Hydroxylation Of Carbon Research Articles

Production of derivatives of α-terpineol by bacterial CYP102A1 enzymes.

The monooxygenase activity of engineered CYP102A1 on α-terpineol was investigated. CYP102A1 M850 mutant (F11Y/R47L/D68G/F81I/F87V/E143G/L188Q/E267V/H408R) showed the highest catalytic activity toward α-terpineol among the engineered mutants produced by random mutagenesis. The major product (P1) of α-terpineol, p-menth-1-ene-3,8-diol, was characterized by high-performance liquid chromatography, gas-chromatography mass spectrometry, and nuclear magnetic resonance spectroscopy. Three minor products (P2-P4) of α-terpineol were considered as 6-hydroxy-α,α,4-trimethyl-3-cyclohexene-1-methanol (P2), trans-sobrerol (P3), and carvone hydrate (P4). Optimal conditions for product formation were determined as pH 7.0 and 30 °C. Production of p-menth-1-ene-3,8-diol was 0.87mM at 1h. Structure modeling using PyMOL and CAVER Web 1.2 server indicated that several mutations of CYP102A1 M850 were involved in access tunnels and active sites, resulting in increased activity toward α-terpineol. The major product, p-menth-1-ene-3,8-diol, of α-terpineol was produced by engineered CYP102A1 M850 via regioselective carbon hydroxylation. The engineered CYP102A1 could be a suitable biocatalyst for producing α-terpineol derivatives.

Promoting Electrocatalytic Reduction of CO2 to C2 H4 Production by Inhibiting C2 H5 OH Desorption from Cu2 O/C Composite.

The electrochemical CO2 reduction reaction (CO2 RR) has great potential in realizing carbon recycling while storing sustainable electricity as hydrocarbon fuels. However, it is still a challenge to enhance the selectivity of the CO2 RR to single multi-carbon (C2+ ) product, such as C2 H4 . Here, an effective method is proposed to improve C2 H4 selectivity by inhibiting the production of the other competitive C2 products, namely C2 H5 OH, from Cu2 O/C composite. Density functional theory indicates that the heterogeneous structure between Cu2 O and carbon is expected to inhibit C2 H5 OH production and promote CC coupling, which facilitates C2 H4 production. To prove this, a composite electrode containing octahedral Cu2 O nanoparticles (NPs) (o-Cu2 O) with {111} facets and carbon NPs is constructed, which experimentally inhibits C2 H5 OH production while strongly enhancing C2 H4 selectivity compared with o-Cu2 O electrode. Furthermore, the surface hydroxylation of carbon can further improve the C2 H4 production of o-Cu2 O/C electrode, exhibiting a high C2 H4 Faradaic efficiency of 67% and a high C2 H4 current density of 45mA cm-2 at -1.1 Vin a near-neutral electrolyte. This work provides a new idea to improve C2+ selectivity by controlling products desorption.

Dioxybenzone triggers enhanced estrogenic effect via metabolic activation: in silico, invitro and invivo investigation.

Dioxybenzone is widely used in cosmetics and personal care products and frequently detected in multiple environmental media and human samples. However, the current understanding of the metabolic susceptibility of dioxybenzone and the potential endocrine disruption through its metabolites in mimicking human estrogens remains largely unclear. Here we investigated the invitro metabolism of dioxybenzone, detected the residue of metabolites in rats, and determined the estrogenic disrupting effects of these metabolites toward estrogen receptor α (ERα). Invitro metabolism revealed two major metabolites from dioxybenzone, i.e., M1 through the demethylation of methoxy moiety and M2 through hydroxylation of aromatic carbon. M1 and M2 were both rapidly detected in rat plasma upon exposure to dioxybenzone, which were then distributed into organs of rats in the order of livers>kidneys>uteri>ovaries. The 100 ns molecular dynamics simulation revealed that M1 and M2 formed hydrogen bond to residue Leu387 and Glu353, respectively, on ERα ligand binding domain, leading to a reduced binding free energy. M1 and M2 also significantly induced estrogenic effect in comparison to dioxybenzone as validated by the recombinant ERα yeast two-hybrid assay and uterotrophic assay. Overall, our study revealed the potential of metabolic activation of dioxybenzone to induce estrogenic disrupting effects, suggesting the need for incorporating metabolic evaluation into the health risk assessment of benzophenones and their structurally similar analogs.

ATP-dependent hydroxylation of an unactivated primary carbon with water

Enzymatic hydroxylation of unactivated primary carbons is generally associated with the use of molecular oxygen as co-substrate for monooxygenases. However, in anaerobic cholesterol-degrading bacteria such as Sterolibacterium denitrificans the primary carbon of the isoprenoid side chain is oxidised to a carboxylate in the absence of oxygen. Here, we identify an enzymatic reaction sequence comprising two molybdenum-dependent hydroxylases and one ATP-dependent dehydratase that accomplish the hydroxylation of unactivated primary C26 methyl group of cholesterol with water: (i) hydroxylation of C25 to a tertiary alcohol, (ii) ATP-dependent dehydration to an alkene via a phosphorylated intermediate, (iii) hydroxylation of C26 to an allylic alcohol that is subsequently oxidised to the carboxylate. The three-step enzymatic reaction cascade divides the high activation energy barrier of primary C–H bond cleavage into three biologically feasible steps. This finding expands our knowledge of biological C–H activations beyond canonical oxygenase-dependent reactions.

Vanillic Acid Restores Coenzyme Q Biosynthesis and ATP Production in Human Cells Lacking COQ6.

Coenzyme Q (CoQ), a redox-active lipid, is comprised of a quinone group and a polyisoprenoid tail. It is an electron carrier in the mitochondrial respiratory chain, a cofactor of other mitochondrial dehydrogenases, and an essential antioxidant. CoQ requires a large set of enzymes for its biosynthesis; mutations in genes encoding these proteins cause primary CoQ deficiency, a clinically and genetically heterogeneous group of diseases. Patients with CoQ deficiency often respond to oral CoQ10 supplementation. Treatment is however problematic because of the low bioavailability of CoQ10 and the poor tissue delivery. In recent years, bypass therapy using analogues of the precursor of the aromatic ring of CoQ has been proposed as a promising alternative. We have previously shown using a yeast model that vanillic acid (VA) can bypass mutations of COQ6, a monooxygenase required for the hydroxylation of the C5 carbon of the ring. In this work, we have generated a human cell line lacking functional COQ6 using CRISPR/Cas9 technology. We show that these cells cannot synthesize CoQ and display severe ATP deficiency. Treatment with VA can recover CoQ biosynthesis and ATP production. Moreover, these cells display increased ROS production, which is only partially corrected by exogenous CoQ, while VA restores ROS to normal levels. Furthermore, we show that these cells accumulate 3-decaprenyl-1,4-benzoquinone, suggesting that in mammals, the decarboxylation and C1 hydroxylation reactions occur before or independently of the C5 hydroxylation. Finally, we show that COQ6 isoform c (transcript NM_182480) does not encode an active enzyme. VA can be produced in the liver by the oxidation of vanillin, a nontoxic compound commonly used as a food additive, and crosses the blood-brain barrier. These characteristics make it a promising compound for the treatment of patients with CoQ deficiency due to COQ6 mutations.

On the trail of steroid aromatase: The work of Kenneth J. Ryan

On the trail of steroid aromatase: The work of Kenneth J. Ryan

Study of the in vitro metabolic profile of a new α2-adrenergic agonist in rat and human liver microsomes by using liquid chromatography-multiple-stage mass spectrometry and nuclear magnetic resonance

A potent synthetic α2-adrenergic agonist called PT-31, (3-(2-chloro-6-fluorobenzyl)-imidazolidine-2,4-dione), was recently detected as a potential drug to be used as an adjuvant drug to treat chronic pain. The excellent pharmacological property of PT-31 highlights the importance in elucidating its metabolism, which could provide valuable information about its metabolite profile for further pharmacokinetics studies and additionally to estimate the impact of its metabolites on the efficacy, safety and elimination of PT-31. In this work, the study of the in vitro metabolism of PT-31 was initially carried out by using a liquid chromatography coupled to ion trap multiple-stage mass spectrometer (LC-IT-MSn) and a hybrid triple quadrupole/linear ion trap mass spectrometer (LC-QTrap). The production of at least three unknown oxidative metabolites was observed. Structural identification of the unknown metabolites was carried out by combination of LC–MS experiments, including selected reaction monitoring (SRM) and multi-stage full scan experiments. Further analysis of 1H-NMR led to the structural confirmation of the major metabolite. The results indicated that PT-31 was metabolized by a hydroxylation reaction in the imidazolidine-2,4-dione ring in rat and human liver microsomes, producing the metabolite 3-(2-chloro-6-fluorobenzyl)-5-hydroxyimidazolidine-2,4-dione in rat liver microsomes. A carbon hydroxylation onto the benzyl ring, produced two other minor metabolites of the PT-31 in rat liver microsomes.

Metabolic Susceptibility of 2-Chlorothioxanthone and Its Toxic Effects on mRNA and Protein Expression and Activities of Human CYP1A2 and CYP3A4 Enzymes.

Thioxanthones (TXs) are photoinitiators widely used in UV curable resins and food packaging, and their residues have been frequently detected in human bodies. Our current understanding of the susceptibility of residual TXsto metabolism and their effects on human health is very limited. Thein vitro metabolism of TXs and its toxic effects on cytochrome P450 (CYP) (the key xenobiotic metabolizing enzymes) were examined in this study. 2-Chlorothioxanthone (2-Cl-TX) significantly inhibited the enzymatic activities of CYP1A2 and CYP3A4 with IC50 of 8.36 and 0.86 μM, respectively. The exposure to 2-Cl-TX at 2.5 μM up-regulated the mRNA expression of CYP1A2 and CYP3A4 in human hepatocellular carcinoma cells to 3.03-fold and 2.02-fold, respectively. 2-Cl-TX at 2.5 μM caused 2.19-fold and 1.98-fold overexpression of CYP1A2 and CYP3A4, respectively. In vitro studies revealed that 2-Cl-TX was biotransformed into two metabolites through the sulfoxidation of the sulfur atom, or via the hydroxylation of aromatic carbon. Results from this study, including the metabolic susceptibility of residual 2-Cl-TX, the proposed metabolites and the significant toxic effect on the activities, mRNA, and protein expression of CYP1A2 and CYP3A4, are vital to the human health and safety risk assessment from this ubiquitous xenobiotic.

Two Distinct Mechanisms for C-C Desaturation by Iron(II)- and 2-(Oxo)glutarate-Dependent Oxygenases: Importance of α-Heteroatom Assistance.

Hydroxylation of aliphatic carbons by nonheme Fe(IV)-oxo (ferryl) complexes proceeds by hydrogen-atom (H•) transfer (HAT) to the ferryl and subsequent coupling between the carbon radical and Fe(III)-coordinated oxygen (termed rebound). Enzymes that use H•-abstracting ferryl complexes for other transformations must either suppress rebound or further process hydroxylated intermediates. For olefin-installing C-C desaturations, it has been proposed that a second HAT to the Fe(III)-OH complex from the carbon α to the radical preempts rebound. Deuterium (2H) at the second site should slow this step, potentially making rebound competitive. Desaturations mediated by two related l-arginine-modifying iron(II)- and 2-(oxo)glutarate-dependent (Fe/2OG) oxygenases behave oppositely in this key test, implicating different mechanisms. NapI, the l-Arg 4,5-desaturase from the naphthyridinomycin biosynthetic pathway, abstracts H• first from C5 but hydroxylates this site (leading to guanidine release) to the same modest extent whether C4 harbors 1H or 2H. By contrast, an unexpected 3,4-desaturation of l-homoarginine (l-hArg) by VioC, the l-Arg 3-hydroxylase from the viomycin biosynthetic pathway, is markedly disfavored relative to C4 hydroxylation when C3 (the second hydrogen donor) harbors 2H. Anchimeric assistance by N6 permits removal of the C4-H as a proton in the NapI reaction, but, with no such assistance possible in the VioC desaturation, a second HAT step (from C3) is required. The close proximity (≤3.5 Å) of both l-hArg carbons to the oxygen ligand in an X-ray crystal structure of VioC harboring a vanadium-based ferryl mimic supports and rationalizes the sequential-HAT mechanism. The results suggest that, although the sequential-HAT mechanism is feasible, its geometric requirements may make competing hydroxylation unavoidable, thus explaining the presence of α-heteroatoms in nearly all native substrates for Fe/2OG desaturases.

Snapshots of the catalytic cycle of an O2, pyridoxal phosphate‐dependent hydroxylase

The organic cofactor pyridoxal phosphate (PLP) is used by enzymes to catalyze numerous modifications of amino acid substrates. Recently, the diversity of PLP‐dependent enzymes has been expanded with the discovery of several enzymes that use O2 as a co‐substrate to perform chemically challenging oxidative reactions. The interesting chemistry catalyzed by such O2‐, PLP‐dependent oxidases prompted us to undertake a bioinformatic search for new O2‐, PLP‐dependent enzymes. We identified one widely distributed enzyme, which we named RohP. Through in vitro biochemical studies we characterized the activity of RohP and found that it uses PLP and O2 to catalyze the hydroxylation of an sp3‐hybridized carbon in l‐arginine, giving 4‐hydroxy‐2‐ketoarginine, along with stoichiometric conversion of O2 to H2O2. Surprisingly, we found that the hydroxyl group in the 4‐hydroxy‐2‐ketoarginine product is derived from water, raising questions about the mechanism of RohP. To provide insight into the mechanism behind this hydroxylation, we used X‐ray crystallography to obtain four ~1.5 Å resolution snapshots interpreted to represent RohP at different stages of its catalytic cycle: the holo enzyme, two different PLP‐bound intermediates, and the enzyme in complex with (S)‐4‐hydroxy‐2‐ketoarginine. These structures reveal that the dynamic N‐terminus of RohP, which is required for catalysis, becomes ordered upon substrate binding, sealing off the active site from the bulk solvent. Concurrently, several conformational changes of residues in the active site help to properly orient the substrate for catalysis. Through our structural work we also identify a conserved histidine, which we demonstrate is essential for the later steps in catalysis, and we suggest that this residue may be key to catalyzing a stereospecific alkene hydration. Collectively, our biochemical and X‐ray crystallographic investigation provides insights into how O2‐, PLP‐dependent oxidases can catalyze the challenging hydroxylation of an unactivated sp3‐hybridized carbon in l‐arginine and sets the stage for further mechanistic studies on this exciting group of enzymes.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Mechanism of Water Oxidation Catalyzed by a Mononuclear Iron Complex with a Square Polypyridine Ligand: A DFT Study.

The mononuclear [Cl-FeIII(dpa)-Cl]+ (1Cl) complex containing a square planar tetradentate polypyridine ligand has been reported to catalyze water oxidation in pH = 1 aqueous medium with ceric ammonium nitrate (CAN) as a chemical oxidant. The reaction mechanism of the oxygen evolution driven by this catalyst was investigated by means of density functional calculations. The results showed that one chloride ligand of 1Cl has to exchange with a water molecule to generate 1, [Cl-FeIII(dpa)-OH2]2+, as the starting species of the catalytic cycle. The initial one-electron oxidation of 1 is coupled with the release of two protons, generating [Cl-FeIV(dpa)═O]+ (2). Another one-electron transfer from 2 leads to the formation of an FeV═O complex [Cl-FeV(dpa)═O]2+ (3), which triggers the critical O-O bond formation. The electronic structure of 3 was found to be very similar to that of the high-valent heme-iron center of P450 enzymes, termed Compound I, in which a π-cation radical ligand is believed to support a formal iron(IV)-oxo core. More importantly, 3 and Compound I share the same tendency toward electrophilic reactions. Two competing pathways were suggested for the O-O bond formation based on the present calculations. One is the nitrate nucleophilic attack on the iron(V)-oxo moiety with a total barrier of 12.3 kcal mol-1. In this case, nitrate functions as a co-catalyst for the dioxygen formation. The other is the water nucleophilic attack on iron(V)-oxo with a greater barrier of 16.5 kcal mol-1. In addition, ligand degradation via methyl hydrogen abstraction was found to have a barrier similar to that of the O-O bond formation, while the aromatic carbon hydroxylation has a higher barrier.

Side chain removal from corticosteroids by unspecific peroxygenase

Two unspecific peroxygenases (UPO, EC 1.11.2.1) from the basidiomycetous fungi Marasmius rotula and Marasmius wettsteinii oxidized steroids with hydroxyacetyl and hydroxyl functionalities at C17 - such as cortisone, Reichstein's substance S and prednisone - via stepwise oxygenation and final fission of the side chain. The sequential oxidation started with the hydroxylation of the terminal carbon (C21) leading to a stable geminal alcohol (e.g. cortisone 21-gem-diol) and proceeded via a second oxygenation resulting in the corresponding α-ketocarboxylic acid (e.g. cortisone 21-oic acid). The latter decomposed under formation of adrenosterone (4-androstene-3,11,17-trione) as well as formic acid and carbonic acid (that is in equilibrium with carbon dioxide); fission products comprising two carbon atoms such as glycolic acid or glyoxylic acid were not detected. Protein models based on the crystal structure data of MroUPO (Marasmius rotula unspecific peroxygenase) revealed that the bulky cortisone molecule suitably fits into the enzyme's access channel, which enables the heme iron to come in close contact to the carbons (C21, C20) of the steroidal side chain. ICP-MS analysis of purified MroUPO confirmed the presence of magnesium supposedly stabilizing the porphyrin ring system.

Snapshots of the Catalytic Cycle of an O2, Pyridoxal Phosphate-Dependent Hydroxylase.

Enzymes that catalyze hydroxylation of unactivated carbons normally contain heme and nonheme iron cofactors. By contrast, how a pyridoxal phosphate (PLP)-dependent enzyme could catalyze such a hydroxylation was unknown. Here, we investigate RohP, a PLP-dependent enzyme that converts l-arginine to ( S)-4-hydroxy-2-ketoarginine. We determine that the RohP reaction consumes oxygen with stoichiometric release of H2O2. To understand this unusual chemistry, we obtain ∼1.5 Å resolution structures that capture intermediates along the catalytic cycle. Our data suggest that RohP carries out a four-electron oxidation and a stereospecific alkene hydration to give the ( S)-configured product. Together with our earlier studies on an O2, PLP-dependent l-arginine oxidase, our work suggests that there is a shared pathway leading to both oxidized and hydroxylated products from l-arginine.

A novel eremophilane lactone inhibits FcεRI-dependent release of pro-inflammatory mediators: structure-dependent bioactivity.

Allergic inflammation is primarily mediated by immune effector cells such as mast cells and basophils that release proinflammatory cytokines. Both mast cells and basophils are activated via their high affinity IgE receptor (FcεRI) which initiates the release of proinflammatory mediators such as histamine and tumor necrosis factor (TNF). Considerable effort has been focused on finding an effective basophil stabilizer that inhibits the activation of FcεRI-activated mediator release. Recently, eremophilane lactones, a novel family of sesquiterpene compound originally isolated from Petasites japonicas (Sieb. et Zucc.), have been described, and it has been postulated that they may have anti-inflammatory activity, particularly in allergic disease. Our objective was to determine the effect of two eremophilane lactones derived from 6β-angeloyloxy-3β,8-dihydroxyeremophil-7(11)-en-12,8β-olide (F-1) on immunoglobulin E (IgE)-dependent release of pro-inflammatory mediators by a basophil cell line, RBL-2H3, a model system for FcεRI-mediated activation of pro-inflammatory mediator release. The parent compound (F-1) was chemically modified to produce F-1a [6β-angeloyloxy-3β-benzoyloxy-8-hydroxyeremophil-7(11)-en-12,8β-olide] and F-1b [6β-angeloyloxy-3β,8-diacetoxyeremophil-7(11)-en-12,8β-olide]. RBL-2H3 cells were sensitized with DNP-specific IgE and then activated with DNP-BSA. The effect of these compounds on IgE-dependent basophil degranulation was assessed by measuring the release of β-hexosaminidase (b-hex). In addition, TNF release was measured via ELISA. The phenylacetyl reaction modified C-8 and produced F-1a whereas acetylation of F-1 produced F-1b. F-1a was not cytotoxic to RBL-2H3 cells even at 50 μM, but F-1b was slightly cytotoxic at 50 μM, reducing viability of the cells by approximately 15 %. Neither F-1a nor F-1b inhibited FcεRI-dependent activation of RBL-2H3 cells when the cells were pretreated for only 30 min with the compounds. However, 24 h pretreatment with F-1a inhibited antigen-dependent degranulation by as much as 60 % and TNF production by as much as 90 %. F-1b had no effect on RBL-2H3 activation via FcεRI. These results indicate that F-1a inhibits degranulation of RBL-2H3 cells activated via the high affinity IgE receptor, FcεRI, and that this effect is dependent upon hydroxylation of the third carbon.

ETEM observation of Pt/C electrode catalysts in a moisturized cathode atmosphere

There have been reports of challenges in designing platinum carbon (Pt/C) electrode catalysts for PEMFC. Pt/C electrode catalysts deactivate much faster on the cathode (in moisturized O2) than on the anode (in H2). To understand influences of moisture and oxygen on the deactivation of the Pt/C catalysts in proton-exchange-membrane fuel cells (PEMFCs), spherical-aberration-corrected environmental transmission electron microscopy (AC-ETEM) was applied with a high-speed CCD camera. Structural changes of the Pt/C electrode catalysts were dynamically recorded in moisturized nitrogen, oxygen and hydrogen. The mass spectrometry confirmed the moisture content (between 5 to 30 %) of nitrogen driving gas through a humidifier. Coalescence of platinum nanoparticles (D = 3.24 nm) was carefully evaluated in pure N2 and moisturized N2 atmosphere. The Pt/C showed considerable structural weakness in a moisturized N2 atmosphere. Comparable results obtained by AC-ETEM in different gas atmospheres also suggested ways to improve the oxygen reduction reaction (ORR). In this paper, the deactivation process due to moisture (hydroxylation) of carbon supports is discussed using for comparison the movement of platinum nanoparticles measured in moisturized nitrogen and pure nitrogen atmospheres.

Photocatalytic degradation of tetracycline in aqueous solution by nanosized TiO2

Tetracyclines are widely-used antibiotics in the world. Due to their poor absorption by human beings, or poultry and livestocks, most of them are excreted into the environment, causing growing concern about their potential impact, while photodegradation has been found to dominate their sequestration and bioavailability. Coupling with high-performance liquid chromatography–mass spectroscopy (HPLC–MS), gas chromatography–mass spectroscopy (GC–MS) and electron spin resonance (ESR), the mechanism of photocatalytic degradation of TC in aqueous solution by nanosized TiO2 (P25) under UV irradiation was investigated. The photocatalysis eliminated 95% of TC and 60% of total organic carbon (TOC) after 60min irradiation, and NH4+ ion was found to be one of the end-products. Bioluminescence assay showed that the toxicity of TC solution reached the maximum after 20min irradiation and then gradually decreased. The degradation of TC included electron transfer, hydroxylation, open-ring reactions and cleavage of the central carbon. A possible photocatalytic degradation pathway of TC was proposed on the basis of the identified intermediates. Overall, the TiO2 photocatalysis was found to be a promising process for removing TC and its intermediates.

Direct hydroxylation of primary carbons in small alkanes by wild-type cytochrome P450BM3 containing perfluorocarboxylic acids as decoy molecules

Cytochrome P450BM3 (P450BM3) is a long-alkyl-chain fatty acid hydroxylase that shows an extremely high catalytic turnover rate and high coupling efficiency of NADPH for product formation (product formation rate per NADPH consumption rate). Although P450BM3 exclusively hydroxylates long-alkyl-chain fatty acids, we have found that simple addition of perfluorocarboxylic acids (PFs) as inert dummy substrates (decoy molecules) turns P450BM3 into a small alkane hydroxylase. For example, PF-bound P450BM3 oxidizes propane and butane to 2-propanol and 2-butanol, respectively. The coupling efficiency of small alkane hydroxylation, however, is very low compared with that of long-alkyl-chain fatty acid hydroxylation. In this study, we examined the experimental conditions for small alkane hydroxylation in an effort to improve the coupling efficiency and to realize the hydroxylation of their primary carbons. To increase the concentration of gaseous substrates in the reaction mixture, we performed reactions under the high-pressure condition of 0.5 MPa small alkanes. Propane hydroxylation under high-pressure conditions significantly improved the coupling efficiency to 48%. Furthermore, 1-propanol, which has never been observed under lower-pressure conditions, was produced. It is noteworthy that a detectable amount of ethanol was observed in the ethane hydroxylation under the pressure condition of 0.5 MPa, whereas methane was not hydroxylated. These results indicate that by increasing the concentration of small alkanes, the “P450BM3–decoy molecule system” can catalyze hydroxylation reactions of the primary carbons in small alkanes.

Mechanism of Benzylic Hydroxylation by 4-Hydroxymandelate Synthase. A Computational Study

Hydroxymandelate synthase (HMS) and 4-hydroxyphenylpyruvate dioxygenase (HPPD) are highly related enzymes using the same substrates but catalyzing hydroxylation reactions yielding different products. The first steps of the HMS and HPPD catalytic reactions are believed to proceed in the same way and lead to an Fe(IV)═O-hydroxyphenylacetate (HPA) intermediate. Further down the catalytic cycles, HMS uses Fe(IV)═O to perform hydroxylation of the benzylic carbon, whereas in HPPD, the reactive oxoferryl intermediate attacks the aromatic ring of HPA. This study focuses on this part of the HMS catalytic cycle that starts from the oxoferryl intermediate and aims to identify interactions within the active site that are responsible for enzyme specificity. To this end, a HMS-Fe(IV)═O-HPA complex was modeled with molecular dynamics simulations. On the basis of the molecular dynamics-equilibrated structure, an active site model suitable for quantum chemical investigations was constructed and used for density functional theory (B3LYP) calculations of the mechanism of the native reaction of HMS, i.e., benzylic hydroxylation, and the alternative electrophilic attack on the ring, which is a step of the HPPD catalytic cycle. The most important result of this study is the finding that the conformation of the Ser201 side chain in the second coordination shell has a key role in directing the reaction of Fe(IV)═O into either the HMS or the HPPD channel.

Nicotine and 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone Binding and Access Channel in Human Cytochrome P450 2A6 and 2A13 Enzymes

Cytochromes P450 (CYP) from the 2A subfamily are known for their roles in the metabolism of nicotine, the addictive agent in tobacco, and activation of the tobacco procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Although both the hepatic CYP2A6 and respiratory CYP2A13 enzymes metabolize these compounds, CYP2A13 does so with much higher catalytic efficiency, but the structural basis for this has been unclear. X-ray structures of nicotine complexes with CYP2A13 (2.5 Å) and CYP2A6 (2.3 Å) yield a structural rationale for the preferential binding of nicotine to CYP2A13. Additional structures of CYP2A13 with NNK reveal either a single NNK molecule in the active site with orientations corresponding to metabolites known to form DNA adducts and initiate lung cancer (2.35 Å) or with two molecules of NNK bound (2.1 Å): one in the active site and one in a more distal staging site. Finally, in contrast to prior CYP2A structures with enclosed active sites, CYP2A13 conformations were solved that adopt both open and intermediate conformations resulting from an ∼2.5 Å movement of the F to G helices. This channel occurs in the same region where the second, distal NNK molecule is bound, suggesting that the channel may be used for ligand entry and/or exit from the active site. Altogether these structures provide multiple new snapshots of CYP2A13 conformations that assist in understanding the binding and activation of an important human carcinogen, as well as critical comparisons in the binding of nicotine, one of the most widely used and highly addictive drugs in human use.

Hydroxylation of azomethine carbon: isolation of complexes of η5 and η6-cyclic hydrocarbon platinum group metals with a new Schiff-base ligand

A new Schiff base, (pyridin-2-yl)-N-(3,5-di(pyridin-2-yl)-4H-1,2,4-triazol-4-yl)methanimine, (L), was synthesized. Reaction of [(η6-arene)Ru(µ-Cl)Cl]2 and [Cp*M(µ-Cl)Cl]2 (M = Rh and Ir) with one equivalent of L in the presence of NH4PF6 in methanol yielded dinuclear complexes, [(η6-arene)2Ru2(L-OH)Cl](PF6)2 {arene = C6H6 (1), p-iPrC6H4Me (p-cymene) (2) and C6Me6 (3)}, and [Cp*2M2(L-OH)Cl](PF6)2 [M = Rh (4) and Ir (5)], respectively, leading to the formation of five new chiral complexes with –OH on the azomethine carbon. L is a pentadentate ligand where one of the metal centers is coordinated to two nitrogen atoms in a bidentate chelating fashion while the other metal is bonded tridentate to three nitrogen atoms. Although the ligand is neutral before coordination, after complexation it is anionic (uni-negative) with negative charge on the azo nitrogen {see the structures: N(5) in 2[PF6]2 and N(3) for 4[PF6]2}. The complexes have been characterized by various spectroscopic methods including infrared and 1H NMR and the molecular structures of the representative complexes are established by single-crystal X-ray diffraction studies.