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The gut microbiota synthesize hundreds of molecules, many of which influence host physiology. Among the most abundant metabolites are the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA), which accumulate at concentrations of around 500 [mu]M and are known to block the growth of Clostridium difficile.sup.1, promote hepatocellular carcinoma.sup.2 and modulate host metabolism via the G-protein-coupled receptor TGR5 (ref. .sup.3). More broadly, DCA, LCA and their derivatives are major components of the recirculating pool of bile acids.sup.4; the size and composition of this pool are a target of therapies for primary biliary cholangitis and nonalcoholic steatohepatitis. Nonetheless, despite the clear impact of DCA and LCA on host physiology, an incomplete knowledge of their biosynthetic genes and a lack of genetic tools to enable modification of their native microbial producers limit our ability to modulate secondary bile acid levels in the host. Here we complete the pathway to DCA and LCA by assigning and characterizing enzymes for each of the steps in its reductive arm, revealing a strategy in which the A-B rings of the steroid core are transiently converted into an electron acceptor for two reductive steps carried out by Fe-S flavoenzymes. Using anaerobic in vitro reconstitution, we establish that a set of six enzymes is necessary and sufficient for the eight-step conversion of cholic acid to DCA. We then engineer the pathway into Clostridium sporogenes, conferring production of DCA and LCA on a nonproducing commensal and demonstrating that a microbiome-derived pathway can be expressed and controlled heterologously. These data establish a complete pathway to two central components of the bile acid pool. The biosynthetic pathway that produces the secondary bile acids DCA and LCA in human gut microbes has been fully characterized, engineered into another bacterial host, and used to confer DCA production in germ-free mice--an important proof-of-principle for the engineering of gut microbial pathways., Author(s): Masanori Funabashi [sup.1] [sup.6] , Tyler L. Grove [sup.2] , Min Wang [sup.1] , Yug Varma [sup.1] , Molly E. McFadden [sup.3] , Laura C. Brown [sup.3] , Chunjun [...]

Author(s): Benjamin E. R. Snyder [1]; Pieter Vanelderen [1, 2]; Max L. Bols [2]; Simon D. Hallaert [3]; Lars H. Bttger [1]; Liviu Ungur [3]; Kristine Pierloot [3]; Robert A. [...]

Abstract Cottonseed oil (CSO) was converted into a polymerizable polyol by in-situ epoxidation and hydroxylation in the presence of water as nucleophile and sulfuric acid as catalyst. This hydroxylated CSO [...]

The case reports of two siblings are discussed to understand that a lack of cyclophilin B (CyPB) does not limit the rate of peptidyl-prolyl isomerase B gene (PPIB) for type I collagen folding in vivo. These case reports highlight the need for further research into the function of the 3-hydroxylation modification.

The various mutations taking place in the PPIB genes that lead to the onset of osteogenesis imperfecta (OI) disorder are discussed. The results prove that a dysfunctional complex is more likely to lead to OI, in comparison with the lack of a single proline residue.

The analysis explains the mechanism involved in the aromatic hydroxylation by high-valent iron(IV)-oxo porphyrin [pi]-cation radical complexes. The oxygen found in such oxygenated products is derived from various iron-oxo porphyrin intermediates.

The C-H hydroxylation of camphor by Compound I (Cpd I) of cytochrome [P450.sub.cam] in different electronic states is investigated. This study broadens view on Cpd I reactivity by considering different electronic states and oxidizing agents at the M/MM level.

The partial synthesis of the two naturally occurring compounds, 23-hydroxyoleanolic acid (hederagenin) and 24-hydroxymaslinic acid (hyptatic acid), using a regioselective cyclopalladation is presented. The regioselective cyclopalladation of the axial methyl group on C-4 of maslinic acid afforded the C-24 hydroxymethylene group because of the presence of a C-2-OR substituent while hederagenin was produced in the absence of oxygenated group.

A new type of cross/coupling/cycloaddition cascade is developed and used in the total synthesis of some members of the hydroxylated phenanthridone subclass of the Amaryllidaceae alkaloid family. The alkaloids are assembled by a one-pot Stille/intramolecular Diels-Alder reactioncascade and the resulting cycloadducts are used for the stereocontrolled installation of other functionality present in the C-ring of the target molecules.

The function of RemO as an FAD-dependent monooxygenase involved in resistoflavin biosynthesis is disclosed by in vivo and in vitro experiments. The successful in vitro biotransformation assay using heterologously expressed RemO revealed that the pseudo-boat-shaped pentacyclic ring system of resistoflavin is derived from a stereospecific interior, nonperipheral hydroxylation of the planar polyphenol resistomycin.

The Cartilage Association Protein (CRTAP) which copurifies with a collagen prolyl 3-hydroxylase activity is involved in osteogenesis imperfecta and mutations are associated with brittle bones in patients with a spectrum of mild to severe recessive osteogenesis imperfecta. Hence altered CRTAP function and prolyl 3-hydroxylation contribute to the connective tissue disease.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.07.023 Byline: Hajime Ono (a)(b), Kim F. Rewitz (f), Tetsuro Shinoda (d), Kyo Itoyama (e), Anna Petryk (a)(c), Robert Rybczynski (f), Michael Jarcho (c), James T. Warren (f), Guillermo Marques (a)(b), Mary Jane Shimell (a), Lawrence I. Gilbert (f), Michael B. O'Connor (a)(b) Keywords: Ecdysone; Molting; CYP; p450 Abstract: Ecdysteroids regulate many key developmental events in arthropods including molting and metamorphosis. Recently, members of the Drosophila Halloween group of genes, that are required for embryonic viability and cuticle deposition, have been shown to code for several cytochrome P450 enzymes that catalyze the terminal hydroxylation steps in the conversion of cholesterol to the molting hormone 20-hydroxyecdysone. These P450s are conserved in other insects and each is thought to function throughout development as the sole mediator of a particular biosynthetic step since, where analyzed, each is expressed at all stages of development and shows no closely related homolog in their respective genomes. In contrast, we show here that several dipteran genomes encode two novel, highly related, microsomal P450 enzymes, Cyp307A1 and Cyp307A2, that likely participate as stage-specific components of the ecdysone biosynthetic machinery. This hypothesis comes from the observation that Cyp307A1 is encoded by the Halloween gene spook (spo), but unlike other Halloween class genes, Dmspo is not expressed during the larval stages. In contrast, Cyp307a2, dubbed spookier (spok), is expressed primarily during larval stages within the prothoracic gland cells of the ring gland. RNAi mediated reduction in the expression of this heterochromatin localized gene leads to arrest at the first instar stage which can be rescued by feeding the larva 20E, E or ketodiol but not 7dC. In addition, spok expression is eliminated in larvae carrying mutations in molting defective (mld), a gene encoding a nuclear zinc finger protein that is required for production of ecdysone during Drosophila larval development. Intriguingly, mld is not present in the Bombyx mori genome, and we have identified only one spook homolog in both Bombyx and Manduca that is expressed in both embryos and larva. These studies suggest an evolutionary split between Diptera and Lepidoptera in how the ecdysone biosynthetic pathway is regulated during development. Author Affiliation: (a) The Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN, 55455, USA (b) Howard Hughes Medical Institute, University of Minnesota, Minneapolis, MN, 55455, USA (c) Department of Pediatrics, University of Minnesota, Minneapolis, MN, 55455, USA (d) National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, 305-8634 Japan (e) Akita Prefectural Agricultural Experiment Station, 34-1 Genpachisawa, Aikawa, Yuwa, Kawabe, Akita 010-1231, Japan (f) Department of Biology, University of North Carolina, Chapel Hill, NC 27599-3280, USA Article History: Received 30 March 2006; Revised 7 July 2006; Accepted 9 July 2006

Hybrid quantum-classical (QM-MM) computational techniques were used to investigate the molecular basis of the hydroxylation reaction of the [C.sub.alpha] of a C-terminal glycine catalyzed by peptidylglycine [alpha]-hydroxylating monooxygenase (PHM). Results reveal that O2 binds to [Cu.sub.e] to generate [[Cu.sub.B].sup.II]-[O2.sup.(.-)] followed by electron transfer from [Cu.sub.A] to form [[Cu.sub.B].sup.I]-[O2.sup.(.-)].

The Fe(II)- and [alpha]-ketoglutarate ([alpha]KG)-dependent dioxygenases use mononuclear nonheme iron centers to effect hydroxylation of their substrates and decarboxylation of their cosubstrate, [alpha]KG, to C[O.sub.2] and succinate. Our recent dissection of the mechanism of taurine:[alpha]KG dioxygenase (TauD), a member of this enzyme family, revealed that two transient complexes accumulate during catalysis in the presence of saturating substrates. The first complex contains the long-postulated C-H-cleaving Fe(IV)-oxo intermediate, J, and the second is an enzyme product(s) complex. Here, we demonstrate the accumulation of two transient complexes in the reaction of a prolyl-4-hydroxylase (P4H), a functional homologue of human [alpha]KG-dependent dioxygenases with essential roles in collagen biosynthesis and oxygen sensing. The kinetic and spectroscopic properties of these two P4H complexes suggest that they are homologues of the TauD intermediates. Most notably, the first exhibits optical absorption and Mossbauer spectra similar to those of J and, like J, a large substrate deuterium kinetic isotope on its decay. The close correspondence of the accumulating states in the P4H and TauD reactions supports the hypothesis of a conserved mechanism for substrate hydroxylation by enzymes in this family. Fe(IV)-oxo intermediates | hydroxylation | nonheme iron enzymes | oxygen activation

Studies on hypoxia-sensitive pathways have revealed a series of Fe(II)-dependent dioxygenases that regulate hypoxia-inducible factor (HIF) by prolyl and asparaginyl hydroxylation. The recognition of these unprecedented signaling processes has led to a search for other substrates of the HIF hydroxylases. Here we show that the human HIF asparaginyl hydroxylase, factor inhibiting HIF (FIH), also efficiently hydroxylates specific asparaginyl (Asn)-residues within proteins of the I[kappa]B family. After the identification of a series of ankyrin repeat domain (ARD)-containing proteins in a screen for proteins interacting with FIH, the ARDs of p105 (NFKB1) and I[kappa]B[alpha] were shown to be efficiently hydroxylated by FIH at specific Asn residues in the hairpin loops linking particular ankyrin repeats. The target Asn residue is highly conserved as part of the ankyrin consensus, and peptides derived from a diverse range of ARD-containing proteins supported FIH enzyme activity. These findings demonstrate that this type of protein hydroxylation is not restricted to HIF and strongly suggest that FIH-dependent ARD hydroxylation is a common occurrence, potentially providing an oxygen-sensitive signal to a diverse range of processes. NF-[kappa]B | 2-oxoglutarate-dependent dioxygenase | protein hydroxylation

[sup.29]Si MAS NMR spectroscopy on samples of 10-[Angstrom] phase synthesized from oxides (6.0 GPa/600 [degrees]C/400 h) and from partial transformation of talc (6.5 GPa/650 [degrees]C/12.5 h) reveals that this phase contains [Q.sup.2]-type Si sites in a ratio [Q.sup.3]:[Q.sup.2] of 5.33:1. It is proposed that the [Q.sup.2] arise from adjacent vacancies in the tetrahedral sheets for which charge balance is most likely achieved by hydroxylation via a hydrogamet-like substitution involving the formation of [Q.sup.2] silanol groups. Variable-contact-time [sup.29]Si {[sup.1]H} CP/MAS NMR spectra of the talc/10-[Angstrom] phase product support the assignment of [Q.sup.2] Si to the proposed Si[O.sub.3](OH) groups. Electron microprobe analysis, including oxygen, gives the following empirical formula normalized to three Mg apfu and inferring a hydrogarnet component Si [right arrow] 4H associated with Si vacancies: [Mg.sub.3][Si.sub.3.83(8)][O.sub.9.32][(OH).sub.2.68].1.1(4)[H.sub.2]O. The observed Mg:Si indicates a significant Si deficiency relative to talc. Comparison of the [sup.29]Si MAS NMR and microprobe data indicates that Si vacancies likely occur as single isolated entities, rather than as pairs or clusters, and that between 1 in 18 and 1 in 23 Si sites is vacant. The results suggest new and intriguing possibilities for the incorporation of excess H into the 10-[Angstrom] phase and, potentially, other phyllosilicates under upper-mantle conditions. Keywords: 10-[Angstrom] phase, Si vacancies, [sup.29]Si NMR spectroscopy, silanol

The catalytic mechanisms of phenylalanine and tryptophan hydroxylase from kinetic isotope effects on aromatic hydroxylation are studied. The results suggest that all the aromatic amino acid hydroxylases share the same chemical mechanism but differ in the identity of the rate-limiting step.

The first and key step in alkane metabolism is the terminal hydroxylation of alkanes to 1-alkanols, a reaction catalyzed by a family of integral-membrane diiron enzymes related to Pseudomonas putida Gpo1 AlkB, by a diverse group of methane, propane, and butane monooxygenases and by some membrane-bound cytochrome P450s. Recently, a family of cytoplasmic P450 enzymes was identified in prokaryotes that allow their host to grow on aliphatic alkanes. One member of this family, CYP153A6 from Mycobacterium sp. HXN-1500, hydroxylates medium-chain-length alkanes ([C.sub.6] to [C.sub.11]) to 1-alkanols with a maximal turnover number of 70 [min.sup.-1] and has a regiospecificity of [greater than or equal to] 95% for the terminal carbon atom position. Spectroscopic binding studies showed that [C.sub.6]-to-[C.sub.11] aliphatic alkanes bind in the active site with [K.sub.d] values varying from ~20 [micro]M to 3.7 [micro]M. Longer alkanes bind more strongly than shorter alkanes, while the introduction of sterically hindering groups reduces the affinity. This suggests that the substrate-binding pocket is shaped such that linear alkanes are preferred. Electron paramagnetic resonance spectroscopy in the presence of the substrate showed the formation of an enzyme-substrate complex, which confirmed the binding of substrates observed in optical titrations. To rationalize the experimental observations on a molecular scale, homology modeling of CYP153A6 and docking of substrates were used to provide the first insight into structural features required for terminal alkane hydroxylation.

The mechanism of hydrocarbon hydroxylation mediated by polyhalogenated ruthenium porphyrin catalysts with 2,6-dichloropyridine N-oxide as the oxidant is reported. The analysis has shown that the Ru(II), Ru(IV), and Ru(VI) porphyrin species observed during catalysis act as a reservoir of catalyst precursors and the results have supported a Ru(III)-oxoRu(V) cycle for the 'fast catalysis'.

The interactions of single Pd atoms with gamma-Al(sub 2)O(sub 3) surfaces corresponding to realistic pretreatment conditions by means of density functional theory is reported using well-defined models of gamma-Al(sub 2)O(sub 3) surfaces. The results show that the dependence of structure and interaction energies with selected surfaces are depicted and reveal the low-energy diffusion paths of Pd on these surfaces.

The representatives of the fungal community from an agricultural soil are isolated to study their potential for isoproturon transformation and to identify the transformation products. Among the fungi that were isolated, asco-, basidio-, and zygomycetes were found to transform isoproturon mainly to 1-OH-IPU (N-(-(2-hydroxy-1-methylethyl)phenyl)-N',N'-dimethylurea) and 2-OH-IPU (N-(4-(1-hydroxy-1-methylethyl)phenyl)-N',N'-dimethylurea), thus suggesting that soil fungi could be the source of the hydroxylated metabolites of isoproturon detected in environmental samples.

The crystal-chemistry of 13 elbaite-schorl tourmaline crystals from the Cruzeiro pegmatite (Minas Gerais, Brazil) was studied with a multi-analytical approach (SREF, EMPA, SIMS, MS). Effective cation radii at the Y and Z sites and site populations were refined by a minimization procedure. The results indicate that the crystals belong to the alkali group. Elbaite crystals are [O.sup.2] -free at the W and V sites and show OH content at the O2 site (up to 0.2 apfu). Conversely, schorl crystals always show [O.sup.2-] at the W site. The main substitutional mechanism is the dehydroxylation type: [sup.Y][Fe.sup.2+] + [sup.Y][Fe.sup.3+] + [sup.w]O [right arrow] [sup.Y]Li + [sup.Y]Al + [sup.w][OH+F). The T site is characterized by [sup.T]Si [right arrow] [sup.T]Al substitution. is linearly correlated with vacancy content in crystals with (OH + F) [less than or equal to] or 4, whereas it is almost constant in crystals with OH at the O2 position. Along the series, is inversely correlated with [sup.Y]Al. The Z site is almost fully occupied by [R.sup.3+] (with [sup.Z]Al largely dominant) and the [sup.Z.][Fe.sub.tot] [left and right arrow] [sup.Z]Al substitution explains the inverse correlation of with [sup.Z]Al. In the elbaite compositional range, lattice parameters are functions of , whereas in the schorl range they are essentially functions of . Along the whole elbaite-schorl series, both chemical substitutions and size increase of Y are far larger than those of Z. In spite of this, lattice parameters increase with as much as with . This is due to the role of the [Z[O.sub.6]] polyhedra, which extend along a and c to form the skeleton of the tourmaline structure. Therefore, any change in the size of Z leads to a change in the whole structure.

The C-H hydroxylation and C=C epoxidation of propene by the penta-radical states is reported. The initial energy differences, between the penta-radical and tri-radical states, diminish along the reaction pathway due to the favorable and cumulative exchange stabilization of the more open-shell species.

Quasiclassical trajectories is computed describing the H + O2 OH + O with the O2 reagent vibrationally excited to different levels at different temperatures. The vibrational energy levels are selected by using a semiclassical Einstein-Brillouin-Keller quantization procedure while the relative translational energy is sampled from a Boltzmann weighted distribution.

Shotgun lipidomics is exploited to demonstrate a 4-fold increase in acylcarnitines in diabetic myocardium, which is reversible upon insulin treatment. The analysis of acylcarnitine molecular species in diabetic myocardium identified acylcarnitine, molecular species containing a mass shift of 16 amu in comparison to the anticipated molecular species while synthesis of 3-hydroxy acylcarnitines identified natural products such as the 3-hydroxylated acylcarnitines.

The conformational effects on glycoside reactivity has been described. The results indicate that (super 1)C4 conformers of glucosides are highly reactive species and the axial OH groups are less electron withdrawing than equatorial OH groups.

An examination of the surface structure of sulfated zirconia (SZ) is presented by density-functional theory (DFT) with periodic boundary conditions. The study observed a qualitative change of the prevailing sulfate surface species depending on the sulfatation agent (SO3 or H2O4).

A novel feature identified in catalytic voltammetry of xanthine dehydrogenase (XDH) concerns the potential for the onset of catalysis (ca. 400 mV), which is at least 600 mV more positive than that of the highest potential cofactor. This is explained on the basis of a pterin-associated oxidative switch during voltammetry that precedes catalysis.

The IR spectrum of catechol in CCl4 shows two fairly sharp O-H stretching bands of roughly equal absorbance to the free OH and the intramolecularly H-bonded OH groups. Intermolecular H-bond formation between the free OH and a hydrogen bond acceptor decreases its stretching frequency by several hundred wavenumbers and simultaneously decreases the frequency of the intramolecularly H-bonded OH by a few tens of wavenumbers.

Studies done on the collisions of the gaseous hydroxyl (OH) radical and ozone (O3) with the surfaces of sodium chloride or iodide solutions, and with the surface of neat water by using molecular dynamics simulations is reported. Results of the study reveal that both OH and O3 prefer surface adsorption over bulk solvation in water as well as in salt solutions.

A mechanism of direct benzene hydroxylation over Fe-ZSM-5 zeolite from B3LYP density functional theory computations is discussed. A mononuclear iron model supported at the AlO4(super -) of zeolite that catalyzes the conversion of benzene to phenol with N2O is also demonstrated.

(Super 13)C nuclear magnetic resonance (NMR) spectroscopic studies have been conducted with the hydroxide complex of Pseudomonas aeruginosa heme oxygenase, where OH(super -1) is used as a model of the OOH (super -1) ligand to investigate the ferric hydroperoxide intermediate in heme catabolism at ambient temperatures. Results suggest that if the ligand field strength of the coordinated OOH(super -1) in heme oxygenase is modulated, the resultant large spin density at the meso carbons and nonplanar deformations of the pophyrin ring prime the macrocycle to actively participate in its own hydroxylation.

The first variational transition-state theory rate constant calculations including multidimensional tunneling corrections of the OH + CH3SH reaction and several isotopic variants is presented. It is found that the reaction takes place by forming first a weakly bound complex that presents two hydrogen bonds between the hydroxyl radical and methanethiol from which two reaction paths diverge.

The kinetics of the reaction between OH and pyridine, all three picoline isomers, all six lutidine isomers, and all three ethylpyridine isomers are experimentally investigated. The atmospheric implications of the findings are discussed in terms of the role of pyridinated compounds in the ionic composition of the troposphere are discussed.

The kinetics of hydroxyl radical trapping by some nitrones and the decay of their corresponding spin adduct and correlation of these experimental results with theoretical calculations is reported. The results suggest that hydroxyl radical trapping by 5-d3emethyl -1-pyrroline N-oxide (DMPO) is significantly slower compared to the trapping rate of spin traps 5-ethoxycarbonyl-5-methyl-1-pyrrolilne N-oxide (EMPO) , 5-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BocMPO) and many more.

The study of the peptidylgycine alpha-hydroxylating monooxygenase H172A mutant has been carried out. The role of the Cu (sub)H ligand in the catalytic mechanism in this monooxygenase has been determined via the use of spectroscopic characterization of alpha-hydroxylating monooxygenase and the details are reported.

The quantum chemical methods are employed to investigate gaps in our understanding of the fates of radical intermediates in the OH-initiated degradation of isoprene. The results highlight the uncertainties present in current model assumptions about branching ratios and mechanisms for the OH-initiated degradation of isoprene.

The study explains how CYP119 hydroxylates lauric acid in a reaction supported by putidaredoxin and putidaredoxin reductase. This helps to engineer the mutagenesis of the complementarity of the protein-protein interface.

The quantitative measurements of the complex binding process between rgp 120 and GSL receptors at a model lipid membrane surface are detailed. The total internal reflection fluorescence (TIRF) results demonstrates that a carbohydrate on ceramide is a requirement for rgp120 recognition, and there is little dependence on the chemical composition of the fatty acid chain of ceramide in terms of both hydroxylation and fatty acid chain length.

Research has been conducted on the synthesis of the 1,2,3,4-tetrahydroisoquinoline moiety of tetrazomine. The reaction sequence which utilizes Sharpless asymmetric dihydroxylation to close the heterocyclic ring is described.

Research has been conducted on the toluene 4-monooxygenase from Pseudomonas mendocina that catalyzes the NADH- and dioxygen-dependent toluene hydroxylation to form p-cresol. The solution structure of this monooxygenase has been investigated and the results are discussed.

The rate of reaction, Arrhenius preexponential factors, and activation energies of cyclopropane and difluoromethoxydifluoromethane, reacting with hydroxyl radical were measured by a relative method. Ethane was used as a reference and eight measurements were obtained in the range from 25 to 186 degree C that yielded an Arrhenius fit for the reaction of cyclopropane with hydroxyl radical.

Research has been conducted on the Gloeophyllum striatum strains. The mechanism of the Fenton-type degradation has been examined.

Research has been conducted on the DNA oxidative damage. The role of the leukocyte-generated oxidants has been investigated.