2, and M3. The subtypes show little sequence homology, and their loop sizes (intercysteine amino acid chains) vary significantly. The mini‐Ms isolated from dissected venom contains preferentially hydroxylated proline residues, thus augmenting the structural reach of this conotoxin class. Using 2D‐NMR methods, we have determined the 3D structure of reg3b, an M2 subtype conotoxin, which shows a constrained multi‐turn scaffold. The structural diversity found within mini‐M conotoxin scaffolds of C. regius is indicative of structural hypervariability of the conotoxin M superfamily that is not seen in other superfamilies. These stable minimalistic scaffolds may be investigated for the development of engineered peptides for therapeutic applications. Databases: Sequences are available in GenBank under accession numbers MF588935‐ MF588952. Structural data are available in the RCSB protein database under the accession code 6BX9. [ABSTRACT FROM AUTHOR]

Compositional changes in unleached and acid-leached soda-lime silicate surfaces were tracked with in-vacuo heating and X-ray Photoelectron Spectroscopy. Surface oxygen speciation was determined using a stoichiometry-based algorithm via elemental composition, instead of the typical O 1s peak-fitting approach. Accurate surface hydroxyl quantification is shown to require dehydration at temperatures near 200°C. On the unleached surface, no change in surface hydroxyl density (~2.5 OH/nm²) is observed in the temperature range of 200°C-500°C after the initial dehydration. However, repolymerization in the network (non-bridging oxygen→bridging oxygen) is observed due to volatilization of sodium. The acid-leached surface undergoes sodium out-diffusion from the bulk at sub-Tg temperatures with laterally resolved inhomogeneity and shows a reduction in the concentration of hydroxyls from 4.5 OH/nm² (200°C) to 3.2 OH/nm² (500°C) accompanied by an increase in bridging oxygen. These results suggest that when [OH] > 2.5~3/nm², vicinal OH undergo dehydroxylation with evolution of water, whereas when [OH] < 2.5/nm², most OHs are non-interacting and isolated (at temperatures below Tg). Furthermore, at temperatures exceeding 300°C, sodium has enough thermal energy to desorb in vacuum and diffuse from the bulk (depending on the abundance & local structure). [ABSTRACT FROM AUTHOR]

Sesquiterpene lactones ( STLs) are C15 terpenoid natural products with α-methylene γ-lactone moiety. A large proportion of STLs in Asteraceae species is derived from the central precursor germacrene A acid ( GAA). Formation of the lactone rings depends on the regio-(C6 or C8) and stereoselective (α- or β-)hydroxylations of GAA, producing STLs with four distinct stereo-configurations (12,6α-, 12,6β-, 12,8α-, and 12,8β-olide derivatives of GAA) in nature. Curiously, two configurations of STLs (C12,8α and C12,8β) are simultaneously present in the Chinese medicinal plant, Inula hupehensis. However, how these related yet distinct STL stereo-isomers are co-synthesized in I. hupehensis remains unknown. Here, we describe the functional identification of the I. hupehensis cytochrome P450 ( CYP71 BL6) that can catalyze the hydroxylation of GAA in either 8α- or 8β-configuration, resulting in the synthesis of both 8α- and 8β-hydroxyl GAAs. Of these two products, only 8α-hydroxyl GAA spontaneously lactonizes to the C12,8α- STL while the 8β-hydroxyl GAA remains stable without lactonization. Chemical structures of the C12,8α- STL, named inunolide, and 8β-hydroxyl GAA were fully elucidated by nuclear magnetic resonance analysis and mass spectrometry. The CYP71 BL6 displays 63-66% amino acid identity to the previously reported CYP71 BL1/2 catalyzing GAA 6α- or 8β-hydroxylation, indicating CYP71 BL6 shares the same evolutionary lineage with other stereoselective cytochrome P450s, but catalyzes hydroxylation in a non-stereoselective manner. We observed that the CYP71 BL6 transcript abundance correlates closely to the accumulation of C12,8- STLs in I. hupehensis. The identification of CYP71 BL6 provides an insight into the biosynthesis of STLs in Asteraceae. [ABSTRACT FROM AUTHOR]

LipL and Cpr19 are nonheme, mononuclear Fe( II)-dependent, α-ketoglutarate (α KG): UMP oxygenases that catalyze the formation of CO2, succinate, phosphate, and uridine-5′-aldehyde, the last of which is a biosynthetic precursor for several nucleoside antibiotics that inhibit bacterial translocase I (MraY). To better understand the chemistry underlying this unusual oxidative dephosphorylation and establish a mechanistic framework for LipL and Cpr19, we report herein the synthesis of two biochemical probes-[1′,3′,4′,5′,5′-2H] UMP and the phosphonate derivative of UMP-and their activity with both enzymes. The results are consistent with a reaction coordinate that proceeds through the loss of one 2H atom of [1′,3′,4′,5′,5′-2H] UMP and stereospecific hydroxylation geminal to the phosphoester to form a cryptic intermediate, (5′ R)-5′-hydroxy- UMP. Thus, these enzyme catalysts can additionally be assigned as UMP hydroxylase-phospholyases. [ABSTRACT FROM AUTHOR]

In this study, we report the crystal structure of the cytochrome P450 CYP260A1 (PDB ) from the myxobacterium Sorangium cellulosum So ce56. In addition, we investigated the hydroxylation of 11-deoxycorticosterone by CYP260A1 by reconstituting the enzyme with the surrogate redox partners adrenodoxin and adrenodoxin reductase. The major product of this steroid conversion was identified as 1α-hydroxy-11-deoxycorticosterone, a novel Δ4 C-21 steroidal derivative. Furthermore, we docked the substrate into the crystal structure and replaced Ser326, the residue responsible for substrate orientation, with asparagine and observed that the mutant S326N displayed higher activity and selectivity for the formation of 1α-hydroxy-11-deoxycorticosterone compared to the wild-type CYP260A1. Thus, our findings highlight the usefulness of the obtained crystal structure of CYP260A1 in identifying biotechnologically more efficient reactions. [ABSTRACT FROM AUTHOR]

Methylation of histone H3 lysine 4 is linked to active transcription and can be removed by LSD1 or the JmjC domain-containing proteins by amino-oxidation or hydroxylation, respectively. Here we describe that its deamination can be catalyzed by lysyl oxidase-like 2 protein ( LOXL2), presenting an unconventional chemical mechanism for H3K4 modification. Infrared spectroscopy and mass spectrometry analyses demonstrated that recombinant LOXL2 specifically deaminates trimethylated H3K4. Moreover, by regulating H3K4me3 deamination, LOXL2 activity is linked with the transcriptional control of the CDH1 gene. These results reveal the existence of further H3 modification as well as a novel mechanism for H3K4me3 demethylation. Database The GEO accession number for the data referred to this paper is . [ABSTRACT FROM AUTHOR]

The effects of ethylene oxide (EO), vaporized hydrogen peroxide (VHP), gamma (γ) radiation, and electron-beam (E-beam) on the physiochemical and morphological properties of medical device polymers are investigated. Polymers with ether, carbonate, carboxylic acid, amide and ester functionalities are selected from a family of poly(ethylene glycol) (PEG) containing tyrosine-derived polycarbonates (TyrPCs) to include slow, medium, fast, and ultra-fast degrading polymers. Poly(lactic acid) (PLA) is used for comparison. Molecular weight ( Mw) of all tested polymers decreases upon gamma and E-beam, and this effect becomes more pronounced at higher PEG content. Gamma sterilization increases the glass transition temperature of polymers with high PEG content. EO esterifies the carboxylic acid groups in desaminotyrosol-tyrosine (DT) and causes significant degradation. VHP causes hydroxylation of the phenyl ring, and hydrolytic degradation. This study signifies the importance of the chemical composition when selecting a sterilization method, and provides suggested conditions for each of the sterilization methods. [ABSTRACT FROM AUTHOR]

Myxobacterial CYP260B1 from Sorangium cellulosum was heterologously expressed in Escherichia coli and purified. The in vitro conversion of a small focused substrate library comprised of Δ4 C21-steroids and steroidal drugs using surrogate bovine redox partners shows that CYP260B1 is a novel steroid hydroxylase. CYP260B1 performs the regio- and stereoselective hydroxylation of the glucocorticoid cortodoxone (RSS) to produce 6β-OH-RSS. The substrate-free crystal structure of CYP260B1 (PDB ) was resolved. Docking of the tested ligands into the crystal structure suggested that the C17 hydroxy moiety and the presence of either a keto or a hydroxy group at C11 determine the selectivity of hydroxylation. [ABSTRACT FROM AUTHOR]

The present paper deals with the synthesis of porous, sintered glass-ceramics obtained at temperatures below 1150°C, originating from inorganic polymers based on fayalite slag. Firing led to the evaporation of water, dehydroxylation, and oxidation of Fe2+ above 345°C. For heating >700°C, the Si-O stretching band shifted from the 1160 and 750 cm−1 to the 1255 and 830 cm−1 region, due to a structural reorganization of the amorphous phase, whereas Fe-O bands appeared at 550 cm−1. The final microstructure consisted predominantly of an amorphous phase, hematite, and franklinite. The open porosity and compressive strength decreased and increased, respectively, as the firing temperature increased. The final values suggest properties comparable to that of structural lightweight concrete, still, the materials synthesized herein, are lighter, and made primarily from secondary resources. [ABSTRACT FROM AUTHOR]

This article is a Commentary on Polturak et al., 210: 269‐283. [ABSTRACT FROM AUTHOR]

The article presents abstracts related to chemistry including synthesis of phosphorylated histone, magnetic anisotropy and relaxation dynamics, and enantioselective benzylic hydroxylation.

Key points Sustained hypoxic exposure increases ventilatory sensitivity to hypoxia as part of physiological acclimatisation., Oxygen-sensitive signals are transduced in animal cells by post-translational hydroxylation of transcription factors termed hypoxia-inducible factors (HIFs)., Mice heterozygous for the principal 'oxygen-sensing' HIF hydroxylase PHD2 (prolyl hydroxylase domain 2) show enhanced ventilatory sensitivity to hypoxia., To analyse the underlying mechanisms, functional (hypoxic ventilatory responses, HVRs) and anatomical (cellular proliferation within carotid bodies) responses were studied in genetic models of inducible and constitutive inactivation of PHD2 and its principal hydroxylation substrates, HIF-1α and HIF-2α., Inducible PHD2 inactivation enhanced HVR, similar to constitutive inactivation; both responses were almost entirely compensated for by specific inactivation of HIF-2α., Inducible inactivation of HIF-2α, but not HIF-1α, strikingly reduced ventilatory acclimatisation to hypoxia and associated carotid body cell proliferation., These findings demonstrate a key role for PHD2 and HIF-2α in ventilatory control and carotid body biology., Abstract Ventilatory sensitivity to hypoxia increases in response to continued hypoxic exposure as part of acute acclimatisation. Although this process is incompletely understood, insights have been gained through studies of the hypoxia-inducible factor (HIF) hydroxylase system. Genetic studies implicate these pathways widely in the integrated physiology of hypoxia, through effects on developmental or adaptive processes. In keeping with this, mice that are heterozygous for the principal HIF prolyl hydroxylase, PHD2, show enhanced ventilatory sensitivity to hypoxia and carotid body hyperplasia. Here we have sought to understand this process better through comparative analysis of inducible and constitutive inactivation of PHD2 and its principal targets HIF-1α and HIF-2α. We demonstrate that general inducible inactivation of PHD2 in tamoxifen-treated Phd2f/f;Rosa26+/CreERT2 mice, like constitutive, heterozygous PHD2 deficiency, enhances hypoxic ventilatory responses (HVRs: 7.2 ± 0.6 vs. 4.4 ± 0.4 ml min−1 g−1 in controls, P < 0.01). The ventilatory phenotypes associated with both inducible and constitutive inactivation of PHD2 were strongly compensated for by concomitant inactivation of HIF-2α, but not HIF-1α. Furthermore, inducible inactivation of HIF-2α strikingly impaired ventilatory acclimatisation to chronic hypoxia (HVRs: 4.1 ± 0.5 vs. 8.6 ± 0.5 ml min−1 g−1 in controls, P < 0.0001), as well as carotid body cell proliferation (400 ± 81 vs. 2630 ± 390 bromodeoxyuridine-positive cells mm−2 in controls, P < 0.0001). The findings demonstrate the importance of the PHD2/HIF-2α enzyme-substrate couple in modulating ventilatory sensitivity to hypoxia. [ABSTRACT FROM AUTHOR]

3-Hydroxybenzoate 6-hydroxylase (3 HB6H) from Rhodococcus jostii RHA1 is an NADH-specific flavoprotein monooxygenase that contains FAD as a redox-active cofactor. The enzyme catalyzes para-hydroxylation of 3-hydroxybenzoate (3 HB) to form 2,5-dihydroxybenzoate (2,5- DHB). Based on the enzyme crystal structure, residue His213 is located close to the hydroxyl moiety, whereas Tyr217 is close to the carboxylate group of 3 HB. Y217A and Y217S did not show any perturbation of flavin absorption upon addition of 3 HB, whereas Y217F has a Kd value for 3 HB binding of 7.5 m m, which is ~ 50-fold larger than that found for wild-type enzyme. The results clearly indicate that Tyr217 is necessary for substrate binding. All His213 variants can bind to 3 HB with similar affinity as the wild-type enzyme and form C4a-hydroperoxy intermediate. H213S, H213D and H213E produce 2,5- DHB with yields of 28 ± 5%, 52 ± 7% and 92 ± 6%, respectively, whereas H213A cannot catalyze hydroxylation. The results indicate that the interaction between the hydroxyl group of 3 HB and residue 213 is important for substrate hydroxylation. Interestingly, the hydroxylation rate constant of H213E (35 s−1) is similar to that of wild-type enzyme (36 s−1) and this variant has an efficiency of hydroxylation (92 ± 6%) similar to the wild-type enzyme (86 ± 2%). Difference spectra of enzyme-bound substrate suggest that 3 HB binds to H213E in the phenolic form. The results indicate that His213 and Glu213 in H213E may act as a catalytic base to initiate the substrate deprotonation and facilitate the electrophilic aromatic substitution of 3 HB. [ABSTRACT FROM AUTHOR]

Triterpenoids have diverse chemical structures and bioactivities. Cytochrome P450 monooxygenases play a key role in their structural diversification. In higher plants, CYP716A subfamily enzymes are triterpene oxidases. In this study, Arabidopsis thaliana CYP716A1 and CYP716A2 were characterized by heterologously expressing them in simple triterpene-producing yeast strains. In contrast to the C-28 oxidative activity of CYP716A1 shown in several CYP716A subfamily enzymes, remarkably, CYP716A2 displayed 22α-hydroxylation activity against α-amyrin that has not been previously reported, which produces the cytotoxic triterpenoid, 22α-hydroxy-α-amyrin. Our results contribute to the enrichment of the molecular toolbox that allows for the combinatorial biosynthesis of diverse triterpenoids. [ABSTRACT FROM AUTHOR]

Eribulin mesylate, one of the most synthetically challenging drugs to date, possesses 19 stereocentres in its structure and ascertaining the absolute stereochemistry at every stage of the 64-stage synthesis is crucial. In our quest to synthesize eribulin, we identified two critical building blocks of this molecule, namely 3,4:6,7-di- O-cyclohexylidene-D- glycero-α-L- talo-heptopyranose methanol monosolvate, C19H30O7·CH3OH, and (2 R,3 R,4 R,5 S)-5-allyl-2-[( S)-2,3-dihydroxypropyl]-4-[(phenylsulfonyl)methyl]tetrahydrofuran-3-ol, C17H24O6S, for which two-dimensional NMR (2D-NMR) data were not sufficient to prove the absolute configuration. To ensure structural integrity, single-crystal X-ray diffraction data were obtained to confirm the structures. This information provides useful insights into the structural framework of the large eribulin mesylate molecule. [ABSTRACT FROM AUTHOR]

Accurate information on the interactions between water and silica is critical to the understanding of its properties including mechanical strength under stress and long-term chemical durability of silica and silicate glasses. In this study, interactions between water and nanoporous amorphous silica models were investigated using density functional theory (DFT) based ab initio molecular dynamics (AIMD) simulations which accurately describe bond breakage and formation as well as chemical reactions. AIMD simulations up to 30 ps were performed for systems containing water and nanoporous silica with a wide range of porosities (31%-67%). Partial removal of defects, such as two-membered rings, was observed during the AIMD runs whereas more reactive coordination defects were removed during the initial geometry optimization. The limited two-membered ring removal can be attributed to restricted water-defect movement or the increased stability of rings located on concave surfaces. Two-membered ring removal mechanisms included the formation of an overcoordinated silicon (Si5) intermediate defect from the dynamic simulations. Si5 defects continued to develop throughout the simulations, indicating a thermodynamic drive for two-membered ring removal which is kinetically limited. Changes in the electronic structures, such as atomic charges, and bond length-bond angle correlation functions were monitored during the hydroxylation process. [ABSTRACT FROM AUTHOR]

Since its introduction in 2009, application of the rehydroxylation (RHX) technique for dating fired-clay ceramics has been controversial, with very few satisfactory dating results collected in the interim. The stability and efficiency of this technique has been called into question by several investigators in the last few years, who have struggled to reproduce and validate this new dating method. Based on our new mass gain measurements for ca. 2000-7000 yrs old ceramic artifacts, the reproducibility in the RHX process rate is analyzed and discussed. Timespan analysis was performed, and age uncertainty related to RHX dating technique was evaluated by considering the error propagation. The results show poor reproducibility of the RHX process in the samples of the same origins, which give new evidence for a revision of the RHX protocols. [ABSTRACT FROM AUTHOR]

We present a series of new mass-gain data acquired on ancient pottery following heating at increasing temperatures from 60°C to 590°C to further constrain our understanding of the rehydration and rehydroxylation ( RHX) processes acting within archeological fired-clay artifacts. The ancient pottery, deliberately chosen for its low-porosity, is second century ad Samian ware fragments originating from Lezoux in central France. The mass-gain data, after each temperature step, adequately define a (time)1/ N power law. We demonstrate that values of the 1/ N exponent vary systematically with heating temperature. These results suggest that the sorption of chemisorbed and rehydroxylated water into the clay matrix occurs simultaneously. The 1/ N exponent values obtained are consistent with power law kinetics resulting from the combination of both one-dimensional and Brownian diffusion processes. The balance between these two end-member processes, in particular after heating at 500°C, may depend on the nature of the clay and on the manufacturing conditions of the studied ceramics. The results presented herein confirm previous claims that major complexities vitiate the RHX dating method proposed by Wilson et al. [ABSTRACT FROM AUTHOR]

CYP106A1 from Bacillus megaterium DSM319 was recently shown to catalyze steroid and terpene hydroxylations. Besides producing hydroxylated steroid metabolites at positions 6β, 7β, 9α and 15β, the enzyme displayed previously unknown 11-oxidase activity towards 11β-hydroxysteroids. Novel examples for 11-oxidation were identified and confirmed by 1 H and 13 C NMR for prednisolone, dexamethasone and 11β-hydroxyandrostenedione. However, only 11β-hydroxyandrostenedione formed a single 11-keto product. The latter reaction was chosen to investigate the kinetic solvent isotope effect on the steady-state turnover of the CYP106A1-mediated 11-oxidation. Our results reveal a large inverse kinetic isotope effect (∼0.44) suggesting the involvement of the ferric peroxoanion as a reactive intermediate. [ABSTRACT FROM AUTHOR]

Protein uL2 is essential for the catalytic activity of the ribosome and has a conserved shape in ribosomes from all domains of life. However, the sequence of its unstructured C-terminal loop apex that contacts the conserved 23S/28S rRNA helix (H) 93 near the ribosomal peptidyl transferase center differs in bacteria, archaea and eukaryotes. Eukaryote-specific residue His216 located in this loop in mammalian uL2 is hydroxylated in ribosomes. We used a set of chemical probes to explore the structure of an RNA that mimicked a segment of 28S rRNA domain V containing part of the uL2 binding site including H93, complexed with either natural (hydroxylated) or recombinant (unmodified) human uL2. It was found that both protein forms engage H93 during binding, but only natural uL2 ( uL2n) protects it from hydroxyl radicals. The association of uL2n with RNA leads to changes in its structure at U4532 adjacent to the universally conserved U4531 (U2585, Escherichia coli numbering) involved in peptidyl transferase center formation, and at the universally conserved C4447 (2501) located in the ribosome near A4397 (2451) and C3909 (2063) belonging to the peptidyl transferase center. As a result, both nucleotides become strongly exposed to hydroxyl radicals. Our data argue that the hydroxyl group at His216 in the C-terminal loop apex of mammalian uL2 contributes to stabilization of a protein conformation that is favorable for binding to H93 of 28S rRNA and that this binding induces structural rearrangement in the regions close to the peptidyl transferase center in the mature ribosome. [ABSTRACT FROM AUTHOR]

Determining the absolute chronology of ceramic artifacts has significant implications for archeological and historical research. Wilson, Hall et al. recently suggested a new technique for direct absolute dating of archeological ceramics based on a moisture-induced chemical reaction, called rehydroxylation (RHX) dating. RHX dating proceeds by measuring the mass of chemically combined water in the ceramics in the form of OH hydroxyls, and the mass gain rate at the Effective Lifetime Temperature (ELT) that the ceramics experienced over its lifetime. To date, ELT determinations have been based on estimates of the ceramic's lifetime temperature history; taking into account weather and climate data and the depth at which the artifact was found. The uncertainty in determining the ELT can be a major component of the overall dating uncertainty. Here, we propose an alternative method which relies minimally on weather and climate data, and provides more precise determinations of the ELT and the ceramic age. The proposed method (SAS: Same Age Samples) involves a minimum of four measurements of the RHX mass gain rate constant for two ceramic samples of the same age at two temperatures. We show via simulations that the proposed SAS method can determine the ELT with a precision of 0.2 K which is comparable to the best ELT determination based on lifetime temperature history, and also comparable to available microbalance temperature resolutions of around 0.1 K. The corresponding percent age error is then 1.4%, or 43 yr for a 3000-yr-old ceramic. The proposed SAS method should be tested with ceramic samples of different ages, whose ELT are well-known. [ABSTRACT FROM AUTHOR]

The rehydroxylation dating method of archeological fired-clay artifacts relies on the reliability of the (time)1/4 power law model considered for long-term moisture expansion and mass gain in ceramics. Here, we reanalyze different moisture expansion datasets that were previously used to verify this model. We show that these data obey an accurate (time)1/ N power law, but they reveal a variability in the values of the exponent 1/ N between ~1/2 and ~1/4. It is possible that this value is an intrinsic property of ceramics, naturally varying between a behavior governed by a Brownian process ( t1/2 power law) and a behavior controlled by a one-dimensional diffusion process ( t1/4 power law). [ABSTRACT FROM AUTHOR]

A novel naturally occurring heme-signature variant of CYP267A1 from myxobacterium Sorangium cellulosum So ce56 and its mutant L366F, the actual mimic of the 'conserved' heme-signature of cytochromes P450, were heterologously expressed in Escherichia coli in a soluble form and purified. The UV-visible characteristics of both variants were highly similar. Although leucine replaced the phenylalanine in the heme-signature domain of CYP267A1, EPR measurements of the ligand-free wild-type CYP267A1 and the mutant L366F showed low-spin rhombic species suggesting a conserved heme environment of the P450s. The need of primary redox partners for the orphan P450 was sustained by the bovine redox system and a class-I electron transfer path was provided during fatty acid hydroxylation. CYP267A1 showed higher activity and produced more diverse ω-hydroxylated products compared with L366F. In both enzymes the regioselectivity of the fatty acid hydroxylation shifted towards the inner carbon atoms of the fatty acid chains with increasing carbon chain lengths. Our docking results in a homology model of the protein showed that longer fatty acids need to be folded to fit into the binding pocket. In the mutant L366F, the ω-1 and ω-2 positions which exhibit the largest electron density of the highest occupied molecular orbital are preferred. It is speculated that the leucine heme-signature variant of P450 might have evolved under selective evolutionary pressure, which confers an increased advantage to generate a broader spectrum of related alcohols and carboxylic acids required for the bacterial homeostasis or metabolism in a particular ecological niche. [ABSTRACT FROM AUTHOR]

Hypoxia causes an increase in pulmonary artery pressure. Gene expression controlled by the hypoxia-inducible factor ( HIF) family of transcription factors plays an important role in the underlying pulmonary vascular responses. The hydroxylase enzymes that regulate HIF are highly sensitive to varying iron availability, and iron status modifies the pulmonary vascular response to hypoxia, possibly through its effects on HIF. Ascorbate (vitamin C) affects HIF hydroxylation in a similar manner to iron and may therefore have similar pulmonary effects. This study investigated the possible contribution of ascorbate availability to hypoxic pulmonary vasoconstriction in humans. Seven healthy volunteers undertook a randomized, controlled, double-blind, crossover protocol which studied the effects of high-dose intravenous ascorbic acid (total 6 g) on the pulmonary vascular response to 5 h of sustained hypoxia. Systolic pulmonary artery pressure ( SPAP) was assessed during hypoxia by Doppler echocardiography. Results were compared with corresponding data from a similar study investigating the effect of intravenous iron, in which SPAP was measured in seven healthy volunteers during 8 h of sustained hypoxia. Consistent with other studies, iron supplementation profoundly inhibited hypoxic pulmonary vasoconstriction ( P < 0.001). In contrast, supraphysiological supplementation of ascorbate did not affect the increase in pulmonary artery pressure induced by several hours of hypoxia ( P = 0.61). We conclude that ascorbate does not interact with hypoxia and the pulmonary circulation in the same manner as iron. Whether the effects of iron are HIF-mediated remains unknown, and the extent to which ascorbate contributes to HIF hydroxylation in vivo is also unclear. [ABSTRACT FROM AUTHOR]