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Sample records for alkane monooxygenase alkb

  1. Expression of an alkane monooxygenase (alkB) gene and methyl tert-butyl ether co-metabolic oxidation in Pseudomonas citronellolis.

    PubMed

    Bravo, Ana Luisa; Sigala, Juan Carlos; Le Borgne, Sylvie; Morales, Marcia

    2015-04-01

    Pseudomonas citronellolis UAM-Ps1 co-metabolically transforms methyl tert-butyl ether (MTBE) to tert-butyl alcohol with n-pentane (2.6 mM), n-octane (1.5 mM) or dicyclopropylketone (DCPK) (4.4 mM), a gratuitous inducer of alkane hydroxylase (AlkB) activity. The reverse transcription quantitative real-time PCR was used to quantify the alkane monooxygenase (alkB) gene expression. The alkB gene was expressed in the presence of n-alkanes and DCPK and MTBE oxidation occurred only in cultures when alkB was transcribed. A correlation between the number of alkB transcripts and MTBE consumption was found (ΜΤΒΕ consumption in μmol = 1.44e(-13) x DNA copies, R(2) = 0.99) when MTBE (0.84 mM) was added. Furthermore, alkB was cloned and expressed into Escherichia coli and the recombinant AlkB had a molecular weight of 42 kDa. This is the first report where the expression of alkB is related to the co-metabolic oxidation of MTBE. PMID:25432418

  2. Monitoring the alkane monooxygenase gene alkB in different soil interfaces during plant litter degradation of C3 and C4 plants

    NASA Astrophysics Data System (ADS)

    Schulz, S.; Munch, J. C.; Schloter, M.

    2009-04-01

    Hydrocarbons like n-alkanes are ubiquitous in the environment as a result of anthropogenic contamination (e.g. oil spills) as well as a part of an ecosystem's biomass. For example n-alkanes become released during plant litter degradation; consequently they become a high abundant carbon source for microorganism. One possibility for the prokaryotic hydrocarbon metabolisation is an aerobic degradation pathway where the initial step is catalysed by the membrane bound alkane monooxygenase alkB. We analysed the influence of alkanes on the abundance of the alkB gene in different interfaces of the litter-soil system during the degradation of maize and pea litter. Therefore soil samples of a sandy and a loamy soil have been incubated with straw of maize and pea plants up to 30 weeks with constant soil moisture and temperature. Using quantitative real-time PCR we were able to monitor the changes of the abundance and the expression rates of alkB. In our experiments we focused on the straw layer, the litter/soil interface and the soil 1 cm below this interface (bulk soil). Our results clearly demonstrate time and space dependent abundance patterns of alkB genes and transcripts in the different layers studied, which are additionally shaped by the soil type used.

  3. MPN- and Real-Time-Based PCR Methods for the Quantification of Alkane Monooxygenase Homologous Genes (alkB) in Environmental Samples

    NASA Astrophysics Data System (ADS)

    Pérez-de-Mora, Alfredo; Schulz, Stephan; Schloter, Michael

    Hydrocarbons are major contaminants of soil ecosystems as a result of uncontrolled oil spills and wastes disposal into the environment. Ecological risk assessment and remediation of affected sites is often constrained due to lack of suitable prognostic and diagnostic tools that provide information of abiotic-biotic interactions occurring between contaminants and biological targets. Therefore, the identification and quantification of genes involved in the degradation of hydrocarbons may play a crucial role for evaluating the natural attenuation potential of contaminated sites and the development of successful bioremediation strategies. Besides other gene clusters, the alk operon has been identified as a major player for alkane degradation in different soils. An oxygenase gene (alkB) codes for the initial step of the degradation of aliphatic alkanes under aerobic conditions. In this work, we present an MPN- and a real-time PCR method for the quantification of the bacterial gene alkB (coding for rubredoxin-dependent alkane monooxygenase) in environmental samples. Both approaches enable a rapid culture-independent screening of the alkB gene in the environment, which can be used to assess the intrinsic natural attenuation potential of a site or to follow up the on-going progress of bioremediation assays.

  4. The Use of a Combination of alkB Primers to Better Characterize the Distribution of Alkane-Degrading Bacteria

    PubMed Central

    Jurelevicius, Diogo; Alvarez, Vanessa Marques; Peixoto, Raquel; Rosado, Alexandre S.; Seldin, Lucy

    2013-01-01

    The alkane monooxygenase AlkB, which is encoded by the alkB gene, is a key enzyme involved in bacterial alkane degradation. To study the alkB gene within bacterial communities, researchers need to be aware of the variations in alkB nucleotide sequences; a failure to consider the sequence variations results in the low representation of the diversity and richness of alkane-degrading bacteria. To minimize this shortcoming, the use of a combination of three alkB-targeting primers to enhance the detection of the alkB gene in previously isolated alkane-degrading bacteria was proposed. Using this approach, alkB-related PCR products were detected in 79% of the strains tested. Furthermore, the chosen set of primers was used to study alkB richness and diversity in different soils sampled in Carmópolis, Brazil and King George Island, Antarctica. The DNA extracted from the different soils was PCR amplified with each set of alkB-targeting primers, and clone libraries were constructed, sequenced and analyzed. A total of 255 alkB phylotypes were detected. Venn diagram analyses revealed that only low numbers of alkB phylotypes were shared among the different libraries derived from each primer pair. Therefore, the combination of three alkB-targeting primers enhanced the richness of alkB phylotypes detected in the different soils by 45% to 139%, when compared to the use of a single alkB-targeting primer. In addition, a dendrogram analysis and beta diversity comparison of the alkB composition showed that each of the sampling sites studied had a particular set of alkane-degrading bacteria. The use of a combination of alkB primers was an efficient strategy for enhancing the detection of the alkB gene in cultivable bacteria and for better characterizing the distribution of alkane-degrading bacteria in different soil environments. PMID:23825163

  5. Regioselective alkane hydroxylation with a mutant AlkB enzyme

    DOEpatents

    Koch, Daniel J.; Arnold, Frances H.

    2012-11-13

    AlkB from Pseudomonas putida was engineered using in-vivo directed evolution to hydroxylate small chain alkanes. Mutant AlkB-BMO1 hydroxylates propane and butane at the terminal carbon at a rate greater than the wild-type to form 1-propanol and 1-butanol, respectively. Mutant AlkB-BMO2 similarly hydroxylates propane and butane at the terminal carbon at a rate greater than the wild-type to form 1-propanol and 1-butanol, respectively. These biocatalysts are highly active for small chain alkane substrates and their regioselectivity is retained in whole-cell biotransformations.

  6. Occurrence of diverse alkane hydroxylase alkB genes in indigenous oil-degrading bacteria of Baltic Sea surface water.

    PubMed

    Viggor, Signe; Jõesaar, Merike; Vedler, Eve; Kiiker, Riinu; Pärnpuu, Liis; Heinaru, Ain

    2015-12-30

    Formation of specific oil degrading bacterial communities in diesel fuel, crude oil, heptane and hexadecane supplemented microcosms of the Baltic Sea surface water samples was revealed. The 475 sequences from constructed alkane hydroxylase alkB gene clone libraries were grouped into 30 OPFs. The two largest groups were most similar to Pedobacter sp. (245 from 475) and Limnobacter sp. (112 from 475) alkB gene sequences. From 56 alkane-degrading bacterial strains 41 belonged to the Pseudomonas spp. and 8 to the Rhodococcus spp. having redundant alkB genes. Together 68 alkB gene sequences were identified. These genes grouped into 20 OPFs, half of them being specific only to the isolated strains. Altogether 543 diverse alkB genes were characterized in the brackish Baltic Sea water; some of them representing novel lineages having very low sequence identities with corresponding genes of the reference strains. PMID:26541986

  7. Novel Alkane Hydroxylase Gene (alkB) Diversity in Sediments Associated with Hydrocarbon Seeps in the Timor Sea, Australia▿

    PubMed Central

    Wasmund, Kenneth; Burns, Kathryn A.; Kurtböke, D. Ipek; Bourne, David G.

    2009-01-01

    Hydrocarbon seeps provide inputs of petroleum hydrocarbons to widespread areas of the Timor Sea. Alkanes constitute the largest proportion of chemical components found in crude oils, and therefore genes involved in the biodegradation of these compounds may act as bioindicators for this ecosystem's response to seepage. To assess alkane biodegradation potential, the diversity and distribution of alkane hydroxylase (alkB) genes in sediments of the Timor Sea were studied. Deduced AlkB protein sequences derived from clone libraries identified sequences only distantly related to previously identified AlkB sequences, suggesting that the Timor Sea maybe a rich reservoir for novel alkane hydroxylase enzymes. Most sequences clustered with AlkB sequences previously identified from marine Gammaproteobacteria though protein sequence identities averaged only 73% (with a range of 60% to 94% sequence identities). AlkB sequence diversity was lower in deep water (>400 m) samples off the continental slope than in shallow water (<100 m) samples on the continental shelf but not significantly different in response to levels of alkanes. Real-time PCR assays targeting Timor Sea alkB genes were designed and used to quantify alkB gene targets. No correlation was found between gene copy numbers and levels of hydrocarbons measured in sediments using sensitive gas chromatography-mass spectrometry techniques, probably due to the very low levels of hydrocarbons found in most sediment samples. Interestingly, however, copy numbers of alkB genes increased substantially in sediments exposed directly to active seepage even though only low or undetectable concentrations of hydrocarbons were measured in these sediments in complementary geochemical analyses due to efficient biodegradation. PMID:19820158

  8. Abundance and diversity of n-alkane-degrading bacteria in a forest soil co-contaminated with hydrocarbons and metals: a molecular study on alkB homologous genes.

    PubMed

    Pérez-de-Mora, Alfredo; Engel, Marion; Schloter, Michael

    2011-11-01

    Unraveling functional genes related to biodegradation of organic compounds has profoundly improved our understanding of biological remediation processes, yet the ecology of such genes is only poorly understood. We used a culture-independent approach to assess the abundance and diversity of bacteria catalyzing the degradation of n-alkanes with a chain length between C(5) and C(16) at a forest site co-contaminated with mineral oil hydrocarbons and metals for nearly 60 years. The alkB gene coding for a rubredoxin-dependent alkane monooxygenase enzyme involved in the initial activation step of aerobic aliphatic hydrocarbon metabolism was used as biomarker. Within the area of study, four different zones were evaluated: one highly contaminated, two intermediately contaminated, and a noncontaminated zone. Contaminant concentrations, hydrocarbon profiles, and soil microbial respiration and biomass were studied. Abundance of n-alkane-degrading bacteria was quantified via real-time PCR of alkB, whereas genetic diversity was examined using molecular fingerprints (T-RFLP) and clone libraries. Along the contamination plume, hydrocarbon profiles and increased respiration rates suggested on-going natural attenuation at the site. Gene copy numbers of alkB were similar in contaminated and control areas. However, T-RFLP-based fingerprints suggested lower diversity and evenness of the n-alkane-degrading bacterial community in the highly contaminated zone compared to the other areas; both diversity and evenness were negatively correlated with metal and hydrocarbon concentrations. Phylogenetic analysis of alkB denoted a shift of the hydrocarbon-degrading bacterial community from Gram-positive bacteria in the control zone (most similar to Mycobacterium and Nocardia types) to Gram-negative genotypes in the contaminated zones (Acinetobacter and alkB sequences with little similarity to those of known bacteria). Our results underscore a qualitative rather than a quantitative response of

  9. Two distinct monooxygenases for alkane oxidation in Nocardioides sp. strain CF8.

    PubMed

    Hamamura, N; Yeager, C M; Arp, D J

    2001-11-01

    Alkane monooxygenases in Nocardioides sp. strain CF8 were examined at the physiological and genetic levels. Strain CF8 can utilize alkanes ranging in chain length from C(2) to C(16). Butane degradation by butane-grown cells was strongly inhibited by allylthiourea, a copper-selective chelator, while hexane-, octane-, and decane-grown cells showed detectable butane degradation activity in the presence of allylthiourea. Growth on butane and hexane was strongly inhibited by 1-hexyne, while 1-hexyne did not affect growth on octane or decane. A specific 30-kDa acetylene-binding polypeptide was observed for butane-, hexane-, octane-, and decane-grown cells but was absent from cells grown with octane or decane in the presence of 1-hexyne. These results suggest the presence of two monooxygenases in strain CF8. Degenerate primers designed for PCR amplification of genes related to the binuclear-iron-containing alkane hydroxylase from Pseudomonas oleovorans were used to clone a related gene from strain CF8. Reverse transcription-PCR and Northern blot analysis showed that this gene encoding a binuclear-iron-containing alkane hydroxylase was expressed in cells grown on alkanes above C(6). These results indicate the presence of two distinct monooxygenases for alkane oxidation in Nocardioides sp. strain CF8. PMID:11679317

  10. Effects of different compost amendments on the abundance and composition of alkB harboring bacterial communities in a soil under industrial use contaminated with hydrocarbons

    PubMed Central

    Wallisch, Stefanie; Gril, Tjasa; Dong, Xia; Welzl, Gerd; Bruns, Christian; Heath, Ester; Engel, Marion; Suhadolc, Marjetka; Schloter, Michael

    2014-01-01

    Alkane degrading microorganisms play an important role for the bioremediation of petrogenic contaminated environments. In this study, we investigated the effects of compost addition on the abundance and diversity of bacteria harboring the alkane monooxygenase gene (alkB) in an oil-contaminated soil originated from an industrial zone in Celje, Slovenia (Technosol). Soil without any amendments (control soil) and soil amended with two composts differing in their maturation stage and nutrient availability, were incubated under controlled conditions in a microcosm experiment and sampled after 0, 6, 12, and 36 weeks of incubation. As expected the addition of compost stimulated the degradation of alkanes in the investigated soil shortly after the addition. By using quantitative real-time PCR higher number of alkB genes were detected in soil samples amended with compost compared to the control soils. To get an insight into the composition of alkB harboring microbial communities, we performed next generation sequencing of amplicons of alkB gene fragment. Richness and diversity of alkB gene harboring prokaryotes was higher in soil mixed with compost compared to control soils with stronger effects of the less maturated, nutrient poor compost. The phylogenetic analysis of communities suggested that the addition of compost stimulated the abundance of alkB harboring Actinobacteria during the experiment independent from the maturation stage of the compost. AlkB harboring γ-proteobacteria like Shewanella or Hydrocarboniphaga as well as α-proteobacteria of the genus Agrobacterium responded also positively to the addition of compost to soil. The amendment of the less maturated, nutrient poor compost resulted in addition in a large increase of alkB harboring bacteria of the Cytophaga group (Microscilla) mainly at the early sampling time points. Our data indicates that compost amendments significantly change abundance and diversity pattern of alkB harboring microbes in Technosol and

  11. Involvement of an Alkane Hydroxylase System of Gordonia sp. Strain SoCg in Degradation of Solid n-Alkanes▿

    PubMed Central

    Lo Piccolo, Luca; De Pasquale, Claudio; Fodale, Roberta; Puglia, Anna Maria; Quatrini, Paola

    2011-01-01

    Enzymes involved in oxidation of long-chain n-alkanes are still not well known, especially those in Gram-positive bacteria. This work describes the alkane degradation system of the n-alkane degrader actinobacterium Gordonia sp. strain SoCg, which is able to grow on n-alkanes from dodecane (C12) to hexatriacontane (C36) as the sole C source. SoCg harbors in its chromosome a single alk locus carrying six open reading frames (ORFs), which shows 78 to 79% identity with the alkane hydroxylase (AH)-encoding systems of other alkane-degrading actinobacteria. Quantitative reverse transcription-PCR showed that the genes encoding AlkB (alkane 1-monooxygenase), RubA3 (rubredoxin), RubA4 (rubredoxin), and RubB (rubredoxin reductase) were induced by both n-hexadecane and n-triacontane, which were chosen as representative long-chain liquid and solid n-alkane molecules, respectively. Biotransformation of n-hexadecane into the corresponding 1-hexadecanol was detected by solid-phase microextraction coupled with gas chromatography-mass spectrometry (SPME/GC-MS) analysis. The Gordonia SoCg alkB was heterologously expressed in Escherichia coli BL21 and in Streptomyces coelicolor M145, and both hosts acquired the ability to transform n-hexadecane into 1-hexadecanol, but the corresponding long-chain alcohol was never detected on n-triacontane. However, the recombinant S. coelicolor M145-AH, expressing the Gordonia alkB gene, was able to grow on n-triacontane as the sole C source. A SoCg alkB disruption mutant that is completely unable to grow on n-triacontane was obtained, demonstrating the role of an AlkB-type AH system in degradation of solid n-alkanes. PMID:21183636

  12. Discrimination of the prochiral hydrogens at the C-2 position of n-alkanes by the methane/ammonia monooxygenase family proteins.

    PubMed

    Miyaji, Akimitsu; Miyoshi, Teppei; Motokura, Ken; Baba, Toshihide

    2015-08-14

    The selectivity of ammonia monooxygenase from Nitrosomonas europaea (AMO-Ne) for the oxidation of C4-C8n-alkanes to the corresponding alcohol isomers was examined to show the ability of AMO-Ne to recognize the n-alkane orientation within the catalytic site. AMO-Ne in whole cells produces 1- and 2-alcohols from C4-C8n-alkanes, and the regioselectivity is dependent on the length of the carbon chain. 2-Alcohols produced from C4-C7n-alkanes were predominantly either the R- or S-enantiomers, while 2-octanol produced from n-octane was racemic. These results indicate that AMO-Ne can discriminate between the prochiral hydrogens at the C-2 position, with the degree of discrimination varying according to the n-alkane. Compared to the particulate methane monooxygenase (pMMO) of Methylococcus capsulatus (Bath) and that of Methylosinus trichosporium OB3b, AMO-Ne showed a distinct ability to discriminate between the orientation of n-butane and n-pentane in the catalytic site. PMID:26138087

  13. Tritiated chiral alkanes as substrates for soluble methane monooxygenase from Methylococcus capsulatus (Bath): Probes for the mechanism of hydroxylation

    SciTech Connect

    Valentine, A.M.; Liu, K.E.; Komar-Panicucci, S.; Lippard, S.J.; Wilkinson, B.; Priestley, N.D.; Floss, H.G.; Williams, P.G.; Morimoto, Hiromi

    1997-02-26

    The tritiated chiral alkanes (S)-[1-{sup 2}H{sub 1}, 1-{sup 3}H]ethane, (R)-[1-{sup 2}H{sub 1},1-{sup 3}H]ethane, (S)-[1-{sup 2}H{sub 1},1-{sup 3}H]butane, (R)-[1-{sup 2}H{sub 1}, 1-{sup 3}H]butane, (S)-[2-{sup 3}H]butane, (R)-[2-{sup 3}H]butane, and racemic [2-{sup 3}H]butane were oxidized by soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath), and the absolute stereochemistry of the resulting product alcohols was determined in order to probe the mechanism of substrate hydroxylation. When the hydroxylations were performed with purified hydroxylase but only a partially purified cellular extract for the coupling and reductase proteins, different product distributions were observed. These apparently anomalous results could be explained by invoking exchange of hydrogen atoms at the {alpha} carbon of the product alcohols. The characteristics of this exchange reaction are discussed. Together with the mechanistic information available from a range of substrate probes, the results are best accounted for by a nonsynchronous concerted process involving attack on the C-H bond by one or more of several pathways discussed in the text. 65 refs., 5 figs., 3 tabs.

  14. Diverse Bacterial Groups Contribute to the Alkane Degradation Potential of Chronically Polluted Subantarctic Coastal Sediments.

    PubMed

    Guibert, Lilian M; Loviso, Claudia L; Borglin, Sharon; Jansson, Janet K; Dionisi, Hebe M; Lozada, Mariana

    2016-01-01

    We aimed to gain insight into the alkane degradation potential of microbial communities from chronically polluted sediments of a subantarctic coastal environment using a combination of metagenomic approaches. A total of 6178 sequences annotated as alkane-1-monooxygenases (EC 1.14.15.3) were retrieved from a shotgun metagenomic dataset that included two sites analyzed in triplicate. The majority of the sequences binned with AlkB described in Bacteroidetes (32 ± 13 %) or Proteobacteria (29 ± 7 %), although a large proportion remained unclassified at the phylum level. Operational taxonomic unit (OTU)-based analyses showed small differences in AlkB distribution among samples that could be correlated with alkane concentrations, as well as with site-specific variations in pH and salinity. A number of low-abundance OTUs, mostly affiliated with Actinobacterial sequences, were found to be only present in the most contaminated samples. On the other hand, the molecular screening of a large-insert metagenomic library of intertidal sediments from one of the sampling sites identified two genomic fragments containing novel alkB gene sequences, as well as various contiguous genes related to lipid metabolism. Both genomic fragments were affiliated with the phylum Planctomycetes, and one could be further assigned to the genus Rhodopirellula due to the presence of a partial sequence of the 23S ribosomal RNA (rRNA) gene. This work highlights the diversity of bacterial groups contributing to the alkane degradation potential and reveals patterns of functional diversity in relation with environmental stressors in a chronically polluted, high-latitude coastal environment. In addition, alkane biodegradation genes are described for the first time in members of Planctomycetes. PMID:26547568

  15. The soluble methane mono-oxygenase of Methylococcus capsulatus (Bath). Its ability to oxygenate n-alkanes, n-alkenes, ethers, and alicyclic, aromatic and heterocyclic compounds.

    PubMed Central

    Colby, J; Stirling, D I; Dalton, H

    1977-01-01

    1. Methane mono-oxygenase of Methylococcus capsulatus (Bath) catalyses the oxidation of various substituted methane derivatives including methanol. 2. It is a very non-specific oxygenase and, in some of its catalytic properties, apparently resembles the analogous enzyme from Methylomonas methanica but differs from those found in Methylosinus trichosporium and Methylomonas albus. 3. CO is oxidized to CO2. 4. C1-C8 n-alkanes are hydroxylated, yielding mixtures of the corresponding 1- and 2-alcohols; no 3- or 4-alcohols are formed. 5. Terminal alkenes yield the corresponding 1,2-epoxides. cis- or trans-but-2-ene are each oxidized to a mixture of 2,3-epoxybutane and but-2-en-1-ol with retention of the cis or trans configuration in both products; 2-butanone is also formed from cis-but-2-ene only. 6. Dimethyl ether is oxidized. Diethyl ether undergoes sub-terminal oxidation, yielding ethanol and ethanal in equimolar amounts. 7. Methane mono-oxygenase also hydroxylates cyclic alkanes and aromatic compounds. However, styrene yields only styrene epoxide and pyridine yields only pyridine N-oxide. 8. Of those compounds tested, only NADPH can replace NADH as electron donor. PMID:411486

  16. Growth of Rhodococcus sp. strain BCP1 on gaseous n-alkanes: new metabolic insights and transcriptional analysis of two soluble di-iron monooxygenase genes

    PubMed Central

    Cappelletti, Martina; Presentato, Alessandro; Milazzo, Giorgio; Turner, Raymond J.; Fedi, Stefano; Frascari, Dario; Zannoni, Davide

    2015-01-01

    Rhodococcus sp. strain BCP1 was initially isolated for its ability to grow on gaseous n-alkanes, which act as inducers for the co-metabolic degradation of low-chlorinated compounds. Here, both molecular and metabolic features of BCP1 cells grown on gaseous and short-chain n-alkanes (up to n-heptane) were examined in detail. We show that propane metabolism generated terminal and sub-terminal oxidation products such as 1- and 2-propanol, whereas 1-butanol was the only terminal oxidation product detected from n-butane metabolism. Two gene clusters, prmABCD and smoABCD—coding for Soluble Di-Iron Monooxgenases (SDIMOs) involved in gaseous n-alkanes oxidation—were detected in the BCP1 genome. By means of Reverse Transcriptase-quantitative PCR (RT-qPCR) analysis, a set of substrates inducing the expression of the sdimo genes in BCP1 were assessed as well as their transcriptional repression in the presence of sugars, organic acids, or during the cell growth on rich medium (Luria–Bertani broth). The transcriptional start sites of both the sdimo gene clusters were identified by means of primer extension experiments. Finally, proteomic studies revealed changes in the protein pattern induced by growth on gaseous- (n-butane) and/or liquid (n-hexane) short-chain n-alkanes as compared to growth on succinate. Among the differently expressed protein spots, two chaperonins and an isocytrate lyase were identified along with oxidoreductases involved in oxidation reactions downstream of the initial monooxygenase reaction step. PMID:26029173

  17. Growth of Rhodococcus sp. strain BCP1 on gaseous n-alkanes: new metabolic insights and transcriptional analysis of two soluble di-iron monooxygenase genes.

    PubMed

    Cappelletti, Martina; Presentato, Alessandro; Milazzo, Giorgio; Turner, Raymond J; Fedi, Stefano; Frascari, Dario; Zannoni, Davide

    2015-01-01

    Rhodococcus sp. strain BCP1 was initially isolated for its ability to grow on gaseous n-alkanes, which act as inducers for the co-metabolic degradation of low-chlorinated compounds. Here, both molecular and metabolic features of BCP1 cells grown on gaseous and short-chain n-alkanes (up to n-heptane) were examined in detail. We show that propane metabolism generated terminal and sub-terminal oxidation products such as 1- and 2-propanol, whereas 1-butanol was the only terminal oxidation product detected from n-butane metabolism. Two gene clusters, prmABCD and smoABCD-coding for Soluble Di-Iron Monooxgenases (SDIMOs) involved in gaseous n-alkanes oxidation-were detected in the BCP1 genome. By means of Reverse Transcriptase-quantitative PCR (RT-qPCR) analysis, a set of substrates inducing the expression of the sdimo genes in BCP1 were assessed as well as their transcriptional repression in the presence of sugars, organic acids, or during the cell growth on rich medium (Luria-Bertani broth). The transcriptional start sites of both the sdimo gene clusters were identified by means of primer extension experiments. Finally, proteomic studies revealed changes in the protein pattern induced by growth on gaseous- (n-butane) and/or liquid (n-hexane) short-chain n-alkanes as compared to growth on succinate. Among the differently expressed protein spots, two chaperonins and an isocytrate lyase were identified along with oxidoreductases involved in oxidation reactions downstream of the initial monooxygenase reaction step. PMID:26029173

  18. n-Alkane assimilation and tert-butyl alcohol (TBA) oxidation capacity in Mycobacterium austroafricanum strains.

    PubMed

    Lopes Ferreira, Nicolas; Mathis, Hugues; Labbé, Diane; Monot, Frédéric; Greer, Charles W; Fayolle-Guichard, Françoise

    2007-06-01

    Mycobacterium austroafricanum IFP 2012, which grows on methyl tert-butyl ether (MTBE) and on tert-butyl alcohol (TBA), the main intermediate of MTBE degradation, also grows on a broad range of n-alkanes (C2 to C16). A single alkB gene copy, encoding a non-heme alkane monooxygenase, was partially amplified from the genome of this bacterium. Its expression was induced after growth on n-propane, n-hexane, n-hexadecane and on TBA but not after growth on LB. The capacity of other fast-growing mycobacteria to grow on n-alkanes (C1 to C16) and to degrade TBA after growth on n-alkanes was compared to that of M. austroafricanum IFP 2012. We studied M. austroafricanum IFP 2012 and IFP 2015 able to grow on MTBE, M. austroafricanum IFP 2173 able to grow on isooctane, Mycobacterium sp. IFP 2009 able to grow on ethyl tert-butyl ether (ETBE), M. vaccae JOB5 (M. austroaafricanum ATCC 29678) able to degrade MTBE and TBA and M. smegmatis mc2 155 with no known degradation capacity towards fuel oxygenates. The M. austroafricanum strains grew on a broad range of n-alkanes and three were able to degrade TBA after growth on propane, hexane and hexadecane. An alkB gene was partially amplified from the genome of all mycobacteria and a sequence comparison demonstrated a close relationship among the M. austroafricanum strains. This is the first report suggesting the involvement of an alkane hydroxylase in TBA oxidation, a key step during MTBE metabolism. PMID:17347817

  19. Controlled oxidation of aliphatic CH bonds in metallo-monooxygenases: mechanistic insights derived from studies on deuterated and fluorinated hydrocarbons.

    PubMed

    Chen, Yao-Sheng; Luo, Wen-I; Yang, Chung-Ling; Tu, Yi-Jung; Chang, Chun-Wei; Chiang, Chih-Hsiang; Chang, Chi-Yao; Chan, Sunney I; Yu, Steve S-F

    2014-05-01

    The control over the regio- and/or stereo-selective aliphatic CH oxidation by metalloenzymes is of great interest to scientists. Typically, these enzymes invoke host-guest chemistry to sequester the substrates within the protein pockets, exploiting sizes, shapes and specific interactions such as hydrogen-bonding, electrostatic forces and/or van der Waals interactions to control the substrate specificity, regio-specificity and stereo-selectivity. Over the years, we have developed a series of deuterated and fluorinated variants of these hydrocarbon substrates as probes to gain insights into the controlled CH oxidations of hydrocarbons facilitated by these enzymes. In this review, we illustrate the application of these designed probes in the study of three monooxygenases: (i) the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath), which oxidizes straight-chain C1-C5 alkanes and alkenes to form their corresponding 2-alcohols and epoxides, respectively; (ii) the recombinant alkane hydroxylase (AlkB) from Pseudomonas putida GPo1, which oxidizes the primary CH bonds of C5-C12 linear alkanes; and (iii) the recombinant cytochrome P450 from Bacillus megaterium, which oxidizes C12-C20 fatty acids at the ω-1, ω-2 or ω-3 CH positions. PMID:24629413

  20. Clay minerals and metal oxides strongly influence the structure of alkane-degrading microbial communities during soil maturation.

    PubMed

    Steinbach, Annelie; Schulz, Stefanie; Giebler, Julia; Schulz, Stephan; Pronk, Geertje J; Kögel-Knabner, Ingrid; Harms, Hauke; Wick, Lukas Y; Schloter, Michael

    2015-07-01

    Clay minerals, charcoal and metal oxides are essential parts of the soil matrix and strongly influence the formation of biogeochemical interfaces in soil. We investigated the role of these parental materials for the development of functional microbial guilds using the example of alkane-degrading bacteria harbouring the alkane monooxygenase gene (alkB) in artificial mixtures composed of different minerals and charcoal, sterile manure and a microbial inoculum extracted from an agricultural soil. We followed changes in abundance and community structure of alkane-degrading microbial communities after 3 and 12 months of soil maturation and in response to a subsequent 2-week plant litter addition. During maturation we observed an overall increasing divergence in community composition. The impact of metal oxides on alkane-degrading community structure increased during soil maturation, whereas the charcoal impact decreased from 3 to 12 months. Among the clay minerals illite influenced the community structure of alkB-harbouring bacteria significantly, but not montmorillonite. The litter application induced strong community shifts in soils, maturated for 12 months, towards functional guilds typical for younger maturation stages pointing to a resilience of the alkane-degradation function potentially fostered by an extant 'seed bank'. PMID:25535940

  1. CYP63A2, a catalytically versatile fungal P450 monooxygenase capable of oxidizing higher-molecular-weight polycyclic aromatic hydrocarbons, alkylphenols, and alkanes

    EPA Science Inventory

    Cytochrome P450 monooxygenases (P450s) are known to oxidize hydrocarbons albeit with limited substrate specificity across classes of these compounds. Here we report a P450 monooxygenase (CYP63A2) from the model ligninolytic white rot fungus Phanerochaete chrysosporium that was fo...

  2. The Genome of the Moderate Halophile Amycolicicoccus subflavus DQS3-9A1T Reveals Four Alkane Hydroxylation Systems and Provides Some Clues on the Genetic Basis for Its Adaptation to a Petroleum Environment

    PubMed Central

    Nie, Yong; Fang, Hui; Li, Yan; Chi, Chang-Qiao; Tang, Yue-Qin; Wu, Xiao-Lei

    2013-01-01

    The moderate halophile Amycolicicoccus subflavus DQS3-9A1T is the type strain of a novel species in the recently described novel genus Amycolicicoccus, which was isolated from oil mud precipitated from oil produced water. The complete genome of A. subflavus DQS3-9A1T has been sequenced and is characteristic of harboring the genes for adaption to the harsh petroleum environment with salinity, high osmotic pressure, and poor nutrient levels. Firstly, it characteristically contains four types of alkane hydroxylases, including the integral-membrane non-heme iron monooxygenase (AlkB) and cytochrome P450 CYP153, a long-chain alkane monooxygenase (LadA) and propane monooxygenase. It also accommodates complete pathways for the response to osmotic pressure. Physiological tests proved that the strain could grow on n-alkanes ranging from C10 to C36 and propane as the sole carbon sources, with the differential induction of four kinds of alkane hydroxylase coding genes. In addition, the strain could grow in 1–12% NaCl with the putative genes responsible for osmotic stresses induced as expected. These results reveal the effective adaptation of the strain DQS3-9A1T to harsh oil environment and provide a genome platform to investigate the global regulation of different alkane metabolisms in bacteria that are crucially important for petroleum degradation. To our knowledge, this is the first report to describe the co-existence of such four types of alkane hydroxylases in a bacterial strain. PMID:23967144

  3. In situ detection of alkB2 gene involved in Alcanivorax borkumensis SK2(T) hydrocarbon biodegradation.

    PubMed

    Matturro, Bruna; Frascadore, Emanuela; Cappello, Simone; Genovese, Mariella; Rossetti, Simona

    2016-09-15

    This study aimed to develop a new assay based on the whole cell hybridization in order to monitor alkane hydroxylase genes (alkB system) of the marine bacterium Alcanivorax borkumensis SK2(T) commonly reported as the predominant microorganism responsible for the biodegradation of n-alkanes which are the major fraction of petroleum hydrocarbons. The assay based on the whole cell hybridization targeting alkB2 gene was successfully developed and calibrated on a pure culture of Alcanivorax borkumensis SK2(T) with a detection efficiency up to 80%. The approach was further successfully validated on hydrocarbon-contaminated seawater and provided cells abundance (6.74E+04alkB2-carryingcellsmL(-1)) higher of about one order of magnitude than those obtained by qPCR (4.96E+03alkB2genecopiesmL(-1)). This study highlights the validity of the assay for the detection at single cell level of key-functional genes involved in the biodegradation of n-alkanes. PMID:27315756

  4. RNAi silencing of a cytochrome P450 monooxygenase disrupts the ability of a filamentous fungus, Graphium sp. to grow on short-chain gaseous alkanes and ethers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Graphium sp. (ATCC 58400), a filamentous fungus, is one of the few eukaryotes that grows on short-chain alkanes and ethers. In this study, we investigated the genetic underpinnings that enable this fungus to catalyze the first step in the alkane and ether oxidation pathway. A gene, CYP52L1, was iden...

  5. GPo1 alkB gene expression for improvement of the degradation of diesel oil by a bacterial consortium

    PubMed Central

    Luo, Qun; He, Ying; Hou, Deng-Yong; Zhang, Jian-Guo; Shen, Xian-Rong

    2015-01-01

    To facilitate the biodegradation of diesel oil, an oil biodegradation bacterial consortium was constructed. The alkane hydroxylase (alkB) gene of Pseudomonas putida GPo1 was constructed in a pCom8 expression vector, and the pCom8-GPo1 alkB plasmid was transformed into Escherichia coli DH5α. The AlkB protein was expressed by diesel oil induction and detected through SDS-polyacrylamide gel electrophoresis. The culture of the recombinant (pCom8-GPo1 alkB/E. coli DH5α) with the oil biodegradation bacterial consortium increased the degradation ratio of diesel oil at 24 h from 31% to 50%, and the facilitation rates were increased as the proportion of pCom8-GPo1 alkB/E. coli DH5α to the consortium increased. The results suggested that the expression of the GPo1 gene in E. coli DH5α could enhance the function of diesel oil degradation by the bacterial consortium. PMID:26413044

  6. SmoXYB1C1Z of Mycobacterium sp. strain NBB4: a soluble methane monooxygenase (sMMO)-like enzyme, active on C2 to C4 alkanes and alkenes.

    PubMed

    Martin, Kiri E; Ozsvar, Jazmin; Coleman, Nicholas V

    2014-09-01

    Monooxygenase (MO) enzymes initiate the aerobic oxidation of alkanes and alkenes in bacteria. A cluster of MO genes (smoXYB1C1Z) of thus-far-unknown function was found previously in the genomes of two Mycobacterium strains (NBB3 and NBB4) which grow on hydrocarbons. The predicted Smo enzymes have only moderate amino acid identity (30 to 60%) to their closest homologs, the soluble methane and butane MOs (sMMO and sBMO), and the smo gene cluster has a different organization from those of sMMO and sBMO. The smoXYB1C1Z genes of NBB4 were cloned into pMycoFos to make pSmo, which was transformed into Mycobacterium smegmatis mc(2)-155. Cells of mc(2)-155(pSmo) metabolized C2 to C4 alkanes, alkenes, and chlorinated hydrocarbons. The activities of mc(2)-155(pSmo) cells were 0.94, 0.57, 0.12, and 0.04 nmol/min/mg of protein with ethene, ethane, propane, and butane as substrates, respectively. The mc(2)-155(pSmo) cells made epoxides from ethene, propene, and 1-butene, confirming that Smo was an oxygenase. Epoxides were not produced from larger alkenes (1-octene and styrene). Vinyl chloride and 1,2-dichloroethane were biodegraded by cells expressing Smo, with production of inorganic chloride. This study shows that Smo is a functional oxygenase which is active against small hydrocarbons. M. smegmatis mc(2)-155(pSmo) provides a new model for studying sMMO-like monooxygenases. PMID:25015887

  7. SmoXYB1C1Z of Mycobacterium sp. Strain NBB4: a Soluble Methane Monooxygenase (sMMO)-Like Enzyme, Active on C2 to C4 Alkanes and Alkenes

    PubMed Central

    Martin, Kiri E.; Ozsvar, Jazmin

    2014-01-01

    Monooxygenase (MO) enzymes initiate the aerobic oxidation of alkanes and alkenes in bacteria. A cluster of MO genes (smoXYB1C1Z) of thus-far-unknown function was found previously in the genomes of two Mycobacterium strains (NBB3 and NBB4) which grow on hydrocarbons. The predicted Smo enzymes have only moderate amino acid identity (30 to 60%) to their closest homologs, the soluble methane and butane MOs (sMMO and sBMO), and the smo gene cluster has a different organization from those of sMMO and sBMO. The smoXYB1C1Z genes of NBB4 were cloned into pMycoFos to make pSmo, which was transformed into Mycobacterium smegmatis mc2-155. Cells of mc2-155(pSmo) metabolized C2 to C4 alkanes, alkenes, and chlorinated hydrocarbons. The activities of mc2-155(pSmo) cells were 0.94, 0.57, 0.12, and 0.04 nmol/min/mg of protein with ethene, ethane, propane, and butane as substrates, respectively. The mc2-155(pSmo) cells made epoxides from ethene, propene, and 1-butene, confirming that Smo was an oxygenase. Epoxides were not produced from larger alkenes (1-octene and styrene). Vinyl chloride and 1,2-dichloroethane were biodegraded by cells expressing Smo, with production of inorganic chloride. This study shows that Smo is a functional oxygenase which is active against small hydrocarbons. M. smegmatis mc2-155(pSmo) provides a new model for studying sMMO-like monooxygenases. PMID:25015887

  8. Probing the hydrophobic pocket of the active site in the particulate methane monooxygenase (pMMO) from Methylococcus capsulatus (Bath) by variable stereoselective alkane hydroxylation and olefin epoxidation.

    PubMed

    Ng, Kok-Yaoh; Tu, Li-Chun; Wang, Yane-Shih; Chan, Sunney I; Yu, Steve S-F

    2008-05-01

    pMMO from M. capsulatus (Bath) oxidizes straight-chain C1-C5 alkanes and alkenes to form their corresponding 2-alcohols and epoxides. According to experiments performed with cryptically chiral ethane and D,L-[2-(2)H(1),3-(2)H(1)]butane, the reactions proceed through the concerted O-atom insertion mechanism. However, when propene and but-1-ene are used as epoxidation substrates, the enantiomeric excesses (ees) of the enzymatic products are only 18 and 37 %, respectively. This relatively poor stereoselectivity in the enzymatic epoxidation presumably reflects low stereochemical differentiation between the re and si faces in the hydrophobic pocket of the active site. Further insights into the reaction mechanism are now provided by studies on trans-but-2-ene, which reveal only the D,L-2,3-dimethyloxirane products, and on cis-but-2-ene, which yield only the meso product. These observations indicate that the enzymatic epoxidation indeed proceeds through electrophilic syn addition. To achieve better facial selectivity, we have also used 3,3,3-trifluoroprop-1-ene as the substrate. The products obtained are 90 % (2S)-oxirane. When 1,1,1-trifluoropropane is the substrate, the hydroxylation at the 2-carbon exhibits an inverse chiral selectivity relative to that seen with normal butane, if we consider the size of the CF(3) group in the fluorinated propane to be comparable to one of the ethyl groups in butane. These experiments are beginning to delineate the factors that influence the orientations of various substrates in the hydrophobic cavity of the active site in the enzyme. PMID:18383583

  9. Diverse alkane hydroxylase genes in microorganisms and environments

    PubMed Central

    Nie, Yong; Chi, Chang-Qiao; Fang, Hui; Liang, Jie-Liang; Lu, She-Lian; Lai, Guo-Li; Tang, Yue-Qin; Wu, Xiao-Lei

    2014-01-01

    AlkB and CYP153 are important alkane hydroxylases responsible for aerobic alkane degradation in bioremediation of oil-polluted environments and microbial enhanced oil recovery. Since their distribution in nature is not clear, we made the investigation among thus-far sequenced 3,979 microbial genomes and 137 metagenomes from terrestrial, freshwater, and marine environments. Hundreds of diverse alkB and CYP153 genes including many novel ones were found in bacterial genomes, whereas none were found in archaeal genomes. Moreover, these genes were detected with different distributional patterns in the terrestrial, freshwater, and marine metagenomes. Hints for horizontal gene transfer, gene duplication, and gene fusion were found, which together are likely responsible for diversifying the alkB and CYP153 genes adapt to the ubiquitous distribution of different alkanes in nature. In addition, different distributions of these genes between bacterial genomes and metagenomes suggested the potentially important roles of unknown or less common alkane degraders in nature. PMID:24829093

  10. Viral AlkB proteins repair RNA damage by oxidative demethylation

    PubMed Central

    van den Born, Erwin; Omelchenko, Marina V.; Bekkelund, Anders; Leihne, Vibeke; Koonin, Eugene V.; Dolja, Valerian V.; Falnes, Pål Ø.

    2008-01-01

    Bacterial and mammalian AlkB proteins are iron(II)- and 2-oxoglutarate-dependent dioxygenases that reverse methylation damage, such as 1-methyladenine and 3-methylcytosine, in RNA and DNA. An AlkB-domain is encoded by the genome of numerous single-stranded, plant-infecting RNA viruses, the majority of which belong to the Flexiviridae family. Our phylogenetic analysis of AlkB sequences suggests that a single plant virus might have acquired AlkB relatively recently, followed by horizontal dissemination among other viruses via recombination. Here, we describe the first functional characterization of AlkB proteins from three plant viruses. The viral AlkB proteins efficiently reactivated methylated bacteriophage genomes when expressed in Escherichia coli, and also displayed robust, iron(II)- and 2-oxoglutarate-dependent demethylase activity in vitro. Viral AlkB proteins preferred RNA over DNA substrates, and thus represent the first AlkBs with such substrate specificity. Our results suggest a role for viral AlkBs in maintaining the integrity of the viral RNA genome through repair of deleterious methylation damage, and support the notion that AlkB-mediated RNA repair is biologically relevant. PMID:18718927

  11. Defective processing of methylated single-stranded DNA by E. coli alkB mutants

    PubMed Central

    Dinglay, Suneet; Trewick, Sarah C.; Lindahl, Tomas; Sedgwick, Barbara

    2000-01-01

    Escherichia coli alkB mutants are very sensitive to DNA methylating agents. Despite these mutants being the subject of many studies, no DNA repair or other function has been assigned to the AlkB protein or to its human homolog. Here, we report that reactivation of methylmethanesulfonate (MMS)-treated single-stranded DNA phages, M13, f1, and G4, was decreased dramatically in alkB mutants. No such decrease occurred when using methylated λ phage or M13 duplex DNA. These data show that alkB mutants have a marked defect in processing methylation damage in single-stranded DNA. Recombinant AlkB protein bound more efficiently to single- than double-stranded DNA. The single-strand damage processed by AlkB was primarily cytotoxic and not mutagenic and was induced by SN2 methylating agents, MMS, DMS, and MeI but not by SN1 agent N-methyl-N-nitrosourea or by γ irradiation. Strains lacking other DNA repair activities, alkA tag, xth nfo, uvrA, mutS, and umuC, were not defective in reactivation of methylated M13 phage and did not enhance the defect of an alkB mutant. A recA mutation caused a small but additive defect. Thus, AlkB functions in a novel pathway independent of these activities. We propose that AlkB acts on alkylated single-stranded DNA in replication forks or at transcribed regions. Consistent with this theory, stationary phase alkB cells were less MMS sensitive than rapidly growing cells. PMID:10950872

  12. Rhein Inhibits AlkB Repair Enzymes and Sensitizes Cells to Methylated DNA Damage.

    PubMed

    Li, Qi; Huang, Yue; Liu, Xichun; Gan, Jianhua; Chen, Hao; Yang, Cai-Guang

    2016-05-20

    The AlkB repair enzymes, including Escherichia coli AlkB and two human homologues, ALKBH2 and ALKBH3, are iron(II)- and 2-oxoglutarate-dependent dioxygenases that efficiently repair N(1)-methyladenine and N(3)-methylcytosine methylated DNA damages. The development of small molecule inhibitors of these enzymes has seen less success. Here we have characterized a previously discovered natural product rhein and tested its ability to inhibit AlkB repair enzymes in vitro and to sensitize cells to methyl methane sulfonate that mainly produces N(1)-methyladenine and N(3)-methylcytosine lesions. Our investigation of the mechanism of rhein inhibition reveals that rhein binds to AlkB repair enzymes in vitro and promotes thermal stability in vivo In addition, we have determined a new structural complex of rhein bound to AlkB, which shows that rhein binds to a different part of the active site in AlkB than it binds to in fat mass and obesity-associated protein (FTO). With the support of these observations, we put forth the hypothesis that AlkB repair enzymes would be effective pharmacological targets for cancer treatment. PMID:27015802

  13. Draft Genome Sequence of Gordonia sihwensis Strain 9, a Branched Alkane-Degrading Bacterium

    PubMed Central

    Brown, Lisa M.; Gunasekera, Thusitha S.; Striebich, Richard C.

    2016-01-01

    Gordonia sihwensis strain 9 is a Gram-positive bacterium capable of efficient aerobic degradation of branched and normal alkanes. The draft genome of G. sihwensis S9 is 4.16 Mb in size, with 3,686 coding sequences and 68.1% G+C content. Alkane monooxygenase and P-450 cytochrome genes required for alkane degradation are predicted in G. sihwensis S9. PMID:27340079

  14. Molecular screening for alkane hydroxylase genes in Gram-negative and Gram-positive strains.

    PubMed

    Smits, T H; Röthlisberger, M; Witholt, B; van Beilen, J B

    1999-08-01

    We have developed highly degenerate oligonucleotides for polymerase chain reaction (PCR) amplification of genes related to the Pseudomonas oleovorans GPo1 and Acinetobacter sp. ADP1 alkane hydroxylases, based on a number of highly conserved sequence motifs. In all Gram-negative and in two out of three Gram-positive strains able to grow on medium- (C6-C11) or long-chain n-alkanes (C12-C16), PCR products of the expected size were obtained. The PCR fragments were cloned and sequenced and found to encode peptides with 43.2-93.8% sequence identity to the corresponding fragment of the P. oleovorans GPo1 alkane hydroxylase. Strains that were unable to grow on n-alkanes did not yield PCR products with homology to alkane hydroxylase genes. The alkane hydroxylase genes of Acinetobacter calcoaceticus EB104 and Pseudomonas putida P1 were cloned using the PCR products as probes. The two genes allow an alkane hydroxylase-negative mutant of Acinetobacter sp. ADP1 and an Escherichia coli recombinant containing all P. oleovorans alk genes except alkB, respectively, to grow on n-alkanes, showing that the cloned genes do indeed encode alkane hydroxylases. PMID:11207749

  15. Adaptive Response Enzyme AlkB Preferentially Repairs 1-Methylguanine and 3-Methylthymine Adducts in Double-Stranded DNA.

    PubMed

    Chen, Fangyi; Tang, Qi; Bian, Ke; Humulock, Zachary T; Yang, Xuedong; Jost, Marco; Drennan, Catherine L; Essigmann, John M; Li, Deyu

    2016-04-18

    The AlkB protein is a repair enzyme that uses an α-ketoglutarate/Fe(II)-dependent mechanism to repair alkyl DNA adducts. AlkB has been reported to repair highly susceptible substrates, such as 1-methyladenine and 3-methylcytosine, more efficiently in ss-DNA than in ds-DNA. Here, we tested the repair of weaker AlkB substrates 1-methylguanine and 3-methylthymine and found that AlkB prefers to repair them in ds-DNA. We also discovered that AlkB and its human homologues, ABH2 and ABH3, are able to repair the aforementioned adducts when the adduct is present in a mismatched base pair. These observations demonstrate the strong adaptability of AlkB toward repairing various adducts in different environments. PMID:26919079

  16. Biodegradation of variable-chain-length n-alkanes in Rhodococcus opacus R7 and the involvement of an alkane hydroxylase system in the metabolism

    PubMed Central

    2014-01-01

    Rhodococcus opacus R7 is a Gram-positive bacterium isolated from a polycyclic aromatic hydrocarbon contaminated soil for its versatile metabolism; indeed the strain is able to grow on naphthalene, o-xylene, and several long- and medium-chain n-alkanes. In this work we determined the degradation of n-alkanes in Rhodococcus opacus R7 in presence of n-dodecane (C12), n-hexadecane (C16), n-eicosane (C20), n-tetracosane (C24) and the metabolic pathway in presence of C12. The consumption rate of C12 was 88%, of C16 was 69%, of C20 was 51% and of C24 it was 78%. The decrement of the degradation rate seems to be correlated to the length of the aliphatic chain of these hydrocarbons. On the basis of the metabolic intermediates determined by the R7 growth on C12, our data indicated that R. opacus R7 metabolizes medium-chain n-alkanes by the primary alcohol formation. This represents a difference in comparison with other Rhodococcus strains, in which a mixture of the two alcohols was observed. By GC-MSD analysis we also identified the monocarboxylic acid, confirming the terminal oxidation. Moreover, the alkB gene cluster from R. opacus R7 was isolated and its involvement in the n-alkane degradation system was investigated by the cloning of this genomic region into a shuttle-vector E. coli-Rhodococcus to evaluate the alkane hydroxylase activity. Our results showed an increased biodegradation of C12 in the recombinant strain R. erythropolis AP (pTipQT1-alkR7) in comparison with the wild type strain R. erythropolis AP. These data supported the involvement of the alkB gene cluster in the n-alkane degradation in the R7 strain. PMID:25401074

  17. Evidence linking the Pseudomonas oleovorans alkane omega-hydroxylase, an integral membrane diiron enzyme, and the fatty acid desaturase family.

    PubMed

    Shanklin, John; Whittle, Edward

    2003-06-19

    Pseudomonas oleovorans alkane omega-hydroxylase (AlkB) is an integral membrane diiron enzyme that shares a requirement for iron and oxygen for activity in a manner similar to that of the non-heme integral membrane desaturases, epoxidases, acetylenases, conjugases, ketolases, decarbonylase and methyl oxidases. No overall sequence similarity is detected between AlkB and these desaturase-like enzymes by computer algorithms; however, they do contain a series of histidine residues in a similar relative positioning with respect to hydrophobic regions thought to be transmembrane domains. To test whether these conserved histidine residues are functionally equivalent to those of the desaturase-like enzymes we used scanning alanine mutagenesis to test if they are essential for activity of AlkB. These experiments show that alanine substitution of any of the eight conserved histidines results in complete inactivation, whereas replacement of three non-conserved histidines in close proximity to the conserved residues, results in only partial inactivation. These data provide the first experimental support for the hypotheses: (i) that the histidine motif in AlkB is equivalent to that in the desaturase-like enzymes and (ii) that the conserved histidine residues play a vital role such as coordinating the Fe ions comprising the diiron active site. PMID:12804773

  18. The AlkB Family of Fe(II)/α-Ketoglutarate-dependent Dioxygenases: Repairing Nucleic Acid Alkylation Damage and Beyond*

    PubMed Central

    Fedeles, Bogdan I.; Singh, Vipender; Delaney, James C.; Li, Deyu; Essigmann, John M.

    2015-01-01

    The AlkB family of Fe(II)- and α-ketoglutarate-dependent dioxygenases is a class of ubiquitous direct reversal DNA repair enzymes that remove alkyl adducts from nucleobases by oxidative dealkylation. The prototypical and homonymous family member is an Escherichia coli “adaptive response” protein that protects the bacterial genome against alkylation damage. AlkB has a wide variety of substrates, including monoalkyl and exocyclic bridged adducts. Nine mammalian AlkB homologs exist (ALKBH1–8, FTO), but only a subset functions as DNA/RNA repair enzymes. This minireview presents an overview of the AlkB proteins including recent data on homologs, structural features, substrate specificities, and experimental strategies for studying DNA repair by AlkB family proteins. PMID:26152727

  19. Differential repair of etheno-DNA adducts by bacterial and human AlkB proteins.

    PubMed

    Zdżalik, Daria; Domańska, Anna; Prorok, Paulina; Kosicki, Konrad; van den Born, Erwin; Falnes, Pål Ø; Rizzo, Carmelo J; Guengerich, F Peter; Tudek, Barbara

    2015-06-01

    AlkB proteins are evolutionary conserved Fe(II)/2-oxoglutarate-dependent dioxygenases, which remove alkyl and highly promutagenic etheno(ɛ)-DNA adducts, but their substrate specificity has not been fully determined. We developed a novel assay for the repair of ɛ-adducts by AlkB enzymes using oligodeoxynucleotides with a single lesion and specific DNA glycosylases and AP-endonuclease for identification of the repair products. We compared the repair of three ɛ-adducts, 1,N(6)-ethenoadenine (ɛA), 3,N(4)-ethenocytosine (ɛC) and 1,N(2)-ethenoguanine (1,N(2)-ɛG) by nine bacterial and two human AlkBs, representing four different structural groups defined on the basis of conserved amino acids in the nucleotide recognition lid, engaged in the enzyme binding to the substrate. Two bacterial AlkB proteins, MT-2B (from Mycobacterium tuberculosis) and SC-2B (Streptomyces coelicolor) did not repair these lesions in either double-stranded (ds) or single-stranded (ss) DNA. Three proteins, RE-2A (Rhizobium etli), SA-2B (Streptomyces avermitilis), and XC-2B (Xanthomonas campestris) efficiently removed all three lesions from the DNA substrates. Interestingly, XC-2B and RE-2A are the first AlkB proteins shown to be specialized for ɛ-adducts, since they do not repair methylated bases. Three other proteins, EcAlkB (Escherichia coli), SA-1A, and XC-1B removed ɛA and ɛC from ds and ssDNA but were inactive toward 1,N(2)-ɛG. SC-1A repaired only ɛA with the preference for dsDNA. The human enzyme ALKBH2 repaired all three ɛ-adducts in dsDNA, while only ɛA and ɛC in ssDNA and repair was less efficient in ssDNA. ALKBH3 repaired only ɛC in ssDNA. Altogether, we have shown for the first time that some AlkB proteins, namely ALKBH2, RE-2A, SA-2B and XC-2B can repair 1,N(2)-ɛG and that ALKBH3 removes only ɛC from ssDNA. Our results also suggest that the nucleotide recognition lid is not the sole determinant of the substrate specificity of AlkB proteins. PMID:25797601

  20. Diversity and abundance of n-alkane degrading bacteria in the near surface soils of a Chinese onshore oil and gas field

    NASA Astrophysics Data System (ADS)

    Xu, K.; Tang, Y.; Ren, C.; Zhao, K.; Sun, Y.

    2012-10-01

    Alkane degrading bacteria have long been used as an important biological indicator for oil and gas prospecting, but their ecological characteristics in hydrocarbon microseep habitats are still poorly understood. In this study, the diversity and abundance of n-alkane degrading bacterial community in the near surface soils of a Chinese onshore oil and gas field were investigated using molecular techniques. Terminal restriction fragment length polymorphism (T-RFLP) analyses in combination with cloning and sequencing of alkB genes revealed that trace amount of volatile hydrocarbons migrated from oil and gas reservoirs caused a shift of the n-alkane degrading bacterial community from Gram-positive bacteria (Mycobacterium and Rhodococcus) to Gram-negative genotypes (Alcanivorax and Acinetobacter). Real-time PCR results furthermore showed that the abundance of alkB genes increased substantially in the surface soils underlying oil and gas reservoirs even though only low or undetectable concentrations of hydrocarbons were measured in these soils due to efficient microbial degradation. Our findings broadened the knowledge on the ecological characteristics of alkane degrading community in hydrocarbon microseeps and may provide a new approach for microbial prospecting for oil and gas (MPOG).

  1. Regulation of the Alkane Hydroxylase CYP153 Gene in a Gram-Positive Alkane-Degrading Bacterium, Dietzia sp. Strain DQ12-45-1b

    PubMed Central

    Liang, Jie-Liang; JiangYang, Jing-Hong

    2015-01-01

    CYP153, one of the most common medium-chain n-alkane hydroxylases belonging to the cytochrome P450 superfamily, is widely expressed in n-alkane-degrading bacteria. CYP153 is also thought to cooperate with AlkB in degrading various n-alkanes. However, the mechanisms regulating the expression of the protein remain largely unknown. In this paper, we studied CYP153 gene transcription regulation by the potential AraC family regulator (CypR) located upstream of the CYP153 gene cluster in a broad-spectrum n-alkane-degrading Gram-positive bacterium, Dietzia sp. strain DQ12-45-1b. We first identified the transcriptional start site and the promoter of the CYP153 gene cluster. Sequence alignment of upstream regions of CYP153 gene clusters revealed high conservation in the −10 and −35 regions in Actinobacteria. Further analysis of the β-galactosidase activity in the CYP153 gene promoter-lacZ fusion cell indicated that the CYP153 gene promoter was induced by n-alkanes comprised of 8 to 14 carbon atoms, but not by derived decanol and decanic acid. Moreover, we constructed a cypR mutant strain and found that the CYP153 gene promoter activities and CYP153 gene transcriptional levels in the mutant strain were depressed compared with those in the wild-type strain in the presence of n-alkanes, suggesting that CypR served as an activator for the CYP153 gene promoter. By comparing CYP153 gene arrangements in Actinobacteria and Proteobacteria, we found that the AraC family regulator is ubiquitously located upstream of the CYP153 gene, suggesting its universal regulatory role in CYP153 gene transcription. We further hypothesize that the observed mode of CYP153 gene regulation is shared by many Actinobacteria. PMID:26567302

  2. Analysis of Pseudomonas putida alkane-degradation gene clusters and flanking insertion sequences: evolution and regulation of the alk genes.

    PubMed

    van Beilen, J B; Panke, S; Lucchini, S; Franchini, A G; Röthlisberger, M; Witholt, B

    2001-06-01

    The Pseudomonas putida GPo1 (commonly known as Pseudomonas oleovorans GPo1) alkBFGHJKL and alkST gene clusters, which encode proteins involved in the conversion of n-alkanes to fatty acids, are located end to end on the OCT plasmid, separated by 9.7 kb of DNA. This DNA segment encodes, amongst others, a methyl-accepting transducer protein (AlkN) that may be involved in chemotaxis to alkanes. In P. putida P1, the alkBFGHJKL and alkST gene clusters are flanked by almost identical copies of the insertion sequence ISPpu4, constituting a class 1 transposon. Other insertion sequences flank and interrupt the alk genes in both strains. Apart from the coding regions of the GPo1 and P1 alk genes (80-92% sequence identity), only the alkB and alkS promoter regions are conserved. Competition experiments suggest that highly conserved inverted repeats in the alkB and alkS promoter regions bind ALKS: PMID:11390693

  3. Diversity and abundance of n-alkane-degrading bacteria in the near-surface soils of a Chinese onshore oil and gas field

    NASA Astrophysics Data System (ADS)

    Xu, K.; Tang, Y.; Ren, C.; Zhao, K.; Sun, Y.

    2013-03-01

    Alkane-degrading bacteria have long been used as an important biological indicator for oil and gas prospecting, but their ecological characteristics in hydrocarbon microseep habitats are still poorly understood. In this study, the diversity and abundance of n-alkane-degrading bacterial community in the near-surface soils of a Chinese onshore oil and gas field were investigated using molecular techniques. Terminal restriction fragment length polymorphism (T-RFLP) analyses in combination with cloning and sequencing of alkB genes revealed that Gram-negative genotypes (Alcanivorax and Acinetobacter) dominated n-alkane-degrading bacterial communities in the near-surface soils of oil and gas reservoirs, while the dominant microbial communities were Gram-positive bacteria (Mycobacterium and Rhodococcus) in background soil. Real-time quantitative polymerase chain reaction (PCR) results furthermore showed that the abundance of alkB genes increased substantially in the surface soils above oil and gas reservoirs even though only low or undetectable concentrations of hydrocarbons were measured in these soils. The results of this study implicate that trace amounts of volatile hydrocarbons migrate from oil and gas reservoirs, and likely result in the changes of microbial communities in the near-surface soil.

  4. Structural and Mutational Analysis of Escherichia coli AlkB Provides Insight into Substrate Specificity and DNA Damage Searching

    SciTech Connect

    Holland, P.; Hollis, T

    2010-01-01

    In Escherichia coli, cytotoxic DNA methyl lesions on the N1 position of purines and N3 position of pyrimidines are primarily repaired by the 2-oxoglutarate (2-OG) iron(II) dependent dioxygenase, AlkB. AlkB repairs 1-methyladenine (1-meA) and 3-methylcytosine (3-meC) lesions, but it also repairs 1-methylguanine (1-meG) and 3-methylthymine (3-meT) at a much less efficient rate. How the AlkB enzyme is able to locate and identify methylated bases in ssDNA has remained an open question. We determined the crystal structures of the E. coli AlkB protein holoenzyme and the AlkB-ssDNA complex containing a 1-meG lesion. We coupled this to site-directed mutagenesis of amino acids in and around the active site, and tested the effects of these mutations on the ability of the protein to bind both damaged and undamaged DNA, as well as catalyze repair of a methylated substrate. A comparison of our substrate-bound AlkB-ssDNA complex with our unliganded holoenzyme reveals conformational changes of residues within the active site that are important for binding damaged bases. Site-directed mutagenesis of these residues reveals novel insight into their roles in DNA damage recognition and repair. Our data support a model that the AlkB protein utilizes at least two distinct conformations in searching and binding methylated bases within DNA: a 'searching' mode and 'repair' mode. Moreover, we are able to functionally separate these modes through mutagenesis of residues that affect one or the other binding state. Finally, our mutagenesis experiments show that amino acid D135 of AlkB participates in both substrate specificity and catalysis.

  5. Detection of genes for alkane and naphthalene catabolism in Rhodococcus sp. strain 1BN.

    PubMed

    Andreoni, V; Bernasconi, S; Colombo, M; van Beilen, J B; Cavalca, L

    2000-10-01

    Rhodococcus sp. 1BN was isolated from a contaminated site and showed various biodegradative capabilities. Besides naphthalene, strain 1BN degraded medium- (C6) and long-chain alkanes (C16-C28), benzene and toluene, alone or when the hydrocarbons were mixed in equal proportions. The nucleotide sequence of an alk polymerase chain reaction (PCR) fragment revealed a 59% nucleotide homology to the Pseudomonas oleovorans alkB gene. The nar fragments were highly homologous to genes coding for large and small subunits of cis-naphthalene 1,2-dioxygenase (narAa and narAb) and to cis-naphthalene dihydrodiol dehydrogenase (narB) from other rhodococci. The oxidation of indene to cis-(1S,2R)-1,2-dihydroxyindan by toluene-induced cells allows to hypothesize that strain 1BN also carries a toluene dioxygenase-like system. PMID:11233165

  6. Exocyclic carbons adjacent to the N6 of adenine are targets for oxidation by the Escherichia coli adaptive response protein AlkB.

    PubMed

    Li, Deyu; Delaney, James C; Page, Charlotte M; Yang, Xuedong; Chen, Alvin S; Wong, Cintyu; Drennan, Catherine L; Essigmann, John M

    2012-05-30

    The DNA and RNA repair protein AlkB removes alkyl groups from nucleic acids by a unique iron- and α-ketoglutarate-dependent oxidation strategy. When alkylated adenines are used as AlkB targets, earlier work suggests that the initial target of oxidation can be the alkyl carbon adjacent to N1. Such may be the case with ethano-adenine (EA), a DNA adduct formed by an important anticancer drug, BCNU, whereby an initial oxidation would occur at the carbon adjacent to N1. In a previous study, several intermediates were observed suggesting a pathway involving adduct restructuring to a form that would not hinder replication, which would match biological data showing that AlkB almost completely reverses EA toxicity in vivo. The present study uses more sensitive spectroscopic methodology to reveal the complete conversion of EA to adenine; the nature of observed additional putative intermediates indicates that AlkB conducts a second oxidation event in order to release the two-carbon unit completely. The second oxidation event occurs at the exocyclic carbon adjacent to the N(6) atom of adenine. The observation of oxidation of a carbon at N(6) in EA prompted us to evaluate N(6)-methyladenine (m6A), an important epigenetic signal for DNA replication and many other cellular processes, as an AlkB substrate in DNA. Here we show that m6A is indeed a substrate for AlkB and that it is converted to adenine via its 6-hydroxymethyl derivative. The observation that AlkB can demethylate m6A in vitro suggests a role for AlkB in regulation of important cellular functions in vivo. PMID:22512456

  7. Characterization of the Medium- and Long-Chain n-Alkanes Degrading Pseudomonas aeruginosa Strain SJTD-1 and Its Alkane Hydroxylase Genes

    PubMed Central

    Liu, Huan; Xu, Jing; Liang, Rubing; Liu, Jianhua

    2014-01-01

    A gram-negative aliphatic hydrocarbon-degrading bacterium SJTD-1 isolated from oil-contaminated soil was identified as Pseudomonas aeruginosa by comparative analyses of the 16S rRNA sequence, phenotype, and physiological features. SJTD-1 could efficiently mineralize medium- and long-chain n-alkanes (C12-C30) as its sole carbon source within seven days, showing the most optimal growth on n-hexadecane, followed by n-octadecane, and n-eicosane. In 36 h, 500 mg/L of tetradecane, hexadecane, and octadecane were transformed completely; and 2 g/L n-hexadecane was degraded to undetectable levels within 72 h. Two putative alkane-degrading genes (gene 3623 and gene 4712) were characterized and our results indicated that their gene products were rate-limiting enzymes involved in the synergetic catabolism of C12–C16 alkanes. On the basis of bioinformatics and transcriptional analysis, two P450 monooxygenases, along with a putative AlmA-like oxygenase, were examined. Genetically defective mutants lacking the characteristic alkane hydroxylase failed to degrade n-octadecane, thereby suggesting a different catalytic mechanism for the microbial transformation of alkanes with chain lengths over C18. PMID:25165808

  8. The role of AlkB protein in repair of 1,N⁶-ethenoadenine in Escherichia coli cells.

    PubMed

    Maciejewska, Agnieszka M; Sokołowska, Beata; Nowicki, Adam; Kuśmierek, Jarosław T

    2011-05-01

    Etheno (ε) DNA adducts, including 1,N(6)-ethenoadenine (εA), are formed by various bifunctional agents of exogenous and endogenous origin. The AT→TA transversion, the most frequent mutation provoked by the presence of εA in DNA, is very common in critical codons of the TP53 and RAS genes in tumours induced by exposure to carcinogenic vinyl compounds. Here, using a method that allows examination of the mutagenic potency of a metabolite of vinyl chloride, chloroacetaldehyde (CAA), but eliminates its cytotoxicity, we studied the participation of alkA, alkB and mug gene products in the repair of εA in Escherichia coli cells. The test system used comprised the pIF105 plasmid bearing the lactose operon of CC105 origin, which allowed monitoring of Lac(+) revertants that arose by AT→TA substitutions due to the modification of adenine by CAA. The plasmid was CAA-modified in vitro and replicated in E.coli of various genetic backgrounds (wt, alkA, alkB, mug, alkAalkB, alkAmug and alkBmug). To modify the levels of the AlkA and AlkB proteins, mutagenesis was studied in E.coli cells induced or not in adaptive response to alkylating agents. Considering the levels of CAA-induced Lac(+) revertants in strains harbouring the CAA-modified pIF105 plasmid and induced or not in adaptive response, we conclude that the AlkB dioxygenase plays a major role in decreasing the level of AT→TA mutations, thus in the repair of εA in E.coli cells. The observed differences of mutation frequencies in the various mutant strains assayed indicate that Mug glycosylase is also engaged in the repair of εA, whereas the role the AlkA glycosylase in this repair is negligible. PMID:21193516

  9. Enzymological and Structural Studies of the Mechanism of Promiscuous Substrate Recognition by the Oxidative DNA Repair Enzyme AlkB

    SciTech Connect

    Yu, B.; Hunt, J

    2009-01-01

    Promiscuous substrate recognition, the ability to catalyze transformations of chemically diverse compounds, is an evolutionarily advantageous, but poorly understood phenomenon. The promiscuity of DNA repair enzymes is particularly important, because it enables diverse kinds of damage to different nucleotide bases to be repaired in a metabolically parsimonious manner. We present enzymological and crystallographic studies of the mechanisms underlying promiscuous substrate recognition by Escherichia coli AlkB, a DNA repair enzyme that removes methyl adducts and some larger alkylation lesions from endocyclic positions on purine and pyrimidine bases. In vitro Michaelis-Menten analyses on a series of alkylated bases show high activity in repairing N1-methyladenine (m1A) and N3-methylcytosine (m3C), comparatively low activity in repairing 1,N6-ethenoadenine, and no detectable activity in repairing N1-methylguanine or N3-methylthymine. AlkB has a substantially higher kcat and Km for m3C compared with m1A. Therefore, the enzyme maintains similar net activity on the chemically distinct substrates by increasing the turnover rate of the substrate with nominally lower affinity. Cocrystal structures provide insight into the structural basis of this 'kcat/Km compensation,' which makes a significant contribution to promiscuous substrate recognition by AlkB. In analyzing a large ensemble of crystal structures solved in the course of these studies, we observed 2 discrete global conformations of AlkB differing in the accessibility of a tunnel hypothesized to control diffusion of the O2 substrate into the active site. Steric interactions between a series of protein loops control this conformational transition and present a plausible mechanism for preventing O2 binding before nucleotide substrate binding.

  10. Structural biology of heme monooxygenases

    SciTech Connect

    Poulos, Thomas L. . E-mail: poulos@uci.edu

    2005-12-09

    Over the past few years the number of crystal structures available for heme monooxygenases has substantially increased. Those most closely related to one another are cytochrome P450, nitric oxide synthase, and heme oxygenase. The present mini-review provides a summary of some recently published work on how crystallography and solution studies have provided new insights on function and especially the oxygen activation process. It now appears that in all three monooxygenases highly ordered solvent in the active site serves as direct proton donors to the iron-linked dioxygen; a requirement for splitting the O-O bond. This is in sharp contrast to the related peroxidase family of enzymes where strategically positioned amino acid side chains serve the function of shuttling protons. The P450cam-oxy-complex as well as various mutants in a complex with either oxygen or carbon monoxide have enabled a fairly detailed picture to be developed on the role of specific amino acids and conformational changes in both electron transfer and oxygen activation.

  11. Squalene monooxygenase - a target for hypercholesterolemic therapy.

    PubMed

    Belter, Agnieszka; Skupinska, Miroslawa; Giel-Pietraszuk, Malgorzata; Grabarkiewicz, Tomasz; Rychlewski, Leszek; Barciszewski, Jan

    2011-12-01

    Squalene monooxygenase catalyzes the epoxidation of C-C double bond of squalene to yield 2,3-oxidosqualene, the key step of sterol biosynthesis pathways in eukaryotes. Sterols are essential compounds of these organisms and squalene epoxidation is an important regulatory point in their synthesis. Squalene monooxygenase downregulation in vertebrates and fungi decreases synthesis of cholesterol and ergosterol, respectively, which makes squalene monooxygenase a potent and attractive target of hypercholesterolemia and antifungal therapies. Currently some fungal squalene monooxygenase inhibitors (terbinafine, naftifine, butenafine) are in clinical use, whereas mammalian enzymes' inhibitors are still under investigation. Research on new squalene monooxygenase inhibitors is important due to the prevalence of hypercholesterolemia and the lack of both sufficient and safe remedies. In this paper we (i) review data on activity and the structure of squalene monooxygenase, (ii) present its inhibitors, (iii) compare current strategies of lowering cholesterol level in blood with some of the most promising strategies, (iv) underline advantages of squalene monooxygenase as a target for hypercholesterolemia therapy, and (v) discuss safety concerns about hypercholesterolemia therapy based on inhibition of cellular cholesterol biosynthesis and potential usage of squalene monooxygenase inhibitors in clinical practice. After many years of use of statins there is some clinical evidence for their adverse effects and only partial effectiveness. Currently they are drugs of choice but are used with many restrictions, especially in case of children, elderly patients and women of childbearing potential. Certainly, for the next few years, statins will continue to be a suitable tool for cost-effective cardiovascular prevention; however research on new hypolipidemic drugs is highly desirable. We suggest that squalene monooxygenase inhibitors could become the hypocholesterolemic agents of the

  12. Evolving P450pyr Monooxygenase for Regio- and Stereoselective Hydroxylations.

    PubMed

    Yang, Yi; Li, Zhi

    2015-01-01

    P450pyr monooxygenase from Sphingomonas sp. HXN-200 catalysed the regio- and stereoselective hydroxylation at a non-activated carbon atom, a useful but challenging reaction in classic chemistry, with unique substrate specificity for a number of alicyclic compounds. New P450pyr mutants were developed by directed evolution with improved catalytic performance, thus significantly extending the application of the P450pyr monooxygenase family in biohydroxylation to prepare useful and valuable chiral alcohols. Directed evolution of P450pyr created new enzymes with improved S-enantioselectivity or R-enantioselectivity for the hydroxylation of N-benzyl pyrrolidine, enhanced regioselectivity for the hydroxylation of N-benzyl pyrrolidinone, and increased enantioselectivity for the hydroxylation of N-benzyl piperidinone, respectively. Directed evolution of P450pyr generated also mutants with fully altered regioselectivity (from terminal to subterminal) and newly created excellent S-enantioselectivity for the biohydroxylation of n-octane and propylbenzene, respectively, providing new opportunities for the regio- and enantioselective alkane functionalization. New P450pyr mutants were engineered as the first catalyst for highly selective terminal hydroxylation of n-butanol to 1,4-butanediol. Several novel, accurate, sensitive, simple, and HTS assays based on colorimetric or MS detection for measuring the enantio- and/or regioselectivity of hydroxylation were developed and proven to be practical in directed evolution. The P450pyr X-ray structure was obtained and used to guide the evolution. In silico modelling and substrate docking provided some insight into the influence of several important amino acid mutations of the engineered P450pyr mutants on the altered or enhanced regio- and enantioselectivity as well as new substrate acceptance. The obtained information and knowledge is useful for further engineering of P450pyr for other hydroxylations and oxidations. PMID:26507217

  13. Identification of a novel Baeyer‐Villiger monooxygenase from Acinetobacter radioresistens: close relationship to the Mycobacterium tuberculosis prodrug activator EtaA

    PubMed Central

    Minerdi, Daniela; Zgrablic, Ivan; Sadeghi, Sheila J.; Gilardi, Gianfranco

    2012-01-01

    Summary This work demonstrates that Acinetobacter radioresistens strain S13 during the growth on medium supplemented with long‐chain alkanes as the sole energy source expresses almA gene coding for a Baeyer‐Villiger monooxygenase (BVMO) involved in alkanes subterminal oxidation. Phylogenetic analysis placed the sequence of this novel BVMO in the same clade of the prodrug activator ethionamide monooxygenase (EtaA) and it bears only a distant relation to the other known class I BVMO proteins. In silico analysis of the 3D model of the S13 BVMO generated by homology modelling also supports the similarities with EtaA by binding ethionamide to the active site. In vitro experiments carried out with the purified enzyme confirm that this novel BVMO is indeed capable of typical Baeyer‐Villiger reactions as well as oxidation of the prodrug ethionamide. PMID:22862894

  14. Cellulose degradation by polysaccharide monooxygenases.

    PubMed

    Beeson, William T; Vu, Van V; Span, Elise A; Phillips, Christopher M; Marletta, Michael A

    2015-01-01

    Polysaccharide monooxygenases (PMOs), also known as lytic PMOs (LPMOs), enhance the depolymerization of recalcitrant polysaccharides by hydrolytic enzymes and are found in the majority of cellulolytic fungi and actinomycete bacteria. For more than a decade, PMOs were incorrectly annotated as family 61 glycoside hydrolases (GH61s) or family 33 carbohydrate-binding modules (CBM33s). PMOs have an unusual surface-exposed active site with a tightly bound Cu(II) ion that catalyzes the regioselective hydroxylation of crystalline cellulose, leading to glycosidic bond cleavage. The genomes of some cellulolytic fungi contain more than 20 genes encoding cellulose-active PMOs, suggesting a diversity of biological activities. PMOs show great promise in reducing the cost of conversion of lignocellulosic biomass to fermentable sugars; however, many questions remain about their reaction mechanism and biological function. This review addresses, in depth, the structural and mechanistic aspects of oxidative depolymerization of cellulose by PMOs and considers their biological function and phylogenetic diversity. PMID:25784051

  15. Protein Dynamics Control the Progression and Efficiency of the Catalytic Reaction Cycle of the Escherichia coli DNA-Repair Enzyme AlkB*

    PubMed Central

    Ergel, Burçe; Gill, Michelle L.; Brown, Lewis; Yu, Bomina; Palmer, Arthur G.; Hunt, John F.

    2014-01-01

    A central goal of enzymology is to understand the physicochemical mechanisms that enable proteins to catalyze complex chemical reactions with high efficiency. Recent methodological advances enable the contribution of protein dynamics to enzyme efficiency to be explored more deeply. Here, we utilize enzymological and biophysical studies, including NMR measurements of conformational dynamics, to develop a quantitative mechanistic scheme for the DNA repair enzyme AlkB. Like other iron/2-oxoglutarate-dependent dioxygenases, AlkB employs a two-step mechanism in which oxidation of 2-oxoglutarate generates a highly reactive enzyme-bound oxyferryl intermediate that, in the case of AlkB, slowly hydroxylates an alkylated nucleobase. Our results demonstrate that a microsecond-to-millisecond time scale conformational transition facilitates the proper sequential order of substrate binding to AlkB. Mutations altering the dynamics of this transition allow generation of the oxyferryl intermediate but promote its premature quenching by solvent, which uncouples 2-oxoglutarate turnover from nucleobase oxidation. Therefore, efficient catalysis by AlkB depends upon the dynamics of a specific conformational transition, establishing another paradigm for the control of enzyme function by protein dynamics. PMID:25043760

  16. Alternative Pathway for the Reaction Catalyzed by DNA Dealkylase AlkB from Ab Initio QM/MM Calculations

    PubMed Central

    2015-01-01

    AlkB is the title enzyme of a family of DNA dealkylases that catalyze the direct oxidative dealkylation of nucleobases. The conventional mechanism for the dealkylation of N1-methyl adenine (1-meA) catalyzed by AlkB after the formation of FeIV–oxo is comprised by a reorientation of the oxo moiety, hydrogen abstraction, OH rebound from the Fe atom to the methyl adduct, and the dissociation of the resulting methoxide to obtain the repaired adenine base and formaldehyde. An alternative pathway with hydroxide as a ligand bound to the iron atom is proposed and investigated by QM/MM simulations. The results show OH– has a small impact on the barriers for the hydrogen abstraction and OH rebound steps. The effects of the enzyme and the OH– ligand on the hydrogen abstraction by the FeIV–oxo moiety are discussed in detail. The new OH rebound step is coupled with a proton transfer to the OH– ligand and results in a novel zwitterion intermediate. This zwitterion structure can also be characterized as Fe–O–C complex and facilitates the formation of formaldehyde. In contrast, for the pathway with H2O bound to iron, the hydroxyl product of the OH rebound step first needs to unbind from the metal center before transferring a proton to Glu136 or other residue/substrate. The consistency between our theoretical results and experimental findings is discussed. This study provides new insights into the oxidative repair mechanism of DNA repair by nonheme FeII and α-ketoglutarate (α-KG) dependent dioxygenases and a possible explanation for the substrate preference of AlkB. PMID:25400523

  17. Chloroacetaldehyde-induced mutagenesis in Escherichia coli: the role of AlkB protein in repair of 3,N(4)-ethenocytosine and 3,N(4)-alpha-hydroxyethanocytosine.

    PubMed

    Maciejewska, Agnieszka M; Ruszel, Karol P; Nieminuszczy, Jadwiga; Lewicka, Joanna; Sokołowska, Beata; Grzesiuk, Elzbieta; Kuśmierek, Jarosław T

    2010-02-01

    Etheno (epsilon) adducts are formed in reaction of DNA bases with various environmental carcinogens and endogenously created products of lipid peroxidation. Chloroacetaldehyde (CAA), a metabolite of carcinogen vinyl chloride, is routinely used to generate epsilon-adducts. We studied the role of AlkB, along with AlkA and Mug proteins, all engaged in repair of epsilon-adducts, in CAA-induced mutagenesis. The test system used involved pIF102 and pIF104 plasmids bearing the lactose operon of CC102 or CC104 origin (Cupples and Miller (1989) [17]) which allowed to monitor Lac(+) revertants, the latter arose by GC-->AT or GC-->TA substitutions, respectively, as a result of modification of guanine and cytosine. The plasmids were CAA-damaged in vitro and replicated in Escherichia coli of various genetic backgrounds. To modify the levels of AlkA and AlkB proteins, mutagenesis was studied in E. coli cells induced or not in adaptive response. Formation of varepsilonC proceeds via a relatively stable intermediate, 3,N(4)-alpha-hydroxyethanocytosine (HEC), which allowed to compare repair of both adducts. The results indicate that all three genes, alkA, alkB and microg, are engaged in alleviation of CAA-induced mutagenesis. The frequency of mutation was higher in AlkA-, AlkB- and Mug-deficient strains in comparison to alkA(+), alkB(+), and microg(+) controls. Considering the levels of CAA-induced Lac(+) revertants in strains harboring the pIF plasmids and induced or not in adaptive response, we conclude that AlkB protein is engaged in the repair of epsilonC and HEC in vivo. Using the modified TTCTT 5-mers as substrates, we confirmed in vitro that AlkB protein repairs epsilonC and HEC although far less efficiently than the reference adduct 3-methylcytosine. The pH optimum for repair of HEC and epsilonC is significantly different from that for 3-methylcytosine. We propose that the protonated form of adduct interact in active site of AlkB protein. PMID:19941873

  18. An improved choline monooxygenase assay

    SciTech Connect

    Lafontaine, P.J.; Hanson, A.D. )

    1991-05-01

    Glycine betaine accumulates in leaves of plants from several angiosperm families in response to drought or salinization. Its synthesis, from the oxidation of choline, is mediated by a two step pathway. In spinach the first enzyme of this pathway is a ferredoxin-dependent choline monooxygenase (CMO). In order to purify this enzyme a sensitive and reliable assay is necessary. Two types of modifications were explored to improve the existing assay. (1) Ferredoxin reduction - one way of providing reduced Fd to CMO is by the addition of isolated spinach thylakoids in the assay mixture. In order to optimize the reduction of Fd two different systems were compared: (a) where only PS is active, by adding DCMU to inhibit electron transport from PS II and DAD as electron donor for PS I; (b) where both PS II and PS I are active. (2) Betaine aldehyde estimation - to simplify this, it is possible to couple the CMO reaction with betaine aldehyde dehydrogenase (BADH) from E. coli. BADH converts betaine aldehyde to betaine as it is formed in the assay, eliminating the need for a chemical oxidation step.

  19. Comparison of Bacillus monooxygenase genes for unique fatty acid production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This paper reviews Bacillus genes encoding monooxygenase enzymes producing unique fatty acid metabolites. Specifically, it examines standard monooxygenase electron transfer schemes and related domain structures of these fused domain enzymes on route to understanding the observed oxygenase activiti...

  20. Starch-degrading polysaccharide monooxygenases.

    PubMed

    Vu, Van V; Marletta, Michael A

    2016-07-01

    Polysaccharide degradation by hydrolytic enzymes glycoside hydrolases (GHs) is well known. More recently, polysaccharide monooxygenases (PMOs, also known as lytic PMOs or LPMOs) were found to oxidatively degrade various polysaccharides via a copper-dependent hydroxylation. PMOs were previously thought to be either GHs or carbohydrate binding modules (CBMs), and have been re-classified in carbohydrate active enzymes (CAZY) database as auxiliary activity (AA) families. These enzymes include cellulose-active fungal PMOs (AA9, formerly GH61), chitin- and cellulose-active bacterial PMOs (AA10, formerly CBM33), and chitin-active fungal PMOs (AA11). These PMOs significantly boost the activity of GHs under industrially relevant conditions, and thus have great potential in the biomass-based biofuel industry. PMOs that act on starch are the latest PMOs discovered (AA13), which has expanded our perspectives in PMOs studies and starch degradation. Starch-active PMOs have many common structural features and biochemical properties of the PMO superfamily, yet differ from other PMO families in several important aspects. These differences likely correlate, at least in part, to the differences in primary and higher order structures of starch and cellulose, and chitin. In this review we will discuss the discovery, structural features, biochemical and biophysical properties, and possible biological functions of starch-active PMOs, as well as their potential application in the biofuel, food, and other starch-based industries. Important questions regarding various aspects of starch-active PMOs and possible economical driving force for their future studies will also be highlighted. PMID:27170366

  1. Liquid-liquid interfaces of semifluorinated alkane diblock copolymers with water, alkanes, and perfluorinated alkanes.

    SciTech Connect

    Perahia, Dvora, Dr.; Pierce, Flint; Tsige, Mesfin; Grest, Gary Stephen, Dr.

    2008-08-01

    The liquid-liquid interface between semifluorinated alkane diblock copolymers of the form F3C(CF2)n-1-(CH2)m-1CH3 and water, protonated alkanes, and perfluorinated alkanes are studied by fully atomistic molecular dynamics simulations. A modified version of the OPLS-AA (Optimized Parameter for Liquid Simulation All-Atom) force field of Jorgensen et al. has been used to study the interfacial behavior of semifluorinated diblocks. Aqueous interfaces are found to be sharp, with correspondingly large values of the interfacial tension. Due to the reduced hydrophobicity of the protonated block compared to the fluorinated block, hydrogen enhancement is observed at the interface. Water dipoles in the interfacial region are found to be oriented nearly parallel to the liquid-liquid interface. A number of protonated alkanes and perfluorinated alkanes are found to be mutually miscible with the semifluorinated diblocks. For these liquids, interdiffusion follows the expected Fickian behavior, and concentration-dependent diffusivities are determined.

  2. Identification of Novel Genes Involved in Long-Chain n-Alkane Degradation by Acinetobacter sp. Strain DSM 17874▿

    PubMed Central

    Throne-Holst, Mimmi; Wentzel, Alexander; Ellingsen, Trond E.; Kotlar, Hans-Kristian; Zotchev, Sergey B.

    2007-01-01

    Acinetobacter sp. strain DSM 17874 is capable of utilizing n-alkanes with chain lengths ranging from that of decane (C10H22) to that of tetracontane (C40H82) as a sole carbon source. Two genes encoding AlkB-type alkane hydroxylase homologues, designated alkMa and alkMb, have been shown to be involved in the degradation of n-alkanes with chain lengths of from 10 to 20 C atoms in this strain. Here, we describe a novel high-throughput screening method and the screening of a transposon mutant library to identify genes involved in the degradation of n-alkanes with C chain lengths longer than 20, which are solid at 30°C, the optimal growth temperature for Acinetobacter sp. strain DSM 17874. A library consisting of approximately 6,800 Acinetobacter sp. strain DSM 17874 transposon mutants was constructed and screened for mutants unable to grow on dotriacontane (C32H66) while simultaneously showing wild-type growth characteristics on shorter-chain n-alkanes. For 23 such mutants isolated, the genes inactivated by transposon insertion were identified. Targeted inactivation and complementation studies of one of these genes, designated almA and encoding a putative flavin-binding monooxygenase, confirmed its involvement in the strain's metabolism of long-chain n-alkanes. To our knowledge, almA represents the first cloned gene shown to be involved in the bacterial degradation of long-chain n-alkanes of 32 C's and longer. Genes encoding AlmA homologues were also identified in other long-chain n-alkane-degrading Acinetobacter strains. PMID:17400787

  3. The Origin and Evolution of Baeyer—Villiger Monooxygenases (BVMOs): An Ancestral Family of Flavin Monooxygenases

    PubMed Central

    Mascotti, Maria Laura; Lapadula, Walter Jesús; Juri Ayub, Maximiliano

    2015-01-01

    The Baeyer—Villiger Monooxygenases (BVMOs) are enzymes belonging to the “Class B” of flavin monooxygenases and are capable of performing exquisite selective oxidations. These enzymes have been studied from a biotechnological perspective, but their physiological substrates and functional roles are widely unknown. Here, we investigated the origin, taxonomic distribution and evolutionary history of the BVMO genes. By using in silico approaches, 98 BVMO encoding genes were detected in the three domains of life: Archaea, Bacteria and Eukarya. We found evidence for the presence of these genes in Metazoa (Hydra vulgaris, Oikopleura dioica and Adineta vaga) and Haptophyta (Emiliania huxleyi) for the first time. Furthermore, a search for other “Class B” monooxygenases (flavoprotein monooxygenases –FMOs – and N-hydroxylating monooxygenases – NMOs) was conducted. These sequences were also found in the three domains of life. Phylogenetic analyses of all “Class B” monooxygenases revealed that NMOs and BVMOs are monophyletic, whereas FMOs form a paraphyletic group. Based on these results, we propose that BVMO genes were already present in the last universal common ancestor (LUCA) and their current taxonomic distribution is the result of differential duplication and loss of paralogous genes. PMID:26161776

  4. Steady-state kinetic analysis of soluble methane mono-oxygenase from Methylococcus capsulatus (Bath).

    PubMed Central

    Green, J; Dalton, H

    1986-01-01

    A steady-state kinetic analysis of purified soluble methane mono-oxygenase of Methylococcus capsulatus (Bath) was performed. The enzyme was found to follow a concerted-substitution mechanism. Methane binds to the enzyme followed by NADH, which reacts to yield reduced enzyme and NAD+. The reduced enzyme-methane complex binds O2 to give a second ternary complex, which breaks down to release water and methanol. In this way the enzyme can control the supply of electrons to the active site to coincide with the arrival of methane. Product-inhibition studies (with propylene as substrate) supported the reaction mechanism proposed. Ki values for NAD+ and propylene oxide are reported. The Km for NADH varied from 25 microM to 300 microM, depending on the nature of the hydrocarbon substrate, and thus supports the proposed reaction sequence. With methane as substrate the Km values for methane, NADH and O2 were shown to be 3 microM, 55.8 microM and 16.8 microM respectively. With propylene as substrate the Km values for propylene, NADH and O2 were 0.94 microM, 25.2 microM and 12.7-15.9 microM respectively. Methane mono-oxygenase was shown to be well adapted to the oxidation of methane compared with other straight-chain alkanes. PMID:3098230

  5. Isolation and characterization of different bacterial strains for bioremediation of n-alkanes and polycyclic aromatic hydrocarbons.

    PubMed

    Guermouche M'rassi, A; Bensalah, F; Gury, J; Duran, R

    2015-10-01

    Crude oil is a common environmental pollutant composed of a large number of both aromatic and aliphatic hydrocarbons. Biodegradation is carried out by microbial communities that are important in determining the fate of pollutants in the environment. The intrinsic biodegradability of the hydrocarbons and the distribution in the environment of competent degrading microorganisms are crucial information for the implementation of bioremediation processes. In the present study, the biodegradation capacities of various bacteria toward aliphatic and aromatic hydrocarbons were determined. The purpose of the study was to isolate and characterize hydrocarbon-degrading bacteria from contaminated soil of a refinery in Arzew, Algeria. A collection of 150 bacterial strains was obtained; the bacterial isolates were identified by 16S rRNA gene sequencing and their ability to degrade hydrocarbon compounds characterized. The isolated strains were mainly affiliated to the Gamma-Proteobacteria class. Among them, Pseudomonas spp. had the ability to metabolize high molecular weight hydrocarbon compounds such as pristane (C19) at 35.11 % by strain LGM22 and benzo[a] pyrene (C20) at 33.93 % by strain LGM11. Some strains were able to grow on all the hydrocarbons tested including octadecane, squalene, phenanthrene, and pyrene. Some strains were specialized degrading only few substrates. In contrast, the strain LGM2 designated as Pseudomonas sp. was found able to degrade both linear and branched alkanes as well as low and high poly-aromatic hydrocarbons (PAHs). The alkB gene involved in alkane degradation was detected in LGM2 and other Pseudomonas-related isolates. The capabilities of the isolated bacterial strains to degrade alkanes and PAHs should be of great practical significance in bioremediation of oil-contaminated environments. PMID:25813636

  6. Microbial Flavoprotein Monooxygenases as Mimics of Mammalian Flavin-Containing Monooxygenases for the Enantioselective Preparation of Drug Metabolites.

    PubMed

    Gul, Turan; Krzek, Marzena; Permentier, Hjalmar P; Fraaije, Marco W; Bischoff, Rainer

    2016-08-01

    Mammalian flavin-containing monooxygenases, which are difficult to obtain and study, play a major role in detoxifying various xenobiotics. To provide alternative biocatalytic tools to generate flavin-containing monooxygenases (FMO)-derived drug metabolites, a collection of microbial flavoprotein monooxygenases, sequence-related to human FMOs, was tested for their ability to oxidize a set of xenobiotic compounds. For all tested xenobiotics [nicotine, lidocaine, 3-(methylthio)aniline, albendazole, and fenbendazole], one or more monooxygenases were identified capable of converting the target compound. Chiral liquid chromatography with tandem mass spectrometry analyses of the conversions of 3-(methylthio)aniline, albendazole, and fenbendazole revealed that the respective sulfoxides are formed in good to excellent enantiomeric excess (e.e.) by several of the tested monooxygenases. Intriguingly, depending on the chosen microbial monooxygenase, either the (R)- or (S)-sulfoxide was formed. For example, when using a monooxygenase from Rhodococcus jostii the (S)-sulfoxide of albendazole (ricobendazole) was obtained with a 95% e.e. whereas a fungal monooxygenase yielded the respective (R)-sulfoxide in 57% e.e. For nicotine and lidocaine, monooxygenases could be identified that convert the amines into their respective N-oxides. This study shows that recombinantly expressed microbial monooxygenases represent a valuable toolbox of mammalian FMO mimics that can be exploited for the production of FMO-associated xenobiotic metabolites. PMID:26984198

  7. Catalytic conversion of light alkanes

    SciTech Connect

    Lyons, J.E.

    1992-06-30

    The second Quarterly Report of 1992 on the Catalytic Conversion of Light Alkanes reviews the work done between April 1, 1992 and June 31, 1992 on the Cooperative Agreement. The mission of this work is to devise a new catalyst which can be used in a simple economic process to convert the light alkanes in natural gas to oxygenate products that can either be used as clean-burning, high octane liquid fuels, as fuel components or as precursors to liquid hydrocarbon uwspomdon fuel. During the past quarter we have continued to design, prepare, characterize and test novel catalysts for the mild selective reaction of light hydrocarbons with air or oxygen to produce alcohols directly. These catalysts are designed to form active metal oxo (MO) species and to be uniquely active for the homolytic cleavage of the carbon-hydrogen bonds in light alkanes producing intermediates which can form alcohols. We continue to investigate three molecular environments for the active catalytic species that we are trying to generate: electron-deficient macrocycles (PHASE I), polyoxometallates (PHASE II), and regular oxidic lattices including zeolites and related structures as well as other molecular surface structures having metal oxo groups (PHASE I).

  8. [Advances in biomolecular machine: methane monooxygenases].

    PubMed

    Lu, Jixue; Wang, Shizhen; Fang, Baishan

    2015-07-01

    Methane monooxygenases (MMO), regarded as "an amazing biomolecular machine", catalyze the oxidation of methane to methanol under aerobic conditions. MMO catalyze the oxidation of methane elaborately, which is a novel way to catalyze methane to methanol. Furthermore, MMO can inspire the biomolecular machine design. In this review, we introduced MMO including structure, gene and catalytic mechanism. The history and the taxonomy of MMO were also introduced. PMID:26647577

  9. Toluene Monooxygenase-Catalyzed Epoxidation of Alkenes

    PubMed Central

    McClay, Kevin; Fox, Brian G.; Steffan, Robert J.

    2000-01-01

    Several toluene monooxygenase-producing organisms were tested for their ability to oxidize linear alkenes and chloroalkenes three to eight carbons long. Each of the wild-type organisms degraded all of the alkenes that were tested. Epoxides were produced during the oxidation of butene, butadiene, and pentene but not hexene or octadiene. A strain of Escherichia coli expressing the cloned toluene-4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 was able to oxidize butene, butadiene, pentene, and hexene but not octadiene, producing epoxides from all of the substrates that were oxidized. A T4MO-deficient variant of P. mendocina KR1 oxidized alkenes that were five to eight carbons long, but no epoxides were detected, suggesting the presence of multiple alkene-degrading enzymes in this organism. The alkene oxidation rates varied widely (ranging from 0.01 to 0.33 μmol of substrate/min/mg of cell protein) and were specific for each organism-substrate pair. The enantiomeric purity of the epoxide products also varied widely, ranging from 54 to >90% of a single epoxide enantiomer. In the absence of more preferred substrates, such as toluene or alkenes, the epoxides underwent further toluene monooxygenase-catalyzed transformations, forming products that were not identified. PMID:10788354

  10. Alkane biohydroxylation: Interests, constraints and future developments.

    PubMed

    Soussan, Laurence; Pen, Nakry; Belleville, Marie-Pierre; Marcano, José Sanchez; Paolucci-Jeanjean, Delphine

    2016-03-20

    Alkanes constitute one of the vastest reserves of raw materials for the production of fine chemicals. This paper focuses on recent advances in alkane biohydroxylation, i.e. the bioactivation of alkanes into their corresponding alcohols. Enzyme and whole-cell biocatalysts have been reviewed. Process considerations to implement such biocatalysts in bioreactors at large scale by coupling the bioconversion with cofactor regeneration and product removal are also discussed. PMID:26853477

  11. The Atomic Resolution Structure of Human AlkB Homolog 7 (ALKBH7), a Key Protein for Programmed Necrosis and Fat Metabolism*

    PubMed Central

    Wang, Guoqiang; He, Qingzhong; Feng, Chong; Liu, Yang; Deng, Zengqin; Qi, Xiaoxuan; Wu, Wei; Mei, Pinchao; Chen, Zhongzhou

    2014-01-01

    ALKBH7 is the mitochondrial AlkB family member that is required for alkylation- and oxidation-induced programmed necrosis. In contrast to the protective role of other AlkB family members after suffering alkylation-induced DNA damage, ALKBH7 triggers the collapse of mitochondrial membrane potential and promotes cell death. Moreover, genetic ablation of mouse Alkbh7 dramatically increases body weight and fat mass. Here, we present crystal structures of human ALKBH7 in complex with Mn(II) and α-ketoglutarate at 1.35 Å or N-oxalylglycine at 2.0 Å resolution. ALKBH7 possesses the conserved double-stranded β-helix fold that coordinates a catalytically active iron by a conserved HX(D/E) … Xn … H motif. Self-hydroxylation of Leu-110 was observed, indicating that ALKBH7 has the potential to catalyze hydroxylation of its substrate. Unlike other AlkB family members whose substrates are DNA or RNA, ALKBH7 is devoid of the “nucleotide recognition lid” which is essential for binding nucleobases, and thus exhibits a solvent-exposed active site; two loops between β-strands β6 and β7 and between β9 and β10 create a special outer wall of the minor β-sheet of the double-stranded β-helix and form a negatively charged groove. These distinct features suggest that ALKBH7 may act on protein substrate rather than nucleic acids. Taken together, our findings provide a structural basis for understanding the distinct function of ALKBH7 in the AlkB family and offer a foundation for drug design in treating cell death-related diseases and metabolic diseases. PMID:25122757

  12. Reaction pathway for alkane dehydrocyclization

    SciTech Connect

    Shi, Buchang; Davis, B.H.

    1996-08-01

    Naphtha reforming to produce high octane gasoline is an important process. Many reaction mechanisms are involved in this process. For example, the study of the fundamentals of this process led to the concept of bi- or poly-functional catalysis. The results of this study provide additional mechanistic information about the dehydrocyclization of an n-alkane to produce aromatics. The reaction coordinate diagram advanced to account for the observation of irreversible adsorption should be modified to account for the present results. 32 refs., 1 fig.

  13. Regulation of alkane degradation pathway by a TetR family repressor via an autoregulation positive feedback mechanism in a Gram-positive Dietzia bacterium.

    PubMed

    Liang, Jie-Liang; Nie, Yong; Wang, Miaoxiao; Xiong, Guangming; Wang, Yi-Ping; Maser, Edmund; Wu, Xiao-Lei

    2016-01-01

    n-Alkanes are ubiquitous in nature and serve as important carbon sources for both Gram-positive and Gram-negative bacteria. Hydroxylation of n-alkanes by alkane monooxygenases is the first and most critical step in n-alkane metabolism. However, regulation of alkane degradation genes in Gram-positive bacteria remains poorly characterized. We therefore explored the transcriptional regulation of an alkB-type alkane hydroxylase-rubredoxin fusion gene, alkW1, from Dietzia sp. DQ12-45-1b. The alkW1 promoter was characterized and so was the putative TetR family regulator, AlkX, located downstream of alkW1 gene. We further identified an unusually long 48 bp inverted repeat upstream of alkW1 and demonstrated the binding of AlkX to this operator. Analytical ultracentrifugation and microcalorimetric results indicated that AlkX formed stable dimers in solution and two dimers bound to one operator in a positive cooperative fashion characterized by a Hill coefficient of 1.64 (± 0.03) [k(D)  = 1.06 (± 0.16) μM, k(D) ' = 0.05 (± 0.01) μM]. However, the DNA-binding affinity was disrupted in the presence of long-chain fatty acids (C10-C24), suggesting that AlkX can sense the concentrations of n-alkane degradation metabolites. A model was therefore proposed where AlkX controls alkW1 expression in a metabolite-dependent manner. Bioinformatic analysis revealed that the alkane hydroxylase gene regulation mechanism may be common among Actinobacteria. PMID:26418273

  14. Alkane-Based Urethane Potting Compounds

    NASA Technical Reports Server (NTRS)

    Morris, D. E.

    1986-01-01

    New low viscosity urethanes easily mixed, molded, and outgassed. Alkane-based urethanes resist hydrolysis and oxidation and have excellent dielectric properties. Low-viscosity alkane-based urethane prepolymer prepared by one-step reaction of either isophorone diisocyanate or methyl-bis (4-cyclohexyl isocyanate) with hydrogenated, hydroxy-terminated polybutadiene (HTPBD).

  15. [Hydrocarbon-Oxidizing potential and the genes for n-alkane biodegradation in a new acidophilic mycobacterial association from sulfur blocks].

    PubMed

    Ivanova, I E; Sukhacheva, M V; Kanat'eva, A Yu; Kravchenko, I K; Kurganov, A A

    2014-01-01

    Capacity of AG(S10), a new aerobic acidophilic (growing within the pH range from 1.3 to 4.5 with the optimum at 2.0-2.5) bacterial association from sulfur blocks of the Astrakhan gas-processing complex (AGC), for oxidation of hydrocarbons of various chemical structure was investigated. A broad spectrum of normal (C10-C21) and iso-alkanes, toluene, naphthalene, andphenanthrene, as well as isoprenoids resistant to microbial degradation, pristane and phytane (components of paraffin oil), and 2,2,4,4,6,8,8,-heptamethylnonane, a branched hydrocarbon, were biodegraded under acidic conditions. Microbiological investigation revealed the dominance of mycobacteria in the AGS10 association, which was confirmed by analysis of the 16S rRNA gene clone library. In the phylogenetic tree, the 16S rRNA sequences formed a branch within the cluster of slow-growing mycobacteria, with 98% homology to the closest species Mycobacterium florentinum. Genomic DNA of AG(S10) culture grown on C14-C17 n-alkanes at pH 2.5 was found to contain the genes of two hydroxylase families, alkB and Cyp 153, indicating their combined involvement in hydrocarbon biodegradation. The high hydrocarbon-oxidizing potential of the AGS10 bacterial association, indicated that further search for the genes responsible for degradation of various hydrocarbons in acidophilic mycobacteria could be promising. PMID:25941716

  16. Monooxygenase activity of type 3 copper proteins.

    PubMed

    Itoh, Shinobu; Fukuzumi, Shunichi

    2007-07-01

    The molecular mechanism of the monooxygenase (phenolase) activity of type 3 copper proteins has been examined in detail both in the model systems and in the enzymatic systems. The reaction of a side-on peroxo dicopper(II) model compound ( A) and neutral phenols proceeds via a proton-coupled electron-transfer (PCET) mechanism to generate phenoxyl radical species, which collapse each other to give the corresponding C-C coupling dimer products. In this reaction, a bis(mu-oxo)dicopper(III) complex ( B) generated by O-O bond homolysis of A is suggested to be a real active species. On the other hand, the reaction of lithium phenolates (deprotonated form of phenols) with the same side-on peroxo dicopper(II) complex proceeds via an electrophilic aromatic substitution mechanism to give the oxygenated products (catechols). The mechanistic difference between these two systems has been discussed on the basis of the Marcus theory of electron transfer and Hammett analysis. Mechanistic details of the monooxygenase activity of tyrosinase have also been examined using a simplified enzymatic reaction system to demonstrate that the enzymatic reaction mechanism is virtually the same as that of the model reaction, that is, an electrophilic aromatic substitution mechanism. In addition, the monooxygenase activity of the oxygen carrier protein hemocyanin has been explored for the first time by employing urea as an additive in the reaction system. In this case as well, the ortho-hydroxylation of phenols to catechols has been demonstrated to involve the same ionic mechanism. PMID:17461541

  17. Structural basis of kynurenine 3-monooxygenase inhibition.

    PubMed

    Amaral, Marta; Levy, Colin; Heyes, Derren J; Lafite, Pierre; Outeiro, Tiago F; Giorgini, Flaviano; Leys, David; Scrutton, Nigel S

    2013-04-18

    Inhibition of kynurenine 3-monooxygenase (KMO), an enzyme in the eukaryotic tryptophan catabolic pathway (that is, kynurenine pathway), leads to amelioration of Huntington's-disease-relevant phenotypes in yeast, fruitfly and mouse models, as well as in a mouse model of Alzheimer's disease. KMO is a flavin adenine dinucleotide (FAD)-dependent monooxygenase and is located in the outer mitochondrial membrane where it converts l-kynurenine to 3-hydroxykynurenine. Perturbations in the levels of kynurenine pathway metabolites have been linked to the pathogenesis of a spectrum of brain disorders, as well as cancer and several peripheral inflammatory conditions. Despite the importance of KMO as a target for neurodegenerative disease, the molecular basis of KMO inhibition by available lead compounds has remained unknown. Here we report the first crystal structure of Saccharomyces cerevisiae KMO, in the free form and in complex with the tight-binding inhibitor UPF 648. UPF 648 binds close to the FAD cofactor and perturbs the local active-site structure, preventing productive binding of the substrate l-kynurenine. Functional assays and targeted mutagenesis reveal that the active-site architecture and UPF 648 binding are essentially identical in human KMO, validating the yeast KMO-UPF 648 structure as a template for structure-based drug design. This will inform the search for new KMO inhibitors that are able to cross the blood-brain barrier in targeted therapies against neurodegenerative diseases such as Huntington's, Alzheimer's and Parkinson's diseases. PMID:23575632

  18. Electron transfer control in soluble methane monooxygenase.

    PubMed

    Wang, Weixue; Iacob, Roxana E; Luoh, Rebecca P; Engen, John R; Lippard, Stephen J

    2014-07-01

    The hydroxylation or epoxidation of hydrocarbons by bacterial multicomponent monooxygenases (BMMs) requires the interplay of three or four protein components. How component protein interactions control catalysis, however, is not well understood. In particular, the binding sites of the reductase components on the surface of their cognate hydroxylases and the role(s) that the regulatory proteins play during intermolecular electron transfer leading to the hydroxylase reduction have been enigmatic. Here we determine the reductase binding site on the hydroxylase of a BMM enzyme, soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath). We present evidence that the ferredoxin domain of the reductase binds to the canyon region of the hydroxylase, previously determined to be the regulatory protein binding site as well. The latter thus inhibits reductase binding to the hydroxylase and, consequently, intermolecular electron transfer from the reductase to the hydroxylase diiron active site. The binding competition between the regulatory protein and the reductase may serve as a control mechanism for regulating electron transfer, and other BMM enzymes are likely to adopt the same mechanism. PMID:24937475

  19. Structural and Kinetic Studies of Novel Cytochrome P450 Small-Alkane Hydroxylases

    SciTech Connect

    Arnold, Frances H.

    2012-02-27

    The goals of this project are to investigate (1) the kinetics and stabilities of engineered cytochrome P450 (P450) small alkane hydroxylases and their evolutionary intermediates, (2) the structural basis for catalytic proficiency on small alkanes of these engineered P450s, and (3) the changes in redox control resulting from protein engineering. To reach these goals, we have established new methods for determining the kinetics and stabilities of multicomponent P450s such as CYP153A6. Using these, we were able to determine that CYP153A6 is proficient for hydroxylation of alkanes as small as ethane, an activity that has never been observed previously in any natural P450. To elucidate the structures of the engineered P450s, we obtained x-ray diffraction data for two variants in the P450PMO (propane monooxygenase) lineage and a preliminary structure for the most evolved variant. This structure shows changes in the substrate binding regions of the enzyme and a reduction in active site volume that are consistent with the observed changes in substrate specificity from fatty acids in the native enzyme to small alkanes in P450PMO. We also constructed semi-rational designed libraries mutating only residues in the enzyme active site that in one round of mutagenesis and screening produced variants that achieved nearly half of the activity of the most evolved enzymes of the P450PMO lineage. Finally, we found that changes in redox properties of the laboratory-evolved P450 alkane hydroxylases did not reflect the improvement in their electron transfer efficiency. The heme redox potential remained constant throughout evolution, while activity increased and coupling efficiency improved from 10% to 90%. The lack of correlation between heme redox potential and enzyme activity and coupling efficiency led us to search for other enzyme properties that could be better predictors for activity towards small alkanes, specifically methane. We investigated the oxidation potential of the radical

  20. Dynamic changes in the structure of microbial communities in Baltic Sea coastal seawater microcosms modified by crude oil, shale oil or diesel fuel.

    PubMed

    Viggor, Signe; Juhanson, Jaanis; Jõesaar, Merike; Mitt, Mario; Truu, Jaak; Vedler, Eve; Heinaru, Ain

    2013-08-25

    The coastal waters of the Baltic Sea are constantly threatened by oil spills, due to the extensive transportation of oil products across the sea. To characterise the hydrocarbon-degrading bacterial community of this marine area, microcosm experiments on diesel fuel, crude oil and shale oil were performed. Analysis of these microcosms, using alkane monooxygenase (alkB) and 16S rRNA marker genes in PCR-DGGE experiments, demonstrated that substrate type and concentration strongly influence species composition and the occurrence of alkB genes in respective oil degrading bacterial communities. Gammaproteobacteria (particularly the genus Pseudomonas) and Alphaproteobacteria were dominant in all microcosms treated with oils. All alkB genes carried by bacterial isolates (40 strains), and 8 of the 11 major DGGE bands from the microcosms, had more than 95% sequence identity with the alkB genes of Pseudomonas fluorescens. However, the closest relatives of the majority of sequences (54 sequences from 79) of the alkB gene library from initially collected seawater DNA were Actinobacteria. alkB gene expression, induced by hexadecane, was recorded in isolated bacterial strains. Thus, complementary culture dependent and independent methods provided a more accurate picture about the complex seawater microbial communities of the Baltic Sea. PMID:23510642

  1. Supported organoiridium catalysts for alkane dehydrogenation

    DOEpatents

    Baker, R. Thomas; Sattelberger, Alfred P.; Li, Hongbo

    2013-09-03

    Solid supported organoiridium catalysts, a process for preparing such solid supported organoiridium catalysts, and the use of such solid supported organoiridium catalysts in dehydrogenation reactions of alkanes is provided. The catalysts can be easily recovered and recycled.

  2. Solar photothermochemical alkane reverse combustion

    PubMed Central

    Chanmanee, Wilaiwan; Islam, Mohammad Fakrul; Dennis, Brian H.; MacDonnell, Frederick M.

    2016-01-01

    A one-step, gas-phase photothermocatalytic process for the synthesis of hydrocarbons, including liquid alkanes, aromatics, and oxygenates, with carbon numbers (Cn) up to C13, from CO2 and water is demonstrated in a flow photoreactor operating at elevated temperatures (180–200 °C) and pressures (1–6 bar) using a 5% cobalt on TiO2 catalyst and under UV irradiation. A parametric study of temperature, pressure, and partial pressure ratio revealed that temperatures in excess of 160 °C are needed to obtain the higher Cn products in quantity and that the product distribution shifts toward higher Cn products with increasing pressure. In the best run so far, over 13% by mass of the products were C5+ hydrocarbons and some of these, i.e., octane, are drop-in replacements for existing liquid hydrocarbons fuels. Dioxygen was detected in yields ranging between 64% and 150%. In principle, this tandem photochemical–thermochemical process, fitted with a photocatalyst better matched to the solar spectrum, could provide a cheap and direct method to produce liquid hydrocarbons from CO2 and water via a solar process which uses concentrated sunlight for both photochemical excitation to generate high-energy intermediates and heat to drive important thermochemical carbon-chain-forming reactions. PMID:26903631

  3. Solar photothermochemical alkane reverse combustion.

    PubMed

    Chanmanee, Wilaiwan; Islam, Mohammad Fakrul; Dennis, Brian H; MacDonnell, Frederick M

    2016-03-01

    A one-step, gas-phase photothermocatalytic process for the synthesis of hydrocarbons, including liquid alkanes, aromatics, and oxygenates, with carbon numbers (Cn) up to C13, from CO2 and water is demonstrated in a flow photoreactor operating at elevated temperatures (180-200 °C) and pressures (1-6 bar) using a 5% cobalt on TiO2 catalyst and under UV irradiation. A parametric study of temperature, pressure, and partial pressure ratio revealed that temperatures in excess of 160 °C are needed to obtain the higher Cn products in quantity and that the product distribution shifts toward higher Cn products with increasing pressure. In the best run so far, over 13% by mass of the products were C5+ hydrocarbons and some of these, i.e., octane, are drop-in replacements for existing liquid hydrocarbons fuels. Dioxygen was detected in yields ranging between 64% and 150%. In principle, this tandem photochemical-thermochemical process, fitted with a photocatalyst better matched to the solar spectrum, could provide a cheap and direct method to produce liquid hydrocarbons from CO2 and water via a solar process which uses concentrated sunlight for both photochemical excitation to generate high-energy intermediates and heat to drive important thermochemical carbon-chain-forming reactions. PMID:26903631

  4. Metathesis of alkanes and related reactions.

    PubMed

    Basset, Jean-Marie; Copéret, Christophe; Soulivong, Daravong; Taoufik, Mostafa; Cazat, Jean Thivolle

    2010-02-16

    The transformation of alkanes remains a difficult challenge because of the relative inertness of the C-H and C-C bonds. The rewards for asserting synthetic control over unfunctionalized, saturated hydrocarbons are considerable, however, because converting short alkanes into longer chain analogues is usually a value-adding process. Alkane metathesis is a novel catalytic and direct transformation of two molecules of a given alkane into its lower and higher homologues; moreover, the process proceeds at relatively low temperature (ambient conditions or higher). It was discovered through the use of a silica-supported tantalum hydride, ([triple bond]SiO)(2)TaH, a multifunctional catalyst with a single site of action. This reaction completes the story of the metathesis reactions discovered over the past 40 years: olefin metathesis, alkyne metathesis, and ene-yne cyclizations. In this Account, we examine the fundamental mechanistic aspects of alkane metathesis as well as the novel reactions that have been derived from its study. The silica-supported tantalum hydride catalyst was developed as the result of systematic and meticulous studies of the interaction between oxide supports and organometallic complexes, a field of study denoted surface organometallic chemistry (SOMC). A careful examination of this surface-supported tantalum hydride led to the later discovery of alumina-supported tungsten hydride, W(H)(3)/Al(2)O(3), which proved to be an even better catalyst for alkane metathesis. Supported tantalum and tungsten hydrides are highly unsaturated, electron-deficient species that are very reactive toward the C-H and C-C bonds of alkanes. They show a great versatility in various other reactions, such as cross-metathesis between methane and alkanes, cross-metathesis between toluene and ethane, or even methane nonoxidative coupling. Moreover, tungsten hydride exhibits a specific ability in the transformation of isobutane into 2,3-dimethylbutane as well as in the metathesis

  5. Enzymes involved in the anaerobic oxidation of n-alkanes: from methane to long-chain paraffins

    PubMed Central

    Callaghan, Amy V.

    2013-01-01

    Anaerobic microorganisms play key roles in the biogeochemical cycling of methane and non-methane alkanes. To date, there appear to be at least three proposed mechanisms of anaerobic methane oxidation (AOM). The first pathway is mediated by consortia of archaeal anaerobic methane oxidizers and sulfate-reducing bacteria (SRB) via “reverse methanogenesis” and is catalyzed by a homolog of methyl-coenzyme M reductase. The second pathway is also mediated by anaerobic methane oxidizers and SRB, wherein the archaeal members catalyze both methane oxidation and sulfate reduction and zero-valent sulfur is a key intermediate. The third AOM mechanism is a nitrite-dependent, “intra-aerobic” pathway described for the denitrifying bacterium, ‘Candidatus Methylomirabilis oxyfera.’ It is hypothesized that AOM proceeds via reduction of nitrite to nitric oxide, followed by the conversion of two nitric oxide molecules to dinitrogen and molecular oxygen. The latter can be used to functionalize the methane via a particulate methane monooxygenase. With respect to non-methane alkanes, there also appear to be novel mechanisms of activation. The most well-described pathway is the addition of non-methane alkanes across the double bond of fumarate to form alkyl-substituted succinates via the putative glycyl radical enzyme, alkylsuccinate synthase (also known as methylalkylsuccinate synthase). Other proposed mechanisms include anaerobic hydroxylation via ethylbenzene dehydrogenase-like enzymes and an “intra-aerobic” denitrification pathway similar to that described for ‘Methylomirabilis oxyfera.’ PMID:23717304

  6. Structural insights into diversity and n-alkane biodegradation mechanisms of alkane hydroxylases

    PubMed Central

    Ji, Yurui; Mao, Guannan; Wang, Yingying; Bartlam, Mark

    2013-01-01

    Environmental microbes utilize four degradation pathways for the oxidation of n-alkanes. Although the enzymes degrading n-alkanes in different microbes may vary, enzymes functioning in the first step in the aerobic degradation of alkanes all belong to the alkane hydroxylases. Alkane hydroxylases are a class of enzymes that insert oxygen atoms derived from molecular oxygen into different sites of the alkane terminus (or termini) depending on the type of enzymes. In this review, we summarize the different types of alkane hydroxylases, their degrading steps, and compare typical enzymes from various classes with regard to their three-dimensional structures, in order to provide insights into how the enzymes mediate their different roles in the degradation of n-alkanes and what determines their different substrate ranges. Through the above analyzes, the degrading mechanisms of enzymes can be elucidated and molecular biological methods can be utilized to expand their catalytic roles in the petrochemical industry or in bioremediation of oil-contaminated environments. PMID:23519435

  7. Cyanobacterial aldehyde deformylase oxygenation of aldehydes yields n-1 aldehydes and alcohols in addition to alkanes

    PubMed Central

    Aukema, Kelly G.; Makris, Thomas M.; Stoian, Sebastian A.; Richman, Jack E.; Münck, Eckard; Lipscomb, John D.; Wackett, Lawrence P.

    2013-01-01

    Aldehyde-deformylating oxygenase (ADO) catalyzes O2-dependent release of the terminal carbon of a biological substrate, octadecanal, to yield formate and heptadecane in a reaction that requires external reducing equivalents. We show here that ADO also catalyzes incorporation of an oxygen atom from O2 into the alkane product to yield alcohol and aldehyde products. Oxygenation of the alkane product is much more pronounced with C9-10 aldehyde substrates, so that use of nonanal as the substrate yields similar amounts of octane, octanal, and octanol products. When using doubly-labeled [1,2-13C]-octanal as the substrate, the heptane, heptanal and heptanol products each contained a single 13C-label in the C-1 carbons atoms. The only one-carbon product identified was formate. [18O]-O2 incorporation studies demonstrated formation of [18O]-alcohol product, but rapid solvent exchange prevented similar determination for the aldehyde product. Addition of [1-13C]-nonanol with decanal as the substrate at the outset of the reaction resulted in formation of [1-13C]-nonanal. No 13C-product was formed in the absence of decanal. ADO contains an oxygen-bridged dinuclear iron cluster. The observation of alcohol and aldehyde products derived from the initially formed alkane product suggests a reactive species similar to that formed by methane monooxygenase (MMO) and other members of the bacterial multicomponent monooxygenase family. Accordingly, characterization by EPR and Mössbauer spectroscopies shows that the electronic structure of the ADO cluster is similar, but not identical, to that of MMO hydroxylase component. In particular, the two irons of ADO reside in nearly identical environments in both the oxidized and fully reduced states, whereas those of MMOH show distinct differences. These favorable characteristics of the iron sites allow a comprehensive determination of the spin Hamiltonian parameters describing the electronic state of the diferrous cluster for the first time for

  8. DNA Damage: Quantum Mechanics/Molecular Mechanics Study on the Oxygen Binding and Substrate Hydroxylation Step in AlkB Repair Enzymes

    PubMed Central

    Quesne, Matthew G; Latifi, Reza; Gonzalez-Ovalle, Luis E; Kumar, Devesh; de Visser, Sam P

    2014-01-01

    AlkB repair enzymes are important nonheme iron enzymes that catalyse the demethylation of alkylated DNA bases in humans, which is a vital reaction in the body that heals externally damaged DNA bases. Its mechanism is currently controversial and in order to resolve the catalytic mechanism of these enzymes, a quantum mechanics/molecular mechanics (QM/MM) study was performed on the demethylation of the N1-methyladenine fragment by AlkB repair enzymes. Firstly, the initial modelling identified the oxygen binding site of the enzyme. Secondly, the oxygen activation mechanism was investigated and a novel pathway was found, whereby the catalytically active iron(IV)–oxo intermediate in the catalytic cycle undergoes an initial isomerisation assisted by an Arg residue in the substrate binding pocket, which then brings the oxo group in close contact with the methyl group of the alkylated DNA base. This enables a subsequent rate-determining hydrogen-atom abstraction on competitive σ-and π-pathways on a quintet spin-state surface. These findings give evidence of different locations of the oxygen and substrate binding channels in the enzyme and the origin of the separation of the oxygen-bound intermediates in the catalytic cycle from substrate. Our studies are compared with small model complexes and the effect of protein and environment on the kinetics and mechanism is explained. PMID:24339041

  9. Substrate radical intermediates in soluble methane monooxygenase

    SciTech Connect

    Liu Aimin; Jin Yi; Zhang Jingyan; Brazeau, Brian J.; Lipscomb, John D. . E-mail: lipsc001@umn.edu

    2005-12-09

    EPR spin-trapping experiments were carried out using the three-component soluble methane monooxygenase (MMO). Spin-traps 5,5-dimethyl-1-pyrroline N-oxide (DMPO), {alpha}-4-pyridyl-1-oxide N-tert-butylnitrone (POBN), and nitrosobenzene (NOB) were used to investigate the possible formation of substrate radical intermediates during catalysis. In contrast to a previous report, the NADH-coupled oxidations of various substrates did not produce any trapped radical species when DMPO or POBN was present. However, radicals were detected by these traps when only the MMO reductase component and NADH were present. DMPO and POBN were found to be weak inhibitors of the MMO reaction. In contrast, NOB is a strong inhibitor for the MMO-catalyzed nitrobenzene oxidation reaction. When NOB was used as a spin-trap in the complete MMO system with or without substrate, EPR signals from an NOB radical were detected. We propose that a molecule of NOB acts simultaneously as a substrate and a spin-trap for MMO, yielding the long-lived radical and supporting a stepwise mechanism for MMO.

  10. The hydrodeoxygenation of bioderived furans into alkanes

    NASA Astrophysics Data System (ADS)

    Sutton, Andrew D.; Waldie, Fraser D.; Wu, Ruilian; Schlaf, Marcel; ‘Pete' Silks, Louis A.; Gordon, John C.

    2013-05-01

    The conversion of biomass into fuels and chemical feedstocks is one part of a drive to reduce the world's dependence on crude oil. For transportation fuels in particular, wholesale replacement of a fuel is logistically problematic, not least because of the infrastructure that is already in place. Here, we describe the catalytic defunctionalization of a series of biomass-derived molecules to provide linear alkanes suitable for use as transportation fuels. These biomass-derived molecules contain a variety of functional groups, including olefins, furan rings and carbonyl groups. We describe the removal of these in either a stepwise process or a one-pot process using common reagents and catalysts under mild reaction conditions to provide n-alkanes in good yields and with high selectivities. Our general synthetic approach is applicable to a range of precursors with different carbon content (chain length). This allows the selective generation of linear alkanes with carbon chain lengths between eight and sixteen carbons.

  11. The hydrodeoxygenation of bioderived furans into alkanes.

    PubMed

    Sutton, Andrew D; Waldie, Fraser D; Wu, Ruilian; Schlaf, Marcel; Silks, Louis A Pete; Gordon, John C

    2013-05-01

    The conversion of biomass into fuels and chemical feedstocks is one part of a drive to reduce the world's dependence on crude oil. For transportation fuels in particular, wholesale replacement of a fuel is logistically problematic, not least because of the infrastructure that is already in place. Here, we describe the catalytic defunctionalization of a series of biomass-derived molecules to provide linear alkanes suitable for use as transportation fuels. These biomass-derived molecules contain a variety of functional groups, including olefins, furan rings and carbonyl groups. We describe the removal of these in either a stepwise process or a one-pot process using common reagents and catalysts under mild reaction conditions to provide n-alkanes in good yields and with high selectivities. Our general synthetic approach is applicable to a range of precursors with different carbon content (chain length). This allows the selective generation of linear alkanes with carbon chain lengths between eight and sixteen carbons. PMID:23609095

  12. Enzymes and genes involved in aerobic alkane degradation

    PubMed Central

    Wang, Wanpeng; Shao, Zongze

    2013-01-01

    Alkanes are major constituents of crude oil. They are also present at low concentrations in diverse non-contaminated because many living organisms produce them as chemo-attractants or as protecting agents against water loss. Alkane degradation is a widespread phenomenon in nature. The numerous microorganisms, both prokaryotic and eukaryotic, capable of utilizing alkanes as a carbon and energy source, have been isolated and characterized. This review summarizes the current knowledge of how bacteria metabolize alkanes aerobically, with a particular emphasis on the oxidation of long-chain alkanes, including factors that are responsible for chemotaxis to alkanes, transport across cell membrane of alkanes, the regulation of alkane degradation gene and initial oxidation. PMID:23755043

  13. Improved Alkane Production in Nitrogen-Fixing and Halotolerant Cyanobacteria via Abiotic Stresses and Genetic Manipulation of Alkane Synthetic Genes.

    PubMed

    Kageyama, Hakuto; Waditee-Sirisattha, Rungaroon; Sirisattha, Sophon; Tanaka, Yoshito; Mahakhant, Aparat; Takabe, Teruhiro

    2015-07-01

    Cyanobacteria possess the unique capacity to produce alkane. In this study, effects of nitrogen deficiency and salt stress on biosynthesis of alkanes were investigated in three kinds of cyanobacteria. Intracellular alkane accumulation was increased in nitrogen-fixing cyanobacterium Anabaena sp. PCC7120, but decreased in non-diazotrophic cyanobacterium Synechococcus elongatus PCC7942 and constant in a halotolerant cyanobacterium Aphanothece halophytica under nitrogen-deficient condition. We also found that salt stress increased alkane accumulation in Anabaena sp. PCC7120 and A. halophytica. The expression levels of two alkane synthetic genes were not upregulated significantly under nitrogen deficiency or salt stress in Anabaena sp. PCC7120. The transformant Anabaena sp. PCC7120 cells with additional alkane synthetic gene set from A. halophytica increased intracellular alkane accumulation level compared to control cells. These results provide a prospect to improve bioproduction of alkanes in nitrogen-fixing halotolerant cyanobacteria via abiotic stresses and genetic engineering. PMID:25971893

  14. 40 CFR 721.10163 - Chloro fluoro alkane (generic).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Chloro fluoro alkane (generic). 721... Substances § 721.10163 Chloro fluoro alkane (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as chloro fluoro alkane (PMN...

  15. 40 CFR 721.10163 - Chloro fluoro alkane (generic).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Chloro fluoro alkane (generic). 721... Substances § 721.10163 Chloro fluoro alkane (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as chloro fluoro alkane (PMN...

  16. In situ detection of anaerobic alkane metabolites in subsurface environments

    PubMed Central

    Agrawal, Akhil; Gieg, Lisa M.

    2013-01-01

    Alkanes comprise a substantial fraction of crude oil and refined fuels. As such, they are prevalent within deep subsurface fossil fuel deposits and in shallow subsurface environments such as aquifers that are contaminated with hydrocarbons. These environments are typically anaerobic, and host diverse microbial communities that can potentially use alkanes as substrates. Anaerobic alkane biodegradation has been reported to occur under nitrate-reducing, sulfate-reducing, and methanogenic conditions. Elucidating the pathways of anaerobic alkane metabolism has been of interest in order to understand how microbes can be used to remediate contaminated sites. Alkane activation primarily occurs by addition to fumarate, yielding alkylsuccinates, unique anaerobic metabolites that can be used to indicate in situ anaerobic alkane metabolism. These metabolites have been detected in hydrocarbon-contaminated shallow aquifers, offering strong evidence for intrinsic anaerobic bioremediation. Recently, studies have also revealed that alkylsuccinates are present in oil and coal seam production waters, indicating that anaerobic microbial communities can utilize alkanes in these deeper subsurface environments. In many crude oil reservoirs, the in situ anaerobic metabolism of hydrocarbons such as alkanes may be contributing to modern-day detrimental effects such as oilfield souring, or may lead to more beneficial technologies such as enhanced energy recovery from mature oilfields. In this review, we briefly describe the key metabolic pathways for anaerobic alkane (including n-alkanes, isoalkanes, and cyclic alkanes) metabolism and highlight several field reports wherein alkylsuccinates have provided evidence for anaerobic in situ alkane metabolism in shallow and deep subsurface environments. PMID:23761789

  17. Substrate Trafficking And Dioxygen Activation in Bacterial Multicomponent Monooxygenases

    SciTech Connect

    Murray, L.J.; Lippard, S.J.

    2009-06-03

    Non-heme carboxylate-bridged diiron centers in the hydroxylase components of the bacterial multicomponent monooxygenases process four substrates during catalysis: electrons, protons, dioxygen, and hydrocarbons. Understanding how protein-protein interactions mediate the transport of these substrates to the diiron center to achieve the selective oxidation of the hydrocarbon is a significant challenge. In this Account, we summarize our current knowledge of these processes with a focus on the methane monooxygenase system. We also describe recent results for the toluene/o-xylene monooxygenase and phenol hydroxylase systems from Pseudomonas sporium OX1. The observation in these latter systems of a diiron(III) oxygenated intermediate having different Moessbauer parameters from analogous species in other carboxylate-bridged diiron proteins is discussed. The results indicate that the ability of the protein framework to tune the reactivity of the diiron center at structurally similar active sites is substantially more complex than previously recognized.

  18. Nitrated metalloporphyrins as catalysts for alkane oxidation

    DOEpatents

    Ellis, Jr., Paul E.; Lyons, James E.

    1994-01-01

    Compositions of matter comprising nitro-substituted metal complexes of porphyrins are catalysts for the oxidation of alkanes. The metal is iron, chromium, manganese, ruthenium, copper or cobalt. The porphyrin ring has nitro groups attached thereto in meso and/or .beta.-pyrrolic positions.

  19. Reflectance spectroscopy of organic compounds: 1. Alkanes

    USGS Publications Warehouse

    Clark, R.N.; Curchin, J.M.; Hoefen, T.M.; Swayze, G.A.

    2009-01-01

    Reflectance spectra of the organic compounds comprising the alkane series are presented from the ultraviolet to midinfrared, 0.35 to 15.5 /??m. Alkanes are hydrocarbon molecules containing only single carbon-carbon bonds, and are found naturally on the Earth and in the atmospheres of the giant planets and Saturn's moon, Titan. This paper presents the spectral properties of the alkanes as the first in a series of papers to build a spectral database of organic compounds for use in remote sensing studies. Applications range from mapping the environment on the Earth, to the search for organic molecules and life in the solar system and throughout the. universe. We show that the spectral reflectance properties of organic compounds are rich, with major diagnostic spectral features throughout the spectral range studied. Little to no spectral change was observed as a function of temperature and only small shifts and changes in the width of absorption bands were observed between liquids and solids, making remote detection of spectral properties throughout the solar system simpler. Some high molecular weight organic compounds contain single-bonded carbon chains and have spectra similar to alkanes even ' when they fall into other families. Small spectral differences are often present allowing discrimination among some compounds, further illustrating the need to catalog spectral properties for accurate remote sensing identification with spectroscopy.

  20. Nitrated metalloporphyrins as catalysts for alkane oxidation

    DOEpatents

    Ellis, Jr., Paul E.; Lyons, James E.

    1992-01-01

    Alkanes are oxidized by contact with oxygen-containing gas in the presence as catalyst of a metalloporphyrin in which hydrogen atoms in the porphyrin ring have been replaced with one or more nitro groups. Hydrogen atoms in the porphyrin ring may also be substituted with halogen atoms.

  1. Nitrated metalloporphyrins as catalysts for alkane oxidation

    DOEpatents

    Ellis, P.E. Jr.; Lyons, J.E.

    1994-01-18

    Compositions of matter comprising nitro-substituted metal complexes of porphyrins are catalysts for the oxidation of alkanes. The metal is iron, chromium, manganese, ruthenium, copper or cobalt. The porphyrin ring has nitro groups attached thereto in meso and/or [beta]-pyrrolic positions.

  2. Parallel and Competitive Pathways for Substrate Desaturation, Hydroxylation and Radical Rearrangement by the Non-heme Diiron Hydroxylase AlkB

    PubMed Central

    Cooper, Harriet L. R.; Mishra, Girish; Huang, Xiongyi; Pender-Cudlip, Marilla; Austin, Rachel N.; Shanklin, John; Groves, John T.

    2012-01-01

    A purified and highly active form of the non-heme diiron hydroxylase AlkB was investigated using the diagnostic probe substrate norcarane. The reaction afforded C2 (26%) and C3 (43%) hydroxylation and desaturation products (31%). Initial C-H cleavage at C2 led to 7% C2 hydroxylation and 19% 3-hydroxymethylcyclohexene, a rearrangement product characteristic of a radical rearrangement pathway. A deuterated substrate analog, 3,3,4,4-norcarane-d4, afforded drastically reduced amounts of C3 alcohol (8%) and desaturation products (5%), while the radical rearranged alcohol was now the major product (65%). This change in product ratios indicates a large kinetic hydrogen isotope effect of ~20 for both the C-H hydroxylation at C3 and the desaturation pathway, with all of the desaturation originating via hydrogen abstraction at C3 and not C2. The data indicates that AlkB reacts with norcarane via initial C-H hydrogen abstraction from C2 or C3 and that the three pathways, C3 hydroxylation, C3 desaturation and C2 hydroxylation/radical rearrangement, are parallel and competitive. Thus, the incipient radical at C3 either reacts with the iron-oxo center to form an alcohol or proceeds along the desaturation pathway via a second H-abstraction to afford both 2-norcarene and 3-norcarene. Subsequent reactions of these norcarenes lead to detectable amounts of hydroxylation products and toluene. By contrast, the 2-norcaranyl radical intermediate leads to C2 hydroxylation and the diagnostic radical rearrangement, but this radical apparently does not afford desaturation products. The results indicate that C-H hydroxylation and desaturation follow analogous stepwise reaction channels via carbon radicals that diverge at the product-forming step. PMID:23157204

  3. Whole-cell bio-oxidation of n-dodecane using the alkane hydroxylase system of P. putida GPo1 expressed in E. coli.

    PubMed

    Grant, Chris; Woodley, John M; Baganz, Frank

    2011-05-01

    The alkane-1-monoxygenase (alkB) complex of Pseudomonas putida GPo1 has been extensively studied in the past and shown to be capable of oxidising aliphatic C(5)-C(12) alkanes to primary alcohols both in the wild-type organism by growth on C(5)-C(12) alkanes as sole carbon source and in vitro. Despite this, successful n-dodecane oxidation for the production of 1-dodecanol or dodecanoic acid has proven elusive in the past when using alkB-expressing recombinants. This article demonstrates, for the first time in vivo, by using the Escherichia coli GEC137 pGEc47ΔJ strain, that n-dodecane oxidation using this enzyme for the production of primary alcohols and carboxylic acids is feasible and in fact potentially more promising than n-octane oxidation due to lower product and substrate toxicity. Yields are reported of 1-dodecanol of up to 2 g/L(organic) and dodecanoic acid up to 19.7 g/L(organic) in a 2 L stirred tank reactor with 1L aqueous phase and 200 mL of n-dodecane as a second phase. The maximum volumetric rate of combined alcohol and acid production achieved was 1.9 g/L(organic)/h (0.35 g/L(total)/h). The maximum specific activity of combined alcohol and acid production was 7-fold lower on n-dodecane (3.5 μmol/min/g(dcw)) than on n-octane (21 μmol/min/g(dcw)); similar to the 5-fold difference observed between wild-type growth rates using the two respective alkanes as sole carbon source. Despite this, both total volumetric rate and final yield exceeded n-octane oxidation by 3.5-fold under the same conditions, due to the lower toxicity of n-dodecane and its oxidation products to E. coli compared to the 8-carbon equivalents. Substrate access limitations and the overoxidation of 1-dodecanol to dodecanoic acid were identified as the most important limitations to be addressed. PMID:22113020

  4. Hydroxylation of methane through component interactions in soluble methane monooxygenases.

    PubMed

    Lee, Seung Jae

    2016-04-01

    Methane hydroxylation through methane monooxygenases (MMOs) is a key aspect due to their control of the carbon cycle in the ecology system and recent applications of methane gas in the field of bioenergy and bioremediation. Methanotropic bacteria perform a specific microbial conversion from methane, one of the most stable carbon compounds, to methanol through elaborate mechanisms. MMOs express particulate methane monooxygenase (pMMO) in most strains and soluble methane monooxygenase (sMMO) under copper-limited conditions. The mechanisms of MMO have been widely studied from sMMO belonging to the bacterial multicomponent monooxygenase (BMM) superfamily. This enzyme has diiron active sites where different types of hydrocarbons are oxidized through orchestrated hydroxylase, regulatory and reductase components for precise control of hydrocarbons, oxygen, protons, and electrons. Recent advances in biophysical studies, including structural and enzymatic achievements for sMMO, have explained component interactions, substrate pathways, and intermediates of sMMO. In this account, oxidation of methane in sMMO is discussed with recent progress that is critical for understanding the microbial applications of C-H activation in one-carbon substrates. PMID:27033202

  5. Regulation of cytochrome P-450 monooxygenases in the mouse

    SciTech Connect

    Kelley, M.F.

    1986-01-01

    Recently, the compound 1,4-bis(2-(3,4-dichloropyridyloxy)) benzene (TCPOBOP) has been identified as a highly potent phenobabital-like agonist in mice. This finding has led to the suggestion that a receptor-mediated process may govern the induction of cytochrome P-450 monooxygenases by phenobarbital and phenobarbital-like agonists. This dissertation examines: (1) the effects of structural alterations of the TCPOBOP molecule on enzyme induction activity, (2) the induction response to phenobarbital and TCPOBOP among inbred mouse strains, (3) the spectrum of monooxygenase activities induced by phenobarbital and TCPOBOP compared to 3-methylcholanthrene, isosafrole and pregnenolone 16..cap alpha..-carbonitrile (PCN) and (4) the binding of (/sup 3/H) TCPOBOP in hepatic cytosol. Changes in the structure of the pyridyloxy or benzene rings markedly affect enzyme induction activity and provide additional indirect evidence for a receptor-mediated response. An evaluation of monooxygenase induction by TCPOBOP for 27 inbred mouse strains and by phenobarbital for 15 inbred mouse strains failed to identify a strain which was completely nonresponsive to these compounds, although several strains exhibited decreased responsiveness for select monooxygenase reactions. TCPOBOP, PCN and phenobarbital were all found to significantly increase the rate of hydroxylation of testosterone at the 2..cap alpha..-, 6..beta..- and 15..beta..- positions but only TCPOBOP and phenobarbital dramatically increased the rate of pentoxyresorufin O-dealkylation. The results demonstrates that TCPOBOP most closely resembles phenobarbital in its mode of monooxygenase induction in mice. Sucrose density gradient analysis of (/sup 3/H) TCPOBOP-hepatic cytosol incubations failed to identify specific, saturable binding of (/sup 3/H) TCPOBOP to cytosolic marcomolecular elements.

  6. Density functional steric analysis of linear and branched alkanes.

    PubMed

    Ess, Daniel H; Liu, Shubin; De Proft, Frank

    2010-12-16

    Branched alkane hydrocarbons are thermodynamically more stable than straight-chain linear alkanes. This thermodynamic stability is also manifest in alkane bond separation energies. To understand the physical differences between branched and linear alkanes, we have utilized a novel density functional theory (DFT) definition of steric energy based on the Weizäcker kinetic energy. Using the M06-2X functional, the total DFT energy was partitioned into a steric energy term (E(s)[ρ]), an electrostatic energy term (E(e)[ρ]), and a fermionic quantum energy term (E(q)[ρ]). This analysis revealed that branched alkanes have less (destabilizing) DFT steric energy than linear alkanes. The lower steric energy of branched alkanes is mitigated by an equal and opposite quantum energy term that contains the Pauli component of the kinetic energy and exchange-correlation energy. Because the steric and quantum energy terms cancel, this leaves the electrostatic energy term that favors alkane branching. Electrostatic effects, combined with correlation energy, explains why branched alkanes are more stable than linear alkanes. PMID:21086970

  7. Density Functional Steric Analysis of Linear and Branched Alkanes

    SciTech Connect

    Ess, Daniel H.; Liu, Shubin; De Proft, Frank

    2010-11-18

    Branched alkane hydrocarbons are thermodynamically more stable than straight-chain linear alkanes. This thermodynamic stability is also manifest in alkane bond separation energies. To understand the physical differences between branched and linear alkanes, we have utilized a novel density functional theory (DFT) definition of steric energy based on the Weizäcker kinetic energy. Using the M06-2X functional, the total DFT energy was partitioned into a steric energy term (Ee[[ρ]), an electrostatic energy term (Ee[ρ]), and a fermionic quantum energy term (Eq[[ρ]). This analysis revealed that branched alkanes have less (destabilizing) DFT steric energy than linear alkanes. The lower steric energy of branched alkanes is mitigated by an equal and opposite quantum energy term that contains the Pauli component of the kinetic energy and exchange-correlation energy. Because the steric and quantum energy terms cancel, this leaves the electrostatic energy term that favors alkane branching. Electrostatic effects, combined with correlation energy, explains why branched alkanes are more stable than linear alkanes.

  8. Oxidative dehydrogenation of alkanes to unsaturated hydrocarbons

    DOEpatents

    Kung, Harold H.; Chaar, Mohamed A.

    1988-01-01

    Oxidative dehydrogenation of alkanes to unsaturated hydrocarbons is carried out over metal vanadate catalysts under oxidizing conditions. The vanadate catalysts are represented by the formulas M.sub.3 (VO.sub.4).sub.2 and MV.sub.2 O.sub.6, M representing Mg, Zn, Ca, Pb, or Cd. The reaction is carried out in the presence of oxygen, but the formation of oxygenate by-products is suppressed.

  9. Oxidative dehydrogenation of alkanes to unsaturated hydrocarbons

    DOEpatents

    Kung, H.H.; Chaar, M.A.

    1988-10-11

    Oxidative dehydrogenation of alkanes to unsaturated hydrocarbons is carried out over metal vanadate catalysts under oxidizing conditions. The vanadate catalysts are represented by the formulas M[sub 3](VO[sub 4])[sub 2] and MV[sub 2]O[sub 6], M representing Mg, Zn, Ca, Pb, or Cd. The reaction is carried out in the presence of oxygen, but the formation of oxygenate by-products is suppressed.

  10. Alkane metathesis by tandem alkane-dehydrogenation-olefin-metathesis catalysis and related chemistry.

    PubMed

    Haibach, Michael C; Kundu, Sabuj; Brookhart, Maurice; Goldman, Alan S

    2012-06-19

    Methods for the conversion of both renewable and non-petroleum fossil carbon sources to transportation fuels that are both efficient and economically viable could greatly enhance global security and prosperity. Currently, the major route to convert natural gas and coal to liquids is Fischer-Tropsch catalysis, which is potentially applicable to any source of synthesis gas including biomass and nonconventional fossil carbon sources. The major desired products of Fischer-Tropsch catalysis are n-alkanes that contain 9-19 carbons; they comprise a clean-burning and high combustion quality diesel, jet, and marine fuel. However, Fischer-Tropsch catalysis also results in significant yields of the much less valuable C(3) to C(8)n-alkanes; these are also present in large quantities in oil and gas reserves (natural gas liquids) and can be produced from the direct reduction of carbohydrates. Therefore, methods that could disproportionate medium-weight (C(3)-C(8)) n-alkanes into heavy and light n-alkanes offer great potential value as global demand for fuel increases and petroleum reserves decrease. This Account describes systems that we have developed for alkane metathesis based on the tandem operation of catalysts for alkane dehydrogenation and olefin metathesis. As dehydrogenation catalysts, we used pincer-ligated iridium complexes, and we initially investigated Schrock-type Mo or W alkylidene complexes as olefin metathesis catalysts. The interoperability of the catalysts typically represents a major challenge in tandem catalysis. In our systems, the rate of alkane dehydrogenation generally limits the overall reaction rate, whereas the lifetime of the alkylidene complexes at the relatively high temperatures required to obtain practical dehydrogenation rates (ca. 125 -200 °C) limits the total turnover numbers. Accordingly, we have focused on the development and use of more active dehydrogenation catalysts and more stable olefin-metathesis catalysts. We have used thermally

  11. Synthesis of chiral 2-alkanols from n-alkanes by a P. putida whole-cell biocatalyst.

    PubMed

    Tieves, Florian; Erenburg, Isabelle N; Mahmoud, Osama; Urlacher, Vlada B

    2016-09-01

    The cytochrome P450 monooxygenase CYP154A8 from Nocardia farcinica was previously found to catalyze hydroxylation of linear alkanes (C7 -C9 ) with a high regio- and stereoselectivity. The objective of this study was to integrate CYP154A8 along with suitable redox partners into a whole-cell system for the production of chiral 2-alkanols starting from alkanes. Both recombinant Escherichia coli and Pseudomonas putida whole-cell biocatalysts tested for this purpose showed the ability to produce chiral alkanols, but a solvent tolerant P. putida strain demonstrated several advantages in the applied biphasic reaction system. The optimized P. putida whole-cell system produced ∼16 mM (S)-2-octanol with 87% ee from octane, which is more than sevenfold higher than the previously described system with isolated enzymes. The achieved enantiopurity of the product could further be increased up to 99% ee by adding an alcohol dehydrogenase (ADH) to the alkane-oxidizing P. putida whole-cell systems. By using this setup for the individual conversions of heptane, octane or nonane, 2.6 mM (S)-2-heptanol with 91% ee, 5.4 mM (S)-2-octanol with 97% ee, or 5.5 mM (S)-2-nonanol with 97% ee were produced, respectively. The achieved concentrations of chiral 2-alkanols are the highest reported for a P450-based whole-cell system so far. Biotechnol. Bioeng. 2016;113: 1845-1852. © 2016 Wiley Periodicals, Inc. PMID:26887569

  12. Stimulation of Lipase Production During Bacterial Growth on Alkanes

    PubMed Central

    Breuil, Colette; Shindler, D. B.; Sijher, J. S.; Kushner, D. J.

    1978-01-01

    Acinetobacter lwoffi strain O16, a facultative psychrophile, can grow on crude oil, hexadecane, octadecane, and most alkanes when tested at 20 but not at 30°C. Growth occurred on a few alkanes at 30°C but after a longer lag than at 20°C. Cells grown on alkanes as sole carbon sources had high levels of cell-bound lipase. In contrast, previous work has shown that those grown on complex medium produced cell-free lipase and those grown on defined medium without alkanes produced little or no lipase. Low concentrations of the detergent Triton X-100 caused the liberation of most of the lipase activity of alkane-grown cells and increased total lipase activity. When ethanol and hexadecane were both present in a mineral medium, diauxic growth occurred; until the ethanol was completely used up, hexadecane was not utilized, and the lipase activity was very low. When growth on hexadecane began, lipase activity increased, reaching a level 50- to 100-fold higher than that of cells growing on ethanol. A similar pattern of lipase formation and hexadecane utilization was observed with Pseudomonas aeruginosa. Whenever A. lwoffi and other bacteria degraded alkanes they exhibited substantial lipase activity. Not all bacteria that produced lipase, however, could attack alkanes. Bacteria that could not produce lipase did not attack alkanes. The results suggest that a correlation may exist between lipase formation and alkane utilization. PMID:627533

  13. Novel cyclohexane monooxygenase from Acidovorax sp. CHX100.

    PubMed

    Salamanca, Diego; Karande, Rohan; Schmid, Andreas; Dobslaw, Daniel

    2015-08-01

    Acidovorax sp. CHX100 has a remarkable ability for growth on short cycloalkanes (C5-C8) as a sole source of carbon and energy under aerobic conditions via an uncharacterized mechanism. Transposon mutagenesis of Acidovorax sp. CHX100 revealed a novel cytochrome P450 monooxygenase (CYP450chx) which catalyzed the transformation of cyclohexane to cyclohexanol. Primer walking methods categorized CYP450chx as cytochrome P450 class I taking into account its operon structure: monooxygenase, FAD oxidoreductase, and ferredoxin. CYP450chx was successfully cloned and expressed in Escherichia coli JM109. The activity of CYP450chx was demonstrated by means of the indole co-oxidation. Biotransformation capability of CYP450chx was confirmed through the catalysis of cycloalkanes (C5-C8) to their respective cyclic alcohols. PMID:25935342

  14. Conformation of liquid N-alkanes.

    PubMed Central

    Goodsaid-Zalduondo, F; Engelman, D M

    1981-01-01

    The conformations of liquid n-alkanes have been studied using neutron scattering techniques to better understand the conformational forces present in membrane lipid interiors. We have studied hydrocarbon chains having lengths comparable to those found for esterified membrane lipid fatty acids, and find that the steric constraints of packing in the liquid state do not change the conformational distributions of hydrocarbon chains from those imposed by the intrachain forces present in the gas phase. It follows that the central region of membranes containing lipids in the disordered state should contain hydrocarbon chain conformations determined primarily by intrachain forces. PMID:7272453

  15. Analysis of Genes for Succinoyl Trehalose Lipid Production and Increasing Production in Rhodococcus sp. Strain SD-74

    PubMed Central

    Inaba, Tomohiro; Tokumoto, Yuta; Miyazaki, Yusuke; Inoue, Naoyuki; Maseda, Hideaki; Nakajima-Kambe, Toshiaki; Uchiyama, Hiroo

    2013-01-01

    Succinoyl trehalose lipids (STLs) are promising glycolipid biosurfactants produced from n-alkanes that are secreted by Rhodococcus species bacteria. These compounds not only exhibit unique interfacial properties but also demonstrate versatile biochemical actions. In this study, three novel types of genes involved in the biosynthesis of STLs, including a putative acyl coenzyme A (acyl-CoA) transferase (tlsA), fructose-bisphosphate aldolase (fda), and alkane monooxygenase (alkB), were identified. The predicted functions of these genes indicate that alkane metabolism, sugar synthesis, and the addition of acyl groups are important for the biosynthesis of STLs. Based on these results, we propose a biosynthesis pathway for STLs from alkanes in Rhodococcus sp. strain SD-74. By overexpressing tlsA, we achieved a 2-fold increase in the production of STLs. This study advances our understanding of bacterial glycolipid production in Rhodococcus species. PMID:24038682

  16. Methane monooxygenase: functionalizing methane at iron and copper.

    PubMed

    Sazinsky, Matthew H; Lippard, Stephen J

    2015-01-01

    Methane monooxygenases (MMOs) catalyze the conversion of methane to methanol as the first committed step in the assimilation of this hydrocarbon into biomass and energy by methanotrophs, thus playing a significant role in the biogeochemistry of this potent greenhouse gas. Two distinct enzymes, a copper-dependent membrane protein, particulate methane monooxygenase (pMMO), and an iron-dependent cytosolic protein, soluble methane monooxygenase (sMMO), carry out this transformation using large protein scaffolds that help to facilitate the timely transport of hydrocarbon, O₂, proton, and electron substrates to buried dimetallic active sites. For both enzymes, reaction of the reduced metal centers with O₂leads to intermediates that activate the relatively inert C-H bonds of hydrocarbons to yield oxidized products. Among synthetic and biological catalysts, MMOs are unique because they are the only ones known to hydroxylate methane at ambient temperatures. As a need for new industrial catalysts and green chemical transformations increases, understanding how the different MMO metal centers efficiently accomplish this challenging chemistry has become the focus of intense study. This chapter examines current understanding of the sMMO and pMMO protein structures, their methods for substrate channeling, and mechanisms for the dimetallic activation of O₂and C-H bonds. PMID:25707469

  17. Monophenol monooxygenase and lincomysin biosynthesis in Streptomyces lincolnensis.

    PubMed

    Michalik, J; Emilianowicz-Czerska, W; Switalski, L; Raczyńska-Bojanowska, K

    1975-11-01

    Monophenol monooxygenase (monophenol, dihydroxyphenylalanine:oxygen oxidoreductase EC 1.14.18.1) was studied in melanin-positive and melanin-negative mutants of Streptomyces lincolnensis NCIB 9413, varying in the lincomycin synthesizing ability. The activities of laccase and tyrosine phenol lyase (EC 4.1.99.2) are absent in this organism. The monophenol monooxygenase catalyzes hydroxylation of monophenols (K(m) and V(max) for l-tyrosine, 2 x 10(-4) M and 8.0 nmol of O(2)/min per ml, respectively) at a slower rate than it dehydrogenates diphenols to o-quinones (K(m) and V(max) for l-3,4-dihydroxyphenylalanine, 7 x 10(-5) M and 51.7 nmol of O(2)/min per ml, respectively. It is inhibited by KCN, beta-mercaptoethanol, ethylenediaminetetraacetate, dipyridyl, thiourea, p-aminobenzoic acids and by some tryptophan metabolites. Changes in the activity of monophenol monooxygenase caused by mutation or by inhibitors are reflected in the synthesis of the antibiotic. Its participation in the biogenesis of the propylhygric moiety of lincomycin is discussed. PMID:813570

  18. Products of Chemistry: Alkanes: Abundant, Pervasive, Important, and Essential.

    ERIC Educational Resources Information Center

    Seymour, Raymond B.

    1989-01-01

    Discusses the history and commercialization of alkanes. Examines the nomenclature and uses of alkanes. Studies polymerization and several types of polyethylenes: low-density, high-density, low-molecular-weight, cross-linked, linear low-density, and ultrahigh-molecular-weight. Includes a glossary of hydrocarbon terms. (MVL)

  19. 40 CFR 721.535 - Halogenated alkane (generic).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.535 Halogenated alkane (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as halogenated alkane (PMN P-01-433) is...

  20. 40 CFR 721.536 - Halogenated phenyl alkane.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.536 Halogenated phenyl alkane. (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as halogenated phenyl alkane (PMN P-89-867)...

  1. 40 CFR 721.535 - Halogenated alkane (generic).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.535 Halogenated alkane (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as halogenated alkane (PMN P-01-433) is...

  2. Thomas Reiche Kuhn populations in alkanes

    NASA Astrophysics Data System (ADS)

    Lazzeretti, P.; Caputo, M. C.; Ferraro, M. B.

    1999-07-01

    Atomic populations in a molecule have been defined via the Thomas-Reiche-Kuhn sum rule for oscillator strengths written within the acceleration gauge. These atomic populations are related to nuclear electric shieldings, i.e., to geometrical derivatives of electric dipole moment, and can therefore be connected with observable infrared intensities. A number of relationships can be considered to test a priori the quality of calculated electronic charges and to assess their physical meaning. It is shown via extended numerical tests on the first members of the alkane series that the Thomas-Reiche-Kuhn populations are consistent with a (small) polarity C +-H - of carbon-hydrogen bond in methane, for which a bond dipole moment can be exactly defined. Although the idea of bond dipole cannot be extended to the C-H fragments belonging to other alkane molecules in the absence of local C3 v symmetry, the calculations prove that the same electron charge polarization should characterize the whole homologous series.

  3. Alkanes in benthic organisms from the Buccaneer oil field

    SciTech Connect

    Middleditch, B.S.; Basile, B.

    1980-06-01

    About 200 g per day of alkanes are present in brine discharged from each of two production platforms in the Buccaneer oil field in the NW Gulf of Mexico. These alkanes disperse rapidly in the water column, so that seawater concentrations of petroleum alkanes in this region are generally very low. They can be taken up to some extent by plankton, fish, and barnacles, but the petroleum alkane concentrations in these organisms are also relatively low. The largest pool of petroleum alkanes is in the surficial sediments, where concentrations of up to 25 ppM are observed, with concentration gradients extending more than 20 m from the production platforms. Organisms are examined which are exposed to these sediments and, for comparison, other specimens from control sites around structures from which there are no discharges.

  4. Utilization of n-Alkanes by Cladosporium resinae

    PubMed Central

    Teh, J. S.; Lee, K. H.

    1973-01-01

    Four different isolates of Cladosporium resinae from Australian soils were tested for their ability to utilize liquid n-alkanes ranging from n-hexane to n-octadecane under standard conditions. The isolates were unable to make use of n-hexane, n-heptane, and n-octane for growth. In fact, these hydrocarbons, particularly n-hexane, exerted an inhibitory effect on spore germination and mycelial growth. All higher n-alkanes from n-nonane to n-octadecane were assimilated by the fungus, although only limited growth occurred on n-nonane and n-decane. The long chain n-alkanes (C14 to C18) supported good growth of all isolates, but there was no obvious correlation between cell yields and chain lengths of these n-alkanes. Variation in growth responses to individual n-alkane among the different isolates was also observed. The cause of this variation is unknown. PMID:4735447

  5. Sophorolipids from Torulopsis bombicola: possible relation to alkane uptake.

    PubMed Central

    Ito, S; Inoue, S

    1982-01-01

    Torulopsis bombicola produces extracellular sophorolipids when it is grown on water-insoluble alkanes. Sophorolipids and related model compounds, which were not themselves used for growth, were found to stimulate markedly the growth of T. bombicola on alkanes. This stimulatory effect was restricted to growth on C10 to C20 alkanes, whereas no significantly influence was observed for growth on fatty alcohols, fatty acids, glucose, or glycerol. The nonionic methyl ester of the glycolipid supported the greatest cell yield. However, a number of synthetic nonionic surfactants were unable to replace the glycolipid. When organisms were grown on hexadecane, stimulation of growth by sophorolipids was observed almost exclusively with strains of Torulopsis yeasts. In contrast, the growth of other typical alkane-utilizing yeasts, such as candida and Pichia strains, was inhibited or not affected. It appears that sophorolipids are involved in alkane dissimilation by T. bombicola through an undetermined mechanism. PMID:7201782

  6. Active site models for the Cu(A) site of peptidylglycine α-hydroxylating monooxygenase and dopamine β-monooxygenase.

    PubMed

    Kunishita, Atsushi; Ertem, Mehmed Z; Okubo, Yuri; Tano, Tetsuro; Sugimoto, Hideki; Ohkubo, Kei; Fujieda, Nobutaka; Fukuzumi, Shunichi; Cramer, Christopher J; Itoh, Shinobu

    2012-09-01

    A mononuclear copper(II) superoxo species has been invoked as the key reactive intermediate in aliphatic substrate hydroxylation by copper monooxygenases such as peptidylglycine α-hydroxylating monooxygenase (PHM), dopamine β-monooxygenase (DβM), and tyramine β-monooxygenase (TβM). We have recently developed a mononuclear copper(II) end-on superoxo complex using a N-[2-(2-pyridyl)ethyl]-1,5-diazacyclooctane tridentate ligand, the structure of which is similar to the four-coordinate distorted tetrahedral geometry of the copper-dioxygen adduct found in the oxy-form of PHM (Prigge, S. T.; Eipper, B. A.; Mains, R. E.; Amzel, L. M. Science2004, 304, 864-867). In this study, structures and physicochemical properties as well as reactivity of the copper(I) and copper(II) complexes supported by a series of tridentate ligands having the same N-[2-(2-pyridyl)ethyl]-1,5-diazacyclooctane framework have been examined in detail to shed light on the chemistry dictated in the active sites of mononuclear copper monooxygenases. The ligand exhibits unique feature to stabilize the copper(I) complexes in a T-shape geometry and the copper(II) complexes in a distorted tetrahedral geometry. Low temperature oxygenation of the copper(I) complexes generated the mononuclear copper(II) end-on superoxo complexes, the structure and spin state of which have been further characterized by density functional theory (DFT) calculations. Detailed kinetic analysis on the O(2)-adduct formation reaction gave the kinetic and thermodynamic parameters providing mechanistic insights into the association and dissociation processes of O(2) to the copper complexes. The copper(II) end-on superoxo complex thus generated gradually decomposed to induce aliphatic ligand hydroxylation. Kinetic and DFT studies on the decomposition reaction have suggested that C-H bond abstraction occurs unimolecularly from the superoxo complex with subsequent rebound of the copper hydroperoxo species to generate the oxygenated

  7. Spectroscopy of the tilde A state of NO-alkane complexes (alkane = methane, ethane, propane, and n-butane)

    NASA Astrophysics Data System (ADS)

    Tamé-Reyes, Victor M.; Gardner, Adrian M.; Harris, Joe P.; McDaniel, Jodie; Wright, Timothy G.

    2012-12-01

    We have recorded (1+1) resonance-enhanced multiphoton ionization spectra of complexes formed between NO and the alkanes: CH4, C2H6, C3H8, and n-C4H10. The spectra correspond to the tilde A ← tilde X transition, which is a NO-localized 3s ← 2pπ* transition. In line with previous work, the spectrum for NO-CH4 has well-defined structure, but this is only partially resolved for the other complexes. The spectra recorded in the NO+-alkane mass channels all show a slowly rising onset, followed by a sharp offset, which is associated with dissociation of NO-alkane, from which binding energies in the tilde X and tilde A states are deduced. Beyond this sharp offset, there is a further rise in signal, which is attributed to fragmentation of higher complexes, NO-(alkane)n. Analysis of these features allows binding energies for (NO-alkane) ... alkane to be estimated, and these suggest that in the NO-(alkane)2 complexes, the second alkane molecule is bound to the first, rather than to NO. Calculated structures for the 1:1 complexes are reported, as well as binding energies.

  8. Kinetic study of asphaltene dissolution in amphiphile/alkane solutions

    SciTech Connect

    Permsukarome, P.; Chang, C.; Fogler, H.S.

    1997-09-01

    The kinetics of dissolution of pentane-insoluble solid asphaltene precipitates by amphiphile/alkane solutions were investigated using a differential reactor flow system. Two amphiphiles, dodecylbenzenesulfonic acid and nonylphenol, and five alkane solvents, ranging from hexane to hexadecane, were used. Results showed that the rate of asphaltene dissolution in amphiphile/alkane fluids could be approximated with a first-order kinetics with respect to the undissolved asphaltene mass in solution. The specific dissolution rate constant, k, varied with the concentration of amphiphiles, the type of alkane solvents, the temperature, and the fluid flow rate. The rate of asphaltene dissolution displayed a Langmuir-Hinshelwood kinetics with respect to the concentration of amphiphiles. Increasing the temperature of amphiphile/alkane fluids also enhanced the rate of asphaltene dissolution. The apparent activation energy for asphaltene dissolution was approximated to be 4--7 kcal/mol. The rate of asphaltene dissolution was also greater in amphiphile solutions containing lighter alkanes, such as hexane, with lower viscosities. These trends suggest that both surface reaction and mass transfer processes are important to the rate of asphaltene dissolution in amphiphile/alkane fluids.

  9. Long-chain alkane production by the yeast Saccharomyces cerevisiae.

    PubMed

    Buijs, Nicolaas A; Zhou, Yongjin J; Siewers, Verena; Nielsen, Jens

    2015-06-01

    In the past decade industrial-scale production of renewable transportation biofuels has been developed as an alternative to fossil fuels, with ethanol as the most prominent biofuel and yeast as the production organism of choice. However, ethanol is a less efficient substitute fuel for heavy-duty and maritime transportation as well as aviation due to its low energy density. Therefore, new types of biofuels, such as alkanes, are being developed that can be used as drop-in fuels and can substitute gasoline, diesel, and kerosene. Here, we describe for the first time the heterologous biosynthesis of long-chain alkanes by the yeast Saccharomyces cerevisiae. We show that elimination of the hexadecenal dehydrogenase Hfd1 and expression of a redox system are essential for alkane biosynthesis in yeast. Deletion of HFD1 together with expression of an alkane biosynthesis pathway resulted in the production of the alkanes tridecane, pentadecane, and heptadecane. Our study provides a proof of principle for producing long-chain alkanes in the industrial workhorse S. cerevisiae, which was so far limited to bacteria. We anticipate that these findings will be a key factor for further yeast engineering to enable industrial production of alkane based drop-in biofuels, which can allow the biofuel industry to diversify beyond bioethanol. PMID:25545362

  10. Mechanistic Studies on the Hydroxylation of Methane by Methane Monooxygenase

    SciTech Connect

    Baik, Mu-Hyun; Newcomb, Martin; Friesner, Richard A.; Lippard, Stephen J.

    2003-05-17

    Methanotrophs are bacteria that live on methane as their only source of carbon.1 The first step in their utilization of this simplest of all hydrocarbons is its selective conversion to methanol. Subsequent biochemical pathways transform methanol to formaldehyde, which in turn is processed into biomass. Further oxidation of formaldehyde to carbon dioxide provides energy that is stored for later use as NADH.2 The conversion of methane to methanol is catalyzed at the active site of a metalloenzyme known as methane monooxygenase, or MMO.3-9

  11. On the catalytic mechanisms of lytic polysaccharide monooxygenases.

    PubMed

    Walton, Paul H; Davies, Gideon J

    2016-04-01

    Lytic polysaccharide monooxygenases (LPMOs) are recently discovered copper-containing oxygenases. LPMOs oxidise recalcitrant polysaccharides such as chitin and cellulose, thereby making these substrates more tractable to canonical chitinase or cellulase action. As such, LPMOs are attracting much attention not only for their capacity to greatly increase the efficiency of production of cellulosic-based biofuels, but also for the new questions they pose about the mechanisms of biological oxidation of recalcitrant substrates. This review draws together the current thinking on the catalytic mechanisms of LPMOs and other copper catalysed oxygenations and provides a blueprint for further investigation into the mechanisms of action of these intriguing enzymes. PMID:27094791

  12. The framework of polysaccharide monooxygenase structure and chemistry.

    PubMed

    Span, Elise A; Marletta, Michael A

    2015-12-01

    Polysaccharide monooxygenases, or PMOs (also known as lytic PMOs or LPMOs), are a group of enzymes discovered in recent years to catalyze the oxidative degradation of carbohydrate polymers. The PMO catalytic domain has a β-sandwich fold that bears a strong resemblance to both immunoglobulin (Ig) and fibronectin type III (FnIII) domains. PMOs are secreted by fungi and bacteria, and there is recent evidence for their roles in pathogenesis, in addition to biomass processing. This review addresses the biological origins and functions of emerging PMO families, as well as describes the aspects of PMO structure that support the chemistry of copper-catalyzed, oxidative polysaccharide degradation. PMID:26615470

  13. Bacterial expression of human kynurenine 3-monooxygenase: solubility, activity, purification.

    PubMed

    Wilson, K; Mole, D J; Binnie, M; Homer, N Z M; Zheng, X; Yard, B A; Iredale, J P; Auer, M; Webster, S P

    2014-03-01

    Kynurenine 3-monooxygenase (KMO) is an enzyme central to the kynurenine pathway of tryptophan metabolism. KMO has been implicated as a therapeutic target in several disease states, including Huntington's disease. Recombinant human KMO protein production is challenging due to the presence of transmembrane domains, which localise KMO to the outer mitochondrial membrane and render KMO insoluble in many in vitro expression systems. Efficient bacterial expression of human KMO would accelerate drug development of KMO inhibitors but until now this has not been achieved. Here we report the first successful bacterial (Escherichia coli) expression of active FLAG™-tagged human KMO enzyme expressed in the soluble fraction and progress towards its purification. PMID:24316190

  14. The molecular basis of polysaccharide cleavage by lytic polysaccharide monooxygenases.

    PubMed

    Frandsen, Kristian E H; Simmons, Thomas J; Dupree, Paul; Poulsen, Jens-Christian N; Hemsworth, Glyn R; Ciano, Luisa; Johnston, Esther M; Tovborg, Morten; Johansen, Katja S; von Freiesleben, Pernille; Marmuse, Laurence; Fort, Sébastien; Cottaz, Sylvain; Driguez, Hugues; Henrissat, Bernard; Lenfant, Nicolas; Tuna, Floriana; Baldansuren, Amgalanbaatar; Davies, Gideon J; Lo Leggio, Leila; Walton, Paul H

    2016-04-01

    Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that oxidatively break down recalcitrant polysaccharides such as cellulose and chitin. Since their discovery, LPMOs have become integral factors in the industrial utilization of biomass, especially in the sustainable generation of cellulosic bioethanol. We report here a structural determination of an LPMO-oligosaccharide complex, yielding detailed insights into the mechanism of action of these enzymes. Using a combination of structure and electron paramagnetic resonance spectroscopy, we reveal the means by which LPMOs interact with saccharide substrates. We further uncover electronic and structural features of the enzyme active site, showing how LPMOs orchestrate the reaction of oxygen with polysaccharide chains. PMID:26928935

  15. The metal centres of particulate methane mono-oxygenase.

    PubMed

    Rosenzweig, Amy C

    2008-12-01

    pMMO (particulate methane mono-oxygenase) is an integral membrane metalloenzyme that catalyses the oxidation of methane to methanol. The pMMO metal active site has not been identified, precluding detailed investigation of the reaction mechanism. Models for the metal centres proposed by various research groups have evolved as crystallographic and spectroscopic data have become available. The present review traces the evolution of these active-site models before and after the 2005 Methylococcus capsulatus (Bath) pMMO crystal structure determination. PMID:19021511

  16. Alkanes-filled photonic crystal fibers as sensor transducers

    NASA Astrophysics Data System (ADS)

    Marć, P.; Przybysz, N.; Stasiewicz, K.; Jaroszewicz, L. R.

    2015-09-01

    In this paper we propose alkanes-filled PCFs as the new class of transducers for optical fiber sensors. We investigated experimentally thermo-optic properties of a commercially available LMA8 partially filled with different alkanes with a higher number of carbon atoms. A partially filled PCF spliced with standard SMFs constitutes one of the newest type transducer. We have selected a group of eight alkanes which have melting points in different temperatures. An analysis of temperature spectral characteristics of these samples will allow to design an optical fiber sensor with different temperature thresholds at specific wavelengths.

  17. Abnormal carbon and hydrogen isotopes of alkane gases from the Qingshen gas field, Songliao Basin, China, suggesting abiogenic alkanes?

    NASA Astrophysics Data System (ADS)

    Liu, Quanyou; Dai, Jinxing; Jin, Zhijun; Li, Jian; Wu, Xiaoqi; Meng, Qingqiang; Yang, Chun; Zhou, Qinghua; Feng, Zihui; Zhu, Dongya

    2016-01-01

    It is great debate that the alkane gases of abiogenic origin would constitute a major portion of the commercial accumulation of the Qingshen gas field, Songliao Basin, China. In this study, abiogenic gases characterized by heavy δ13C1 values, reversal of the usual carbon isotopic trend of C1-C5 alkanes, very narrow variation in δ2HC1 values, and low CH4/3He ratios associated with high R/Ra values (>1) were identified. The hydrocarbon gas in the Qingshen gas field is a mixture of thermogenic alkanes derived from Cretaceous mudstone (type I kerogen) or Jurassic coal (type III kerogen) and abiogenic alkanes (mainly CH4) from mantle degassing. A quantitative estimation of abiogenic alkanes contribution to the Qingshen gas field is made based on a δ13C1 vs. δ13C2 plot: about 30-40% of alkane gases in the Qingshen gas field, along with its helium, are estimated to be derived from the mantle via magmatic activity. Particularly, the abiogenic formation of CH4 generated from the reduction of CO2 by hydrothermal activity may contribute. Our study suggests that abiogenic alkane gases in certain geological settings could be more widespread than previously thought, and may accumulate into economic reservoirs.

  18. Role of flavin-containing monooxygenase in drug development.

    PubMed

    Cashman, John R

    2008-12-01

    This review summarizes some recent observations and information related to the role of the flavin-containing monooxygenase (FMO) in preclinical drug development. Flavin-containing monooxygenase is a complimentary enzyme system to the cytochrome P450 (CYP) family of enzymes and oxygenates several soft, highly polarizable nucleophilic heteroatom-containing chemicals and drugs. The products of FMO-mediated metabolism are generally benign and highly polar, readily excreted materials. There may be some advantages in designing drugs that are metabolized in part by FMO and not exclusively by CYP. In this review, I describe the practical aspects for the participation of FMO in drug and chemical metabolism including: i) the study of FMO using in vitro preparations; ii) some observations about metabolism of drugs and chemicals by FMO in vivo; and iii) the consequences of studying FMO-related metabolism in various small animal models. Some of the preclinical research and development areas related to FMO are not fully mature areas and there are certain gaps in our knowledge. However, I include discussion of these areas to stimulate further work and invite further discussion. PMID:19040327

  19. The methane monooxygenase intrinsic activity of kinds of methanotrophs.

    PubMed

    Zhang, Yingxin; Xin, Jiaying; Chen, Linlin; Xia, Chungu

    2009-06-01

    Methanotrophs have promising applications in the epoxidation of some alkenes and some chlorinated hydrocarbons and in the production of a biopolymer, poly-beta-hydroxybutyrate (poly-3-hydroxybutyrate; PHB). In contrast with methane monooxygenase (MMO) activity and ability of PHB synthesis of four kinds of methanotrophic bacteria Methylosinus trichosporium OB3b, M. trichosporium IMV3011, Methylococcus capsulatus HD6T, Methylomonas sp. GYJ3, and the mixture of the four kinds of strains, M. trichosporium OB3b is the highest of the four in the activity of propene epoxidation (10.72 nmol/min mg dry weight of cell [dwc]), the activity of naphthalene oxidation (22.7 mmol/mg dwc), and ability in synthesis of PHB(11% PHB content in per gram dry weight of cell in 84 h). It could be feasible to improve the MMO activity by mixing four kinds of methanotrophs. The MMO activity dramatically decreased when the cellular PHB accumulated in the second stage. The reason for this may be the dilution of the MMO system in the cells with increasing PHB contents. It has been found that the PHB contents at the level of 1-5% are beneficial to the cells for maintenance of MMO epoxidation activity when enough PHB have been accumulated. Moreover, it was also found that high particulate methane monooxygenase activity may contribute to the synthesis of PHB in the cell, which could be used to improve the yield of PHB in methanotrophs. PMID:19052919

  20. Process for converting light alkanes to higher hydrocarbons

    DOEpatents

    Noceti, Richard P.; Taylor, Charles E.

    1988-01-01

    A process is disclosed for the production of aromatic-rich, gasoline boiling range hydrocarbons from the lower alkanes, particularly from methane. The process is carried out in two stages. In the first, alkane is reacted with oxygen and hydrogen chloride over an oxyhydrochlorination catalyst such as copper chloride with minor proportions of potassium chloride and rare earth chloride. This produces an intermediate gaseous mixture containing water and chlorinated alkanes. The chlorinated alkanes are contacted with a crystalline aluminosilicate catalyst in the hydrogen or metal promoted form to produce gasoline range hydrocarbons with a high proportion of aromatics and a small percentage of light hydrocarbons (C.sub.2 -C.sub.4). The light hydrocarbons can be recycled for further processing over the oxyhydrochlorination catalyst.

  1. Contribution of cyanobacterial alkane production to the ocean hydrocarbon cycle.

    PubMed

    Lea-Smith, David J; Biller, Steven J; Davey, Matthew P; Cotton, Charles A R; Perez Sepulveda, Blanca M; Turchyn, Alexandra V; Scanlan, David J; Smith, Alison G; Chisholm, Sallie W; Howe, Christopher J

    2015-11-01

    Hydrocarbons are ubiquitous in the ocean, where alkanes such as pentadecane and heptadecane can be found even in waters minimally polluted with crude oil. Populations of hydrocarbon-degrading bacteria, which are responsible for the turnover of these compounds, are also found throughout marine systems, including in unpolluted waters. These observations suggest the existence of an unknown and widespread source of hydrocarbons in the oceans. Here, we report that strains of the two most abundant marine cyanobacteria, Prochlorococcus and Synechococcus, produce and accumulate hydrocarbons, predominantly C15 and C17 alkanes, between 0.022 and 0.368% of dry cell weight. Based on global population sizes and turnover rates, we estimate that these species have the capacity to produce 2-540 pg alkanes per mL per day, which translates into a global ocean yield of ∼ 308-771 million tons of hydrocarbons annually. We also demonstrate that both obligate and facultative marine hydrocarbon-degrading bacteria can consume cyanobacterial alkanes, which likely prevents these hydrocarbons from accumulating in the environment. Our findings implicate cyanobacteria and hydrocarbon degraders as key players in a notable internal hydrocarbon cycle within the upper ocean, where alkanes are continually produced and subsequently consumed within days. Furthermore we show that cyanobacterial alkane production is likely sufficient to sustain populations of hydrocarbon-degrading bacteria, whose abundances can rapidly expand upon localized release of crude oil from natural seepage and human activities. PMID:26438854

  2. Contribution of cyanobacterial alkane production to the ocean hydrocarbon cycle

    PubMed Central

    Lea-Smith, David J.; Biller, Steven J.; Davey, Matthew P.; Cotton, Charles A. R.; Perez Sepulveda, Blanca M.; Turchyn, Alexandra V.; Scanlan, David J.; Smith, Alison G.; Chisholm, Sallie W.; Howe, Christopher J.

    2015-01-01

    Hydrocarbons are ubiquitous in the ocean, where alkanes such as pentadecane and heptadecane can be found even in waters minimally polluted with crude oil. Populations of hydrocarbon-degrading bacteria, which are responsible for the turnover of these compounds, are also found throughout marine systems, including in unpolluted waters. These observations suggest the existence of an unknown and widespread source of hydrocarbons in the oceans. Here, we report that strains of the two most abundant marine cyanobacteria, Prochlorococcus and Synechococcus, produce and accumulate hydrocarbons, predominantly C15 and C17 alkanes, between 0.022 and 0.368% of dry cell weight. Based on global population sizes and turnover rates, we estimate that these species have the capacity to produce 2–540 pg alkanes per mL per day, which translates into a global ocean yield of ∼308–771 million tons of hydrocarbons annually. We also demonstrate that both obligate and facultative marine hydrocarbon-degrading bacteria can consume cyanobacterial alkanes, which likely prevents these hydrocarbons from accumulating in the environment. Our findings implicate cyanobacteria and hydrocarbon degraders as key players in a notable internal hydrocarbon cycle within the upper ocean, where alkanes are continually produced and subsequently consumed within days. Furthermore we show that cyanobacterial alkane production is likely sufficient to sustain populations of hydrocarbon-degrading bacteria, whose abundances can rapidly expand upon localized release of crude oil from natural seepage and human activities. PMID:26438854

  3. Analysis of the gene cluster encoding toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1

    SciTech Connect

    Bertoni, G.; Martino, M.; Galli, E.; Barbieri, P.

    1998-10-01

    The toluene/o-xylene monooxygenase cloned from Pseudomonas stutzeri OX1 displays a very broad range of substrates and a very peculiar regioselectivity, because it is able to hydroxylate more than one position on the aromatic ring of several hydrocarbons and phenols. The nucleotide sequence of the gene cluster coding for this enzymatic system has been determined. The sequence analysis revealed the presence of six open reading frames (ORFs) homologous to other genes clustered in operons coding for multicomponent monooxygenases found in benzene- and toluene-degradative pathways cloned from Pseudomonas strains. Significant similarities were also found with multicomponent monooxygenase systems for phenol, methane, alkene, and dimethyl sulfide cloned from different bacterial strains. The knockout of each ORF and complementation with the wild-type allele indicated that all six ORFs are essential for the full activity of the toluene/o-xylene monooxygenase in Escherichia coli. This analysis also shows that despite its activity on both hydrocarbons and phenols, toluene/o-xylene monooxygenase belongs to a toluene multicomponent monooxygenase subfamily rather than to the monooxygenases active on phenols.

  4. Identification and Characterization of the CYP52 Family of Candida tropicalis ATCC 20336, Important for the Conversion of Fatty Acids and Alkanes to α,ω-Dicarboxylic Acids

    PubMed Central

    Craft, David L.; Madduri, Krishna M.; Eshoo, Mark; Wilson, C. Ron

    2003-01-01

    Candida tropicalis ATCC 20336 excretes α,ω-dicarboxylic acids as a by-product when cultured on n-alkanes or fatty acids as the carbon source. Previously, a β-oxidation-blocked derivative of ATCC 20336 was constructed which showed a dramatic increase in the production of dicarboxylic acids. This paper describes the next steps in strain improvement, which were directed toward the isolation and characterization of genes encoding the ω-hydroxylase enzymes catalyzing the first step in the ω-oxidation pathway. Cytochrome P450 monooxygenase (CYP) and the accompanying NADPH cytochrome P450 reductase (NCP) constitute the hydroxylase complex responsible for the first and rate-limiting step of ω-oxidation of n-alkanes and fatty acids. 10 members of the alkane-inducible P450 gene family (CYP52) of C. tropicalis ATCC20336 as well as the accompanying NCP were cloned and sequenced. The 10 CYP genes represent four unique genes with their putative alleles and two unique genes for which no allelic variant was identified. Of the 10 genes, CYP52A13 and CYP52A14 showed the highest levels of mRNA induction, as determined by quantitative competitive reverse transcription-PCR during fermentation with pure oleic fatty acid (27-fold increase), pure octadecane (32-fold increase), and a mixed fatty acid feed, Emersol 267 (54-fold increase). The allelic pair CYP52A17 and CYP52A18 was also induced under all three conditions but to a lesser extent. Moderate induction of CYP52A12 was observed. These results identify the CYP52 and NCP genes as being involved in α,ω-dicarboxylic acid production by C. tropicalis and provide the foundation for biocatalyst improvement. PMID:14532053

  5. Determinants of regioselective hydroxylation in the fungal polysaccharide monooxygenases.

    PubMed

    Vu, Van V; Beeson, William T; Phillips, Christopher M; Cate, Jamie H D; Marletta, Michael A

    2014-01-15

    The ubiquitous fungal polysaccharide monooxygenases (PMOs) (also known as GH61 proteins, LPMOs, and AA9 proteins) are structurally related but have significant variation in sequence. A heterologous expression method in Neurospora crassa was developed as a step toward connecting regioselectivity of the chemistry to PMO phylogeny. Activity assays, as well as sequence and phylogenetic analyses, showed that the majority of fungal PMOs fall into three major groups with distinctive active site surface features. PMO1s and PMO2s hydroxylate glycosidic positions C1 and C4, respectively. PMO3s hydroxylate both C1 and C4. A subgroup of PMO3s (PMO3*) hydroxylate C1. Mutagenesis studies showed that an extra subdomain of about 12 amino acids contribute to C4 oxidation in the PMO3 family. PMID:24350607

  6. Quantum Chemical Studies of Methane Monooxygenase: Comparison with P450

    SciTech Connect

    Guallar, Victor; Gherman, Benjamin F.; Lippard, Stephen J.; Friesner, Richard A.

    2002-04-01

    The catalytic pathways of soluble methane monooxygenase (sMMO) and cytochrome P450CAM, iron-containing enzymes, are described and compared. Recent extensive density functional ab initio electronic structure calculations have revealed many similarities in a number of the key catalytic steps, as well as some important differences. A particularly interesting and significant contrast is the role played by the protein in each system. For sMMO, the protein stabilizes various species in the catalytic cycle through a series of carboxylate shifts. This process is adequately described by a relatively compact model of the active site (similar to100 atoms), providing a reasonable description of the energetics of hydrogen atom abstraction. For P450CAM, in contrast, the inclusion of the full protein is necessary for an accurate description of the hydrogen atom abstraction.

  7. [Association of kynurenine-3-monooxygenase gene with schizophrenia].

    PubMed

    Golimbet, V E; Lezheiko, T V; Alfimova, M V; Abramova, L I; Kondrat'ev, N V

    2014-06-01

    Neurotoxic products produced during tryptophan metabolism via the kynurenine pathway could be involved in schizophrenia pathogenesis. It has been shown that kynurenine-3-monooxygenase (KMO) is indirectly involved in these products' formation. KMO polymorphic loci rs2275163 (C/T) and rs1053230 (A/G) were examined in 187 schizophrenia patients and 229 healthy subjects. A genetic combination of allele T and genotype GG was observed more often in a patient group compared with healthy controls (p = 0.003, OR 2.0 (95% CI 1.2-2.9). In the latter group, this combination was associated with schizophrenia endophenotype (p = 0.04), which manifested in a higher expression of schizotypal personality traits assessed using the MMPI test. PMID:25715464

  8. Kynurenine-3-monooxygenase: a review of structure, mechanism, and inhibitors.

    PubMed

    Smith, Jason R; Jamie, Joanne F; Guillemin, Gilles J

    2016-02-01

    Kynurenine monooxygenase (KMO) is an enzyme of the kynurenine (Kyn) pathway (KP), which is the major catabolic route of tryptophan. Kyn represents a branch point of the KP, being converted into the neurotoxin 3-hydroxykynurenine via KMO, neuroprotectant kynurenic acid, and anthranilic acid. As a result of this branch point, KMO is an attractive drug target for several neurodegenerative and/or neuroinflammatory diseases, especially Huntington's (HD), Alzheimer's (AD), and Parkinson's (PD) diseases. Although a neurological target, administration of KMO inhibitors in the periphery has demonstrated promising pharmacological results. In light of a recent crystal structure release and reports of preclinical candidates, here we provide a concise yet comprehensive update on the current state of research into the enzymology of KMO and related drug discovery efforts, highlighting areas where further work is required. PMID:26589832

  9. Acinetobacter cyclohexanone monooxygenase: gene cloning and sequence determination.

    PubMed Central

    Chen, Y C; Peoples, O P; Walsh, C T

    1988-01-01

    The gene coding for cyclohexanone monooxygenase from Acinetobacter sp. strain NCIB 9871 was isolated by immunological screening methods. We located and determined the nucleotide sequence of the gene. The structural gene is 1,626 nucleotides long and codes for a polypeptide of 542 amino acids; 389 nucleotides 5' and 108 nucleotides 3' of the coding region are also reported. The complete amino acid sequence of the enzyme was derived by translation of the nucleotide sequence. From a comparison of the amino acid sequence with consensus sequences of nucleotide-binding folds, we identified a potential flavin-binding site at the NH2 terminus of the enzyme (residues 6 to 18) and a potential nicotinamide-binding site extending from residue 176 to residue 208 of the protein. An overproduction system for the gene to facilitate genetic manipulations was also constructed by using the tac promoter vector pKK223-3 in Escherichia coli. Images PMID:3338974

  10. Aryl hydrocarbon mono-oxygenase activity in human lymphocytes

    SciTech Connect

    Griffin, G.D.; Schuresko, D.D.

    1981-06-01

    Aryl hydrocarbon mono-oxygenase (AHM), an enzyme of key importance in metabolism of xenobiotic chemicals such as polynuclear aromatic hydrocarbons (PNA), is present in human lymphocytes. Studies investing the relation of activity of AHM in human lymphocytes to parameters such as disease state, PNA exposure, in vitro mitogen stimulation, etc. have been summarized in this report. Some studies have demonstrated increased AHM activity in lymphocytes from cigarette smokers (compared to nonsmokers), and in lung cancer patients when compared to appropriate control groups. These observations are confused by extreme variability in human lymphocyte AHM activities, such variability arising from factors such as genetic variation in AHM activity, variation in in vitro culture conditions which affect AHM activity, and the problematical relationship of common AHM assays to actual PNA metabolism taking place in lymphocytes. If some of the foregoing problems can be adequately addressed, lymphocyte AHM activity could hold the promise of being a useful biomarker system for human PNA exposure.

  11. Selective hydroxylation of alkanes by an extracellular fungal peroxygenase.

    PubMed

    Peter, Sebastian; Kinne, Matthias; Wang, Xiaoshi; Ullrich, René; Kayser, Gernot; Groves, John T; Hofrichter, Martin

    2011-10-01

    Fungal peroxygenases are novel extracellular heme-thiolate biocatalysts that are capable of catalyzing the selective monooxygenation of diverse organic compounds, using only H(2)O(2) as a cosubstrate. Little is known about the physiological role or the catalytic mechanism of these enzymes. We have found that the peroxygenase secreted by Agrocybe aegerita catalyzes the H(2)O(2)-dependent hydroxylation of linear alkanes at the 2-position and 3-position with high efficiency, as well as the regioselective monooxygenation of branched and cyclic alkanes. Experiments with n-heptane and n-octane showed that the hydroxylation proceeded with complete stereoselectivity for the (R)-enantiomer of the corresponding 3-alcohol. Investigations with a number of model substrates provided information about the route of alkane hydroxylation: (a) the hydroxylation of cyclohexane mediated by H(2)(18)(2) resulted in complete incorporation of (18)O into the hydroxyl group of the product cyclohexanol; (b) the hydroxylation of n-hexane-1,1,1,2,2,3,3-D(7) showed a large intramolecular deuterium isotope effect [(k(H)/k(D))(obs)] of 16.0 ± 1.0 for 2-hexanol and 8.9 ± 0.9 for 3-hexanol; and (c) the hydroxylation of the radical clock norcarane led to an estimated radical lifetime of 9.4 ps and an oxygen rebound rate of 1.06 × 10(11) s(-1). These results point to a hydrogen abstraction and oxygen rebound mechanism for alkane hydroxylation. The peroxygenase appeared to lack activity on long-chain alkanes (> C(16)) and highly branched alkanes (e.g. tetramethylpentane), but otherwise exhibited a broad substrate range. It may accordingly have a role in the bioconversion of natural and anthropogenic alkane-containing structures (including alkyl chains of complex biomaterials) in soils, plant litter, and wood. PMID:21812933

  12. Monooxygenase Substrates Mimic Flavin to Catalyze Cofactorless Oxygenations.

    PubMed

    Machovina, Melodie M; Usselman, Robert J; DuBois, Jennifer L

    2016-08-19

    Members of the antibiotic biosynthesis monooxygenase family catalyze O2-dependent oxidations and oxygenations in the absence of any metallo- or organic cofactor. How these enzymes surmount the kinetic barrier to reactions between singlet substrates and triplet O2 is unclear, but the reactions have been proposed to occur via a flavin-like mechanism, where the substrate acts in lieu of a flavin cofactor. To test this model, we monitored the uncatalyzed and enzymatic reactions of dithranol, a substrate for the nogalamycin monooxygenase (NMO) from Streptomyces nogalater As with flavin, dithranol oxidation was faster at a higher pH, although the reaction did not appear to be base-catalyzed. Rather, conserved asparagines contributed to suppression of the substrate pKa The same residues were critical for enzymatic catalysis that, consistent with the flavoenzyme model, occurred via an O2-dependent slow step. Evidence for a superoxide/substrate radical pair intermediate came from detection of enzyme-bound superoxide during turnover. Small molecule and enzymatic superoxide traps suppressed formation of the oxygenation product under uncatalyzed conditions, whereas only the small molecule trap had an effect in the presence of NMO. This suggested that NMO both accelerated the formation and directed the recombination of a superoxide/dithranyl radical pair. These catalytic strategies are in some ways flavin-like and stand in contrast to the mechanisms of urate oxidase and (1H)-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase, both cofactor-independent enzymes that surmount the barriers to direct substrate/O2 reactivity via markedly different means. PMID:27307041

  13. Effects of bromocriptine on hepatic cytochrome P-450 monooxygenase system.

    PubMed

    Moochhala, S M; Lee, E J; Hu, G T; Koh, O S; Becket, G

    1989-02-01

    We have evaluated the in vitro effects of bromocriptine (Br), on the hepatic cytochrome P-450 monooxygenase system of rats pretreated with saline phenobarbitone (PB) and beta-naphthoflavone (BNF). Br inhibited ethoxyresorufin O-dealkylase (EROD) activity in liver microsomes of rats pretreated with saline and PB but not in BNF pretreated animals. Maximum inhibition of EROD activity by Br in the microsomes of saline and PB pretreated rats were 50%-60% of the control. In contrast, a dual effect was observed on aminopyrine N-demethylase activity (APD) by Br in microsomes of saline, PB and BNF pretreated rats. At a low concentration (25 microM), Br inhibited the activity of APD to a similar extent in all pretreatment groups; however, with higher concentrations of Br (50 microM to 300 microM), enhancement of APD activity was observed. Br (300 microM) increased the APD activity to 2-3 times the control level in microsomes of rats pretreated with saline, PB or BNF. Spectral studies revealed a Type II binding of Br to cytochrome P-450 from microsomes of saline and PB pretreated rats. A reverse type I binding was observed for BNF induced microsomes. In addition, Br also enhanced NADPH cytochrome c (P-450) reductase activity to a similar extent in all pretreatment groups. These results suggest that the inhibition of EROD activity may be due to direct binding by Br to certain isozymes of cytochrome P-450 and that the enhancing effect of Br on APD activity may be in part due to the activation of the NADPH cytochrome c reductase component of the cytochrome P-450 monooxygenase system. PMID:2499727

  14. Variation in n-Alkane Distributions of Modern Plants: Questioning Applications of n-Alkanes in Chemotaxonomy and Paleoecology

    NASA Astrophysics Data System (ADS)

    Bush, R. T.; McInerney, F. A.

    2010-12-01

    Long chain n-alkanes (n-C21 to n-C37) are synthesized as part of the epicuticular leaf wax of terrestrial plants and are among the most recognizable and widely used plant biomarkers. n-Alkane distributions have been utilized in previous studies on modern plant chemotaxonomy, testing whether taxa can be identified based on characteristic n-alkane profiles. Dominant n-alkanes (e.g. n-C27 or n-C31) have also been ascribed to major plant groups (e.g. trees or grasses respectively) and have been used in paleoecology studies to reconstruct fluctuations in plant functional types. However, many of these studies have been based on relatively few modern plant data; with the wealth of modern n-alkane studies, a more comprehensive analysis of n-alkanes in modern plants is now possible and can inform the usefulness of n-alkane distributions as paleoecological indicators. The work presented here is a combination of measurements made using plant leaves collected from the Chicago Botanic Garden and a compilation of published literature data from six continents. We categorized plants by type: angiosperms, gymnosperms, woody plants, forbs, grasses, ferns and pteridophytes, and mosses. We then quantified n-alkane distribution parameters such as carbon preference index (CPI), average chain length (ACL), and dispersion (a measure of the spread of the profile over multiple chain lengths) and used these to compare plant groups. Among all plants, one of the emergent correlations is a decrease in dispersion with increasing CPI. Within and among plant groups, n-alkane distributions show a very large range of variation, and the results show little or no correspondence between broad plant groups and a single dominant n-alkane or a ratio of n-alkanes. These findings are true both when data from six continents are combined and when plants from a given region are compared (North America). We also compared the n-alkane distributions of woody angiosperms, woody gymnosperms, and grasses with one

  15. Metabolism of Hydrocarbons in n-Alkane-Utilizing Anaerobic Bacteria.

    PubMed

    Wilkes, Heinz; Buckel, Wolfgang; Golding, Bernard T; Rabus, Ralf

    2016-01-01

    The glycyl radical enzyme-catalyzed addition of n-alkanes to fumarate creates a C-C-bond between two concomitantly formed stereogenic carbon centers. The configurations of the two diastereoisomers of the product resulting from n-hexane activation by the n-alkane-utilizing denitrifying bacterium strain HxN1, i.e. (1-methylpentyl)succinate, were assigned as (2S,1'R) and (2R,1'R). Experiments with stereospecifically deuterated n-(2,5-2H2)hexanes revealed that exclusively the pro-S hydrogen atom is abstracted from C2 of the n-alkane by the enzyme and later transferred back to C3 of the alkylsuccinate formed. These results indicate that the alkylsuccinate-forming reaction proceeds with an inversion of configuration at the carbon atom (C2) of the n-alkane forming the new C-C-bond, and thus stereochemically resembles a SN2-type reaction. Therefore, the reaction may occur in a concerted manner, which may avoid the highly energetic hex-2-yl radical as an intermediate. The reaction is associated with a significant primary kinetic isotope effect (kH/kD ≥3) for hydrogen, indicating that the homolytic C-H-bond cleavage is involved in the first irreversible step of the reaction mechanism. The (1-methylalkyl)succinate synthases of n-alkane-utilizing anaerobic bacteria apparently have very broad substrate ranges enabling them to activate not only aliphatic but also alkyl-aromatic hydrocarbons. Thus, two denitrifiers and one sulfate reducer were shown to convert the nongrowth substrate toluene to benzylsuccinate and further to the dead-end product benzoyl-CoA. For this purpose, however, the modified β-oxidation pathway known from alkylbenzene-utilizing bacteria was not employed, but rather the pathway used for n-alkane degradation involving CoA ligation, carbon skeleton rearrangement and decarboxylation. Furthermore, various n-alkane- and alkylbenzene-utilizing denitrifiers and sulfate reducers were found to be capable of forming benzyl alcohols from diverse alkylbenzenes

  16. Detailed chemical kinetic models for large n-alkanes and iso-alkanes found in conventional and F-T diesel fuels

    SciTech Connect

    Westbrook, C K; Pitz, W J; Curran, H J; Mehl, M

    2008-12-15

    Detailed chemical kinetic models are needed to simulate the combustion of current and future transportation fuels. These models should represent the various chemical classes in these fuels. Conventional diesel fuels are composed of n-alkanes, iso-alkanes, cycloalkanes and aromatics (Farrell et al. 2007). For future fuels, there is a renewed interest in Fischer-Tropsch (F-T) processes which can be used to synthesize diesel and other transportation fuels from biomass, coal and natural gas. F-T diesel fuels are expected to be similar to F-T jet fuels which are commonly comprised of iso-alkanes with some n-alkanes (Smith and Bruno, 2008). Thus, n-alkanes and iso-alkanes are common chemical classes in these conventional and future fuels. This paper reports on the development of chemical kinetic models of large n-alkanes and iso-alkanes to represent these chemical classes in conventional and future fuels. Two large iso-alkanes are 2,2,4,4,6,8,8-heptamethylnonane, which is a primary reference fuel for diesel, and isooctane, a primary reference fuel for gasoline. Other iso-alkanes are branched alkanes with a single methyl side chain, typical of most F-T fuels. The chemical kinetic models are then used to predict the effect of these fuel components on ignition characteristics under conditions found in internal combustion engines.

  17. Alkane inducible proteins in Geobacillus thermoleovorans B23

    PubMed Central

    2009-01-01

    Background Initial step of β-oxidation is catalyzed by acyl-CoA dehydrogenase in prokaryotes and mitochondria, while acyl-CoA oxidase primarily functions in the peroxisomes of eukaryotes. Oxidase reaction accompanies emission of toxic by-product reactive oxygen molecules including superoxide anion, and superoxide dismutase and catalase activities are essential to detoxify them in the peroxisomes. Although there is an argument about whether primitive life was born and evolved under high temperature conditions, thermophilic archaea apparently share living systems with both bacteria and eukaryotes. We hypothesized that alkane degradation pathways in thermophilic microorganisms could be premature and useful to understand their evolution. Results An extremely thermophilic and alkane degrading Geobacillus thermoleovorans B23 was previously isolated from a deep subsurface oil reservoir in Japan. In the present study, we identified novel membrane proteins (P16, P21) and superoxide dismutase (P24) whose production levels were significantly increased upon alkane degradation. Unlike other bacteria acyl-CoA oxidase and catalase activities were also increased in strain B23 by addition of alkane. Conclusion We first suggested that peroxisomal β-oxidation system exists in bacteria. This eukaryotic-type alkane degradation pathway in thermophilic bacterial cells might be a vestige of primitive living cell systems that had evolved into eukaryotes. PMID:19320977

  18. Diffusion of Benzene and Alkylbenzenes in n-Alkanes.

    PubMed

    Kowert, Bruce A; Register, Paul M

    2015-10-01

    The translational diffusion constants, D, of benzene and a series of alkylbenzenes have been determined in four n-alkanes at room temperature using capillary flow techniques. The alkylbenzenes are toluene, ethylbenzene, 1-phenylpropane, 1-phenylpentane, 1-phenyloctane, 1-phenylundecane, 1-phenyltetradecane, and 1-phenylheptadecane. The n-alkanes are n-nonane, n-decane, n-dodecane, and n-pentadecane. Ratios of the solutes' D values are independent of solvent and in general agreement with the predictions of diffusion models for cylinders and lollipops. For the latter, an alkylbenzene's phenyl ring is the lollipop's candy; the alkyl chain is its handle. A model that considers the solutes to be spheres with volumes determined by the van der Waals increments of their constituent atoms is not in agreement with experiment. The diffusion constants of 1-alkene and n-alkane solutes in n-alkane solvents also are compared with the cylinder model; reasonably good agreement is found. The n-alkanes are relatively extended, and this appears to be the case for the alkyl chains of the 1-alkenes and alkylbenzenes as well. PMID:26417941

  19. Trichloroethylene oxidation by purified toluene 2-monooxygenase: products, kinetics, and turnover-dependent inactivation.

    PubMed Central

    Newman, L M; Wackett, L P

    1997-01-01

    Trichloroethylene is oxidized by several types of nonspecific bacterial oxygenases. Toluene 2-monooxygenase from Burkholderia cepacia G4 is implicated in trichloroethylene oxidation and is uniquely suggested to be resistant to turnover-dependent inactivation in vivo. In this work, the oxidation of trichloroethylene was studied with purified toluene 2-monooxygenase. All three purified toluene 2-monooxygenase protein components and NADH were required to reconstitute full trichloroethylene oxidation activity in vitro. The apparent Km and Vmax were 12 microM and 37 nmol per min per mg of hydroxylase component, respectively. Ten percent of the full activity was obtained when the small-molecular-weight enzyme component was omitted. The stable oxidation products, accounting for 84% of the trichloroethylene oxidized, were carbon monoxide, formic acid, glyoxylic acid, and covalently modified oxygenase proteins that constituted 12% of the reacted [14C]trichloroethylene. The stable oxidation products may all derive from the unstable intermediate trichloroethylene epoxide that was trapped by reaction with 4-(p-nitrobenzyl)pyridine. Chloral hydrate and dichloroacetic acid were not detected. This finding differs from that with soluble methane monooxygenase and cytochrome P-450 monooxygenase, which produce chloral hydrate. Trichloroethylene-dependent inactivation of toluene 2-monooxygenase activity was observed. All of the protein components were covalently modified during the oxidation of trichloroethylene. The addition of cysteine to reaction mixtures partially protected the enzyme system against inactivation, most notably protecting the NADH-oxidoreductase component. This suggested the participation of diffusible intermediates in the inactivation of the oxidoreductase. PMID:8981984

  20. Alkanes in shrimp from the Buccaneer Oil Field

    SciTech Connect

    Middleditch, B.S.; Basile, B.; Chang, E.S.

    1982-07-01

    A total of 36 samples of shrimp were examined from the region of the Buccaneer oil field, eighteen of which were representatives of the commercial species Penaeus aztecus and the rest were various other species: Penaeus duorarum (pink shrimp), Trachypenaeus duorarum (sugar shrimp), Squilla empusa (mantis shrimp), and Sicyonia dorsalis (chevron shrimp). The alkanes and deuteriated alkanes were completely separated by GC, so a mass spectrometer was not required for their detection and quantitation. To confirm the identities of individual compounds, however, some samples were examined by combined gas chromatography-mass spectrometry. Results show that only thirteen of the forty shrimp collected from the region of the Buccaneer oil field contained petroleum alkanes, and the majority of these were obtained from trawls immediately adjacent to the production platforms. It appears that shrimp caught in the region of the Buccaneer oil field are not appreciably tainted with hydrocarbons discharged from the production platforms. (JMT)

  1. High Temperature Chemical Kinetic Combustion Modeling of Lightly Methylated Alkanes

    SciTech Connect

    Sarathy, S M; Westbrook, C K; Pitz, W J; Mehl, M

    2011-03-01

    Conventional petroleum jet and diesel fuels, as well as alternative Fischer-Tropsch (FT) fuels and hydrotreated renewable jet (HRJ) fuels, contain high molecular weight lightly branched alkanes (i.e., methylalkanes) and straight chain alkanes (n-alkanes). Improving the combustion of these fuels in practical applications requires a fundamental understanding of large hydrocarbon combustion chemistry. This research project presents a detailed high temperature chemical kinetic mechanism for n-octane and three lightly branched isomers octane (i.e., 2-methylheptane, 3-methylheptane, and 2,5-dimethylhexane). The model is validated against experimental data from a variety of fundamental combustion devices. This new model is used to show how the location and number of methyl branches affects fuel reactivity including laminar flame speed and species formation.

  2. BIODEGRADATION AND GAS-EXCHANGE OF GASEOUS ALKANES IN MODEL ESTUARINE ECOSYSTEMS

    EPA Science Inventory

    Gas exchange-biodegradation experiments conducted in model estuarine ecosystems indicate that the ease of degradation of gaseious normal alkanes increases with chain length. The behavior of gaseous perhalogenated alkanes can be explained by gas exchange alone with no degradation....

  3. Modeling of alkane emissions from a wood stain

    SciTech Connect

    Chang, J.C.S.; Guo, Z.

    1993-01-01

    The article discusses full-scale residential house tests to evaluate the effects of organic emissions from a wood finishing product--wood stain--on indoor air quality (IAQ). The test house concentrations of three alkane species, nonane, decane, and undecane, were measured as a function of time after the application of the wood stain. It was found that the test house concentrations can be simulated by an integrated IAQ model which takes into consideration source, sink, and ventilation effects. The alkane emissions were controlled by an evaporation-like process.

  4. Catalytic, mild, and selective oxyfunctionalization of linear alkanes: current challenges.

    PubMed

    Bordeaux, Mélanie; Galarneau, Anne; Drone, Jullien

    2012-10-22

    Selective catalysts for sustainable oxidation of alkanes are highly demanded because of the abundance of these molecules in the environment, the possibility to transform them into higher-value compounds, such as chemicals or synthetic fuels, and the fact that, kinetically speaking, this is a difficult reaction. Numerous chemical and biological catalysts have been developed in the lasts decades for this purpose, rendering the overview over this field of chemistry difficult. After giving a definition of the ideal catalyst for alkane oxyfunctionalization, this review aims to present the catalysts available today that are closest to ideal. PMID:22996726

  5. A nonequilibrium molecular dynamics study of the rheology of alkanes

    SciTech Connect

    Gupta, S.A.; Cui, S.T.; Cummings, P.T.; Cochran, H.D. |

    1996-05-01

    We examine the rheological properties of four different alkanes: n-decane, n-hexadecane, n-tetracosane, and squalane. Simulations of Couette flow are performed for a range of shear rates with 100 molecules in each case using a replicated data version of our code. Number of interaction sites ranges from 1000 to 3000. We have performed extremely long simulations required to obtain acceptable statistics at low shear rates. The alkanes show a transition from non-Newtonian to Newtonian behavior as the shear rate decreases to low values. 1 tab, 1 fig, 17 refs.

  6. Assimilation of chlorinated alkanes by hydrocarbon-utilizing fungi

    SciTech Connect

    Murphy, G.L.; Perry, J.J.

    1984-12-01

    The fatty acid compositions of two filamentous fungi (Cunninghamella elegans and Penicillium zonatum) and a yeast (Candida lipolytica) were determined after the organisms were grown on 1-chlorohexadecane or 1-chlorooctadecane. These organisms utilized the chlorinated alkanes as sole sources of carbon and energy. Analyses of the fatty acids present after growth on the chlorinated alkanes indicated that 60 to 70% of the total fatty acids in C. elegans were chlorinated. Approximately 50% of the fatty acids in C. lipolytica were also chlorinated. P. zonatum contained 20% 1-chlorohexadecanoic acid after growth on either substrate but did not incorporate C/sub 18/ chlorinated fatty acids.

  7. Coupling Oxygen Consumption with Hydrocarbon Oxidation in Bacterial Multicomponent Monooxygenases.

    PubMed

    Wang, Weixue; Liang, Alexandria D; Lippard, Stephen J

    2015-09-15

    A fundamental goal in catalysis is the coupling of multiple reactions to yield a desired product. Enzymes have evolved elegant approaches to address this grand challenge. A salient example is the biological conversion of methane to methanol catalyzed by soluble methane monooxygenase (sMMO), a member of the bacterial multicomponent monooxygenase (BMM) superfamily. sMMO is a dynamic protein complex of three components: a hydroxylase, a reductase, and a regulatory protein. The active site, a carboxylate-rich non-heme diiron center, is buried inside the 251 kDa hydroxylase component. The enzyme processes four substrates: O2, protons, electrons, and methane. To couple O2 activation to methane oxidation, timely control of substrate access to the active site is critical. Recent studies of sMMO, as well as its homologues in the BMM superfamily, have begun to unravel the mechanism. The emerging and unifying picture reveals that each substrate gains access to the active site along a specific pathway through the hydroxylase. Electrons and protons are delivered via a three-amino-acid pore located adjacent to the diiron center; O2 migrates via a series of hydrophobic cavities; and hydrocarbon substrates reach the active site through a channel or linked set of cavities. The gating of these pathways mediates entry of each substrate to the diiron active site in a timed sequence and is coordinated by dynamic interactions with the other component proteins. The result is coupling of dioxygen consumption with hydrocarbon oxidation, avoiding unproductive oxidation of the reductant rather than the desired hydrocarbon. To initiate catalysis, the reductase delivers two electrons to the diiron(III) center by binding over the pore of the hydroxylase. The regulatory component then displaces the reductase, docking onto the same surface of the hydroxylase. Formation of the hydroxylase-regulatory component complex (i) induces conformational changes of pore residues that may bring protons to the

  8. 40 CFR 721.785 - Halogenated alkane aromatic compound (generic name).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Halogenated alkane aromatic compound... Specific Chemical Substances § 721.785 Halogenated alkane aromatic compound (generic name). (a) Chemical... as a halogenated alkane aromatic compound (PMN P-94-1747) is subject to reporting under this...

  9. 40 CFR 721.785 - Halogenated alkane aromatic compound (generic name).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Halogenated alkane aromatic compound... Specific Chemical Substances § 721.785 Halogenated alkane aromatic compound (generic name). (a) Chemical... as a halogenated alkane aromatic compound (PMN P-94-1747) is subject to reporting under this...

  10. 40 CFR 721.785 - Halogenated alkane aromatic compound (generic name).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Halogenated alkane aromatic compound... Specific Chemical Substances § 721.785 Halogenated alkane aromatic compound (generic name). (a) Chemical... as a halogenated alkane aromatic compound (PMN P-94-1747) is subject to reporting under this...

  11. 40 CFR 721.785 - Halogenated alkane aromatic compound (generic name).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Halogenated alkane aromatic compound... Specific Chemical Substances § 721.785 Halogenated alkane aromatic compound (generic name). (a) Chemical... as a halogenated alkane aromatic compound (PMN P-94-1747) is subject to reporting under this...

  12. 40 CFR 721.785 - Halogenated alkane aromatic compound (generic name).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Halogenated alkane aromatic compound... Specific Chemical Substances § 721.785 Halogenated alkane aromatic compound (generic name). (a) Chemical... as a halogenated alkane aromatic compound (PMN P-94-1747) is subject to reporting under this...

  13. Kynurenine 3-Monooxygenase: An Influential Mediator of Neuropathology

    PubMed Central

    Parrott, Jennifer M.; O’Connor, Jason C.

    2015-01-01

    Mounting evidence demonstrates that kynurenine metabolism may play an important pathogenic role in the development of multiple neurological and neuropsychiatric disorders. The kynurenine pathway consists of two functionally distinct branches that generate both neuroactive and oxidatively reactive metabolites. In the brain, the rate-limiting enzyme for one of these branches, kynurenine 3-monooxygenase (KMO), is predominantly expressed in microglia and has emerged as a pivotal point of metabolic regulation. KMO substrate and expression levels are upregulated by pro-inflammatory cytokines and altered by functional genetic mutations. Increased KMO metabolism results in the formation of metabolites that activate glutamate receptors and elevate oxidative stress, while recent evidence has revealed neurodevelopmental consequences of reduced KMO activity. Together, the evidence suggests that KMO is positioned at a critical metabolic junction to influence the development or trajectory of a myriad of neurological diseases. Understanding the mechanism(s) by which alterations in KMO activity are able to impair neuronal function, and viability will enhance our knowledge of related disease pathology and provide insight into novel therapeutic opportunities. This review will discuss the influence of KMO on brain kynurenine metabolism and the current understanding of molecular mechanisms by which altered KMO activity may contribute to neurodevelopment, neurodegenerative, and neuropsychiatric diseases. PMID:26347662

  14. Structural basis for pregnenolone biosynthesis by the mitochondrial monooxygenase system

    SciTech Connect

    Strushkevich, Natallia; MacKenzie, Farrell; Cherkesova, Tatyana; Grabovec, Irina; Usanov, Sergey; Park, Hee-Won

    2011-09-06

    In humans, the precursor to all steroid hormones, pregnenolone, is synthesized from cholesterol by an enzyme complex comprising adrenodoxin reductase (AdR), adrenodoxin (Adx), and a cytochrome P450 (P450scc or CYP11A1). This complex not only plays a key role in steroidogenesis, but also has long been a model to study electron transfer, multistep catalysis, and C-C bond cleavage performed by monooxygenases. Detailed mechanistic understanding of these processes has been hindered by a lack of structural information. Here we present the crystal structure of the complex of human Adx and CYP11A1 - the first of a complex between a eukaryotic CYP and its redox partner. The structures with substrate and a series of reaction intermediates allow us to define the mechanism underlying sequential hydroxylations of the cholesterol and suggest the mechanism of C-C bond cleavage. In the complex the [2Fe-2S] cluster of Adx is positioned 17.4 {angstrom} away from the heme iron of CYP11A1. This structure suggests that after an initial protein-protein association driven by electrostatic forces, the complex adopts an optimized geometry between the redox centers. Conservation of the interaction interface suggests that this mechanism is common for all mitochondrial P450s.

  15. Diversity and evolution of cytochrome P450 monooxygenases in Oomycetes

    PubMed Central

    Sello, Mopeli Marshal; Jafta, Norventia; Nelson, David R; Chen, Wanping; Yu, Jae-Hyuk; Parvez, Mohammad; Kgosiemang, Ipeleng Kopano Rosinah; Monyaki, Richie; Raselemane, Seiso Caiphus; Qhanya, Lehlohonolo Benedict; Mthakathi, Ntsane Trevor; Sitheni Mashele, Samson; Syed, Khajamohiddin

    2015-01-01

    Cytochrome P450 monooxygenases (P450s) are heme-thiolate proteins whose role as drug targets against pathogens, as well as in valuable chemical production and bioremediation, has been explored. In this study we performed comprehensive comparative analysis of P450s in 13 newly explored oomycete pathogens. Three hundred and fifty-six P450s were found in oomycetes. These P450s were grouped into 15 P450 families and 84 P450 subfamilies. Among these, nine P450 families and 31 P450 subfamilies were newly found in oomycetes. Research revealed that oomycetes belonging to different orders contain distinct P450 families and subfamilies in their genomes. Evolutionary analysis and sequence homology data revealed P450 family blooms in oomycetes. Tandem arrangement of a large number of P450s belonging to the same family indicated that P450 family blooming is possibly due to its members’ duplications. A unique combination of amino acid patterns was observed at EXXR and CXG motifs for the P450 families CYP5014, CYP5015 and CYP5017. A novel P450 fusion protein (CYP5619 family) with an N-terminal P450 domain fused to a heme peroxidase/dioxygenase domain was discovered in Saprolegnia declina. Oomycete P450 patterns suggested host influence in shaping their P450 content. This manuscript serves as reference for future P450 annotations in newly explored oomycetes. PMID:26129850

  16. Kynurenine 3-Monooxygenase: An Influential Mediator of Neuropathology.

    PubMed

    Parrott, Jennifer M; O'Connor, Jason C

    2015-01-01

    Mounting evidence demonstrates that kynurenine metabolism may play an important pathogenic role in the development of multiple neurological and neuropsychiatric disorders. The kynurenine pathway consists of two functionally distinct branches that generate both neuroactive and oxidatively reactive metabolites. In the brain, the rate-limiting enzyme for one of these branches, kynurenine 3-monooxygenase (KMO), is predominantly expressed in microglia and has emerged as a pivotal point of metabolic regulation. KMO substrate and expression levels are upregulated by pro-inflammatory cytokines and altered by functional genetic mutations. Increased KMO metabolism results in the formation of metabolites that activate glutamate receptors and elevate oxidative stress, while recent evidence has revealed neurodevelopmental consequences of reduced KMO activity. Together, the evidence suggests that KMO is positioned at a critical metabolic junction to influence the development or trajectory of a myriad of neurological diseases. Understanding the mechanism(s) by which alterations in KMO activity are able to impair neuronal function, and viability will enhance our knowledge of related disease pathology and provide insight into novel therapeutic opportunities. This review will discuss the influence of KMO on brain kynurenine metabolism and the current understanding of molecular mechanisms by which altered KMO activity may contribute to neurodevelopment, neurodegenerative, and neuropsychiatric diseases. PMID:26347662

  17. Inhibition of ammonia monooxygenase in Nitrosomonas europaea by carbon disulfide.

    PubMed Central

    Hyman, M R; Kim, C Y; Arp, D J

    1990-01-01

    Carbon disulfide has long been recognized as a potent inhibitor of nitrification, and it is the likely active component in several nitrification inhibitors suitable for field use. The effects of this compound on Nitrosomonas europaea have been investigated, and the site of action has been determined. Low concentrations of CS2 (less than 400 microM) produced a time-dependent inhibition of ammonia-dependent O2 uptake but did not inhibit hydrazine-oxidizing activity. CS2 also produced distinct changes in difference spectra of whole cells. These results suggest that ammonia monooxygenase (AMO) is the site of action of CS2. Unlike the case for thiourea and acetylene, saturating concentrations of CS2 did not fully inhibit AMO, and the inhibition resulted in a low but significant rate of ammonia-dependent O2 uptake. The effects of CS2 were not competitive with respect to ammonia concentration, and the inhibition by CS2 did not require the turnover of AMO to take effect. The ability of CS2-treated cells to incorporate [14C]acetylene into the 28-kilodalton polypeptide of AMO was used to demonstrate that the effects of CS2 are compatible with a mode of action which involves a reduction of the rate of turnover of AMO without effects on the catalytic mechanism. It is proposed that CS2 may act on AMO by reversibly reacting with a suitable nucleophilic amino acid in close proximity to the active site copper. Images PMID:2118501

  18. Mechanism of Action of a Flavin-Containing Monooxygenase

    SciTech Connect

    Eswaramoorthy,S.; Bonanno, J.; Burley, S.; Swaminathan, S.

    2006-01-01

    Elimination of nonnutritional and insoluble compounds is a critical task for any living organism. Flavin-containing monooxygenases (FMOs) attach an oxygen atom to the insoluble nucleophilic compounds to increase solubility and thereby increase excretion. Here we analyze the functional mechanism of FMO from Schizosaccharomyces pombe using the crystal structures of the wild type and protein-cofactor and protein-substrate complexes. The structure of the wild-type FMO revealed that the prosthetic group FAD is an integral part of the protein. FMO needs NADPH as a cofactor in addition to the prosthetic group for its catalytic activity. Structures of the protein-cofactor and protein-substrate complexes provide insights into mechanism of action. We propose that FMOs exist in the cell as a complex with a reduced form of the prosthetic group and NADPH cofactor, readying them to act on substrates. The 4{alpha}-hydroperoxyflavin form of the prosthetic group represents a transient intermediate of the monooxygenation process. The oxygenated and reduced forms of the prosthetic group help stabilize interactions with cofactor and substrate alternately to permit continuous enzyme turnover.

  19. Architecture and active site of particulate methane monooxygenase

    PubMed Central

    Culpepper, Megen A.; Rosenzweig, Amy C.

    2012-01-01

    Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria, organisms that live on methane gas as their sole carbon source. Understanding pMMO function has important implications for bioremediation applications and for the development of new, environmentally friendly catalysts for the direct conversion of methane to methanol. Crystal structures of pMMOs from three different methanotrophs reveal a trimeric architecture, consisting of three copies each of the pmoB, pmoA, and pmoC subunits. There are three distinct metal centers in each protomer of the trimer, mononuclear and dinuclear copper sites in the periplasmic regions of pmoB and a mononuclear site within the membrane that can be occupied by copper or zinc. Various models for the pMMO active site have been proposed within these structural constraints, including dicopper, tricopper, and diiron centers. Biochemical and spectroscopic data on pMMO and recombinant soluble fragments, denoted spmoB proteins, indicate that the active site involves copper and is located at the site of the dicopper center in the pmoB subunit. Initial spectroscopic evidence for O2 binding at this site has been obtained. Despite these findings, questions remain about the active site identity and nuclearity and will be the focus of future studies. PMID:22725967

  20. Effects of zinc on particulate methane monooxygenase activity and structure.

    PubMed

    Sirajuddin, Sarah; Barupala, Dulmini; Helling, Stefan; Marcus, Katrin; Stemmler, Timothy L; Rosenzweig, Amy C

    2014-08-01

    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. PMID:24942740

  1. Effects of Zinc on Particulate Methane Monooxygenase Activity and Structure*

    PubMed Central

    Sirajuddin, Sarah; Barupala, Dulmini; Helling, Stefan; Marcus, Katrin; Stemmler, Timothy L.; Rosenzweig, Amy C.

    2014-01-01

    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. PMID:24942740

  2. Assay, Purification, and Partial Characterization of Choline Monooxygenase from Spinach.

    PubMed Central

    Burnet, M.; Lafontaine, P. J.; Hanson, A. D.

    1995-01-01

    The osmoprotectant glycine betaine is synthesized via the path-way choline -> betaine aldehyde -> glycine betaine. In spinach (Spinacia oleracea), the first step is catalyzed by choline monooxygenase (CMO), and the second is catalyzed by betaine aldehyde dehydrogenase. Because betaine aldehyde is unstable and not easily detected, we developed a coupled radiometric assay for CMO. [14C]Choline is used as substrate; NAD+ and betaine aldehyde dehydrogenase prepared from Escherichia coli are added to oxidize [14C]betaine aldehyde to [14C]glycine betaine, which is isolated by ion exchange. The assay was used in the purification of CMO from leaves of salinized spinach. The 10-step procedure included polyethylene glycol precipitation, polyethyleneimine precipitation, hydrophobic interaction, anion exchange on choline-Sepharose, dimethyldiethanolamine-Sepharose, and Mono Q, hydroxyapatite, gel filtration, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Following gel filtration, overall purification was about 600-fold and recovery of activity was 0.5%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a polypeptide with a molecular mass of 45 kD. Taken with the value of 98 kD estimated for native CMO (R. Brouquisse, P. Weigel, D. Rhodes, C.F. Yocum, A.D. Hanson [1989] Plant Physiol 90: 322-329), this indicates that CMO is a homodimer. CMO preparations were red-brown, showed absorption maxima at 329 and 459 nm, and lost color upon dithionite addition, suggesting that CMO is an iron-sulfur protein. PMID:12228495

  3. Kynurenine 3-monooxygenase inhibition in blood ameliorates neurodegeneration

    PubMed Central

    Zwilling, Daniel; Huang, Shao-Yi; Sathyasaikumar, Korrapati V.; Notarangelo, Francesca M.; Guidetti, Paolo; Wu, Hui-Qiu; Lee, Jason; Truong, Jennifer; Andrews-Zwilling, Yaisa; Hsieh, Eric W.; Louie, Jamie Y.; Wu, Tiffany; Scearce-Levie, Kimberly; Patrick, Christina; Adame, Anthony; Giorgini, Flaviano; Moussaoui, Saliha; Laue, Grit; Rassoulpour, Arash; Flik, Gunnar; Huang, Yadong; Muchowski, Joseph M.; Masliah, Eliezer; Schwarcz, Robert; Muchowski, Paul J.

    2011-01-01

    SUMMARY Metabolites in the kynurenine pathway of tryptophan degradation are thought to play an important role in neurodegenerative disorders such as Alzheimer’s disease and Huntington’s disease. Metabolites that cause glutamate receptor-mediated excitotoxicity and free radical formation are elevated in the blood and vulnerable brain regions in these diseases, while levels of the neuroprotective metabolite kynurenic acid are often decreased. Here we describe the synthesis and characterization of JM6, a novel small-molecule pro-drug inhibitor of kynurenine 3-monooxygenase (KMO). JM6 raises kynurenic acid and reduces extracellular glutamate in the brain after chronic oral administration by inhibiting KMO in blood. In a transgenic mouse model of Alzheimer’s disease, JM6 prevented spatial memory deficits, anxiety-related behavior, and synaptic loss. JM6 also extended life span, prevented synaptic loss, and decreased microglial activation in a mouse model of Huntington’s disease. These findings support a critical link between blood cells and neurodegeneration that is mediated by KMO and the kynurenine pathway. PMID:21640374

  4. Activation of enzymatic chitin degradation by a lytic polysaccharide monooxygenase.

    PubMed

    Hamre, Anne Grethe; Eide, Kristine B; Wold, Hanne H; Sørlie, Morten

    2015-04-30

    For decades, the enzymatic conversion of recalcitrant polysaccharides such as cellulose and chitin was thought to solely rely on the synergistic action of hydrolytic enzymes, but recent work has shown that lytic polysaccharide monooxygenases (LPMOs) are important contributors to this process. Here, we have examined the initial rate enhancement an LPMO (CBP21) has on the hydrolytic enzymes (ChiA, ChiB, and ChiC) of the chitinolytic machinery of Serratia marcescens through determinations of apparent k(cat) (k(cat)(app)) values on a β-chitin substrate. k(cat)(app) values were determined to be 1.7±0.1 s(-1) and 1.7±0.1 s(-1) for the exo-active ChiA and ChiB, respectively and 1.2±0.1 s(-1) for the endo-active ChiC. The addition of CBP21 boosted the k(cat)(app) values of ChiA and ChiB giving values of 11.1±1.5 s(-1) and 13.9±1.4 s(-1), while there was no effect on ChiC (0.9±0.1 s(-1)). PMID:25812992

  5. Cyano- and polycyanometallo-porphyrins as catalysts for alkane oxidation

    DOEpatents

    Ellis, P.E. Jr.; Lyons, J.E.

    1993-05-18

    New compositions of matter comprising cyano-substituted metal complexes of porphyrins are catalysts for the oxidation of alkanes. The metal is iron, chromium, manganese, ruthenium, copper or cobalt. The porphyrin ring has cyano groups attached thereto in meso- and/or [beta]-pyrrolic positions.

  6. Cyano- and polycyanometallo-porphyrins as catalysts for alkane oxidation

    DOEpatents

    Ellis, Jr., Paul E.; Lyons, James E.

    1993-01-01

    New compositions of matter comprising cyano-substituted metal complexes of porphyrins are catalysts for the oxidation of alkanes. The metal is iron, chromium, manganese, ruthenium, copper or cobalt. The porphyrin ring has cyano groups attached thereto in meso and/or .beta.-pyrrolic positions.

  7. Cyano- and polycyanometallo-porphyrins as catalysts for alkane oxidation

    DOEpatents

    Ellis, P.E. Jr.; Lyons, J.E.

    1995-01-17

    New compositions of matter comprising cyano-substituted metal complexes of porphyrins are catalysts for the oxidation of alkanes. The metal is iron, chromium, manganese, ruthenium, copper or cobalt. The porphyrin ring has cyano groups attached thereto in meso and/or [beta]-pyrrolic positions.

  8. Cyano- and polycyanometallo-porphyrins as catalysts for alkane oxidation

    DOEpatents

    Ellis, Jr., Paul E.; Lyons, James E.

    1995-01-01

    New compositions of matter comprising cyano-substituted metal complexes of porphyrins are catalysts for the oxidation of alkanes. The metal is iron, chromium, manganese, ruthenium, copper or cobalt. The porphyrin ring has cyano groups attached thereto in meso and/or .beta.-pyrrolic positions.

  9. MODELING OF ALKANE EMISSIONS FROM A WOOD STAIN

    EPA Science Inventory

    The article discusses full-scale residential house tests to evaluate the effects of organic emissions from a wood finishing product--wood stain--on indoor air quality (IAQ). The test house concentrations of three alkane species, nonane, decane, and undecane, were measured as a fu...

  10. Analysis of the vibrational bandwidths of alkane-urea clathrates

    NASA Astrophysics Data System (ADS)

    Wood, Kurt A.; Snyder, Robert G.; Strauss, Herbert L.

    1989-11-01

    The only large amplitude motion possible for an n-alkane molecule in urea-inclusion compounds is libration-torsion about the long axis of the chain. We present a quantitative model that incorporates the effect of this motion on the widths of the alkane vibrational bands. This model explains the difference in the widths of the different vibrations of the alkanes and their temperature dependence. Two effects are combined: (1) a modulation of the angles between the components of the polarizability in the space and the molecule-fixed frames for Raman spectra or between the components of the dipole moment for the infrared spectra, and (2) a modulation of the frequency of the alkane vibration via anharmonic coupling terms with the libration-torsion. The first effect gives rise to a distinctly non-Lorentzian band shape, which is convoluted with the approximately Lorentzian band of the second effect to produce the final result. The libration-torsional motion is modeled as that of a Brownian harmonic oscillator. Most of the parameters that enter the calculation are obtained from data other than that involving the bandwidths themselves. The libration-torsion relaxation time of about 1 ps obtained from fitting the observed bandwidths agrees with the value obtained from recent quasielastic neutron scattering experiments. Other bandwidth mechanisms that have been proposed are evaluated and it is shown that site hopping is too slow to account for the observations.

  11. Roaming radical pathways for the decomposition of alkanes.

    SciTech Connect

    Harding, L. B.; Klippenstein, S. J.

    2010-01-01

    CASPT2 calculations predict the existence of roaming radical pathways for the decomposition of propane, n-butane, isobutane and neopentane. The roaming radical paths lead to the formation of an alkane and an alkene instead of the expected radical products. The predicted barriers for the roaming radical paths lie {approx}1 kcal/mol below the corresponding radical asymptotes.

  12. Improving alkane synthesis in Escherichia coli via metabolic engineering.

    PubMed

    Song, Xuejiao; Yu, Haiying; Zhu, Kun

    2016-01-01

    Concerns about energy security and global petroleum supply have made the production of renewable biofuels an industrial imperative. The ideal biofuels are n-alkanes in that they are chemically and structurally identical to the fossil fuels and can "drop in" to the transportation infrastructure. In this work, an Escherichia coli strain that produces n-alkanes was constructed by heterologous expression of acyl-acyl carrier protein (ACP) reductase (AAR) and aldehyde deformylating oxygenase (ADO) from Synechococcus elongatus PCC7942. The accumulation of alkanes ranged from 3.1 to 24.0 mg/L using different expressing strategies. Deletion of yqhD, an inherent aldehyde reductase in E. coli, or overexpression of fadR, an activator for fatty acid biosynthesis, exhibited a nearly twofold increase in alkane titers, respectively. Combining yqhD deletion and fadR overexpression resulted in a production titer of 255.6 mg/L in E. coli, and heptadecene was the most abundant product. PMID:26476644

  13. Cyano- and polycyanometalloporphyrins as catalysts for alkane oxidation

    DOEpatents

    Ellis, Jr., Paul E.; Lyons, James E.

    1992-01-01

    Alkanes are oxidized by contact with oxygen-containing gas in the presence as catalyst of a metalloporphyrin in which hydrogen atoms in the porphyrin ring have been substituted with one or more cyano groups. Hydrogen atoms in the porphyrin ring may also be substituted with halogen atoms.

  14. Integrated process for preparing a carboxylic acid from an alkane

    DOEpatents

    Benderly, Abraham; Chadda, Nitin; Sevon, Douglass

    2011-12-20

    The present invention relates to an integrated process for producing unsaturated carboxylic acids from the corresponding C.sub.2-C.sub.4 alkane. The process begins with performance of thermally integrated dehydrogenation reactions which convert a C.sub.2-C.sub.4 alkane to its corresponding C.sub.2-C.sub.4 alkene, and which involve exothermically converting a portion of an alkane to its corresponding alkene by oxidative dehydrogenation in an exothermic reaction zone, in the presence of oxygen and a suitable catalyst, and then feeding the products of the exothermic reaction zone to an endothermic reaction zone wherein at least a portion of the remaining unconverted alkane is endothermically dehydrogenated to form an additional quantity of the same corresponding alkene, in the presence of carbon dioxide and an other suitable catalyst. The alkene products of the thermally integrated dehydrogenation reactions are then provided to a catalytic vapor phase partial oxidation process for conversion of the alkene to the corresponding unsaturated carboxylic acid or nitrile. Unreacted alkene and carbon dioxide are recovered from the oxidation product stream and recycled back to the thermally integrated dehydrogenation reactions.

  15. Modular and selective biosynthesis of gasoline-range alkanes.

    PubMed

    Sheppard, Micah J; Kunjapur, Aditya M; Prather, Kristala L J

    2016-01-01

    Typical renewable liquid fuel alternatives to gasoline are not entirely compatible with current infrastructure. We have engineered Escherichia coli to selectively produce alkanes found in gasoline (propane, butane, pentane, heptane, and nonane) from renewable substrates such as glucose or glycerol. Our modular pathway framework achieves carbon-chain extension by two different mechanisms. A fatty acid synthesis route is used to generate longer chains heptane and nonane, while a more energy efficient alternative, reverse-β-oxidation, is used for synthesis of propane, butane, and pentane. We demonstrate that both upstream (thiolase) and intermediate (thioesterase) reactions can act as control points for chain-length specificity. Specific free fatty acids are subsequently converted to alkanes using a broad-specificity carboxylic acid reductase and a cyanobacterial aldehyde decarbonylase (AD). The selectivity obtained by different module pairings provides a foundation for tuning alkane product distribution for desired fuel properties. Alternate ADs that have greater activity on shorter substrates improve observed alkane titer. However, even in an engineered host strain that significantly reduces endogenous conversion of aldehyde intermediates to alcohol byproducts, AD activity is observed to be limiting for all chain lengths. Given these insights, we discuss guiding principles for pathway selection and potential opportunities for pathway improvement. PMID:26556131

  16. Diffusion of squalene in n-alkanes and squalane.

    PubMed

    Kowert, Bruce A; Watson, Michael B; Dang, Nhan C

    2014-02-27

    Squalene, an intermediate in the biosynthesis of cholesterol, has a 24-carbon backbone with six methyl groups and six isolated double bonds. Capillary flow techniques have been used to determine its translational diffusion constant, D, at room temperature in squalane, n-C16, and three n-C8-squalane mixtures. The D values have a weaker dependence on viscosity, η, than predicted by the Stokes-Einstein relation, D = kBT/(6πηr). A fit to the modified relation, D/T = ASE/η(p), gives p = 0.820 ± 0.028; p = 1 for the Stokes-Einstein limit. The translational motion of squalene appears to be much like that of n-alkane solutes with comparable chain lengths; their D values show similar deviations from the Stokes-Einstein model. The n-alkane with the same carbon chain length as squalene, n-C24, has a near-equal p value of 0.844 ± 0.018 in n-alkane solvents. The values of the hydrodynamic radius, r, for n-C24, squalene, and other n-alkane solutes decrease as the viscosity increases and have a common dependence on the van der Waals volumes of the solute and solvent. The possibility of studying squalene in lipid droplets and membranes is discussed. PMID:24528091

  17. Catalytic oxidation of light alkanes in presence of a base

    DOEpatents

    Bhinde, Manoj V.; Bierl, Thomas W.

    1998-01-01

    The presence of a base in the reaction mixture in a metal-ligand catalyzed partial oxidation of alkanes results in sustained catalyst activity, and in greater percent conversion as compared with oxidation in the absence of base, while maintaining satisfactory selectivity for the desired oxidation, for example the oxidation of isobutane to isobutanol.

  18. Catalytic oxidation of light alkanes in presence of a base

    DOEpatents

    Bhinde, M.V.; Bierl, T.W.

    1998-03-03

    The presence of a base in the reaction mixture in a metal-ligand catalyzed partial oxidation of alkanes results in sustained catalyst activity, and in greater percent conversion as compared with oxidation in the absence of base, while maintaining satisfactory selectivity for the desired oxidation, for example the oxidation of isobutane to isobutanol. 1 fig.

  19. MODELING OF ALKANE EMISSIONS FROM A WOOD STAIN

    EPA Science Inventory

    The article discusses full-scale residential house tests to evaluate the effects of organic emissions from a wood finishing product--wood stain--on indoor air quality (IAQ). he test house concentrations of three alkane species, nonane, decane, and undecane, were measured as a fun...

  20. A superoleophobic textile repellent towards impacting drops of alkanes

    NASA Astrophysics Data System (ADS)

    Artus, Georg R. J.; Zimmermann, Jan; Reifler, Felix A.; Brewer, Stuart A.; Seeger, Stefan

    2012-02-01

    A commercially available polyester fabric has been rendered superoleophobic by coating with silicone nanofilaments and subsequent plasma fluorination. The treated samples show outstanding oil-repellency. They achieve the highest possible oil-repellency grade of 8, repel impacting drops of alkanes and show a plastron layer in hexadecane. The oil repellency is shown to depend on the topography of the silicone nanofilament coating.

  1. Isolating the non-polar contributions to the intermolecular potential for water-alkane interactions

    NASA Astrophysics Data System (ADS)

    Ballal, Deepti; Venkataraman, Pradeep; Fouad, Wael A.; Cox, Kenneth R.; Chapman, Walter G.

    2014-08-01

    Intermolecular potential models for water and alkanes describe pure component properties fairly well, but fail to reproduce properties of water-alkane mixtures. Understanding interactions between water and non-polar molecules like alkanes is important not only for the hydrocarbon industry but has implications to biological processes as well. Although non-polar solutes in water have been widely studied, much less work has focused on water in non-polar solvents. In this study we calculate the solubility of water in different alkanes (methane to dodecane) at ambient conditions where the water content in alkanes is very low so that the non-polar water-alkane interactions determine solubility. Only the alkane-rich phase is simulated since the fugacity of water in the water rich phase is calculated from an accurate equation of state. Using the SPC/E model for water and TraPPE model for alkanes along with Lorentz-Berthelot mixing rules for the cross parameters produces a water solubility that is an order of magnitude lower than the experimental value. It is found that an effective water Lennard-Jones energy ɛW/k = 220 K is required to match the experimental water solubility in TraPPE alkanes. This number is much higher than used in most simulation water models (SPC/E—ɛW/k = 78.2 K). It is surprising that the interaction energy obtained here is also higher than the water-alkane interaction energy predicted by studies on solubility of alkanes in water. The reason for this high water-alkane interaction energy is not completely understood. Some factors that might contribute to the large interaction energy, such as polarizability of alkanes, octupole moment of methane, and clustering of water at low concentrations in alkanes, are examined. It is found that, though important, these factors do not completely explain the anomalously strong attraction between alkanes and water observed experimentally.

  2. Secondary organic aerosol composition from C12 alkanes.

    PubMed

    Schilling Fahnestock, Katherine A; Yee, Lindsay D; Loza, Christine L; Coggon, Matthew M; Schwantes, Rebecca; Zhang, Xuan; Dalleska, Nathan F; Seinfeld, John H

    2015-05-14

    The effects of structure, NOx conditions, relative humidity, and aerosol acidity on the chemical composition of secondary organic aerosol (SOA) are reported for the photooxidation of three C12 alkanes: n-dodecane, cyclododecane, and hexylcyclohexane. Acidity was modified through seed particle composition: NaCl, (NH4)2SO4, and (NH4)2SO4 + H2SO4. Off-line analysis of SOA was carried out by solvent extraction and gas chromatography-mass spectrometry (GC/MS) and direct analysis in real-time mass spectrometry. We report here 750 individual masses of SOA products identified from these three alkane systems and 324 isomers resolved by GC/MS analysis. The chemical compositions for each alkane system provide compelling evidence of particle-phase chemistry, including reactions leading to oligomer formation. Major oligomeric species for alkane SOA are peroxyhemiacetals, hemiacetals, esters, and aldol condensation products. Furans, dihydrofurans, hydroxycarbonyls, and their corresponding imine analogues are important participants in these oligomer-producing reactions. Imines are formed in the particle phase from the reaction of the ammonium sulfate seed aerosol with carbonyl-bearing compounds present in all the SOA systems. Under high-NO conditions, organonitrate products can lead to an increase of aerosol volume concentration by up to a factor of 5 over that in low-NO conditions. Structure was found to play a key role in determining the degree of functionalization and fragmentation of the parent alkane, influencing the mean molecular weight of the SOA produced and the mean atomic O:C ratio. PMID:24814371

  3. Transcriptional Regulation of the Grape Cytochrome P450 Monooxygenase Gene CYP736B Expression in Response to Xylella fastidiosa Infection

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant cytochrome P450 monooxygenases are a group of versatile redox proteins that mediate the biosynthesis of lignins, terpenes, alkaloids, and a variety of other secondary compounds which act as plant defense agents. To determine if cytochrome P450 monooxygenases are involved in defense response to...

  4. Selective Usage of Transcription Initiation and Polyadenylation Sites in Grape Cytochrome P450 Monooxygenase Gene CYP736B Expression

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant cytochrome P450 monooxygenases are versatile redox proteins that mediate biosynthesis of lignins, terpenes, alkaloids, and a variety of other secondary compounds as plant defense agents against a range of pathogens and insects. To determine if cytochrome P450 monooxygenases are involved in the...

  5. THE BIOCHEMICAL CHARACTERIZATION OF FERRET CAROTENE-9', 10'-MONOOXYGENASE CATALYZING CLEAVAGE OF CAROTENOIDS IN VITRO AND IN VIVO

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Previous studies have shown that beta -carotene 15,15'-monooxygenase (CMO1) catalyzes the cleavage of beta -carotene at the central carbon 15, 15’-double bond, but cleaves lycopene with much lower activity. However, expressing the mouse carotene-9’,10’-monooxygenase (CMO2) in beta-carotene/lycopene...

  6. A family of starch-active polysaccharide monooxygenases

    PubMed Central

    Vu, Van V.; Beeson, William T.; Span, Elise A.; Farquhar, Erik R.; Marletta, Michael A.

    2014-01-01

    The recently discovered fungal and bacterial polysaccharide monooxygenases (PMOs) are capable of oxidatively cleaving chitin, cellulose, and hemicelluloses that contain β(1→4) linkages between glucose or substituted glucose units. They are also known collectively as lytic PMOs, or LPMOs, and individually as AA9 (formerly GH61), AA10 (formerly CBM33), and AA11 enzymes. PMOs share several conserved features, including a monocopper center coordinated by a bidentate N-terminal histidine residue and another histidine ligand. A bioinformatic analysis using these conserved features suggested several potential new PMO families in the fungus Neurospora crassa that are likely to be active on novel substrates. Herein, we report on NCU08746 that contains a C-terminal starch-binding domain and an N-terminal domain of previously unknown function. Biochemical studies showed that NCU08746 requires copper, oxygen, and a source of electrons to oxidize the C1 position of glycosidic bonds in starch substrates, but not in cellulose or chitin. Starch contains α(1→4) and α(1→6) linkages and exhibits higher order structures compared with chitin and cellulose. Cellobiose dehydrogenase, the biological redox partner of cellulose-active PMOs, can serve as the electron donor for NCU08746. NCU08746 contains one copper atom per protein molecule, which is likely coordinated by two histidine ligands as shown by X-ray absorption spectroscopy and sequence analysis. Results indicate that NCU08746 and homologs are starch-active PMOs, supporting the existence of a PMO superfamily with a much broader range of substrates. Starch-active PMOs provide an expanded perspective on studies of starch metabolism and may have potential in the food and starch-based biofuel industries. PMID:25201969

  7. A family of starch-active polysaccharide monooxygenases.

    PubMed

    Vu, Van V; Beeson, William T; Span, Elise A; Farquhar, Erik R; Marletta, Michael A

    2014-09-23

    The recently discovered fungal and bacterial polysaccharide monooxygenases (PMOs) are capable of oxidatively cleaving chitin, cellulose, and hemicelluloses that contain β(1→4) linkages between glucose or substituted glucose units. They are also known collectively as lytic PMOs, or LPMOs, and individually as AA9 (formerly GH61), AA10 (formerly CBM33), and AA11 enzymes. PMOs share several conserved features, including a monocopper center coordinated by a bidentate N-terminal histidine residue and another histidine ligand. A bioinformatic analysis using these conserved features suggested several potential new PMO families in the fungus Neurospora crassa that are likely to be active on novel substrates. Herein, we report on NCU08746 that contains a C-terminal starch-binding domain and an N-terminal domain of previously unknown function. Biochemical studies showed that NCU08746 requires copper, oxygen, and a source of electrons to oxidize the C1 position of glycosidic bonds in starch substrates, but not in cellulose or chitin. Starch contains α(1→4) and α(1→6) linkages and exhibits higher order structures compared with chitin and cellulose. Cellobiose dehydrogenase, the biological redox partner of cellulose-active PMOs, can serve as the electron donor for NCU08746. NCU08746 contains one copper atom per protein molecule, which is likely coordinated by two histidine ligands as shown by X-ray absorption spectroscopy and sequence analysis. Results indicate that NCU08746 and homologs are starch-active PMOs, supporting the existence of a PMO superfamily with a much broader range of substrates. Starch-active PMOs provide an expanded perspective on studies of starch metabolism and may have potential in the food and starch-based biofuel industries. PMID:25201969

  8. Discovery and industrial applications of lytic polysaccharide mono-oxygenases.

    PubMed

    Johansen, Katja S

    2016-02-01

    The recent discovery of copper-dependent lytic polysaccharide mono-oxygenases (LPMOs) has opened up a vast area of research covering several fields of application. The biotech company Novozymes A/S holds patents on the use of these enzymes for the conversion of steam-pre-treated plant residues such as straw to free sugars. These patents predate the correct classification of LPMOs and the striking synergistic effect of fungal LPMOs when combined with canonical cellulases was discovered when fractions of fungal secretomes were evaluated in industrially relevant enzyme performance assays. Today, LPMOs are a central component in the Cellic CTec enzyme products which are used in several large-scale plants for the industrial production of lignocellulosic ethanol. LPMOs are characterized by an N-terminal histidine residue which, together with an internal histidine and a tyrosine residue, co-ordinates a single copper atom in a so-called histidine brace. The mechanism by which oxygen binds to the reduced copper atom has been reported and the general mechanism of copper-oxygen-mediated activation of carbon is being investigated in the light of these discoveries. LPMOs are widespread in both the fungal and the bacterial kingdoms, although the range of action of these enzymes remains to be elucidated. However, based on the high abundance of LPMOs expressed by microbes involved in the decomposition of organic matter, the importance of LPMOs in the natural carbon-cycle is predicted to be significant. In addition, it has been suggested that LPMOs play a role in the pathology of infectious diseases such as cholera and to thus be relevant in the field of medicine. PMID:26862199

  9. Terpene hydroxylation with microbial cytochrome P450 monooxygenases.

    PubMed

    Janocha, Simon; Schmitz, Daniela; Bernhardt, Rita

    2015-01-01

    Terpenoids comprise a highly diverse group of natural products. In addition to their basic carbon skeleton, they differ from one another in their functional groups. Functional groups attached to the carbon skeleton are the basis of the terpenoids' diverse properties. Further modifications of terpene olefins include the introduction of acyl-, aryl-, or sugar moieties and usually start with oxidations catalyzed by cytochrome P450 monooxygenases (P450s, CYPs). P450s are ubiquitously distributed throughout nature, involved in essential biological pathways such as terpenoid biosynthesis as well as the tailoring of terpenoids and other natural products. Their ability to introduce oxygen into nonactivated C-H bonds is unique and makes P450s very attractive for applications in biotechnology. Especially in the field of terpene oxidation, biotransformation methods emerge as an attractive alternative to classical chemical synthesis. For this reason, microbial P450s depict a highly interesting target for protein engineering approaches in order to increase selectivity and activity, respectively. Microbial P450s have been described to convert industrial and pharmaceutically interesting terpenoids such as ionones, limone, valencene, resin acids, and triterpenes (including steroids) as well as vitamin D3. Highly selective and active mutants have been evolved by applying classical site-directed mutagenesis as well as directed evolution of proteins. As P450s usually depend on electron transfer proteins, mutagenesis has also been applied to improve the interactions between P450s and their respective redox partners. This chapter provides an overview of terpenoid hydroxylation reactions catalyzed by bacterial P450s and highlights the achievements made by protein engineering to establish productive hydroxylation processes. PMID:25682070

  10. Membrane-associated methane monooxygenase from Methylococcus capsulatus (Bath).

    PubMed Central

    Zahn, J A; DiSpirito, A A

    1996-01-01

    An active preparation of the membrane-associated methane monooxygenase (pMMO) from Methylococcus capsulatus Bath was isolated by ion-exchange and hydrophobic interaction chromatography using dodecyl beta-D-maltoside as the detergent. The active preparation consisted of three major polypeptides with molecular masses of 47,000, 27,000, and 25,000 Da. Two of the three polypeptides (those with molecular masses of 47,000 and 27,000 Da) were identified as the polypeptides induced when cells expressing the soluble MMO are switched to culture medium in which the pMMO is expressed. The 27,000-Da polypeptide was identified as the acetylene-binding protein. The active enzyme complex contained 2.5 iron atoms and 14.5 copper atoms per 99,000 Da. The electron paramagnetic resonance spectrum of the enzyme showed evidence for a type 2 copper center (g perpendicular = 2.057, g parallel = 2.24, and magnitude of A parallel = 172 G), a weak high-spin iron signal (g = 6.0), and a broad low-field (g = 12.5) signal. Treatment of the pMMO with nitric oxide produced the ferrous-nitric oxide derivative observed in the membrane fraction of cells expressing the pMMO. When duroquinol was used as a reductant, the specific activity of the purified enzyme was 11.1 nmol of propylene oxidized.min-1.mg of protein-1, which accounted for approximately 30% of the cell-free propylene oxidation activity. The activity was stimulated by ferric and cupric metal ions in addition to the cytochrome b-specific inhibitors myxothiazol and 2-heptyl-4-hydroxyquinoline-N-oxide. PMID:8576034

  11. Membrane-associated methane monooxygenase from Methylococcus capsulatus (Bath).

    PubMed

    Zahn, J A; DiSpirito, A A

    1996-02-01

    An active preparation of the membrane-associated methane monooxygenase (pMMO) from Methylococcus capsulatus Bath was isolated by ion-exchange and hydrophobic interaction chromatography using dodecyl beta-D-maltoside as the detergent. The active preparation consisted of three major polypeptides with molecular masses of 47,000, 27,000, and 25,000 Da. Two of the three polypeptides (those with molecular masses of 47,000 and 27,000 Da) were identified as the polypeptides induced when cells expressing the soluble MMO are switched to culture medium in which the pMMO is expressed. The 27,000-Da polypeptide was identified as the acetylene-binding protein. The active enzyme complex contained 2.5 iron atoms and 14.5 copper atoms per 99,000 Da. The electron paramagnetic resonance spectrum of the enzyme showed evidence for a type 2 copper center (g perpendicular = 2.057, g parallel = 2.24, and magnitude of A parallel = 172 G), a weak high-spin iron signal (g = 6.0), and a broad low-field (g = 12.5) signal. Treatment of the pMMO with nitric oxide produced the ferrous-nitric oxide derivative observed in the membrane fraction of cells expressing the pMMO. When duroquinol was used as a reductant, the specific activity of the purified enzyme was 11.1 nmol of propylene oxidized.min-1.mg of protein-1, which accounted for approximately 30% of the cell-free propylene oxidation activity. The activity was stimulated by ferric and cupric metal ions in addition to the cytochrome b-specific inhibitors myxothiazol and 2-heptyl-4-hydroxyquinoline-N-oxide. PMID:8576034

  12. Oxidation Products of Semi-volatile Alkanes by Hydroxyl Radicals

    NASA Astrophysics Data System (ADS)

    Zhang, H.; Worton, D. R.; Nah, T.; Goldstein, A. H.; Wilson, K. R.

    2013-12-01

    Alkanes are ubiquitous in the atmosphere and are important components that influence atmospheric chemistry. Semi-volatile alkanes are partitioned between the gas- and the particle-phases and can be readily oxidized in both phases. Previous studies have demonstrated that reaction rates and the products of OH oxidation are very different for organic compounds in the gas- and particle phases. In the present study, n-octadecane (C18H38), n-eicosane (C20H42), n-docosane (C22H46), n-tricosane (C24H50), and n-pentadecylcyclohexane (C21H42) were chosen as model compounds for semi-volatile alkanes to examine their OH-initiated oxidation reactions in a flow tube reactor. OH exposure was varied in the experiments, equivalent to oxidation of up to one week in the atmosphere. Oxidation products were collected on filters and analyzed using two-dimensional gas chromatography coupled to a high-resolution time-of-flight electron impact ionization and vacuum ultraviolet photoionization mass spectrometer. Most of the oxygenated higher molecular weight isomers were separated and quantified. Our results suggest that aerosol samples formed in the n-octadecane experiment were more oxidized than the other model compounds (i.e., functionalization products with three oxygen atoms per molecule compared to two oxygen atoms per molecule) at similar OH exposures and aerosol mass loadings. This is likely due to the concentration of n-octadecane in the gas phase where oxidation is more rapid. We find that the first-generation gas-phase oxidation products quickly partition to the particle phase after which higher-generation oxidation likely occurs in the particle phase. Interestingly, functionalized carbonyl isomers for the normal alkanes were only observed on the 4 carbon positions closest to the molecule end in all cases, which is in contrast to structure-reactivity relationship (SRR) predictions for gas-phase reactions. For n-octadecane, the concentrations of first-generation functionalization

  13. Expression and Characterization of CYP52 Genes Involved in the Biosynthesis of Sophorolipid and Alkane Metabolism from Starmerella bombicola

    PubMed Central

    Huang, Fong-Chin; Peter, Alyssa

    2014-01-01

    Three cytochrome P450 monooxygenase CYP52 gene family members were isolated from the sophorolipid-producing yeast Starmerella bombicola (former Candida bombicola), namely, CYP52E3, CYP52M1, and CYP52N1, and their open reading frames were cloned into the pYES2 vector for expression in Saccharomyces cerevisiae. The functions of the recombinant proteins were analyzed with a variety of alkane and fatty acid substrates using microsome proteins or a whole-cell system. CYP52M1 was found to oxidize C16 to C20 fatty acids preferentially. It converted oleic acid (C18:1) more efficiently than stearic acid (C18:0) and linoleic acid (C18:2) and much more effectively than α-linolenic acid (C18:3). No products were detected when C10 to C12 fatty acids were used as the substrates. Moreover, CYP52M1 hydroxylated fatty acids at their ω- and ω-1 positions. CYP52N1 oxidized C14 to C20 saturated and unsaturated fatty acids and preferentially oxidized palmitic acid, oleic acid, and linoleic acid. It only catalyzed ω-hydroxylation of fatty acids. Minor ω-hydroxylation activity against myristic acid, palmitic acid, palmitoleic acid, and oleic acid was shown for CYP52E3. Furthermore, the three P450s were coassayed with glucosyltransferase UGTA1. UGTA1 glycosylated all hydroxyl fatty acids generated by CYP52E3, CYP52M1, and CYP52N1. The transformation efficiency of fatty acids into glucolipids by CYP52M1/UGTA1 was much higher than those by CYP52N1/UGTA1 and CYP52E3/UGTA1. Taken together, CYP52M1 is demonstrated to be involved in the biosynthesis of sophorolipid, whereas CYP52E3 and CYP52N1 might be involved in alkane metabolism in S. bombicola but downstream of the initial oxidation steps. PMID:24242247

  14. A study of the hepatic microsomal monooxygenase of sea birds and its relationship to organochlorine pollutants.

    PubMed

    Knight, G C; Walker, C H

    1982-01-01

    1. The levels of hepatic microsomal monooxygenase in sea birds were determined using organochlorine substrates. Levels of cytochrome P450 and organochlorine residues were also measured. 2. The razorbill (Alca torda) and puffin (Fratercula arctica) showed highly variable activities which were resolved into multiple peaks on frequency diagrams. 3. The most active individuals amongst razorbills were early season females with large ovaries. 4. The properties of monooxygenase from individuals of low and high activity were compared. 5. The results are discussed in relation to PCB pollution. PMID:6128175

  15. Toluene 2-Monooxygenase-Dependent Growth of Burkholderia cepacia G4/PR1 on Diethyl Ether

    PubMed Central

    Hur, H.; Newman, L. M.; Wackett, L. P.; Sadowsky, M. J.

    1997-01-01

    Aerobic bacterial growth on aromatic hydrocarbons typically requires oxygenase enzymes, which are known to fortuitously oxidize nongrowth substrates. In this study, we found that oxidation of diethyl ether by toluene 2-monooxygenase supported more rapid growth of Burkholderia cepacia G4/PR1 than did the aromatic substrates n-propylbenzene and o-xylene. The wild-type Burkholderia cepacia G4 failed to grow on diethyl ether. Purified toluene 2-monooxygenase protein components oxidized diethyl ether stoichiometrically to ethanol and acetaldehyde. Butyl methyl ether, diethyl sulfide, and 2-chloroethyl ethyl ether were oxidized by B. cepacia G4/PR1. PMID:16535583

  16. The vibrational spectrum of water in liquid alkanes.

    PubMed Central

    Conrad, M P; Strauss, H L

    1985-01-01

    The water wire hypothesis of hydrogen-ion transport in lipid bilayers has prompted a search for water aggregates in bulk hydrocarbons. The asymmetric stretching vibration of the water dissolved in n-decane and in a number of other alkanes and alkenes has been observed. The water band in the alkanes is very wide and fits to the results of a J-diffusion calculation for the water rotation. This implies that the water is freely rotating between collisions with the solvent and certainly not hydrogen bonded to anything. The existence of water aggregates is thus most unlikely. In contrast, water in an alkene is hydrogen bonded to the solvent molecules (although not to other water molecules) and shows an entirely different spectrum. PMID:4016205

  17. Site isolation in vanadium phosphorus oxide alkane oxidation

    SciTech Connect

    Thompson, M R; Ebner, J R

    1991-06-01

    Single crystal X-ray diffraction studies of vanadyl pyrophosphate indicate that at least two polytypical structures exists for this active and selective alkane oxidation catalyst. The crystal structures of these materials differ with respect to the symmetry and direction of columns of vanadyl groups within the unit cell. Single crystals of vanadyl pyrophosphate have been generated at extreme temperatures not often experienced by microcrystalline catalysts. The crystallography of the system suggests that other crystalline modifications or disordered phases might also exist. Zeroth-order models of crystal surface termination of vanadyl pyrophosphate have been constructed which conceptually illustrate the ability of vanadyl pyrophosphate to accommodate varying amounts of surface phosphorus parallel to (1,0,0), (0,1,0) and (0,2,4). Pyrophosphate termination of surfaces parallel to (1,0,0) likely results in the isolation of clusters of reactive centers and limits overoxidation of the alkane substrate. 23 refs., 6 figs.

  18. Flash Points of Secondary Alcohol and n-Alkane Mixtures.

    PubMed

    Esina, Zoya N; Miroshnikov, Alexander M; Korchuganova, Margarita R

    2015-11-19

    The flash point is one of the most important characteristics used to assess the ignition hazard of mixtures of flammable liquids. To determine the flash points of mixtures of secondary alcohols with n-alkanes, it is necessary to calculate the activity coefficients. In this paper, we use a model that allows us to obtain enthalpy of fusion and enthalpy of vaporization data of the pure components to calculate the liquid-solid equilibrium (LSE) and vapor-liquid equilibrium (VLE). Enthalpy of fusion and enthalpy of vaporization data of secondary alcohols in the literature are limited; thus, the prediction of these characteristics was performed using the method of thermodynamic similarity. Additionally, the empirical models provided the critical temperatures and boiling temperatures of the secondary alcohols. The modeled melting enthalpy and enthalpy of vaporization as well as the calculated LSE and VLE flash points were determined for the secondary alcohol and n-alkane mixtures. PMID:26491811

  19. Adsorption of n-alkane vapours at the water surface.

    PubMed

    Biscay, Frédéric; Ghoufi, Aziz; Malfreyt, Patrice

    2011-06-21

    Monte Carlo simulations are reported here to predict the surface tension of the liquid-vapour interface of water upon adsorption of alkane vapours (methane to hexane). A decrease of the surface tension has been established from n-pentane. A correlation has been evidenced between the decrease of the surface tension and the absence of specific arrangement at the water surface for n-pentane and n-hexane. The thermodynamic stability of the adsorption layer and the absence of film for longer alkanes have been checked through the calculation of a potential of mean force. This complements the work recently published [Ghoufi et al., Phys. Chem. Chem. Phys., 2010, 12, 5203] concerning the adsorption of methane at the water surface. The decrease of the surface tension has been interpreted in terms of the degree of hydrogen bonding of water molecules at the liquid-vapour interface upon adsorption. PMID:21584320

  20. Cyanobacterial alkane biosynthesis further expands the catalytic repertoire of the ferritin-like “di-iron-carboxylate” proteins

    PubMed Central

    Krebs, Carsten; Bollinger, J. Martin; Booker, Squire J.

    2011-01-01

    Summary Enzymes that activate dioxygen at carboxylate-bridged non-heme diiron clusters residing within ferritin-like, four-helix-bundle protein architectures have crucial roles in, among other processes, the global carbon cycle (e.g., soluble methane monooxygenase), fatty acid biosynthesis [plant fatty acyl-acyl carrier protein (ACP) desaturases], DNA biosynthesis [the R2 or β2 subunits of class Ia ribonucleotide reductases (RNRs)], and cellular iron trafficking (ferritins). Classic studies on class Ia RNRs showed long ago how this obligatorily oxidative di-iron/O2 chemistry can be used to activate an enzyme for even a reduction reaction, and more recent investigations of class Ib and Ic RNRs, coupled with earlier studies on dimanganese catalases, have shown that members of this protein family can also incorporate either one or two Mn ions and use them in place of iron for redox catalysis. These two strategies – oxidative activation for non-oxidative reactions and use of alternative metal ions – expand the catalytic repertoire of the family, probably to include activities that remain to be discovered. Indeed, a recent study has suggested that fatty aldehyde decarbonylases (ADs) from cyanobacteria, purported to catalyze a redox-neutral cleavage of a Cn aldehyde to the Cn−1 alkane (or alkene) and CO, also belong to this enzyme family and are most similar in structure to two other members with heterodinuclear (Mn-Fe) cofactors. Here, we first briefly review both the chemical principles underlying the O2-dependent oxidative chemistry of the “classical” di-iron-carboxylate proteins and the two aforementioned strategies that have expanded their functional range, and then consider what metal ion(s) and what chemical mechanism(s) might be employed by the newly discovered cyanobacterial ADs. PMID:21440485

  1. Alkane Biosynthesis Genes in Cyanobacteria and Their Transcriptional Organization

    PubMed Central

    Klähn, Stephan; Baumgartner, Desirée; Pfreundt, Ulrike; Voigt, Karsten; Schön, Verena; Steglich, Claudia; Hess, Wolfgang R.

    2014-01-01

    In cyanobacteria, alkanes are synthesized from a fatty acyl-ACP by two enzymes, acyl–acyl carrier protein reductase and aldehyde deformylating oxygenase. Despite the great interest in the exploitation for biofuel production, nothing is known about the transcriptional organization of their genes or the physiological function of alkane synthesis. The comparison of 115 microarray datasets indicates the relatively constitutive expression of aar and ado genes. The analysis of 181 available genomes showed that in 90% of the genomes both genes are present, likely indicating their physiological relevance. In 61% of them they cluster together with genes encoding acetyl-CoA carboxyl transferase and a short-chain dehydrogenase, strengthening the link to fatty acid metabolism and in 76% of the genomes they are located in tandem, suggesting constraints on the gene arrangement. However, contrary to the expectations for an operon, we found in Synechocystis sp. PCC 6803 specific promoters for the two genes, sll0208 (ado) and sll0209 (aar), which give rise to monocistronic transcripts. Moreover, the upstream located ado gene is driven by a proximal as well as a second, distal, promoter, from which a third transcript, the ~160 nt sRNA SyR9 is transcribed. Thus, the transcriptional organization of the alkane biosynthesis genes in Synechocystis sp. PCC 6803 is of substantial complexity. We verified all three promoters to function independently from each other and show a similar promoter arrangement also in the more distant Nodularia spumigena, Trichodesmium erythraeum, Anabaena sp. PCC 7120, Prochlorococcus MIT9313, and MED4. The presence of separate regulatory elements and the dominance of monocistronic mRNAs suggest the possible autonomous regulation of ado and aar. The complex transcriptional organization of the alkane synthesis gene cluster has possible metabolic implications and should be considered when manipulating the expression of these genes in cyanobacteria. PMID

  2. Hydroxylation of alkanes using sodium hypochlorite catalyzed by iron porphyrins

    SciTech Connect

    Sorokin, A.B.; Khenkin, A.M.

    1988-10-01

    This communication presents data about the oxidation of alkanes to alcohols with hypochlorite in the presence of Fe(III) phenylporphyrin derivatives in the system water-benzene. We used as catalysts the following compounds: tetraphenylporphyrin iron chloride, tetramesitylporphyrin iron chloride, tetra(2-fluorophenyl)porphyrin from chloride, and tetra (2-ntrophenyl)porphyrin iron chloride. The reaction products were analyzed by gas-liquid chromatography. The efficiency of the reaction was determined by the structure of the porphyrin used.

  3. Modeling SOA production from the oxidation of intermediate volatility alkanes

    NASA Astrophysics Data System (ADS)

    Aumont, B.; Mouchel-Vallon, C.; Camredon, M.; Lee-Taylor, J.; Madronich, S.

    2012-12-01

    Secondary Organic Aerosols (SOA) production and ageing is a multigenerational oxidation process involving the formation of successive organic compounds with higher oxidation degree and lower vapour pressure. This process was investigated using the explicit oxidation model GECKO-A (Generator for Explicit Chemistry and Kinetics of Organics in the Atmosphere). Results for the C8-C24 n-alkane series show the expected trends, i.e. (i) SOA yield grows with the carbon backbone of the parent hydrocarbon, (ii) SOA yields decreases with the decreasing pre-existing organic aerosol concentration, (iii) the number of generations required to describe SOA production increases when the pre-existing organic aerosol concentration decreases. Most SOA contributors were found to be not oxidized enough to be categorized as highly oxygenated organic aerosols (OOA) but reduced enough to be categorized as hydrocarbon like organic aerosols (HOA). Branched alkanes are more prone to fragment in the early stage of the oxidation than their corresponding linear analogues. Fragmentation is expected to alter both the yield and the mean oxidation state of the SOA. Here, GECKO-A is applied to generate highly detailed oxidation schemes for various series of branched and cyclised alkanes. Branching and cyclisation effects on SOA yields and oxidation states will be examined.

  4. Nanoscale Trapping and Squeeze-Out of Confined Alkane Monolayers.

    PubMed

    Gosvami, N N; O'Shea, S J

    2015-12-01

    We present combined force curve and conduction atomic force microscopy (AFM) data for the linear alkanes CnH2n+2 (n = 10, 12, 14, 16) confined between a gold-coated AFM tip and a graphite surface. Solvation layering is observed in the force curves for all liquids, and conduction AFM is used to study in detail the removal of the confined (mono)layer closest to the graphite surface. The squeeze-out behavior of the monolayer can be very different depending upon the temperature. Below the monolayer melting transition temperatures the molecules are in an ordered state on the graphite surface, and fast and complete removal of the confined molecules is observed. However, above the melting transition temperature the molecules are in a disordered state, and even at large applied pressure a few liquid molecules are trapped within the tip-sample contact zone. These findings are similar to a previous study for branched alkanes [ Gosvami Phys. Rev. Lett. 2008, 100, 076101 ], but the observation for the linear alkane homologue series demonstrates clearly the dependence of the squeeze-out and trapping on the state of the confined material. PMID:26529283

  5. Dielectric constant of liquid alkanes and hydrocarbon mixtures

    NASA Technical Reports Server (NTRS)

    Sen, A. D.; Anicich, V. G.; Arakelian, T.

    1992-01-01

    The complex dielectric constants of n-alkanes with two to seven carbon atoms have been measured. The measurements were conducted using a slotted-line technique at 1.2 GHz and at atmospheric pressure. The temperature was varied from the melting point to the boiling point of the respective alkanes. The real part of the dielectric constant was found to decrease with increasing temperature and correlate with the change in the molar volume. An upper limit to all the loss tangents was established at 0.001. The complex dielectric constants of a few mixtures of liquid alkanes were also measured at room temperature. For a pentane-octane mixture the real part of the dielectric constant could be explained by the Clausius-Mosotti theory. For the mixtures of n-hexane-ethylacetate and n-hexane-acetone the real part of the dielectric constants could be explained by the Onsager theory extended to mixtures. The dielectric constant of the n-hexane-acetone mixture displayed deviations from the Onsager theory at the highest fractions of acetone. The dipole moments of ethylacetate and acetone were determined for dilute mixtures using the Onsager theory and were found to be in agreement with their accepted gas-phase values. The loss tangents of the mixtures exhibited a linear relationship with the volume fraction for low concentrations of the polar liquids.

  6. Alkane production from biomass: chemo-, bio- and integrated catalytic approaches.

    PubMed

    Deneyer, Aron; Renders, Tom; Van Aelst, Joost; Van den Bosch, Sander; Gabriëls, Dries; Sels, Bert F

    2015-12-01

    Linear, branched and cyclic alkanes are important intermediates and end products of the chemical industry and are nowadays mainly obtained from fossil resources. In search for alternatives, biomass feedstocks are often presented as a renewable carbon source for the production of fuels, chemicals and materials. However, providing a complete market for all these applications seems unrealistic due to both financial and logistic issues. Despite the very large scale of current alkane-based fuel applications, biomass definitely has the potential to offer a partial solution to the fuel business. For the smaller market of chemicals and materials, a transition to biomass as main carbon source is more realistic and even probably unavoidable in the long term. The appropriate use and further development of integrated chemo- and biotechnological (catalytic) process strategies will be crucial to successfully accomplish this petro-to-bio feedstock transition. Furthermore, a selection of the most promising technologies from the available chemo- and biocatalytic tool box is presented. New opportunities will certainly arise when multidisciplinary approaches are further explored in the future. In an attempt to select the most appropriate biomass sources for each specific alkane-based application, a diagram inspired by van Krevelen is applied, taking into account both the C-number and the relative functionality of the product molecules. PMID:26360875

  7. Biochemical studies on the metabolic activation of halogenated alkanes.

    PubMed Central

    Cheeseman, K H; Albano, E F; Tomasi, A; Slater, T F

    1985-01-01

    This paper reviews recent investigations by Slater and colleagues into the metabolic activation of halogenated alkanes in general and carbon tetrachloride in particular. It is becoming increasingly accepted that free radical intermediates are involved in the toxicity of many such compounds through mechanisms including lipid peroxidation, covalent binding, and cofactor depletion. Here we describe the experimental approaches that are used to establish that halogenated alkanes are metabolized in animal tissues to reactive free radicals. Electron spin resonance spectroscopy is used to identify free-radical products, often using spin-trapping compounds. The generation of specific free radicals by radiolytic methods is useful in the determination of the precise reactivity of radical intermediates postulated to be injurious to the cell. The enzymic mechanism of the production of such free radicals and their subsequent reactions with biological molecules is studied with specific metabolic inhibitors and free-radical scavengers. These combined techniques provide considerable insight into the process of metabolic activation of halogenated compounds. It is readily apparent, for instance, that the local oxygen concentration at the site of activation is of crucial importance to the subsequent reactions; the formation of peroxy radical derivatives from the primary free-radical product is shown to be of great significance in relation to carbon tetrachloride and may be of general importance. However, while these studies have provided much information on the biochemical mechanisms of halogenated alkane toxicity, it is clear that many problems remain to be solved. PMID:3007102

  8. Intermediate P* from Soluble Methane Monooxygenase Contains a Diferrous Cluster

    PubMed Central

    Banerjee, Rahul; Meier, Katlyn K.; Münck, Eckard; Lipscomb, John D.

    2013-01-01

    During a single turnover of the hydroxylase component (MMOH) of soluble methane monooxygenase from Methylosinus trichosporium OB3b, several discrete intermediates are formed. The diiron cluster of MMOH is first reduced to the FeIIFeII state (Hred). O2 binds rapidly at a site away from the cluster to form the FeIIFeII intermediate O, which converts to an FeIIIFeIII-peroxo intermediate P and finally to the FeIVFeIV intermediate Q. Q binds and reacts with methane to yield methanol and water. The rate constants for these steps are increased by a regulatory protein, MMOB. Previously reported transient kinetic studies have suggested that an intermediate P* forms between O and P in which the g = 16 EPR signal characteristic of the reduced diiron cluster of Hred and O is lost. This was interpreted as signaling oxidation of the cluster, but low accumulation of P* prevented further characterization. In this study, three methods to directly detect and trap P* are applied together to allow its spectroscopic and kinetic characterization. First, the MMOB mutant His33Ala is used to specifically slow the decay of P* without affecting its formation rate, leading to its nearly quantitative accumulation. Second, spectra-kinetic data collection is used to provide a sensitive measure of the formation and decay rate constants of intermediates as well as their optical spectra. Finally, the substrate furan is included to react with Q and quench its strong chromophore. The optical spectrum of P* closely mimics those of Hred and O, but it is distinctly different from that of P. The reaction cycle rate constants allowed prediction of the times for maximal accumulation of the intermediates. Mössbauer spectra of rapid freeze quench samples at these times show that the intermediates are formed at almost exactly the predicted levels. The Mössbauer spectra show that the diiron cluster of P*, quite unexpectedly, is in the FeIIFeII state. Thus, the loss of the g = 16 EPR results from a change of

  9. Biocatalytic conversion of ethylene to ethylene oxide using an engineered toluene monooxygenase

    SciTech Connect

    Carlin, DA; Bertolani, SJ; Siegel, JB

    2015-01-01

    Mutants of toluene o-xylene monooxygenase are demonstrated to oxidize ethylene to ethylene oxide in vivo at yields of >99%. The best mutant increases ethylene oxidation activity by >5500-fold relative to the native enzyme. This is the first report of a recombinant enzyme capable of carrying out this industrially significant chemical conversion.

  10. A fluorescence polarization binding assay to identify inhibitors of flavin-dependent monooxygenases.

    PubMed

    Qi, Jun; Kizjakina, Karina; Robinson, Reeder; Tolani, Karishma; Sobrado, Pablo

    2012-06-01

    N-Hydroxylating monooxygenases (NMOs) are essential for pathogenesis in fungi and bacteria. NMOs catalyze the hydroxylation of sine and ornithine in the biosynthesis of hydroxamate-containing siderophores. Inhibition of kynurenine monooxygenase (KMO), which catalyzes the conversion of kynurenine to 3-hydroxykynurenine, alleviates neurodegenerative disorders such as Huntington's and Alzheimer's diseases and brain infections caused by the parasite Trypanosoma brucei. These enzymes are examples of flavin-dependent monooxygenases, which are validated drug targets. Here, we describe the development and optimization of a fluorescence polarization assay to identify potential inhibitors of flavin-dependent monooxygenases. Fluorescently labeled ADP molecules were synthesized and tested. An ADP-TAMRA chromophore bound to KMO with a K(d) value of 0.60 ± 0.05 μM and to the NMOs from Aspergillus fumigatus and Mycobacterium smegmatis with K(d) values of 2.1 ± 0.2 and 4.0 ± 0.2 μM, respectively. The assay was tested in competitive binding experiments with substrates and products of KMO and an NMO. Furthermore, we show that this assay can be used to identify inhibitors of NMOs. A Z' factor of 0.77 was calculated, and we show that the assay exhibits good tolerance to temperature, incubation time, and dimethyl sulfoxide concentration. PMID:22410281

  11. Flavoprotein monooxygenases for oxidative biocatalysis: recombinant expression in microbial hosts and applications

    PubMed Central

    Ceccoli, Romina D.; Bianchi, Dario A.; Rial, Daniela V.

    2014-01-01

    External flavoprotein monooxygenases comprise a group of flavin-dependent oxidoreductases that catalyze the insertion of one atom of molecular oxygen into an organic substrate and the second atom is reduced to water. These enzymes are involved in a great number of metabolic pathways both in prokaryotes and eukaryotes. Flavoprotein monooxygenases have attracted the attention of researchers for several decades and the advent of recombinant DNA technology caused a great progress in the field. These enzymes are subjected to detailed biochemical and structural characterization and some of them are also regarded as appealing oxidative biocatalysts for the production of fine chemicals and valuable intermediates toward active pharmaceutical ingredients due to their high chemo-, stereo-, and regioselectivity. Here, we review the most representative reactions catalyzed both in vivo and in vitro by prototype flavoprotein monooxygenases, highlighting the strategies employed to produce them recombinantly, to enhance the yield of soluble proteins, and to improve cofactor regeneration in order to obtain versatile biocatalysts. Although we describe the most outstanding features of flavoprotein monooxygenases, we mainly focus on enzymes that were cloned, expressed and used for biocatalysis during the last years. PMID:24567729

  12. Cloning, expression and characterization of a eukaryotic cycloalkanone monooxygenase from Cylindrocarpon radicicola ATCC 11011.

    PubMed

    Leipold, Friedemann; Wardenga, Rainer; Bornscheuer, Uwe T

    2012-05-01

    In this study, we have cloned and characterized a cycloalkanone monooxygenase (CAMO) from the ascomycete Cylindrocarpon radicicola ATCC 11011 (identical to Cylindrocarpon destructans DSM 837). The primary structure of this Baeyer-Villiger monooxygenase (BMVO) revealed 531 residues with around 45% sequence identity to known cyclohexanone monooxygenases. The enzyme was functionally overexpressed in Escherichia coli and investigated with respect to substrate spectrum and kinetic parameters. Substrate specificity studies revealed that a large variety of cycloaliphatic and bicycloaliphatic ketones are converted by this CAMO. A high catalytic efficiency against cyclobutanone was observed and seems to be a particular property of this BVMO. The thus produced butyrolactone derivatives are valuable building blocks for the synthesis of a variety of natural products and bioactive compounds. Furthermore, the enzyme revealed activity against open-chain ketones such as cyclobutyl, cyclopentyl and cyclohexyl methyl ketone which have not been reported to be accepted by typical cyclohexanone monooxygenases. These results suggest that the BVMO from C. radicicola indeed might be rather unique and since no BVMOs originating from eukaryotic organisms have been produced recombinantly so far, this study provides the first example for such an enzyme. PMID:22075635

  13. Expression of Individual Copies of Methylococcus capsulatus Bath Particulate Methane Monooxygenase Genes

    PubMed Central

    Stolyar, Sergei; Franke, Marion; Lidstrom, Mary E.

    2001-01-01

    The expression of the two gene clusters encoding the particulate methane monooxygenase (pMMO) in Methylococcus capsulatus Bath was assessed by analysis of transcripts and by use of chromosomal gene fusions. The results suggest that the two clusters are functionally redundant but that relative expression alters depending on the copper levels available for growth. PMID:11160118

  14. Some properties of a soluble methane mono-oxygenase from Methylococcus capsulatus strain Bath.

    PubMed Central

    Colby, J; Dalton, H

    1976-01-01

    Soluble extracts of Methylococcus capsulatus (Bath), obtained by centrifugation of crude extracts at 160000g for 1h, catalyse the NAD(P)H- and O2-dependent disappearance of bromomethane, and also the formation of methanol from methane. Soluble methane mono-oxygenase is not inhibited by chelating agents or by most electron-transport inhibitors, and is a multicomponent enzyme. PMID:962879

  15. Expression of individual copies of Methylococcus capsulatus bath particulate methane monooxygenase genes.

    PubMed

    Stolyar, S; Franke, M; Lidstrom, M E

    2001-03-01

    The expression of the two gene clusters encoding the particulate methane monooxygenase (pMMO) in Methylococcus capsulatus Bath was assessed by analysis of transcripts and by use of chromosomal gene fusions. The results suggest that the two clusters are functionally redundant but that relative expression alters depending on the copper levels available for growth. PMID:11160118

  16. Identification of Cytochrome P450 monooxygenases in Diaphornia citri, an economically important psyllid

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Twenty P450’s in the CYP monooxygenases were identified in the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae). The psyllid is responsible for the transmission of Candidatus Liberibacter asiaticus, the causative agent of huanglongbing ( HLB), also known as citrus greening dise...

  17. Identification of a microsomal retinoic acid synthase as a microsomal cytochrome P-450-linked monooxygenase system.

    PubMed

    Tomita, S; Tsujita, M; Matsuo, Y; Yubisui, T; Ichikawa, Y

    1993-12-01

    1. To characterize an enzyme which metabolizes retinal in liver microsomes, several properties of the enzymatic reaction from retinal to retinoic acid were investigated using rabbit liver microsomes. 2. The maximum pH of the reaction in the liver microsomes was 7.6. 3. The Km and Vmax values for all-trans, 9-cis and 13-cis-retinals were determined. 4. The reaction proceeded in the presence of NADPH and molecular oxygen. 5. The incorporation of one atom of molecular oxygen into retinal was confirmed by using oxygen-18, showing that the reaction comprised monooxygenation, not dehydrogenation. 6. The monooxygenase activity was inhibited by carbon monoxide, phenylisocyanide and anti-NADPH-cytochrome P-450 reductase IgG, but not by anti-cytochrome b5 IgG. 7. The enzymatic activity inhibited by carbon monoxide was photoreversibly restored by light of a wavelength of around 450 nm. 8. The retinal-induced spectra of liver microsomes with three isomeric retinals were type I spectra. 9. The microsomal monooxygenase activity induced by phenobarbital or ethanol were more effective than that by 3-methylcholanthrene, clotrimazole or beta-naphthoflavone. 10. These results showed that the monooxygenase reaction from retinal to retinoic acid in liver microsomes is catalyzed by a cytochrome P-450-linked monooxygenase system. PMID:8138015

  18. Modeling the role of alkanes, polycyclic aromatic hydrocarbons, and their oligomers in secondary organic aerosol formation.

    PubMed

    Pye, Havala O T; Pouliot, George A

    2012-06-01

    A computationally efficient method to treat secondary organic aerosol (SOA) from various length and structure alkanes as well as SOA from polycyclic aromatic hydrocarbons (PAHs) is implemented in the Community Multiscale Air Quality (CMAQ) model to predict aerosol concentrations over the United States. Oxidation of alkanes is predicted to produce more aerosol than oxidation of PAHs driven by relatively higher alkane emissions. SOA from alkanes and PAHs, although small in magnitude, can be a substantial fraction of the SOA from anthropogenic hydrocarbons, particularly in winter, and could contribute more if emission inventories lack intermediate volatility alkanes (>C(13)) or if the vehicle fleet shifts toward diesel-powered vehicles. The SOA produced from oxidation of alkanes correlates well with ozone and odd oxygen in many locations, but the lower correlation of anthropogenic oligomers with odd oxygen indicates that models may need additional photochemically dependent pathways to low-volatility SOA. PMID:22568386

  19. Fundamental Flame Velocities of Pure Hydrocarbons I : Alkanes, Alkenes, Alkynes Benzene, and Cyclohexane

    NASA Technical Reports Server (NTRS)

    Gerstein, Melvin; Levine, Oscar; Wong, Edgar L

    1950-01-01

    The flame velocities of 37 pure hydrocarbons including normal and branched alkanes, alkenes, and alkynes; as well as benzene and cyclohexane, together with the experimental technique employed are presented. The normal alkanes have about the same flame velocity from ethane through heptane with methane being about 16 percent lower. Unsaturation increases the flame velocity in the order of alkanes, alkenes, and alkynes. Branching reduces the flame velocity.

  20. Photochemical dimerization and functionalization of alkanes, ethers, primary alcohols and silanes

    DOEpatents

    Crabtree, Robert H.; Brown, Stephen H.

    1988-01-01

    The space-time yield and/or the selectivity of the photochemical dimerization of alkanes, ethers, primary alcohols and tertiary silanes with Hg and U.V. light is enhanced by refluxing the substrate in the irradiated reaction zone at a temperature at which the dimer product condenses and remains condensed promptly upon its formation. Cross-dimerization of the alkanes, ethers and silanes with primary alcohols is disclosed, as is the functionalization to aldehydes of the alkanes with carbon monoxide.

  1. Photochemical dimerization and functionalization of alkanes, ethers, primary alcohols and silanes

    DOEpatents

    Crabtree, R.H.; Brown, S.H.

    1988-02-16

    The space-time yield and/or the selectivity of the photochemical dimerization of alkanes, ethers, primary alcohols and tertiary silanes with Hg and U.V. light is enhanced by refluxing the substrate in the irradiated reaction zone at a temperature at which the dimer product condenses and remains condensed promptly upon its formation. Cross-dimerization of the alkanes, ethers and silanes with primary alcohols is disclosed, as is the functionalization to aldehydes of the alkanes with carbon monoxide.

  2. Cloning and characterization of the genes encoding nitrilotriacetate monooxygenase of Chelatobacter heintzii ATCC 29600.

    PubMed Central

    Knobel, H R; Egli, T; van der Meer, J R

    1996-01-01

    A 6.2-kb DNA fragment containing the genes for the nitrilotriacetate (NTA) monooxygenase of Chelatobacter heintzii ATCC 29600 was cloned and characterized by DNA sequencing and expression studies. The nucleotide sequence contained three major open reading frames (ORFs). Two of the ORFs, which were oriented divergently with an intergenic region of 307 bp, could be assigned to the NTA monooxygenase components A and B. The predicted N-terminal amino acid sequences of these ORFs were identical with those determined for the purified components. We therefore named these genes ntaA (for component A of NTA monooxygenase) and ntaB (for component B). The ntaA and ntaB genes could be expressed in Escherichia coli DH5alpha, and the gene products were visualized after Western blotting (immunoblotting) and incubation with polyclonal antibodies against component A or B. By mixing overproduced NtaB from E. coli and purified component A from C. heintzii ATCC 29600, reconstitution of a functional NTA monooxygenase complex was possible. The deduced gene product of ntaA showed only significant homology to SoxA (involved in dibenzothiophene degradation) and to SnaA (involved in pristamycin synthesis); that of ntaB shared weak homologies in one domain with other NADH:flavine mononucleotide oxidoreductases. These homologies provide no conclusive answer as to the possible evolutionary origin of the NTA monooxygenase. The deduced gene product of the third ORF (ORF1) had homology in the N-terminal region with the GntR class of bacterial regulator proteins and therefore may encode a regulator protein, possibly involved in regulation of ntaA and ntaB expression. PMID:8892809

  3. Gas-Phase Reactions of Atomic Gold Cations with Linear Alkanes (C2-C9).

    PubMed

    Zhang, Ting; Li, Zi-Yu; Zhang, Mei-Qi; He, Sheng-Gui

    2016-06-30

    To develop proper ionization methods for alkanes, the reactivity of bare or ligated transition metal ions toward alkanes has attracted increasing interests. In this study, the reactions of the gold cations with linear alkanes from ethane up to nonane (CnH2n+2, n = 2-9) under mild conditions have been characterized by mass spectrometry and density functional theory calculations. When reacting with Au(+), small alkanes (n = 2-6) were confirmed to follow specific reaction channels of dehydrogenation for ethane and hydride transfer for others to generate product ions characteristic of the original alkanes, which indicates that Au(+) can act as a reagent ion to ionize alkanes from ethane to n-hexane. Strong dependence of the chain length of alkanes was observed for the rate constants and reaction efficiencies. Extensive fragmentation took place for larger alkanes (n > 6). Theoretical results show that the fragmentation induced by the hydride transfer occurs after the release of AuH. Moreover, the fragmentation of n-heptane was successfully avoided when the reaction took place in a high-pressure reactor. This implies that Au(+) is a potential reagent ion to ionize linear and even the branched alkanes. PMID:27266670

  4. Identification and use of an alkane transporter plug-in for applications in biocatalysis and whole-cell biosensing of alkanes

    NASA Astrophysics Data System (ADS)

    Grant, Chris; Deszcz, Dawid; Wei, Yu-Chia; Martínez-Torres, Rubéns Julio; Morris, Phattaraporn; Folliard, Thomas; Sreenivasan, Rakesh; Ward, John; Dalby, Paul; Woodley, John M.; Baganz, Frank

    2014-07-01

    Effective application of whole-cell devices in synthetic biology and biocatalysis will always require consideration of the uptake of molecules of interest into the cell. Here we demonstrate that the AlkL protein from Pseudomonas putida GPo1 is an alkane import protein capable of industrially relevant rates of uptake of C7-C16 n-alkanes. Without alkL expression, native E.coli n-alkane uptake was the rate-limiting step in both the whole-cell bioconversion of C7-C16 n-alkanes and in the activation of a whole-cell alkane biosensor by C10 and C11 alkanes. By coexpression of alkL as a transporter plug-in, specific yields improved by up to 100-fold for bioxidation of >C12 alkanes to fatty alcohols and acids. The alkL protein was shown to be toxic to the host when overexpressed but when expressed from a vector capable of controlled induction, yields of alkane oxidation were improved a further 10-fold (8 g/L and 1.7 g/g of total oxidized products). Further testing of activity on n-octane with the controlled expression vector revealed the highest reported rates of 120 μmol/min/g and 1 g/L/h total oxidized products. This is the first time AlkL has been shown to directly facilitate enhanced uptake of C10-C16 alkanes and represents the highest reported gain in product yields resulting from its use.

  5. Preliminary assessment of halogenated alkanes as vapor-phase tracers

    SciTech Connect

    Adams, Michael C.; Moore, Joseph N.; Hirtz, Paul

    1991-01-01

    New tracers are needed to evaluate the efficiency of injection strategies in vapor-dominated environments. One group of compounds that seems to meet the requirements for vapor-phase tracing are the halogenated alkanes (HCFCs). HCFCs are generally nontoxic, and extrapolation of tabulated thermodynamic data indicate that they will be thermally stable and nonreactive in a geothermal environment. The solubilities and stabilities of these compounds, which form several homologous series, vary according to the substituent ratios of fluorine, chlorine, and hydrogen. Laboratory and field tests that will further define the suitability of HCFCs as vapor-phase tracers are under way.

  6. Transporter engineering for improved tolerance against alkane biofuels in Saccharomyces cerevisiae

    PubMed Central

    2013-01-01

    Background Hydrocarbon alkanes, components of major fossil fuels, are considered as next-generation biofuels because their biological production has recently been shown to be possible. However, high-yield alkane production requires robust host cells that are tolerant against alkanes, which exhibit cytotoxicity. In this study, we aimed to improve alkane tolerance in Saccharomyces cerevisiae, a key industrial microbial host, by harnessing heterologous transporters that potentially pump out alkanes. Results To this end, we attempted to exploit ABC transporters in Yarrowia lipolytica based on the observation that it utilizes alkanes as a carbon source. We confirmed the increased transcription of ABC2 and ABC3 transporters upon exposure to a range of alkanes in Y. lipolytica. We then showed that the heterologous expression of ABC2 and ABC3 transporters significantly increased tolerance against decane and undecane in S. cerevisiae through maintaining lower intracellular alkane level. In particular, ABC2 transporter increased the tolerance limit of S. cerevisiae about 80-fold against decane. Furthermore, through site-directed mutagenesis for glutamate (E988 for ABC2, and E989 for ABC3) and histidine (H1020 for ABC2, and H1021 for ABC3), we provided the evidence that glutamate was essential for the activity of ABC2 and ABC3 transporters, with ATP most likely to be hydrolyzed by a catalytic carboxylate mechanism. Conclusions Here, we demonstrated that transporter engineering through expression of heterologous efflux pumps led to significantly improved tolerance against alkane biofuels in S. cerevisiae. We believe that our results laid the groundwork for developing robust alkane-producing yeast cells through transporter engineering, which will greatly aid in next-generation alkane biofuel production and recovery. PMID:23402697

  7. A New Biocatalyst for Production of Optically Pure Aryl Epoxides by Styrene Monooxygenase from Pseudomonas fluorescens ST

    PubMed Central

    Di Gennaro, Patrizia; Colmegna, Andrea; Galli, Enrica; Sello, Guido; Pelizzoni, Francesca; Bestetti, Giuseppina

    1999-01-01

    We developed a biocatalyst by cloning the styrene monooxygenase genes (styA and styB) from Pseudomonas fluorescens ST responsible for the oxidation of styrene to its corresponding epoxide. Recombinant Escherichia coli was able to oxidize different aryl vinyl and aryl ethenyl compounds to their corresponding optically pure epoxides. The results of bioconversions indicate the broad substrate preference of styrene monooxygenase and its potential for the production of several fine chemicals. PMID:10347083

  8. Nucleotide sequence analysis of genes encoding a toluene/benzene-2-monooxygenase from pseudomonas sp. strain JS150

    SciTech Connect

    Johnson, G.R.; Olsen, R.H.

    1995-09-01

    Pseudomonas sp. strain JS150 metabolizes benzene and alkyl- and chloro-substituted benzenes by using dioxygenase-initiated pathways coupled with multiple downstream metabolic pathways to accommodate catechol metabolism. By cloning genes encoding benzene-degradative enzymes, strain JS150 was also found to carry genes for a toluene/benzene-2-monooxygenase. The gene cluster encoding a 2-monooxygenase and its cognate regulator was cloned from a plasmid carried by strain JS150. Oxygen ({sup 18}O{sub 2}) incorporation experiments using Pseudomonas aeruginosa strains carrying the cloned genes confirmed toluene hydroxylation was catalyzed through an authentic monooxygenase reaction to yield ortho-cresol. Encoding the toluene-2-monooxygenase and regulatory gene product was localized in two regions of the cloned fragment. The nucleotide sequence of the toluene/benzene-2-monooxygenase locus was determined, revealing six open reading frames that were then designated tbmA, tbmB, tbmC, tbmD, tbmE, and tbmF. The deduced amino acid sequences for these genes showed the presence of motifs similar to well-conserved functional domains of multicomponent oxygenases. This analysis allowed the tentative identification of two terminal oxygenase subunits (TbmB and TbmD) and an electron transport protein (TbmF) for the monooxygenase enzyme. All the tbm polypeptides shared significant homology with protein components from other bacterial multicomponent monooxygenases. Overall, the tbm gene products shared greater similarity with polypeptides from the phenol hydroxylases of Pseudomo-KR1 and Burkholderia (Pseudomonas) picketti PKO1. The relationship found between the phenol hydroxlases and a toluene-2-monooxygenase, characterized in this study for the first time at the nucleotide sequence level, suggested DNA probes used for surveys of environmental populations should be carefully selected to reflect DNA sequences corresponding to the metabolic pathway of interest. 58 refs., 8 figs., 1 tab.

  9. Effect of alkane chain length and counterion on the freezing transition of cationic surfactant adsorbed film at alkane mixture - water interfaces.

    PubMed

    Tokiwa, Yuhei; Sakamoto, Hiroyasu; Takiue, Takanori; Aratono, Makoto; Matsubara, Hiroki

    2015-05-21

    Penetration of alkane molecules into the adsorbed film gives rise to a surface freezing transition of cationic surfactant at the alkane-water interface. To examine the effect of the alkane chain length and counterion on the surface freezing, we employed interfacial tensiometry and ellipsometry to study the interface of cetyltrimethylammonium bromide and cetyltrimethylammonium chloride aqueous solutions against dodecane, tetradecane, hexadecane, and their mixtures. Applying theoretical equations to the experimental results obtained, we found that the alkane molecules that have the same chain length as the surfactant adsorb preferentially into the surface freezing film. Furthermore, we demonstrated that the freezing transition temperature of cationic surfactant adsorbed film was independent of the kind of counterion. PMID:25932500

  10. 40 CFR 721.4464 - Mixture of hydrofluoro alkanes and hydrofluoro alkene.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... hydrofluoro alkene. 721.4464 Section 721.4464 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.4464 Mixture of hydrofluoro alkanes and hydrofluoro alkene. (a) Chemical... as a mixture of hydrofluoro alkanes and hydrofluoro alkene (PMNs P-96-945/946/947/948) are subject...

  11. 40 CFR 721.4464 - Mixture of hydrofluoro alkanes and hydrofluoro alkene.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... hydrofluoro alkene. 721.4464 Section 721.4464 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.4464 Mixture of hydrofluoro alkanes and hydrofluoro alkene. (a) Chemical... as a mixture of hydrofluoro alkanes and hydrofluoro alkene (PMNs P-96-945/946/947/948) are subject...

  12. 40 CFR 721.4464 - Mixture of hydrofluoro alkanes and hydrofluoro alkene.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... hydrofluoro alkene. 721.4464 Section 721.4464 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.4464 Mixture of hydrofluoro alkanes and hydrofluoro alkene. (a) Chemical... as a mixture of hydrofluoro alkanes and hydrofluoro alkene (PMNs P-96-945/946/947/948) are subject...

  13. 40 CFR 721.4464 - Mixture of hydrofluoro alkanes and hydrofluoro alkene.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... hydrofluoro alkene. 721.4464 Section 721.4464 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.4464 Mixture of hydrofluoro alkanes and hydrofluoro alkene. (a) Chemical... as a mixture of hydrofluoro alkanes and hydrofluoro alkene (PMNs P-96-945/946/947/948) are subject...

  14. 40 CFR 721.4464 - Mixture of hydrofluoro alkanes and hydrofluoro alkene.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... hydrofluoro alkene. 721.4464 Section 721.4464 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Specific Chemical Substances § 721.4464 Mixture of hydrofluoro alkanes and hydrofluoro alkene. (a) Chemical... as a mixture of hydrofluoro alkanes and hydrofluoro alkene (PMNs P-96-945/946/947/948) are subject...

  15. 40 CFR 721.2625 - Reaction product of alkane-diol and epichlorohydrin.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ...) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.2625 Reaction product of alkane-diol and epichlorohydrin. (a) Chemical... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Reaction product of alkane-diol...

  16. 40 CFR 721.2625 - Reaction product of alkane-diol and epichlorohydrin.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ...) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.2625 Reaction product of alkane-diol and epichlorohydrin. (a) Chemical... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Reaction product of alkane-diol...

  17. 40 CFR 721.2625 - Reaction product of alkane-diol and epichlorohydrin.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ...) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.2625 Reaction product of alkane-diol and epichlorohydrin. (a) Chemical... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Reaction product of alkane-diol...

  18. 40 CFR 721.2625 - Reaction product of alkane-diol and epichlorohydrin.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ...) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.2625 Reaction product of alkane-diol and epichlorohydrin. (a) Chemical... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Reaction product of alkane-diol...

  19. 40 CFR 721.2625 - Reaction product of alkane-diol and epichlorohydrin.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ...) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES Significant New Uses for Specific Chemical Substances § 721.2625 Reaction product of alkane-diol and epichlorohydrin. (a) Chemical... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Reaction product of alkane-diol...

  20. Measurement of n-alkanals and hydroxyalkenals in biological samples.

    PubMed

    Holley, A E; Walker, M K; Cheeseman, K H; Slater, T F

    1993-09-01

    A modified method was developed to measure nM levels of a range of n-alkanals and hydroxyalkenals in biological samples such as blood plasma and tissue homogenates and also in Folch lipid extracts of these samples. Butylated hydroxytoluene (BHT) and desferrioxamine (Desferal) were added to samples to prevent artifactual peroxidation. Aldehydes were reacted with 1,3-cyclohexanedione (CHD), cleaned up by solid-phase extraction on a Sep-Pak C18 cartridge and the fluorescent decahydroacridine derivatives resolved by reverse-phase high-performance liquid chromatography (HPLC) with gradient elution. A wider range of aldehydes was detected in lipid extracts of plasma and liver homogenate compared to whole (unextracted) samples. Human plasma contained nM levels of acetaldehyde, propanal, butanal, pentanal, hexanal, and heptanal. 4-Hydroxynonenal (0.93 nmol/g) and alkanals with two to six carbons (up to 7.36 nmol/g) were detected in rat liver. Recovery of aldehydes added to whole plasma or to lipid extracts of plasma was dependent on carbon chain length, varying from 95% for acetaldehyde to 8% for decanal. Recovery from biological samples was significantly less than that of standards taken through the Sep-Pak clean-up procedure, suggesting that aldehydes can bind to plasma protein and lipid components. PMID:8406128

  1. Direct hydrodeoxygenation of raw woody biomass into liquid alkanes.

    PubMed

    Xia, Qineng; Chen, Zongjia; Shao, Yi; Gong, Xueqing; Wang, Haifeng; Liu, Xiaohui; Parker, Stewart F; Han, Xue; Yang, Sihai; Wang, Yanqin

    2016-01-01

    Being the only sustainable source of organic carbon, biomass is playing an ever-increasingly important role in our energy landscape. The conversion of renewable lignocellulosic biomass into liquid fuels is particularly attractive but extremely challenging due to the inertness and complexity of lignocellulose. Here we describe the direct hydrodeoxygenation of raw woods into liquid alkanes with mass yields up to 28.1 wt% over a multifunctional Pt/NbOPO4 catalyst in cyclohexane. The superior performance of this catalyst allows simultaneous conversion of cellulose, hemicellulose and, more significantly, lignin fractions in the wood sawdust into hexane, pentane and alkylcyclohexanes, respectively. Investigation on the molecular mechanism reveals that a synergistic effect between Pt, NbOx species and acidic sites promotes this highly efficient hydrodeoxygenation of bulk lignocellulose. No chemical pretreatment of the raw woody biomass or separation is required for this one-pot process, which opens a general and energy-efficient route for converting raw lignocellulose into valuable alkanes. PMID:27025898

  2. Multiple sources of alkanes in Quaternary oceanic sediment of Antarctica

    USGS Publications Warehouse

    Kvenvolden, K.A.; Rapp, J.B.; Golan-Bac, M.; Hostettler, F.D.

    1987-01-01

    Normal alkanes (n-C13n-C36), isoprenoid hydrocarbons (i-C15, i-C16, i-C18, i-C19, and i-C20) triterpanes (C27C32), and (C27C29) are present in low concentrations offshore Antarctica in near-surface, Quaternary sediment of the Wilkes Land continental margin and of the western Ross Sea. The distributions of these hydrocarbons are interpreted relative to possible sources and processes. The hydrocarbons appear to be mixtures of primary and recycled material from marine and terrigenous sources. The n-alkanes are most abundant and are characterized by two distinct populations, one of probable marine origin and the other likely from terrigenous, vascular plant sources. Because the continent of Antarctica today is devoid of higher plants, the plant-derived hydrocarbons in these offshore sediments probably came from wind-blown material and recycled Antarctic sediment that contains land-plant remains from an earlier period of time. Isoprenoid hydrocarbons are partially recycled and mainly of marine origin; the dominance of pristane over phytane suggests oxic paleoenvironmental conditions. Both modern and ancient triterpanes and steranes are present, and the distribution of these indicates a mixture of primary and recycled bacterial, algal, and possible higher-plant materials. Although the sampled sediments were deposited during the Quaternary, they apparently contain a significant component of hydrocarbons of pre-Quaternary age. ?? 1987.

  3. Direct hydrodeoxygenation of raw woody biomass into liquid alkanes

    PubMed Central

    Xia, Qineng; Chen, Zongjia; Shao, Yi; Gong, Xueqing; Wang, Haifeng; Liu, Xiaohui; Parker, Stewart F.; Han, Xue; Yang, Sihai; Wang, Yanqin

    2016-01-01

    Being the only sustainable source of organic carbon, biomass is playing an ever-increasingly important role in our energy landscape. The conversion of renewable lignocellulosic biomass into liquid fuels is particularly attractive but extremely challenging due to the inertness and complexity of lignocellulose. Here we describe the direct hydrodeoxygenation of raw woods into liquid alkanes with mass yields up to 28.1 wt% over a multifunctional Pt/NbOPO4 catalyst in cyclohexane. The superior performance of this catalyst allows simultaneous conversion of cellulose, hemicellulose and, more significantly, lignin fractions in the wood sawdust into hexane, pentane and alkylcyclohexanes, respectively. Investigation on the molecular mechanism reveals that a synergistic effect between Pt, NbOx species and acidic sites promotes this highly efficient hydrodeoxygenation of bulk lignocellulose. No chemical pretreatment of the raw woody biomass or separation is required for this one-pot process, which opens a general and energy-efficient route for converting raw lignocellulose into valuable alkanes. PMID:27025898

  4. Geologic seepage of methane and light alkanes in Los Angeles

    NASA Astrophysics Data System (ADS)

    Doezema, L. A.; Chang, K.; Baril, R.; Nwachuku, I.; Contreras, P.; Marquez, A.; Howard, D.

    2013-12-01

    Natural geologic seepage of methane from underground oil and natural gas reservoirs has been suggested to be an underreported part of the global methane budget. Other light alkanes are also given off in combination with the methane seepage, making it possible that geologic seepage is also a potentially significant global source of these light alkanes. This study reports C1-C5 findings from geologic seepage made in the Los Angeles region. Microseepage, invisible escape of gases, was measured primarily at Kenneth Hahn Regional Park, while macroseepage, the visible release of gases, was measured at the La Brea Tar Pits. Samples were collected using stainless steel canisters and flux chambers and were analyzed using gas chromatography with flame ionization detectors (GC-FID). Average microseepage flux rates of 0.95 μg m-2 h-1 for ethane and 0.51 μg m-2 h-1 were found for propane, while average macroseepage rates for methane, ethane, and propane were 664, 19.8, and 18.1 mg m-2 h-1 respectively. Relationships between microseepage flux rate and location of underground oil and natural deposit and earthquake fault lines are presented. Additionally, the relative importance of findings in context with global budgets and local air quality is discussed.

  5. Geomicrobiological linkages between short-chain alkane consumption and sulfate reduction rates in seep sediments

    PubMed Central

    Bose, Arpita; Rogers, Daniel R.; Adams, Melissa M.; Joye, Samantha B.; Girguis, Peter R.

    2013-01-01

    Marine hydrocarbon seeps are ecosystems that are rich in methane, and, in some cases, short-chain (C2–C5) and longer alkanes. C2–C4 alkanes such as ethane, propane, and butane can be significant components of seeping fluids. Some sulfate-reducing microbes oxidize short-chain alkanes anaerobically, and may play an important role in both the competition for sulfate and the local carbon budget. To better understand the anaerobic oxidation of short-chain n-alkanes coupled with sulfate-reduction, hydrocarbon-rich sediments from the Gulf of Mexico (GoM) were amended with artificial, sulfate-replete seawater and one of four n-alkanes (C1–C4) then incubated under strict anaerobic conditions. Measured rates of alkane oxidation and sulfate reduction closely follow stoichiometric predictions that assume the complete oxidation of alkanes to CO2 (though other sinks for alkane carbon likely exist). Changes in the δ13C of all the alkanes in the reactors show enrichment over the course of the incubation, with the C3 and C4 incubations showing the greatest enrichment (4.4 and 4.5‰, respectively). The concurrent depletion in the δ13C of dissolved inorganic carbon (DIC) implies a transfer of carbon from the alkane to the DIC pool (−3.5 and −6.7‰ for C3 and C4 incubations, respectively). Microbial community analyses reveal that certain members of the class Deltaproteobacteria are selectively enriched as the incubations degrade C1–C4 alkanes. Phylogenetic analyses indicate that distinct phylotypes are enriched in the ethane reactors, while phylotypes in the propane and butane reactors align with previously identified C3–C4 alkane-oxidizing sulfate-reducers. These data further constrain the potential influence of alkane oxidation on sulfate reduction rates (SRRs) in cold hydrocarbon-rich sediments, provide insight into their contribution to local carbon cycling, and illustrate the extent to which short-chain alkanes can serve as electron donors and govern microbial

  6. Diversity of soluble methane monooxygenase-containing methanotrophs isolated from polluted environments.

    PubMed

    McDonald, Ian R; Miguez, Carlos B; Rogge, Gerlinde; Bourque, Denis; Wendlandt, Karin D; Groleau, Denis; Murrell, J Colin

    2006-02-01

    Methanotrophs were enriched and isolated from polluted environments in Canada and Germany. Enrichments in low copper media were designed to specifically encourage growth of soluble methane monooxygenase (sMMO) containing organisms. The 10 isolates were characterized physiologically and genetically with one type I and nine type II methanotrophs being identified. Three key genes: 16S rRNA; pmoA and mmoX, encoding for the particulate and soluble methane monooxygenases respectively, were cloned from the isolates and sequenced. Phylogenetic analysis of these sequences identified strains, which were closely related to Methylococcus capsulatus, Methylocystis sp., Methylosinus sporium and Methylosinus trichosporium. Diversity of sMMO-containing methanotrophs detected in this and previous studies was rather narrow, both genetically and physiologically, suggesting possible constraints on genetic diversity of sMMO due to essential conservation of enzyme function. PMID:16448499

  7. Enhancing Indigo Production by Over-Expression of the Styrene Monooxygenase in Pseudomonas putida.

    PubMed

    Cheng, Lei; Yin, Sheng; Chen, Min; Sun, Baoguo; Hao, Shuai; Wang, Chengtao

    2016-08-01

    As an important traditional blue dye, indigo has been used in food and textile industry for centuries, which can be produced via the styrene oxygenation pathway in Pseudomonas putida. Hence, the styrene monooxygenase gene styAB and oxide isomerase gene styC are over-expressed in P. putida to investigate their roles in indigo biosynthesis. RT-qPCR analysis indicated that transcriptions of styA and styB were increased by 2500- and 750-folds in the styAB over-expressed strain B4-01, compared with the wild-type strain B4, consequently significantly enhancing the indole monooxygenase activity. Transcription of styC was also increased by 100-folds in the styC over-expressed strain B4-02. Besides, styAB over-expression slightly up-regulated the transcription of styC in B4-01, while styC over-expression hardly exerted an effect on the transcriptional levels of styA and styB and indole monooxygenase activity in B4-02. Furthermore, shaking flask experiments showed that indigo production in B4-01 reached 52.13 mg L(-1) after 24 h, which was sevenfold higher than that in B4. But no obvious increase in indigo yield was observed in B4-02. Over-expression of styAB significantly enhanced the indigo production, revealing that the monooxygenase STYAB rather than oxide isomerase STYC probably acted as the key rate-limiting enzyme in the indigo biosynthesis pathway in P. putida. This work provided a new strategy for enhancing indigo production in Pseudomonas. PMID:27154464

  8. Factors limiting aliphatic chlorocarbon degradation by Nitrosomonas europaea: Cometabolic inactivation of ammonia monooxygenase and substrate specificity

    SciTech Connect

    Rasche, M.E.; Hyman, M.R.; Arp, D.J. )

    1991-10-01

    The soil nitrifying bacterium Nitrosomonas europaea is capable of degrading trichloroethylene (TCE) and other halogenated hydrocarbons. TCE cometabolism by N. europaea resulted in an irreversible loss of TCE biodegradative capacity, ammonia-oxidizing activity, and ammonia-dependent O{sub 2} uptake by the cells. Inactivation was not observed in the presence of allylthiourea, a specific inhibitor of enzyme ammonia monooxygenase, or under anaerobic conditions, indicating that the TCE-mediated inactivation required ammonia monooxygenase activity. When N. europaea cells were incubated with ({sup 14}C)TCE under conditions which allowed turnover of ammonia monooxygenase, a number of cellular proteins were covalently labeled with {sup 14}C. Treatment of cells with allylthiourea or acetylene prior to incubation with ({sup 14}C)TCE prevented incorporation of {sup 14}C into proteins. The ammonia-oxidizing activity of cells inactivated in the presence of TCE could be recovered through a process requiring de novo protein synthesis. In addition to TCE, a series of chlorinated methanes, ethanes, and other ethylenes were screened as substrates for ammonia monooxygenase and for their ability to inactivate the ammonia-oxidizing system of N. europaea. The chlorocarbons would be divided into three classes depending on their biodegradability and inactivating potential: (1) compounds which were not biodegradable by N. europaea and which had no toxic effect on the cells (2) compounds which were cooxidized by N. europaea and had little or no toxic effect on the cells; and (3) compounds which were cooxidized and produced a turnover-dependent inactivation of ammonia oxidation by N. europaea.

  9. Induction of cytochrome P450 1A1 and monooxygenase activity in Tilapia by sediment extract

    SciTech Connect

    Ueng, Y.F.; Ueng, T.H.; Liu, T.Y.

    1995-01-01

    Cytochrome P450 (P450)-dependent monooxygenases of fishes are inducible by a variety of environmental pollutants including polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs). Induction of fish monoxygenases may serve as a biological monitor for PAH- and PCB-types of environmental chemicals. Many studies have demonstrated environmental induction of fish monooxygenases using various experimental approaches. However, relatively few studies have been conducted using fish treated with contaminated river sediment extracts. Damsui River is the largest river in the north of Taiwan. The lower section of the river in the Taipei Metropolitan area is heavily polluted by industrial and municipal wastes. Tilapia (Oreochromis mossambicus) is one of the few species of fish that occur in the polluted river. Previous field studies showed that the levels of P450 1A1, benzo(a)pyrene hydroxylase and 7-ethoxyresorufin O-deethylase activities in tilapia collected at Fu-Ho Bridge, a polluted section of Damsui River, were higher than respective levels in fish collected from an unpolluted section. These results suggested that tilapia caught at the polluted site were exposed to substances similar in action to PAHs and PCBs, because these chemical pollutants are potent inducers of P450 1A1. PAHs and PCBs are persistent compounds that can accumulate in sediment. Tilapia are occasionally associated with the bottom and could ingest chemically contaminated sediment. In the present study, we determined the induction properties of monooxygenases using tilapia treated with extract of sediment collected from a polluted section of Damsui River. The present study demonstrates that Damsui River sediment extract has the ability to induce hepatic P450 1A1 and dependent monooxygenase activities in tilapia. 17 refs., 2 figs., 2 tabs.

  10. Crystallization and initial crystallographic characterization of the Corynebacterium glutamicum nitrilotriacetate monooxygenase component A

    SciTech Connect

    Kim, Kyung-Jin; Kim, Sujin; Lee, Sujin; Kang, Beom Sik; Lee, Heung-Soo; Oh, Tae-Kwang; Kim, Myung Hee

    2006-11-01

    The Corynebacterium glutamicum NTA monooxygenase component A protein, which plays the central role in NTA biodegradation, was crystallized. The initial X-ray crystallographic characterization is reported. Safety and environmental concerns have recently dictated the proper disposal of nitrilotriacetate (NTA). Biodegradation of NTA is initiated by NTA monooxygenase, which is composed of two proteins: component A and component B. The NTA monooxygenase component A protein from Corynebacterium glutamicum was crystallized using the sitting-drop vapour-diffusion method in the presence of ammonium sulfate as the precipitant. X-ray diffraction data were collected to a maximum resolution of 2.5 Å on a synchrotron beamline. The crystal belongs to the monoclinic space group C2, with unit-cell parameters a = 111.04, b = 98.51, c = 171.61 Å, β = 101.94°. The asymmetric unit consists of four molecules, corresponding to a packing density of 2.3 Å{sup 3} Da{sup −1}. The structure was solved by molecular replacement. Structure refinement is in progress.

  11. Catalytic residues and a predicted structure of tetrahydrobiopterin-dependent alkylglycerol mono-oxygenase.

    PubMed

    Watschinger, Katrin; Fuchs, Julian E; Yarov-Yarovoy, Vladimir; Keller, Markus A; Golderer, Georg; Hermetter, Albin; Werner-Felmayer, Gabriele; Hulo, Nicolas; Werner, Ernst R

    2012-04-01

    Alkylglycerol mono-oxygenase (EC 1.14.16.5) forms a third, distinct, class among tetrahydrobiopterin-dependent enzymes in addition to aromatic amino acid hydroxylases and nitric oxide synthases. Its protein sequence contains the fatty acid hydroxylase motif, a signature indicative of a di-iron centre, which contains eight conserved histidine residues. Membrane enzymes containing this motif, including alkylglycerol mono-oxygenase, are especially labile and so far have not been purified to homogeneity in active form. To obtain a first insight into structure-function relationships of this enzyme, we performed site-directed mutagenesis of 26 selected amino acid residues and expressed wild-type and mutant proteins containing a C-terminal Myc tag together with fatty aldehyde dehydrogenase in Chinese-hamster ovary cells. Among all of the acidic residues within the eight-histidine motif, only mutation of Glu137 to alanine led to an 18-fold increase in the Michaelis-Menten constant for tetrahydrobiopterin, suggesting a role in tetrahydrobiopterin interaction. A ninth additional histidine residue essential for activity was also identified. Nine membrane domains were predicted by four programs: ESKW, TMHMM, MEMSAT and Phobius. Prediction of a part of the structure using the Rosetta membrane ab initio method led to a plausible suggestion for a structure of the catalytic site of alkylglycerol mono-oxygenase. PMID:22220568

  12. Kinetic evidence for an anion binding pocket in the active site of nitronate monooxygenase.

    PubMed

    Francis, Kevin; Gadda, Giovanni

    2009-10-01

    A series of monovalent, inorganic anions and aliphatic aldehydes were tested as inhibitors for Hansenula mrakii and Neurospora crassa nitronate monooxygenase, formerly known as 2-nitropropane dioxygenase, to investigate the structural features that contribute to the binding of the anionic nitronate substrates to the enzymes. A linear correlation between the volumes of the inorganic anions and their effectiveness as competitive inhibitors of the enzymes was observed in a plot of pK(is)versus the ionic volume of the anion with slopes of 0.041+/-0.001 mM/A(3) and 0.027+/-0.001 mM/A(3) for the H. mrakii and N. crassa enzymes, respectively. Aliphatic aldehydes were weak competitive inhibitors of the enzymes, with inhibition constants that are independent of their alkyl chain lengths. The reductive half reactions of H. mrakii nitronate monooxygenase with primary nitronates containing two to four carbon atoms all showed apparent K(d) values of approximately 5 mM. These results are consistent with the presence of an anion binding pocket in the active site of nitronate monooxygenase that interacts with the nitro group of the substrate, and suggest a minimal contribution of the hydrocarbon chain of the nitronates to the binding of the ligands to the enzyme. PMID:19683782

  13. A Flexible Glutamine Regulates the Catalytic Activity of Toluene o-Xylene Monooxygenase

    PubMed Central

    2015-01-01

    Toluene/o-xylene monooxygenase (ToMO) is a bacterial multicomponent monooxygenase capable of oxidizing aromatic substrates. The carboxylate-rich diiron active site is located in the hydroxylase component of ToMO (ToMOH), buried 12 Å from the surface of the protein. A small, hydrophilic pore is the shortest pathway between the diiron active site and the protein exterior. In this study of ToMOH from Pseudomonas sp. OX1, the functions of two residues lining this pore, N202 and Q228, were investigated using site-directed mutagenesis. Steady-state characterization of WT and the three mutant enzymes demonstrates that residues N202 and Q228 are critical for turnover. Kinetic isotope effects and pH profiles reveal that these residues govern the kinetics of water egress and prevent quenching of activated oxygen intermediates formed at the diiron active site. We propose that this activity arises from movement of these residues, opening and closing the pore during catalysis, as seen in previous X-ray crystallographic studies. In addition, N202 and Q228 are important for the interactions of the reductase and regulatory components to ToMOH, suggesting that they bind competitively to the hydroxylase. The role of the pore in the hydroxylase components of other bacterial multicomponent monooxygenases within the superfamily is discussed in light of these conclusions. PMID:24873259

  14. Glucosinolate Biosynthesis (Further Characterization of the Aldoxime-Forming Microsomal Monooxygenases in Oilseed Rape Leaves).

    PubMed Central

    Bennett, R. N.; Hick, A. J.; Dawson, G. W.; Wallsgrove, R. M.

    1995-01-01

    The initial steps in glucosinolate biosynthesis are thought to proceed from amino acids, via N-hydroxy amino acids, to aldoximes. We showed previously that microsomes from green leaves of oilseed rape (Brassica napus cv Bienvenu) contain two distinct monooxygenases that catalyze the conversion of homophenylalanine and dihomomethionine to their respective aldoximes. Further characterization of these enzymes has now demonstrated that the latter enzyme catalyzes the NADPH-dependent oxidative decarboxylation of two higher homologs of methionine, in addition to dihomomethionine. No activity was found for either enzyme with L-methionine, DL-homomethionine, L-phenylalanine, L-tyrosine, or L-tryptophan. Both of these rape monooxygenase activities are dependent on O2, not requiring any other O2 species or radical. The presence of an unoxidized sulfur atom and its relative position in the side chain of the aliphatic substrates are important for binding to the active site of the methionine-homolog enzyme. Neither enzyme has any characteristics of a cytochrome P450-type enzyme, and antiserum raised against cytochrome P450 reductase did not significantly inhibit monooxygenase activity. PMID:12228596

  15. Mammalian flavin-containing monooxygenases: structure/function, genetic polymorphisms and role in drug metabolism

    PubMed Central

    Krueger, Sharon K.; Williams, David E.

    2005-01-01

    Flavin-containing monooxygenase (FMO) oxygenates drugs and xenobiotics containing a “soft-nucleophile”, usually nitrogen or sulfur. FMO, like cytochrome P450 (CYP), is a monooxygenase, utilizing the reducing equivalents of NADPH to reduce 1 atom of molecular oxygen to water, while the other atom is used to oxidize the substrate. FMO and CYP also exhibit similar tissue and cellular location, molecular weight, substrate specificity, and exist as multiple enzymes under developmental control. The human FMO functional gene family is much smaller (5 families each with a single member) than CYP. FMO does not require a reductase to transfer electrons from NADPH and the catalytic cycle of the 2 monooxygenases is strikingly different. Another distinction is the lack of induction of FMOs by xenobiotics. In general, CYP is the major contributor to oxidative xenobiotic metabolism. However, FMO activity may be of significance in a number of cases and should not be overlooked. FMO and CYP have overlapping substrate specificities, but often yield distinct metabolites with potentially significant toxicological/pharmacological consequences. The physiological function(s) of FMO are poorly understood. Three of the 5 expressed human FMO genes, FMO1, FMO2 and FMO3, exhibit genetic polymorphisms. The most studied of these is FMO3 (adult human liver) in which mutant alleles contribute to the disease known as trimethylaminuria. The consequences of these FMO genetic polymorphisms in drug metabolism and human health are areas of research requiring further exploration. PMID:15922018

  16. Catalytic residues and a predicted structure of tetrahydrobiopterin-dependent alkylglycerol mono-oxygenase

    PubMed Central

    Watschinger, Katrin; Fuchs, Julian E.; Yarov-Yarovoy, Vladimir; Keller, Markus A.; Golderer, Georg; Hermetter, Albin; Werner-Felmayer, Gabriele; Hulo, Nicolas; Werner, Ernst R.

    2012-01-01

    Alkylglycerol mono-oxygenase (EC 1.14.16.5) forms a third, distinct, class among tetrahydrobiopterin-dependent enzymes in addition to aromatic amino acid hydroxylases and nitric oxide synthases. Its protein sequence contains the fatty acid hydroxylase motif, a signature indicative of a di-iron centre, which contains eight conserved histidine residues. Membrane enzymes containing this motif, including alkylglycerol mono-oxygenase, are especially labile and so far have not been purified to homogeneity in active form. To obtain a first insight into structure–function relationships of this enzyme, we performed site-directed mutagenesis of 26 selected amino acid residues and expressed wild-type and mutant proteins containing a C-terminal Myc tag together with fatty aldehyde dehydrogenase in Chinese-hamster ovary cells. Among all of the acidic residues within the eight-histidine motif, only mutation of Glu137 to alanine led to an 18-fold increase in the Michaelis–Menten constant for tetrahydrobiopterin, suggesting a role in tetrahydrobiopterin interaction. A ninth additional histidine residue essential for activity was also identified. Nine membrane domains were predicted by four programs: ESKW, TMHMM, MEMSAT and Phobius. Prediction of a part of the structure using the Rosetta membrane ab initio method led to a plausible suggestion for a structure of the catalytic site of alkylglycerol mono-oxygenase. PMID:22220568

  17. Crystal structure of a phenol-coupling P450 monooxygenase involved in teicoplanin biosynthesis

    SciTech Connect

    Li, Zhi; Rupasinghe, Sanjeewa G.; Schuler, Mary A.; Nair, Satish K.

    2012-02-08

    The lipoglycopeptide antibiotic teicoplanin has proven efficacy against gram-positive pathogens. Teicoplanin is distinguished from the vancomycin-type glycopeptide antibiotics, by the presence of an additional cross-link between the aromatic amino acids 1 and 3 that is catalyzed by the cytochrome P450 monooxygenase Orf6* (CYP165D3). As a goal towards understanding the mechanism of this phenol-coupling reaction, we have characterized recombinant Orf6* and determined its crystal structure to 2.2-{angstrom} resolution. Although the structure of Orf6* reveals the core fold common to other P450 monooxygenases, there are subtle differences in the disposition of secondary structure elements near the active site cavity necessary to accommodate its complex heptapeptide substrate. Specifically, the orientation of the F and G helices in Orf6* results in a more closed active site than found in the vancomycin oxidative enzymes OxyB and OxyC. In addition, Met226 in the I helix replaces the more typical Gly/Ala residue that is positioned above the heme porphyrin ring, where it forms a hydrogen bond with a heme iron-bound water molecule. Sequence comparisons with other phenol-coupling P450 monooxygenases suggest that Met226 plays a role in determining the substrate regiospecificity of Orf6*. These features provide further insights into the mechanism of the cross-linking mechanisms that occur during glycopeptide antibiotics biosynthesis.

  18. Inducible Monooxygenase Activities and 3-Methylcholanthrene-Initiated Tumorigenesis in Mouse Recombinant Inbred Sublines

    PubMed Central

    Atlas, Steven A.; Taylor, Benjamin A.; Diwan, Bhalchandra A.; Nebert, Daniel W.

    1976-01-01

    The induction of a certain group of hepatic monooxygenase activities by polycyclic aromatic compounds is regulated by the same locus or gene cluster controlling the formation of cytochrome P1–450 (P–448) in mice. Certain inbred strains of mice are "responsive" (Ahb) to such induction, whereas others are "nonresponsive" (Ahd). A pair of closely related sublines that differ with respect to the Ah locus (for aromatic hydrocarbon responsiveness) were used to identify or confirm the pleiotropic effects of this gene. The lines were derived by sibling-mating without selection from (C57L/J x AKR/J)F 2 mice; the two sublines were separated at the F12 generation. Ten microsomal monooxygenase activities and one cytosol enzyme activity known to be associated with the Ah locus were similarly associated with cytochrome P1–450 formation in these recombinant inbred sublines as well. Nine additional hepatic monooxygenase activities studied were found not to be associated with the Ah locus; certain of these activities were increased slightly, following treatment of nonresponsive as well as responsive mice with polycyclic aromatic compounds. The Ahb-containing subline was highly susceptible to 3-methylcholanthrene-induced subcutaneous sarcomas, whereas the Ah-d-containing subline was relatively resistant. These results emphasize the potential importance of this particular enzyme for the study of coordinated regulation in mammals. PMID:955403

  19. The oxygenating constituent of 3,6-diketocamphane monooxygenase from the CAM plasmid of Pseudomonas putida: the first crystal structure of a type II Baeyer–Villiger monooxygenase

    SciTech Connect

    Isupov, Michail N.; Schröder, Ewald; Gibson, Robert P.; Beecher, Jean; Donadio, Giuliana; Saneei, Vahid; Dcunha, Stephlina A.; McGhie, Emma J.; Sayer, Christopher; Davenport, Colin F.; Lau, Peter C.; Hasegawa, Yoshie; Iwaki, Hiroaki; Kadow, Maria; Balke, Kathleen; Bornscheuer, Uwe T.; Bourenkov, Gleb; Littlechild, Jennifer A.

    2015-10-31

    The first crystal structure of a type II Baeyer–Villiger monooxygenase reveals a different ring orientation of its FMN cofactor compared with other related bacterial luciferase-family enzymes. The three-dimensional structures of the native enzyme and the FMN complex of the overexpressed form of the oxygenating component of the type II Baeyer–Villiger 3,6-diketocamphane monooxygenase have been determined to 1.9 Å resolution. The structure of this dimeric FMN-dependent enzyme, which is encoded on the large CAM plasmid of Pseudomonas putida, has been solved by a combination of multiple anomalous dispersion from a bromine crystal soak and molecular replacement using a bacterial luciferase model. The orientation of the isoalloxazine ring of the FMN cofactor in the active site of this TIM-barrel fold enzyme differs significantly from that previously observed in enzymes of the bacterial luciferase-like superfamily. The Ala77 residue is in a cis conformation and forms a β-bulge at the C-terminus of β-strand 3, which is a feature observed in many proteins of this superfamily.

  20. Copper-Catalyzed Intermolecular Amidation and Imidation of Unactivated Alkanes

    PubMed Central

    2015-01-01

    We report a set of rare copper-catalyzed reactions of alkanes with simple amides, sulfonamides, and imides (i.e., benzamides, tosylamides, carbamates, and phthalimide) to form the corresponding N-alkyl products. The reactions lead to functionalization at secondary C–H bonds over tertiary C–H bonds and even occur at primary C–H bonds. [(phen)Cu(phth)] (1-phth) and [(phen)Cu(phth)2] (1-phth2), which are potential intermediates in the reaction, have been isolated and fully characterized. The stoichiometric reactions of 1-phth and 1-phth2 with alkanes, alkyl radicals, and radical probes were investigated to elucidate the mechanism of the amidation. The catalytic and stoichiometric reactions require both copper and tBuOOtBu for the generation of N-alkyl product. Neither 1-phth nor 1-phth2 reacted with excess cyclohexane at 100 °C without tBuOOtBu. However, the reactions of 1-phth and 1-phth2 with tBuOOtBu afforded N-cyclohexylphthalimide (Cy-phth), N-methylphthalimide, and tert-butoxycyclohexane (Cy-OtBu) in approximate ratios of 70:20:30, respectively. Reactions with radical traps support the intermediacy of a tert-butoxy radical, which forms an alkyl radical intermediate. The intermediacy of an alkyl radical was evidenced by the catalytic reaction of cyclohexane with benzamide in the presence of CBr4, which formed exclusively bromocyclohexane. Furthermore, stoichiometric reactions of [(phen)Cu(phth)2] with tBuOOtBu and (Ph(Me)2CO)2 at 100 °C without cyclohexane afforded N-methylphthalimide (Me-phth) from β-Me scission of the alkoxy radicals to form a methyl radical. Separate reactions of cyclohexane and d12-cyclohexane with benzamide showed that the turnover-limiting step in the catalytic reaction is the C–H cleavage of cyclohexane by a tert-butoxy radical. These mechanistic data imply that the tert-butoxy radical reacts with the C–H bonds of alkanes, and the subsequent alkyl radical combines with 1-phth2 to form the corresponding N-alkyl imide product

  1. Understanding plant-microbe interactions for phytoremediation of petroleum-polluted soil.

    PubMed

    Nie, Ming; Wang, Yijing; Yu, Jiayi; Xiao, Ming; Jiang, Lifen; Yang, Ji; Fang, Changming; Chen, Jiakuan; Li, Bo

    2011-01-01

    Plant-microbe interactions are considered to be important processes determining the efficiency of phytoremediation of petroleum pollution, however relatively little is known about how these interactions are influenced by petroleum pollution. In this experimental study using a microcosm approach, we examined how plant ecophysiological traits, soil nutrients and microbial activities were influenced by petroleum pollution in Phragmites australis, a phytoremediating species. Generally, petroleum pollution reduced plant performance, especially at early stages of plant growth. Petroleum had negative effects on the net accumulation of inorganic nitrogen from its organic forms (net nitrogen mineralization (NNM)) most likely by decreasing the inorganic nitrogen available to the plants in petroleum-polluted soils. However, abundant dissolved organic nitrogen (DON) was found in petroleum-polluted soil. In order to overcome initial deficiency of inorganic nitrogen, plants by dint of high colonization of arbuscular mycorrhizal fungi might absorb some DON for their growth in petroleum-polluted soils. In addition, through using a real-time polymerase chain reaction method, we quantified hydrocarbon-degrading bacterial traits based on their catabolic genes (i.e. alkB (alkane monooxygenase), nah (naphthalene dioxygenase) and tol (xylene monooxygenase) genes). This enumeration of target genes suggests that different hydrocarbon-degrading bacteria experienced different dynamic changes during phytoremediation and a greater abundance of alkB was detected during vegetative growth stages. Because phytoremediation of different components of petroleum is performed by different hydrocarbon-degrading bacteria, plants' ability of phytoremediating different components might therefore vary during the plant life cycle. Phytoremediation might be most effective during the vegetative growth stages as greater abundances of hydrocarbon-degrading bacteria containing alkB and tol genes were observed at

  2. Understanding Plant-Microbe Interactions for Phytoremediation of Petroleum-Polluted Soil

    PubMed Central

    Nie, Ming; Wang, Yijing; Yu, Jiayi; Xiao, Ming; Jiang, Lifen; Yang, Ji; Fang, Changming; Chen, Jiakuan; Li, Bo

    2011-01-01

    Plant-microbe interactions are considered to be important processes determining the efficiency of phytoremediation of petroleum pollution, however relatively little is known about how these interactions are influenced by petroleum pollution. In this experimental study using a microcosm approach, we examined how plant ecophysiological traits, soil nutrients and microbial activities were influenced by petroleum pollution in Phragmites australis, a phytoremediating species. Generally, petroleum pollution reduced plant performance, especially at early stages of plant growth. Petroleum had negative effects on the net accumulation of inorganic nitrogen from its organic forms (net nitrogen mineralization (NNM)) most likely by decreasing the inorganic nitrogen available to the plants in petroleum-polluted soils. However, abundant dissolved organic nitrogen (DON) was found in petroleum-polluted soil. In order to overcome initial deficiency of inorganic nitrogen, plants by dint of high colonization of arbuscular mycorrhizal fungi might absorb some DON for their growth in petroleum-polluted soils. In addition, through using a real-time polymerase chain reaction method, we quantified hydrocarbon-degrading bacterial traits based on their catabolic genes (i.e. alkB (alkane monooxygenase), nah (naphthalene dioxygenase) and tol (xylene monooxygenase) genes). This enumeration of target genes suggests that different hydrocarbon-degrading bacteria experienced different dynamic changes during phytoremediation and a greater abundance of alkB was detected during vegetative growth stages. Because phytoremediation of different components of petroleum is performed by different hydrocarbon-degrading bacteria, plants’ ability of phytoremediating different components might therefore vary during the plant life cycle. Phytoremediation might be most effective during the vegetative growth stages as greater abundances of hydrocarbon-degrading bacteria containing alkB and tol genes were observed

  3. Modeling of Alkane Oxidation Using Constituents and Species

    NASA Technical Reports Server (NTRS)

    Bellan, Jasette; Harstad, Kenneth G.

    2010-01-01

    It is currently not possible to perform simulations of turbulent reactive flows due in particular to complex chemistry, which may contain thousands of reactions and hundreds of species. This complex chemistry results in additional differential equations, making the numerical solution of the equation set computationally prohibitive. Reducing the chemical kinetics mathematical description is one of several important goals in turbulent reactive flow modeling. A chemical kinetics reduction model is proposed for alkane oxidation in air that is based on a parallel methodology to that used in turbulence modeling in the context of the Large Eddy Simulation. The objective of kinetic modeling is to predict the heat release and temperature evolution. This kinetic mechanism is valid over a pressure range from atmospheric to 60 bar, temperatures from 600 K to 2,500 K, and equivalence ratios from 0.125 to 8. This range encompasses diesel, HCCI, and gas-turbine engines, including cold ignition. A computationally efficient kinetic reduction has been proposed for alkanes that has been illustrated for n-heptane using the LLNL heptane mechanism. This model is consistent with turbulence modeling in that scales were first categorized into either those modeled or those computed as progress variables. Species were identified as being either light or heavy. The heavy species were decomposed into defined 13 constituents, and their total molar density was shown to evolve in a quasi-steady manner. The light species behave either in a quasi-steady or unsteady manner. The modeled scales are the total constituent molar density, Nc, and the molar density of the quasi-steady light species. The progress variables are the total constituent molar density rate evolution and the molar densities of the unsteady light species. The unsteady equations for the light species contain contributions of the type gain/loss rates from the heavy species that are modeled consistent with the developed mathematical

  4. Equilibrium thermophysical properties of alkanes at very high temperatures

    SciTech Connect

    Arunachalam, C.; Bozkurt, B.; Eubank, P.T.

    1996-01-01

    In order to perform calculations for thermal plasmas, sparks, and arcs, as in the thermal arc and electrical discharge machining (EDM) processes, thermophysical properties, such as the density, enthalpy, and heat capacity, of the original ambient dielectric liquid are required at very high temperatures and often pressures in the plasma state. A statistical model has been developed to provide these properties. At high temperatures, these hydrocarbons undergo a series of reactions to first dissociate and then to ionize to produce a plasma. The partition functions of each of the species generated are calculated and sued to determine the equilibrium mole fractions or particle fractions of each constituent of the resultant plasma. Only the hydrogen-to-carbon ratio matters so mixtures of alkanes can also be used. Specifically, tables of particles fractions, densities, enthalpies, and specific heat capacities are provided for methane and for hexadecane to 60,000 K and 10 kbar.

  5. Thermal analysis of n-alkane phase change material mixtures

    SciTech Connect

    Chio, Y.I.; Choi, E.; Lorsch, H.G.

    1991-03-31

    Tests were performed to characterize the thermal behavior of it number of n-alkanes to be used as phase change materials (PCMs) in district cooling applications. Hexadecane and tetradecane were mixed in different fractions, and their thermal behavior was experimentally evaluated. Test results for melting temperature and fusion energy for laboratory grade hexadecane and tetradecane showed good agreement with datain the literature. However, values for commercial grade hexadecane were found to be considerably lower. In the range of temperatures of interest for district cooling, mixtures of tetradecane and hexadecane can be treated as homogeneous substances. However, their heats of fusion are slightly lower than those of the pure substances. Their melting temperatures are also lower by an amount that can be predicted.

  6. Cometabolism of Methyl tertiary Butyl Ether and Gaseous n-Alkanes by Pseudomonas mendocina KR-1 Grown on C5 to C8 n-Alkanes

    PubMed Central

    Smith, Christy A.; O'Reilly, Kirk T.; Hyman, Michael R.

    2003-01-01

    Pseudomonas mendocina KR-1 grew well on toluene, n-alkanes (C5 to C8), and 1° alcohols (C2 to C8) but not on other aromatics, gaseous n-alkanes (C1 to C4), isoalkanes (C4 to C6), 2° alcohols (C3 to C8), methyl tertiary butyl ether (MTBE), or tertiary butyl alcohol (TBA). Cells grown under carbon-limited conditions on n-alkanes in the presence of MTBE (42 μmol) oxidized up to 94% of the added MTBE to TBA. Less than 3% of the added MTBE was oxidized to TBA when cells were grown on either 1° alcohols, toluene, or dextrose in the presence of MTBE. Concentrated n-pentane-grown cells oxidized MTBE to TBA without a lag phase and without generating tertiary butyl formate (TBF) as an intermediate. Neither TBF nor TBA was consumed by n-pentane-grown cells, while formaldehyde, the expected C1 product of MTBE dealkylation, was rapidly consumed. Similar Ks values for MTBE were observed for cells grown on C5 to C8 n-alkanes (12.95 ± 2.04 mM), suggesting that the same enzyme oxidizes MTBE in cells grown on each n-alkane. All growth-supporting n-alkanes (C5 to C8) inhibited MTBE oxidation by resting n-pentane-grown cells. Propane (Ki = 53 μM) and n-butane (Ki = 16 μM) also inhibited MTBE oxidation, and both gases were also consumed by cells during growth on n-pentane. Cultures grown on C5 to C8 n-alkanes also exhibited up to twofold-higher levels of growth in the presence of propane or n-butane, whereas no growth stimulation was observed with methane, ethane, MTBE, TBA, or formaldehyde. The results are discussed in terms of their impacts on our understanding of MTBE biodegradation and cometabolism. PMID:14660389

  7. Enrichment and Characterization of a Psychrotolerant Consortium Degrading Crude Oil Alkanes Under Methanogenic Conditions.

    PubMed

    Ding, Chen; Ma, Tingting; Hu, Anyi; Dai, Lirong; He, Qiao; Cheng, Lei; Zhang, Hui

    2015-08-01

    Anaerobic alkane degradation via methanogenesis has been intensively studied under mesophilic and thermophilic conditions. While there is a paucity of information on the ability and composition of anaerobic alkane-degrading microbial communities under low temperature conditions. In this study, we investigated the ability of consortium Y15, enriched from Shengli oilfield, to degrade hydrocarbons under different temperature conditions (5-35 °C). The consortium could use hexadecane over a low temperature range (15-30 °C). No growth was detected below 10 °C and above 35 °C, indicating the presence of cold-tolerant species capable of alkane degradation. The preferential degradation of short chain n-alkanes from crude oil was observed by this consortium. The structure and dynamics of the microbial communities were examined using terminal restriction fragment length polymorphism (T-RFLP) fingerprinting and Sanger sequencing of 16S rRNA genes. The core archaeal communities were mainly composed of aceticlastic Methanosaeta spp. Syntrophaceae-related microorganisms were always detected during consecutive transfers and dominated the bacterial communities, sharing 94-96 % sequence similarity with Smithella propionica strain LYP(T). Phylogenetic analysis of Syntrophaceae-related clones in diverse methanogenic alkane-degrading cultures revealed that most of them were clustered into three sublineages. Syntrophaceae clones retrieved from this study were mainly clustered into sublineage I, which may represent psychrotolerant, syntrophic alkane degraders. These results indicate the wide geographic distribution and ecological function of syntrophic alkane degraders. PMID:25783218

  8. MIR and NIR group spectra of n-alkanes and 1-chloroalkanes

    NASA Astrophysics Data System (ADS)

    Kwaśniewicz, Michał; Czarnecki, Mirosław A.

    2015-05-01

    Numerous attempts were undertaken to resolve the absorption originating from different parts of alkanes. The separation of the contributions from the terminal and midchain methylene units was observed only in the spectra of solid alkanes at low temperatures. On the other hand, for liquid alkanes this effect was not reported as yet. In this study, ATR-IR, Raman and NIR spectra of eight n-alkanes and seven 1-chloroalkanes in the liquid phase were measured from 1000 to 12,000 cm-1. The spectra were analyzed by using two-dimensional (2D) correlation approach and chemometrics methods. It was shown that in 2D asynchronous contour plots, constructed from the spectra of n-alkanes and 1-chloroalkanes, the methylene band was resolved into two components. These two components were assigned to the terminal and midchain methylene groups. For the first time, the contributions from these two molecular fragments were resolved in the spectra of liquid n-alkanes and 1-chloroalkanes. MCR-ALS resolved these spectra into two components that were assigned to the ethyl and midchain methylene groups. These components represent the group spectra that can be used for assignment, spectral analysis and prediction of unknown spectra. The spectral prediction based on the group spectra provides very good results for n-alkanes, especially in the first and second overtone regions.

  9. Adsorption of alkanes on stoichiometric and oxygen-rich RuO2(110).

    PubMed

    Li, Tao; Kim, Minkyu; Rai, Rahul; Liang, Zhu; Asthagiri, Aravind; Weaver, Jason F

    2016-08-10

    We investigated the molecular adsorption of methane, ethane, propane and n-butane on stoichiometric and oxygen-rich RuO2(110) surfaces using temperature-programmed desorption (TPD) and dispersion-corrected density functional theory (DFT-D3) calculations. We find that each alkane adsorbs strongly on the coordinatively-unsaturated Ru (Rucus) atoms of s-RuO2(110), with desorption from this state producing a well-defined TPD peak at low alkane coverage. As the coverage increases, we find that alkanes first form a compressed layer on the Rucus atoms and subsequently adsorb on the bridging O atoms of the surface until the monolayer saturates. DFT-D3 calculations predict that methane preferentially adsorbs on top of a Rucus atom and that the C2 to C4 alkanes preferentially adopt bidentate configurations in which each molecule aligns parallel to the Rucus atom row and datively bonds to neighboring Rucus atoms. DFT-D3 predicts binding energies that agree quantitatively with our experimental estimates for alkane σ-complexes on RuO2(110). We find that oxygen atoms adsorbed on top of Rucus atoms (Oot atoms) stabilize the adsorbed alkane complexes that bind in a given configuration, while also blocking the sites needed for σ-complex formation. This site blocking causes the coverage of the most stable, bidentate alkane complexes to decrease sharply with increasing Oot coverage. Concurrently, we find that a new peak develops in the C2 to C4 alkane TPD spectra with increasing Oot coverage, and that the desorption yield in this TPD feature passes through a maximum at Oot coverages between ∼50% and 60%. We present evidence that the new TPD peak arises from C2 to C4 alkanes that adsorb in upright, monodentate configurations on stranded Rucus sites located within the Oot layer. PMID:27477390

  10. Biodegradation of variable-chain-length alkanes at low temperatures by a psychrotrophic Rhodococcus sp.

    SciTech Connect

    Whyte, L.G.; Hawari, J.; Zhou, E.; Bourbonniere, L.; Greer, C.W.; Inniss, W.E.

    1998-07-01

    The psychrotroph Rhodococcus sp. strain Q15 was examined for its ability to degrade individual n-alkanes and diesel fuel at low temperatures, and its alkane catabolic pathway was investigated by biochemical and genetic techniques. At 0 and 5 C, Q15 mineralized the short-chain alkanes dodecane and hexadecane to a greater extent than that observed for the long-chain alkanes octacosane and dotriacontane. Q15 utilized a broad range of aliphatics (C{sub 10} to C{sub 21} alkanes, branched alkanes, and a substituted cyclohexane) present in diesel fuel at 5 C. Mineralization of hexadecane at 5 C was significantly greater in both hydrocarbon-contaminated and pristine soil microcosms seeded with Q15 cells than in uninoculated control soil microcosms. The detection of hexadecane and dodecane metabolic intermediates (1-hexadecanol and 2-hexadecanol and 1-do-decanol and 2-dodecanone, respectively) by solid-phase microextraction-gas chromatography-mass spectrometry and the utilization of potential metabolic intermediates indicated that Q15 oxidizes alkanes by both the terminal oxidation pathway and the subterminal oxidation pathway. Genetic characterization by PCR and nucleotide sequence analysis indicated that Q15 possesses an aliphatic aldehyde dehydrogenase gene highly homologous to the Rhodococcus erythropolis thcA gene. Rhodococcus sp. strain Q15 possessed two large plasmids of approximately 90 and 115 kb (shown to mediate Cd resistance) which were not required for alkane mineralization, although the 90-kb plasmid enhanced mineralization of some alkanes and growth on diesel oil at both 5 and 25 C.

  11. Compound-specific hydrogen isotope composition of n-alkanes in combustion residuals of fossil fuels

    NASA Astrophysics Data System (ADS)

    Bai, Huiling; Peng, Lin; Li, Zhongping; Liu, Xiaofeng; Song, Chongfang; Mu, Ling

    2014-11-01

    The hydrogen isotope compositions (δD) of n-alkanes present in the combustion residuals of fossil fuels (coal, gasoline, and diesel) were measured using GC-IRMS to distinguish between coal soot and vehicle exhaust. The n-alkane δD values of industrial and domestic coal soot ranged from -95.3‰ to -219.6‰ and -128.1‰ to -188.6‰, respectively, exhibiting similar tendencies. The δD values of the C15-C18n-alkanes in both types of coal soot were nearly consistent, and the δD values of the C19-C24n-alkanes exhibited a zigzag profile. The δD values of C16-C22n-alkanes in gasoline exhaust exhibited a saw-tooth distribution, decreased with the carbon number, and were more positive than the δD values of C16-C22n-alkanes in diesel exhaust, which increased with the carbon number. However, the δD values of the C23-C29n-alkanes in gasoline and diesel vehicle exhaust were mostly consistent. The weighted average δD values of the C16-C19n-alkanes in industrial and domestic coal soot were similar to the average δD values in gasoline and diesel vehicle exhausts; however, the average δD values of the C21-C29n-alkanes in vehicle exhausts were richer in D than those in coal soot.

  12. Characterization of cyclic and acyclic alkanes in Forties and Kuwait petroleum crudes

    SciTech Connect

    Jones, D.W. ); Pakdel, H. ); Bartle, K.D. )

    1990-01-01

    Alkane hydrocarbon fractions from Forties (North Sea) and Kuwait petroleum crudes, separated by distillation, solvent extraction and silicagel column chromatography and sub-fractionated by molecular-sieve adsorption, have been examined by gas chromatography (GC), {sup 1}H and {sup 13}C NMR spectroscopy, GC-mass spectrometry (MS) and field desorption (FD)MS. GC indicates that Forties contains rather more acyclic isoprenoids and cyclic alkanes than Kuwait; FDMS of Kuwait shows molecular-weight ranges for mono-, di-, tri-, tetra-, and pentacyclic alkanes. {sup 13}C NMR spectra provide evidence of higher aromatic carbon, C{sub A}, in Forties than Kuwait and longer T{sub 1} relaxation times.

  13. Adsorption and dissociation kinetics of alkanes on CaO(100)

    NASA Astrophysics Data System (ADS)

    Chakradhar, A.; Liu, Y.; Schmidt, J.; Kadossov, E.; Burghaus, U.

    2011-08-01

    The adsorption kinetics of ethane, butane, pentane, and hexane on CaO(100) have been studied by multi-mass thermal desorption (TDS) spectroscopy. The sample cleanliness was checked by Auger electron spectroscopy. A molecular and dissociative adsorption pathway was evident for the alkanes, except for ethane, which does not undergo bond activation. Two TDS peaks appeared when recording the parent mass, which are assigned to different adsorption sites/configurations of the molecularly adsorbed alkanes. Bond activation leads to desorption of hydrogen and several alkane fragments assigned to methane and ethylene formation. Only one TDS feature is seen in this case. Formation of carbon residuals was absent.

  14. Photochemical dimerization and functionalization of alkanes, ethers, primary and secondary alcohols, phosphine oxides and silanes

    DOEpatents

    Crabtree, Robert H.; Brown, Stephen H.

    1989-01-01

    The space-time yield and/or the selectivity of the photochemical dimerization of alkanes, ethers, primary and secondary alcohols, phosphine oxides and primary, secondary and tertiary silanes with Hg and U.V. light is enhanced by refluxing the substrate in the irradiated reaction zone at a temperature at which the dimer product condenses and remains condensed promptly upon its formation. Cross-dimerization of the alkanes, ethers and silanes with primary alcohols is disclosed, as is the functionalization to aldehydes of the alkanes with carbon monoxide.

  15. Photochemical dimerization and functionalization of alkanes, ethers, primary and secondary alcohols, phosphine oxides and silanes

    DOEpatents

    Crabtree, R.H.; Brown, S.H.

    1989-10-17

    The space-time yield and/or the selectivity of the photochemical dimerization of alkanes, ethers, primary and secondary alcohols, phosphine oxides and primary, secondary and tertiary silanes with Hg and U.V. light is enhanced by refluxing the substrate in the irradiated reaction zone at a temperature at which the dimer product condenses and remains condensed promptly upon its formation. Cross-dimerization of the alkanes, ethers and silanes with primary alcohols is disclosed, as is the functionalization to aldehydes of the alkanes with carbon monoxide.

  16. Effect of n-alkanes on asphaltene structuring in petroleum oils.

    PubMed

    Stachowiak, Christian; Viguié, Jean-Romain; Grolier, Jean-Pierre E; Rogalski, Marek

    2005-05-24

    The interactions between asphaltenes and short- to medium-chain n-alkanes were studied using titration microcalorimetry and inverse chromatography. The exothermic heat effects observed upon mixing of asphaltenes and n-alkanes were interpreted in terms of assembling of the two types of compounds into mixed structures. We show that the energy of the interactions between n-alkanes and the asphaltene hydrocarbon chains is close to the energy of the interactions between the asphaltene chains. We propose that the latter interactions are responsible for the formation of the asphaltene aggregates and are the driving force of the aggregate assembly into higher structures. PMID:15896019

  17. Light alkane conversion processes - Suprabiotic catalyst systems for selective oxidation of light alkane gases to fuel oxygenates.

    SciTech Connect

    Lyons, J.E.

    1992-07-01

    The objective of the work presented in this paper is to develop new, efficient catalysts for the selective transformation of the light alkanes in natural gas to alcohols for use as liquid transportation fuels, fuel precursors and chemical products. There currently exists no DIRECT one-step catalytic air-oxidation process to convert these substrates to alcohols. Such a one-step route would represent superior useful technology for the utilization of natural gas and similar refinery-derived light hydrocarbon streams. Processes for converting natural gas or its components (methane, ethane, propane, and the butanes) to alcohols for use as motor fuels, fuel additives or fuel precursors will not only add a valuable alternative to crude oil but will produce a clean-burning, high octane alternative to conventional gasoline.

  18. Light alkane conversion processes - Suprabiotic catalyst systems for selective oxidation of light alkane gases to fuel oxygenates

    SciTech Connect

    Lyons, J.E.

    1992-01-01

    The objective of the work presented in this paper is to develop new, efficient catalysts for the selective transformation of the light alkanes in natural gas to alcohols for use as liquid transportation fuels, fuel precursors and chemical products. There currently exists no DIRECT one-step catalytic air-oxidation process to convert these substrates to alcohols. Such a one-step route would represent superior useful technology for the utilization of natural gas and similar refinery-derived light hydrocarbon streams. Processes for converting natural gas or its components (methane, ethane, propane, and the butanes) to alcohols for use as motor fuels, fuel additives or fuel precursors will not only add a valuable alternative to crude oil but will produce a clean-burning, high octane alternative to conventional gasoline.

  19. The oxygenating constituent of 3,6-diketocamphane monooxygenase from the CAM plasmid of Pseudomonas putida: the first crystal structure of a type II Baeyer–Villiger monooxygenase

    PubMed Central

    Isupov, Michail N.; Schröder, Ewald; Gibson, Robert P.; Beecher, Jean; Donadio, Giuliana; Saneei, Vahid; Dcunha, Stephlina A.; McGhie, Emma J.; Sayer, Christopher; Davenport, Colin F.; Lau, Peter C.; Hasegawa, Yoshie; Iwaki, Hiroaki; Kadow, Maria; Balke, Kathleen; Bornscheuer, Uwe T.; Bourenkov, Gleb; Littlechild, Jennifer A.

    2015-01-01

    The three-dimensional structures of the native enzyme and the FMN complex of the overexpressed form of the oxygenating component of the type II Baeyer–Villiger 3,6-diketocamphane monooxygenase have been determined to 1.9 Å resolution. The structure of this dimeric FMN-dependent enzyme, which is encoded on the large CAM plasmid of Pseudomonas putida, has been solved by a combination of multiple anomalous dispersion from a bromine crystal soak and molecular replacement using a bacterial luciferase model. The orientation of the isoalloxazine ring of the FMN cofactor in the active site of this TIM-barrel fold enzyme differs significantly from that previously observed in enzymes of the bacterial luciferase-like superfamily. The Ala77 residue is in a cis conformation and forms a β-bulge at the C-terminus of β-strand 3, which is a feature observed in many proteins of this superfamily. PMID:26527149

  20. STRUCTURE-REACTIVITY RELATIONSHIPS IN DEHYDROHALOGENATION REACTIONS OF POLYCHLORINATED AND POLYBROMINATED ALKANES

    EPA Science Inventory

    Current information is inadequate to predict the rates at which polyhalogenated alkanes undergo dehydrohalogenation rations under environmental conditions, forming olefins that are frequently more toxic and more recalcitrant than the products of substitution reactions. o permit e...

  1. Modeling the Role of Alkanes, Polycyclic Aromatic Hydrocarbons, and Their Oligomers in Secondary Organic Aerosol Formation

    EPA Science Inventory

    A computationally efficient method to treat secondary organic aerosol (SOA) from various length and structure alkanes as well as SOA from polycyclic aromatic hydrocarbons (PAHs) is implemented in the Community Multiscale Air Quality (CMAQ) model to predict aerosol concentrations ...

  2. Draft Genome Sequence of the Versatile Alkane-Degrading Bacterium Aquabacterium sp. Strain NJ1

    PubMed Central

    Shiwa, Yuh; Yoshikawa, Hirofumi; Zylstra, Gerben J.

    2014-01-01

    The draft genome sequence of a soil bacterium, Aquabacterium sp. strain NJ1, capable of utilizing both liquid and solid alkanes, was deciphered. This is the first report of an Aquabacterium genome sequence. PMID:25477416

  3. 40 CFR 721.10148 - Acryloxy alkanoic alkane derivative with mixed metal oxides (generic).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... PROTECTION AGENCY (CONTINUED) TOXIC SUBSTANCES CONTROL ACT SIGNIFICANT NEW USES OF CHEMICAL SUBSTANCES... mixed metal oxides (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as acryloxy alkanoic alkane derivative with mixed...

  4. Production of Liquid Alkanes by Aqueous-Phase Processing of Biomass-Derived Carbohydrates

    NASA Astrophysics Data System (ADS)

    Huber, George W.; Chheda, Juben N.; Barrett, Christopher J.; Dumesic, James A.

    2005-06-01

    Liquid alkanes with the number of carbon atoms ranging from C7 to C15 were selectively produced from biomass-derived carbohydrates by acid-catalyzed dehydration, which was followed by aldol condensation over solid base catalysts to form large organic compounds. These molecules were then converted into alkanes by dehydration/hydrogenation over bifunctional catalysts that contained acid and metal sites in a four-phase reactor, in which the aqueous organic reactant becomes more hydrophobic and a hexadecane alkane stream removes hydrophobic species from the catalyst before they go on further to form coke. These liquid alkanes are of the appropriate molecular weight to be used as transportation fuel components, and they contain 90% of the energy of the carbohydrate and H2 feeds.

  5. Photocatalytic acceptorless alkane dehydrogenation: scope, mechanism, and conquering deactivation with carbon dioxide.

    PubMed

    Chowdhury, Abhishek Dutta; Julis, Jennifer; Grabow, Kathleen; Hannebauer, Bernd; Bentrup, Ursula; Adam, Martin; Franke, Robert; Jackstell, Ralf; Beller, Matthias

    2015-01-01

    Alkane dehydrogenation is of special interest for basic science but also offers interesting opportunities for industry. The existing dehydrogenation methodologies make use of heterogeneous catalysts, which suffer from harsh reaction conditions and a lack of selectivity, whereas homogeneous methodologies rely mostly on unsolicited waste generation from hydrogen acceptors. Conversely, acceptorless photochemical alkane dehydrogenation in the presence of trans-Rh(PMe3 )2 (CO)Cl can be regarded as a more benign and atom efficient alternative. However, this methodology suffers from catalyst deactivation over time. Herein, we provide a detailed investigation of the trans-Rh(PMe3 )2 (CO)Cl-photocatalyzed alkane dehydrogenation using spectroscopic and theoretical investigations. These studies inspired us to utilize CO2 to prevent catalyst deactivation, which leads eventually to improved catalyst turnover numbers in the dehydrogenation of alkanes that include liquid organic hydrogen carriers. PMID:25346450

  6. Alkane oxidation with porphyrins and metal complexes thereof having haloalkyl side chains

    DOEpatents

    Wijesekera, T.; Lyons, J.E.; Ellis, P.E. Jr.; Bhinde, M.V.

    1998-06-23

    Transition metal complexes of meso-haloalkylporphyrins are disclosed, wherein the haloalkyl groups contain 2 to 8 carbon atoms have been found to be highly effective catalysts for oxidation of alkanes and for the decomposition of hydroperoxides. 7 figs.

  7. Alkane oxidation with porphyrins and metal complexes thereof having haloalkyl side chains

    DOEpatents

    Wijesekera, Tilak; Lyons, James E.; Ellis, Jr., Paul E.; Bhinde, Manoj V.

    1998-01-01

    Transition metal complexes of meso-haloalkylporphyrins, wherein the haloalkyl groups contain 2 to 8 carbon atoms have been found to be highly effective catalysts for oxidation of alkanes and for the decomposition of hydroperoxides.

  8. Structural and Catalytic Characterization of a Fungal Baeyer-Villiger Monooxygenase

    PubMed Central

    Ferroni, Felix Martin; Tolmie, Carmien; Smit, Martha Sophia; Opperman, Diederik Johannes

    2016-01-01

    Baeyer-Villiger monooxygenases (BVMOs) are biocatalysts that convert ketones to esters. Due to their high regio-, stereo- and enantioselectivity and ability to catalyse these reactions under mild conditions, they have gained interest as alternatives to chemical Baeyer-Villiger catalysts. Despite their widespread occurrence within the fungal kingdom, most of the currently characterized BVMOs are from bacterial origin. Here we report the catalytic and structural characterization of BVMOAFL838 from Aspergillus flavus. BVMOAFL838 converts linear and aryl ketones with high regioselectivity. Steady-state kinetics revealed BVMOAFL838 to show significant substrate inhibition with phenylacetone, which was more pronounced at low pH, enzyme and buffer concentrations. Para substitutions on the phenyl group significantly improved substrate affinity and increased turnover frequencies. Steady-state kinetics revealed BVMOAFL838 to preferentially oxidize aliphatic ketones and aryl ketones when the phenyl group are separated by at least two carbons from the carbonyl group. The X-ray crystal structure, the first of a fungal BVMO, was determined at 1.9 Å and revealed the typical overall fold seen in type I bacterial BVMOs. The active site Arg and Asp are conserved, with the Arg found in the “in” position. Similar to phenylacetone monooxygenase (PAMO), a two residue insert relative to cyclohexanone monooxygenase (CHMO) forms a bulge within the active site. Approximately half of the “variable” loop is folded into a short α-helix and covers part of the active site entry channel in the non-NADPH bound structure. This study adds to the current efforts to rationalize the substrate scope of BVMOs through comparative catalytic and structural investigation of different BVMOs. PMID:27472055

  9. A stopped-flow kinetic study of soluble methane mono-oxygenase from Methylococcus capsulatus (Bath).

    PubMed Central

    Green, J; Dalton, H

    1989-01-01

    1. The roles of the three protein components of soluble methane mono-oxygenase were investigated by the use of rapid-reaction techniques. The transfer of electrons through the enzyme complex from NADH to methane/O2 was also investigated. 2. Electron transfer from protein C, the reductase component, to protein A, the hydroxylase component, was demonstrated. Protein C was shown to undergo a three-electron--one-electron catalytic cycle. The interaction of protein C with NADH was investigated. Reduction of protein C was shown to be rapid, and a charge-transfer interaction between reduced FAD and NAD+ was observed; this intermediate was also found in static titration experiments. Thus the binding of NADH, the reduction of protein C and the intramolecular transfer of electrons through protein C were shown to be much more rapid than the turnover rate of methane mono-oxygenase. 3. The rate of transfer of electrons from protein C to protein A was shown to be lower than the reduction of protein C but higher than the turnover rate of methane mono-oxygenase. Association of the proteins was not rate-limiting. The amount of protein A present in the system had a small effect on the rate of reduction of protein C, indicating some co-operativity between the two proteins. 4. Protein B was shown to prevent electron transfer between protein C and protein A in the absence of methane. On addition of saturating concentrations of methane electron transfer was restored. With saturating concentrations of methane and O2 the observed rate constant for the conversion of methane into methanol was 0.26 s-1 at 18 degrees C. 5. By the use of [2H4]methane it was demonstrated that C-H-bond breakage is likely to be the rate-limiting step in the conversion of methane into methanol. PMID:2497729

  10. Structural and Catalytic Characterization of a Fungal Baeyer-Villiger Monooxygenase.

    PubMed

    Ferroni, Felix Martin; Tolmie, Carmien; Smit, Martha Sophia; Opperman, Diederik Johannes

    2016-01-01

    Baeyer-Villiger monooxygenases (BVMOs) are biocatalysts that convert ketones to esters. Due to their high regio-, stereo- and enantioselectivity and ability to catalyse these reactions under mild conditions, they have gained interest as alternatives to chemical Baeyer-Villiger catalysts. Despite their widespread occurrence within the fungal kingdom, most of the currently characterized BVMOs are from bacterial origin. Here we report the catalytic and structural characterization of BVMOAFL838 from Aspergillus flavus. BVMOAFL838 converts linear and aryl ketones with high regioselectivity. Steady-state kinetics revealed BVMOAFL838 to show significant substrate inhibition with phenylacetone, which was more pronounced at low pH, enzyme and buffer concentrations. Para substitutions on the phenyl group significantly improved substrate affinity and increased turnover frequencies. Steady-state kinetics revealed BVMOAFL838 to preferentially oxidize aliphatic ketones and aryl ketones when the phenyl group are separated by at least two carbons from the carbonyl group. The X-ray crystal structure, the first of a fungal BVMO, was determined at 1.9 Å and revealed the typical overall fold seen in type I bacterial BVMOs. The active site Arg and Asp are conserved, with the Arg found in the "in" position. Similar to phenylacetone monooxygenase (PAMO), a two residue insert relative to cyclohexanone monooxygenase (CHMO) forms a bulge within the active site. Approximately half of the "variable" loop is folded into a short α-helix and covers part of the active site entry channel in the non-NADPH bound structure. This study adds to the current efforts to rationalize the substrate scope of BVMOs through comparative catalytic and structural investigation of different BVMOs. PMID:27472055

  11. The Number of High-Energy Bands in the Photoelectron Spectrum of Alkanes

    NASA Astrophysics Data System (ADS)

    Merris, Russell; Gutman, Ivan

    2000-12-01

    It was observed that within the Bieri-Dill-Heilbronner-Schmelzer model for the calculation of the ion-ization energies of alkanes CnH2n+2, there are exactly n C2s -electron energy levels lying below the degenerate α-ß manifold. We now show that, indeed, this regularity is obeyed by practically all alkane species. Exceptions do exist, but they must possess a (chemically infeasible) group of more than six mutually connected quaternary carbon atoms.

  12. Measuring long chain alkanes in diesel engine exhaust by thermal desorption PTR-MS

    NASA Astrophysics Data System (ADS)

    Erickson, M. H.; Gueneron, M.; Jobson, B. T.

    2014-01-01

    A method using thermal desorption sampling and analysis by proton transfer reaction mass spectrometry (PTR-MS) to measure long chain alkanes (C12-C18) and other larger organics associated with diesel engine exhaust emissions is described. Long chain alkanes undergo dissociative proton transfer reactions forming a series of fragment ions with formula CnH2n+1. The PTR-MS is insensitive to n-alkanes less than C8 but displays an increasing sensitivity for larger alkanes. Fragment ion distribution and sensitivity is a function of drift conditions. At 80 Td the most abundant ion fragments from C10 to C16 n-alkanes were m/z 57, 71 and 85. The mass spectrum of gasoline and diesel fuel at 80 Td displayed ion group patterns that can be related to known fuel constituents, such as alkanes, alkylbenzenes and cycloalkanes, and other compound groups that are inferred from molecular weight distributions such as dihydronapthalenes and naphthenic monoaromatics. It is shown that thermal desorption sampling of gasoline and diesel engine exhausts at 80 Td allows for discrimination against volatile organic compounds, allowing for quantification of long chain alkanes from the abundance of CnH2n+1 fragment ions. The total abundance of long chain alkanes in diesel engine exhaust was measured to be similar to the total abundance of C1-C4 alkylbenzene compounds. The abundance patterns of compounds determined by thermal desorption sampling may allow for emission profiles to be developed to better quantify the relative contributions of diesel and gasoline exhaust emissions on organic compounds concentrations in urban air.

  13. The quantitative significance of Syntrophaceae and syntrophic partnerships in methanogenic degradation of crude oil alkanes

    PubMed Central

    Gray, N D; Sherry, A; Grant, R J; Rowan, A K; Hubert, C R J; Callbeck, C M; Aitken, C M; Jones, D M; Adams, J J; Larter, S R; Head, I M

    2011-01-01

    Libraries of 16S rRNA genes cloned from methanogenic oil degrading microcosms amended with North Sea crude oil and inoculated with estuarine sediment indicated that bacteria from the genera Smithella (Deltaproteobacteria, Syntrophaceace) and Marinobacter sp. (Gammaproteobacteria) were enriched during degradation. Growth yields and doubling times (36 days for both Smithella and Marinobacter) were determined using qPCR and quantitative data on alkanes, which were the predominant hydrocarbons degraded. The growth yield of the Smithella sp. [0.020 g(cell-C)/g(alkane-C)], assuming it utilized all alkanes removed was consistent with yields of bacteria that degrade hydrocarbons and other organic compounds in methanogenic consortia. Over 450 days of incubation predominance and exponential growth of Smithella was coincident with alkane removal and exponential accumulation of methane. This growth is consistent with Smithella's occurrence in near surface anoxic hydrocarbon degrading systems and their complete oxidation of crude oil alkanes to acetate and/or hydrogen in syntrophic partnership with methanogens in such systems. The calculated growth yield of the Marinobacter sp., assuming it grew on alkanes, was [0.0005 g(cell-C)/g(alkane-C)] suggesting that it played a minor role in alkane degradation. The dominant methanogens were hydrogenotrophs (Methanocalculus spp. from the Methanomicrobiales). Enrichment of hydrogen-oxidizing methanogens relative to acetoclastic methanogens was consistent with syntrophic acetate oxidation measured in methanogenic crude oil degrading enrichment cultures. qPCR of the Methanomicrobiales indicated growth characteristics consistent with measured rates of methane production and growth in partnership with Smithella. PMID:21914097

  14. Oxidative cleavage of cellulose by fungal copper-dependent polysaccharide monooxygenases.

    PubMed

    Beeson, William T; Phillips, Christopher M; Cate, Jamie H D; Marletta, Michael A

    2012-01-18

    Fungal-derived, copper-dependent polysaccharide monooxygenases (PMOs), formerly known as GH61 proteins, have recently been shown to catalyze the O(2)-dependent oxidative cleavage of recalcitrant polysaccharides. Different PMOs isolated from Neurospora crassa were found to generate oxidized cellodextrins modified at the reducing or nonreducing ends upon incubation with cellulose and cellobiose dehydrogenase. Here we show that the nonreducing end product formed by an N. crassa PMO is a 4-ketoaldose. Together with isotope labeling experiments, further support is provided for a mechanism involving oxygen insertion and subsequent elimination to break glycosidic bonds in crystalline cellulose. PMID:22188218

  15. Overexpression and purification of the particulate methane monooxygenase from Methylococcus capsulatus (Bath).

    PubMed

    Chan, Sunney I; Nguyen, H-Hoa T; Chen, Kelvin H-C; Yu, Steve S-F

    2011-01-01

    The particulate methane monooxygenase (pMMO) is a multi-copper enzyme that mediates the facile conversion of methane to methanol in methanotrophic bacteria. As a membrane-bound multi-subunit metalloprotein, the highly active protein has been difficult to isolate and purify to homogeneity for biochemical and biophysical studies. In this chapter, we describe a method to overexpress pMMO with good specific activity in high yields in the intracytoplasmic membranes of the host organism, together with two protocols to isolate and purify the enzyme from pMMO-enriched membranes without loss of the copper cofactors and enzymatic activity. PMID:21419922

  16. Metal reconstitution of particulate methane monooxygenase and heterologous expression of the pmoB subunit.

    PubMed

    Smith, Stephen M; Balasubramanian, Ramakrishnan; Rosenzweig, Amy C

    2011-01-01

    Particulate methane monooxygenase (pMMO) is a multisubunit metalloenzyme complex used by methanotrophic bacteria to oxidize methane in the first step of carbon assimilation and energy production. In this chapter, we detail methods to prepare metal free (apo) membrane-bound pMMO and to reconstitute apo pMMO with metal ions. We also describe protocols to clone, express, and refold metal-loaded soluble domain constructs of the pmoB subunit. These approaches were used to address fundamental questions concerning the metal content and location of the pMMO active site. PMID:21419923

  17. Structure and boosting activity of a starch-degrading lytic polysaccharide monooxygenase.

    PubMed

    Lo Leggio, Leila; Simmons, Thomas J; Poulsen, Jens-Christian N; Frandsen, Kristian E H; Hemsworth, Glyn R; Stringer, Mary A; von Freiesleben, Pernille; Tovborg, Morten; Johansen, Katja S; De Maria, Leonardo; Harris, Paul V; Soong, Chee-Leong; Dupree, Paul; Tryfona, Theodora; Lenfant, Nicolas; Henrissat, Bernard; Davies, Gideon J; Walton, Paul H

    2015-01-01

    Lytic polysaccharide monooxygenases (LPMOs) are recently discovered enzymes that oxidatively deconstruct polysaccharides. LPMOs are fundamental in the effective utilization of these substrates by bacteria and fungi; moreover, the enzymes have significant industrial importance. We report here the activity, spectroscopy and three-dimensional structure of a starch-active LPMO, a representative of the new CAZy AA13 family. We demonstrate that these enzymes generate aldonic acid-terminated malto-oligosaccharides from retrograded starch and boost significantly the conversion of this recalcitrant substrate to maltose by β-amylase. The detailed structure of the enzyme's active site yields insights into the mechanism of action of this important class of enzymes. PMID:25608804

  18. Degradation of trichloroethene by a linear-plasmid-encoded alkene monooxygenase in Rhodococcus corallinus (Nocardia corallina) B-276.

    PubMed

    Saeki, H; Akira, M; Furuhashi, K; Averhoff, B; Gottschalk, G

    1999-07-01

    Rhodococcus corallinus (formerly Nocardia corallina) B-276, isolated with propene as sole carbon and energy source, is able to oxidize trichloroethene (TCE). Glucose- or propene-grown R. corallinus B-276 cells exhibited no difference in TCE degradation efficiency. TCE degradation was found to be growth-phase-dependent and maximum rates were monitored with stationary-phase cells. K(m) and Vmax values for TCE degradation of R. corallinus B-276 grown in nutrient broth medium in the presence of glucose were 187 microM and 2.4 nmol min-1 (mg protein)-1, respectively. Escherichia coli recombinants harbouring and expressing the alkene monooxygenase genes of R. corallinus B-276 exhibited the ability to degrade TCE. This result provides clear evidence that the alkene monooxygenase of R. corallinus B-276 catalyses TCE oxidation. R. corallinus B-276 was shown to contain four linear plasmids, pNC10 (70 kb), pNC20 (85 kb), pNC30 (185 kb) and pNC40 (235 kb). The observation that pNC30-deficient strains had lost the ability to grow on propene suggested that the genes of the propene degradation pathway are encoded by the linear plasmid pNC30. Southern blot analysis with cloned alkene monooxygenase genes from R. corallinus B-276 revealed a positive hybridization signal with the linear plasmid pNC30. This result clearly shows that the alkene monooxygenase is encoded by the linear plasmid pNC30. Eleven short-chain-alkene-oxidizing strains were screened for the presence of linear plasmids. Among these, four propene-oxidizing Rhodococcus strains and one ethene-oxidizing Mycobacterium strain were found to contain linear megaplasmids. Southern blot analysis with the alkene monooxygenase revealed positive signals with linear plasmids of two propene-oxidizing Rhodococcus ruber strains. These results indicate that homologous alkene monooxygenases are encoded by linear plasmids in R. ruber strains. PMID:10439411

  19. Marine hydrocarbonoclastic bacteria as whole-cell biosensors for n-alkanes

    PubMed Central

    Sevilla, Emma; Yuste, Luis; Rojo, Fernando

    2015-01-01

    Whole-cell biosensors offer potentially useful, cost-effective systems for the in-situ monitoring of seawater for hydrocarbons derived from accidental spills. The present work compares the performance of a biosensor system for the detection of alkanes in seawater, hosted in either Escherichia coli (commonly employed in whole-cell biosensors but not optimized for alkane assimilation) or different marine bacteria specialized in assimilating alkanes. The sensor system was based on the Pseudomonas putida AlkS regulatory protein and the PalkB promoter fused to a gene encoding the green fluorescent protein. While the E. coli sensor provided the fastest response to pure alkanes (25-fold induction after 2 h under the conditions used), a sensor based on Alcanivorax borkumensis was slower, requiring 3–4 h to reach similar induction values. However, the A. borkumensis sensor showed a fourfold lower detection threshold for octane (0.5 μM), and was also better at sensing the alkanes present in petrol. At petrol concentrations of 0.0125%, the A. borkumensis sensor rendered a sevenfold induction, while E. coli sensor showed no response. We discuss possible explanations to this behaviour in terms of the cellular adaptations to alkane uptake and the basal fluorescence produced by each bacterial strain, which was lowest for A. borkumensis. PMID:25874658

  20. Alkanes in flower surface waxes of Momordica cochinchinensis influence attraction to Aulacophora foveicollis Lucas (Coleoptera: Chrysomelidae).

    PubMed

    Mukherjee, A; Sarkar, N; Barik, A

    2013-08-01

    Extraction, thin-layer chromatography, and gas chromatography-mass spectrophotometry analyses revealed 15 alkanes representing 97.14% of the total alkanes in the surface waxes of Momordica cochinchinensis Spreng flowers. Nonacosane was the prevailing alkane followed by hexatriacontane, nonadecane, heptacosane, and hentriacontane, accounting for 39.08%, 24.24%, 13.52%, 6.32%, and 5.12%, respectively. The alkanes from flower surface waxes followed by a synthetic mixture of alkanes mimicking alkanes of flower surface waxes elicited attraction of the female insect, Aulacophora foveicollis Lucas (Coleoptera: Chrysomelidae) between 2 and 10-μg/mL concentrations in a Y-shaped glass tube olfactometer bioassay under laboratory conditions. Synthetic nonadecane from 178.28-891.37 ng, heptacosane from 118.14-590.72 ng, and nonacosane at 784.73 ng showed attraction of the insect. A synthetic mixture of 534.82 ng nonadecane, 354.43 ng heptacosane, and 2,354.18 ng nonacosane elicited highest attraction of A. foveicollis. PMID:23949856

  1. Combustion Characteristics of Liquid Normal Alkane Fuels in a Model Combustor of Supersonic Combustion Ramjet Engine

    NASA Astrophysics Data System (ADS)

    今村, 宰; 石川, 雄太; 鈴木, 俊介; 福本, 皓士郎; 西田, 俊介; 氏家, 康成; 津江, 光洋

    Effect of kinds of one-component n-alkane liquid fuels on combustion characteristics was investigated experimentally using a model combustor of scramjet engine. The inlet condition of a model combustor is 2.0 of Mach number, up to 2400K of total temperature, and 0.38MPa of total pressure. Five kinds of n-alkane are tested, of which carbon numbers are 7, 8, 10, 13, and 16. They are more chemically active and less volatile with an increase of alkane carbon number. Fuels are injected to the combustor in the upstream of cavity with barbotage nitrogen gas and self-ignition performance was investigated. The result shows that self-ignition occurs with less equivalence ratio when alkane carbon number is smaller. This indicates that physical characteristic of fuel, namely volatile of fuel, is dominant for self-ignition behavior. Effect on flame-holding performance is also examined with adding pilot hydrogen and combustion is kept after cutting off pilot hydrogen with the least equivalence ratio where alkane carbon number is from 8 to 10. These points are discussed qualitatively from the conflict effect of chemical and physical properties on alkane carbon number.

  2. Biodegradation of tetrahydrofuran and 1,4-dioxane by soluble diiron monooxygenase in Pseudonocardia sp. strain ENV478.

    PubMed

    Masuda, Hisako; McClay, Kevin; Steffan, Robert J; Zylstra, Gerben J

    2012-01-01

    1,4-Dioxane is an important groundwater contaminant. Pseudonocardia sp. strain ENV478 degrades 1,4-dioxane via cometabolism after the growth on tetrahydrofuran (THF) and other carbon sources. Here, we have identified a THF monooxygenase (thm) in ENV478. The thm genes are transcribed constitutively and are induced to higher levels by THF. Decreased translation of the thmB gene encoding one of the monooxygenase subunits by antisense RNA resulted in the loss of its ability to degrade THF and 1,4-dioxane. This is the first study to link thm genes to THF degradation, as well as the cometabolic oxidation of 1,4-dioxane. PMID:23147387

  3. Increased monooxygenase activity associated with resistance to permethrin in Pediculus humanus capitis (Anoplura: Pediculidae) from Argentina.

    PubMed

    González Audino, P; Barrios, S; Vassena, C; Mougabure Cueto, G; Zerba, E; Picollo, M I

    2005-05-01

    We studied the profile of permethrin resistance in populations of head lice infesting children 6-12 yr old in schools and their homes in and around Buenos Aires, Argentina. Five permethrin-resistant populations with different levels of resistance were collected: Hogar Loyola (HL), Republica de Turquia (RT), Hogar Mitre (HM), Guardia de Honor (GH), and Ricardo Guiraldes (RG). One susceptible population, Bandera Argentina (BA), also was collected. Their level of resistance was evaluated, and results showed resistance ratios of 13 for HL, 16 for RT, 22 for HM, 61 for GH, and 69 for RG. To elucidate the possible involvement of the cytochrome P450 monooxygenase system in conferring permethrin resistance, ethoxycoumarin-O-deethylase (ECOD) activity was measured in abdomens of individual third instars and adults by using a fluorometric assay. The ECOD activity was lower in the susceptible BA population (4.7 ng per louse) than in the resistant ones (13.7 ng per louse for RG, 12.3 ng per louse for GH, 8.6 ng per louse for RT, and 8.2 ng per louse for HL). ECOD activity was significantly correlated with the level of resistance in the field populations (r = 0.97, P = 0.0009), suggesting a role for cytochrome monooxygenase P450 system in permethrin resistance by head louse, Pediculus humanus capitis De Geer. PMID:15962785

  4. P450monooxygenases (P450ome) of the model white rot fungus Phanerochaete chrysosporium

    PubMed Central

    Syed, Khajamohiddin; Yadav, Jagjit S

    2012-01-01

    Phanerochaete chrysosporium, the model white rot fungus, has been the focus of research for the past about four decades for understanding the mechanisms and processes of biodegradation of the natural aromatic polymer lignin and a broad range of environmental toxic chemicals. The ability to degrade this vast array of xenobiotic compounds was originally attributed to its lignin-degrading enzyme system (LDS), mainly the extracellular peroxidases. However, subsequent physiological, biochemical, and/or genetic studies by us and others identified the involvement of a peroxidase-independent oxidoreductase system, the cytochrome P450 monooxygenase system. The whole genome sequence revealed an extraordinarily large P450 contingent (P450ome) with an estimated 149 P450s in this organism. This review focuses on the current status of understanding on the P450 monooxygenase system of P. chrysosporium in terms of pre-genomic and post-genomic identification, structural and evolutionary analysis, transcriptional regulation, redox partners, and functional characterization for its biodegradative potential. Future research on this catalytically diverse oxidoreductase enzyme system and its major role as a newly emerged player in xenobiotic metabolism/degradation is discussed. PMID:22624627

  5. Contribution to catalysis of ornithine binding residues in ornithine N5-monooxygenase.

    PubMed

    Robinson, Reeder; Qureshi, Insaf A; Klancher, Catherine A; Rodriguez, Pedro J; Tanner, John J; Sobrado, Pablo

    2015-11-01

    The SidA ornithine N5-monooxygenase from Aspergillus fumigatus is a flavin monooxygenase that catalyzes the NADPH-dependent hydroxylation of ornithine. Herein we report a mutagenesis study targeting four residues that contact ornithine in crystal structures of SidA: Lys107, Asn293, Asn323, and Ser469. Mutation of Lys107 to Ala abolishes activity as measured in steady-state oxygen consumption and ornithine hydroxylation assays, indicating that the ionic interaction of Lys107 with the carboxylate of ornithine is essential for catalysis. Mutation of Asn293, Asn323, or Ser469 individually to Ala results in >14-fold increases in Km values for ornithine. Asn323 to Ala also increases the rate constant for flavin reduction by NADPH by 18-fold. Asn323 is unique among the four ornithine binding residues in that it also interacts with NADPH by forming a hydrogen bond with the nicotinamide ribose. The crystal structure of N323A complexed with NADP(+) and ornithine shows that the nicontinamide riboside group of NADP is disordered. This result suggests that the increase in flavin reduction rate results from an increase in conformational space available to the enzyme-bound NADP(H). Asn323 thus facilitates ornithine binding at the expense of hindering flavin reduction, which demonstrates the delicate balance that exists within protein-ligand interaction networks in enzyme active sites. PMID:26375201

  6. Electron-transfer reactions and functionalization of cytochrome P450cam monooxygenase system in reverse micelles.

    PubMed

    Ichinose, Hirofumi; Michizoe, Junji; Maruyama, Tatsuo; Kamiya, Noriho; Goto, Masahiro

    2004-06-22

    Enzyme-based electron-transfer reactions involved in the cytochrome P450 monooxygenase system were investigated in nanostructural reverse micelles. A bacterial flavoprotein, putidaredoxin reductase (PdR), was activated and shown to be capable of catalyzing the electron transport from NADH to electron-carrier proteins such as cytochrome b5 (tCyt-b5) and putidaredoxin (Pdx) in reverse micelles. Ferric tCyt-b5 in reverse micelles was effectively converted to its ferrous form by the exogenous addition of separately prepared reverse micellar solution harboring PdR and NADH. The fact that direct interactions of macromolecular proteins should be possible in the reverse micellar system encouraged us to functionalize a multicomponent monooxygenase system composed of the bacterial cytochrome P450cam (P450cam), putidaredoxin (Pdx), and PdR in reverse micelles. The successful camphor hydroxylation reaction catalyzed by P450cam was significantly dependent on the coexistence of Pdx, PdR, and NADH but not H2O2, suggesting that the oxygen-transfer reactions proceeded via a "monooxygenation" mechanism. This is the first report of a multicomponent cytochrome P450 system exhibiting enzymatic activity in organic media. PMID:15986701

  7. Component Interactions and Electron Transfer in Toluene/o-Xylene Monooxygenase

    PubMed Central

    2015-01-01

    The multicomponent protein toluene/o-xylene monooxygenase (ToMO) activates molecular oxygen to oxidize aromatic hydrocarbons. Prior to dioxygen activation, two electrons are injected into each of two diiron(III) units of the hydroxylase, a process that involves three redox active proteins: the ToMO hydroxylase (ToMOH), Rieske protein (ToMOC), and an NADH oxidoreductase (ToMOF). In addition to these three proteins, a small regulatory protein is essential for catalysis (ToMOD). Through steady state and pre-steady state kinetics studies, we show that ToMOD attenuates electron transfer from ToMOC to ToMOH in a concentration-dependent manner. At substoichiometric concentrations, ToMOD increases the rate of turnover, which we interpret to be a consequence of opening a pathway for oxygen transport to the catalytic diiron center in ToMOH. Excess ToMOD inhibits steady state catalysis in a manner that depends on ToMOC concentration. Through rapid kinetic assays, we demonstrate that ToMOD attenuates formation of the ToMOC–ToMOH complex. These data, coupled with protein docking studies, support a competitive model in which ToMOD and ToMOC compete for the same binding site on the hydroxylase. These results are discussed in the context of other studies of additional proteins in the superfamily of bacterial multicomponent monooxygenases. PMID:25402597

  8. The reduced flavin-dependent monooxygenase SfnG converts dimethylsulfone to methanesulfinate.

    PubMed

    Wicht, Denyce K

    2016-08-15

    The biochemical pathway through which sulfur may be assimilated from dimethylsulfide (DMS) is proposed to proceed via oxidation of DMS to dimethylsulfoxide (DMSO) and subsequent conversion of DMSO to dimethylsulfone (DMSO2). Analogous chemical oxidation processes involving biogenic DMS in the atmosphere result in the deposition of DMSO2 into the terrestrial environment. Elucidating the enzymatic pathways that involve DMSO2 contribute to our understanding of the global sulfur cycle. Dimethylsulfone monooxygenase SfnG and flavin mononucleotide (FMN) reductase MsuE from the genome of the aerobic soil bacterium Pseudomonas fluorescens Pf0-1 were produced in Escherichia coli, purified, and biochemically characterized. The enzyme MsuE functions as a reduced nicotinamide adenine dinucleotide (NADH)-dependent FMN reductase with apparent steady state kinetic parameters of Km = 69 μM and kcat/Km = 9 min(-1) μM (-1) using NADH as the variable substrate, and Km = 8 μM and kcat/Km = 105 min(-1) μM (-1) using FMN as the variable substrate. The enzyme SfnG functions as a flavoprotein monooxygenase and converts DMSO2 to methanesulfinate in the presence of FMN, NADH, and MsuE, as evidenced by (1)H and (13)C nuclear magnetic resonance (NMR) spectroscopy. The results suggest that methanesulfinate is a biochemical intermediate in sulfur assimilation. PMID:27392454

  9. NMR structure of the flavin domain from soluble methane monooxygenase reductase from Methylococcus capsulatus (Bath).

    PubMed

    Chatwood, Lisa L; Müller, Jens; Gross, John D; Wagner, Gerhard; Lippard, Stephen J

    2004-09-28

    Soluble methane monooxygenase (sMMO) catalyzes the hydroxylation of methane by dioxygen to methanol, the first step in carbon assimilation by methanotrophs. This multicomponent system transfers electrons from NADH through a reductase component to the non-heme diiron center in the hydroxylase where O(2) is activated. The reductase component comprises three distinct domains, a [2Fe-2S] ferredoxin domain along with FAD- and NADH-binding domains. We report the solution structure of the reduced 27.6 kDa FAD- and NADH-binding domains (MMOR-FAD) of the reductase from Methylococcus capsulatus (Bath). The FAD-binding domain consists of a six-stranded antiparallel beta-barrel and one alpha-helix, with the first 10 N-terminal residues unstructured. In the interface between the two domains, the FAD cofactor is tightly bound in an unprecedented extended conformation. The NADH-binding domain consists of a five-stranded parallel beta-sheet with four alpha-helices packing closely around this sheet. MMOR-FAD is structurally homologous to other FAD-containing oxidoreductases, and we expect similar structures for the FAD/NADH-binding domains of reductases that occur in other multicomponent monooxygenases. PMID:15379538

  10. Characterization and structural analysis of an active particulate methane monooxygenase trimer from Methylococcus capsulatus (Bath).

    PubMed

    Kitmitto, Ashraf; Myronova, Natalia; Basu, Piku; Dalton, Howard

    2005-08-23

    The oxidation of methane to methanol in methanotrophs is catalyzed by the enzyme methane monooxygenase (MMO). Two distinct forms of this enzyme exist, a soluble cytoplasmic MMO (sMMO) and a membrane-bound particulate form (pMMO). We describe here the biochemical characterization of a stable and active purified pMMO hydroxylase (pMMO-H) and report a three-dimensional (3D) structure, determined by electron microscopy and single-particle analysis at 23 A resolution. Both biochemical and structural data indicate that pMMO hydroxylase is trimeric, with each monomer unit comprised of three polypeptides of 47, 26, and 23 kDa. Comparison of the recent crystal structure [Lieberman, R. L., and Rosenzweig, A. C. (2005) Nature 434, 177] of an uncharacterized pMMO-H complex with the three-dimensional (3D) structure determined here yielded a good match between the principal features and the organization of the enzyme monomers into trimers. The data presented here advance our current understanding of particulate methane monooxygenase function by the characterization of an active form of the enzyme and the corresponding 3D structure. PMID:16101279

  11. Structural and biochemical characterization of EDTA monooxygenase and its physical interaction with a partner flavin reductase.

    PubMed

    Jun, Se-Young; Lewis, Kevin M; Youn, Buhyun; Xun, Luying; Kang, ChulHee

    2016-06-01

    Ethylenediaminetetraacetate (EDTA) is currently the most abundant organic pollutant due to its recalcitrance and extensive use. Only a few bacteria can degrade it, using EDTA monooxygenase (EmoA) to initiate the degradation. EmoA is an FMNH2 -dependent monooxygenase that requires an NADH:FMN oxidoreductase (EmoB) to provide FMNH2 as a cosubstrate. Although EmoA has been identified from Chelativorans (ex. Mesorhizobium) sp. BNC1, its catalytic mechanism is unknown. Crystal structures of EmoA revealed a domain-like insertion into a TIM-barrel, which might serve as a flexible lid for the active site. Docking of MgEDTA(2-) into EmoA identified an intricate hydrogen bond network connected to Tyr(71) , which should potentially lower its pKa. Tyr(71) , along with nearby Glu(70) and a peroxy flavin, facilitates a keto-enol transition of the leaving acetyl group of EDTA. Further, for the first time, the physical interaction between EmoA and EmoB was observed by ITC, molecular docking and enzyme kinetic assay, which enhanced both EmoA and EmoB activities probably through coupled channelling of FMNH2 . PMID:26928990

  12. Process development for oxidations of hydrophobic compounds applying cytochrome P450 monooxygenases in-vitro.

    PubMed

    Brummund, Jan; Müller, Monika; Schmitges, Thomas; Kaluzna, Iwona; Mink, Daniel; Hilterhaus, Lutz; Liese, Andreas

    2016-09-10

    Cytochrome P450 monooxygenases are a unique family of enzymes that are able to catalyze regio- and stereospecific oxidations for a broad substrate range. However, due to limited enzyme activities and stabilities, hydrophobicity of substrates, as well as the necessity of a continuous electron and oxygen supply the implementation of P450s for industrial processes remains challenging. Aim of this study was to point out key aspects for the development of an efficient synthesis concept for cytochrome P450 catalyzed oxidations. In order to regenerate the natural cofactor NADPH, a glucose dehydrogenase was applied. The low water soluble terpene α-ionone was used as substrate for the model reaction system. The studies reveal that an addition of surfactants in combination with low volumetric amounts of co-solvent can significantly increase substrate availability and reaction rates. Furthermore, these additives facilitated a reliable sampling procedure during the process. Another key factor for the process design was the oxygen supply. Based on various investigations, a bubble-aerated stirred tank reactor in batch mode represents a promising reactor concept for P450 oxidations. Main restriction of the investigated reaction system was the low process stability of the P450 monooxygenase, characterized by maximum total turnover numbers of ∼4100molα-ionone/molP450. PMID:27396939

  13. Investigation of the enzymology and pharmacology of novel substrates and inhibitors of dopamine beta-monooxygenase

    SciTech Connect

    Roberts, S.F.

    1987-01-01

    Dopamine beta-monooxygenase (DBM) was shown to catalyze the selenoxidation of 2-(phenylseleno)ethylamines, selenium-containing analogues of dopamine, by the normal monooxygenase pathway. The compounds 2-(phenylseleno)-ethylamine (PAESe), 2-(4'-hydroxyphenylseleno)ethylamine (pOH PAESe), and 1-(phenylseleno)-2-propylamine (Me PAESe) were synthesized and fully characterized as DBM substrates. Two other classes of compounds were investigated as potential alternate substrates for DBM. The possibility of stereoselective sulfonylation of 2-(phenylsulfenyl)- ethylamine (PAESO) was considered. A unique class of compounds, 2-(phenylthio)ethanols were designed and synthesized as DBM substrates but were found to be a novel class of potent competitive inhibitors of DBM with respect to tyramine. Preliminary experiments were also performed in an effort to demonstrate that the potent antihypertensive and indirect-acting sympathomimetic activity of 2-(phenylthio)ethylamine (PAES) was a result of DBM-oxygenation of this compound in vivo. The specific reserpine-sensitive uptake of (/sup 3/H)-norepinephrine into rat brain synaptosomes was demonstrated as was the synaptosomal conversion of (/sup 3/H)-dopamine to (/sup 3/H)-norepinephrine.

  14. Control of substrate access to the active site in methane monooxygenase.

    PubMed

    Lee, Seung Jae; McCormick, Michael S; Lippard, Stephen J; Cho, Uhn-Soo

    2013-02-21

    Methanotrophs consume methane as their major carbon source and have an essential role in the global carbon cycle by limiting escape of this greenhouse gas to the atmosphere. These bacteria oxidize methane to methanol by soluble and particulate methane monooxygenases (MMOs). Soluble MMO contains three protein components, a 251-kilodalton hydroxylase (MMOH), a 38.6-kilodalton reductase (MMOR), and a 15.9-kilodalton regulatory protein (MMOB), required to couple electron consumption with substrate hydroxylation at the catalytic diiron centre of MMOH. Until now, the role of MMOB has remained ambiguous owing to a lack of atomic-level information about the MMOH-MMOB (hereafter termed H-B) complex. Here we remedy this deficiency by providing a crystal structure of H-B, which reveals the manner by which MMOB controls the conformation of residues in MMOH crucial for substrate access to the active site. MMOB docks at the α(2)β(2) interface of α(2)β(2)γ(2) MMOH, and triggers simultaneous conformational changes in the α-subunit that modulate oxygen and methane access as well as proton delivery to the diiron centre. Without such careful control by MMOB of these substrate routes to the diiron active site, the enzyme operates as an NADH oxidase rather than a monooxygenase. Biological catalysis involving small substrates is often accomplished in nature by large proteins and protein complexes. The structure presented in this work provides an elegant example of this principle. PMID:23395959

  15. Inactivation of peptidylglycine α-hydroxylating monooxygenase by cinnamic acid analogs.

    PubMed

    McIntyre, Neil R; Lowe, Edward W; Battistini, Matthew R; Leahy, James W; Merkler, David J

    2016-08-01

    Peptidylglycine α-amidating monooxygenase (PAM) is a bifunctional enzyme that catalyzes the final reaction in the maturation of α-amidated peptide hormones. Peptidylglycine α-hydroxylating monooxygenase (PHM) is the PAM domain responsible for the copper-, ascorbate- and O2-dependent hydroxylation of a glycine-extended peptide. Peptidylamidoglycolate lyase is the PAM domain responsible for the Zn(II)-dependent dealkylation of the α-hydroxyglycine-containing precursor to the final α-amidated peptide. We report herein that cinnamic acid and cinnamic acid analogs are inhibitors or inactivators of PHM. The inactivation chemistry exhibited by the cinnamates exhibits all the attributes of a suicide-substrate. However, we find no evidence for the formation of an irreversible linkage between cinnamate and PHM in the inactivated enzyme. Our data support the reversible formation of a Michael adduct between an active site nucleophile and cinnamate that leads to inactive enzyme. Our data are of significance given that cinnamates are found in foods, perfumes, cosmetics and pharmaceuticals. PMID:26024288

  16. Effects of nutrition and alcohol on the microsomal monooxygenase system (MMS) of rat kidney

    SciTech Connect

    Ronis, M.; Huang, J.; Ingelman-Sundberg, M.; Badger, T.M. Arkansas Children's Hospital Research Center, Little Rock )

    1991-03-15

    Ethanol is a known inducer of the hepatic cytochrome P450 dependent microsomal monooxygenase system (MMS). As a consequence, ethanol intake affects the clearance and metabolism of many drugs and other xenobiotics including acetaminophen, enflurane, carbon tetrachloride and ethanol itself. The major ethanol inducible cytochrome P450 isozyme in the rat liver, CYP 2E1, has been well characterized. Much less is known concerning extrahepatic effects of ethanol on the monooxygenase system. In the current study, the effects of diet and alcohol were examined on MMS activities and cytochrome P450 expression in the kidneys of adult male Sprague-Dawley rats. Three diets containing no ethanol and two diets containing ethanol at 35% of total calories were studied. Renal MMS activities were measured using enzyme specific substrates and isozyme apoprotein levels were determined by Western blot analysis using antibodies directed against rat hepatic cytochrome P450s CYP 2E1, CYP 2A1 and CYP 3A2. Several diet and alcohol induced effects were observed, including a 5-fold diet-independent ethanol induction of CYP 2E1 cross reactive protein. No diet or ethanol effects were observed in levels of CYP 2A1 or CYP 3A2 cross reactive proteins.

  17. Conversion of alkanes to linear alkylsilanes using an iridium-iron-catalysed tandem dehydrogenation-isomerization-hydrosilylation

    NASA Astrophysics Data System (ADS)

    Jia, Xiangqing; Huang, Zheng

    2016-02-01

    The conversion of inexpensive, saturated hydrocarbon feedstocks into value-added speciality chemicals using regiospecific, catalytic functionalization of alkanes is a major goal of organometallic chemistry. Linear alkylsilanes represent one such speciality chemical—they have a wide range of applications, including release coatings, silicone rubbers and moulding products. Direct, selective, functionalization of alkanes at primary C-H bonds is difficult and, to date, methods for catalytically converting alkanes into linear alkylsilanes are unknown. Here, we report a well-defined, dual-catalyst system for one-pot, two-step alkane silylations. The system comprises a pincer-ligated Ir catalyst for alkane dehydrogenation and an Fe catalyst that effects a subsequent tandem olefin isomerization-hydrosilylation. This method exhibits exclusive regioselectivity for the production of terminally functionalized alkylsilanes. This dual-catalyst strategy has also been applied to regioselective alkane borylations to form linear alkylboronate esters.

  18. [Sources, Migration and Conversion of Dissolved Alkanes, Dissolved Fatty Acids in a Karst Underground River Water, in Chongqing Area].

    PubMed

    Liang, Zuo-bing; Sun, Yu-chuan; Wang, Zun-bo; Shi, Yang; Jiang, Ze-li; Zhang, Mei; Xie, Zheng-Lan; Liao, Yu

    2015-09-01

    Dissolved alkanes and dissolved fatty acids were collected from Qingmuguan underground river in July, October 2013. By gas chromatography-mass spectrometer (GC-MS), alkanes and fatty acids were quantitatively analyzed. The results showed that average contents of alkanes and fatty acids were 1 354 ng.L-1, 24203 ng.L-1 in July, and 667 ng.L-1, 2526 ng.L-1 in October respectively. With the increasing migration distance of dissolved alkanes and dissolved fatty acids in underground river, their contents decreased. Based on the molecular characteristic indices of alkanes, like CPI, OEP, Paq and R, dissolved alkanes were mainly originated from microorganisms in July, and aquatic plants in October. Saturated straight-chain fatty acid had the highest contents in all samples with the dominant peak in C16:0, combined with the characteristics of carbon peak, algae or bacteria might be the dominant source of dissolved fatty acids. PMID:26717680

  19. Conversion of alkanes to linear alkylsilanes using an iridium-iron-catalysed tandem dehydrogenation-isomerization-hydrosilylation.

    PubMed

    Jia, Xiangqing; Huang, Zheng

    2016-02-01

    The conversion of inexpensive, saturated hydrocarbon feedstocks into value-added speciality chemicals using regiospecific, catalytic functionalization of alkanes is a major goal of organometallic chemistry. Linear alkylsilanes represent one such speciality chemical-they have a wide range of applications, including release coatings, silicone rubbers and moulding products. Direct, selective, functionalization of alkanes at primary C-H bonds is difficult and, to date, methods for catalytically converting alkanes into linear alkylsilanes are unknown. Here, we report a well-defined, dual-catalyst system for one-pot, two-step alkane silylations. The system comprises a pincer-ligated Ir catalyst for alkane dehydrogenation and an Fe catalyst that effects a subsequent tandem olefin isomerization-hydrosilylation. This method exhibits exclusive regioselectivity for the production of terminally functionalized alkylsilanes. This dual-catalyst strategy has also been applied to regioselective alkane borylations to form linear alkylboronate esters. PMID:26791899

  20. Berberine cation: A fluorescent chemosensor for alkanes and other low-polarity compounds. An explanation of this phenomenon

    PubMed

    Cossio; Arrieta; Cebolla; Membrado; Vela; Garriga; Domingo

    2000-07-27

    Alkanes in the presence of berberine sulfate provide an enhancement of fluorescent signal, which depends on alkane concentration and structure, when the system is irradiated with monochromatic UV light. Computational analysis suggests that an ion-induced dipole between alkanes and berberine sulfate is responsible for this phenomenon. This interaction can properly model the experimentally obtained fluorescent response. The proposed explanation allows other interacting systems to be designed, which have been experimentally confirmed. PMID:10930271

  1. The anaerobic degradation of gaseous, nonmethane alkanes — From in situ processes to microorganisms

    PubMed Central

    Musat, Florin

    2015-01-01

    The short chain, gaseous alkanes ethane, propane, n- and iso-butane are released in significant amounts into the atmosphere, where they contribute to tropospheric chemistry and ozone formation. Biodegradation of gaseous alkanes by aerobic microorganisms, mostly bacteria and fungi isolated from terrestrial environments, has been known for several decades. The first indications for short chain alkane anaerobic degradation were provided by geochemical studies of deep-sea environments around hydrocarbon seeps, and included the uncoupling of the sulfate-reduction and anaerobic oxidation of methane rates, the consumption of gaseous alkanes in anoxic sediments, or the enrichment in 13C of gases in interstitial water vs. the source gas. Microorganisms able to degrade gaseous alkanes were recently obtained from deep-sea and terrestrial sediments around hydrocarbon seeps. Up to date, only sulfate-reducing pure or enriched cultures with ethane, propane and n-butane have been reported. The only pure culture presently available, strain BuS5, is affiliated to the Desulfosarcina–Desulfococcus cluster of the Deltaproteobacteria. Other phylotypes involved in gaseous alkane degradation have been identified based on stable-isotope labeling and whole-cell hybridization. Under anoxic conditions, propane and n-butane are activated similar to the higher alkanes, by homolytic cleavage of the C—H bond of a subterminal carbon atom, and addition of the ensuing radical to fumarate, yielding methylalkylsuccinates. An additional mechanism of activation at the terminal carbon atoms was demonstrated for propane, which could in principle be employed also for the activation of ethane. PMID:25904994

  2. Anaerobic alkane biodegradation by cultures enriched from oil sands tailings ponds involves multiple species capable of fumarate addition.

    PubMed

    Tan, BoonFei; Semple, Kathleen; Foght, Julia

    2015-05-01

    A methanogenic short-chain alkane-degrading culture (SCADC) was enriched from oil sands tailings and transferred several times with a mixture of C6, C7, C8 and C10 n-alkanes as the predominant organic carbon source, plus 2-methylpentane, 3-methylpentane and methylcyclopentane as minor components. Cultures produced ∼40% of the maximum theoretical methane during 18 months incubation while depleting the n-alkanes, 2-methylpentane and methylcyclopentane. Substrate depletion correlated with detection of metabolites characteristic of fumarate activation of 2-methylpentane and methylcyclopentane, but not n-alkane metabolites. During active methanogenesis with the mixed alkanes, reverse-transcription PCR confirmed the expression of functional genes (assA and bssA) associated with hydrocarbon addition to fumarate. Pyrosequencing of 16S rRNA genes amplified during active alkane degradation revealed enrichment of Clostridia (particularly Peptococcaceae) and methanogenic Archaea (Methanosaetaceae and Methanomicrobiaceae). Methanogenic cultures transferred into medium containing sulphate produced sulphide, depleted n-alkanes and produced the corresponding succinylated alkane metabolites, but were slow to degrade 2-methylpentane and methylcyclopentane; these cultures were enriched in Deltaproteobacteria rather than Clostridia. 3-Methylpentane was not degraded by any cultures. Thus, nominally methanogenic oil sands tailings harbour dynamic and versatile hydrocarbon-degrading fermentative syntrophs and sulphate reducers capable of degrading n-, iso- and cyclo-alkanes by addition to fumarate. PMID:25873461

  3. [Distribution Characteristics and Source Apportionment of n-Alkanes in Water from Yellow River in Henan Section].

    PubMed

    Feng, Jing-lan; Xi, Nan-nan; Zhang, Fei; Liu, Shu-hui; Sun, Jian-hui

    2016-03-15

    To investigate the distributions and possible sources of n-alkanes in water and suspended particulate matter from Yellow River in Henan section, 26 water and suspended particulate matter samples were collected in August 2010 and 22 n-alkanes (C₁₄-C₃₆) were quantitatively determined by gas chromatography-mass spectrometer (GC-MS). Potential sources of n-alkanes were analyzed using different characteristic parameters. The results indicated that total concentrations of 22 n-alkanes were 521-5,843 ng · L⁻¹ with a mean concentration of 1,409 ng · L⁻¹, while the total amounts of n-alkanes in the suspended particulate matter were 463-11,142 ng · L⁻¹ with a mean value of 1,951 ng · L⁻¹. The composition profiles of n-alkanes in water showed unimodal distribution with a peak at C₂₅ in water. However, the composition characteristics of n-alkanes in SPM were of bimodal type, but still with the advantage of high carbon hydrocarbons peak at C₂₅. Results of characteristic parameters including CPI, TAR, OEP and % WaxCn showed that n-alkanes in the studied area were derived mainly from combustion of fossil fuel, while terrestrial higher plant played a role in the existence of n-alkanes in water and suspended particulate matter from Yellow River in Henan section. PMID:27337879

  4. Whole genome co-expression analysis of soybean cytochrome P450 genes identifies nodulation-specific P450 monooxygenases

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cytochrome P450 monooxygenases (P450s) catalyze oxidation of various substrates using oxygen and NAD(P)H. Plant P450s are involved in the biosynthesis of primary and secondary metabolites performing diverse biological functions. The recent availability of soybean genome sequence allows us to ident...

  5. Leaf wax n-alkane distributions in and across modern plants: Implications for paleoecology and chemotaxonomy

    NASA Astrophysics Data System (ADS)

    Bush, Rosemary T.; McInerney, Francesca A.

    2013-09-01

    Long chain (C21 to C37) n-alkanes are among the most long-lived and widely utilized terrestrial plant biomarkers. Dozens of studies have examined the range and variation of n-alkane chain-length abundances in modern plants from around the world, and n-alkane distributions have been used for a variety of purposes in paleoclimatology and paleoecology as well as chemotaxonomy. However, most of the paleoecological applications of n-alkane distributions have been based on a narrow set of modern data that cannot address intra- and inter-plant variability. Here, we present the results of a study using trees from near Chicago, IL, USA, as well as a meta-analysis of published data on modern plant n-alkane distributions. First, we test the conformity of n-alkane distributions in mature leaves across the canopy of 38 individual plants from 24 species as well as across a single growing season and find no significant differences for either canopy position or time of leaf collection. Second, we compile 2093 observations from 86 sources, including the new data here, to examine the generalities of n-alkane parameters such as carbon preference index (CPI), average chain length (ACL), and chain-length ratios for different plant groups. We show that angiosperms generally produce more n-alkanes than do gymnosperms, supporting previous observations, and furthermore that CPI values show such variation in modern plants that it is prudent to discard the use of CPI as a quantitative indicator of n-alkane degradation in sediments. We also test the hypotheses that certain n-alkane chain lengths predominate in and therefore can be representative of particular plant groups, namely, C23 and C25 in Sphagnum mosses, C27 and C29 in woody plants, and C31 in graminoids (grasses). We find that chain-length distributions are highly variable within plant groups, such that chemotaxonomic distinctions between grasses and woody plants are difficult to make based on n-alkane abundances. In contrast

  6. Ubiquitous Presence and Novel Diversity of Anaerobic Alkane Degraders in Cold Marine Sediments

    PubMed Central

    Gittel, Antje; Donhauser, Johanna; Røy, Hans; Girguis, Peter R.; Jørgensen, Bo B.; Kjeldsen, Kasper U.

    2015-01-01

    Alkanes are major constituents of crude oil and are released to the marine environment by natural seepage and from anthropogenic sources. Due to their chemical inertness, their removal from anoxic marine sediments is primarily controlled by the activity of anaerobic alkane-degrading microorganisms. To facilitate comprehensive cultivation-independent surveys of the diversity and distribution of anaerobic alkane degraders, we designed novel PCR primers that cover all known diversity of the 1-methylalkyl succinate synthase gene (masD/assA), which catalyzes the initial activation of alkanes. We studied masD/assA gene diversity in pristine and seepage-impacted Danish coastal sediments, as well as in sediments and alkane-degrading enrichment cultures from the Middle Valley (MV) hydrothermal vent system in the Pacific Northwest. MasD/assA genes were ubiquitously present, and the primers captured the diversity of both known and previously undiscovered masD/assA gene diversity. Seepage sediments were dominated by a single masD/assA gene cluster, which is presumably indicative of a substrate-adapted community, while pristine sediments harbored a diverse range of masD/assA phylotypes including those present in seepage sediments. This rare biosphere of anaerobic alkane degraders will likely increase in abundance in the event of seepage or accidental oil spillage. Nanomolar concentrations of short-chain alkanes (SCA) were detected in pristine and seepage sediments. Interestingly, anaerobic alkane degraders closely related to strain BuS5, the only SCA degrader in pure culture, were found in mesophilic MV enrichments, but not in cold sediments from Danish waters. We propose that the new masD/assA gene lineages in these sediments represent novel phylotypes that are either fueled by naturally occurring low levels of SCA or that metabolize medium- to long-chain alkanes. Our study highlights that masD/assA genes are a relevant diagnostic marker to identify seepage and microseepage, e

  7. Ubiquitous Presence and Novel Diversity of Anaerobic Alkane Degraders in Cold Marine Sediments.

    PubMed

    Gittel, Antje; Donhauser, Johanna; Røy, Hans; Girguis, Peter R; Jørgensen, Bo B; Kjeldsen, Kasper U

    2015-01-01

    Alkanes are major constituents of crude oil and are released to the marine environment by natural seepage and from anthropogenic sources. Due to their chemical inertness, their removal from anoxic marine sediments is primarily controlled by the activity of anaerobic alkane-degrading microorganisms. To facilitate comprehensive cultivation-independent surveys of the diversity and distribution of anaerobic alkane degraders, we designed novel PCR primers that cover all known diversity of the 1-methylalkyl succinate synthase gene (masD/assA), which catalyzes the initial activation of alkanes. We studied masD/assA gene diversity in pristine and seepage-impacted Danish coastal sediments, as well as in sediments and alkane-degrading enrichment cultures from the Middle Valley (MV) hydrothermal vent system in the Pacific Northwest. MasD/assA genes were ubiquitously present, and the primers captured the diversity of both known and previously undiscovered masD/assA gene diversity. Seepage sediments were dominated by a single masD/assA gene cluster, which is presumably indicative of a substrate-adapted community, while pristine sediments harbored a diverse range of masD/assA phylotypes including those present in seepage sediments. This rare biosphere of anaerobic alkane degraders will likely increase in abundance in the event of seepage or accidental oil spillage. Nanomolar concentrations of short-chain alkanes (SCA) were detected in pristine and seepage sediments. Interestingly, anaerobic alkane degraders closely related to strain BuS5, the only SCA degrader in pure culture, were found in mesophilic MV enrichments, but not in cold sediments from Danish waters. We propose that the new masD/assA gene lineages in these sediments represent novel phylotypes that are either fueled by naturally occurring low levels of SCA or that metabolize medium- to long-chain alkanes. Our study highlights that masD/assA genes are a relevant diagnostic marker to identify seepage and microseepage, e

  8. Biodegradation of C7 and C8 iso-alkanes under methanogenic conditions.

    PubMed

    Abu Laban, Nidal; Dao, Anh; Semple, Kathleen; Foght, Julia

    2015-12-01

    Iso-alkanes comprise a substantial proportion of petroleum and refined products that impact the environment, but their fate is cryptic under methanogenic conditions. We investigated methanogenic biodegradation of C7 and C8 iso-alkanes found in naphtha, specifically 2-methylhexane, 3-methylhexane, 2-methylheptane, 4-methylheptane and 3-ethylhexane. These were incubated as a mixture or individually with enrichment cultures derived from oil sands tailings ponds that generate methane from naphtha components; substrate depletion and methane production were monitored for up to 663 days. 3-Methylhexane and 4-methylheptane were degraded both singly and in the mixture, whereas 2-methylhexane and 2-methylheptane resisted degradation as single substrates but were depleted in the iso-alkane mixture, suggesting co-metabolism. 3-Ethylhexane was degraded neither singly nor with co-substrates. Putative metabolites consistent with succinylated C7 and C8 were detected, suggesting activation by addition of iso-alkanes to fumarate and corresponding to detection of alkylsuccinate synthase-like genes. 454 pyrotag sequencing, cloning and terminal restriction fragment length polymorphism of 16S rRNA genes revealed predominance of a novel member of the family Peptococcaceae (order Clostridiales) and Archaea affiliated with Methanoregula and Methanosaeta. We report here isomer-specific metabolism of C7 -C8 iso-alkanes under methanogenic conditions and propose their activation by a novel Peptococcaceae via addition to fumarate. PMID:25331365

  9. Effects of fuel properties on the burning characteristics of collision-merged alkane/water droplets

    SciTech Connect

    Wang, C.H.; Pan, K.L.; Huang, W.C.; Wen, H.C.; Yang, J.Y.; Law, C.K.

    2008-04-15

    The combustion characteristics of freely falling droplets, individually generated by the merging of colliding alkane and water droplets, were experimentally investigated. The outcome of the collision droplets was first studied and then the subsequent burning processes such as the flame appearance, ignition and burning behaviors were recorded, through either visual observation or microphotography with the aid of stroboscopic lighting. If the merged droplets were exhibited in an insertive manner, while the water droplet inserted into the alkane droplet, these yield the burning behaviors prior to the end of flame were very much similar to that of pure alkane. The burning was ended with droplet extinction for lower-C alkane, and with either droplet ''flash vaporization'' or extinction for hexadecane. And if the merged droplets were in adhesive manner, for hexadecane with large water content, they either could not be ignited for the large merged droplets, or be ignited with a much prolonged ignition delay, followed by a soot-reducing flame and an ending of droplet extinction for the small merged droplets. ''Homogeneous'' explosion was not observed in any of the tests, and ''heterogeneous'' explosion, induced by trapped air bubbles, occasionally occurred for merged droplets with C-atom in alkane is higher than dodecane. And the sudden disappearance of droplet definitely decreased the burning time and thus enhanced the burning intensity. Besides, the fuel mass consumption rates were increased, even in the cases that having droplet extinction, because of the enlargement of the surface area due to the stuffing of water droplet. (author)

  10. Whole‐cell bacterial bioreporter for actively searching and sensing of alkanes and oil spills

    PubMed Central

    Zhang, Dayi; He, Yi; Wang, Yun; Wang, Hui; Wu, Lin; Aries, Eric; Huang, Wei E.

    2012-01-01

    Summary Acinetobacter baylyi ADP1 was found to tolerate seawater and have a special ability of adhering to an oil–water interface of 10–80 µm emulsified mineral and crude oil droplets. These properties make ADP1 an ideal bacterial chassis for constructing bioreporters that are able to actively search and sense oil spill in water and soils. Acinetobacter baylyi bioreporter ADPWH_alk was developed and applied to the detection of alkanes and alkenes in water, seawater and soils. Bioreporter ADPWH_alk was able to detect a broad range of alkanes and alkenes with carbon chain length from C7 to C36. So far, ADPWH_alk is the only bioreporter that is able to detect alkane with carbon chain length greater than C18. This bioreporter responded to the alkanes in about 30 min and it was independent to the cell growth phase because of two point mutations in alkM promoter recognized by alkane regulatory protein ALKR. ADPWH_alk was applied to detect mineral oil, Brent, Chestnut and Sirri crude oils in water and seawater in the range 0.1–100 mg l−1, showing that the bioreporter oil detection was semi‐quantitative. This study demonstrates that ADPWH_alk is a rapid, sensitive and semi‐quantitative bioreporter that can be useful for environmental monitoring and assessment of oil spills in seawater and soils. PMID:21951420

  11. Toward aldehyde and alkane production by removing aldehyde reductase activity in Escherichia coli

    PubMed Central

    Rodriguez, Gabriel M.; Atsumi, Shota

    2015-01-01

    Advances in synthetic biology and metabolic engineering have enabled the construction of novel biological routes to valuable chemicals using suitable microbial hosts. Aldehydes serve as chemical feedstocks in the synthesis of rubbers, plastics, and other larger molecules. Microbial production of alkanes is dependent on the formation of a fatty aldehyde intermediate which is converted to an alkane by an aldehyde deformylating oxygenase (ADO). However, microbial hosts such as Escherichia coli are plagued by many highly active endogenous aldehyde reductases (ALRs) that convert aldehydes to alcohols, which greatly complicates strain engineering for aldehyde and alkane production. It has been shown that the endogenous ALR activity outcompetes the ADO enzyme for fatty aldehyde substrate. The large degree of ALR redundancy coupled with an incomplete database of ALRs represents a significant obstacle in engineering E. coli for either aldehyde or alkane production. In this study, we identified 44 ALR candidates encoded in the E. coli genome using bioinformatics tools, and undertook a comprehensive screening by measuring the ability of these enzymes to produce isobutanol. From the pool of 44 candidates, we found five new ALRs using this screening method (YahK, DkgA, GldA, YbbO, and YghA). Combined deletions of all 13 known ALRs resulted in a 90–99% reduction in endogenous ALR activity for a wide range of aldehyde substrates (C2–C12). Elucidation of the ALRs found in E. coli could guide one in reducing competing alcohol formation during alkane or aldehyde production. PMID:25108218

  12. Biogeographic variation of foliar n-alkanes of Juniperus communis var. saxatilis Pallas from the Balkans.

    PubMed

    Rajčević, Nemanja; Janaćković, Pedja; Dodoš, Tanja; Tešević, Vele; Marin, Petar D

    2014-12-01

    The composition of the epicuticular n-alkanes isolated from the leaves of ten populations of Juniperus communis L. var. saxatilis Pallas from central (continental) and western (coastal) areas of the Balkan Peninsula was characterized by GC-FID and GC/MS analyses. In the leaf waxes, 14 n-alkane homologues with chain-lengths ranging from C22 to C35 were identified. All samples were dominated by n-tritriacontane (C33 ), but differences in two other dominant n-alkanes allowed separating the coastal from the continental populations. Several statistical methods (ANOVA, principal component, discriminant, and cluster analyses as well as the Mantel test) were deployed to analyze the diversity and variability of the epicuticular-leaf-n-alkane patterns of the ten natural populations of J. communis var. saxatilis and their relation to different geographic and bioclimatic parameters. Cluster analysis showed a high correlation of the leaf-n-alkane patterns with the geographical distribution of the investigated samples, differentiating the coastal from the continental populations of this taxon. Several bioclimatic parameters related to aridity were highly correlated with this differentiation. PMID:25491336

  13. Using tyrosinase as a monophenol monooxygenase: A combined strategy for effective inhibition of melanin formation.

    PubMed

    Lee, Sang-Hyuk; Baek, Kiheon; Lee, Ju-Eun; Kim, Byung-Gee

    2016-04-01

    Tyrosinase is a binuclear copper-containing metalloprotein that leads the fast and regio-selective o-hydroxylation of monophenols to o-diphenols. However, the subsequent second oxidation to produce o-quinones, i.e., melanin precursors, from the o-diphenols has restricted its use to the production of functional o-diphenol derivatives. Herein, we present a combined strategy for the effective inhibition of melanin formation in tyrosinase reaction, which allows the use of tyrosinase as a monophenol monooxygenase. The o-diphenolic products were protected from being oxidized in the tyrosinase reaction by borate ions and L-ascorbic acid (LAA). Borate-o-diphenol complexes were favorable formed at high pH and consequentially protected the o-diphenolic products from the catecholase activity of tyrosinase. LAA not only directly reduced the byproduct, o-quinones, into o-diphenols but also assisted the completion of the tyrosinase reaction cycle by removing a hydroxyl group attached to the copper metal cluster at the active site of the met-form tyrosinase. The regio-selective o-hydroxylation of 7,4'-dihydroxyisoflavone (daidzein) to produce 7,3',4'-trihydroxyisoflavone (3'-ODI) was successfully carried out by whole E. coli cell biotransformation with heterologously expressed tyrosinase from Bacillus megaterium. The yield of this o-hydroxylation of 5 mM daidzein in one-pot 400 mL reaction was ca. 100% in 90 min and the productivity was 16.3 mg 3'-ODI · L(-1)  ·  h(-1)  ·  DCW mg(-1) , which is considerably higher than that of other monooxygenases. The method effectively abolished melanin synthesis, so that the o-diphenolic product remained stable without enzyme inactivation. Other monophenolic phytochemicals such as resveratrol and genistein could be subjected to the same strategy. After 1 h, 1 mM of genistein and resveratrol were both converted to orobol and piceatannol, respectively, with ca. 95% conversion yield. These results support the strong

  14. Anaerobic oxidation of short-chain alkanes in hydrothermal sediments: potential influences on sulfur cycling and microbial diversity

    SciTech Connect

    Adams, MM; Hoarfrost, AL; Bose, A; Joye, SB; Girguis, PR

    2013-05-14

    Short-chain alkanes play a substantial role in carbon and sulfur cycling at hydrocarbon-rich environments globally, yet few studies have examined the metabolism of ethane (C-2), propane (C-3), and butane (C-4) in anoxic sediments in contrast to methane (C-1). In hydrothermal vent systems, short-chain alkanes are formed over relatively short geological time scales via thermogenic processes and often exist at high concentrations. The sediment-covered hydrothermal vent systems at Middle Valley (MV Juan de Fuca Ridge) are an ideal site for investigating the anaerobic oxidation of C-1-C-4 alkanes, given the elevated temperatures and dissolved hydrocarbon species characteristic of these metalliferous sediments. We examined whether MV microbial communities oxidized C-1-C-4 alkanes under mesophilic to thermophilic sulfate-reducing conditions. Here we present data from discrete temperature (25, 55, and 75 degrees C) anaerobic batch reactor incubations of MV sediments supplemented with individual alkanes. Co-registered alkane consumption and sulfate reduction (SR) measurements provide clear evidence for C-1-C-4 alkane oxidation linked to SR over time and across temperatures. In these anaerobic batch reactor sediments, 16S ribosomal RNA pyrosequencing revealed that Deltaproteobacteria, particularly a novel sulfate-reducing lineage, were the likely phylotypes mediating the oxidation of C-2-C-4 alkanes. Maximum C-1-C-4 alkane oxidation rates occurred at 55 degrees C, which reflects the mid-core sediment temperature profile and corroborates previous studies of rate maxima for the anaerobic oxidation of methane (AOM). Of the alkanes investigated, C-3 was oxidized at the highest rate over time, then C-4, C-2, and C-1, respectively. The implications of these results are discussed with respect to the potential competition between the anaerobic oxidation of C-2-C(4)alkanes with AOM for available oxidants and the influence on the fate of C-1 derived from these hydrothermal systems.

  15. Catalytic dehydroaromatization of n-alkanes by pincer-ligated iridium complexes

    NASA Astrophysics Data System (ADS)

    Ahuja, Ritu; Punji, Benudhar; Findlater, Michael; Supplee, Carolyn; Schinski, William; Brookhart, Maurice; Goldman, Alan S.

    2011-02-01

    Aromatic hydrocarbons are among the most important building blocks in the chemical industry. Benzene, toluene and xylenes are obtained from the high temperature thermolysis of alkanes. Higher alkylaromatics are generally derived from arene-olefin coupling, which gives branched products—that is, secondary alkyl arenes—with olefins higher than ethylene. The dehydrogenation of acyclic alkanes to give alkylaromatics can be achieved using heterogeneous catalysts at high temperatures, but with low yields and low selectivity. We present here the first catalytic conversion of n-alkanes to alkylaromatics using homogeneous or molecular catalysts—specifically ‘pincer’-ligated iridium complexes—and olefinic hydrogen acceptors. For example, the reaction of n-octane affords up to 86% yield of aromatic product, primarily o-xylene and secondarily ethylbenzene. In the case of n-decane and n-dodecane, the resulting alkylarenes are exclusively unbranched (that is, n-alkyl-substituted), with selectivity for the corresponding o-(n-alkyl)toluene.

  16. Dehydrogenation of n-alkanes catalyzed by iridium ``pincer`` complexes: Regioselective formation of {alpha}-olefins

    SciTech Connect

    Liu, F.; Singh, B.; Goldman, A.S.; Pak, E.B.; Jensen, C.M.

    1999-04-28

    The development of methods for the functionalization of alkanes is of cardinal importance in catalytic chemistry. A specific functionalization of particularly great potential value is the conversion of n-alkanes to the corresponding 1-alkenes ({alpha}-olefins) since these serve as precursors for a wide range of commodity-scale chemicals (>2 {times} 10{sup 9} kg/yr). Such a conversion is also an intriguing challenge as viewed from a fundamental perspective. n-Alkanes are the simplest organic molecules with the potential to undergo regioselective transformations; {alpha}-olefins are the thermodynamically least stable of the corresponding double-bond isomers and any mechanism for their formation must presumably involve activation of the strongest bond (primary C-{single_bond}H) in the molecule.

  17. Oxidation of alkanes by cobalt(II) salts of weakly coordinating anions

    SciTech Connect

    Goldstein, A.S.; Drago, R.S. )

    1991-11-27

    Catalysts which effect the selective oxidation of alkanes under mild reaction conditions are highly desired. Commercial processes exist which involve the oxidation of alkanes by O{sub 2} with cobalt carboxylate catalysts. Elevated temperatures and pressures are required, and the metal ion function is to decompose hydroperoxides formed in a radical-chain process. The authors have demonstrated that a weakly solvated cobalt-acetonitrile complex (Co(NCCH{sub 3}){sub 4})(PF{sub 6}){sub 2}, with a weakly coordinating anion catalyzes the air oxidation of alkanes under mild conditions (75C and 3 atm). Cyclohexane and adamantane are converted to the corresponding alcohol and ketone products. The commercial catalyst for cyclohexane oxidation does not function under these milder conditions. Experiments indicate a mechanism in which the metal ion functions both as an initiator and as a hydroperoxide decomposition catalyst.

  18. Distribution, activity and function of short-chain alkane degrading phylotypes in hydrothermal vent sediments

    NASA Astrophysics Data System (ADS)

    Adams, M. M.; Joye, S. B.; Hoarfrost, A.; Girguis, P. R.

    2012-12-01

    Global geochemical analyses suggest that C2-C4 short chain alkanes are a common component of the utilizable carbon pool in deep-sea sediments worldwide and have been found in diverse ecosystems. From a thermodynamic standpoint, the anaerobic microbial oxidation of these aliphatic hydrocarbons is more energetically yielding than the anaerobic oxidation of methane (AOM). Therefore, the preferential degradation of these hydrocarbons may compete with AOM for the use of oxidants such as sulfate, or other potential oxidants. Such processes could influence the fate of methane in the deep-sea. Sulfate-reducing bacteria (SRB) from hydrocarbon seep sediments of the Gulf of Mexico and Guaymas Basin have previously been enriched that anaerobically oxidize short chain alkanes to generate CO2 with the preferential utilization of 12C-enriched alkanes (Kniemeyer et al. 2007). Different temperature regimens along with multiple substrates were tested and a pure culture (deemed BuS5) was isolated from mesophilic enrichments with propane or n-butane as the sole carbon source. Through comparative sequence analysis, strain BuS5 was determined to cluster with the metabolically diverse Desulfosarcina / Desulfococcus cluster, which also contains the SRB found in consortia with anaerobic, methane-oxidizing archaea in seep sediments. Enrichments from a terrestrial, low temperature sulfidic hydrocarbon seep also corroborated that propane degradation occurred with most bacterial phylotypes surveyed belonging to the Deltaproteobacteria, particularly Desulfobacteraceae (Savage et al. 2011). To date, no microbes capable of ethane oxidation or anaerobic C2-C4 alkane oxidation at thermophilic temperature have been isolated. The sediment-covered, hydrothermal vent systems found at Middle Valley (Juan de Fuca Ridge, eastern Pacific Ocean) are a prime environment for investigating mesophilic to thermophilic anaerobic oxidation of short-chain alkanes, given the elevated temperatures and dissolved

  19. The low temperature phase transition in octane and its possible generalisation to other n-alkanes

    NASA Astrophysics Data System (ADS)

    Neumann, M. A.; Johnson, M. R.; Radaelli, P. G.

    2001-05-01

    A neutron powder diffraction study of three n-alkanes, octane, nonane and pentadecane, down to 2 K is presented. The temperature dependence of the octane diffraction pattern reveals a solid state phase transition between 40 and 55 K, which involves a doubling of the unit cell in the b direction, the space group remaining P 1¯. Confirmation of the phase transition, which results in a doubling of the number of crystallographically inequivalent methyl groups, is sought in the published NMR, tunnelling data and neutron scattering, vibrational data. Density functional theory and force field techniques are used to simulate spectroscopic data based on the measured structures. While no unequivocal evidence is found in spectroscopic data, the published data does not rule out the existence of energetically inequivalent methyl groups. Indeed close inspection of the spectroscopic data for other n-alkanes suggests that the phase transition may be common to many alkanes.

  20. The synergetic effect of starch and alpha amylase on the biodegradation of n-alkanes.

    PubMed

    Karimi, M; Biria, D

    2016-06-01

    The impact of adding soluble starch on biodegradation of n-alkanes (C10-C14) by Bacillus subtilis TB1 was investigated. Gas chromatography was employed to measure the residual hydrocarbons in the system. It was observed that the efficiency of biodegradation improved with the presence of starch and the obtained residual hydrocarbons in the system were 53% less than the samples without starch. The produced bacterial enzymes were studied through electrophoresis and reverse zymography for explaining the observations. The results indicated that the produced amylase by the bacteria can degrade hydrocarbons and the same was obtained by the application of a commercial alpha amylase sample. In addition, in silico docking of alpha-amylase with n-alkanes with different molecular weights was studied by Molegro virtual docker which showed high negative binding energies and further substantiated the experimental observations. Overall, the findings confirmed the catalytic effect of alpha amylase on n-alkanes degradation. PMID:26971168

  1. n-Alkanes in surficial sediments of Visakhapatnam harbour, east coast of India

    NASA Astrophysics Data System (ADS)

    Punyu, V. R.; Harji, R. R.; Bhosle, N. B.; Sawant, S. S.; Venkat, K.

    2013-04-01

    Surface sediments collected from 19 stations along Visakhapatnam harbour were analysed for organic carbon (OC), δ 13Coc, total lipids (TL), total hydrocarbon (THC), n-alkane concentration and composition. OC, δ 13Coc, TL and THC ranged from 0.6% to 7.6%, -29.3 to -23.8‰, 300 to 14,948 \\upmu g g - 1 dw, and 0.2 to 2,277 \\upmu g g - 1 dw, respectively. Predominance of even carbon numbers n-alkanes C12-C21 with carbon preference index (CPI) of <1 suggests major microbial influence. Fair abundance of odd carbon number n-alkanes in the range of C15-C22 and C23-C33 indicates some input from phytoplankton and terrestrial sources, respectively. Petrogenic input was evident from the presence of hopanes and steranes. The data suggest that organic matter (OM) sources varied spatially and were mostly derived from mixed source.

  2. Recent insights into copper-containing lytic polysaccharide mono-oxygenases.

    PubMed

    Hemsworth, Glyn R; Davies, Gideon J; Walton, Paul H

    2013-10-01

    Recently the role of oxidative enzymes in the degradation of polysaccharides by saprophytic bacteria and fungi was uncovered, challenging the classical model of polysaccharide degradation of being solely via a hydrolytic pathway. 3D structural analyses of lytic polysaccharide mono-oxygenases of both bacterial AA10 (formerly CBM33) and fungal AA9 (formerly GH61) enzymes revealed structures with β-sandwich folds containing an active site with a metal coordinated by an N-terminal histidine. Following some initial confusion about the identity of the metal ion it has now been shown that these enzymes are copper-dependent oxygenases. Here we assess recent developments in the academic literature, focussing on the structures of the copper active sites. We provide critical comparisons with known small-molecules studies of copper-oxygen complexes and with copper methane monoxygenase, another of nature's powerful copper oxygenases. PMID:23769965

  3. Cell Non-Autonomous Activation of Flavin-containing Monooxygenase Promotes Longevity and Healthspan

    PubMed Central

    Leiser, Scott F.; Fletcher, Marissa; Leonard, Alison; Primitivo, Melissa; Rintala, Nicholas; Ramos, Fresnida J.; Miller, Dana L.; Kaeberlein, Matt

    2016-01-01

    Stabilization of the hypoxia-inducible factor-1 (HIF-1) increases lifespan and healthspan in nematodes through an unknown mechanism. We report that neuronal stabilization of HIF-1 mediates these effects in C. elegans through a cell non-autonomous signal to the intestine resulting in activation of the xenobiotic detoxification enzyme flavin-containing monooxygenase-2 (FMO-2). This pro-longevity signal requires the serotonin biosynthetic enzyme TPH-1 in neurons and the serotonin receptor SER-7 in the intestine. Intestinal FMO-2 is also activated by dietary restriction (DR) and necessary for DR-mediated lifespan extension, suggesting that this enzyme represents a point of convergence for two distinct longevity pathways. FMOs are conserved in eukaryotes and induced by multiple lifespan-extending interventions in mice, suggesting that these enzymes may play a critical role in promoting health and longevity across phyla. PMID:26586189

  4. Single-domain flavoenzymes trigger lytic polysaccharide monooxygenases for oxidative degradation of cellulose

    PubMed Central

    Garajova, Sona; Mathieu, Yann; Beccia, Maria Rosa; Bennati-Granier, Chloé; Biaso, Frédéric; Fanuel, Mathieu; Ropartz, David; Guigliarelli, Bruno; Record, Eric; Rogniaux, Hélène; Henrissat, Bernard; Berrin, Jean-Guy

    2016-01-01

    The enzymatic conversion of plant biomass has been recently revolutionized by the discovery of lytic polysaccharide monooxygenases (LPMOs) that carry out oxidative cleavage of polysaccharides. These very powerful enzymes are abundant in fungal saprotrophs. LPMOs require activation by electrons that can be provided by cellobiose dehydrogenases (CDHs), but as some fungi lack CDH-encoding genes, other recycling enzymes must exist. We investigated the ability of AA3_2 flavoenzymes secreted under lignocellulolytic conditions to trigger oxidative cellulose degradation by AA9 LPMOs. Among the flavoenzymes tested, we show that glucose dehydrogenase and aryl-alcohol quinone oxidoreductases are catalytically efficient electron donors for LPMOs. These single-domain flavoenzymes display redox potentials compatible with electron transfer between partners. Our findings extend the array of enzymes which regulate the oxidative degradation of cellulose by lignocellulolytic fungi. PMID:27312718

  5. Rv1894c Is a Novel Hypoxia-Induced Nitronate Monooxygenase Required for Mycobacterium tuberculosis Virulence

    PubMed Central

    Klinkenberg, Lee G.; Karakousis, Petros C.

    2013-01-01

    Tuberculosis is difficult to cure, requiring a minimum of 6 months of treatment with multiple antibiotics. Small numbers of organisms are able to tolerate the antibiotics and persist in the lungs of infected humans, but they still require some metabolic activity to survive. We studied the role of the hypoxia-induced Rv1894c gene in Mycobacterium tuberculosis virulence in guinea pigs, which develop hypoxic, necrotic granulomas histologically resembling those in humans and found this gene to be necessary for full bacillary growth and survival. We characterized the function of the encoded enzyme as a nitronate monooxygenase, which is needed to prevent the buildup of toxic products during hypoxic metabolism and is negatively regulated by the transcriptional repressor KstR. Future studies will focus on developing small-molecule inhibitors that target Rv1894c and its homologs, with the goal of killing persistent bacteria, thereby shortening the time needed to treat tuberculosis. PMID:23408846

  6. Nitronate monooxygenase, a model for anionic flavin semiquinone intermediates in oxidative catalysis.

    PubMed

    Gadda, Giovanni; Francis, Kevin

    2010-01-01

    Nitronate monooxygenase (NMO), formerly referred to as 2-nitropropane dioxygenase, is an FMN-dependent enzyme that uses molecular oxygen to oxidize (anionic) alkyl nitronates and, in the case of the enzyme from Neurospora crassa, (neutral) nitroalkanes to the corresponding carbonyl compounds and nitrite. Over the past 5 years, a resurgence of interest on the enzymology of NMO has driven several studies aimed at the elucidation of the mechanistic and structural properties of the enzyme. This review article summarizes the knowledge gained from these studies on NMO, which has been emerging as a model system for the investigation of anionic flavosemiquinone intermediates in the oxidative catalysis of organic molecules, and for the effect that branching of reaction intermediates has on both the kinetic parameters and isotope effects associated with enzymatic reactions. A comparison of the catalytic mechanism of NMO with other flavin-dependent enzymes that oxidize nitroalkane and nitronates is also presented. PMID:19577534

  7. Structure and boosting activity of a starch-degrading lytic polysaccharide monooxygenase

    PubMed Central

    Lo Leggio, Leila; Simmons, Thomas J.; Poulsen, Jens-Christian N.; Frandsen, Kristian E. H.; Hemsworth, Glyn R.; Stringer, Mary A.; von Freiesleben, Pernille; Tovborg, Morten; Johansen, Katja S.; De Maria, Leonardo; Harris, Paul V.; Soong, Chee-Leong; Dupree, Paul; Tryfona, Theodora; Lenfant, Nicolas; Henrissat, Bernard; Davies, Gideon J.; Walton, Paul H.

    2015-01-01

    Lytic polysaccharide monooxygenases (LPMOs) are recently discovered enzymes that oxidatively deconstruct polysaccharides. LPMOs are fundamental in the effective utilization of these substrates by bacteria and fungi; moreover, the enzymes have significant industrial importance. We report here the activity, spectroscopy and three-dimensional structure of a starch-active LPMO, a representative of the new CAZy AA13 family. We demonstrate that these enzymes generate aldonic acid-terminated malto-oligosaccharides from retrograded starch and boost significantly the conversion of this recalcitrant substrate to maltose by β-amylase. The detailed structure of the enzyme’s active site yields insights into the mechanism of action of this important class of enzymes. PMID:25608804

  8. Searching for monooxygenases and hydrolases in bacteria from an extreme environment.

    PubMed

    da Cruz, Georgiana F; Angolini, Célio F F; de Oliveira, Luciana G; Lopes, Patrícia F; de Vasconcellos, Suzan P; Crespim, Elaine; de Oliveira, Valéria M; dos Santos Neto, Eugênio V; Marsaioli, Anita J

    2010-06-01

    Microbial oxidation potentials of extremophiles recovered from Pampo Sul oil field, Campos Basin, Brazil, in pure culture or in consortia, were investigated using high-throughput screening (HTS) and multibioreactions. Camphor (1), cis-jasmone (2), 2-methyl-cyclohexanone (3), 1,2-epoxyoctane (4), phenylethyl acetate (5), phenylethyl propionate (6), and phenylethyl octanoate (7) were used to perform multibioreaction assays. Eighty-two bacterial isolates were recovered from oil and formation water samples and those presenting outstanding activities in HTS assays were identified by sequencing their 16S rRNA genes. These results revealed that most microorganisms belonged to the genus Bacillus and presented alcohol dehydrogenase, monooxygenase, epoxide hydrolase, esterase, and lipase activities. PMID:20204614

  9. Structural basis for substrate targeting and catalysis by fungal polysaccharide monooxygenases.

    PubMed

    Li, Xin; Beeson, William T; Phillips, Christopher M; Marletta, Michael A; Cate, Jamie H D

    2012-06-01

    The use of cellulases remains a major cost in the production of renewable fuels and chemicals from lignocellulosic biomass. Fungi secrete copper-dependent polysaccharide monooxygenases (PMOs) that oxidatively cleave crystalline cellulose and improve the effectiveness of cellulases. However, the means by which PMOs recognize and cleave their substrates in the plant cell wall remain unclear. Here, we present structures of Neurospora crassa PMO-2 and PMO-3 at 1.10 and 1.37 Å resolution, respectively. In the structures, dioxygen species are found in the active sites, consistent with the proposed cleavage mechanism. Structural and sequence comparisons between PMOs also reveal that the enzyme substrate-binding surfaces contain highly varied aromatic amino acid and glycosylation positions. The structures reported here provide evidence for a wide range of PMO substrate recognition patterns in the plant cell wall, including binding modes that traverse multiple glucan chains. PMID:22578542

  10. Expression and characterization of a lytic polysaccharide monooxygenase from Bacillus thuringiensis.

    PubMed

    Zhang, Huiyan; Zhao, Yong; Cao, Hailong; Mou, Guangqing; Yin, Heng

    2015-08-01

    Lytic polysaccharide monooxygenases (LPMOs) are recently discovered oxidative enzymes that are capable of oxidative cleavage of recalcitrant polysaccharides such as chitin or cellulose. Despite the importance of LPMOs in biomass conversion and the large number of lpmo genes in microorganisms, only a few LPMOs have been well studied, and further characterization of these proteins is thus of interest. In this study, a chitin-active AA10 family LPMO from Bacillus thuringiensis subsp. kurstaki, BtLPMO10A, is described. This enzyme generates even-numbered oxidized oligosaccharides as the dominated products from crystalline chitin, however, interestingly, when colloidal chitin is used as the substrate, a ladder of oxidized oligosaccharides is observed. These results provide new insights into the action mode of LPMOs that may be affected by the substrates. PMID:25936286

  11. Discovery and characterization of a new family of lytic polysaccharide monooxygenases.

    PubMed

    Hemsworth, Glyn R; Henrissat, Bernard; Davies, Gideon J; Walton, Paul H

    2014-02-01

    Lytic polysaccharide monooxygenases (LPMOs) are a recently discovered class of enzymes capable of oxidizing recalcitrant polysaccharides. They are attracting considerable attention owing to their potential use in biomass conversion, notably in the production of biofuels. Previous studies have identified two discrete sequence-based families of these enzymes termed AA9 (formerly GH61) and AA10 (formerly CBM33). Here, we report the discovery of a third family of LPMOs. Using a chitin-degrading exemplar from Aspergillus oryzae, we show that the three-dimensional structure of the enzyme shares some features of the previous two classes of LPMOs, including a copper active center featuring the 'histidine brace' active site, but is distinct in terms of its active site details and its EPR spectroscopy. The newly characterized AA11 family expands the LPMO clan, potentially broadening both the range of potential substrates and the types of reactive copper-oxygen species formed at the active site of LPMOs. PMID:24362702

  12. Direct electrochemistry of the hydroxylase of soluble methane monooxygenase from Methylococcus capsulatus (Bath).

    PubMed

    Kazlauskaite, J; Hill, H A; Wilkins, P C; Dalton, H

    1996-10-15

    The redox properties of the hydroxylase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath) have been thoroughly investigated. Previous studies used redox indicator titrations and spectroscopic methods for the determination of the concentrations of reduced species. Herein we report, for the first time, direct electrochemistry (i.e. without the use of mediators) of the diiron centers of the hydroxylase from M. capsulatus (Bath) at a modified gold electrode giving rise to two waves at 4(+/- 10) mV and -386(+/- 14) mV versus saturated calomel electrode (SCE). In addition, the effects of proteins B and B' on the redox reactions were determined. The redox potentials of the complex with protein B are -25(+/- 14) mV and -433(+/- 8) mV versus SCE whereas protein B' had no effect though it did alter the effect of protein B on the redox potentials. PMID:8917455

  13. Fourier transform infrared characterization of the azido complex of methane monooxygenase hydroxylase from Methylococcus capsulatus (Bath).

    PubMed

    Lu, Shen; Sazinsky, Matthew H; Whittaker, James W; Lippard, Stephen J; Moënne-Loccoz, Pierre

    2005-03-30

    The azido complex formed in oxidized methane monooxygenase from Methylococcus capsulatus (Bath) was investigated with resonance Raman and FTIR techniques. These experiments show the presence of a nuas(NNN) at approximately 2077 cm-1 which splits to two components at 2059 and 2073 cm-1 with 15N14N2. The vibrational data are assigned to an azido complex bound terminally to one iron(III) at the diiron center. When the azido complex is illuminated at 15 K, a new nuas(NNN) is observed at 2136 cm-1 which is assigned to a photodissociated HN3 within the substrate pocket. We propose a model where an aqua ligand engages a hydrogen bond interaction with the 1N atom of the azido group and acts as at a proton donor during the photolysis process. PMID:15783178

  14. Crystal Structure of Dicamba Monooxygenase: A Rieske Nonheme Oxygenase that Catalyzes Oxidative Demethylation

    SciTech Connect

    Dumitru, Razvan; Jiang, Wen Zhi; Weeks, Donald P.; Wilson, Mark A.

    2009-08-28

    Dicamba (3,6-dichloro-2-methoxybenzoic acid) is a widely used herbicide that is efficiently degraded by soil microbes. These microbes use a novel Rieske nonheme oxygenase, dicamba monooxygenase (DMO), to catalyze the oxidative demethylation of dicamba to 3,6-dichlorosalicylic acid (DCSA) and formaldehyde. We have determined the crystal structures of DMO in the free state, bound to its substrate dicamba, and bound to the product DCSA at 2.10-1.75 {angstrom} resolution. The structures show that the DMO active site uses a combination of extensive hydrogen bonding and steric interactions to correctly orient chlorinated, ortho-substituted benzoic-acid-like substrates for catalysis. Unlike other Rieske aromatic oxygenases, DMO oxygenates the exocyclic methyl group, rather than the aromatic ring, of its substrate. This first crystal structure of a Rieske demethylase shows that the Rieske oxygenase structural scaffold can be co-opted to perform varied types of reactions on xenobiotic substrates.

  15. Structure, dynamics, and function of the monooxygenase P450 BM-3: insights from computer simulations studies

    NASA Astrophysics Data System (ADS)

    Roccatano, Danilo

    2015-07-01

    The monooxygenase P450 BM-3 is a NADPH-dependent fatty acid hydroxylase enzyme isolated from soil bacterium Bacillus megaterium. As a pivotal member of cytochrome P450 superfamily, it has been intensely studied for the comprehension of structure-dynamics-function relationships in this class of enzymes. In addition, due to its peculiar properties, it is also a promising enzyme for biochemical and biomedical applications. However, despite the efforts, the full understanding of the enzyme structure and dynamics is not yet achieved. Computational studies, particularly molecular dynamics (MD) simulations, have importantly contributed to this endeavor by providing new insights at an atomic level regarding the correlations between structure, dynamics, and function of the protein. This topical review summarizes computational studies based on MD simulations of the cytochrome P450 BM-3 and gives an outlook on future directions.

  16. Flavin-containing monooxygenase 3 as a potential player in diabetes-associated atherosclerosis.

    PubMed

    Miao, Ji; Ling, Alisha V; Manthena, Praveen V; Gearing, Mary E; Graham, Mark J; Crooke, Rosanne M; Croce, Kevin J; Esquejo, Ryan M; Clish, Clary B; Vicent, David; Biddinger, Sudha B

    2015-01-01

    Despite the well-documented association between insulin resistance and cardiovascular disease, the key targets of insulin relevant to the development of cardiovascular disease are not known. Here, using non-biased profiling methods, we identify the enzyme flavin-containing monooxygenase 3 (Fmo3) to be a target of insulin. FMO3 produces trimethylamine N-oxide (TMAO), which has recently been suggested to promote atherosclerosis in mice and humans. We show that FMO3 is suppressed by insulin in vitro, increased in obese/insulin resistant male mice and increased in obese/insulin-resistant humans. Knockdown of FMO3 in insulin-resistant mice suppresses FoxO1, a central node for metabolic control, and entirely prevents the development of hyperglycaemia, hyperlipidemia and atherosclerosis. Taken together, these data indicate that FMO3 is required for FoxO1 expression and the development of metabolic dysfunction. PMID:25849138

  17. Hepatic microsomal monooxygenase activity in black-crowned night herons (BCNHS) from the Chesapeake basin

    USGS Publications Warehouse

    Melancon, M.J.; Rattner, B.A.; Rice, C.P.; Hines, R.K.; Eisemann, J.

    1992-01-01

    In a continuation of our studies on the use of hepatic cytochromes P450 as a biomarker for contaminant exposure, BCNH eggs were collected from Baltimore Harbor (BH) (n = 20), Washington National Zoo (WNZ) (n = 13) and Chincoteague National Wildlife Refuge (CNWR) (reference location) (n = 20). Eggs were artificially incubated and sacrificed at pipping. Livers were snap frozen in liquid nitrogen and stored at -80?C until assay. Hepatic microsomes were prepared by differential centrifugation of homogenates and assayed for protein, benzyloxy-resorufin-O-dealkylase, (BROD) ethoxyresorufinO-dealkylase (EROD) and pentoxyresorufin-O-dealkylase (PROD). Monooxygenase assays were run in triplicate using a computer-coupled fluorometric microwell plate scanner. Values for EROD and BROD, but not PROD, from BH and WNZ were significantly greater (approximately double) than those from CNWR. Organochlorine pesticide residues were much higher in carcasses from BH and WNZ as compared to CNWR. Carcasses are presently being analyzed for PCB congeners.

  18. Single-domain flavoenzymes trigger lytic polysaccharide monooxygenases for oxidative degradation of cellulose.

    PubMed

    Garajova, Sona; Mathieu, Yann; Beccia, Maria Rosa; Bennati-Granier, Chloé; Biaso, Frédéric; Fanuel, Mathieu; Ropartz, David; Guigliarelli, Bruno; Record, Eric; Rogniaux, Hélène; Henrissat, Bernard; Berrin, Jean-Guy

    2016-01-01

    The enzymatic conversion of plant biomass has been recently revolutionized by the discovery of lytic polysaccharide monooxygenases (LPMOs) that carry out oxidative cleavage of polysaccharides. These very powerful enzymes are abundant in fungal saprotrophs. LPMOs require activation by electrons that can be provided by cellobiose dehydrogenases (CDHs), but as some fungi lack CDH-encoding genes, other recycling enzymes must exist. We investigated the ability of AA3_2 flavoenzymes secreted under lignocellulolytic conditions to trigger oxidative cellulose degradation by AA9 LPMOs. Among the flavoenzymes tested, we show that glucose dehydrogenase and aryl-alcohol quinone oxidoreductases are catalytically efficient electron donors for LPMOs. These single-domain flavoenzymes display redox potentials compatible with electron transfer between partners. Our findings extend the array of enzymes which regulate the oxidative degradation of cellulose by lignocellulolytic fungi. PMID:27312718

  19. Fungal lytic polysaccharide monooxygenases bind starch and β-cyclodextrin similarly to amylolytic hydrolases.

    PubMed

    Nekiunaite, Laura; Isaksen, Trine; Vaaje-Kolstad, Gustav; Abou Hachem, Maher

    2016-08-01

    Starch-binding modules of family 20 (CBM20) are present in 60% of lytic polysaccharide monooxygenases (LPMOs) catalyzing the oxidative breakdown of starch, which highlights functional importance in LPMO activity. The substrate-binding properties of starch-active LMPOs, however, are currently unexplored. Affinities and binding-thermodynamics of two recombinant fungal LPMOs toward starch and β-cyclodextrin were shown to be similar to fungal CBM20s. Amplex Red assays showed ascorbate and Cu-dependent activity, which was inhibited in the presence of β-cylodextrin and amylose. Phylogenetically, the clustering of CBM20s from starch-targeting LPMOs and hydrolases was in accord with taxonomy and did not correlate to appended catalytic activity. Altogether, these results demonstrate that the CBM20-binding scaffold is retained in the evolution of hydrolytic and oxidative starch-degrading activities. PMID:27397613

  20. Linking Low-Level Stable Isotope Fractionation to Expression of the Cytochrome P450 Monooxygenase-Encoding ethB Gene for Elucidation of Methyl tert-Butyl Ether Biodegradation in Aerated Treatment Pond Systems▿ †

    PubMed Central

    Jechalke, Sven; Rosell, Mònica; Martínez-Lavanchy, Paula M.; Pérez-Leiva, Paola; Rohwerder, Thore; Vogt, Carsten; Richnow, Hans H.

    2011-01-01

    Multidimensional compound-specific stable isotope analysis (CSIA) was applied in combination with RNA-based molecular tools to characterize methyl tertiary (tert-) butyl ether (MTBE) degradation mechanisms occurring in biofilms in an aerated treatment pond used for remediation of MTBE-contaminated groundwater. The main pathway for MTBE oxidation was elucidated by linking the low-level stable isotope fractionation (mean carbon isotopic enrichment factor [ɛC] of −0.37‰ ± 0.05‰ and no significant hydrogen isotopic enrichment factor [ɛH]) observed in microcosm experiments to expression of the ethB gene encoding a cytochrome P450 monooxygenase able to catalyze the oxidation of MTBE in biofilm samples both from the microcosms and directly from the ponds. 16S rRNA-specific primers revealed the presence of a sequence 100% identical to that of Methylibium petroleiphilum PM1, a well-characterized MTBE degrader. However, neither expression of the mdpA genes encoding the alkane hydroxylase-like enzyme responsible for MTBE oxidation in this strain nor the related MTBE isotope fractionation pattern produced by PM1 could be detected, suggesting that this enzyme was not active in this system. Additionally, observed low inverse fractionation of carbon (ɛC of +0.11‰ ± 0.03‰) and low fractionation of hydrogen (ɛH of −5‰ ± 1‰) in laboratory experiments simulating MTBE stripping from an open surface water body suggest that the application of CSIA in field investigations to detect biodegradation may lead to false-negative results when volatilization effects coincide with the activity of low-fractionating enzymes. As shown in this study, complementary examination of expression of specific catabolic genes can be used as additional direct evidence for microbial degradation activity and may overcome this problem. PMID:21148686

  1. Methane monooxygenase gene expression mediated by methanobactin in the presence of mineral copper sources

    PubMed Central

    Knapp, Charles W.; Fowle, David A.; Kulczycki, Ezra; Roberts, Jennifer A.; Graham, David W.

    2007-01-01

    Methane is a major greenhouse gas linked to global warming; however, patterns of in situ methane oxidation by methane-oxidizing bacteria (methanotrophs), nature's main biological mechanism for methane suppression, are often inconsistent with laboratory predictions. For example, one would expect a strong relationship between methanotroph ecology and Cu level because methanotrophs require Cu to sustain particulate methane monooxygenase (pMMO), the most efficient enzyme for methane oxidation. However, no correlation has been observed in nature, which is surprising because methane monooxygenase (MMO) gene expression has been unequivocally linked to Cu availability. Here we provide a fundamental explanation for this lack of correlation. We propose that MMO expression in nature is largely controlled by solid-phase Cu geochemistry and the relative ability of Cu acquisition systems in methanotrophs, such as methanobactins (mb), to obtain Cu from mineral sources. To test this hypothesis, RT-PCR expression assays were developed for Methylosinus trichosporium OB3b (which produces mb) to quantify pMMO, soluble MMO (the alternate MMO expressed when Cu is “unavailable”), and 16S-rRNA gene expression under progressively more stringent Cu supply conditions. When Cu was provided as CuCl2, pMMO transcript levels increased significantly consistent with laboratory work. However, when Cu was provided as Cu-doped iron oxide, pMMO transcript levels increased only when mb was also present. Finally, when Cu was provided as Cu-doped borosilicate glass, pMMO transcription patterns varied depending on the ambient mb:Cu supply ratio. Cu geochemistry clearly influences MMO expression in terrestrial systems, and, as such, local Cu mineralogy might provide an explanation for methane oxidation patterns in the natural environment. PMID:17615240

  2. Transformation yields of chlorinated ethenes by a methanotrophic mixed culture expressing particulate methane monooxygenase.

    PubMed Central

    Anderson, J E; McCarty, P L

    1997-01-01

    Transformation yields for the aerobic cometabolic degradation of five chlorinated ethenes were determined by using a methanotrophic mixed culture expressing particulate methane monooxygenase (pMMO). Transformation yields (expressed as moles of chlorinated ethene degraded per mole of methane consumed) were 0.57, 0.25, 0.058, 0.0019, and 0.00022 for trans-1,2-dichloroethylene (t-DCE), vinyl chloride (VC), cis-1,2-dichloroethylene (c-DCE), trichloroethylene (TCE), and 1,1-dichloroethylene (1,1-DCE), respectively. Degradation of t-DCE and VC was observed only in the presence of formate or methane, sources of reducing energy necessary for cometabolism. The t-DCE and VC transformation yields represented 35 and 15%, respectively, of the theoretical maximum yields, based on reducing-energy availability from methane dissimilation to carbon dioxide, exclusive of all other processes that require reducing energy. The yields for t-DCE and VC were 20 times greater than the yields reported by others for cells expressing soluble methane monooxygenase (sMMO). Transformation yields for c-DCE, TCE, and 1,1-DCE were similar to or less than those for cultures expressing sMMO. Although methanotrophic biotreatment systems have typically been designed to incorporate cultures expressing sMMO, these results suggest that pMMO expression may be highly advantageous for degradation of t-DCE or VC. It may also be much easier to maintain pMMO expression in treatment systems, because pMMO is expressed by all methanotrophs whereas sMMO is expressed only by type II methanotrophs under copper-limited conditions. PMID:9023946

  3. Soluble expression and purification of the oxidoreductase component of toluene 4-monooxygenase.

    PubMed

    Bailey, Lucas J; Elsen, Nathaniel L; Pierce, Brad S; Fox, Brian G

    2008-01-01

    Toluene 4-monooxygenase (T4MO) is a member of the bacterial multicomponent monooxygenases, an enzyme family that utilizes a soluble diiron hydroxylase to oxidize a variety of hydrocarbons as the initial step in their metabolism. The hydroxylases obtain reducing equivalents from NAD(P)H via an electron transfer chain that is initiated by an oxidoreductase containing an N-terminal ferredoxin domain and C-terminal flavin- and NAD-binding domains. T4moF, the NADH oxidoreductase of T4MO, was expressed as a soluble protein in Escherichia coli BL21(DE3) from the pUC-derived expression vector pRS205. This vector contains a lac promoter instead of a T7 promoter. A three step purification from the soluble cell lysate yielded approximately 1 mg of T4moF per gram of wet cell paste with greater than 90% purity. The purified protein contained 1 mol of FAD and 2 mol of Fe per mol of T4moF; quantitative EPR spectroscopy showed approximately 1 mol of the S=1/2 signal from the reduced [2Fe-2S] cluster per mol of T4moF. Steady state kinetic analysis of p-cresol formation activity treating T4moF as the variable substrate while all other proteins and substrates were held constant gave apparent K(M-) and apparent k(cat)-values of 0.15 microM and 3.0 s(-1), respectively. This expression system and purification allows for the recovery of the soluble oxidoreductase in yields that facilitate further biochemical and structural characterizations. PMID:17964805

  4. Insights into the different dioxygen activation pathways of methane and toluene monooxygenase hydroxylases.

    PubMed

    Bochevarov, Arteum D; Li, Jianing; Song, Woon Ju; Friesner, Richard A; Lippard, Stephen J

    2011-05-18

    The methane and toluene monooxygenase hydroxylases (MMOH and TMOH, respectively) have almost identical active sites, yet the physical and chemical properties of their oxygenated intermediates, designated P*, H(peroxo), Q, and Q* in MMOH and ToMOH(peroxo) in a subclass of TMOH, ToMOH, are substantially different. We review and compare the structural differences in the vicinity of the active sites of these enzymes and discuss which changes could give rise to the different behavior of H(peroxo) and Q. In particular, analysis of multiple crystal structures reveals that T213 in MMOH and the analogous T201 in TMOH, located in the immediate vicinity of the active site, have different rotatory configurations. We study the rotational energy profiles of these threonine residues with the use of molecular mechanics (MM) and quantum mechanics/molecular mechanics (QM/MM) computational methods and put forward a hypothesis according to which T213 and T201 play an important role in the formation of different types of peroxodiiron(III) species in MMOH and ToMOH. The hypothesis is indirectly supported by the QM/MM calculations of the peroxodiiron(III) models of ToMOH and the theoretically computed Mössbauer spectra. It also helps explain the formation of two distinct peroxodiiron(III) species in the T201S mutant of ToMOH. Additionally, a role for the ToMOD regulatory protein, which is essential for intermediate formation and protein functioning in the ToMO system, is advanced. We find that the low quadrupole splitting parameter in the Mössbauer spectrum observed for a ToMOH(peroxo) intermediate can be explained by protonation of the peroxo moiety, possibly stabilized by the T201 residue. Finally, similarities between the oxygen activation mechanisms of the monooxygenases and cytochrome P450 are discussed. PMID:21517016

  5. Flavin-dependent monooxygenases as a detoxification mechanism in insects: new insights from the arctiids (lepidoptera).

    PubMed

    Sehlmeyer, Sven; Wang, Linzhu; Langel, Dorothee; Heckel, David G; Mohagheghi, Hoda; Petschenka, Georg; Ober, Dietrich

    2010-01-01

    Insects experience a wide array of chemical pressures from plant allelochemicals and pesticides and have developed several effective counterstrategies to cope with such toxins. Among these, cytochrome P450 monooxygenases are crucial in plant-insect interactions. Flavin-dependent monooxygenases (FMOs) seem not to play a central role in xenobiotic detoxification in insects, in contrast to mammals. However, the previously identified senecionine N-oxygenase of the arctiid moth Tyria jacobaeae (Lepidoptera) indicates that FMOs have been recruited during the adaptation of this insect to plants that accumulate toxic pyrrolizidine alkaloids. Identification of related FMO-like sequences of various arctiids and other Lepidoptera and their combination with expressed sequence tag (EST) data and sequences emerging from the Bombyx mori genome project show that FMOs in Lepidoptera form a gene family with three members (FMO1 to FMO3). Phylogenetic analyses suggest that FMO3 is only distantly related to lepidopteran FMO1 and FMO2 that originated from a more recent gene duplication event. Within the FMO1 gene cluster, an additional gene duplication early in the arctiid lineage provided the basis for the evolution of the highly specific biochemical, physiological, and behavioral adaptations of these butterflies to pyrrolizidine-alkaloid-producing plants. The genes encoding pyrrolizidine-alkaloid-N-oxygenizing enzymes (PNOs) are transcribed in the fat body and the head of the larvae. An N-terminal signal peptide mediates the transport of the soluble proteins into the hemolymph where PNOs efficiently convert pro-toxic pyrrolizidine alkaloids into their non-toxic N-oxide derivatives. Heterologous expression of a PNO of the generalist arctiid Grammia geneura produced an N-oxygenizing enzyme that shows noticeably expanded substrate specificity compared with the related enzyme of the specialist Tyria jacobaeae. The data about the evolution of FMOs within lepidopteran insects and the

  6. Crystal Structure of Albaflavenone Monooxygenase Containing a Moonlighting Terpene Synthase Active Site

    SciTech Connect

    Zhao, Bin; Lei, Li; Vassylyev, Dmitry G.; Lin, Xin; Cane, David E.; Kelly, Steven L.; Yuan, Hang; Lamb, David C.; Waterman, Michael R.

    2010-01-08

    Albaflavenone synthase (CYP170A1) is a monooxygenase catalyzing the final two steps in the biosynthesis of this antibiotic in the soil bacterium, Streptomyces coelicolor A3(2). Interestingly, CYP170A1 shows no stereo selection forming equal amounts of two albaflavenol epimers, each of which is oxidized in turn to albaflavenone. To explore the structural basis of the reaction mechanism, we have studied the crystal structures of both ligand-free CYP170A1 (2.6 {angstrom}) and complex of endogenous substrate (epi-isozizaene) with CYP170A1 (3.3 {angstrom}). The structure of the complex suggests that the proximal epi-isozizaene molecules may bind to the heme iron in two orientations. In addition, much to our surprise, we have found that albaflavenone synthase also has a second, completely distinct catalytic activity corresponding to the synthesis of farnesene isomers from farnesyl diphosphate. Within the cytochrome P450 {alpha}-helical domain both the primary sequence and x-ray structure indicate the presence of a novel terpene synthase active site that is moonlighting on the P450 structure. This includes signature sequences for divalent cation binding and an {alpha}-helical barrel. This barrel is unusual because it consists of only four helices rather than six found in all other terpene synthases. Mutagenesis establishes that this barrel is essential for the terpene synthase activity of CYP170A1 but not for the monooxygenase activity. This is the first bifunctional P450 discovered to have another active site moonlighting on it and the first time a terpene synthase active site is found moonlighting on another protein.

  7. Eukaryotic formylglycine-generating enzyme catalyses a monooxygenase type of reaction.

    PubMed

    Peng, Jianhe; Alam, Sarfaraz; Radhakrishnan, Karthikeyan; Mariappan, Malaiyalam; Rudolph, Markus Georg; May, Caroline; Dierks, Thomas; von Figura, Kurt; Schmidt, Bernhard

    2015-09-01

    C α-formylglycine (FGly) is the catalytic residue of sulfatases in eukaryotes. It is generated by a unique post-translational modification catalysed by the FGly-generating enzyme (FGE) in the endoplasmic reticulum. FGE oxidizes a cysteine residue within the conserved CxPxR sequence motif of nascent sulfatase polypeptides to FGly. Here we show that this oxidation is strictly dependent on molecular oxygen (O2) and consumes 1 mol O2 per mol FGly formed. For maximal activity FGE requires an O2 concentration of 9% (105 μM). Sustained FGE activity further requires the presence of a thiol-based reductant such as DTT. FGly is also formed in the absence of DTT, but its formation ceases rapidly. Thus inactivated FGE accumulates in which the cysteine pair Cys336/Cys341 in the catalytic site is oxidized to form disulfide bridges between either Cys336 and Cys341 or Cys341 and the CxPxR cysteine of the sulfatase. These results strongly suggest that the Cys336/Cys341 pair is directly involved in the O2 -dependent conversion of the CxPxR cysteine to FGly. The available data characterize eukaryotic FGE as a monooxygenase, in which Cys336/Cys341 disulfide bridge formation donates the electrons required to reduce one oxygen atom of O2 to water while the other oxygen atom oxidizes the CxPxR cysteine to FGly. Regeneration of a reduced Cys336/Cys341 pair is accomplished in vivo by a yet unknown reductant of the endoplasmic reticulum or in vitro by DTT. Remarkably, this monooxygenase reaction utilizes O2 without involvement of any activating cofactor. PMID:26077311

  8. The biochemical mechanism of auxin biosynthesis by an arabidopsis YUCCA flavin-containing monooxygenase.

    PubMed

    Dai, Xinhua; Mashiguchi, Kiyoshi; Chen, Qingguo; Kasahara, Hiroyuki; Kamiya, Yuji; Ojha, Sunil; DuBois, Jennifer; Ballou, David; Zhao, Yunde

    2013-01-18

    Auxin regulates every aspect of plant growth and development. Previous genetic studies demonstrated that YUCCA (YUC) flavin-containing monooxygenases (FMOs) catalyze a rate-limiting step in auxin biosynthesis and that YUCs are essential for many developmental processes. We proposed that YUCs convert indole-3-pyruvate (IPA) to indole-3-acetate (IAA). However, the exact biochemical mechanism of YUCs has remained elusive. Here we present the biochemical characterization of recombinant Arabidopsis YUC6. Expressed in and purified from Escherichia coli, YUC6 contains FAD as a cofactor, which has peaks at 448 nm and 376 nm in the UV-visible spectrum. We show that YUC6 uses NADPH and oxygen to convert IPA to IAA. The first step of the YUC6-catalyzed reaction is the reduction of the FAD cofactor to FADH(-) by NADPH. Subsequently, FADH(-) reacts with oxygen to form a flavin-C4a-(hydro)peroxy intermediate, which we show has a maximum absorbance at 381 nm in its UV-visible spectrum. The final chemical step is the reaction of the C4a-intermediate with IPA to produce IAA. Although the sequences of the YUC enzymes are related to those of the mammalian FMOs, which oxygenate nucleophilic substrates, YUC6 oxygenates an electrophilic substrate (IPA). Nevertheless, both classes of enzymes form quasi-stable C4a-(hydro)peroxyl FAD intermediates. The YUC6 intermediate has a half-life of ∼20 s whereas that of some FMOs is >30 min. This work reveals the catalytic mechanism of the first known plant flavin monooxygenase and provides a foundation for further investigating how YUC activities are regulated in plants. PMID:23188833

  9. Form Follows Function: Structural and Catalytic Variation in the Class A Flavoprotein Monooxygenases

    PubMed Central

    Crozier-Reabe, Karen; Moran, Graham R.

    2012-01-01

    Flavoprotein monooxygenases (FPMOs) exhibit an array of mechanistic solutions to a common chemical objective; the monooxygenation of a target substrate. Each FPMO efficiently couples reduction of a flavin cofactor by NAD(P)H to oxygenation of the target substrate via a (hydro)peroxyflavin intermediate. This purpose of this review is to describe in detail the Class A flavoprotein hydroxylases (FPMO) in the context of the other FPMO classes (B–F). Both one and two component FPMOs are found in nature. Two-component enzymes require, in addition to the monooxygenase, the involvement of a reductase that first catalyzes the reduction of the flavin by NAD(P)H. The Class A and B FPMOs are single-component and manage to orchestrate the same net reaction within a single peptide. The Class A enzymes have, by some considerable margin, the most complete research record. These enzymes use choreographed movements of the flavin ring that facilitate access of the organic substrates to the active site, provide a means for interaction of NADPH with the flavin, offer a mechanism to sequester the dioxygen reduction chemistry from solvent and a means to release the product. The majority of the discrete catalytic events of the catalytic cycle can be observed directly in exquisite detail using spectrophotometric kinetic methods and many of the key mechanistic conclusions are further supported by structural data. This review attempts to compile each of the key observations made for both paradigm and newly discovered examples of Class A FPMOs into a complete catalytic description of one enzymatic turnover. PMID:23443084

  10. Influence of kynurenine 3-monooxygenase (KMO) gene polymorphism on cognitive function in schizophrenia✰,✰✰

    PubMed Central

    Wonodi, Ikwunga; McMahon, Robert P.; Krishna, Nithin; Mitchell, Braxton D.; Liu, Judy; Glassman, Matthew; Hong, L. Elliot; Gold, James M.

    2015-01-01

    Background Cognitive deficits compromise quality of life and productivity for individuals with schizophrenia and have no effective treatments. Preclinical data point to the kynurenine pathway of tryptophan metabolism as a potential target for pro-cognitive drug development. We have previously demonstrated association of a kynurenine 3-monooxygenase (KMO) gene variant with reduced KMO gene expression in postmortem schizophrenia cortex, and neurocognitive endophenotypic deficits in a clinical sample. KMO encodes kynurenine 3-monooxygenase (KMO), the rate-limiting microglial enzyme of cortical kynurenine metabolism. Aberration of the KMO gene might be the proximal cause of impaired cortical kynurenine metabolism observed in schizophrenia. However, the relationship between KMO variation and cognitive function in schizophrenia is unknown. This study examined the effects of the KMO rs2275163C>T C (risk) allele on cognitive function in schizophrenia. Methods We examined the association of KMO polymorphisms with general neuropsychological performance and P50 gating in a sample of 150 schizophrenia and 95 healthy controls. Results Consistent with our original report, the KMO rs2275163C>T C (risk) allele was associated with deficits in general neuropsychological performance, and this effect was more marked in schizophrenia compared with controls. Additionally, the C (Arg452) allele of the missense rs1053230C>T variant (KMO Arg452Cys) showed a trend effect on cognitive function. Neither variant affected P50 gating. Conclusions These data suggest that KMO variation influences a range of cognitive domains known to predict functional outcome. Extensive molecular characterization of this gene would elucidate its role in cognitive function with implications for vertical integration with basic discovery. PMID:25464917

  11. Structure and Ligand Binding Properties of the Epoxidase Component of Styrene Monooxygenase

    SciTech Connect

    Ukaegbu, Uchechi E.; Kantz, Auric; Beaton, Michelle; Gassner, George T.; Rosenzweig, Amy C.

    2010-07-23

    Styrene monooxygenase (SMO) is a two-component flavoprotein monooxygenase that transforms styrene to styrene oxide in the first step of the styrene catabolic and detoxification pathway of Pseudomonas putida S12. The crystal structure of the N-terminally histidine-tagged epoxidase component of this system, NSMOA, determined to 2.3 {angstrom} resolution, indicates the enzyme exists as a homodimer in which each monomer forms two distinct domains. The overall architecture is most similar to that of p-hydroxybenzoate hydroxylase (PHBH), although there are some significant differences in secondary structure. Structural comparisons suggest that a large cavity open to the surface forms the FAD binding site. At the base of this pocket is another cavity that likely represents the styrene binding site. Flavin binding and redox equilibria are tightly coupled such that reduced FAD binds apo NSMOA {approx}8000 times more tightly than the oxidized coenzyme. Equilibrium fluorescence and isothermal titration calorimetry data using benzene as a substrate analogue indicate that the oxidized flavin and substrate analogue binding equilibria of NSMOA are linked such that the binding affinity of each is increased by 60-fold when the enzyme is saturated with the other. A much weaker {approx}2-fold positive cooperative interaction is observed for the linked binding equilibria of benzene and reduced FAD. The low affinity of the substrate analogue for the reduced FAD complex of NSMOA is consistent with a preferred reaction order in which flavin reduction and reaction with oxygen precede the binding of styrene, identifying the apoenzyme structure as the key catalytic resting state of NSMOA poised to bind reduced FAD and initiate the oxygen reaction.

  12. Isolation of the monooxygenase complex from Rhipicephalus (Boophilus) microplus - clues to understanding acaricide resistance.

    PubMed

    Graham, Kirsty M; Sparagano, Olivier A E; Finn, Robert D

    2016-06-01

    The monooxygenase complex is composed of three key proteins, a cytochrome P450 (CYP), the cytochrome P450 oxidoreductase (CPR) and cytochrome b5 and plays a key role in the metabolism and detoxification of xenobiotic substances, including pesticides. In addition, overexpression of these components has been linked to pesticide resistance in several important vectors of disease. Despite this, the monooxygenase complex has not been isolated from the Southern cattle tick Rhipicephalus (Boophilus) microplus, a major disease vector in livestock. Using bioinformatics 115 transcriptomic sequences were analyzed to identify putative pesticide metabolizing CYPs. RACE-PCR was used to amplify the full length sequence of one CYP; CYP3006G8 which displays a high degree of homology to members of the CYP6 and 9 subfamilies, known to metabolize pyrethroids. mRNA expression levels of CYP3006G8 were investigated in 11 strains of R. microplus with differing resistance profiles by qPCR, the results of which indicated a correlation with pyrethroid metabolic resistance. In addition to this gene, the sequences for CPR and cytochrome b5 were also identified and subsequently isolated from R. microplus using PCR. CYP3006G8 is only the third CYP gene isolated from R. microplus and the first to putatively metabolize pesticides. The initial results of expression analysis suggest that CYP3006G8 metabolizes pyrethroids but further biochemical characterization is required to confirm this. Differences in the kinetic parameters of human and mosquito CPR in terms of NADPH binding have been demonstrated and could potentially be used to design species specific pesticides. Similar differences in the tick CPR would confirm that this is a characteristic of heamatophagous arthropods. PMID:26850353

  13. Monooxygenase, peroxidase and peroxygenase properties and reaction mechanisms of cytochrome P450 enzymes.

    PubMed

    Hrycay, Eugene G; Bandiera, Stelvio M

    2015-01-01

    This review examines the monooxygenase, peroxidase and peroxygenase properties and reaction mechanisms of cytochrome P450 (CYP) enzymes in bacterial, archaeal and mammalian systems. CYP enzymes catalyze monooxygenation reactions by inserting one oxygen atom from O2 into an enormous number and variety of substrates. The catalytic versatility of CYP stems from its ability to functionalize unactivated carbon-hydrogen (C-H) bonds of substrates through monooxygenation. The oxidative prowess of CYP in catalyzing monooxygenation reactions is attributed primarily to a porphyrin π radical ferryl intermediate known as Compound I (CpdI) (Por•+FeIV=O), or its ferryl radical resonance form (FeIV-O•). CYP-mediated hydroxylations occur via a consensus H atom abstraction/oxygen rebound mechanism involving an initial abstraction by CpdI of a H atom from the substrate, generating a highly-reactive protonated Compound II (CpdII) intermediate (FeIV-OH) and a carbon-centered alkyl radical that rebounds onto the ferryl hydroxyl moiety to yield the hydroxylated substrate. CYP enzymes utilize hydroperoxides, peracids, perborate, percarbonate, periodate, chlorite, iodosobenzene and N-oxides as surrogate oxygen atom donors to oxygenate substrates via the shunt pathway in the absence of NAD(P)H/O2 and reduction-oxidation (redox) auxiliary proteins. It has been difficult to isolate the historically elusive CpdI intermediate in the native NAD(P)H/O2-supported monooxygenase pathway and to determine its precise electronic structure and kinetic and physicochemical properties because of its high reactivity, unstable nature (t½~2 ms) and short life cycle, prompting suggestions for participation in monooxygenation reactions of alternative CYP iron-oxygen intermediates such as the ferric-peroxo anion species (FeIII-OO-), ferric-hydroperoxo species (FeIII-OOH) and FeIII-(H2O2) complex. PMID:26002730

  14. Identification of a Novel Self-Sufficient Styrene Monooxygenase from Rhodococcus opacus 1CP▿ †

    PubMed Central

    Tischler, Dirk; Eulberg, Dirk; Lakner, Silvia; Kaschabek, Stefan R.; van Berkel, Willem J. H.; Schlömann, Michael

    2009-01-01

    Sequence analysis of a 9-kb genomic fragment of the actinobacterium Rhodococcus opacus 1CP led to identification of an open reading frame encoding a novel fusion protein, StyA2B, with a putative function in styrene metabolism via styrene oxide and phenylacetic acid. Gene cluster analysis indicated that the highly related fusion proteins of Nocardia farcinica IFM10152 and Arthrobacter aurescens TC1 are involved in a similar physiological process. Whereas 413 amino acids of the N terminus of StyA2B are highly similar to those of the oxygenases of two-component styrene monooxygenases (SMOs) from pseudomonads, the residual 160 amino acids of the C terminus show significant homology to the flavin reductases of these systems. Cloning and functional expression of His10-StyA2B revealed for the first time that the fusion protein does in fact catalyze two separate reactions. Strictly NADH-dependent reduction of flavins and highly enantioselective oxygenation of styrene to (S)-styrene oxide were shown. Inhibition studies and photometric analysis of recombinant StyA2B indicated the absence of tightly bound heme and flavin cofactors in this self-sufficient monooxygenase. StyA2B oxygenates a spectrum of aromatic compounds similar to those of two-component SMOs. However, the specific activities of the flavin-reducing and styrene-oxidizing functions of StyA2B are one to two orders of magnitude lower than those of StyA/StyB from Pseudomonas sp. strain VLB120. PMID:19482928

  15. Escherichia coli Overexpressing a Baeyer-Villiger Monooxygenase from Acinetobacter radioresistens Becomes Resistant to Imipenem

    PubMed Central

    Minerdi, Daniela; Zgrablic, Ivan; Castrignanò, Silvia; Catucci, Gianluca; Medana, Claudio; Terlizzi, Maria Elena; Gribaudo, Giorgio; Gilardi, Gianfranco

    2015-01-01

    Antimicrobial resistance is a global issue currently resulting in the deaths of hundreds of thousands of people a year worldwide. Data present in the literature illustrate the emergence of many bacterial species that display resistance to known antibiotics; Acinetobacter spp. are a good example of this. We report here that Acinetobacter radioresistens has a Baeyer-Villiger monooxygenase (Ar-BVMO) with 100% amino acid sequence identity to the ethionamide monooxygenase of multidrug-resistant (MDR) Acinetobacter baumannii. Both enzymes are only distantly phylogenetically related to other canonical bacterial BVMO proteins. Ar-BVMO not only is capable of oxidizing two anticancer drugs metabolized by human FMO3, danusertib and tozasertib, but also can oxidize other synthetic drugs, such as imipenem. The latter is a member of the carbapenems, a clinically important antibiotic family used in the treatment of MDR bacterial infections. Susceptibility tests performed by the Kirby-Bauer disk diffusion method demonstrate that imipenem-sensitive Escherichia coli BL21 cells overexpressing Ar-BVMO become resistant to this antibiotic. An agar disk diffusion assay proved that when imipenem reacts with Ar-BVMO, it loses its antibiotic property. Moreover, an NADPH consumption assay with the purified Ar-BVMO demonstrates that this antibiotic is indeed a substrate, and its product is identified by liquid chromatography-mass spectrometry to be a Baeyer-Villiger (BV) oxidation product of the carbonyl moiety of the β-lactam ring. This is the first report of an antibiotic-inactivating BVMO enzyme that, while mediating its usual BV oxidation, also operates by an unprecedented mechanism of carbapenem resistance. PMID:26459905

  16. The Toluene o-Xylene Monooxygenase Enzymatic Activity for the Biosynthesis of Aromatic Antioxidants

    PubMed Central

    Pizzo, Elio; Notomista, Eugenio; Pezzella, Alessandro; Di Cristo, Carlo; De Lise, Federica; Di Donato, Alberto; Izzo, Viviana

    2015-01-01

    Monocyclic phenols and catechols are important antioxidant compounds for the food and pharmaceutic industries; their production through biotransformation of low-added value starting compounds is of major biotechnological interest. The toluene o-xylene monooxygenase (ToMO) from Pseudomonas sp. OX1 is a bacterial multicomponent monooxygenase (BMM) that is able to hydroxylate a wide array of aromatic compounds and has already proven to be a versatile biochemical tool to produce mono- and dihydroxylated derivatives of aromatic compounds. The molecular determinants of its regioselectivity and substrate specificity have been thoroughly investigated, and a computational strategy has been developed which allows designing mutants able to hydroxylate non-natural substrates of this enzyme to obtain high-added value compounds of commercial interest. In this work, we have investigated the use of recombinant ToMO, expressed in cells of Escherichia coli strain JM109, for the biotransformation of non-natural substrates of this enzyme such as 2-phenoxyethanol, phthalan and 2-indanol to produce six hydroxylated derivatives. The hydroxylated products obtained were identified, isolated and their antioxidant potential was assessed both in vitro, using the DPPH assay, and on the rat cardiomyoblast cell line H9c2. Incubation of H9c2 cells with the hydroxylated compounds obtained from ToMO-catalyzed biotransformation induced a differential protective effect towards a mild oxidative stress induced by the presence of sodium arsenite. The results obtained confirm once again the versatility of the ToMO system for oxyfunctionalization reactions of biotechnological importance. Moreover, the hydroxylated derivatives obtained possess an interesting antioxidant potential that encourages the use of the enzyme for further functionalization reactions and their possible use as scaffolds to design novel bioactive molecules. PMID:25915063

  17. The Contribution of Non-catalytic Carbohydrate Binding Modules to the Activity of Lytic Polysaccharide Monooxygenases*

    PubMed Central

    Crouch, Lucy I.; Labourel, Aurore; Walton, Paul H.; Davies, Gideon J.; Gilbert, Harry J.

    2016-01-01

    Lignocellulosic biomass is a sustainable industrial substrate. Copper-dependent lytic polysaccharide monooxygenases (LPMOs) contribute to the degradation of lignocellulose and increase the efficiency of biofuel production. LPMOs can contain non-catalytic carbohydrate binding modules (CBMs), but their role in the activity of these enzymes is poorly understood. Here we explored the importance of CBMs in LPMO function. The family 2a CBMs of two monooxygenases, CfLPMO10 and TbLPMO10 from Cellulomonas fimi and Thermobispora bispora, respectively, were deleted and/or replaced with CBMs from other proteins. The data showed that the CBMs could potentiate and, surprisingly, inhibit LPMO activity, and that these effects were both enzyme-specific and substrate-specific. Removing the natural CBM or introducing CtCBM3a, from the Clostridium thermocellum cellulosome scaffoldin CipA, almost abolished the catalytic activity of the LPMOs against the cellulosic substrates. The deleterious effect of CBM removal likely reflects the importance of prolonged presentation of the enzyme on the surface of the substrate for efficient catalytic activity, as only LPMOs appended to CBMs bound tightly to cellulose. The negative impact of CtCBM3a is in sharp contrast with the capacity of this binding module to potentiate the activity of a range of glycoside hydrolases including cellulases. The deletion of the endogenous CBM from CfLPMO10 or the introduction of a family 10 CBM from Cellvibrio japonicus LPMO10B into TbLPMO10 influenced the quantity of non-oxidized products generated, demonstrating that CBMs can modulate the mode of action of LPMOs. This study demonstrates that engineered LPMO-CBM hybrids can display enhanced industrially relevant oxygenations. PMID:26801613

  18. Identification of a flavin-containing S-oxygenating monooxygenase involved in alliin biosynthesis in garlic.

    PubMed

    Yoshimoto, Naoko; Onuma, Misato; Mizuno, Shinya; Sugino, Yuka; Nakabayashi, Ryo; Imai, Shinsuke; Tsuneyoshi, Tadamitsu; Sumi, Shin-ichiro; Saito, Kazuki

    2015-09-01

    S-Alk(en)yl-l-cysteine sulfoxides are cysteine-derived secondary metabolites highly accumulated in the genus Allium. Despite pharmaceutical importance, the enzymes that contribute to the biosynthesis of S-alk-(en)yl-l-cysteine sulfoxides in Allium plants remain largely unknown. Here, we report the identification of a flavin-containing monooxygenase, AsFMO1, in garlic (Allium sativum), which is responsible for the S-oxygenation reaction in the biosynthesis of S-allyl-l-cysteine sulfoxide (alliin). Recombinant AsFMO1 protein catalyzed the stereoselective S-oxygenation of S-allyl-l-cysteine to nearly exclusively yield (RC SS )-S-allylcysteine sulfoxide, which has identical stereochemistry to the major natural form of alliin in garlic. The S-oxygenation reaction catalyzed by AsFMO1 was dependent on the presence of nicotinamide adenine dinucleotide phosphate (NADPH) and flavin adenine dinucleotide (FAD), consistent with other known flavin-containing monooxygenases. AsFMO1 preferred S-allyl-l-cysteine to γ-glutamyl-S-allyl-l-cysteine as the S-oxygenation substrate, suggesting that in garlic, the S-oxygenation of alliin biosynthetic intermediates primarily occurs after deglutamylation. The transient expression of green fluorescent protein (GFP) fusion proteins indicated that AsFMO1 is localized in the cytosol. AsFMO1 mRNA was accumulated in storage leaves of pre-emergent nearly sprouting bulbs, and in various tissues of sprouted bulbs with green foliage leaves. Taken together, our results suggest that AsFMO1 functions as an S-allyl-l-cysteine S-oxygenase, and contributes to the production of alliin both through the conversion of stored γ-glutamyl-S-allyl-l-cysteine to alliin in storage leaves during sprouting and through the de novo biosynthesis of alliin in green foliage leaves. PMID:26345717

  19. Listeria monocytogenes has a functional chitinolytic system and an active lytic polysaccharide monooxygenase.

    PubMed

    Paspaliari, Dafni K; Loose, Jennifer S M; Larsen, Marianne H; Vaaje-Kolstad, Gustav

    2015-03-01

    Chitinases and chitin-active lytic polysaccharide monooxygenases (LPMOs) are most commonly associated with chitin metabolism, but are also reported as virulence factors in pathogenic bacteria. Listeria monocytogenes, a well-known virulent bacterium, possesses two chitinases (ChiA and ChiB) and a multi-modular lytic polysaccharide monooxygenase (LmLPMO10). These enzymes have been related to virulence and their role in chitin metabolism is poorly understood. It is thus of interest to functionally characterize the individual enzymes in order to shed light on their roles in vivo. Our results demonstrate that L. monocytogenes has a fully functional chitinolytic system. Both chitinases show substrate degradation rates similar to those of the nonprocessive endo-chitinase SmChiC from Serratia marcescens. Compared to the S. marcescens LPMO chitin-binding protein CBP21, LmLPMO10 shows a similar rate but different product profiles depending on the substrate. In LPMO-chitinase synergy experiments, CBP21 is able to boost the activity of both ChiA and ChiB more than LmLPMO10. Product analysis of the synergy assays revealed that the chitinases were unable to efficiently hydrolyse the LPMO products (chitooligosaccharide aldonic acids) with a degree of polymerization below four (ChiA and SmChiC) or three (ChiB). Gene transcription and protein expression analysis showed that LmLPMO10 is neither highly transcribed, nor abundantly secreted during the growth of L. monocytogenes in a chitin-containing medium. The chitinases on the other hand are both abundantly secreted in the presence of chitin. Although LmLPMO10 is shown to promote chitin degradation in tandem with the chitinases in vitro, the secretome and transcription data question whether this is the primary role of LmLPMO10 in vivo. PMID:25565565

  20. Leaf-wax n-alkanes record the plant–water environment at leaf flush

    PubMed Central

    Tipple, Brett J.; Berke, Melissa A.; Doman, Christine E.; Khachaturyan, Susanna; Ehleringer, James R.

    2013-01-01

    Leaf-wax n-alkanes 2H/1H ratios are widely used as a proxy in climate reconstruction. Although the broad nature of the relationship between n-alkanes δ2H values and climate is appreciated, the quantitative details of the proxy remain elusive. To examine these details under natural environmental conditions, we studied a riparian broadleaf angiosperm species, Populus angustifolia, growing on water with a constant δ2H value and monitored the δ2H values of leaf-wax n-alkanes and of stem, leaf, stream, and atmospheric waters throughout the entire growing season. Here we found the δ2H values of leaf-wax n-alkanes recorded only a 2-wk period during leaf flush and did not vary for the 19 weeks thereafter when leaves remained active. We found δ2H values of leaf-wax n-alkanes of P. angustifolia record conditions earlier in the season rather than fully integrating the entire growing season. Using these data, we modeled precipitation δ2H values during the time of wax synthesis. We observed that the isotope ratios of this precipitation generally were 2H-enriched compared with mean annual precipitation. This model provides a mechanistic basis of the often-observed 2H-enrichment from the expected fractionation values in studies of broadleaf angiosperm leaf-wax δ2H. In addition, these findings may have implications for the spatial and temporal uses of n-alkane δ2H values in paleoapplications; when both plant community and growth form are known, this study allows the isolation of the precipitation dynamics of individual periods of the growing season. PMID:23359675

  1. Structure and Mass Transport Characteristics at the Intrinsic Liquid-Vapor Interfaces of Alkanes.

    PubMed

    Chilukoti, Hari Krishna; Kikugawa, Gota; Ohara, Taku

    2016-07-28

    In this paper, an instantaneous interface definition has been used to study the intrinsic structure and self-diffusion coefficient in the vicinity of the liquid-vapor interfaces of decane and tetracosane at three different temperatures using molecular dynamics simulations, and the results have been compared with those obtained on the basis of the conventional Gibbs dividing surface (time- and space-averaged interface). The alkane molecules were modeled using the united atom NERD force field. Partial layered structures of alkane molecules at the liquid-vapor interface are observed as a pinned structure of alkane liquids based on the intrinsic interface. This kind of characteristic has not been observed in the density profiles obtained based on the Gibbs dividing surface. By examining the orientation order parameter and radius of gyration of the alkane molecules, it was observed that the alkane molecules were preferentially oriented to be more parallel to the intrinsic interface than to the Gibbs dividing surface, and the shape of the alkane molecules is slightly changed in the vicinity of the liquid-vapor interfaces. The self-diffusion coefficient parallel to the intrinsic interface was examined using the Green-Kubo relation, where the projection of the velocity in the parallel direction to the local intrinsic interface is used in the velocity correlation function. It was found that the self-diffusion coefficient in the direction parallel to the intrinsic interface changes as the position approaches the interface in a more obvious manner as compared with the self-diffusion coefficient obtained with respect to the Gibbs dividing surface. These results suggest that the use of an instantaneous interface definition allowed us to capture sharp variations in transport properties which are originating due to steeper structure at the liquid-vapor interfaces. PMID:27387788

  2. Isomerization of alkanes on sulfated zirconia: Promotion by Pt and by adamantyl hydride transfer species

    SciTech Connect

    Iglesia, E.; Soled, S.L.; Kramer, G.M. )

    1993-11-01

    The work shows that hydride transfer species, such as adamantane, increase isomerization rates and inhibit C-C scission reactions. n-Heptane isomerization rates show positive hydrogen kinetic orders, suggesting that the reaction proceeds on Pt/ZrO[sub 2]-SO[sub 4] via chain transfer pathways, in which carbenium ions propagate, after a chain initiation step involvings loss of hydrogen from alkanes, by hydride transfer from neutral species to carbonations. These pathways contrast with those involved in the bifunctional (metal-acid) catalytic sequences usually required for alkane isomerization, in which metal sites catalyze alkane dehydrogenation and acid sites catalyze skeletal rearrangements of alkenes. Rate-limiting hydride transfer steps are consistent with the strong influence of molecular hydride transfer agents such as adamantane, which act as co-catalysts and increase isomerization rate and selectivity. The addition of small amounts of adamantane (0.1-0.8 wt%) to n-heptane increases isomerizations rates by a factor of 3 and inhibits undesirable cracking reactions. Adamantane increases hydride transfer and carbenium ion termination rates, thus reducing the surface residence time required for a catalytic turnover. As a result, desorption occurs before secondary cracking of isomerized carbenium ions. Less effective hydride transfer agents (n-alkanes, isoalkanes) also increase n-alkanes isomerization rate and selectivity, but require much higher concentrations than adamantane. Dihydrogen also acts as a hydride source in alkane isomerization catalysis, but it requires the additional presence of metals or reducible oxides, which catalyze H[sub 2] dissociation and the formation of hydridic and protonic forms of hydrogen. 40 refs., 10 figs., 4 tabs.

  3. 40 CFR 721.10103 - Naphtha (Fischer-Tropsch), C4-11-alkane, branched and linear.

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 32 2012-07-01 2012-07-01 false Naphtha (Fischer-Tropsch), C4-11... Significant New Uses for Specific Chemical Substances § 721.10103 Naphtha (Fischer-Tropsch), C4-11-alkane... substance identified as naphtha (fischer-tropsch), C4-11-alkane, branched and linear (PMN P-04-235; CAS...

  4. 40 CFR 721.10103 - Naphtha (Fischer-Tropsch), C4-11-alkane, branched and linear.

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Naphtha (Fischer-Tropsch), C4-11... Significant New Uses for Specific Chemical Substances § 721.10103 Naphtha (Fischer-Tropsch), C4-11-alkane... substance identified as naphtha (fischer-tropsch), C4-11-alkane, branched and linear (PMN P-04-235; CAS...

  5. 40 CFR 721.10103 - Naphtha (Fischer-Tropsch), C4-11-alkane, branched and linear.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 31 2011-07-01 2011-07-01 false Naphtha (Fischer-Tropsch), C4-11... Significant New Uses for Specific Chemical Substances § 721.10103 Naphtha (Fischer-Tropsch), C4-11-alkane... substance identified as naphtha (fischer-tropsch), C4-11-alkane, branched and linear (PMN P-04-235; CAS...

  6. 40 CFR 721.10103 - Naphtha (Fischer-Tropsch), C4-11-alkane, branched and linear.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Naphtha (Fischer-Tropsch), C4-11... Significant New Uses for Specific Chemical Substances § 721.10103 Naphtha (Fischer-Tropsch), C4-11-alkane... substance identified as naphtha (fischer-tropsch), C4-11-alkane, branched and linear (PMN P-04-235; CAS...

  7. 40 CFR 721.10103 - Naphtha (Fischer-Tropsch), C4-11-alkane, branched and linear.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 30 2010-07-01 2010-07-01 false Naphtha (Fischer-Tropsch), C4-11... Significant New Uses for Specific Chemical Substances § 721.10103 Naphtha (Fischer-Tropsch), C4-11-alkane... substance identified as naphtha (fischer-tropsch), C4-11-alkane, branched and linear (PMN P-04-235; CAS...

  8. 40 CFR 721.10625 - Distillation bottoms, alkylated benzene by-product, brominated and bromo diphenyl alkane (generic).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Distillation bottoms, alkylated... Distillation bottoms, alkylated benzene by-product, brominated and bromo diphenyl alkane (generic). (a... generically as distillation bottoms, alkylated benzene by-product, brominated and bromo diphenyl alkane...

  9. 40 CFR 721.10625 - Distillation bottoms, alkylated benzene by-product, brominated and bromo diphenyl alkane (generic).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Distillation bottoms, alkylated... Distillation bottoms, alkylated benzene by-product, brominated and bromo diphenyl alkane (generic). (a... generically as distillation bottoms, alkylated benzene by-product, brominated and bromo diphenyl alkane...

  10. Regioselective alkane hydroxylation with a mutant CYP153A6 enzyme

    DOEpatents

    Koch, Daniel J.; Arnold, Frances H.

    2013-01-29

    Cytochrome P450 CYP153A6 from Myobacterium sp. strain HXN1500 was engineered using in-vivo directed evolution to hydroxylate small-chain alkanes regioselectively. Mutant CYP153A6-BMO1 selectively hydroxylates butane and pentane at the terminal carbon to form 1-butanol and 1-pentanol, respectively, at rates greater than wild-type CYP153A6 enzymes. This biocatalyst is highly active for small-chain alkane substrates and the regioselectivity is retained in whole-cell biotransformations.

  11. Thermodynamic functions of formation of n-alkane complexes with crystalline urea

    SciTech Connect

    Tolmachev, V.V.; Semenov, L.V.; Gaile, A.A.; Proskuryakov, V.A.

    1987-07-10

    For optimization of the conditions of deparaffination of petroleum fractions with the aid of urea, with the composition of the feedstock taken into account, it is important to know the equilibrium constants of formation of complexes of urea with n-alkanes differing in the number of carbon atoms in their molecules, as functions of temperature. In this investigation they obtained experimental data necessary for calculating the thermodynamic functions of formation of n-alkane complexes with crystalline urea up to the decomposition temperature, using Kirchhoff's equations.

  12. Monocarboxylic acids from oxidation of acyclic isoprenoid alkanes by Mycobacterium fortuitum

    NASA Technical Reports Server (NTRS)

    Cox, R. E.; Maxwell, J. R.; Myers, R. N.

    1976-01-01

    Mycobacterium fortuitum utilizes certain stereoisomeric mixtures of individual multimethyl branched alkanes as sole carbon source, including 2,6(R), 10(S), 14(RS)-tetramethylhexadecane; 2,6(R), 10(S), 14(RS)-tetramethylheptadecane; 2,6(RS), 10(RS)-trimethyltetradecane, and 2,6(R), 10(S)-trimethylpentadecane. Products of oxidation isolated from the bacterial lipids were acids derived predominantly from oxidation of the isopropyl terminus of each alkane, except in the case of 2,6(RS), 10(RS)-trimethyltetradecane. With the latter, acids from oxidation at either terminus were detected in comparable proportions.

  13. Mass effect on the Soret coefficient in n-alkane mixtures.

    PubMed

    Alonso de Mezquia, David; Bou-Ali, M Mounir; Madariaga, J Antonio; Santamaría, Carlos

    2014-02-28

    We have determined the Soret coefficient of different equimolar and non equimolar n-alkane mixtures from measurements of the molecular diffusion and thermal diffusion coefficients. It is shown that equimolar mixtures behave as isotopic-like mixtures in which only the mass effect contributes to the Soret effect. In non equimolar mixtures, a small linear dependence with the molar fraction is observed. Finally, we have obtained a new correlation, which allows the determination of the Soret coefficient of n-alkane mixtures using the data of viscosity, the thermal expansion coefficient of the pure components, and the density of the equimolar mixture. PMID:24588181

  14. Determination of n-alkane content in middle and heavy distillates by gas chromatography

    SciTech Connect

    Fadeev, V.S.; Shteingardt, N.S.

    1987-07-01

    The authors have modified a procedure of determination of n-alkane content in middle and heavy distillates by gas chromatography. The zeolite is replaced by a surface-layer absorbent consisting of grains of diatomite on which there has been deposited a surface layer of MgA zeolite particles, and the helium is replaced by hydrogen. A special chromatograph attachment is described and the chromatograms are calculated on the basis of the heights or areas of the peaks of the charge and the impurity hydrocarbons which are not n-alkanes.

  15. Detailed chemical kinetic models for large n-alkanes and iso-alkanes found in conventional and F-T diesel fuels

    SciTech Connect

    Westbrook, C K; Pitz, W J; Mehl, M; Curran, H J

    2009-03-09

    n-Hexadecane and 2,2,4,4,6,8,8-heptamethylnonane represent the primary reference fuels for diesel that are used to determine cetane number, a measure of the ignition property of diesel fuel. With the development of chemical kinetics models for both primary reference fuels, a new capability is now available to model diesel fuel ignition. Additionally, we have developed chemical kinetic models for a whole series of large n-alkanes and a large iso-alkane to represent these chemical classes in fuel surrogates for conventional and future fuels. These chemical kinetic models are used to predict the effect of the aforementioned fuel components on ignition characteristics under conditions found in internal combustion engines.

  16. Enzymatic formation of apo-carotenoids from the xanthophyll carotenoids lutein, zeaxanthin and b-cryptoxanthin by ferret carotene-9, 10-monooxygenase

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Xanthophyll carotenoids, such as lutein, zeaxanthin and b-cryptoxanthin, may provide potential health benefits against chronic and degenerative diseases. Investigating pathways of xanthophyll metabolism are important to understanding their biological functions. Carotene-15,150-monooxygenase (CMO1) h...

  17. Enhanced translocation and growth of Rhodococcus erythropolis PR4 in the alkane phase of aqueous-alkane two phase cultures were mediated by GroEL2 overexpression.

    PubMed

    Takihara, Hayato; Ogihara, Jun; Yoshida, Takao; Okuda, Shujiro; Nakajima, Mutsuyasu; Iwabuchi, Noriyuki; Sunairi, Michio

    2014-01-01

    We previously reported that R. erythropolis PR4 translocated from the aqueous to the alkane phase, and then grew in two phase cultures to which long-chain alkanes had been added. This was considered to be beneficial for bioremediation. In the present study, we investigated the proteins involved in the translocation of R. erythropolis PR4. The results of our proteogenomic analysis suggested that GroEL2 was upregulated more in cells that translocated inside of the pristane (C19) phase than in those located at the aqueous-alkane interface attached to the n-dodecane (C12) surface. PR4 (pK4-EL2-1) and PR4 (pK4-ΔEL2-1) strains were constructed to confirm the effects of the upregulation of GroEL2 in translocated cells. The expression of GroEL2 in PR4 (pK4-EL2-1) was 15.5-fold higher than that in PR4 (pK4-ΔEL2-1) in two phase cultures containing C12. The growth and cell surface lipophilicity of PR4 were enhanced by the introduction of pK4-EL2-1. These results suggested that the plasmid overexpression of groEL2 in PR4 (pK4-EL2-1) led to changes in cell localization, enhanced growth, and increased cell surface lipophilicity. Thus, we concluded that the overexpression of GroEL2 may play an important role in increasing the organic solvent tolerance of R. erythropolis PR4 in aqueous-alkane two phase cultures. PMID:25311591

  18. Adiabatic Coupling Constant of Nitrobenzene- n-Alkane Critical Mixtures. Evidence from Ultrasonic Spectra and Thermodynamic Data

    NASA Astrophysics Data System (ADS)

    Mirzaev, Sirojiddin Z.; Kaatze, Udo

    2016-09-01

    Ultrasonic spectra of mixtures of nitrobenzene with n-alkanes, from n-hexane to n-nonane, are analyzed. They feature up to two Debye-type relaxation terms with discrete relaxation times and, near the critical point, an additional relaxation term due to the fluctuations in the local concentration. The latter can be well represented by the dynamic scaling theory. Its amplitude parameter reveals the adiabatic coupling constant of the mixtures of critical composition. The dependence of this thermodynamic parameter upon the length of the n-alkanes corresponds to that of the slope in the pressure dependence of the critical temperature and is thus taken another confirmation of the dynamic scaling model. The change in the variation of the coupling constant and of several other mixture parameters with alkane length probably reflects a structural change in the nitrobenzene- n-alkane mixtures when the number of carbon atoms per alkane exceeds eight.

  19. Changes in iso- and n-alkane distribution during biodegradation of crude oil under nitrate and sulphate reducing conditions.

    PubMed

    Hasinger, Marion; Scherr, Kerstin E; Lundaa, Tserennyam; Bräuer, Leopold; Zach, Clemens; Loibner, Andreas Paul

    2012-02-20

    Crude oil consists of a large number of hydrocarbons with different susceptibility to microbial degradation. The influence of hydrocarbon structure and molecular weight on hydrocarbon biodegradation under anaerobic conditions is not fully explored. In this study oxygen, nitrate and sulphate served as terminal electron acceptors (TEAs) for the microbial degradation of a paraffin-rich crude oil in a freshly contaminated soil. During 185 days of incubation, alkanes from n-C11 to n-C39, three n- to iso-alkane ratios commonly used as weathering indicators and the unresolved complex mixture (UCM) were quantified and statistically analyzed. The use of different TEAs for hydrocarbon degradation resulted in dissimilar degradative patterns for n- and iso-alkanes. While n-alkane biodegradation followed well-established patterns under aerobic conditions, lower molecular weight alkanes were found to be more recalcitrant than mid- to high-molecular weight alkanes under nitrate-reducing conditions. Biodegradation with sulphate as the TEA was most pronounced for long-chain (n-C32 to n-C39) alkanes. The observation of increasing ratios of n-C17 to pristane and of n-C18 to phytane provides first evidence of the preferential degradation of branched over normal alkanes under sulphate reducing conditions. The formation of distinctly different n- and iso-alkane biodegradation fingerprints under different electron accepting conditions may be used to assess the occurrence of specific degradation processes at a contaminated site. The use of n- to iso-alkane ratios for this purpose may require adjustment if applied for anaerobic sites. PMID:22001845

  20. 40 CFR 721.10148 - Acryloxy alkanoic alkane derivative with mixed metal oxides (generic).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... with mixed metal oxides (generic). 721.10148 Section 721.10148 Protection of Environment ENVIRONMENTAL... mixed metal oxides (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as acryloxy alkanoic alkane derivative with mixed...

  1. 40 CFR 721.10148 - Acryloxy alkanoic alkane derivative with mixed metal oxides (generic).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... with mixed metal oxides (generic). 721.10148 Section 721.10148 Protection of Environment ENVIRONMENTAL... mixed metal oxides (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as acryloxy alkanoic alkane derivative with mixed...

  2. A Microporous Metal-Organic Framework for Gas Chromatographic Separation of Alkanes

    SciTech Connect

    Chen, Banglin; Liang, Chengdu; Yang, Jun; Contreras, Damacio; Clancy, Yvette; Lobkovsky, Emil B.; Yaghi, Omar; Dai, Sheng

    2006-01-01

    A zinc-based metal-organic framework (MOF) can be transformed reversibly from an open (a) to a dense (b) configuration. The microporous solid is the first example of a MOF that is highly selective in the gas-chromatographic separation of alkanes.

  3. Modeling the influence of alkane molecular structure on secondary organic aerosol formation.

    PubMed

    Aumont, Bernard; Camredon, Marie; Mouchel-Vallon, Camille; La, Stéphanie; Ouzebidour, Farida; Valorso, Richard; Lee-Taylor, Julia; Madronich, Sasha

    2013-01-01

    Secondary Organic Aerosols (SOA) production and ageing is a multigenerational oxidation process involving the formation of successive organic compounds with higher oxidation degree and lower vapor pressure. Intermediate Volatility Organic Compounds (IVOC) emitted to the atmosphere are expected to be a substantial source of SOA. These emitted IVOC constitute a complex mixture including linear, branched and cyclic alkanes. The explicit gas-phase oxidation mechanisms are here generated for various linear and branched C10-C22 alkanes using the GECKO-A (Generator for Explicit Chemistry and Kinetics of Organics in the Atmosphere) and SOA formation is investigated for various homologous series. Simulation results show that both the size and the branching of the carbon skeleton are dominant factors driving the SOA yield. However, branching appears to be of secondary importance for the particle oxidation state and composition. The effect of alkane molecular structure on SOA yields appears to be consistent with recent laboratory observations. The simulated SOA composition shows, however, an unexpected major contribution from multifunctional organic nitrates. Most SOA contributors simulated for the oxidation of the various homologous series are far too reduced to be categorized as highly oxygenated organic aerosols (OOA). On a carbon basis, the OOA yields never exceeded 10% regardless of carbon chain length, molecular structure or ageing time. This version of the model appears clearly unable to explain a large production of OOA from alkane precursors. PMID:24600999

  4. Stable isotopic studies of n-alkane metabolism by a sulfate-reducing bacterial enrichment culture.

    PubMed

    Davidova, Irene A; Gieg, Lisa M; Nanny, Mark; Kropp, Kevin G; Suflita, Joseph M

    2005-12-01

    Gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy were used to study the metabolism of deuterated n-alkanes (C6 to C12) and 1-13C-labeled n-hexane by a highly enriched sulfate-reducing bacterial culture. All substrates were activated via fumarate addition to form the corresponding alkylsuccinic acid derivatives as transient metabolites. Formation of d14-hexylsuccinic acid in cell extracts from exogenously added, fully deuterated n-hexane confirmed that this reaction was the initial step in anaerobic alkane metabolism. Analysis of resting cell suspensions amended with 1-13C-labeled n-hexane confirmed that addition of the fumarate occurred at the C-2 carbon of the parent substrate. Subsequent metabolism of hexylsuccinic acid resulted in the formation of 4-methyloctanoic acid, and 3-hydroxy-4-methyloctanoic acid was tentatively identified. We also found that 13C nuclei from 1-13C-labeled n-hexane became incorporated into the succinyl portion of the initial metabolite in a manner that indicated that 13C-labeled fumarate was formed and recycled during alkane metabolism. Collectively, the findings obtained with a sulfate-reducing culture using isotopically labeled alkanes augment and support the previously proposed pathway (H. Wilkes, R. Rabus, T. Fischer, A. Armstroff, A. Behrends, and F. Widdel, Arch. Microbiol. 177:235-243, 2002) for metabolism of deuterated n-hexane by a denitrifying bacterium. PMID:16332800

  5. Biosurfactant-mediated biodegradation of straight and methyl-branched alkanes by Pseudomonas aeruginosa ATCC 55925

    PubMed Central

    2011-01-01

    Accidental oil spills and waste disposal are important sources for environmental pollution. We investigated the biodegradation of alkanes by Pseudomonas aeruginosa ATCC 55925 in relation to a rhamnolipid surfactant produced by the same bacterial strain. Results showed that the linear C11-C21 compounds in a heating oil sample degraded from 6% to 100%, whereas the iso-alkanes tended to be recalcitrant unless they were exposed to the biosurfactant; under such condition total biodegradation was achieved. Only the biodegradation of the commercial C12-C19 alkanes could be demonstrated, ranging from 23% to 100%, depending on the experimental conditions. Pristane (a C19 branched alkane) only biodegraded when present alone with the biosurfactant and when included in an artificial mixture even without the biosurfactant. In all cases the biosurfactant significantly enhanced biodegradation. The electron scanning microscopy showed that cells depicted several adaptations to growth on hydrocarbons, such as biopolymeric spheres with embedded cells distributed over different layers on the spherical surfaces and cells linked to each other by extracellular appendages. Electron transmission microscopy revealed transparent inclusions, which were associated with hydrocarbon based-culture cells. These patterns of hydrocarbon biodegradation and cell adaptations depended on the substrate bioavailability, type and length of hydrocarbon. PMID:21906343

  6. Determination of Alkane Content in Fresh Fecal Samples to Estimate Intake on Pasture

    Technology Transfer Automated Retrieval System (TEKTRAN)

    External markers of wax alkanes C32 and C36 are effective tools for determining intake of grazing animals. The technique requires daily dosing of markers which is impractical under extensive grazing conditions, so controlled release capsules (CRC) have been used. However, consistency of payout from ...

  7. Ordered Self-assembled Alkane Monolayer on Graphite and Graphene Surface

    NASA Astrophysics Data System (ADS)

    Su, Yudan; Han, Huiling; Wang, Feng; Cai, Qun; Tian, Chuanshan; Shen, Y. R.

    2015-03-01

    The 2D self-assembly of long chain alkane molecule on graphite and graphene had been studied with phase-sensitive sum-frequency vibrational spectroscopy (PS-SFVS) and scanning tunneling microscopy (STM). The spectrum of Imχs(2) (ωIR) which directly characterizes the surface resonances, shows 10-cm-1 red-shift of the symmetric-stretch frequency of the CH2 groups pointing towards graphite (or graphene) surface indicating Van der Waals interaction in between. The Gibbs adsorption energy of polyethylene (PE, n ~ 140) on graphite from chloroform solution was determined to be -42kJ/mol per molecule or -0.6 kJ/mol per CH2 unit. This large adsorption energy drives the long alkane chain to form an ordered self-assembled monolayer on graphite (or graphene). The sum frequency spectra suggest the orientation of carbon skeleton plane of alkane is predominately perpendicular to the graphite/graphene surface. Our STM result also provides clear evidence for the proposed molecular adsorption model. These results explain the large amount residual of long chain alkane on polystyrene (PS) or poly(methyl methacrylate) (PMMA) transferred graphene, and facilitate a better way to fabricate cleaner large-size graphene.

  8. Cool-flame Extinction During N-Alkane Droplet Combustion in Microgravity

    NASA Technical Reports Server (NTRS)

    Nayagam, Vedha; Dietrich, Daniel L.; Hicks, Michael C.; Williams, Forman A.

    2014-01-01

    Recent droplet combustion experiments onboard the International Space Station (ISS) have revealed that large n-alkane droplets can continue to burn quasi-steadily following radiative extinction in a low-temperature regime, characterized by negative-temperaturecoefficient (NTC) chemistry. In this study we report experimental observations of n-heptane, n-octane, and n-decane droplets of varying initial sizes burning in oxygen/nitrogen/carbon dioxide and oxygen/helium/nitrogen environments at 1.0, 0.7, and 0.5 atmospheric pressures. The oxygen concentration in these tests varied in the range of 14% to 25% by volume. Large n-alkane droplets exhibited quasi-steady low-temperature burning and extinction following radiative extinction of the visible flame while smaller droplets burned to completion or disruptively extinguished. A vapor-cloud formed in most cases slightly prior to or following the "cool flame" extinction. Results for droplet burning rates in both the hot-flame and cool-flame regimes as well as droplet extinction diameters at the end of each stage are presented. Time histories of radiant emission from the droplet captured using broadband radiometers are also presented. Remarkably the "cool flame" extinction diameters for all the three n-alkanes follow a trend reminiscent of the ignition delay times observed in previous studies. The similarities and differences among the n-alkanes during "cool flame" combustion are discussed using simplified theoretical models of the phenomenon

  9. Improved GC/MS method for quantitation of n-Alkanes in plant and fecal material

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A gas chromatography-mass spectrometry (GC/MS) method for the quantitation of n-alkanes (carbon backbones ranging from 21 to 36 carbon atoms) in forage and fecal samples has been developed. Automated solid-liquid extraction using elevated temperature and pressure minimized extraction time to 30 min...

  10. Petrodiesel-like straight chain alkane and fatty alcohol production by the microalga Chlorella sorokiniana.

    PubMed

    Yang, Xuewei; Dai, Xin; Guo, Hui; Geng, Shu; Wang, Gangyi

    2013-05-01

    This study was to investigate the composition and characteristics of long-straight chain alkane and fatty alcohols from the microalga Chlorella Sorokiniana 21, isolated from the coastal water of Pearl River Delta, China. Under the optimized aeration growth condition, this strain yielded up to 1.44 g L(-1) biomass and 24.90% extracts of dry weight. The major compounds of the extracts were identified to be alkanes (35.93%) and alcohols (53.73%). Of the extracts, long-straight chain alkanes accounted for 30.54% with heptadecane (21.13%) as a predominant component. Furthermore, a large amount of fatty alcohols (53.73%) were identified in the algal extracts with 29.09% of 3-(2-methoxyethyl)-1-nonanol. Thus, this algal species is a promising feedstock for the production of supplement for petrodiesel-like fuels and biochemicals used in the cosmetics and food industries. This study represents the first report of long-straight chain alkane and fatty alcohols from microalgae isolated from coastal water of the region. PMID:23567672

  11. Anaerobic biodegradation of long-chain n-alkanes under sulfate-reducing conditions

    SciTech Connect

    Caldwell, M.E.; Suflita, J.M.; Garrett, R.M.; Prince, R.C.

    1998-07-15

    The ability of anaerobic microorganisms to degrade a wide variety of crude oil components was investigated using chronically hydrocarbon-contaminated marine sediments as the source of inoculum. When sulfate reduction was the predominant electron-accepting process, gas chromatographic analysis revealed almost complete n-alkane removal (C{sub 15}-C{sub 34}) from a weathered oil within 201 d of incubation. No alteration of the oil was detected in sterile control incubations or when nitrate served as an alternate electron acceptor. The amount of sulfate reduced in the oil-amended nonsterile incubations was more than enough to account for the complete mineralization of the n-alkane fraction of the oil; no loss of this anion was observed in sterile control incubations. The mineralization of the alkanes was confirmed using {sup 14}C-14,15-octacosane (C{sub 28}H{sub 58}), with 97% of the radioactivity recovered as {sup 14}CO{sub 2}. These findings extend the range of hydrocarbons known to be amenable to anaerobic biodegradation. Moreover, the rapid and extensive alteration in the n-alkanes can no longer be considered a defining characteristic of aerobic oil biodegradation processes alone.

  12. Technical Note: n-Alkane lipid biomarkers in loess: post-sedimentary or syn-sedimentary?

    NASA Astrophysics Data System (ADS)

    Zech, M.; Kreutzer, S.; Goslar, T.; Meszner, S.; Krause, T.; Faust, D.; Fuchs, M.

    2012-07-01

    There is an ongoing discussion whether n-alkane biomarkers - and organic matter (OM) from loess in general - reflect a syn-sedimentary paleoenvironmental and paleoclimate signal or whether they are significantly a post-sedimentary feature contaminated by root-derived OM. We present first radiocarbon data for the n-alkane fraction of lipid extracts and for the first time luminescence ages for the Middle to Late Weichselian loess-paleosol sequence of Gleina in Saxony, Germany. Comparison of these biomarker ages with sedimentation ages as assessed by optically stimulated luminescence (OSL) dating shows that one n-alkane sample features a syn-sedimentary age (14C: 29.2 ± 1.4 kyr cal BP versus OSL: 27.3 ± 3.0 kyr). By contrast, the 14C ages derived from the other n-alkane samples are clearly younger (20.3 ± 0.7 kyr cal BP, 22.1 ± 0.7 kyr cal BP and 29.8 ± 1.4 kyr cal BP) than the corresponding OSL ages (26.6 ± 3.1 kyr, 32.0 ± 3.5 kyr and 45.6 ± 5.3 kyr). This finding suggests that a post-sedimentary n-alkane contamination presumably by roots has occurred. In order to estimate the post-sedimentary n-alkane contamination more quantitatively, we applied a 14C mass balance calculation based on the measured pMC (percent modern carbon) values, the calculated syn-sedimentary pMC values and pMC values suspected to reflect likely time points of post-sedimentary contamination (current, modern, 3 kyr, 6 kyr and 9 kyr). Accordingly, current and modern root-contamination would account for up to 7%, a 3 kyr old root-contamination for up to 10%, and an Early and Middle Holocene root-contamination for up to 20% of the total sedimentary n-alkane pool. We acknowledge and encourage that these first radiocarbon results need further confirmation both from other loess-paleosol sequences and for different biomarkers, e.g. carboxylic acids or alcohols as further lipid biomarkers.

  13. Investigating C4 Grass Contributions to N-alkane Based Paleoclimate Reconstructions

    NASA Astrophysics Data System (ADS)

    Doman, C. E.; Enders, S. K.; Chadwick, O.; Freeman, K. H.

    2014-12-01

    Plant wax n-alkanes are long-chain, saturated hydrocarbons contained within the protective waxy cuticle on leaves. These lipids are pervasive and persistent in soils and sediments and thus are ideal biomarkers of ancient terrestrial organic matter. In ecosystems dominated by C3 plants, the relationship between the carbon isotopic value of whole leaves and lipids is fairly well documented, but this relationship has not been fully investigated for plants that use C4 photosynthesis. In both cases, it is unclear if the isotopic relationships are sensitive to environmental conditions, or reflect inherited characteristics. This study used a natural climate gradient on the Kohala peninsula of Hawaii to investigate relationships between climate and the δ13C and δ2H values of n-alkanes in C3 and C4 plants. δ13C of C3 leaves and lipids decreased 5 ‰ from the driest to the wettest sites, consistent with published data. Carbon isotope values of C4 plants showed no relationship to moisture up to 1000 mm mean annual precipitation (MAP). Above this threshold, δ 13C values were around 10‰ more depleted, likely due to a combination of canopy effects and C4 grasses growing in an uncharacteristically wet and cold environment. In C3 plants, the fractionation between leaf and lipid carbon isotopes did not vary with MAP, which allows estimations of δ13C leaf to be made from alkanes preserved in ancient sediments. Along this transect, C3 plants produce around twice the quantity of n-alkanes as C4 grasses. C4 grasses produce longer carbon chains. As a result, n-alkanes in the geologic record will be biased towards C3 plants, but the presence of alkanes C33 and C35 indicate the contributions of C4 grasses. In both C3 and C4 plants, average chain length increased with mean annual precipitation, but the taxonomic differences in chain length were greater than environmental differences. Hydrogen isotopes of n-alkanes show no trends with MAP, but do show clear differences between plant

  14. n-Alkane lipid biomarkers in loess: post-sedimentary or syn-sedimentary?

    NASA Astrophysics Data System (ADS)

    Zech, Michael; Kreutzer, Sebastian; Goslar, Tomasz; Meszner, Sascha; Krause, Tobias; Faust, Dominik; Fuchs, Markus

    2013-04-01

    There is an ongoing discussion whether n-alkane biomarkers - and organic matter (OM) from loess in general - reflect a syn-sedimentary paleoenvironmental and paleoclimate signal or whether they are significantly a post-sedimentary feature contaminated by root-derived OM (Zech et al., 2012, 2013; Wiesenberg and Gocke, 2013). We present first radiocarbon data for the n-alkane fraction of lipid extracts and for the first time luminescence ages for the Middle to Late Weichselian loess-paleosol sequence of Gleina in Saxony, Germany. Comparison of these biomarker ages with sedimentation ages as assessed by optically stimulated luminescence (OSL) dating shows that one n-alkane sample features a syn-sedimentary age (14C: 29.2 ± 1.4 kyr calBP versus OSL: 27.3 ± 3.0 kyr). By contrast, the 14C ages derived from the other n-alkane samples are clearly younger (20.3 ± 0.7 kyr calBP, 22.1 ± 0.7 kyr calBP and 29.8 ± 1.4 kyr calBP) than the corresponding OSL ages (26.6 ± 3.1 kyr, 32.0 ± 3.5 kyr and 45.6 ± 5.3 kyr). This finding suggests that a post-sedimentary n-alkane contamination presumably by roots has occurred. In order to estimate the post-sedimentary n-alkane contamination more quantitatively, we applied a 14C mass balance calculation based on the measured pMC (percent modern carbon) values, the calculated syn-sedimentary pMC values and pMC values suspected to reflect likely time points of post-sedimentary contamination (modern, last decades, 3 kyr, 6 kyr and 9 kyr). Accordingly, modern and last decadal root-contamination would account for up to 7%, a 3 kyr old root-contamination for up to 10%, and an Early and Middle Holocene root-contamination for up to 20% of the total sedimentary n-alkane pool. We acknowledge and encourage that these first radiocarbon results need further confirmation both from other loess-paleosol sequences and for different biomarkers, e.g. carboxylic acids or alcohols as further lipid biomarkers. Zech, M., Kreutzer, S., Goslar, T., Meszner, S

  15. Widespread distribution of soluble di-iron monooxygenase (SDIMO) genes in Arctic groundwater impacted by 1,4-dioxane.

    PubMed

    Li, Mengyan; Mathieu, Jacques; Yang, Yu; Fiorenza, Stephanie; Deng, Ye; He, Zhili; Zhou, Jizhong; Alvarez, Pedro J J

    2013-09-01

    Soluble di-iron monooxygenases (SDIMOs), especially group-5 SDIMOs (i.e., tetrahydrofuran and propane monooxygenases), are of significant interest due to their potential role in the initiation of 1,4-dioxane (dioxane) degradation. Functional gene array (i.e., GeoChip) analysis of Arctic groundwater exposed to dioxane since 1980s revealed that various dioxane-degrading SDIMO genes were widespread, and PCR-DGGE analysis showed that group-5 SDIMOs were present in every tested sample, including background groundwater with no known dioxane exposure history. A group-5 thmA-like gene was enriched (2.4-fold over background, p < 0.05) in source-zone samples with higher dioxane concentrations, suggesting selective pressure by dioxane. Microcosm assays with (14)C-labeled dioxane showed that the highest mineralization capacity (6.4 ± 0.1% (14)CO2 recovery during 15 days, representing over 60% of the amount degraded) corresponded to the source area, which was presumably more acclimated and contained a higher abundance of SDIMO genes. Dioxane mineralization ceased after 7 days and was resumed by adding acetate (0.24 mM) as an auxiliary substrate to replenish NADH, a key coenzyme for the functioning of monoxygenases. Acetylene inactivation tests further corroborated the vital role of monooxygenases in dioxane degradation. This is the first report of the prevalence of oxygenase genes that are likely involved in dioxane degradation and suggests their usefulness as biomarkers of dioxane natural attenuation. PMID:23909410

  16. Molecular Determinants of the Regioselectivity of Toluene/o-Xylene Monooxygenase from Pseudomonas sp. Strain OX1▿ †

    PubMed Central

    Notomista, Eugenio; Cafaro, Valeria; Bozza, Giuseppe; Di Donato, Alberto

    2009-01-01

    Bacterial multicomponent monooxygenases (BMMs) are a heterogeneous family of di-iron monooxygenases which share the very interesting ability to hydroxylate aliphatic and/or aromatic hydrocarbons. Each BMM possesses defined substrate specificity and regioselectivity which match the metabolic requirements of the strain from which it has been isolated. Pseudomonas sp. strain OX1, a strain able to metabolize o-, m-, and p-cresols, produces the BMM toluene/o-xylene monooxygenase (ToMO), which converts toluene to a mixture of o-, m-, and p-cresol isomers. In order to investigate the molecular determinants of ToMO regioselectivity, we prepared and characterized 15 single-mutant and 3 double-mutant forms of the ToMO active site pocket. Using the Monte Carlo approach, we prepared models of ToMO-substrate and ToMO-reaction intermediate complexes which allowed us to provide a molecular explanation for the regioselectivities of wild-type and mutant ToMO enzymes. Furthermore, using binding energy values calculated by energy analyses of the complexes and a simple mathematical model of the hydroxylation reaction, we were able to predict quantitatively the regioselectivities of the majority of the variant proteins with good accuracy. The results show not only that the fine-tuning of ToMO regioselectivity can be achieved through a careful alteration of the shape of the active site but also that the effects of the mutations on regioselectivity can be quantitatively predicted a priori. PMID:19074607

  17. Molecular determinants of the regioselectivity of toluene/o-xylene monooxygenase from Pseudomonas sp. strain OX1.

    PubMed

    Notomista, Eugenio; Cafaro, Valeria; Bozza, Giuseppe; Di Donato, Alberto

    2009-02-01

    Bacterial multicomponent monooxygenases (BMMs) are a heterogeneous family of di-iron monooxygenases which share the very interesting ability to hydroxylate aliphatic and/or aromatic hydrocarbons. Each BMM possesses defined substrate specificity and regioselectivity which match the metabolic requirements of the strain from which it has been isolated. Pseudomonas sp. strain OX1, a strain able to metabolize o-, m-, and p-cresols, produces the BMM toluene/o-xylene monooxygenase (ToMO), which converts toluene to a mixture of o-, m-, and p-cresol isomers. In order to investigate the molecular determinants of ToMO regioselectivity, we prepared and characterized 15 single-mutant and 3 double-mutant forms of the ToMO active site pocket. Using the Monte Carlo approach, we prepared models of ToMO-substrate and ToMO-reaction intermediate complexes which allowed us to provide a molecular explanation for the regioselectivities of wild-type and mutant ToMO enzymes. Furthermore, using binding energy values calculated by energy analyses of the complexes and a simple mathematical model of the hydroxylation reaction, we were able to predict quantitatively the regioselectivities of the majority of the variant proteins with good accuracy. The results show not only that the fine-tuning of ToMO regioselectivity can be achieved through a careful alteration of the shape of the active site but also that the effects of the mutations on regioselectivity can be quantitatively predicted a priori. PMID:19074607

  18. Five monomeric hemocyanin subunits from Portunus trituberculatus: purification, spectroscopic characterization, and quantitative evaluation of phenol monooxygenase activity.

    PubMed

    Fujieda, Nobutaka; Yakiyama, Aki; Itoh, Shinobu

    2010-11-01

    Five kinds of monomeric subunits of arthropod hemocyanin have been isolated from swimming crab Portunus trituberculatus hemolymph. The copper centers holding a peroxo species, [(μ-η2:η2-peroxo)dicopper(II)], of these subunits exhibited almost the same UV-vis and visible region CD spectroscopic properties, indicating that they have a similar copper coordination geometry and an electronic structure. Under anaerobic conditions, the oxy-forms of the monomeric subunits were stable in 0.5 M borate buffer (pH 9.0) and reacted with 4-methylphenol (p-cresol) to show the phenolases (cresolase/phenol monooxygenase) activity in the presence of urea. To compare the phenolase (monooxygenase) reactivity, the reactivity of the isolated subunits has been examined quantitatively by using a simplified catalytic system, where the initial product catechol is trapped with borate anion of the buffer solution to prevent following catecholase reaction (Yamazaki and Itoh, 2003). The far-UV region CD spectra were measured in order to clarify the relationship between the content of the secondary structure and the phenolase reactivity. Even though the monomeric subunits exhibit a weak catalytic phenol monooxygenase activity, addition of urea (3 M) significantly enhances their catalytic activity. The differences of the phenolase activity among the monomeric subunits has been discussed on the basis of the spectroscopic analysis and reactivity studies in order to shed light on the enzymatic function of the arthropod hemocyanin in vivo. PMID:20727990

  19. Microbial Communities in Methane- and Short Chain Alkane-Rich Hydrothermal Sediments of Guaymas Basin.

    PubMed

    Dowell, Frederick; Cardman, Zena; Dasarathy, Srishti; Kellermann, Matthias Y; Lipp, Julius S; Ruff, S Emil; Biddle, Jennifer F; McKay, Luke J; MacGregor, Barbara J; Lloyd, Karen G; Albert, Daniel B; Mendlovitz, Howard; Hinrichs, Kai-Uwe; Teske, Andreas

    2016-01-01

    The hydrothermal sediments of Guaymas Basin, an active spreading center in the Gulf of California (Mexico), are rich in porewater methane, short-chain alkanes, sulfate and sulfide, and provide a model system to explore habitat preferences of microorganisms, including sulfate-dependent, methane- and short chain alkane-oxidizing microbial communities. In this study, hot sediments (above 60°C) covered with sulfur-oxidizing microbial mats surrounding a hydrothermal mound (termed "Mat Mound") were characterized by porewater geochemistry of methane, C2-C6 short-chain alkanes, sulfate, sulfide, sulfate reduction rate measurements, in situ temperature gradients, bacterial and archaeal 16S rRNA gene clone libraries and V6 tag pyrosequencing. The most abundantly detected groups in the Mat mound sediments include anaerobic methane-oxidizing archaea of the ANME-1 lineage and its sister clade ANME-1Guaymas, the uncultured bacterial groups SEEP-SRB2 within the Deltaproteobacteria and the separately branching HotSeep-1 Group; these uncultured bacteria are candidates for sulfate-reducing alkane oxidation and for sulfate-reducing syntrophy with ANME archaea. The archaeal dataset indicates distinct habitat preferences for ANME-1, ANME-1-Guaymas, and ANME-2 archaea in Guaymas Basin hydrothermal sediments. The bacterial groups SEEP-SRB2 and HotSeep-1 co-occur with ANME-1 and ANME-1Guaymas in hydrothermally active sediments underneath microbial mats in Guaymas Basin. We propose the working hypothesis that this mixed bacterial and archaeal community catalyzes the oxidation of both methane and short-chain alkanes, and constitutes a microbial community signature that is characteristic for hydrothermal and/or cold seep sediments containing both substrates. PMID:26858698

  20. Microbial Communities in Methane- and Short Chain Alkane-Rich Hydrothermal Sediments of Guaymas Basin

    PubMed Central

    Dowell, Frederick; Cardman, Zena; Dasarathy, Srishti; Kellermann, Matthias Y.; Lipp, Julius S.; Ruff, S. Emil; Biddle, Jennifer F.; McKay, Luke J.; MacGregor, Barbara J.; Lloyd, Karen G.; Albert, Daniel B.; Mendlovitz, Howard; Hinrichs, Kai-Uwe; Teske, Andreas

    2016-01-01

    The hydrothermal sediments of Guaymas Basin, an active spreading center in the Gulf of California (Mexico), are rich in porewater methane, short-chain alkanes, sulfate and sulfide, and provide a model system to explore habitat preferences of microorganisms, including sulfate-dependent, methane- and short chain alkane-oxidizing microbial communities. In this study, hot sediments (above 60°C) covered with sulfur-oxidizing microbial mats surrounding a hydrothermal mound (termed “Mat Mound”) were characterized by porewater geochemistry of methane, C2–C6 short-chain alkanes, sulfate, sulfide, sulfate reduction rate measurements, in situ temperature gradients, bacterial and archaeal 16S rRNA gene clone libraries and V6 tag pyrosequencing. The most abundantly detected groups in the Mat mound sediments include anaerobic methane-oxidizing archaea of the ANME-1 lineage and its sister clade ANME-1Guaymas, the uncultured bacterial groups SEEP-SRB2 within the Deltaproteobacteria and the separately branching HotSeep-1 Group; these uncultured bacteria are candidates for sulfate-reducing alkane oxidation and for sulfate-reducing syntrophy with ANME archaea. The archaeal dataset indicates distinct habitat preferences for ANME-1, ANME-1-Guaymas, and ANME-2 archaea in Guaymas Basin hydrothermal sediments. The bacterial groups SEEP-SRB2 and HotSeep-1 co-occur with ANME-1 and ANME-1Guaymas in hydrothermally active sediments underneath microbial mats in Guaymas Basin. We propose the working hypothesis that this mixed bacterial and archaeal community catalyzes the oxidation of both methane and short-chain alkanes, and constitutes a microbial community signature that is characteristic for hydrothermal and/or cold seep sediments containing both substrates. PMID:26858698

  1. Anaerobic n-Alkane Metabolism by a Sulfate-Reducing Bacterium, Desulfatibacillum aliphaticivorans Strain CV2803T

    PubMed Central

    Cravo-Laureau, Cristiana; Grossi, Vincent; Raphel, Danielle; Matheron, Robert; Hirschler-Réa, Agnès

    2005-01-01

    The alkane-degrading, sulfate-reducing bacterium Desulfatibacillum aliphaticivorans strain CV2803T, recently isolated from marine sediments, was investigated for n-alkane metabolism. The total cellular fatty acids of this strain had predominantly odd numbers of carbon atoms (C odd) when the strain was grown on a C-odd alkane (pentadecane) and even numbers of carbon atoms (C even) when it was grown on a C-even alkane (hexadecane). Detailed analyses of those fatty acids by gas chromatography/mass spectrometry allowed us to identify saturated 2-, 4-, 6-, and 8-methyl- and monounsaturated 6-methyl-branched fatty acids, with chain lengths that specifically correlated with those of the alkane. Growth of D. aliphaticivorans on perdeuterated hexadecane demonstrated that those methyl-branched fatty acids were directly derived from the substrate. In addition, cultures on pentadecane and hexadecane produced (1-methyltetradecyl)succinate and (1-methylpentadecyl)succinate, respectively. These results indicate that D. aliphaticivorans strain CV2803T oxidizes n-alkanes into fatty acids anaerobically, via the addition of fumarate at C-2. Based on our observations and on literature data, a pathway for anaerobic n-alkane metabolism by D. aliphaticivorans is proposed. This involves the transformation of the initial alkylsuccinate into a 4-methyl-branched fatty acid which, in addition to catabolic reactions, can alternatively undergo chain elongation and desaturation to form storage fatty acids. PMID:16000749

  2. A Flavin-dependent Monooxygenase from Mycobacterium tuberculosis Involved in Cholesterol Catabolism*

    PubMed Central

    Dresen, Carola; Lin, Leo Y.-C.; D'Angelo, Igor; Tocheva, Elitza I.; Strynadka, Natalie; Eltis, Lindsay D.

    2010-01-01

    Mycobacterium tuberculosis (Mtb) and Rhodococcus jostii RHA1 have similar cholesterol catabolic pathways. This pathway contributes to the pathogenicity of Mtb. The hsaAB cholesterol catabolic genes have been predicted to encode the oxygenase and reductase, respectively, of a flavin-dependent mono-oxygenase that hydroxylates 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3-HSA) to a catechol. An hsaA deletion mutant of RHA1 did not grow on cholesterol but transformed the latter to 3-HSA and related metabolites in which each of the two keto groups was reduced: 3,9-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-17-one (3,9-DHSA) and 3,17-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9-one (3,17-DHSA). Purified 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione 4-hydroxylase (HsaAB) from Mtb had higher specificity for 3-HSA than for 3,17-DHSA (apparent kcat/Km = 1000 ± 100 m−1 s−1 versus 700 ± 100 m−1 s−1). However, 3,9-DHSA was a poorer substrate than 3-hydroxybiphenyl (apparent kcat/Km = 80 ± 40 m−1 s−1). In the presence of 3-HSA the Kmapp for O2 was 100 ± 10 μm. The crystal structure of HsaA to 2.5-Å resolution revealed that the enzyme has the same fold, flavin-binding site, and catalytic residues as p-hydroxyphenyl acetate hydroxylase. However, HsaA has a much larger phenol-binding site, consistent with the enzyme's substrate specificity. In addition, a second crystal form of HsaA revealed that a C-terminal flap (Val367–Val394) could adopt two conformations differing by a rigid body rotation of 25° around Arg366. This rotation appears to gate the likely flavin entrance to the active site. In docking studies with 3-HSA and flavin, the closed conformation provided a rationale for the enzyme's substrate specificity. Overall, the structural and functional data establish the physiological role of HsaAB and provide a basis to further investigate an important class of monooxygenases as well as the bacterial catabolism of steroids. PMID

  3. A flavin-dependent monooxygenase from Mycobacterium tuberculosis involved in cholesterol catabolism.

    PubMed

    Dresen, Carola; Lin, Leo Y-C; D'Angelo, Igor; Tocheva, Elitza I; Strynadka, Natalie; Eltis, Lindsay D

    2010-07-16

    Mycobacterium tuberculosis (Mtb) and Rhodococcus jostii RHA1 have similar cholesterol catabolic pathways. This pathway contributes to the pathogenicity of Mtb. The hsaAB cholesterol catabolic genes have been predicted to encode the oxygenase and reductase, respectively, of a flavin-dependent mono-oxygenase that hydroxylates 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3-HSA) to a catechol. An hsaA deletion mutant of RHA1 did not grow on cholesterol but transformed the latter to 3-HSA and related metabolites in which each of the two keto groups was reduced: 3,9-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-17-one (3,9-DHSA) and 3,17-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9-one (3,17-DHSA). Purified 3-hydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione 4-hydroxylase (HsaAB) from Mtb had higher specificity for 3-HSA than for 3,17-DHSA (apparent k(cat)/K(m) = 1000 +/- 100 M(-1) s(-1) versus 700 +/- 100 M(-1) s(-1)). However, 3,9-DHSA was a poorer substrate than 3-hydroxybiphenyl (apparent k(cat)/K(m) = 80 +/- 40 M(-1) s(-1)). In the presence of 3-HSA the K(m)(app) for O(2) was 100 +/- 10 microM. The crystal structure of HsaA to 2.5-A resolution revealed that the enzyme has the same fold, flavin-binding site, and catalytic residues as p-hydroxyphenyl acetate hydroxylase. However, HsaA has a much larger phenol-binding site, consistent with the enzyme's substrate specificity. In addition, a second crystal form of HsaA revealed that a C-terminal flap (Val(367)-Val(394)) could adopt two conformations differing by a rigid body rotation of 25 degrees around Arg(366). This rotation appears to gate the likely flavin entrance to the active site. In docking studies with 3-HSA and flavin, the closed conformation provided a rationale for the enzyme's substrate specificity. Overall, the structural and functional data establish the physiological role of HsaAB and provide a basis to further investigate an important class of monooxygenases as well as the bacterial

  4. Preferential methanogenic biodegradation of short-chain n-alkanes by microbial communities from two different oil sands tailings ponds.

    PubMed

    Shahimin, Mohd Faidz Mohamad; Foght, Julia M; Siddique, Tariq

    2016-05-15

    Oil sands tailings ponds harbor diverse anaerobic microbial communities capable of methanogenic biodegradation of solvent hydrocarbons entrained in the tailings. Mature fine tailings (MFT) from two operators (Albian and CNRL) that use different extraction solvents were incubated with mixtures of either two (n-pentane and n-hexane) or four (n-pentane, n-hexane, n-octane and n-decane) n-alkanes under methanogenic conditions for ~600 d. Microbes in Albian MFT began methane production by ~80 d, achieving complete depletion of n-pentane and n-hexane in the two-alkane mixture and their preferential biodegradation in the four-alkane mixture. Microbes in CNRL MFT preferentially metabolized n-octane and n-decane in the four-alkane mixture after a ~80 d lag but exhibited a lag of ~360 d before commencing biodegradation of n-pentane and n-hexane in the two-alkane mixture. 16S rRNA gene pyrosequencing revealed Peptococcaceae members as key bacterial n-alkane degraders in all treatments except CNRL MFT amended with the four-alkane mixture, in which Anaerolineaceae, Desulfobacteraceae (Desulfobacterium) and Syntrophaceae (Smithella) dominated during n-octane and n-decane biodegradation. Anaerolineaceae sequences increased only in cultures amended with the four-alkane mixture and only during n-octane and n-decane biodegradation. The dominant methanogens were acetoclastic Methanosaetaceae. These results highlight preferential n-alkane biodegradation by microbes in oil sands tailings from different producers, with implications for tailings management and reclamation. PMID:26925736

  5. Localization of genes encoding three distinct flavin-containing monooxygenases to human chromosome 1q

    SciTech Connect

    Shephard, E.A.; Fox, M.F.; Povey, S. ); Dolphin, C.T.; Phillips, I.R.; Smith, R. )

    1993-04-01

    The authors have used the polymerase chain reaction to map the gene encoding human flavin-containing monooxygenase (FMO) form II (N. Lomri, Q. Gu, and J. R. Cashman, 1992, Proc. Natl. Acad. Sci. USA 89: 1685--1689) to chromosome 1. They propose the designation FMO3 for this gene as it is the third FMO gene to be mapped. The two other human FMO genes identified to date, FMO1 and FMO2, are also located on chromosome 1 (C. Dolphin, E. A. Shephard, S. Povey, C. N. A. Palmer, D. M. Ziegler, R. Ayesh, R. L. Smith, and 1. R. Phillips, 1991, J. Biol. Chem. 266: 12379--12385; C. Dolphin, E. A. Shephard, S. F. Povey, R. L. Smith, and I. R. Phillips, 1992, Biochem. J. 286: 261--267). The localization of FMO1, FMO2, and FMO3 has been refined to the long arm of chromosome 1. Analysis of human metaphase chromosomes by in situ hybridization confirmed the mapping of FMO1 and localized this gene more precisely to 1 q23-q25. 28 refs., 3 figs., 2 tabs.

  6. Kynurenine–3–monooxygenase inhibition prevents multiple organ failure in rodent models of acute pancreatitis

    PubMed Central

    Mole, Damian J; Webster, Scott P; Uings, Iain; Zheng, Xiaozhong; Binnie, Margaret; Wilson, Kris; Hutchinson, Jonathan P; Mirguet, Olivier; Walker, Ann; Beaufils, Benjamin; Ancellin, Nicolas; Trottet, Lionel; Bénéton, Véronique; Mowat, Christopher G; Wilkinson, Martin; Rowland, Paul; Haslam, Carl; McBride, Andrew; Homer, Natalie ZM; Baily, James E; Sharp, Matthew GF; Garden, O James; Hughes, Jeremy; Howie, Sarah EM; Holmes, Duncan S; Liddle, John; Iredale, John P

    2015-01-01

    Acute pancreatitis (AP) is a common and devastating inflammatory condition of the pancreas that is considered to be a paradigm of sterile inflammation leading to systemic multiple organ dysfunction syndrome (MODS) and death1,2 Acute mortality from AP-MODS exceeds 20%3 and for those who survive the initial episode, their lifespan is typically shorter than the general population4. There are no specific therapies available that protect individuals against AP-MODS. Here, we show that kynurenine-3-monooxygenase (KMO), a key enzyme of tryptophan metabolism5, is central to the pathogenesis of AP-MODS. We created a mouse strain deficient for Kmo with a robust biochemical phenotype that protected against extrapancreatic tissue injury to lung, kidney and liver in experimental AP-MODS. A medicinal chemistry strategy based on modifications of the kynurenine substrate led to the discovery of GSK180 as a potent and specific inhibitor of KMO. The binding mode of the inhibitor in the active site was confirmed by X-ray co-crystallography at 3.2 Å resolution. Treatment with GSK180 resulted in rapid changes in levels of kynurenine pathway metabolites in vivo and afforded therapeutic protection against AP-MODS in a rat model of AP. Our findings establish KMO inhibition as a novel therapeutic strategy in the treatment of AP-MODS and open up a new area for drug discovery in critical illness. PMID:26752518

  7. Purification and properties of the NADH reductase component of alkene monooxygenase from Mycobacterium strain E3.

    PubMed Central

    Weber, F J; van Berkel, W J; Hartmans, S; de Bont, J A

    1992-01-01

    Alkene monooxygenase, a multicomponent enzyme system which catalyzes the epoxidation of short-chain alkenes, is induced in Mycobacterium strain E3 when it is grown on ethene. We purified the NADH reductase component of this enzyme system to homogeneity. Recovery of the enzyme was 19%, with a purification factor of 920-fold. The enzyme is a monomer with a molecular mass of 56 kDa as determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is yellow-red with absorption maxima at 384, 410, and 460 nm. Flavin adenine dinucleotide (FAD) was identified as a prosthetic group at a FAD-protein ratio of 1:1. Tween 80 prevented irreversible dissociation of FAD from the enzyme during chromatographic purification steps. Colorimetric analysis revealed 2 mol each of iron and acid-labile sulfide, indicating the presence of a [2Fe-2S] cluster. The presence of this cluster was confirmed by electron paramagnetic resonance spectroscopy (g values at 2.011, 1.921, and 1.876). Anaerobic reduction of the reductase by NADH resulted in formation of a flavin semiquinone. Images PMID:1315734

  8. Trichloroethylene degradation using recombinant bacteria expressing the soluble methane monooxygenase from methylosinus trichosporium OB3b

    SciTech Connect

    Jahng, D.; Kim, C.; Wood, T.K.

    1995-12-01

    Soluble methane monooxygenase (sMMO) from M. trichosporium OB3b has the ability to degrade many halogenated aliphatic compounds that are found in contaminated soil and groundwater. For efficient trichloroethylene (TCE) degradation in a foreign host, efforts are being made to improve inconsistent and low sMMO activity of the recombinant strain constructed previously (Pseudomonas putida F1/pSMMO20). Additional smmo-containing recombinant strains have been constructed including various Pseudomonas, Agrobacterium, and Rhizobium strains. Recombinant facultative methylotrophs containing the smmo locus were also constructed through electroporation and tri-parental mating using a new plasmid pSMMO50. TCE degradation by these recombinant strains was examined. The effect of metal ions on in vitro sMMO activity was also discerned to optimize the expression medium. Among the metal ions examined, Cu(I), Cu(II), Ni(II), and Zn(II) inhibited sMMO purified from trichosporium OB3b, and the effect of the metal ions on each of the components of sMMO will also be discussed. In addition, the post-segregational killing locus (hok/sok) from E. coli plasmid R1 was inserted downstream of the smmo locus to stabilize the recombinant plasmid in these host cells, and chemostat cultures were used to optimize expression of active sMMO by varying the growth rate.

  9. Cellulose surface degradation by a lytic polysaccharide monooxygenase and its effect on cellulase hydrolytic efficiency.

    PubMed

    Eibinger, Manuel; Ganner, Thomas; Bubner, Patricia; Rošker, Stephanie; Kracher, Daniel; Haltrich, Dietmar; Ludwig, Roland; Plank, Harald; Nidetzky, Bernd

    2014-12-26

    Lytic polysaccharide monooxygenase (LPMO) represents a unique principle of oxidative degradation of recalcitrant insoluble polysaccharides. Used in combination with hydrolytic enzymes, LPMO appears to constitute a significant factor of the efficiency of enzymatic biomass depolymerization. LPMO activity on different cellulose substrates has been shown from the slow release of oxidized oligosaccharides into solution, but an immediate and direct demonstration of the enzyme action on the cellulose surface is lacking. Specificity of LPMO for degrading ordered crystalline and unordered amorphous cellulose material of the substrate surface is also unknown. We show by fluorescence dye adsorption analyzed with confocal laser scanning microscopy that a LPMO (from Neurospora crassa) introduces carboxyl groups primarily in surface-exposed crystalline areas of the cellulosic substrate. Using time-resolved in situ atomic force microscopy we further demonstrate that cellulose nano-fibrils exposed on the surface are degraded into shorter and thinner insoluble fragments. Also using atomic force microscopy, we show that prior action of LPMO enables cellulases to attack otherwise highly resistant crystalline substrate areas and that it promotes an overall faster and more complete surface degradation. Overall, this study reveals key characteristics of LPMO action on the cellulose surface and suggests the effects of substrate morphology on the synergy between LPMO and hydrolytic enzymes in cellulose depolymerization. PMID:25361767

  10. Carbon Isotope Fractionations Associated with Methanotrophic Growth with the Soluble and Particulate Methane Monooxygenases

    NASA Technical Reports Server (NTRS)

    Jahnke, Linda L.; Summons, Roger E.; Chang, Sherwood (Technical Monitor)

    1996-01-01

    Growth experiments with the RuMP-type methanotroph, Methylococcus capsulatus (Bath), have demonstrated that biomass and lipid biomarkers are significantly depleted in C-13 compared to the substrate methane and that the extent of fractionation is dependent on whether cells express the soluble (s) or particulate (p) methane monooxygenase (MMO). The presence or absence of the characteristic sMMO subunits was monitored using SDS-polyacrylamide gels. In M. capsulatus grown with no Cu supplementation, the characteristic sMMO subunits were observed in the soluble fraction throughout the entire growth period and biomass was depleted in C-13 by approximately 14,700 relative to substrate methane. In cells grown with 5uM Cu, no sMMO bands were observed and a greater fractionation of approximately 27,700 in resultant biomass was obtained. Methanol growth experiments with M. capsulatus and with a RuMP methylotroph, Methylophilus methylotrophus, in which biomass measurements yielded depletions in C-13 of 9 and 5%(sub o), respectively, suggest that oxidation of methane is the major fractionation step. Growth of M. capsulatus at a low level of oxygen, approximately 0.5%, had no significant effect on carbon isotope fractionation by either sMMO or pMMO. These observations are significant for identification of molecular biomarkers; and methanotrophic contributions to carbon isotope composition in natural environments.

  11. Sterol homeostasis requires regulated degradation of squalene monooxygenase by the ubiquitin ligase Doa10/Teb4

    PubMed Central

    Foresti, Ombretta; Ruggiano, Annamaria; Hannibal-Bach, Hans K; Ejsing, Christer S; Carvalho, Pedro

    2013-01-01

    Sterol homeostasis is essential for the function of cellular membranes and requires feedback inhibition of HMGR, a rate-limiting enzyme of the mevalonate pathway. As HMGR acts at the beginning of the pathway, its regulation affects the synthesis of sterols and of other essential mevalonate-derived metabolites, such as ubiquinone or dolichol. Here, we describe a novel, evolutionarily conserved feedback system operating at a sterol-specific step of the mevalonate pathway. This involves the sterol-dependent degradation of squalene monooxygenase mediated by the yeast Doa10 or mammalian Teb4, a ubiquitin ligase implicated in a branch of the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway. Since the other branch of ERAD is required for HMGR regulation, our results reveal a fundamental role for ERAD in sterol homeostasis, with the two branches of this pathway acting together to control sterol biosynthesis at different levels and thereby allowing independent regulation of multiple products of the mevalonate pathway. DOI: http://dx.doi.org/10.7554/eLife.00953.001 PMID:23898401

  12. Chopping and Changing: the Evolution of the Flavin-dependent Monooxygenases.

    PubMed

    Mascotti, Maria Laura; Juri Ayub, Maximiliano; Furnham, Nicholas; Thornton, Janet M; Laskowski, Roman A

    2016-07-31

    Flavin-dependent monooxygenases play a variety of key physiological roles and are also very powerful biotechnological tools. These enzymes have been classified into eight different classes (A-H) based on their sequences and biochemical features. By combining structural and sequence analysis, and phylogenetic inference, we have explored the evolutionary history of classes A, B, E, F, and G and demonstrate that their multidomain architectures reflect their phylogenetic relationships, suggesting that the main evolutionary steps in their divergence are likely to have arisen from the recruitment of different domains. Additionally, the functional divergence within in each class appears to have been the result of other mechanisms such as a complex set of single-point mutations. Our results reinforce the idea that a main constraint on the evolution of cofactor-dependent enzymes is the functional binding of the cofactor. Additionally, a remarkable feature of this family is that the sequence of the key flavin adenine dinucleotide-binding domain is split into at least two parts in all classes studied here. We propose a complex set of evolutionary events that gave rise to the origin of the different classes within this family. PMID:27423402

  13. Immobilization of P450 monooxygenase and chloroplast for use in light-driven bioreactors.

    PubMed

    Hara, M; Iazvovskaia, S; Ohkawa, H; Asada, Y; Miyake, J

    1999-01-01

    P450 monooxygenases exhibit great potential for application to bioreactors for the decomposition of various hydrophobic chemicals including pollutant compounds. P450-containing microsomes were immobilized in spinach chloroplasts for use in light-driven bioreactors. We tested three methods (entrapment, adsorption and cross-linking) to immobilize chloroplasts and yeast microsomes containing a genetically engineered fusion enzyme between rat P450 1A1 and yeast P450 reductase. Entrapment in agarose gave the best activity for the conversion of 7-ethoxycoumarin to 7-hydroxycoumarin under illumination of 6200 lx. We then tested three light-driven bioreactors (two column-type and one batch-type reactors developed) using the immobilized gels. A two-phase column-type reactor with separately immobilized microsomes and chloroplasts showed a higher conversion rate than a reactor with co-immobilization of both components. The reactor showed a turnover rate of 6.32 mol product/mol P450/min after a 40-min run, and 2.49 after a 180-min run. These turnover rates are higher than the values reported by others using immobilized microsomal P450s. PMID:16232556

  14. Structure of T4moF, the Toluene 4-Monooxygenase Ferredoxin Oxidoreductase.

    PubMed

    Acheson, Justin F; Moseson, Hannah; Fox, Brian G

    2015-09-29

    The 1.6 Å crystal structure of toluene 4-monooxygenase reductase T4moF is reported. The structure includes ferredoxin, flavin, and NADH binding domains. The position of the ferredoxin domain relative to the other two domains represents a new configuration for the iron-sulfur flavoprotein family. Close contacts between the C8 methyl group of FAD and [2Fe-2S] ligand Cys36-O represent a plausible pathway for electron transfer between the redox cofactors. Energy-minimized docking of NADH and calculation of hingelike motions between domains suggest how simple coordinated shifts of residues at the C-terminus of the enzyme could expose the N5 position of FAD for productive interaction with the nicotinamide ring. The domain configuration revealed by the T4moF structure provides an excellent steric and electrostatic match to the obligate electron acceptor, Rieske-type [2Fe-2S] ferredoxin T4moC. Protein-protein docking and energy minimization of the T4moFC complex indicate that T4moF [2Fe-2S] ligand Cys41 and T4moC [2Fe-2S] ligand His67, along with other electrostatic interactions between the protein partners, form the functional electron transfer interface. PMID:26309236

  15. (+)-Abscisic Acid 8′-Hydroxylase Is a Cytochrome P450 Monooxygenase1

    PubMed Central

    Krochko, Joan E.; Abrams, Garth D.; Loewen, Mary K.; Abrams, Suzanne R.; Cutler, Adrian J.

    1998-01-01

    Abscisic acid (ABA) 8′-hydroxylase catalyzes the first step in the oxidative degradation of (+)-ABA. The development of a robust in vitro assay has now permitted detailed examination and characterization of this enzyme. Although several factors (buffer, cofactor, and source tissue) were critical in developing the assay, the most important of these was the identification of a tissue displaying high amounts of in vivo enzyme activity (A.J. Cutler, T.M. Squires, M.K. Loewen, J.J. Balsevich [1997] J Exp Bot 48: 1787–1795). (+)-ABA 8′-hydroxylase is an integral membrane protein that is localized to the microsomal fraction in suspension-cultured maize (Zea mays) cells. (+)-ABA metabolism requires both NADPH and molecular oxygen. NADH was not an effective cofactor, although there was substantial stimulation of activity (synergism) when it was included at rate-limiting NADPH concentrations. The metabolism of (+)-ABA was progressively inhibited at O2 concentrations less than 10% (v/v) and was very low (less than 5% of control) under N2. (+)-ABA 8′-hydroxylase activity was inhibited by tetcyclacis (50% inhibition at 10−6 m), cytochrome c (oxidized form), and CO. The CO inhibition was reversible by light from several regions of the visible spectrum, but most efficiently by blue and amber light. These data strongly support the contention that (+)-ABA 8′-hydroxylase is a cytochrome P450 monooxygenase. PMID:9808729

  16. Two Cytochrome P450 Monooxygenases Catalyze Early Hydroxylation Steps in the Potato Steroid Glycoalkaloid Biosynthetic Pathway.

    PubMed

    Umemoto, Naoyuki; Nakayasu, Masaru; Ohyama, Kiyoshi; Yotsu-Yamashita, Mari; Mizutani, Masaharu; Seki, Hikaru; Saito, Kazuki; Muranaka, Toshiya

    2016-08-01

    α-Solanine and α-chaconine, steroidal glycoalkaloids (SGAs) found in potato (Solanum tuberosum), are among the best-known secondary metabolites in food crops. At low concentrations in potato tubers, SGAs are distasteful; however, at high concentrations, SGAs are harmful to humans and animals. Here, we show that POTATO GLYCOALKALOID BIOSYNTHESIS1 (PGA1) and PGA2, two genes that encode cytochrome P450 monooxygenases (CYP72A208 and CYP72A188), are involved in the SGA biosynthetic pathway, respectively. The knockdown plants of either PGA1 or PGA2 contained very little SGA, yet vegetative growth and tuber production were not affected. Analyzing metabolites that accumulated in the plants and produced by in vitro enzyme assays revealed that PGA1 and PGA2 catalyzed the 26- and 22-hydroxylation steps, respectively, in the SGA biosynthetic pathway. The PGA-knockdown plants had two unique phenotypic characteristics: The plants were sterile and tubers of these knockdown plants did not sprout during storage. Functional analyses of PGA1 and PGA2 have provided clues for controlling both potato glycoalkaloid biosynthesis and tuber sprouting, two traits that can significantly impact potato breeding and the industry. PMID:27307258

  17. A magnetic bead-based ligand binding assay to facilitate human kynurenine 3-monooxygenase drug discovery.

    PubMed

    Wilson, Kris; Mole, Damian J; Homer, Natalie Z M; Iredale, John P; Auer, Manfred; Webster, Scott P

    2015-02-01

    Human kynurenine 3-monooxygenase (KMO) is emerging as an important drug target enzyme in a number of inflammatory and neurodegenerative disease states. Recombinant protein production of KMO, and therefore discovery of KMO ligands, is challenging due to a large membrane targeting domain at the C-terminus of the enzyme that causes stability, solubility, and purification difficulties. The purpose of our investigation was to develop a suitable screening method for targeting human KMO and other similarly challenging drug targets. Here, we report the development of a magnetic bead-based binding assay using mass spectrometry detection for human KMO protein. The assay incorporates isolation of FLAG-tagged KMO enzyme on protein A magnetic beads. The protein-bound beads are incubated with potential binding compounds before specific cleavage of the protein-compound complexes from the beads. Mass spectrometry analysis is used to identify the compounds that demonstrate specific binding affinity for the target protein. The technique was validated using known inhibitors of KMO. This assay is a robust alternative to traditional ligand-binding assays for challenging protein targets, and it overcomes specific difficulties associated with isolating human KMO. PMID:25296660

  18. Lead discovery for human kynurenine 3-monooxygenase by high-throughput RapidFire mass spectrometry.

    PubMed

    Lowe, Denise M; Gee, Michelle; Haslam, Carl; Leavens, Bill; Christodoulou, Erica; Hissey, Paul; Hardwicke, Philip; Argyrou, Argyrides; Webster, Scott P; Mole, Damian J; Wilson, Kris; Binnie, Margaret; Yard, Beverley A; Dean, Tony; Liddle, John; Uings, Iain; Hutchinson, Jonathan P

    2014-04-01

    Kynurenine 3-monooxygenase (KMO) is a therapeutically important target on the eukaryotic tryptophan catabolic pathway, where it converts L-kynurenine (Kyn) to 3-hydroxykynurenine (3-HK). We have cloned and expressed the human form of this membrane protein as a full-length GST-fusion in a recombinant baculovirus expression system. An enriched membrane preparation was used for a directed screen of approximately 78,000 compounds using a RapidFire mass spectrometry (RF-MS) assay. The RapidFire platform provides an automated solid-phase extraction system that gives a throughput of approximately 7 s per well to the mass spectrometer, where direct measurement of both the substrate and product allowed substrate conversion to be determined. The RF-MS methodology is insensitive to assay interference, other than where compounds have the same nominal mass as Kyn or 3-HK and produce the same mass transition on fragmentation. These instances could be identified by comparison with the product-only data. The screen ran with excellent performance (average Z' value 0.8) and provided several tractable hit series for further investigation. PMID:24381207

  19. C. elegans flavin-containing monooxygenase-4 is essential for osmoregulation in hypotonic stress

    PubMed Central

    Hirani, Nisha; Westenberg, Marcel; Seed, Paul T.; Petalcorin, Mark I. R.; Dolphin, Colin T.

    2016-01-01

    ABSTRACT Studies in Caenorhabditis elegans have revealed osmoregulatory systems engaged when worms experience hypertonic conditions, but less is known about measures employed when faced with hypotonic stress. Inactivation of fmo-4, which encodes flavin-containing monooxygenase-4, results in dramatic hypoosmotic hypersensitivity; worms are unable to prevent overwhelming water influx and swell rapidly, finally rupturing due to high internal hydrostatic pressure. fmo-4 is expressed prominently in hypodermis, duct and pore cells but is excluded from the excretory cell. Thus, FMO-4 plays a crucial osmoregulatory role by promoting clearance of excess water that enters during hypotonicity, perhaps by synthesizing an osmolyte that acts to establish an osmotic gradient from excretory cell to duct and pore cells. C. elegans FMO-4 contains a C-terminal extension conserved in all nematode FMO-4s. The coincidently numbered human FMO4 also contains an extended C-terminus with features similar to those of FMO-4. Although these shared sequence characteristics suggest potential orthology, human FMO4 was unable to rescue the fmo-4 osmoregulatory defect. Intriguingly, however, mammalian FMO4 is expressed predominantly in the kidney – an appropriate site if it too is, or once was, involved in osmoregulation. PMID:27010030

  20. Electron transfer reactions in the alkene mono-oxygenase complex from Nocardia corallina B-276.

    PubMed Central

    Gallagher, S C; Cammack, R; Dalton, H

    1999-01-01

    Nocardia corallina B-276 possesses a multi-component enzyme, alkene mono-oxygenase (AMO), that catalyses the stereoselective epoxygenation of alkenes. The reductase component of this system has been shown by EPR and fluorescence spectroscopy to contain two prosthetic groups, an FAD centre and a [2Fe-2S] cluster. The role of these centres in the epoxygenation reaction was determined by midpoint potential measurements and electron transfer kinetics. The order of potentials of the prosthetic groups of the reductase were FAD/FAD.=-216 mV, [2Fe-2S]/[2Fe-2S].=-160 mV and FAD./FAD.=-134 mV. Combined, these data implied that the reductase component supplied the energy required for the epoxygenation reaction and allowed a prediction of the mechanism of electron transfer within the AMO complex. The FAD moiety was reduced by bound NADH in a two-electron reaction. The electrons were then transported to the [2Fe-2S] centre one at a time, which in turn reduced the di-iron centre of the epoxygenase. Reduction of the di-iron centre is required for oxygen binding and substrate oxidation. PMID:10085230

  1. Suppressed expression of choline monooxygenase in sugar beet on the accumulation of glycine betaine.

    PubMed

    Yamada, Nana; Takahashi, Hiroyuki; Kitou, Kunihide; Sahashi, Kosuke; Tamagake, Hideto; Tanaka, Yoshito; Takabe, Teruhiro

    2015-11-01

    Glycine betaine (GB) is an important osmoprotectant and synthesized by two-step oxidation of choline. Choline monooxygenase (CMO) catalyzes the first step of the pathway and is believed to be a rate limiting step for GB synthesis. Recent studies have shown the importance of choline-precursor supply for GB synthesis. In order to investigate the role of CMO for GB accumulation in sugar beet (Beta vulgaris), transgenic plants carrying the antisense BvCMO gene were developed. The antisense BvCMO plants showed the decreased activity of GB synthesis from choline compared to wild-type (WT) plants which is well related to the suppressed level of BvCMO protein. However, GB contents were similar between transgenic and WT plants with the exception of young leaves and storage roots. Transgenic plants showed enhanced susceptibility to salt stress than WT plants. These results suggest the importance of choline-precursor-supply for GB accumulation, and young leaves and storage root are sensitive sites for GB accumulation. PMID:26302482

  2. Regulation of betaine synthesis by precursor supply and choline monooxygenase expression in Amaranthus tricolor.

    PubMed

    Bhuiyan, Nazmul H; Hamada, Akira; Yamada, Nana; Rai, Vandna; Hibino, Takashi; Takabe, Teruhiro

    2007-01-01

    In plants, betaine is synthesized upon abiotic stress via choline oxidation, in which choline monooxygenase (CMO) is a key enzyme. Although it had been thought that betaine synthesis is well regulated to protect abiotic stress, it is shown here that an exogenous supply of precursors such as choline, serine, and glycine in the betaine-accumulating plant Amaranthus tricolor further enhances the accumulation of betaine under salt stress, but not under normal conditions. Addition of isonicotinic acid hydrazide, an inhibitor of glycine decarboxylase, inhibited the salinity-induced accumulation of betaine. Salt-induced accumulation of A. tricolor CMO (AmCMO) and betaine was much slower in roots than in leaves, and a transient accumulation of proline was observed in the roots. Antisense expression of AmCMO mRNA suppressed the salt-induced accumulation of AmCMO and betaine, but increased the level of choline approximately 2- 3-fold. This indicates that betaine synthesis is highly regulated by AmCMO expression. The genomic DNA, including the upstream region (1.6 kbp), of AmCMO was isolated. Deletion analysis of the AmCMO promoter region revealed that the 410 bp fragment upstream of the translation start codon contains the sequence responsive to salt stress. These data reveal that the promoter sequence of CMO, in addition to precursor supply, is important for the accumulation of betaine in the betaine-accumulating plant A. tricolor. PMID:18182425

  3. Kynurenine-3-monooxygenase inhibition prevents multiple organ failure in rodent models of acute pancreatitis.

    PubMed

    Mole, Damian J; Webster, Scott P; Uings, Iain; Zheng, Xiaozhong; Binnie, Margaret; Wilson, Kris; Hutchinson, Jonathan P; Mirguet, Olivier; Walker, Ann; Beaufils, Benjamin; Ancellin, Nicolas; Trottet, Lionel; Bénéton, Véronique; Mowat, Christopher G; Wilkinson, Martin; Rowland, Paul; Haslam, Carl; McBride, Andrew; Homer, Natalie Z M; Baily, James E; Sharp, Matthew G F; Garden, O James; Hughes, Jeremy; Howie, Sarah E M; Holmes, Duncan S; Liddle, John; Iredale, John P

    2016-02-01

    Acute pancreatitis (AP) is a common and devastating inflammatory condition of the pancreas that is considered to be a paradigm of sterile inflammation leading to systemic multiple organ dysfunction syndrome (MODS) and death. Acute mortality from AP-MODS exceeds 20% (ref. 3), and the lifespans of those who survive the initial episode are typically shorter than those of the general population. There are no specific therapies available to protect individuals from AP-MODS. Here we show that kynurenine-3-monooxygenase (KMO), a key enzyme of tryptophan metabolism, is central to the pathogenesis of AP-MODS. We created a mouse strain that is deficient for Kmo (encoding KMO) and that has a robust biochemical phenotype that protects against extrapancreatic tissue injury to the lung, kidney and liver in experimental AP-MODS. A medicinal chemistry strategy based on modifications of the kynurenine substrate led to the discovery of the oxazolidinone GSK180 as a potent and specific inhibitor of KMO. The binding mode of the inhibitor in the active site was confirmed by X-ray co-crystallography at 3.2 Å resolution. Treatment with GSK180 resulted in rapid changes in the levels of kynurenine pathway metabolites in vivo, and it afforded therapeutic protection against MODS in a rat model of AP. Our findings establish KMO inhibition as a novel therapeutic strategy in the treatment of AP-MODS, and they open up a new area for drug discovery in critical illness. PMID:26752518

  4. Yeast flavin-containing monooxygenase is induced by the unfolded protein response

    PubMed Central

    Suh, Jung-Keun; Robertus, Jon D.

    2000-01-01

    Flavin-containing monooxygenase from yeast (yFMO) carries out the O2- and NADPH-dependent oxidation of biological thiols, including oxidizing glutathione to glutathione disulfide. FMO provides a large fraction of the oxidizing necessary for proper folding of disulfide bond-containing proteins; deletion of the enzyme reduces proper folding of endogenous carboxypeptidase Y by about 40%. The enzyme is not essential to cell viability because other enzymes can generate a significant fraction of the oxidizing equivalents required by the cell. However, yFMO is vital to the yeast response to reductive stress. FMO1 deletion mutants grow poorly under reductive stress, and carboxypeptidase Y activity is less than 10% of that in a stressed wild type. The FMO1 gene appears to be under control of an unfolded protein response element and is inducible by factors, such as reductive stress, that elicit the unfolded protein response. Reductive stress can increase yFMO activity at least 6-fold. This increased activity allows the cell to process endogenous disulfide bond-containing proteins and also to allow correct folding of disulfide-bonded proteins expressed from multicopy plasmids. The unfolded protein response is mediated by the Hac1p transcription factor that mediates virtually all of the induction of yFMO triggered by exogenous reducing agents. PMID:10618381

  5. Molecular and dietary regulation of beta,beta-carotene 15,15'-monooxygenase 1 (BCMO1).

    PubMed

    Lietz, Georg; Lange, Jennifer; Rimbach, Gerald

    2010-10-01

    beta,beta-Carotene 15,15'-monooxygenase-1 (BCMO1) is a key enzyme in vitamin A metabolism in mammals. Various dietary components such as non-pro-vitamin A carotenoids, fat, and polyphenols have been shown to influence the intestinal absorption and conversion of pro-vitamin A carotenoids. Furthermore, vitamin A deficiency has been shown to induce BCMO1 expression, whereas supplementation with vitamin A or its active metabolites, all-trans and 9-cis retinoic acid, dose-dependently reverse these effects. A diet-responsive regulatory network involving the intestine specific homeodomain transcription factor ISX has been shown to regulate the intestinal vitamin A uptake and production via a negative feedback control. Furthermore, non-synonymous single nucleotide polymorphisms in the human BCMO1 gene have been discovered causing observably reduced BCMO1 activity. Detailed knowledge about BCMO1 regulation as well as genetic variations causing variable cleavage activities may provide a background, on which individual and/or population based dietary recommendations for beta-carotene and vitamin A intake could be established. PMID:20599666

  6. A C4-oxidizing Lytic Polysaccharide Monooxygenase Cleaving Both Cellulose and Cello-oligosaccharides*

    PubMed Central

    Isaksen, Trine; Westereng, Bjørge; Aachmann, Finn L.; Agger, Jane W.; Kracher, Daniel; Kittl, Roman; Ludwig, Roland; Haltrich, Dietmar; Eijsink, Vincent G. H.; Horn, Svein J.

    2014-01-01

    Lignocellulosic biomass is a renewable resource that significantly can substitute fossil resources for the production of fuels, chemicals, and materials. Efficient saccharification of this biomass to fermentable sugars will be a key technology in future biorefineries. Traditionally, saccharification was thought to be accomplished by mixtures of hydrolytic enzymes. However, recently it has been shown that lytic polysaccharide monooxygenases (LPMOs) contribute to this process by catalyzing oxidative cleavage of insoluble polysaccharides utilizing a mechanism involving molecular oxygen and an electron donor. These enzymes thus represent novel tools for the saccharification of plant biomass. Most characterized LPMOs, including all reported bacterial LPMOs, form aldonic acids, i.e., products oxidized in the C1 position of the terminal sugar. Oxidation at other positions has been observed, and there has been some debate concerning the nature of this position (C4 or C6). In this study, we have characterized an LPMO from Neurospora crassa (NcLPMO9C; also known as NCU02916 and NcGH61–3). Remarkably, and in contrast to all previously characterized LPMOs, which are active only on polysaccharides, NcLPMO9C is able to cleave soluble cello-oligosaccharides as short as a tetramer, a property that allowed detailed product analysis. Using mass spectrometry and NMR, we show that the cello-oligosaccharide products released by this enzyme contain a C4 gemdiol/keto group at the nonreducing end. PMID:24324265

  7. A C4-oxidizing lytic polysaccharide monooxygenase cleaving both cellulose and cello-oligosaccharides.

    PubMed

    Isaksen, Trine; Westereng, Bjørge; Aachmann, Finn L; Agger, Jane W; Kracher, Daniel; Kittl, Roman; Ludwig, Roland; Haltrich, Dietmar; Eijsink, Vincent G H; Horn, Svein J

    2014-01-31

    Lignocellulosic biomass is a renewable resource that significantly can substitute fossil resources for the production of fuels, chemicals, and materials. Efficient saccharification of this biomass to fermentable sugars will be a key technology in future biorefineries. Traditionally, saccharification was thought to be accomplished by mixtures of hydrolytic enzymes. However, recently it has been shown that lytic polysaccharide monooxygenases (LPMOs) contribute to this process by catalyzing oxidative cleavage of insoluble polysaccharides utilizing a mechanism involving molecular oxygen and an electron donor. These enzymes thus represent novel tools for the saccharification of plant biomass. Most characterized LPMOs, including all reported bacterial LPMOs, form aldonic acids, i.e., products oxidized in the C1 position of the terminal sugar. Oxidation at other positions has been observed, and there has been some debate concerning the nature of this position (C4 or C6). In this study, we have characterized an LPMO from Neurospora crassa (NcLPMO9C; also known as NCU02916 and NcGH61-3). Remarkably, and in contrast to all previously characterized LPMOs, which are active only on polysaccharides, NcLPMO9C is able to cleave soluble cello-oligosaccharides as short as a tetramer, a property that allowed detailed product analysis. Using mass spectrometry and NMR, we show that the cello-oligosaccharide products released by this enzyme contain a C4 gemdiol/keto group at the nonreducing end. PMID:24324265

  8. Discovery and characterization of a new family of lytic polysaccharide mono-oxygenases

    PubMed Central

    Hemsworth, Glyn R.; Henrissat, Bernard; Davies, Gideon J.; Walton, Paul H.

    2014-01-01

    Lytic polysaccharide mono-oxygenases (LPMOs) are a recently discovered class of enzymes capable of oxidizing recalcitrant polysaccharides. They currently attract much attention due to their potential use in biomass conversion, notably in the production of biofuels. Past work has identified two discrete sequence-based families of these enzymes termed AA9 (formerly GH61) and AA10 (formerly CBM33). Here we report the discovery of a third family of LPMOs. Using a chitin-degrading exemplar from Aspergillus oryzae, we show that the 3-D structure of the enzyme shares some features of the previous two classes of LPMOs, including a copper active centre featuring the histidine brace active site, but is distinct in terms of its active site details and its EPR spectroscopy. The new AA11 family expands the LPMO clan with the potential to broaden both the range of potential substrates and the types of reactive copper-oxygen species formed at the active site of LPMOs. PMID:24362702

  9. Production of four Neurospora crassa lytic polysaccharide monooxygenases in Pichia pastoris monitored by a fluorimetric assay

    PubMed Central

    2012-01-01

    Background Recent studies demonstrate that enzymes from the glycosyl hydrolase family 61 (GH61) show lytic polysaccharide monooxygenase (PMO) activity. Together with cellobiose dehydrogenase (CDH) an enzymatic system capable of oxidative cellulose cleavage is formed, which increases the efficiency of cellulases and put PMOs at focus of biofuel research. Large amounts of purified PMOs, which are difficult to obtain from the native fungal producers, are needed to study their reaction kinetics, structure and industrial application. In addition, a fast and robust enzymatic assay is necessary to monitor enzyme production and purification. Results Four pmo genes from Neurospora crassa were expressed in P. pastoris under control of the AOX1 promoter. High yields were obtained for the glycosylated gene products PMO-01867, PMO-02916 and PMO-08760 (>300 mg L-1), whereas the yield of non-glycosylated PMO-03328 was moderate (~45 mg L-1). The production and purification of all four enzymes was specifically followed by a newly developed, fast assay based on a side reaction of PMO: the production of H2O2 in the presence of reductants. While ascorbate is a suitable reductant for homogeneous PMO preparations, fermentation samples require the specific electron donor CDH. Conclusions P. pastoris is a high performing expression host for N. crassa PMOs. The pmo genes under control of the native signal sequence are correctly processed and active. The novel CDH-based enzyme assay allows fast determination of PMO activity in fermentation samples and is robust against interfering matrix components. PMID:23102010

  10. Novel hydrocarbon monooxygenase genes in the metatranscriptome of a natural deep-sea hydrocarbon plume.

    PubMed

    Li, Meng; Jain, Sunit; Baker, Brett J; Taylor, Chris; Dick, Gregory J

    2014-01-01

    Particulate membrane-associated hydrocarbon monooxygenases (pHMOs) are critical components of the aerobic degradation pathway for low molecular weight hydrocarbons, including the potent greenhouse gas methane. Here, we analysed pHMO gene diversity in metagenomes and metatranscriptomes of hydrocarbon-rich hydrothermal plumes in the Guaymas Basin (GB) and nearby background waters in the deep Gulf of California. Seven distinct phylogenetic groups of pHMO were present and transcriptionally active in both plume and background waters, including several that are undetectable with currently available polymerase chain reaction (PCR) primers. The seven groups of pHMOs included those related to a putative ethane oxidizing Methylococcaceae-like group, a group of the SAR324 Deltaproteobacteria, three deep-sea clades (Deep sea-1/symbiont-like, Deep sea-2/PS-80 and Deep sea-3/OPU3) within gammaproteobacterial methanotrophs, one clade related to Group Z and one unknown group. Differential abundance of pHMO gene transcripts in plume and background suggests niche differentiation between groups. Corresponding 16S rRNA genes reflected similar phylogenetic and transcriptomic abundance trends. The novelty of transcriptionally active pHMOs we recovered from a hydrocarbon-rich hydrothermal plume suggests there are significant gaps in our knowledge of the diversity and function of these enzymes in the environment. PMID:23826624

  11. Modulation of MICAL Monooxygenase Activity by its Calponin Homology Domain: Structural and Mechanistic Insights

    PubMed Central

    Alqassim, Saif S.; Urquiza, Mauricio; Borgnia, Eitan; Nagib, Marc; Amzel, L. Mario; Bianchet, Mario A.

    2016-01-01

    MICALs (Molecule Interacting with CasL) are conserved multidomain enzymes essential for cytoskeletal reorganization in nerve development, endocytosis, and apoptosis. In these enzymes, a type-2 calponin homology (CH) domain always follows an N-terminal monooxygenase (MO) domain. Although the CH domain is required for MICAL-1 cellular localization and actin-associated function, its contribution to the modulation of MICAL activity towards actin remains unclear. Here, we present the structure of a fragment of MICAL-1 containing the MO and the CH domains—determined by X-ray crystallography and small angle scattering—as well as kinetics experiments designed to probe the contribution of the CH domain to the actin-modification activity. Our results suggest that the CH domain, which is loosely connected to the MO domain by a flexible linker and is far away from the catalytic site, couples F-actin to the enhancement of redox activity of MICALMO-CH by a cooperative mechanism involving a trans interaction between adjacently bound molecules. Binding cooperativity is also observed in other proteins regulating actin assembly/disassembly dynamics, such as ADF/Cofilins. PMID:26935886

  12. Crystal Structure and Characterization of Particulate Methane Monooxygenase from Methylocystis species Strain M

    PubMed Central

    Smith, Stephen M.; Rawat, Swati; Telser, Joshua; Hoffman, Brian M.; Stemmler, Timothy L.; Rosenzweig, Amy C.

    2012-01-01

    Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. Previous biochemical and structural studies of pMMO have focused on preparations from Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b. A pMMO from a third organism, Methylocystis species strain M, has been isolated and characterized. Both membrane-bound and solubilized Methylocystis sp. strain M pMMO contain ~2 copper ions per 100 kDa protomer and exhibit copper-dependent propylene epoxidation activity. Spectroscopic data indicate that Methylocystis sp. strain M pMMO contains a mixture of CuI and CuII, of which the latter exhibits two distinct type 2 CuII electron paramagnetic resonance (EPR) signals. Extended X-ray absorption fine structure (EXAFS) data are best fit with a mixture of Cu–O/N and Cu–Cu ligand environments with a Cu–Cu interaction at 2.52–2.64 Å. The crystal structure of Methylocystis sp. strain M pMMO was determined to 2.68 Å resolution and is the best quality pMMO structure obtained to date. It provides a revised model for the pmoA and pmoC subunits and has led to an improved model of M. capsulatus (Bath) pMMO. In these new structures, the intramembrane zinc/copper binding site has a different coordination environment from that in previous models. PMID:22013879

  13. Reconstitution of {beta}-carotene hydroxylase activity of thermostable CYP175A1 monooxygenase

    SciTech Connect

    Momoi, Kyoko; Hofmann, Ute; Schmid, Rolf D.; Urlacher, Vlada B. . E-mail: itbvkha@po.uni-stuttgart.de

    2006-01-06

    CYP175A1 is a thermostable P450 Monooxygenase from Thermus thermophilus HB27, demonstrating in vivo activity towards {beta}-carotene. Activity of CYP175A1 was reconstituted in vitro using artificial electron transport proteins. First results were obtained in the mixture with a crude Escherichia coli cell extract at 37 {sup o}C. In this system, {beta}-carotene was hydroxylated to {beta}-cryptoxanthin. The result indicated the presence of electron transport enzymes among the E. coli proteins, which are suitable for CYP175A1. However, upon in vitro reconstitution of CYP175A1 activity with purified recombinant flavodoxin and flavodoxin reductase from E. coli, only very low {beta}-cryptoxanthin production was observed. Remarkably, with another artificial electron transport system, putidaredoxin and putidaredoxin reductase from Pseudomonas putida, purified CYP175A1 enzyme hydroxylated {beta}-carotene at 3- and also 3'-positions, resulting in {beta}-cryptoxanthin and zeaxanthin. Under the optimal reaction conditions, the turnover rate of the enzyme reached 0.23 nmol {beta}-cryptoxanthin produced per nmol P450 per min.

  14. Copper-dependent reciprocal transcriptional regulation of methane monooxygenase genes in Methylococcus capsulatus and Methylosinus trichosporium.

    PubMed

    Nielsen, A K; Gerdes, K; Murrell, J C

    1997-07-01

    The methanotrophic bacteria Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b convert methane to methanol using the enzyme, methane monooxygenase (MMO). These bacteria are able to express two distinct MMOs: a cytoplasmic or soluble form (sMMO) and a membrane-bound or particulate form (pMMO). Differential expression of sMMO and pMMO is regulated by the amount of copper ions available to the cells; sMMO is expressed at low copper-biomass ratios, whereas pMMO is expressed at high copper-biomass ratios. In both methanotrophs, transcription of the sMMO gene cluster is negatively regulated by copper ions. Data suggest that transcription of the M. trichosporium OB3b sMMO gene cluster is directed from a sigma54-like and a sigma70-like promoter. The pMMO (pmo) genes of M. capsulatus (Bath) are transcribed into a polycistronic mRNA of 3.3 kb. The synthesis of this mRNA was activated by copper ions. Activation of pmo transcription by copper ions was concomitant with repression of sMMO gene transcription in both methanotrophs. This suggests that a common regulatory pathway may be involved in the transcriptional switch between sMMO and pMMO gene expression. PMID:9282751

  15. Oxidation of ultrafast radical clock substrate probes by the soluble methane monooxygenase from Methylococcus capsulatus (Bath).

    PubMed

    Valentine, A M; LeTadic-Biadatti, M H; Toy, P H; Newcomb, M; Lippard, S J

    1999-04-16

    Radical clock substrate probes were used to assess the viability of a discrete substrate radical species in the mechanism of hydrocarbon oxidation by the soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath). New substituted cyclopropane probes were used with very fast ring-opening rate constants and other desirable attributes, such as the ability to discriminate between radical and cationic intermediates. Oxidation of these substrates by a reconstituted sMMO system resulted in no rearranged products, allowing an upper limit of 150 fs to be placed on the lifetime of a putative radical species. This limit strongly suggests that there is no such substrate radical intermediate. The two enantiomers of trans-1-methyl-2-phenyl-cyclopropane were prepared, and the regioselectivity of their oxidation to the corresponding cyclopropylmethanol and cyclopropylphenol products was determined. The results are consistent with selective orientation of the two enantiomeric substrates in the hydrophobic cavity at the active site of sMMO, specific models for which were examined by molecular modeling. PMID:10196150

  16. Effects of the compounds 2-methoxynaphthoquinone, 2-propoxynaphthoquinone, and 2-isopropoxynaphthoquinone on ecdysone 20-monooxygenase activity.

    PubMed

    Mitchell, Martin J; Brescia, Aaron I; Smith, Stan L; Morgan, E David

    2007-09-01

    The effects of the natural compound 2-methoxy-1,4-naphthoquinone, isolated from the leaves of Impatiens glandulifera and the synthetic compounds 2-propoxy-1,4-naphthoquinone and 2-isopropoxy-1,4-naphthoquinone on ecdysone 20-monooxygenase (E-20-M) activity were examined in three insect species. Homogenates of wandering stage third instar larvae of Drosophila melanogaster, or abdomens from adult female Aedes aegypti, or fat body or midgut from fifth instar larvae of Manduca sexta were incubated with radiolabelled ecdysone and increasing concentrations (from 1 x 10(-8) to 1 x 10(-3) M) of the three compounds. All three compounds were found to inhibit in a dose-dependent fashion the E-20-M activity in the three insect species. The concentration of these compounds required to elicit a 50% inhibition of this steroid hydroxylase activity in the three insect species examined ranged from approximately 3 x 10(-5) to 7 x 10(-4) M. PMID:17694563

  17. Flavin containing monooxygenase 3 exerts broad effects on glucose and lipid metabolism and atherosclerosis.

    PubMed

    Shih, Diana M; Wang, Zeneng; Lee, Richard; Meng, Yonghong; Che, Nam; Charugundla, Sarada; Qi, Hannah; Wu, Judy; Pan, Calvin; Brown, J Mark; Vallim, Thomas; Bennett, Brian J; Graham, Mark; Hazen, Stanley L; Lusis, Aldons J

    2015-01-01

    We performed silencing and overexpression studies of flavin containing monooxygenase (FMO) 3 in hyperlipidemic mouse models to examine its effects on trimethylamine N-oxide (TMAO) levels and atherosclerosis. Knockdown of hepatic FMO3 in LDL receptor knockout mice using an antisense oligonucleotide resulted in decreased circulating TMAO levels and atherosclerosis. Surprisingly, we also observed significant decreases in hepatic lipids and in levels of plasma lipids, ketone bodies, glucose, and insulin. FMO3 overexpression in transgenic mice, on the other hand, increased hepatic and plasma lipids. Global gene expression analyses suggested that these effects of FMO3 on lipogenesis and gluconeogenesis may be mediated through the PPARα and Kruppel-like factor 15 pathways. In vivo and in vitro results were consistent with the concept that the effects were mediated directly by FMO3 rather than trimethylamine/TMAO; in particular, overexpression of FMO3 in the human hepatoma cell line, Hep3B, resulted in significantly increased glucose secretion and lipogenesis. Our results indicate a major role for FMO3 in modulating glucose and lipid homeostasis in vivo, and they suggest that pharmacologic inhibition of FMO3 to reduce TMAO levels would be confounded by metabolic interactions. PMID:25378658

  18. Flavin containing monooxygenase 3 exerts broad effects on glucose and lipid metabolism and atherosclerosis[S

    PubMed Central

    Shih, Diana M.; Wang, Zeneng; Lee, Richard; Meng, Yonghong; Che, Nam; Charugundla, Sarada; Qi, Hannah; Wu, Judy; Pan, Calvin; Brown, J. Mark; Vallim, Thomas; Bennett, Brian J.; Graham, Mark; Hazen, Stanley L.; Lusis, Aldons J.

    2015-01-01

    We performed silencing and overexpression studies of flavin containing monooxygenase (FMO) 3 in hyperlipidemic mouse models to examine its effects on trimethylamine N-oxide (TMAO) levels and atherosclerosis. Knockdown of hepatic FMO3 in LDL receptor knockout mice using an antisense oligonucleotide resulted in decreased circulating TMAO levels and atherosclerosis. Surprisingly, we also observed significant decreases in hepatic lipids and in levels of plasma lipids, ketone bodies, glucose, and insulin. FMO3 overexpression in transgenic mice, on the other hand, increased hepatic and plasma lipids. Global gene expression analyses suggested that these effects of FMO3 on lipogenesis and gluconeogenesis may be mediated through the PPARα and Kruppel-like factor 15 pathways. In vivo and in vitro results were consistent with the concept that the effects were mediated directly by FMO3 rather than trimethylamine/TMAO; in particular, overexpression of FMO3 in the human hepatoma cell line, Hep3B, resulted in significantly increased glucose secretion and lipogenesis. Our results indicate a major role for FMO3 in modulating glucose and lipid homeostasis in vivo, and they suggest that pharmacologic inhibition of FMO3 to reduce TMAO levels would be confounded by metabolic interactions. PMID:25378658

  19. Crystallization and preliminary structural analysis of dibenzothiophene monooxygenase (DszC) from Rhodococcus erythropolis

    PubMed Central

    Duan, Xiaolu; Zhang, Liang; Zhou, Daming; Ji, Kaihua; Ma, Ting; Shui, Wenqing; Li, Guoqiang; Li, Xin

    2013-01-01

    Dibenzothiophene (DBT) and its derivatives are typical sulfur compounds found in fossil fuels. These compounds show resistance to the hydrodesulfuriz­ation treatment that is commonly used in industry. Dibenzothiophene monooxygenase (DszC) is responsible for the oxidation of DBT, which is the first and the rate-limiting step in the DBT enzymatic desulfurization 4S pathway. In this study, the crystal structure of DszC from Rhodococcus erythropolis DS-3 is reported. The crystal of native DszC belonged to space group P1, with unit-cell parameters a = 96.16, b = 96.27, c = 98.56 Å, α = 81.03, β = 67.57, γ = 85.84°. To determine the phase, SAD X-ray diffraction data were collected from a SeMet-derivative DszC crystal, which also belonged to space group P1, with unit-cell parameters a = 95.379, b = 95.167, c = 94.891 Å, α = 87.046, β = 70.536, γ = 79.738°. Further structural analysis of DszC is in progress. PMID:23722833

  20. Taxol biosynthesis: taxane 13 alpha-hydroxylase is a cytochrome P450-dependent monooxygenase.

    PubMed

    Jennewein, S; Rithner, C D; Williams, R M; Croteau, R B

    2001-11-20

    A central feature in the biosynthesis of Taxol is oxygenation at multiple positions of the taxane core structure, reactions that are considered to be mediated by cytochrome P450-dependent monooxygenases. A PCR-based differential display-cloning approach, using Taxus (yew) cells induced for Taxol production, yielded a family of related cytochrome P450 genes, one of which was assigned as a taxane 10 beta-hydroxylase by functional expression in yeast. The acquired clones that did not function in yeast were heterologously expressed by using the Spodoptera fugiperda-baculovirus-based system and were screened for catalytic capability by using taxa-4(20),11(12)-dien-5 alpha-ol and its acetate ester as test substrates. This approach allowed identification of one of the cytochrome P450 clones (which bore 63% deduced sequence identity to the aforementioned taxane 10 beta-hydroxylase) as a taxane 13 alpha-hydroxylase by chromatographic and spectrometric characterization of the corresponding recombinant enzyme product. The demonstration of a second relevant hydroxylase from the induced family of cytochrome P450 genes validates this strategy for elucidating the oxygenation steps of taxane diterpenoid (taxoid) metabolism. Additionally, substrate specificity studies with the available cytochrome P450 hydroxylases now indicate that there is likely more than one biosynthetic route to Taxol in yew species. PMID:11707604

  1. Influence of liver disease and environmental factors on hepatic monooxygenase activity in vitro.

    PubMed

    Brodie, M J; Boobis, A R; Bulpitt, C J; Davies, D S

    1981-01-01

    The effects of liver disease and environmental factors on hepatic microsomal cytochrome P-450 content, NADPH-cytochrome c reductase (reductase) activity and aryl hydrocarbon hydroxylase (AHH) activity have been simultaneously investigated in 70 patients undergoing diagnostic liver biopsy. The activity of reductase was not significantly affected by the presence of liver disease or any of the environmental factors studied. Cytochrome P-450 content decreased with increasing severity of liver disease whereas AHH activity was only significantly reduced in biopsies showing hepatocellular destruction. None of the parameters of monooxygenase activity varied significantly with the age or sex of the patients. Alcohol excess was associated with decreased cytochrome P-450 content and AHH activity and this effect was independent of the histological status of the biopsy. Both high caffeine intake and cigarette smoking increased AHH activity in the absence of any change in cytochrome P-450 content. There was a positive correlation between the number of meat meals eaten per week and cytochrome P-450 content. Chronic treatment with enzyme-inducing anticonvulsants appeared to increase both cytochrome P-450 content and AHH activity. Despite differential effects of liver disease and environmental influences on cytochrome P-450 content and AHH activity there was a highly significant correlation between the two parameters. The results of the present study correlate well with the known effects of disease and environment on drug metabolism in vivo. PMID:7308271

  2. Monooxygenase mediating catecholestrogen formation by rat anterior pituitary is an estrogen-4-hydroxylase.

    PubMed

    Bui, Q D; Weisz, J

    1989-02-01

    Microsomes from rat anterior pituitaries (AP) were incubated with (3H)estradiol under conditions previously shown to support catecholestrogen (CE) formation by placental microsomes via an NADPH- or an organic hydroperoxide-dependent, peroxidatic mechanism. Under conditions optimized for monooxygenase activity (pH 8.0, 5 mM NADPH), 4-hydroxylation predominated (apparent Vmax = 65 pmol and 13 pmol/mg protein/30 min for 4- and 2-hydroxy-E2, respectively). Under conditions optimized for peroxidatic activity (pH 6.0, 50 mM cumene hydroperoxide) 2- and 4-hydroxylated-E2 were produced in similar amounts. Thus in the AP, unlike in other target tissues studied, NADPH-dependent CE synthetase is a 4-hydroxylase and significant 2-hydroxylation occurs only via the peroxidatic mechanism. We propose that 4-hydroxylated CEs, which are both potent, long acting estrogens and catechols, serve as local mediators of actions of phenolic estrogens on the AP. PMID:2536311

  3. Purification and characterization of chlorophenol 4-monooxygenase from Burkholderia cepacia AC1100.

    PubMed Central

    Xun, L

    1996-01-01

    Burkholderia (formerly Pseudomonas) cepacia AC1100 mineralizes the herbicide 2,4,5-trichlorophenoxyacetate (2,4,5-T), and the first intermediate of 2,4,5-T degradation is 2,4,5-trichlorophenol. Chlorophenol 4-monooxygenase activity responsible for 2,4,5-trichlorophenol degradation was detected in the cell extract. The enzyme consisted of two components separated during purification, and both were purified to more than 95% homogeneity. The reconstituted enzyme catalyzed the hydroxylation of several tested chlorophenols with the coconsumption of NADH and oxygen. In addition to chlorophenols, the enzyme also hydroxylated some chloro-p-hydroquinones with the coconsumption of NADH and oxygen. Apparently, the single enzyme was responsible for converting 2,4,5-trichlorophenol to 2,5-dichloro-p-hydroquinone and then to 5-chlorohydroxyquinol (5-chloro-1,2,4-trihydroxybenzene). Component A had a molecular weight of 22,000 and contained flavin adenine dinucleotide. Component A alone catalyzed NADH-dependent cytochrome c reduction, indicating that it had reductase activity. Component B had a molecular weight of 58,000, and no catalytic activity has yet been shown by itself. PMID:8626333

  4. Bacterial dioxygenase- and monooxygenase-catalysed sulfoxidation of benzo[b]thiophenes.

    PubMed

    Boyd, Derek R; Sharma, Narain D; McMurray, Brian; Haughey, Simon A; Allen, Christopher C R; Hamilton, John T G; McRoberts, W Colin; O'Ferrall, Rory A More; Nikodinovic-Runic, Jasmina; Coulombel, Lydie A; O'Connor, Kevin E

    2012-01-28

    Asymmetric heteroatom oxidation of benzo[b]thiophenes to yield the corresponding sulfoxides was catalysed by toluene dioxygenase (TDO), naphthalene dioxygenase (NDO) and styrene monooxygenase (SMO) enzymes present in P. putida mutant and E. coli recombinant whole cells. TDO-catalysed oxidation yielded the relatively unstable benzo[b]thiophene sulfoxide; its dimerization, followed by dehydrogenation, resulted in the isolation of stable tetracyclic sulfoxides as minor products with cis-dihydrodiols being the dominant metabolites. SMO mainly catalysed the formation of enantioenriched benzo[b]thiophene sulfoxide and 2-methyl benzo[b]thiophene sulfoxides which racemized at ambient temperature. The barriers to pyramidal sulfur inversion of 2- and 3-methyl benzo[b]thiophene sulfoxide metabolites, obtained using TDO and NDO as biocatalysts, were found to be ca.: 25-27 kcal mol(-1). The absolute configurations of the benzo[b]thiophene sulfoxides were determined by ECD spectroscopy, X-ray crystallography and stereochemical correlation. A site-directed mutant E. coli strain containing an engineered form of NDO, was found to change the regioselectivity toward preferential oxidation of the thiophene ring rather than the benzene ring. PMID:22134441

  5. Evidence for oxygen binding at the active site of particulate methane monooxygenase.

    PubMed

    Culpepper, Megen A; Cutsail, George E; Hoffman, Brian M; Rosenzweig, Amy C

    2012-05-01

    Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that converts methane to methanol in methanotrophic bacteria. The enzyme consists of three subunits, pmoB, pmoA, and pmoC, organized in an α(3)β(3)γ(3) trimer. Studies of intact pMMO and a recombinant soluble fragment of the pmoB subunit (denoted as spmoB) indicate that the active site is located within the soluble region of pmoB at the site of a crystallographically modeled dicopper center. In this work, we have investigated the reactivity of pMMO and spmoB with oxidants. Upon reduction and treatment of spmoB with O(2) or H(2)O(2) or pMMO with H(2)O(2), an absorbance feature at 345 nm is generated. The energy and intensity of this band are similar to those of the μ-η(2):η(2)-peroxo-Cu(II)(2) species formed in several dicopper enzymes and model compounds. The feature is not observed in inactive spmoB variants in which the dicopper center is disrupted, consistent with O(2) binding to the proposed active site. Reaction of the 345 nm species with CH(4) results in the disappearance of the spectroscopic feature, suggesting that this O(2) intermediate is mechanistically relevant. Taken together, these observations provide strong new support for the identity and location of the pMMO active site. PMID:22540911

  6. Crystal structure and characterization of particulate methane monooxygenase from Methylocystis species strain M.

    PubMed

    Smith, Stephen M; Rawat, Swati; Telser, Joshua; Hoffman, Brian M; Stemmler, Timothy L; Rosenzweig, Amy C

    2011-11-29

    Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. Previous biochemical and structural studies of pMMO have focused on preparations from Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b. A pMMO from a third organism, Methylocystis species strain M, has been isolated and characterized. Both membrane-bound and solubilized Methylocystis sp. strain M pMMO contain ~2 copper ions per 100 kDa protomer and exhibit copper-dependent propylene epoxidation activity. Spectroscopic data indicate that Methylocystis sp. strain M pMMO contains a mixture of Cu(I) and Cu(II), of which the latter exhibits two distinct type 2 Cu(II) electron paramagnetic resonance (EPR) signals. Extended X-ray absorption fine structure (EXAFS) data are best fit with a mixture of Cu-O/N and Cu-Cu ligand environments with a Cu-Cu interaction at 2.52-2.64 Å. The crystal structure of Methylocystis sp. strain M pMMO was determined to 2.68 Å resolution and is the best quality pMMO structure obtained to date. It provides a revised model for the pmoA and pmoC subunits and has led to an improved model of M. capsulatus (Bath) pMMO. In these new structures, the intramembrane zinc/copper binding site has a different coordination environment from that in previous models. PMID:22013879

  7. Soluble methane monooxygenase component B gene probe for identification of methanotrophs that rapidly degrade trichloroethylene

    SciTech Connect

    Hsienchyang Tsien; Hanson, R.S. )

    1992-03-01

    Restriction fragment length polymorphisms, Western blot (immunoblot) analysis, and fluorescence-labelled signature probes were used for the characterization of methanotrophic bacteria as well as for the identification of methanotrophs which contained the soluble methane monooxygenase (MMO) gene and were able to degrade trichloroethylene (TCE). The gene encoding a soluble MMO component B protein from Methylosinus trichosporium OB3b was cloned. It contained a 2.2-kb EcoRI fragment. With this cloned component B gene as probe, methanotroph types I, II, and X and environmental and bioreactor samples were screened for the presence of the gene encoding soluble MMO. Among twelve pure or mixed cultures, DNA fragments of seven methanotrophs hybridized with the soluble MMO B gene probe. When grown in media with limited copper, all of these bacteria degraded TCE. All of them are type II methanotrophs. The soluble MMO component B gene of the type X methanotroph, Methylococcus capsulatus Bath, did not hybridize to the M. trichosporium OB3b soluble MMO component B gene probe, although M. capsulatus Baath also produces a soluble MMO.

  8. Crystal Structure and Characterization of Particulate Methane Monooxygenase from Methylocystis species Strain M

    SciTech Connect

    Smith, Stephen M.; Rawat, Swati; Telser, Joshua; Hoffman, Brian M.; Stemmler, Timothy L.; Rosenzweig, Amy C.

    2012-02-08

    Particulate methane monooxygenase (pMMO) is an integral membrane metalloenzyme that oxidizes methane to methanol in methanotrophic bacteria. Previous biochemical and structural studies of pMMO have focused on preparations from Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b. A pMMO from a third organism, Methylocystis species strain M, has been isolated and characterized. Both membrane-bound and solubilized Methylocystis sp. strain M pMMO contain {approx}2 copper ions per 100 kDa protomer and exhibit copper-dependent propylene epoxidation activity. Spectroscopic data indicate that Methylocystis sp. strain M pMMO contains a mixture of Cu{sup I} and Cu{sup II}, of which the latter exhibits two distinct type 2 Cu{sup II} electron paramagnetic resonance (EPR) signals. Extended X-ray absorption fine structure (EXAFS) data are best fit with a mixture of Cu-O/N and Cu-Cu ligand environments with a Cu-Cu interaction at 2.52-2.64 {angstrom}. The crystal structure of Methylocystis sp. strain M pMMO was determined to 2.68 {angstrom} resolution and is the best quality pMMO structure obtained to date. It provides a revised model for the pmoA and pmoC subunits and has led to an improved model of M. capsulatus (Bath) pMMO. In these new structures, the intramembrane zinc/copper binding site has a different coordination environment from that in previous models.

  9. Bacteriohemerythrin bolsters the activity of the particulate methane monooxygenase (pMMO) in Methylococcus capsulatus (Bath).

    PubMed

    Chen, Kelvin H-C; Wu, Hsin-Hui; Ke, Si-Fu; Rao, Ya-Ting; Tu, Chia-Ming; Chen, Yu-Ping; Kuei, Kuo-Hsuan; Chen, Ying-Siao; Wang, Vincent C-C; Kao, Wei-Chun; Chan, Sunney I

    2012-06-01

    Recently, a native bacteriohemerythrin (McHr) has been identified in Methylococcus capsulatus (Bath). Both the particulate methane monooxygenase (pMMO) and McHr are over-expressed in cells of this bacterium when this strain of methanotroph is cultured and grown under high copper to biomass conditions. It has been suggested that the role of the McHr is to provide a shuttle to transport dioxygen from the cytoplasm of the cell to the intra-cytoplasmic membranes for consumption by the pMMO. Indeed, McHr enhances the activity of the pMMO when pMMO-enriched membranes are used to assay the enzyme activity. We find that McHr can dramatically improve the activity of pMMO toward the epoxidation of propylene to propylene oxide. The maximum activity is observed at a pMMO to McHr concentration ratio of 4:1, where we have obtained specific activities of 103.7nmol propylene oxide/min/mg protein and 122.8nmol propylene oxide/min/mg protein at 45°C when the turnover is driven by NADH and duroquinol, respectively. These results are consistent with the suggestion that the bacterium requires McHr to deliver dioxygen to the pMMO in the intra-cytoplasmic membranes to accomplish efficient catalysis of methane oxidation when the enzyme is over-expressed in the cells. PMID:22484247

  10. Functional characterization of choline monooxygenase, an enzyme for betaine synthesis in plants.

    PubMed

    Hibino, Takashi; Waditee, Rungaroon; Araki, Etsuko; Ishikawa, Hiroshi; Aoki, Kenji; Tanaka, Yoshito; Takabe, Teruhiro

    2002-11-01

    In plants, the first step in betaine synthesis was shown to be catalyzed by a novel Rieske-type iron-sulfur enzyme, choline monooxygenase (CMO). Although CMO so far has been found only in Chenopodiaceae and Amaranthaceae, the recent genome sequence suggests the presence of a CMO-like gene in Arabidopsis, a betaine non-accumulating plant. Here, we examined the functional properties of CMO expressed in Escherichia coli, cyanobacterium, and Arabidopsis thaliana. We found that E. coli cells in which choline dehydrogenase (CDH) was replaced with spinach CMO accumulate betaine and complement the salt-sensitive phenotype of the CDH-deleted E. coli mutant. Changes of Cys-181 in spinach CMO to Ser, Thr, and Ala and His-287 to Gly, Val, and Ala abolished the accumulation of betaine. The Arabidopsis CMO-like gene was transcribed in Arabidopsis, but its protein was not detected. When the Arabidopsis CMO-like gene was expressed in E. coli, the protein was detected but was found not to promote betaine sysnthesis. Overexpression of spinach CMO in E. coli, Synechococcus sp. PCC7942, and Arabidopsis conferred resistance to abiotic stress. These facts clearly indicate that CMO, but not the CMO-like protein, could oxidize choline and that Cys-181 and His-287 are involved in the binding of Fe-S cluster and Fe, respectively. PMID:12192001

  11. Involvement of cytochrome P450 monooxygenases in the response of mosquito larvae to dietary plant xenobiotics.

    PubMed

    David, J P; Boyer, S; Mesneau, A; Ball, A; Ranson, H; Dauphin-Villemant, C

    2006-05-01

    The response of mosquito larvae to plant toxins found in their breeding sites was investigated by using Aedes aegypti larvae and toxic arborescent leaf litter as experimental models. The relation between larval tolerance to toxic leaf litter and cytochrome P450 monooxygenases (P450s) was examined at the toxicological, biochemical and molecular levels. Larvae pre-exposed to toxic leaf litter show a higher tolerance to those xenobiotics together with a strong increase in P450 activity levels. This enzymatic response is both time- and dose-dependent. The use of degenerate primers from various P450 genes (CYPs) allowed us to isolate 16 new CYP genes belonging to CYP4, CYP6 and CYP9 families. Expression studies revealed a 2.3-fold over-expression of 1 CYP gene (CYP6AL1) after larval pre-exposure to toxic leaf litter, this gene being expressed at a high level in late larval and pupal stages and in fat bodies and midgut. The CYP6AL1 protein has a high level of identity with other insect's CYPs involved in xenobiotic detoxification. The role of CYP genes in tolerance to natural xenobiotics and the importance of such adaptive responses in the capacity of mosquitoes to colonize new habitats and to develop insecticide resistance mechanisms are discussed. PMID:16651188

  12. C. elegans flavin-containing monooxygenase-4 is essential for osmoregulation in hypotonic stress.

    PubMed

    Hirani, Nisha; Westenberg, Marcel; Seed, Paul T; Petalcorin, Mark I R; Dolphin, Colin T

    2016-01-01

    Studies in Caenorhabditis elegans have revealed osmoregulatory systems engaged when worms experience hypertonic conditions, but less is known about measures employed when faced with hypotonic stress. Inactivation of fmo-4, which encodes flavin-containing monooxygenase-4, results in dramatic hypoosmotic hypersensitivity; worms are unable to prevent overwhelming water influx and swell rapidly, finally rupturing due to high internal hydrostatic pressure. fmo-4 is expressed prominently in hypodermis, duct and pore cells but is excluded from the excretory cell. Thus, FMO-4 plays a crucial osmoregulatory role by promoting clearance of excess water that enters during hypotonicity, perhaps by synthesizing an osmolyte that acts to establish an osmotic gradient from excretory cell to duct and pore cells. C. elegans FMO-4 contains a C-terminal extension conserved in all nematode FMO-4s. The coincidently numbered human FMO4 also contains an extended C-terminus with features similar to those of FMO-4. Although these shared sequence characteristics suggest potential orthology, human FMO4 was unable to rescue the fmo-4 osmoregulatory defect. Intriguingly, however, mammalian FMO4 is expressed predominantly in the kidney - an appropriate site if it too is, or once was, involved in osmoregulation. PMID:27010030

  13. Reaction Mechanism of the Bicopper Enzyme Peptidylglycine α-Hydroxylating Monooxygenase*

    PubMed Central

    Abad, Enrique; Rommel, Judith B.; Kästner, Johannes

    2014-01-01

    Peptidylglycine α-hydroxylating monooxygenase is a noninteracting bicopper enzyme that stereospecifically hydroxylates the terminal glycine of small peptides for its later amidation. Neuroendocrine messengers, such as oxytocin, rely on the biological activity of this enzyme. Each catalytic turnover requires one oxygen molecule, two protons from the solvent, and two electrons. Despite this enzyme having been widely studied, a consensus on the reaction mechanism has not yet been found. Experiments and theoretical studies favor a pro-S abstraction of a hydrogen atom followed by the rebinding of an OH group. However, several hydrogen-abstracting species have been postulated; because two protons are consumed during the reaction, several protonation states are available. An electron transfer between the copper atoms could play a crucial role for the catalysis as well. This leads to six possible abstracting species. In this study, we compare them on equal footing. We perform quantum mechanics/molecular mechanics calculations, considering the glycine hydrogen abstraction. Our results suggest that the most likely mechanism is a protonation of the abstracting species before the hydrogen abstraction and another protonation as well as a reduction before OH rebinding. PMID:24668808

  14. Interactions of a fungal lytic polysaccharide monooxygenase with β-glucan substrates and cellobiose dehydrogenase.

    PubMed

    Courtade, Gaston; Wimmer, Reinhard; Røhr, Åsmund K; Preims, Marita; Felice, Alfons K G; Dimarogona, Maria; Vaaje-Kolstad, Gustav; Sørlie, Morten; Sandgren, Mats; Ludwig, Roland; Eijsink, Vincent G H; Aachmann, Finn Lillelund

    2016-05-24

    Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze oxidative cleavage of glycosidic bonds using molecular oxygen and an external electron donor. We have used NMR and isothermal titration calorimetry (ITC) to study the interactions of a broad-specificity fungal LPMO, NcLPMO9C, with various substrates and with cellobiose dehydrogenase (CDH), a known natural supplier of electrons. The NMR studies revealed interactions with cellohexaose that center around the copper site. NMR studies with xyloglucans, i.e., branched β-glucans, showed an extended binding surface compared with cellohexaose, whereas ITC experiments showed slightly higher affinity and a different thermodynamic signature of binding. The ITC data also showed that although the copper ion alone hardly contributes to affinity, substrate binding is enhanced for metal-loaded enzymes that are supplied with cyanide, a mimic of O2 (-) Studies with CDH and its isolated heme b cytochrome domain unambiguously showed that the cytochrome domain of CDH interacts with the copper site of the LPMO and that substrate binding precludes interaction with CDH. Apart from providing insights into enzyme-substrate interactions in LPMOs, the present observations shed new light on possible mechanisms for electron supply during LPMO action. PMID:27152023

  15. Cellulose Surface Degradation by a Lytic Polysaccharide Monooxygenase and Its Effect on Cellulase Hydrolytic Efficiency*

    PubMed Central

    Eibinger, Manuel; Ganner, Thomas; Bubner, Patricia; Rošker, Stephanie; Kracher, Daniel; Haltrich, Dietmar; Ludwig, Roland; Plank, Harald; Nidetzky, Bernd

    2014-01-01

    Lytic polysaccharide monooxygenase (LPMO) represents a unique principle of oxidative degradation of recalcitrant insoluble polysaccharides. Used in combination with hydrolytic enzymes, LPMO appears to constitute a significant factor of the efficiency of enzymatic biomass depolymerization. LPMO activity on different cellulose substrates has been shown from the slow release of oxidized oligosaccharides into solution, but an immediate and direct demonstration of the enzyme action on the cellulose surface is lacking. Specificity of LPMO for degrading ordered crystalline and unordered amorphous cellulose material of the substrate surface is also unknown. We show by fluorescence dye adsorption analyzed with confocal laser scanning microscopy that a LPMO (from Neurospora crassa) introduces carboxyl groups primarily in surface-exposed crystalline areas of the cellulosic substrate. Using time-resolved in situ atomic force microscopy we further demonstrate that cellulose nano-fibrils exposed on the surface are degraded into shorter and thinner insoluble fragments. Also using atomic force microscopy, we show that prior action of LPMO enables cellulases to attack otherwise highly resistant crystalline substrate areas and that it promotes an overall faster and more complete surface degradation. Overall, this study reveals key characteristics of LPMO action on the cellulose surface and suggests the effects of substrate morphology on the synergy between LPMO and hydrolytic enzymes in cellulose depolymerization. PMID:25361767

  16. 40 CFR 721.10626 - 1,4-Butanediol, polymer with substituted alkane and substituted methylene biscarbomonocycle, 2...

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... activities. Requirements as specified in § 721.80(o) and (y)(1). (ii) (b) Specific requirements. The... substituted alkane and substituted methylene biscarbomonocycle, 2-hydroxyalkyl acrylate-blocked (PMN...

  17. n-Alkane hydroconversion on Zeogrid and colloidal ZSM-5 assembled from aluminosilicate nanoslabs of MFI framework type.

    PubMed

    Aerts, Alexander; Huybrechts, Ward; Kremer, Sebastien P B; Kirschhock, Christine E A; Theunissen, Elisabeth; Van Isacker, Annabel; Denayer, Joeri F M; Baron, Gino V; Thybaut, Joris W; Marin, Guy B; Jacobs, Pierre A; Martens, Johan A

    2003-08-01

    n-Alkane hydroisomerisation and hydrocracking experiments reveal that ZSM-5 materials synthesized by self-assembly of nanoslabs show different molecular shape selectivity than ZSM-5 synthesized by hydrothermal methods. PMID:12932017

  18. n-Alkane biosynthetic hydrogen isotope fractionation is not constant throughout the growing season in the riparian tree Salix viminalis

    NASA Astrophysics Data System (ADS)

    Newberry, Sarah L.; Kahmen, Ansgar; Dennis, Paul; Grant, Alastair

    2015-09-01

    Compound-specific δ2H values of leaf wax n-alkanes have emerged as a potentially powerful paleohydrological proxy. Research suggests terrestrial plant n-alkane δ2H values are strongly correlated with meteoric water δ2H values, and may provide information on temperature, relative humidity, evaporation, and precipitation. This is based upon several assumptions, including that biosynthetic fractionation of n-alkanes during synthesis is constant within a single species. Here we present a multi-isotope study of the n-alkanes of riparian Salix viminalis growing in Norwich, UK. We measured n-alkane δ2H, leaf water δ2H, xylem water δ2H, and bulk foliar δ13C and evaluated the variability of n-alkane δ2H values and net biosynthetic fractionation (εlw-wax) over a whole growing season. S. viminalis n-alkane δ2H values decreased by 40‰ between the start of the growing season in April and the time when they stabilized in July. Variation in leaf and xylem water δ2H did not explain this variability. εlw-wax varied from -116‰ during leaf expansion in April to -156‰ during the stable phase. This suggests that differential biosynthetic fractionation was responsible for the strong seasonal trends in S. viminalis n-alkane δ2H values. We suggest that variability in εlw-wax is driven by seasonal differences in the carbohydrate source and thus the NADPH used in n-alkane biosynthesis, with stored carbohydrates utilized during spring and recent occurring growing season assimilates used later in the season. This is further supported by bulk foliar δ13C values, which are 13C-enriched during the period of leaf flush, relative to the end of the growing season. Our results challenge the assumption that biosynthetic fractionation is constant for a given species, and suggest that 2H-enriched stored assimilates are an important source for n-alkane biosynthesis early in the growing season. These findings have implications for the interpretation of sedimentary n-alkanes and call

  19. Revisiting Mt. Kilimanjaro: Do n-alkane biomarkers in soils reflect the δ2H isotopic composition of precipitation?

    NASA Astrophysics Data System (ADS)

    Zech, M.; Zech, R.; Rozanski, K.; Hemp, A.; Gleixner, G.; Zech, W.

    2014-06-01

    During the last decade compound-specific deuterium (δ2H) analysis of plant leaf wax-derived n-alkanes has become a promising and popular tool in paleoclimate research. This is based on the widely accepted assumption that n-alkanes in soils and sediments generally reflect δ2H of precipitation (δ2Hprec). Recently, several authors suggested that δ2H of n-alkanes (δ2H,sub>n-alkanes) can also be used as proxy in paleoaltimetry studies. Here we present results from a δ2H transect study (~1500 to 4000 m a.s.l.) carried out on precipitation and soil samples taken from the humid southern slopes of Mt. Kilimanjaro. Contrary to earlier suggestions, a distinct altitude effect in δ2Hprec is present above ~2000 m a.s.l., i.e. δ2Hprec values become more negative with increasing altitude. The compound-specific δ2H values of nC27 and nC29 do not confirm this altitudinal trend, but rather become more positive both in the O-layers (organic layers) and the Ah-horizons (mineral topsoils). Although our δ2Hn-alkane results are in agreement with previously published results from the southern slopes of Mt. Kilimanjaro (Peterse et al., 2009, BG, 6, 2799-2807), a major re-interpretation is required given that the δ2Hn-alkane results do not reflect the δ2Hprec results. The theoretical framework for this re-interpretation is based on the evaporative isotopic enrichment of leaf water associated with transpiration process. Modelling results show that relative humidity, decreasing considerably along the southern slopes of Mt. Kilimanjaro (from 78% at ~ 2000 m a.s.l. to 51% at 4000 m a.s.l.), strongly controls δ2Hleaf water. The modelled δ2H leaf water enrichment along the altitudinal transect matches well the measured 2H leaf water enrichment as assessed by using the δ2Hprec and δ2Hn-alkane results and biosynthetic fractionation during n-alkane biosynthesis in leaves. Given that our results clearly demonstrate that n-alkanes in soils do not simply reflect δ2Hprec but rather δ2

  20. Anaerobic oxidation of short-chain alkanes in hydrothermal sediments: potential influences on sulfur cycling and microbial diversity

    PubMed Central

    Adams, Melissa M.; Hoarfrost, Adrienne L.; Bose, Arpita; Joye, Samantha B.; Girguis, Peter R.

    2013-01-01

    Short-chain alkanes play a substantial role in carbon and sulfur cycling at hydrocarbon-rich environments globally, yet few studies have examined the metabolism of ethane (C2), propane (C3), and butane (C4) in anoxic sediments in contrast to methane (C1). In hydrothermal vent systems, short-chain alkanes are formed over relatively short geological time scales via thermogenic processes and often exist at high concentrations. The sediment-covered hydrothermal vent systems at Middle Valley (MV, Juan de Fuca Ridge) are an ideal site for investigating the anaerobic oxidation of C1–C4 alkanes, given the elevated temperatures and dissolved hydrocarbon species characteristic of these metalliferous sediments. We examined whether MV microbial communities oxidized C1–C4 alkanes under mesophilic to thermophilic sulfate-reducing conditions. Here we present data from discrete temperature (25, 55, and 75°C) anaerobic batch reactor incubations of MV sediments supplemented with individual alkanes. Co-registered alkane consumption and sulfate reduction (SR) measurements provide clear evidence for C1–C4 alkane oxidation linked to SR over time and across temperatures. In these anaerobic batch reactor sediments, 16S ribosomal RNA pyrosequencing revealed that Deltaproteobacteria, particularly a novel sulfate-reducing lineage, were the likely phylotypes mediating the oxidation of C2–C4 alkanes. Maximum C1–C4 alkane oxidation rates occurred at 55°C, which reflects the mid-core sediment temperature profile and corroborates previous studies of rate maxima for the anaerobic oxidation of methane (AOM). Of the alkanes investigated, C3 was oxidized at the highest rate over time, then C4, C2, and C1, respectively. The implications of these results are discussed with respect to the potential competition between the anaerobic oxidation of C2–C4alkanes with AOM for available oxidants and the influence on the fate of C1 derived from these hydrothermal systems. PMID:23717305