Improvement of the purification method for retaining the activity of the particulate methane monooxygenase from Methylosinus trichosporium OB3b

The purification method of particulate methane monooxygenase (pMMO) from Methylosinus trichosporium OB3b was improved, and purified pMMO retained its activity with duroquinol as a reductant. n-Dodecyl-,d-maltoside was used for the solubilization of pMMO and Brij 58 was used for the purification for anion exchange chromatography. Compared to the original pMMO activity in the membrane fraction, 88% of the activity was now retained in the purified material. The purified pMMO monomer (94 kDa) contained only two copper atoms and did not contain iron. Both copper ions showed only a typical type II copper EPR signal with a superhyperfine structure at the g region, indicating that the type II copper ions play an important role as the active site of methane hydroxylation in pMMO.     

Ethane and n-pentane in exhaled breath are biomarkers of exposure not effect

The relationship of exhaled ethane and n-pentane to exhaled NO, carbonylated proteins, and indoor/outdoor atmospheric pollutants were examined in order to evaluate ethane and n-pentane as potential markers of airway inflammation and/or oxidative stress. Exhaled NO and carbonylated proteins were found to have no significant associations with either ethane (p = 0.96 and p = 0.81, respectively) or n-pentane (p = 0.44 and 0.28, respectively) when outliers were included. In the case where outliers were removed n-pentane was found to be inversely associated with carbonylated proteins. Exhaled hydrocarbons adjusted for indoor hydrocarbon concentrations were instead found to be positively associated with air pollutants (NO, NO₂ and CO), suggesting pollutant exposure is driving exhaled hydrocarbon concentrations. Given these findings, ethane and n-pentane do not appear to be markers of airway inflammation or oxidative stress.     

Isolation and Identification of n-Butane-assimilating Bacterium

A bacterial strain capable of assimilating gaseous n-alkanes was newly isolated from activated sludge by enrichment culture technique using n-butane as the sole carbon source. The strain was identified as Pseudomonas butanovora sp. nov. It utilised n-alkanes of C₂~C₉, primary alcohols and carboxylic acids for growth, but did not utilize sugars and C₁ compounds. The cell yields on gaseous n-alkanes, such as ethane, propane and n-butane, were 80% or more. The maximum specific growth rate on n-butane was 0.22 hr^?1 at 30°C, pH 7.0. Dried cells of this new isolate grown on n-butane contained 73% pure protein.     

Gas chromatographic method using photoionization detection for the determination of breath pentane

Lipid peroxidation is thought to be an important event in the pathogenesis of atherosclerosis. It has been suggested that pentane, which can be formed during the oxidation of ω-6 fatty acids, is a marker of lipid peroxidation. Previous studies have reported elevated breath pentane and serum markers of lipid peroxidation in smokers. However, chromatographic separation of pentane from isoprene in virtually all of these studies was incomplete and the methods used did not resolve pentane into its isomers, n-pentane and isopentane. Additionally, most current methods are complicated, requiring trapping and concentrating steps to obtain adequate sensitivity prior to hydrocarbon analysis. The purpose of the current study was to develop a gas chromatographic system to analyze breath pentane, that addressses the above technical problems and that would provide a simple in vivo method for measuring lipid. n-Pentane and isopentane standards were easily separated from isoprene with a Al₂O₃/KCl capillary column contained in a portable gas chromatograph equipped with a photoionization detector. The analysis of repeated measures showed a low coefficient of variation for measurements of n-pentane (10%) and isopentane (9%). We measured breath pentane in 27 subjects (15 smokers, 12 non-smokers). There were no significant difference between the baseline and 4 week interval measurements of n-pentane for smokers both before and after cigarette smoking. The within-subject variability data showed that the assay is highly reproducible for both low and high pentane levels in smokers. Smokers were found to have higher levels of both n-pentane and isopentane than non-smokers (P<0.001). In addition, smokers had further significant elevation of pentane levels 10 min after smoking (P<0.001), which returned to baseline by 1 h. These studies demonstrate that measurement of breath pentane, using a gas chromatograph with a photoionization detector, is simple and reproducible. Additionally, these results suggest that pentane elevation associated with smoking is secondary to the oxidant effects of cigarette smoke and an important temporal relationship exists between cigarette smoking and breath sample analysis.     

Is n-Pentane Really an Index of Lipid Peroxidation in Humans and Animals? A Methodological Reevaluation

Volatile hydrocarbons such as ethane and n-pentane are known to originate from peroxidation of polyunsaturated fatty acids in membrane lipids and they are accepted as a sensitive and direct index of lipid peroxidation both in vitro and in vivo. Until now, an appropriate and commonly accepted method for the analysis of volatile hydrocarbons in exhalation air has not been described. We therefore developed a methodology for routine application in humans that is based on cryofocusing in combination with gas chromatography and is adaptable to mass spectrometry. The samples may be stored in stainless steel bombs up to 3 weeks, and sample volumes necessary to analysis are variable and can be adapted to analytical requirements. The interference by water and carbon dioxide, always present in excess, is strongly reduced. Mass spectroscopic analysis of exhalation air in human control subjects demonstrates, however, the presence of isoprene as the major constituent hitherto identified as n-pentane. The commonly used columns fail to separate n-pentane and isoprene. Based upon studies of the diverse methodologies reported in literature, it must be assumed that the reported responses of the gas chromatographic "n-pentane" peak in exhalation air of humans and animals, hitherto identified exclusively by authentic reference gases, are actually responses to isoprene or, at least, a mixture of both n-pentane and isoprene.     

Structural conservation of the B subunit in the ammonia monooxygenase/particulate methane monooxygenase superfamily

The ammonia monooxygenase (AMO)/particulate methane monooxygenase (pMMO) superfamily is a diverse group of membrane‐bound enzymes of which only pMMO has been characterized on the molecular level. The pMMO active site is believed to reside in the soluble N‐terminal region of the pmoB subunit. To understand the degree of structural conservation within this superfamily, the crystal structure of the corresponding domain of an archaeal amoB subunit from Nitrosocaldus yellowstonii has been determined to 1.8 Å resolution. The structure reveals a remarkable conservation of overall fold and copper binding site location as well as several notable differences that may have implications for function and stability. Proteins 2014; 82:2263–2267. © 2014 Wiley Periodicals, Inc.     

Effects of Zinc on Particulate Methane Monooxygenase Activity and Structure

Particulate methane monooxygenase (pMMO) is a membrane-bound metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. Zinc is a known inhibitor of pMMO, but the details of zinc binding and the mechanism of inhibition are not understood. Metal binding and activity assays on membrane-bound pMMO from Methylococcus capsulatus (Bath) reveal that zinc inhibits pMMO at two sites that are distinct from the copper active site. The 2.6 Å resolution crystal structure of Methylocystis species strain Rockwell pMMO reveals two previously undetected bound lipids, and metal soaking experiments identify likely locations for the two zinc inhibition sites. The first is the crystallographic zinc site in the pmoC subunit, and zinc binding here leads to the ordering of 10 previously unobserved residues. A second zinc site is present on the cytoplasmic side of the pmoC subunit. Parallels between these results and zinc inhibition studies of several respiratory complexes suggest that zinc might inhibit proton transfer in pMMO.     

CDP840

We present the in vitro characterization of a novel phosphodiesterase type 4 inhibitor, CDP840 (R-[+]-4-[2-{3-cyclopentyloxy-4-methoxyphenyl}-2-phenylethyl]pyridine), which has shown efficacy in a phase II allergen challenge study in asthmatics without adverse effects. CDP840 potently inhibits PDE-4 isoenzymes (IC₅₀ 2–30 nM) without any effect on PDE-1, 2, 3, 5, and 7 (IC₅₀>100 μM). It exhibited no significant selectivity in inhibiting human recombinant isoenzymes PDE-4A, B, C or D and was equally active against the isoenzymes lacking UCR1 (PDE-4B2 and PDE-4D2). In contrast to rolipram, CDP840 acted as a simple competitive inhibitor of all PDE-4 isoenzymes. Studies with rolipram indicated a heterogeneity within all the preparations of PDE-4 isoenzymes, indicative of rolipram inhibiting the catalytic activity of PDE-4 with both a low or high affinity. These observations were confirmed by the use of a PDE-4A variant, PDE-4A_330–886, which rolipram inhibited with low affinity (IC₅₀=1022 nM). CDP840 in contrast inhibited this PDE-4A variant with similar potency (IC₅₀=3.9 nM), which was in good agreement with theK _d of 4.8 nM obtained from [³H]-CDP840 binding studies. Both CDP840 and rolipram inhibited the high-affinity binding of [³H]-rolipram binding to PDE-4A, B, C and D with similarK _{d app} (7–19 nM and 3–5 nM, respectively). Thus, the activity of CDP840 at the [³H]-rolipram binding site was in agreement with the inhibitor’s activity at the catalytic site. However, rolipram was ∼100-fold more potent than CDP840 at inhibiting the binding of [³H]-rolipram to mouse brain in vivo. These data clearly demonstrate that CDP840 is a potent selective inhibitor of all PDE-4 isoenzymes. In contrast to rolipram, CDP840 was well-tolerated in humans. This difference, however, cannot at present be attributed to either isoenzyme selectivity or lack of activity in vitro at the high-affinity rolipram binding site (Sr).     

Polyunsaturated C18 fatty acids derivatized with Gly and Ile as an additional tool for studies of the catalytic evolution of fungal 8- and 9-dioxygenases

The fungal linoleate diol synthase (LDS) family contains over twenty characterized 8-, 9-, and 10-dioxygenases (DOX), usually fused to catalytically competent cytochromes P450. Crystal structures are not available, but indirect evidence suggests that linoleic acid enters the active site of 8R-DOX-LDS headfirst and enters 9S-DOX-allene oxide synthase (AOS) with the ω-end (tail) first. Fatty acids derivatized with amino acids can conceivably be used to study oxidation in tail first position by enzymes, which bind natural fatty acids headfirst. The results might reveal catalytic similarities of homologous enzymes. 8R-DOX-5,8-LDS oxidize 18:2n-6-Ile and 18:2n-6-Gly in tail first position to 9S-hydroperoxy metabolites, albeit with less position and stereo specificity than 9S-DOX-AOS. The oxygenation mechanism of 9S-DOX-AOS with antarafacial hydrogen abstraction at C-11 and oxygen insertion at C-9 was also retained. Two homologues, 8R-DOX-7,8-LDS and 8R-DOX-AOS, oxidized 18:2n-6-Ile and 18:2n-6-Gly at C-9, suggesting a conserved feature of 8R-DOX domains. 9R-DOX-AOS, with 54% sequence identity to 9S-DOX-AOS, did not oxidize the derivatized C₁₈ fatty acids. 9Z,12Z-16:2, two carbon shorter than 18:n-6 from the ω-end, was rapidly metabolized to an α-ketol, but 7Z,10Z-16:2 was not a substrate. An unsaturated carbon chain from C-1 to C-8 was apparently more important than the configuration at the ω-end. 8R-DOX-LDS and 9R-DOX-AOS may thus bind 18:2n-6 in the same orientation. The oxidation of 18:2n-6 in straight or reverse head-to-tail positions illustrates evolutionary traits between 8- and 9-DOX domains. Fatty acids derivatized with amino acids provide a complementary tool for the analysis of evolution of enzymes.     

High-performance liquid chromatographic assay of mepivacaine enantiomers in human plasma in the nanogram per milliliter range

A method enabling quantification of R-(−)- and S-(+)-mepivacaine in human plasma in the low nanogram per milliliter range is described. The procedure involves extraction from plasma with diethyl ether, centrifugation, back-extraction into an acidified aqueous solution, washing with a mixture of pentane and isoamylalcohol, alkalinisation, followed by extraction with a mixture of n-pentane and isoamylalcohol. After evaporation of the organic phase, the residue is redissolved in the mobile phase used for the HPLC analysis, which consists of a 6.8:93.2 (v/v) isopropanol-sodium hydrogenphosphate buffer solution with the pH adjusted to 6.8 using phosphoric acid. The HPLC method has been described previously. Separation of the enantiomers is achieved with an α₁-AGP column and the UV detection wavelength is 210 nm. The minimal detectable concentration is ca. 3 ng/ml and the lower limit of quantification is 5 ng/ml for each enantiomer. For both enantiomers r is >0.9995 over the plasma enantiomeric concentration range of 10.5–1054 ng/ml.     

Hydrogen peroxide as an effecter on the inactivation of particulate methane monooxygenase under aerobic conditions

Particulate methane monooxygenase (pMMO), a copper-containing membrane protein, catalyzes methane hydroxylation under aerobic conditions. We found that the activity of pMMO was increased by catalase, implying that hydrogen peroxide (H₂O₂) is generated by pMMO with duroquinol, an electron donor for pMMO, and that the generated H₂O₂ inhibits pMMO activity. In addition, reversible inhibition of pMMO with H₂O₂ was observed upon treatment of pMMO with H₂O₂ followed by the addition of catalase, and H₂O₂ formation by pMMO with duroquinol was detected using a fluorescence probe. The redox behavior of type 2 copper in pMMO measured by the electron paramagnetic resonance revealed that H₂O₂ re-oxidizes the type 2 copper in pMMO reduced with duroquinol.     

The Rheology of 6-Propyl Duodecane and 5-Dibutyl Nonane by Non-Equilibrium Molecular Dynamics Simulations

Abstract

In recent papers, we reported non-equilibrium molecular dynamics (NEMD) simulations of planar Couette flow for liquid n- and i-butane, and liquid n-decane and 4-propyl heptane, using two collapsed atom models and an atomistically detailed model. It was found that the collapsed atom models predict the viscosities of the n-butane and n-decane quite well, and that the atomistically detailed model does not yield quantitative agreement with the viscosity of the n-alkanes or the branched alkanes, but it does have the one positive feature that the calculated viscosities of the branched alkanes are higher than these of the n-alkanes. In the present paper, we report results of NEMD simulations of planar Couette flow for liquid 6-propyl duodecane and 5-dibutyl nonane at 296 K and 0.782 g/cc, using an expanded collapsed atom model for simplicity. The strain rate dependent viscosity shows three different regions—two shear thinning ones and a Newtonian one. The slopes of the log-log plot for the branched-chain alkanes at high strain rate are different from those at intermediate strain rate, which is characterized as a rheological behavior of branched-chain alkanes. The Newtonian viscosity of the branched-chain alkanes can be extrapolated from the plateau value of the strain rate dependent viscosity at low strain rate as for straight-chain alkanes [J. Chem. Phys., 105, 1214 (1996)]. The results indicate that more-branched alkanes have a larger viscosity than less-branched C₁₇ alkanes.     

Molecular characterization of Penicillium oxalicum 6R,8R-linoleate diol synthase with new regiospecificity

Diol synthase-derived metabolites are involved in the sexual and asexual life cycles of fungi. A putative diol synthase from Penicillium oxalicum was found to convert palmitoleic acid (16:1n-7), oleic acid (18:1n-9), linoleic acid (18:2n-6), and α-linolenic acid (18:3n-3) to 6S,8R-dihydroxy-9(Z)-hexadecenoic acid, 6R,8R-dihydroxy-9(Z)-octadecenoic acid, 6R,8R-dihydroxy-9,12(Z,Z)-octadecadienoic acid, and 6S,8R-dihydroxy-9,12,15(Z,Z,Z)-octadecatrienoic acid, respectively, which were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR) spectroscopy analyses. The specific activity and catalytic efficiency of P. oxalicum 6,8-diol synthase were the highest for 18:2n-6, indicating that the enzyme is a 6R,8R-linoleate diol synthase (6R,8R-LDS) with new regiospecificity. This is the first report of a 6R,8R-LDS. LDS is a fusion protein consisting of a dioxygenase domain at the N-terminus and a cytochrome P450/hydroperoxide isomerase (P450/HPI) domain at the C-terminus. The putative active-site residues in the C-terminal domain of P. oxalicum 6R,8R-LDS were proposed based on a substrate-docking homology model. The results of the site-directed mutagenesis within C-terminal P450 domain suggested that Asn⁸⁸⁶, Arg⁷⁰⁷, and Arg⁹³⁴, are catalytic importance and belong to the catalytic groove. Phe⁷⁹⁴ and Gln⁸⁸⁹ were found to be involved in the regiospecific rearrangement of hydroperoxide, while the F794E and Q889A variants of P. oxalicum 6,8-LDS acted as 7,8- and 8,11-LDSs, respectively. All these mutations critically affected the HPI activity of P. oxalicum 6R,8R-LDS.     

Stereoselective Inhibition of Cholesterol Esterase by Enantiomers of <Emphasis Type="Italic">exo</Emphasis>- and <Emphasis Type="Italic">endo</Emphasis>-2-Norbornyl-<Emphasis Type="Italic">N</Emphasis>–<Emphasis Type="Italic">n</Emphasis>-butylcarbamates

Four stereoisomers of 2-norbornyl-N–n-butylcarbamates are characterized as the pseudo substrate inhibitors of cholesterol esterase. Cholesterol esterase shows enantioselective inhibition for enantiomers of exo- and endo-2-norbornyl-N–n-butylcarbamates. For the inhibitions by (R)-(+)- and (S)-(−)-exo-2-norbornyl-N–n-butylcarbamates, the R-enantiomer is 6.8 times more potent than the S-enantiomer. For the inhibitions by (R)-(+)- and (S)-(−)-endo-2-norbornyl-N–n-butyl-carbamates, the S-enantiomer is 4.6 times more potent than the R-enantiomer. The enzyme-inhibitor complex models have been proposed to explain these different enantioselectivities.     

Uptake of volatilen-alkanes byPseudomonas PG-I

Using a bacterial speciesPseudomonas PG-1, evidence has been obtained which indicates that uptake ofn-pentane ton-octane by microbial cells takes place primarily from the gas phase either directly orvia the aqueous phase. Specific growth rate increased along with the increase in substrate concentration but above the alkane concentration of 0.3% by volume, specific growth rate decreased indicating substrate inhibition of growth. In the case of less volatile alkanes,n-nonane andn-decane, substrate transfer is predominantly through substrate solubilization system elaborated by the cells. EDTA, a strong inhibitor of hydrocarbon solubilization by the cells, inhibited growth on these two alkanes but had negligible effect on growth onn-pentane ton-octane.     

Effects of EGTA and calmodulin,neutral thiol proteinases and protein kinase C inhibitors on loss of chicken pineal serotoninn-acetyltransferase activity

Summary This paper shows that the loss of chicken pineal serotoninn-acetyltransferase activity in crude homogenates of the pineal gland during preincubation at 37°C is a complex process which seems to involve protein kinase C, calmodulin and calcium-activated neutral protease. All three compounds are strongly related to free calcium levels, and hence EGTA effectively prevents this loss of activity. It is proposed that the loss of serotoninn-acetyltransferase activity in crude chicken pineal homogenates is due to a series of molecular events, probably triggered by loss of the calcium gradient present in the intact gland by the homogenization process, leading to rapid serotoninn-acetyltransferase deactivation. In these homogenates two calmodulin inhibitors, a protein kinase C inhibitor and a neutral thiol proteinase inhibitor, and EGTA were found to markedly reduce the rate of serotoninn-acetyltransferase deactivation.Abbreviations SNAT Serotoninn-Acetyltransferase (acetylcoenzyme A: arylaminen-acetyltransferase (EC 2.3.1.5) - PKC protein kinase C - CANP calcium-activated neutral protease - CGS 9343B 1,3-Dihydro-1- 1-(4-methyl-4H, 6H-pyrrolo 1,2-a 4,1-benzoxazepin-4-yl) methyl-4-piperidinyl-2H-benzimidazol-2-one (11) maleate - H-7 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine - E-64 trans-epoxysuccinyl-l-leucylamido-(4-guanidinio)-butane     

Structure determination and refinement of the Al complex of the D254,256E mutant of Arthrobacter -xylose isomerase at 2.40 Å resolution. Further evidence for inhibitor-induced metal ion movement

The structure of the D254,256E double mutant of Arthrobacter xylose isomerase with Al³⁺ at both metal-binding sites was determined by the molecular replacement method at a conventional R-factor of 0.179. Binding of the two Al³⁺ does not alter the overall structure significantly. However, there are local rearrangements in the octahedral co-ordination sphere of the Al³⁺. The inhibitor molecule moves somewhat away from the active site. Furthermore, evidence was revealed for metal ion movement from site 2₁ to site 2₂ upon double mutation. Xylose isomerase requires two divalent metal cations for activation. The catalytic metal ion is translocated 1.8 Å away from its initial position during the catalytic reaction. The fact that both activating and inactivating metals (including Al³⁺) were found exclusively at a single location in the double mutant was an indication that the consequently missing shuttle may account for the crippled catalytic efficiency.     

Manganese lipoxygenase of F. oxysporum and the structural basis for biosynthesis of distinct 11-hydroperoxy stereoisomers

The biosynthesis of jasmonates in plants is initiated by 13S-lipoxygenase (LOX), but details of jasmonate biosynthesis by fungi, including Fusarium oxysporum, are unknown. The genome of F. oxysporum codes for linoleate 13S-LOX (FoxLOX) and for F. oxysporum manganese LOX (Fo-MnLOX), an uncharacterized homolog of 13R-MnLOX of Gaeumannomyces graminis. We expressed Fo-MnLOX and compared its properties to Cg-MnLOX from Colletotrichum gloeosporioides. Electron paramagnetic resonance and metal analysis showed that Fo-MnLOX contained catalytic Mn. Fo-MnLOX oxidized 18:2n-6 mainly to 11R-hydroperoxyoctadecadienoic acid (HPODE), 13S-HPODE, and 9(S/R)-HPODE, whereas Cg-MnLOX produced 9S-, 11S-, and 13R-HPODE with high stereoselectivity. The 11-hydroperoxides did not undergo the rapid β-fragmentation earlier observed with 13R-MnLOX. Oxidation of [11S-²H]18:2n-6 by Cg-MnLOX was accompanied by loss of deuterium and a large kinetic isotope effect (>30). The Fo-MnLOX-catalyzed oxidation occurred with retention of the ²H-label. Fo-MnLOX also oxidized 1-lineoyl-2-hydroxy-glycero-3-phosphatidylcholine. The predicted active site of all MnLOXs contains Phe except for Ser³⁴⁸ in this position of Fo-MnLOX. The Ser348Phe mutant of Fo-MnLOX oxidized 18:2n-6 to the same major products as Cg-MnLOX. Our results suggest that Fo-MnLOX, with support of Ser³⁴⁸, binds 18:2n-6 so that the proR rather than the proS hydrogen at C-11 interacts with the metal center, but retains the suprafacial oxygenation mechanism observed in other MnLOXs.