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Sample records for alpha-amidating monooxygenase mrna

  1. Alternative mRNA splicing generates multiple forms of peptidyl-glycine alpha-amidating monooxygenase in rat atrium.

    PubMed Central

    Stoffers, D A; Green, C B; Eipper, B A

    1989-01-01

    Peptidyl-glycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the conversion of a variety of glycine-extended peptides into biologically active alpha-amidated product peptides in a reaction dependent on copper, ascorbate, and molecular oxygen. We have isolated and sequenced cDNAs representing the two major classes of PAM mRNA in the adult rat heart atrium. The two types of cDNA, rPAM-1 and rPAM-2, are identical except for the deletion of a 315-base-pair segment within the protein coding region in rPAM-2, suggesting that rPAM-1 and rPAM-2 arise by alternative splicing. Northern analysis using a cDNA probe derived from within the 315-base-pair region deleted in rPAM-2 visualized the larger of the PAM mRNAs in adult rat atrium and not the smaller, indicating that the presence or absence of this 315-nucleotide segment is a major feature distinguishing the two size forms of PAM mRNA. The 105 amino acid segment that distinguishes the two forms of atrial PAM contains a consensus N-glycosylation site and a paired basic amino acid site of potential importance in endoproteolytic processing. Comparison of the nucleotide sequences of rat, frog, and bovine PAM cDNAs reveals an extremely well conserved segment in the 3' untranslated region. The high degree of conservation in amino acid sequence throughout the catalytic, intragranular, and cytoplasmic domains of rat atrium, bovine pituitary, and frog skin PAM suggests that both the catalytic and noncatalytic domains of the protein subserve important functions. Images PMID:2911604

  2. Effect of cortisol on neurophysin I/oxytocin and peptidyl glycine-alpha-amidating mono-oxygenase mRNA expression in bovine luteal and granulosa cells.

    PubMed

    Ziolkowska, A; Mlynarczuk, J; Kotwica, J

    2013-01-01

    Cortisol stimulates the synthesis and secretion of oxytocin (OT) from bovine granulosa and luteal cells, but the molecular mechanisms of cortisol action remain unknown. In this study, granulosa cells or luteal cells from days 1-5 and 11-15 of the oestrous cycle were incubated for 4 or 8 h with cortisol (1 x 10(-5), 1 x 10(-7) M). After testing cell viability and hormone secretion (OT, progesterone, estradiol), we studied the effect of cortisol on mRNA expression for precursor of OT (NP-I/OT) and peptidyl glycine-alpha-amidating mono-oxygenase (PGA). The influence of RU 486 (1 x 10(-5) M), a progesterone receptor blocker and inhibitor of the glucocorticosteroid receptor (GR), on the expression for both genes was tested. Cortisol increased the mRNA expression for NP-I/OT and PGA in granulosa cells and stimulated the expression for NP-I/OT mRNA in luteal cells obtained from days 1-5 and days 11-15 of the oestrous cycle. Expression for PGA mRNA was increased only in luteal cells from days 11-15 of the oestrous cycle. In addition, RU 486 blocked the cortisol-stimulated mRNA expression for NP-I/OT and PGA in both types of cells. These data suggest that cortisol affects OT synthesis and secretion in bovine ovarian cells, by acting on the expression of key genes, that may impair ovary

  3. Novel substrates and inhibitors of peptidylglycine alpha-amidating monooxygenase.

    PubMed

    Katopodis, A G; May, S W

    1990-05-15

    Peptidylglycine alpha-amidating monooxygenase (PAM, EC 1.14.17.3) catalyzes the formation of alpha-amidated peptides from their glycine-extended precursors, thus playing a key role in the processing of peptide neurohormones. We now report that PAM readily catalyzes three alternate monooxygenase reactions--sulfoxidation, amine N-dealkylation, and O-dealkylation. Thus, (4-nitrobenzyl)thioacetic acid is converted to the analogous sulfoxide, N-(4-nitrobenzyl)glycine is converted to 4-nitrobenzylamine and glyoxylate, and [(4-nitrobenzyl)oxy]acetic acid is converted to 4-nitrobenzyl alcohol and glyoxylate. All these new activities display the characteristics expected for the normal PAM-catalyzed reductive oxygenation pathway and produce an equimolar amount of glyoxylate together with the heteroatom-containing dealkylation products. The ester [(4-methoxybenzoyl)oxy]acetic acid is not a PAM substrate, but is instead a good competitive inhibitor (KI = 0.48 mM). In addition, we report that the olefinic substrate analogues trans-benzoylacrylic acid and 4-phenyl-3-butenoic acid are potent time-dependent inactivators of PAM, with inactivation exhibiting the characteristics expected for mechanism-based inhibition. Monoethyl fumarate is also a time-dependent inactivator of PAM. Finally, we introduce several small non-peptide substrates for PAM by demonstrating that PAM catalyzes the transformation of hippuric acid and several ring-substituted derivatives to the corresponding benzamides and glyoxylic acid, with the most facile substrate of this class being 4-nitrohippuric acid. These compounds are the smallest amide substrates yet reported for PAM, and it is thus apparent that only the minimal structure of an acylglycine is required for PAM-catalyzed oxygenative amidation.

  4. Manipulation of neuropeptide biosynthesis through the expression of antisense RNA for peptidylglycine alpha-amidating monooxygenase.

    PubMed

    Mains, R E; Bloomquist, B T; Eipper, B A

    1991-02-01

    Stable cell lines with significantly elevated or diminished levels of a key neuropeptide processing enzyme, peptidylglycine alpha-amidating monooxygenase (PAM), were generated by transfection of a mouse pituitary cell line with expression vectors containing PAM cDNA in the sense or antisense orientation. By evaluating the ability of these cell lines to alpha-amidate endogenous neuropeptides, a rate-limiting role for PAM in neuropeptide alpha-amidation was demonstrated. Overexpression of either the full-length PAM precursor with its trans-membrane domain or a soluble protein containing only the monooxygenase domain of PAM led to increased alpha-amidation of endogenous neuropeptides. Overexpression of the full-length PAM led to an unexpected decrease in the endoproteolytic processing of endogenous prohormone; conversely, underexpression of PAM led to significantly enhanced endoproteolytic processing of endogenous prohormone. These data suggest that PAM may have additional functions in peptide processing.

  5. Alternative splicing governs sulfation of tyrosine or oligosaccharide on peptidylglycine alpha-amidating monooxygenase.

    PubMed

    Yun, H Y; Keutmann, H T; Eipper, B A

    1994-04-08

    Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the COOH-terminal alpha-amidation of neuro-endocrine peptides through the sequential action of monooxygenase and lyase domains contained within this bifunctional protein. Alternative splicing leads to the expression of soluble and integral membrane bifunctional PAM proteins as well as a soluble monofunctional monooxygenase. In order to determine how alternative splicing affects post-translational modification of PAM proteins, we investigated the sulfation of PAM proteins expressed in stably transfected hEK-293 cells. Metabolic labeling with [35S]SO4(2-) or [35S]methionine and immunoprecipitation demonstrated that [35S]SO4(2-) was efficiently incorporated into PAM proteins that have the noncatalytic exon A region following the monooxygenase domain (PAM-1 and PAM-4) and into a soluble bifunctional PAM protein (PAM-3). Alkaline hydrolysis, radiosequencing, and deglycosylation experiments demonstrated the presence of a sulfated tyrosine (Tyr965) in the COOH-terminal domain of PAM-3 and multiple sulfated O-glycans in the exon A region of PAM-1 and PAM-4. A mutant PAM-3 protein in which Tyr965 was changed to Ala965 (PAM-3/Y965A) was not sulfated and exhibited monooxygenase and lyase activities similar to those of wild type PAM-3. Pulse-chase and temperature block experiments showed that the PAM-3/Y965A protein exits the trans-Golgi network faster than wild type PAM-3. Thus inclusion of exon A results in the sulfation of O-glycans, while elimination of the transmembrane domain results in the sulfation of Tyr965.

  6. Signaling mediated by the cytosolic domain of peptidylglycine alpha-amidating monooxygenase.

    PubMed

    Alam, M R; Steveson, T C; Johnson, R C; Bäck, N; Abraham, B; Mains, R E; Eipper, B A

    2001-03-01

    The luminal domains of membrane peptidylglycine alpha-amidating monooxygenase (PAM) are essential for peptide alpha-amidation, and the cytosolic domain (CD) is essential for trafficking. Overexpression of membrane PAM in corticotrope tumor cells reorganizes the actin cytoskeleton, shifts endogenous adrenocorticotropic hormone (ACTH) from mature granules localized at the tips of processes to the TGN region, and blocks regulated secretion. PAM-CD interactor proteins include a protein kinase that phosphorylates PAM (P-CIP2) and Kalirin, a Rho family GDP/GTP exchange factor. We engineered a PAM protein unable to interact with either P-CIP2 or Kalirin (PAM-1/K919R), along with PAM proteins able to interact with Kalirin but not with P-CIP2. AtT-20 cells expressing PAM-1/K919R produce fully active membrane enzyme but still exhibit regulated secretion, with ACTH-containing granules localized to process tips. Immunoelectron microscopy demonstrates accumulation of PAM and ACTH in tubular structures at the trans side of the Golgi in AtT-20 cells expressing PAM-1 but not in AtT-20 cells expressing PAM-1/K919R. The ability of PAM to interact with P-CIP2 is critical to its ability to block exit from the Golgi and affect regulated secretion. Consistent with this, mutation of its P-CIP2 phosphorylation site alters the ability of PAM to affect regulated secretion.

  7. Membrane-associated forms of peptidylglycine alpha-amidating monooxygenase activity in rat pituitary. Tissue specificity.

    PubMed

    May, V; Cullen, E I; Braas, K M; Eipper, B A

    1988-06-05

    Membrane-associated peptidylglycine alpha-amidating monooxygenase (PAM) activity was investigated in rat anterior and neurointermediate pituitary tissues and in pituitary AtT-20/D-16v and GH3 cell lines. A substantial fraction of total pituitary PAM activity was found to be membrane-associated. Triton X-100, N-octyl-beta-D-glucopyranoside, and Zwittergent were effective in solubilizing PAM activity from crude pituitary membranes. The distribution of enzyme activity between soluble and membrane-associated forms was tissue-specific. In the anterior pituitary lobe and pituitary cell lines, 40-60% of total PAM activity was membrane-associated while only 10% of the alpha-amidating activity in the neurointermediate lobe was membrane-associated. Soluble and membrane-associated forms of PAM shared nearly identical characteristics with respect to copper and ascorbate requirements, pH optima, and Km values. Upon subcellular fractionation of anterior and neurointermediate pituitary lobe homogenates on Percoll gradients, 12-18% of total PAM activity was found in the rough endoplasmic reticulum/Golgi fractions and 42-60% was localized to secretory granule fractions. For both tissues, membrane-associated PAM activity was enriched in the rough endoplasmic reticulum/Golgi pool, whereas most of the secretory granule-associated enzyme activity was soluble.

  8. Isolation and functional expression of human pancreatic peptidylglycine alpha-amidating monooxygenase.

    PubMed

    Tateishi, K; Arakawa, F; Misumi, Y; Treston, A M; Vos, M; Matsuoka, Y

    1994-11-30

    Pancreastatin (PST) is processed from chromogranin A and the C-terminal amide of the peptide is an absolute requirement for biological activities. Human pancreatic carcinoma cells QGP-1 which produce both chromogranin A and PST were used to isolate cDNAs encoding two forms of peptidylglycine alpha-amidating monooxygenase (PAM). The two forms are a full length bifunctional enzyme and a variant lacking the transmembrane domain-coding region. When the cDNAs of these two forms were expressed in COS-7 cells, cells transfected with the predicted soluble form released into the culture medium a very much higher amidating activity which converts human chromogranin A-(273-302) to PST-29. The optimal pH for amidating activity was 5.4 and Cu2+, ascorbate and catalase were required as cofactors for the both forms of PAM. Km values for the membrane-bound and the soluble forms of PAM were 15.7 +/- 3.1 microM and 12.4 +/- 1.6 microM, respectively. These results demonstrate that both forms of PAM can function in the posttranslational processing of chromogranin A to PST in the environment of a secretory vesicle.

  9. Menkes protein contributes to the function of peptidylglycine alpha-amidating monooxygenase.

    PubMed

    Steveson, Tami C; Ciccotosto, Giuseppe D; Ma, Xin-Ming; Mueller, Gregory P; Mains, Richard E; Eipper, Betty A

    2003-01-01

    Menkes protein (ATP7A) is a P-type ATPase involved in copper uptake and homeostasis. Disturbed copper homeostasis occurs in patients with Menkes disease, an X-linked disorder characterized by mental retardation, neurodegeneration, connective tissue disorders, and early childhood death. Mutations in ATP7A result in malfunction of copper-requiring enzymes, such as tyrosinase and copper/zinc superoxide dismutase. The first step of the two-step amidation reaction carried out by peptidylglycine alpha-amidating monooxygenase (PAM) also requires copper. We used tissue from wild-type rats and mice and an ATP7A-specific antibody to determine that ATP7A is expressed at high levels in tissues expressing high levels of PAM. ATP7A is largely localized to the trans Golgi network in pituitary endocrine cells. The Atp7a mouse, bearing a mutation in the Atp7a gene, is an excellent model system for examining the consequences of ATP7A malfunction. Despite normal levels of PAM protein, levels of several amidated peptides were reduced in pituitary and brain extracts of Atp7a mice, demonstrating that PAM function is compromised when ATP7A is inactive. Based on these results, we conclude that a reduction in the ability of PAM to produce bioactive end-products involved in neuronal growth and development could contribute to many of the biological effects associated with Menkes disease.

  10. Localization of integral membrane peptidylglycine alpha-amidating monooxygenase in neuroendocrine cells.

    PubMed

    Milgram, S L; Kho, S T; Martin, G V; Mains, R E; Eipper, B A

    1997-03-01

    Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the amidation of glycine-extended peptides in neuroendocrine cells. At steady state, membrane PAM is accumulated in a perinuclear compartment. We examined the distribution of membrane PAM in stably transfected AtT-20 cells and compared its localization to markers for the trans-Golgi network (TGN), endosomes, and lysosomes. At the light microscopic level, the distribution of membrane PAM does not overlap extensively with lysosomal markers but does overlap with TGN38 and with SCAMP, a component of post-Golgi membranes involved in recycling pathways. By immunoelectron microscopy, membrane PAM is present in tubulovesicular structures which constitute the TGN; some of these PAM-containing tubulovesicular structures are more distal to the Golgi stacks and do not contain TGN38. While some POMC-derived peptides are present in tubulovesicular structures like those that contain membrane PAM, the majority of the POMC-derived peptides are present in secretory granules. There is little overlap between the steady state distribution of membrane PAM and internalized FITC-transferrin in the early endosomes. Few of the perinuclear PAM-containing structures are labeled with HRP or WGA-HRP even following long incubations. Therefore, membrane PAM is localized to perinuclear tubulovesicular structures which are partially devoid of TGN38 and are not all endosomal in origin.

  11. Reversal of physiological deficits caused by diminished levels of peptidylglycine alpha-amidating monooxygenase by dietary copper.

    PubMed

    Bousquet-Moore, D; Ma, X M; Nillni, E A; Czyzyk, T A; Pintar, J E; Eipper, B A; Mains, R E

    2009-04-01

    Amidated peptides are critically involved in many physiological functions. Genetic deletion of peptidylglycine alpha-amidating monooxygenase (PAM), the only enzyme that can synthesize these peptides, is embryonically lethal. The goal of the present study was the identification of physiological functions impaired by haploinsufficiency of PAM. Regulation of the hypothalamic-pituitary-thyroid axis and body temperature, functions requiring contributions from multiple amidated peptides, were selected for evaluation. Based on serum T(4) and pituitary TSH-beta mRNA levels, mice heterozygous for PAM (PAM(+/-)) were euthyroid at baseline. Feedback within the hypothalamic-pituitary-thyroid axis was impaired in PAM(+/-) mice made hypothyroid using a low iodine/propylthiouracil diet. Despite their normal endocrine response to cold, PAM(+/-) mice were unable to maintain body temperature as well as wild-type littermates when kept in a 4 C environment. When provided with additional dietary copper, PAM(+/-) mice maintained body temperature as well as wild-type mice. Pharmacological activation of vasoconstriction or shivering also allowed PAM(+/-) mice to maintain body temperature. Cold-induced vasoconstriction was deficient in PAM(+/-) mice. This deficit was eliminated in PAM(+/-) mice receiving a diet with supplemental copper. These results suggest that dietary deficiency of copper, coupled with genetic deficits in PAM, could result in physiological deficits in humans.

  12. The multifunctional peptidylglycine alpha-amidating monooxygenase gene: exon/intron organization of catalytic, processing, and routing domains.

    PubMed

    Ouafik, L H; Stoffers, D A; Campbell, T A; Johnson, R C; Bloomquist, B T; Mains, R E; Eipper, B A

    1992-10-01

    Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is a multifunctional protein containing two enzymes that act sequentially to catalyze the alpha-amidation of neuroendocrine peptides. Peptidylglycine alpha-hydroxylating monooxygenase (PHM) catalyzes the first step of the reaction and is dependent on copper, ascorbate, and molecular oxygen. Peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) catalyzes the second step of the reaction. Previous studies demonstrated that alternative splicing results in the production of bifunctional PAM proteins that are integral membrane or soluble proteins as well as soluble monofunctional PHM proteins. Rat PAM is encoded by a complex single copy gene that consists of 27 exons and encompasses more than 160 kilobases (kb) of genomic DNA. The 12 exons comprising PHM are distributed over at least 76 kb genomic DNA and range in size from 49-185 base pairs; four of the introns within the PHM domain are over 10 kb in length. Alternative splicing in the PHM region can result in a truncated, inactive PHM protein (rPAM-5), or a soluble, monofunctional PHM protein (rPAM-4) instead of a bifunctional protein. The eight exons comprising PAL are distributed over at least 19 kb genomic DNA. The exons encoding PAL range in size from 54-209 base pairs and have not been found to undergo alternative splicing. The PHM and PAL domains are separated by a single alternatively spliced exon surrounded by lengthy introns; inclusion of this exon results in the production of a form of PAM (rPAM-1) in which endoproteolytic cleavage at a paired basic site can separate the two catalytic domains. The exon following the PAL domain encodes the trans-membrane domain of PAM; alternative splicing at this site produces integral membrane or soluble PAM proteins. The COOH-terminal domain of PAM is comprised of a short exon subject to alternative splicing and a long exon encoding the final 68 amino acids present in all bifunctional PAM proteins along

  13. Functional expression and characterization of a Xenopus laevis peptidylglycine alpha-amidating monooxygenase, AE-II, in insect-cell culture.

    PubMed

    Suzuki, K; Ohta, M; Okamoto, M; Nishikawa, Y

    1993-04-01

    The alpha-amidating reaction of peptide hormones is a two-step process which is catalyzed by peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidylhydroxyglycine N-C lyase (PHL). There are three types of mRNA for these amidating enzymes in Xenopus laevis, namely AE-I, AE-II and AE-III. AE-I encodes only PHM and AE-III encodes both PHM and PHL. AE-II seems to encode subtypes of both PHM and PHL. While AE-II mRNA is present in high amounts in frog skin, the actual enzymes originating from AE-II have not been detected. When we expressed AE-II in cultured insect-cells using the baculovirus expression vector system, the expressed enzyme was specifically localized to the membrane fraction due to its hydrophobic transmembrane domain. Alternatively, when the transmembrane-domain-deleted AE-II (Met1-Ile731) was expressed, the enzyme was secreted into the culture medium; this secreted enzyme was purified to homogeneity by a simple two-step procedure. We have verified that the reaction product of the purified enzyme was the amidated peptide, indicating that AE-II has the ability to catalyze the entire amidating reaction.

  14. Haploinsufficiency in peptidylglycine alpha-amidating monooxygenase leads to altered synaptic transmission in the amygdala and impaired emotional responses.

    PubMed

    Gaier, Eric D; Rodriguiz, Ramona M; Ma, Xin-Ming M; Sivaramakrishnan, Shobhana; Bousquet-Moore, Danielle; Wetsel, William C; Eipper, Betty A; Mains, Richard E

    2010-10-13

    The mammalian amygdala expresses various neuropeptides whose signaling has been implicated in emotionality. Many neuropeptides require amidation for full activation by peptidylglycine α-amidating monooxygenase (PAM), a transmembrane vesicular cuproenzyme and regulator of the secretory pathway. Mice heterozygous for the Pam gene (PAM(+/-)) exhibit physiological and behavioral abnormalities related to specific peptidergic pathways. In the present study, we evaluated emotionality and examined molecular and cellular responses that characterize neurophysiological differences in the PAM(+/-) amygdala. PAM(+/-) mice presented with anxiety-like behaviors in the zero maze that were alleviated by diazepam. PAM(+/-) animals were deficient in short- and long-term contextual and cued fear conditioning and required higher shock intensities to establish fear-potentiated startle than their wild-type littermates. Immunohistochemical analysis of the amygdala revealed PAM expression in pyramidal neurons and local interneurons that synthesize GABA. We performed whole-cell recordings of pyramidal neurons in the PAM(+/-) amygdala to elucidate neurophysiological correlates of the fear behavioral phenotypes. Consistent with these observations, thalamic afferent synapses in the PAM(+/-) lateral nucleus were deficient in long-term potentiation. This deficit was apparent in the absence and presence of the GABA(A) receptor antagonist picrotoxin and was abolished when both GABA(A) and GABA(B) receptors were blocked. Both evoked and spontaneous excitatory signals were enhanced in the PAM(+/-) lateral nucleus. Phasic GABAergic signaling was also augmented in the PAM(+/-) amygdala, and this difference comprised activity-independent and -dependent components. These physiological findings represent perturbations in the PAM(+/-) amygdala that may underlie the aberrant emotional responses in the intact animal.

  15. mRNA differential display in a microbial enrichment culture: simultaneous identification of three cyclohexanone monooxygenases from three species.

    PubMed

    Brzostowicz, Patricia C; Walters, Dana M; Thomas, Stuart M; Nagarajan, Vasantha; Rouvière, Pierre E

    2003-01-01

    mRNA differential display has been used to identify cyclohexanone oxidation genes in a mixed microbial community derived from a wastewater bioreactor. Thirteen DNA fragments randomly amplified from the total RNA of an enrichment subculture exposed to cyclohexanone corresponded to genes predicted to be involved in the degradation of cyclohexanone. Nine of these DNA fragments are part of genes encoding three distinct Baeyer-Villiger cyclohexanone monooxygenases from three different bacterial species present in the enrichment culture. In Arthrobacter sp. strain BP2 and Rhodococcus sp. strain Phi2, the monooxygenase is part of a gene cluster that includes all the genes required for the degradation of cyclohexanone, while in Rhodococcus sp. strain Phi1 the genes surrounding the monooxygenase are not predicted to be involved in this degradation pathway but rather seem to belong to a biosynthetic pathway. Furthermore, in the case of Arthrobacter strain BP2, three other genes flanking the monooxygenase were identified by differential display, demonstrating that the repeated sampling of bacterial operons shown earlier for a pure culture (D. M. Walters, R. Russ, H. Knackmuss, and P. E. Rouvière, Gene 273:305-315, 2001) is also possible for microbial communities. The activity of the three cyclohexanone monooxygenases was confirmed and characterized following their expression in Escherichia coli.

  16. mRNA Differential Display in a Microbial Enrichment Culture: Simultaneous Identification of Three Cyclohexanone Monooxygenases from Three Species

    PubMed Central

    Brzostowicz, Patricia C.; Walters, Dana M.; Thomas, Stuart M.; Nagarajan, Vasantha; Rouvière, Pierre E.

    2003-01-01

    mRNA differential display has been used to identify cyclohexanone oxidation genes in a mixed microbial community derived from a wastewater bioreactor. Thirteen DNA fragments randomly amplified from the total RNA of an enrichment subculture exposed to cyclohexanone corresponded to genes predicted to be involved in the degradation of cyclohexanone. Nine of these DNA fragments are part of genes encoding three distinct Baeyer-Villiger cyclohexanone monooxygenases from three different bacterial species present in the enrichment culture. In Arthrobacter sp. strain BP2 and Rhodococcus sp. strain Phi2, the monooxygenase is part of a gene cluster that includes all the genes required for the degradation of cyclohexanone, while in Rhodococcus sp. strain Phi1 the genes surrounding the monooxygenase are not predicted to be involved in this degradation pathway but rather seem to belong to a biosynthetic pathway. Furthermore, in the case of Arthrobacter strain BP2, three other genes flanking the monooxygenase were identified by differential display, demonstrating that the repeated sampling of bacterial operons shown earlier for a pure culture (D. M. Walters, R. Russ, H. Knackmuss, and P. E. Rouvière, Gene 273:305-315, 2001) is also possible for microbial communities. The activity of the three cyclohexanone monooxygenases was confirmed and characterized following their expression in Escherichia coli. PMID:12514013

  17. Effect of hyperosmotic conditions on flavin-containing monooxygenase activity, protein and mRNA expression in rat kidney

    PubMed Central

    Rodríguez-Fuentes, Gabriela; Coburn, Cary; Currás-Collazo, Margarita; Guillén, Gabriel; Schlenk, Daniel

    2010-01-01

    Flavin-containing monooxigenases (FMOs) are a polymorphic family of drug and pesticide metabolizing enzymes, found in the smooth endoplasmatic reticulum that catalyze the oxidation of soft nucleophilic heteroatom substances to their respective oxides. Previous studies in euryhaline fishes have indicated induction of FMO expression and activity in vivo under hyperosmotic conditions. In this study we evaluated the effect of hypersaline conditions in rat kidney. Male Sprague–Dawley rats were injected intraperitoneal with 3.5 M NaCl at a doses ranging from 0.3 cm3/100 g to 0.6 cm3/100 g in two separate treatments. Three hours after injection, FMO activities and FMO1 protein was examined in the first experiment, and the expression of FMO1 mRNA was measured in the second experiment from kidneys after treatment with NaCl. A positive significant correlation was found between FMO1 protein expression and plasma osmolarity (p < 0.05, r = 0.6193). Methyl-p-tolyl sulfide oxidase showed a statistically significant increase in FMO activity, and a positive correlation was observed between plasma osmolarity and production of FMO1-derived (R)-methyl-p-tolyl sulfoxide (p < 0.05, r = 0.6736). Expression of FMO1 mRNA was also positively correlated with plasma osmolality (p < 0.05, r = 0.8428). Similar to studies in fish, these results suggest that expression and activities of FMOs may be influenced by hyperosmotic conditions in the kidney of rats. PMID:19429252

  18. Effect of PCB 126 on aryl hydrocarbon receptor 1 (AHR1) and AHR1 nuclear translocator 1 (ARNT1) mRNA expression and CYP1 monooxygenase activity in chicken (Gallus domesticus) ovarian follicles.

    PubMed

    Wójcik, Dagmara; Antos, Piotr A; Katarzyńska, Dorota; Hrabia, Anna; Sechman, Andrzej

    2015-12-03

    The aim of the experiment was to study the in vitro effect of 3,3',4,4',5-pentachlorobiphenyl (PCB 126; a coplanar PCB congener) on aryl hydrocarbon receptor (AHR1) and AHR1 nuclear translocator (ARNT1) mRNA expression and the activity of CYP1 family monooxygenases in chicken ovarian follicles. White (1-4 mm) and yellowish (4-8 mm) prehierarchical follicles as well as fragments of the theca and granulosa layers of the 3 largest preovulatory follicles (F3-F1) were incubated in a medium supplemented with 0 (control group), 1, 10 or 100 nM PCB 126. The incubation was carried out for 6 h or 24 h for determination of mRNA expression of AHR1 and ARNT1 genes (real-time qPCR) and CYP1 monooxygenase activity (EROD and MROD fluorometric assays), respectively. It was found that chicken ovarian follicles express mRNA of AHR1 and ARNT1 genes. A modulatory effect of PCB 126 on AHR1 and ARNT1 expression depended not only on the biphenyl concentration but also on the follicular layer and the maturational state of the follicle. EROD and MROD activities appeared predominantly in the granulosa layer of the yellow preovulatory follicles. PCB 126 induced these activities in a dose-dependent manner in all ovarian follicles. The obtained results suggest that ovarian follicles, especially the granulosa layer, are involved in the detoxification process of PCBs in the laying hen. Taking this finding into consideration it can be suggested that the granulosa layer of the yellow hierarchical follicles plays a key role in the protective mechanism which reduces the amount of transferred dioxin-like compounds into the yolk of the oocyte. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  19. A mutation in the promoter of the chicken β,β-carotene 15,15'-monooxygenase 1 gene alters xanthophyll metabolism through a selective effect on its mRNA abundance in the breast muscle.

    PubMed

    Jlali, M; Graulet, B; Chauveau-Duriot, B; Chabault, M; Godet, E; Leroux, S; Praud, C; Le Bihan-Duval, E; Duclos, M J; Berri, C

    2012-12-01

    A polymorphism in the promoter of the β,β-carotene 15,15'-monooxygenase 1 (BCMO1) gene recently was identified in an experimental cross between 2 chicken lines divergently selected on growth rate and found to be associated with variations in the yellow color of the breast meat. In this study, the effects of the polymorphism on several aspects of carotenoid metabolism were evaluated in chickens sharing the same genetic background except for their genotype at the BCMO1 locus. We confirmed that BCMO1 mRNA abundance varied (P < 0.001) between the 2 homozygous genotypes (GG < AA) and in the pectoralis major muscle. By contrast, BCMO1 mRNA expression was not affected (P > 0.05) by the polymorphism in the duodenum, liver, or sartorius muscle. The breast meat of GG chickens was more (P < 0.001) yellow and richer in lutein (P < 0.01) and zeaxanthin (P < 0.05) compared to that of AA chickens whereas these variables did not differ (P > 0.05) in the other tissues tested. The GG were also characterized by reduced (P < 0.01) plasma lutein and zeaxanthin concentrations than AA without affecting plasma and tissue content of fat-soluble vitamins A and E. As lutein and zeaxanthin are usually not considered as substrates of the BCMO1 enzyme, the impact of BCMO1 polymorphism on the activity of other genes involved in carotenoid transport (SCARB1 and CD36 encoding the scavenger receptor class B type I and the cluster determinant 36, respectively) and metabolism (BCDO2 encoding β,β-carotene 9',10'-dioxygenase 2) was evaluated. The BCMO1 polymorphism did not affect mRNA abundance of BCDO2, SCARB1, or CD36, regardless of tissue considered. Taken together, these results indicated that a genetic variant of BCMO1 specifically changes lutein and zeaxanthin content in the chicken plasma and breast muscle without impairing vitamin A and E metabolism.

  20. The influence of polychlorinated biphenyls (PCBs), dichlorodiphenyltrichloroethane (DDT) and its metabolite-dichlorodiphenyldichloroethylene (DDE) on mRNA expression for NP-I/OT and PGA, involved in oxytocin synthesis in bovine granulosa and luteal cells.

    PubMed

    Mlynarczuk, Jaroslaw; Wrobel, Michal H; Kotwica, Jan

    2009-11-01

    The effect of polychlorinated biphenyls (PCBs) congeners (PCB 77, PCB 126, PCB 153) and their technical mixture-Aroclor (Ar) 1248, as well as dichlorodiphenyltrichloroethane (DDT) and its metabolite-dichlorodiphenyldichloroethylene (DDE; two individual isomers p,p'- and o,p'- or their mixture, 95% and 5%, respectively) at the dose of 10 ng/ml each, on the gene expression of (a) oxytocin (OT) precursor-neurophysin-oxytocin (NP-I/OT) and (b) peptidyl glycine-alpha-amidating mono-oxygenase (PGA), the terminal enzyme in the pathway of OT synthesis, was studied. Granulosa cells from follicles >1cm in diameter, collected on days 19-21 of estrous cycle, and luteal cells from corpora lutea (CL) collected on days 8-12 of the estrous cycle were used. The cells were incubated (6h) with these xenobiotics and the expression of NP-I/OT and PGA genes was determined. All PCBs increased (P<0.05) NP-I/OT gene expression in granulosa cells. Similarly, all PCBs but PCB 126 increased (P<0.05) PGA gene expression in these cells. DDT and DDE increased (P<0.05) gene expression of NP-I/OT in granulosa cells, while gene expression of PGA in these cells was stimulated (P<0.05) by DDE only. The mRNA expression for NP-I/OT and PGA in luteal cells was increased (P<0.05) by PCB 77 and PCB 153. Both DDE isomers and mixture also stimulated (P<0.05) of NP-I/OT mRNA expression, while increase (P<0.05) of PGA mRNA expression was elicited by incubation of these cells with DDE mixture and Ar 1248. Obtained data suggest that PCBs, DDT and DDE can affect the mRNA expression for NP-I/OT and PGA in bovine granulosa and luteal cells.

  1. A radiometric kynurenine monooxygenase assay

    SciTech Connect

    Wiseman, J.S.; Nichols, J.S. )

    1990-01-01

    Kynurenine 3-monooxygenase is a flavin-dependent monooxygenase that catalyzes the oxidation of L-kynurenine to 3-hydroxy-L-kynurenine in the kynurenine pathway of tryptophan metabolism. The enzyme requires NADH or NADPH as a cofactor. A discontinuous assay that utilizes L-(3H)kynurenine as substrate is described. The assay offers high precision and a wide range of accessible substrate and cofactor concentrations. The assay was used to measure kinetic isotope effects and the stereospecificity of oxidation of the cofactor. Hydride is transferred from the A-side (pro-R) of NADH and NADPH since primary deuterium isotope effects were observed for both cofactors when they were deuterated on the A-side but not on the B-side. The large isotope effect on Vmax/Km for NADH is sensitive to the concentration of kynurenine, which indicates that NADH can bind before kynurenine.

  2. 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.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  4. Expression of recombinant human bifunctional peptidylglycine α-amidating monooxygenase in CHO cells and its use for insulin analogue modification.

    PubMed

    Zieliński, Marcin; Wójtowicz-Krawiec, Anna; Mikiewicz, Diana; Kęsik-Brodacka, Małgorzata; Cecuda-Adamczewska, Violetta; Marciniak-Rusek, Alina; Sokołowska, Iwona; Łukasiewicz, Natalia; Gurba, Lidia; Odrowąż-Sypniewski, Michał; Baran, Piotr; Płucienniczak, Grażyna; Płucienniczak, Andrzej; Borowicz, Piotr; Szewczyk, Bogusław

    2016-03-01

    The availability of catalytically active peptidylglycine α-amidating monooxygenase (PAM) should provide the means to examine its potential use for the chemienzymatic synthesis of bioactive peptides for the purpose of pharmacological studies. Hypoglycemic activity is one of the most important features of insulin derivatives. Insulin glargine amide was found to show a time/effect profile which is distinctly more flat and thus more advantageous than insulin glargine itself. The aim of the study was to obtain recombinant PAM and use it for insulin analogue amidation. We stably expressed a recombinant PAM in CHO dhfr-cells in culture. Recombinant PAM was partially purified by fractional ammonium sulphate precipitation and ion-exchange chromatography. The enzyme was used to modify glycine-extended A22(G)-B31(K)-B32(R) human insulin analogue (GKR). Alpha-amidated insulin was analyzed by HPLC and mass spectrometry. Hypoglycemic activity of amidated and non-amidated insulin was compared. The pharmacodynamic effect was based on glucose concentration measurement in Wistar rats with hyperglycemia induced by streptozotocin. The overall glycemic profile up to 36 h was evaluated after subcutaneous single dosing at a range of 2.5-7.5 U/kg b.w. The experiment on rats confirmed with a statistical significance (P < 0.05) hypoglycemic activity of GKR-NH2 in comparison to a control group receiving 0.9% NaCl. Characteristics for GKR-NH2 profile was a rather fast beginning of action (0.5-2.0 h) and quite prolonged return to initial values. GKR-NH2 is a candidate for a hypoglycemic drug product in diabetes care. In addition, this work also provides a valuable alternative method for preparing any other recombinant bioactive peptides with C-terminal amidation.

  5. Kinetic isotope effects of peptidylglycine alpha-hydroxylating mono-oxygenase reaction.

    PubMed Central

    Takahashi, K; Onami, T; Noguchi, M

    1998-01-01

    Many bioactive polypeptides or neuropeptides possess a C-terminal alpha-amide group as a critical determinant for their optimal bioactivities. The amide functions are introduced by the sequential actions of peptidylglycine alpha-hydroxylating mono-oxygenase (PHM; EC 1.14.17.3) and peptidylamidoglycollate lyase (PAL; EC 4.3.2.5) from their glycine-extended precursors. In the present study we examined the kinetic isotope effects of the frog PHM reaction by competitive and non-competitive approaches. In the competitive approach we employed the double-label tracer method with D-Tyr-[U-14C]Val-Gly, D-Tyr-[3,4-3H]Val-[2,2-2H2]-Gly, and D-Tyr-Val-(R,S)[2-3H]Gly as substrates, and we determined the deuterium and tritium effects on Vmax/Km as 1.625+/-0.041 (mean+/-S. D.) and 2.71+/-0.16 (mean+/-S.D.), respectively. The intrinsic deuterium isotope effect (Dk) on the glycine hydroxylation reaction was estimated to be 6.5-10.0 (mean 8.1) by the method of Northrop [Northrop (1975) Biochemistry 14, 2644-2651]. In the non-competitive approach with N,N-dimethyl-1,4-phenylenediamine as a reductant, however, the deuterium effect on Vmax (DV) was approximately unity, although the deuterium effect on Vmax/Km (DV/K) was comparable to that obtained by the competitive approach. These results indicated that DV was completely masked by the presence of one or more steps much slower than the glycine hydroxylation step and that DV/K was diminished from Dk by a large forward commitment to catalysis. The addition of PAL, however, increased the apparent DV from 1.0 to 1.2, implying that the product release step was greatly accelerated by PAL. These results suggest that the product release is rate-limiting in the overall PHM reaction. The large Dk indicated that the glycine hydroxylation catalysed by PHM might proceed in a stepwise mechanism similar to that proposed for the dopamine beta-hydroxylase reaction [Miller and Klinman (1983) Biochemistry 22, 3091-3096]. PMID:9806894

  6. 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

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

    PubMed

    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.

  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.

  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. Human phenylalanine monooxygenase and thioether metabolism.

    PubMed

    Boonyapiwat, Boontarika; Panaretou, Barry; Forbes, Ben; Mitchell, Stephen C; Steventon, Glyn B

    2009-01-01

    The substrate specificity of wild-type human phenylalanine monooxygenase (wt-hPAH) has been investigated with respect to the mucoactive drug, S-carboxymethyl-L-cysteine and its thioether metabolites. The ability of wt-hPAH to metabolise other S-substituted cysteines was also examined. Direct assays of PAH activity were by HPLC with fluorescence detection; indirect assays involved following disappearance of the cofactor by UV spectroscopy. wt-hPAH catalysed the S-oxygenation of S-carboxymethyl-L-cysteine, its decarboxylated metabolite, S-methyl-L-cysteine, and both their corresponding N-acetylated forms. However, thiodiglycolic acid was not a substrate. The enzyme profiles for both phenylalanine and S-carboxymethyl-L-cysteine showed allosteric kinetics at low substrate concentrations, with Hill constants of 2.0 and 1.9, respectively, for the substrate-activated wt-hPAH. At higher concentrations, both compounds followed Michaelis-Menten kinetics, with non-competitive substrate inhibition profiles. The thioether compounds, S-ethyl-L-cysteine, S-propyl-L-cysteine and S-butyl-L-cysteine were all found to be substrates for phenylalanine monooxygenase. Phenylalanine monooxygenase may play a wider role outside intermediary metabolism in the biotransformation of dietary-derived substituted cysteines and other exogenous thioether compounds.

  11. Tolerance to Acetaminophen Hepatotoxicity in the Mouse Model of Autoprotection is Associated with Induction of Flavin-containing Monooxygenase-3 (FMO3) in Hepatocytes

    EPA Science Inventory

    Acetaminophen (APAP) pretreatment with a low hepatotoxic dose in mice results in resistance to a second, higher dose of APAP (APAP autoprotection). Recent microarray work by our group showed a drastic induction of liver flavin containing monooxygenase-3 (Fmo3) mRNA expression in...

  12. Tolerance to Acetaminophen Hepatotoxicity in the Mouse Model of Autoprotection is Associated with Induction of Flavin-containing Monooxygenase-3 (FMO3) in Hepatocytes

    EPA Science Inventory

    Acetaminophen (APAP) pretreatment with a low hepatotoxic dose in mice results in resistance to a second, higher dose of APAP (APAP autoprotection). Recent microarray work by our group showed a drastic induction of liver flavin containing monooxygenase-3 (Fmo3) mRNA expression in...

  13. Genetics of particulate methane monooxygenase in methanotrophs

    SciTech Connect

    Peeples, T.L.; Lebron, J.; Costello, A.Y.

    1995-12-01

    The ability of methanotrophs to co-metabolize halocarbons such as trichloroethylene (TCE) by way of the enzyme methane monooxygenase (MMO) is of interest in the development of bioremediation technologies. The presence and the regulation of expression of soluble (sMMO) and membrane-bound (pMMO) forms is important in the design and optimization of such processing schemes. Because the pMMO is found in all methanotrophic species, this form is relevant to in situ remediation technologies. Recently, multiple copies of genes encoding for pMMO have been identified in several species of methanotrophs. Genes encoding the 45 and 27 kDa subunits of the PMMO have been cloned and sequenced. We will discuss the possible implications of multiple copies of pMMO genes in the regulation of pMMO expression. These data may give insights into how MMO activity in natural environments may be manipulated for increased TCE biodegradation.

  14. Structural diversity of lytic polysaccharide monooxygenases.

    PubMed

    Vaaje-Kolstad, Gustav; Forsberg, Zarah; Loose, Jennifer Sm; Bissaro, Bastien; Eijsink, Vincent Gh

    2017-01-10

    Lytic polysaccharide monooxygenases (LPMOs) catalyze the oxidative cleavage of glycosidic bonds and represent a promising resource for development of industrial enzyme cocktails for biomass processing. LPMOs show high sequence and modular diversity and are known, so far, to cleave insoluble substrates such as cellulose, chitin and starch, as well as hemicelluloses such as beta-glucan, xyloglucan and xylan. All LPMOs share a catalytic histidine brace motif to bind copper, but differ strongly when it comes to the nature and arrangement of residues on the substrate-binding surface. In recent years, the number of available LPMO structures has increased rapidly, including the first structure of an enzyme-substrate complex. The insights gained from these structures is reviewed below.

  15. Structural basis of kynurenine 3-monooxygenase inhibition

    PubMed Central

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

    2013-01-01

    Inhibition of kynurenine 3-monooxygenase (KMO), an enzyme in the eukaryotic tryptophan catabolic pathway (i.e. kynurenine pathway), leads to amelioration of Huntington’s disease-relevant phenotypes in yeast, fruit fly, and mouse models1–5, as well as a mouse model of Alzheimer’s disease3. KMO is a 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 disorders6, as well as cancer7,8, and several peripheral inflammatory conditions9. Despite the importance of KMO as a target for neurodegenerative disease, the molecular basis of KMO inhibition by available lead compounds has remained hitherto unknown. Here we report the first crystal structure of 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 kynurenine. Functional assays and targeted mutagenesis revealed 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

  16. A Carbonate-Forming Baeyer-Villiger Monooxygenase

    PubMed Central

    Hu, Youcai; Dietrich, David; Xu, Wei; Patel, Ashay; Thuss, Justin A. J.; Wang, Jingjing; Yin, Wen-Bing; Qiao, Kangjian; Houk, Kendall N.; Vederas, John C.; Tang, Yi

    2014-01-01

    Despite the remarkable versatility displayed by flavin-dependent monooxygenases (FMOs) in natural product biosynthesis, one notably missing activity is the oxidative generation of carbonate functional groups. We describe a multifunctional Baeyer-Villiger monooxygenase CcsB, which catalyzes the formation of an in-line carbonate in the macrocyclic portion of cytochalasin E. This study expands the repertoire of activities of FMOs and provides a possible synthetic strategy for transformation of ketones into carbonates. PMID:24838010

  17. Peptidylglycine α-amidating monooxygenase heterozygosity alters brain copper handling with region specificity.

    PubMed

    Gaier, Eric D; Miller, Megan B; Ralle, Martina; Aryal, Dipendra; Wetsel, William C; Mains, Richard E; Eipper, Betty A

    2013-12-01

    Copper (Cu), an essential trace element present throughout the mammalian nervous system, is crucial for normal synaptic function. Neuronal handling of Cu is poorly understood. We studied the localization and expression of Atp7a, the major intracellular Cu transporter in the brain, and its relation to peptidylglycine α-amidating monooxygenase (PAM), an essential cuproenzyme and regulator of Cu homeostasis in neuroendocrine cells. Based on biochemical fractionation and immunostaining of dissociated neurons, Atp7a was enriched in post-synaptic vesicular fractions. Cu followed a similar pattern, with ~ 20% of total Cu in synaptosomes. A mouse model heterozygous for the Pam gene (PAM+/−) was selectively Cu deficient in the amygdala. As in cortex and hippocampus, Atp7a and PAM expression overlap in the amygdala, with highest expression in interneurons. Messenger RNA levels of Atox-1 and Atp7a, which deliver Cu to the secretory pathway, were reduced in the amygdala but not in the hippocampus in PAM+/− mice, GABAB receptor mRNA levels were similarly affected. Consistent with Cu deficiency, dopamine β-monooxygenase function was impaired as evidenced by elevated dopamine metabolites in the amygdala, but not in the hippocampus, of PAM+/− mice. These alterations in Cu delivery to the secretory pathway in the PAM+/− amygdala may contribute to the physiological and behavioral deficits observed. Atp7a, a Cu-transporting P-type ATPase, is localized to the trans-Golgi network and to vesicles distributed throughout the dendritic arbor. Tissue-specific alterations in Atp7a expression were found in mice heterozygous for peptidylglycine α-amidating monooxygenase (PAM), an essential neuropeptide-synthesizing cuproenzyme. Atp7a and PAM are highly expressed in amygdalar interneurons. Reduced amygdalar expression of Atox-1 and Atp7a in PAM heterozygous mice may lead to reduced synaptic Cu levels, contributing to the behavioral and neurochemical alterations seen in these mice.

  18. Simultaneous Identification of Two Cyclohexanone Oxidation Genes from an Environmental Brevibacterium Isolate Using mRNA Differential Display

    PubMed Central

    Brzostowicz, Patricia C.; Gibson, Katharine L.; Thomas, Stuart M.; Blasko, Mary Sue; Rouvière, Pierre E.

    2000-01-01

    The technique of mRNA differential display was used to identify simultaneously two metabolic genes involved in the degradation of cyclohexanone in a new halotolerant Brevibacterium environmental isolate. In a strategy based only on the knowledge that cyclohexanone oxidation was inducible in this strain, the mRNA population of cells exposed to cyclohexanone was compared to that of control cells using reverse transcription-PCR reactions primed with a collection of 81 arbitrary oligonucleotides. Three DNA fragments encoding segments of flavin monooxygenases were isolated with this technique, leading to the identification of the genes of two distinct cyclohexanone monooxygenases, the enzymes responsible for the oxidation of cyclohexanone. Each monooxygenase was expressed in Escherichia coli and characterized. This work validates the application of mRNA differential display for the discovery of new microbial metabolic genes. PMID:10894733

  19. Substrate radical intermediates in soluble methane monooxygenase.

    PubMed

    Liu, Aimin; Jin, Yi; Zhang, Jingyan; Brazeau, Brian J; Lipscomb, John D

    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.

  20. A tale of two methane monooxygenases

    PubMed Central

    Ross, Matthew O.

    2017-01-01

    Methane monooxygenase (MMO) enzymes activate O2 for oxidation of methane. Two distinct MMOs exist in nature, a soluble form that uses a diiron active site (sMMO) and a membrane-bound form with a catalytic copper center (pMMO). Understanding the reaction mechanisms of these enzymes is of fundamental importance to biologists and chemists, and is also relevant to the development of new biocatalysts. The sMMO catalytic cycle has been elucidated in detail, including O2 activation intermediates and the nature of the methane-oxidizing species. By contrast, many aspects of pMMO catalysis remain unclear, most notably the nuclearity and molecular details of the copper active site. Here, we review the current state of knowledge for both enzymes, and consider pMMO O2 activation intermediates suggested by computational and synthetic studies in the context of existing biochemical data. Further work is needed on all fronts, with the ultimate goal of understanding how these two remarkable enzymes catalyze a reaction not readily achieved by any other metalloenzyme or biomimetic compound. PMID:27878395

  1. Towards practical Baeyer-Villiger-monooxygenases: design of cyclohexanone monooxygenase mutants with enhanced oxidative stability.

    PubMed

    Opperman, Diederik J; Reetz, Manfred T

    2010-12-10

    Baeyer-Villiger monooxygenases (BVMOs) catalyze the conversion of ketones and cyclic ketones into esters and lactones, respectively. Cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871 is known to show an impressive substrate scope as well as exquisite chemo-, regio-, and enantioselectivity in many cases. Large-scale synthetic applications of CHMO are hampered, however, by the instability of the enzyme. Oxidation of cysteine and methionine residues contributes to this instability. Designed mutations of all the methionine and cysteine residues in the CHMO wild type (WT) showed that the amino acids labile towards oxidation are mostly either surface-exposed or located within the active site, whereas the two methionine residues identified for thermostabilization are buried within the folded protein. Combinatorial mutations gave rise to two stabilized mutants with either oxidative or thermal stability, without compromising the activity or stereoselectivity of the enzyme. The most oxidatively stabilized mutant retained nearly 40 % of its activity after incubation with H(2)O(2) (0.2 M), whereas the wild-type enzyme's activity was completely abolished at concentrations as low as 5 mM H(2)O(2). We propose that oxidation-stable mutants might well be a "prerequisite" for thermostabilization, because laboratory-evolved thermostability in CHMO might be masked by a high degree of oxidation instability.

  2. Proposed involvement of a soluble methane monooxygenase homologue in the cyclohexane-dependent growth of a new Brachymonas species.

    PubMed

    Brzostowicz, Patricia C; Walters, Dana M; Jackson, Raymond E; Halsey, Kimberly H; Ni, Hao; Rouvière, Pierre E

    2005-02-01

    High-throughput mRNA differential display (DD) was used to identify genes induced by cyclohexane in Brachymonas petroleovorans CHX, a recently isolated beta-proteobacterium that grows on cyclohexane. Two metabolic gene clusters were identified multiple times in independent reverse transcription polymerase chain reactions (RT-PCR) in the course of this DD experiment. These clusters encode genes believed to be required for cyclohexane metabolism. One gene cluster (8 kb) encodes the subunits of a multicomponent hydroxylase related to the soluble butane of Pseudomonas butanovora and methane monooxygenases (sMMO) of methanotrophs. We propose that this butane monooxygenase homologue carries out the oxidation of cyclohexane into cyclohexanol during growth. A second gene cluster (11 kb) contains almost all the genes required for the oxidation of cyclohexanol to adipic acid. Real-time PCR experiments confirmed that genes from both clusters are induced by cyclohexane. The role of the Baeyer-Villiger cyclohexanone monooxygenase of the second cluster was confirmed by heterologous expression in Escherichia coli.

  3. Hydrocarbon monooxygenase in Mycobacterium: recombinant expression of a member of the ammonia monooxygenase superfamily

    PubMed Central

    Coleman, Nicholas V; Le, Nga B; Ly, Mai A; Ogawa, Hitoha E; McCarl, Victoria; Wilson, Neil L; Holmes, Andrew J

    2012-01-01

    The copper membrane monooxygenases (CuMMOs) are an important group of enzymes in environmental science and biotechnology. Areas of relevance include the development of green chemistry for sustainable exploitation of methane (CH4) reserves, remediation of chlorinated hydrocarbon contamination and monitoring human impact in the biogeochemical cycles of CH4 and nitrogen. Challenges for all these applications are that many aspects of the ecology, physiology and structure–function relationships in the CuMMOs are inadequately understood. Here, we describe genetic and physiological characterization of a novel member of the CuMMO family that has an unusual physiological substrate range (C2–C4 alkanes) and a distinctive bacterial host (Mycobacterium). The Mycobacterial CuMMO genes (designated hmoCAB) were amenable to heterologous expression in M. smegmatis—this is the first example of recombinant expression of a complete and highly active CuMMO enzyme. The apparent specific activity of recombinant cells containing hmoCAB ranged from 2 to 3 nmol min–1 per mg protein on ethane, propane and butane as substrates, and the recombinants could also attack ethene, cis-dichloroethene and 1,2-dichloroethane. No detectable activity of recombinants or wild-type strains was seen with methane. The specific inhibitor allylthiourea strongly inhibited growth of wild-type cells on C2–C4 alkanes, and omission of copper from the medium had a similar effect, confirming the physiological role of the CuMMO for growth on alkanes. The hydrocarbon monooxygenase provides a new model for studying this important enzyme family, and the recombinant expression system will enable biochemical and molecular biological experiments (for example, site-directed mutagenesis) that were previously not possible. PMID:21796219

  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.

  5. Diversity in Butane Monooxygenases among Butane-Grown Bacteria

    PubMed Central

    Hamamura, Natsuko; Storfa, Ryan T.; Semprini, Lewis; Arp, Daniel J.

    1999-01-01

    Butane monooxygenases of butane-grown Pseudomonas butanovora, Mycobacterium vaccae JOB5, and an environmental isolate, CF8, were compared at the physiological level. The presence of butane monooxygenases in these bacteria was indicated by the following results. (i) O2 was required for butane degradation. (ii) 1-Butanol was produced during butane degradation. (iii) Acetylene inhibited both butane oxidation and 1-butanol production. The responses to the known monooxygenase inactivator, ethylene, and inhibitor, allyl thiourea (ATU), discriminated butane degradation among the three bacteria. Ethylene irreversibly inactivated butane oxidation by P. butanovora but not by M. vaccae or CF8. In contrast, butane oxidation by only CF8 was strongly inhibited by ATU. In all three strains of butane-grown bacteria, specific polypeptides were labeled in the presence of [14C]acetylene. The [14C]acetylene labeling patterns were different among the three bacteria. Exposure of lactate-grown CF8 and P. butanovora and glucose-grown M. vaccae to butane induced butane oxidation activity as well as the specific acetylene-binding polypeptides. Ammonia was oxidized by all three bacteria. P. butanovora oxidized ammonia to hydroxylamine, while CF8 and M. vaccae produced nitrite. All three bacteria oxidized ethylene to ethylene oxide. Methane oxidation was not detected by any of the bacteria. The results indicate the presence of three distinct butane monooxygenases in butane-grown P. butanovora, M. vaccae, and CF8. PMID:10508093

  6. Two Structures of an N-Hydroxylating Flavoprotein Monooxygenase

    PubMed Central

    Olucha, Jose; Meneely, Kathleen M.; Chilton, Annemarie S.; Lamb, Audrey L.

    2011-01-01

    The ornithine hydroxylase from Pseudomonas aeruginosa (PvdA) catalyzes the FAD-dependent hydroxylation of the side chain amine of ornithine, which is subsequently formylated to generate the iron-chelating hydroxamates of the siderophore pyoverdin. PvdA belongs to the class B flavoprotein monooxygenases, which catalyze the oxidation of substrates using NADPH as the electron donor and molecular oxygen. Class B enzymes include the well studied flavin-containing monooxygenases and Baeyer-Villiger monooxygenases. The first two structures of a class B N-hydroxylating monooxygenase were determined with FAD in oxidized (1.9 Å resolution) and reduced (3.03 Å resolution) states. PvdA has the two expected Rossmann-like dinucleotide-binding domains for FAD and NADPH and also a substrate-binding domain, with the active site at the interface between the three domains. The structures have NADP(H) and (hydroxy)ornithine bound in a solvent-exposed active site, providing structural evidence for substrate and co-substrate specificity and the inability of PvdA to bind FAD tightly. Structural and biochemical evidence indicates that NADP+ remains bound throughout the oxidative half-reaction, which is proposed to shelter the flavin intermediates from solvent and thereby prevent uncoupling of NADPH oxidation from hydroxylated product formation. PMID:21757711

  7. Kinetic mechanism of phenylacetone monooxygenase from Thermobifida fusca.

    PubMed

    Torres Pazmiño, Daniel E; Baas, Bert-Jan; Janssen, Dick B; Fraaije, Marco W

    2008-04-01

    Phenylacetone monooxygenase (PAMO) from Thermobifida fusca is a FAD-containing Baeyer-Villiger monooxygenase (BVMO). To elucidate the mechanism of conversion of phenylacetone by PAMO, we have performed a detailed steady-state and pre-steady-state kinetic analysis. In the catalytic cycle ( k cat = 3.1 s (-1)), rapid binding of NADPH ( K d = 0.7 microM) is followed by a transfer of the 4( R)-hydride from NADPH to the FAD cofactor ( k red = 12 s (-1)). The reduced PAMO is rapidly oxygenated by molecular oxygen ( k ox = 870 mM (-1) s (-1)), yielding a C4a-peroxyflavin. The peroxyflavin enzyme intermediate reacts with phenylacetone to form benzylacetate ( k 1 = 73 s (-1)). This latter kinetic event leads to an enzyme intermediate which we could not unequivocally assign and may represent a Criegee intermediate or a C4a-hydroxyflavin form. The relatively slow decay (4.1 s (-1)) of this intermediate yields fully reoxidized PAMO and limits the turnover rate. NADP (+) release is relatively fast and represents the final step of the catalytic cycle. This study shows that kinetic behavior of PAMO is significantly different when compared with that of sequence-related monooxygenases, e.g., cyclohexanone monooxygenase and liver microsomal flavin-containing monooxygenase. Inspection of the crystal structure of PAMO has revealed that residue R337, which is conserved in other BVMOs, is positioned close to the flavin cofactor. The analyzed R337A and R337K mutant enzymes were still able to form and stabilize the C4a-peroxyflavin intermediate. The mutants were unable to convert either phenylacetone or benzyl methyl sulfide. This demonstrates that R337 is crucially involved in assisting PAMO-mediated Baeyer-Villiger and sulfoxidation reactions.

  8. 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.

  9. Characterization and Crystal Structure of a Robust Cyclohexanone Monooxygenase.

    PubMed

    Romero, Elvira; Castellanos, J Rubén Gómez; Mattevi, Andrea; Fraaije, Marco W

    2016-12-19

    Cyclohexanone monooxygenase (CHMO) is a promising biocatalyst for industrial reactions owing to its broad substrate spectrum and excellent regio-, chemo-, and enantioselectivity. However, the low stability of many Baeyer-Villiger monooxygenases is an obstacle for their exploitation in industry. Characterization and crystal structure determination of a robust CHMO from Thermocrispum municipale is reported. The enzyme efficiently converts a variety of aliphatic, aromatic, and cyclic ketones, as well as prochiral sulfides. A compact substrate-binding cavity explains its preference for small rather than bulky substrates. Small-scale conversions with either purified enzyme or whole cells demonstrated the remarkable properties of this newly discovered CHMO. The exceptional solvent tolerance and thermostability make the enzyme very attractive for biotechnology.

  10. Characterization and Crystal Structure of a Robust Cyclohexanone Monooxygenase

    PubMed Central

    Romero, Elvira; Castellanos, J. Rubén Gómez

    2016-01-01

    Abstract Cyclohexanone monooxygenase (CHMO) is a promising biocatalyst for industrial reactions owing to its broad substrate spectrum and excellent regio‐, chemo‐, and enantioselectivity. However, the low stability of many Baeyer–Villiger monooxygenases is an obstacle for their exploitation in industry. Characterization and crystal structure determination of a robust CHMO from Thermocrispum municipale is reported. The enzyme efficiently converts a variety of aliphatic, aromatic, and cyclic ketones, as well as prochiral sulfides. A compact substrate‐binding cavity explains its preference for small rather than bulky substrates. Small‐scale conversions with either purified enzyme or whole cells demonstrated the remarkable properties of this newly discovered CHMO. The exceptional solvent tolerance and thermostability make the enzyme very attractive for biotechnology. PMID:27873437

  11. Substrate Specificity and Enantioselectivity of 4-Hydroxyacetophenone Monooxygenase

    PubMed Central

    Kamerbeek, Nanne M.; Olsthoorn, Arjen J. J.; Fraaije, Marco W.; Janssen, Dick B.

    2003-01-01

    The 4-hydroxyacetophenone monooxygenase (HAPMO) from Pseudomonas fluorescens ACB catalyzes NADPH- and oxygen-dependent Baeyer-Villiger oxidation of 4-hydroxyacetophenone to the corresponding acetate ester. Using the purified enzyme from recombinant Escherichia coli, we found that a broad range of carbonylic compounds that are structurally more or less similar to 4-hydroxyacetophenone are also substrates for this flavin-containing monooxygenase. On the other hand, several carbonyl compounds that are substrates for other Baeyer-Villiger monooxygenases (BVMOs) are not converted by HAPMO. In addition to performing Baeyer-Villiger reactions with aromatic ketones and aldehydes, the enzyme was also able to catalyze sulfoxidation reactions by using aromatic sulfides. Furthermore, several heterocyclic and aliphatic carbonyl compounds were also readily converted by this BVMO. To probe the enantioselectivity of HAPMO, the conversion of bicyclohept-2-en-6-one and two aryl alkyl sulfides was studied. The monooxygenase preferably converted (1R,5S)-bicyclohept-2-en-6-one, with an enantiomeric ratio (E) of 20, thus enabling kinetic resolution to obtain the (1S,5R) enantiomer. Complete conversion of both enantiomers resulted in the accumulation of two regioisomeric lactones with moderate enantiomeric excess (ee) for the two lactones obtained [77% ee for (1S,5R)-2 and 34% ee for (1R,5S)-3]. Using methyl 4-tolyl sulfide and methylphenyl sulfide, we found that HAPMO is efficient and highly selective in the asymmetric formation of the corresponding (S)-sulfoxides (ee > 99%). The biocatalytic properties of HAPMO described here show the potential of this enzyme for biotechnological applications. PMID:12514023

  12. The alkene monooxygenase from Xanthobacter strain Py2 is closely related to aromatic monooxygenases and catalyzes aromatic monohydroxylation of benzene, toluene, and phenol

    SciTech Connect

    Zhou, N.Y.; Jenkins, A.; Chan Kwo Chion, C.K.N.; Leak, D.J.

    1999-04-01

    The genes encoding the six polypeptide components of the alkene monooxygenase from Xanthobacter strain Py2 (Xamo) have been located on a 4.9-kb fragment of chromosomal DNA previously cloned in cosmid pNY2. Sequencing and analysis of the predicted amino acid sequences indicate that the components of Xamo are homologous to those of the aromatic monooxygenases, toluene 2-, 3-, and 4-monooxygenase and benzene monooxygenase, and that the gene order is identical. The genes and predicted polypeptides are aamA, encoding the 497-residue oxygenase {alpha}-subunit (XamoA); aamB, encoding the 88-residue oxygenase {gamma}-subunit (XamoB); aamC, encoding the 122-residue ferredoxin (XamoC); aamD, encoding the 101-residue coupling or effector protein (XamoD); aamE, encoding the 341-residue oxygenase {beta}-subunit (XamoE); and aamF, encoding the 327-residue reductase (XamoF). A sequence with > 60% concurrence with the consensus sequence of {sigma}{sup 5.4} (RpoN)-dependent promoters was identified upstream of the aamA gene. Detailed comparison of XamoA with the oxygenase {alpha}-subunits from aromatic monooxygenases, phenol hydroxylases, methane monooxygenase, and the alkene monooxygenase from Rhodococcus rhodochrous B276 showed that, despite the overall similarity to the aromatic monooxygenases, XamoA has some distinctive characteristics of the oxygenases which oxidize aliphatic, and particularly alkene, substrates. On the basis of the similarity between Xamo and the aromatic monooxygenases, Xanthobacter strain Py2 was tested and shown to oxidize benzene, toluene, and phenol, while the alkene monooxygenase-negative mutants NZ1 and NZ2 did not.

  13. Alkane Oxidation: Methane Monooxygenases, Related Enzymes, and Their Biomimetics.

    PubMed

    Wang, Vincent C-C; Maji, Suman; Chen, Peter P-Y; Lee, Hung Kay; Yu, Steve S-F; Chan, Sunney I

    2017-02-16

    Methane monooxygenases (MMOs) mediate the facile conversion of methane into methanol in methanotrophic bacteria with high efficiency under ambient conditions. Because the selective oxidation of methane is extremely challenging, there is considerable interest in understanding how these enzymes carry out this difficult chemistry. The impetus of these efforts is to learn from the microbes to develop a biomimetic catalyst to accomplish the same chemical transformation. Here, we review the progress made over the past two to three decades toward delineating the structures and functions of the catalytic sites in two MMOs: soluble methane monooxygenase (sMMO) and particulate methane monooxygenase (pMMO). sMMO is a water-soluble three-component protein complex consisting of a hydroxylase with a nonheme diiron catalytic site; pMMO is a membrane-bound metalloenzyme with a unique tricopper cluster as the site of hydroxylation. The metal cluster in each of these MMOs harnesses O2 to functionalize the C-H bond using different chemistry. We highlight some of the common basic principles that they share. Finally, the development of functional models of the catalytic sites of MMOs is described. These efforts have culminated in the first successful biomimetic catalyst capable of efficient methane oxidation without overoxidation at room temperature.

  14. Inhibition of human squalene monooxygenase by selenium compounds.

    PubMed

    Gupta, Nisha; Porter, Todd D

    2002-01-01

    Selenosis in animals is characterized by a variety of neurological abnormalities, but the chemical species of selenium and the molecular targets that mediate this neurotoxicity are unknown. We have previously shown that selenite is a potent inhibitor of squalene monooxygenase, the second enzyme in the committed pathway for cholesterol biosynthesis; inhibition of this enzyme by dimethyltellurium leads to a peripheral demyelinating neuropathy similar to that seen in selenosis. To evaluate the role methylation plays in selenium toxicity, we examined the ability of three methylselenium compounds, methylselenol, dimethylselenide, and trimethylselenonium iodide, to inhibit purified recombinant human squalene monooxygenase. IC(50) values for methylselenol (95 microM) and dimethylselenide (680 microM) were greater than that previously obtained for selenite (37 microM), and inhibition by trimethylselenonium iodide was evident only at concentrations above 3 mM. Inhibition by methylselenol as well as by selenite was slow and irreversible, suggestive of covalent binding to the enzyme, and thiol-containing compounds could prevent and reverse this inhibition, indicating that these compounds were reacting with sulfhydryl groups on the protein. Monothiols such as glutathione and beta-mercaptoethanol provided better protection than did dithiols, suggesting that these selenium compounds bind to only one of the two proposed vicinal cysteines on squalene monooxygenase. Unexpectedly, the inhibition by selenite was significantly enhanced by dithiols, indicating that a more toxic species, possibly selenide, was formed in the presence of these dithiol reductants. Copyright 2002 Wiley Periodicals, Inc.

  15. Investigating the coenzyme specificity of phenylacetone monooxygenase from Thermobifida fusca

    PubMed Central

    Dudek, Hanna M.; Torres Pazmiño, Daniel E.; Rodríguez, Cristina; de Gonzalo, Gonzalo; Gotor, Vicente

    2010-01-01

    Type I Baeyer–Villiger monooxygenases (BVMOs) strongly prefer NADPH over NADH as an electron donor. In order to elucidate the molecular basis for this coenzyme specificity, we have performed a site-directed mutagenesis study on phenylacetone monooxygenase (PAMO) from Thermobifida fusca. Using sequence alignments of type I BVMOs and crystal structures of PAMO and cyclohexanone monooxygenase in complex with NADP+, we identified four residues that could interact with the 2′-phosphate moiety of NADPH in PAMO. The mutagenesis study revealed that the conserved R217 is essential for binding the adenine moiety of the nicotinamide coenzyme while it also contributes to the recognition of the 2′-phosphate moiety of NADPH. The substitution of T218 did not have a strong effect on the coenzyme specificity. The H220N and H220Q mutants exhibited a ~3-fold improvement in the catalytic efficiency with NADH while the catalytic efficiency with NADPH was hardly affected. Mutating K336 did not increase the activity of PAMO with NADH, but it had a significant and beneficial effect on the enantioselectivity of Baeyer–Villiger oxidations and sulfoxidations. In conclusion, our results indicate that the function of NADPH in catalysis cannot be easily replaced by NADH. This finding is in line with the complex catalytic mechanism and the vital role of the coenzyme in BVMOs. PMID:20703875

  16. Investigating the coenzyme specificity of phenylacetone monooxygenase from Thermobifida fusca.

    PubMed

    Dudek, Hanna M; Torres Pazmiño, Daniel E; Rodríguez, Cristina; de Gonzalo, Gonzalo; Gotor, Vicente; Fraaije, Marco W

    2010-11-01

    Type I Baeyer-Villiger monooxygenases (BVMOs) strongly prefer NADPH over NADH as an electron donor. In order to elucidate the molecular basis for this coenzyme specificity, we have performed a site-directed mutagenesis study on phenylacetone monooxygenase (PAMO) from Thermobifida fusca. Using sequence alignments of type I BVMOs and crystal structures of PAMO and cyclohexanone monooxygenase in complex with NADP(+), we identified four residues that could interact with the 2'-phosphate moiety of NADPH in PAMO. The mutagenesis study revealed that the conserved R217 is essential for binding the adenine moiety of the nicotinamide coenzyme while it also contributes to the recognition of the 2'-phosphate moiety of NADPH. The substitution of T218 did not have a strong effect on the coenzyme specificity. The H220N and H220Q mutants exhibited a ~3-fold improvement in the catalytic efficiency with NADH while the catalytic efficiency with NADPH was hardly affected. Mutating K336 did not increase the activity of PAMO with NADH, but it had a significant and beneficial effect on the enantioselectivity of Baeyer-Villiger oxidations and sulfoxidations. In conclusion, our results indicate that the function of NADPH in catalysis cannot be easily replaced by NADH. This finding is in line with the complex catalytic mechanism and the vital role of the coenzyme in BVMOs.

  17. Collaborative contribution of six cytochrome P450 monooxygenase genes to fenpropathrin resistance in Tetranychus cinnabarinus (Boisduval).

    PubMed

    Shi, L; Zhang, J; Shen, G; Xu, Z; Xu, Q; He, L

    2016-10-01

    Cytochrome P450 monooxygenases (P450s), as an important family of detoxification enzymes, participate in the metabolism of agrochemicals in almost all agricultural pests and play important roles in the development of insecticide resistance. Two P450 genes (CYP389B1 and CYP392A26) were identified and their expression patterns were investigated in our previous study. In this study, four more P450 gene sequences (CYP391A1, CYP384A1, CYP392D11 and CYP392A28) from the Clan 2, Clan 3 and Clan 4 families were identified and characterized. Quantitative PCR analysis showed that these four P450 genes were highly expressed in a fenpropathrin-resistant (FeR) strain of Tetranychus cinnabarinus. In addition, their expressions were much more sensitive to fenpropathrin induction in the FeR strain than the susceptible strain. Gene-silencing experiments via double-stranded RNA feeding were carried out. The results showed that mRNA levels of these six P450 genes were reduced in the FeR strain and the activities of P450s were decreased. Consequently mite susceptibilities to fenpropathrin were increased. Interestingly, silencing all six P450 genes simultaneously had an even greater effect on resistance than silencing them individually. This study increases our understanding of the molecular mechanisms of insecticide detoxification, suggesting that the overexpression of these six P450 genes might play important roles in fenpropathrin resistance in T. cinnabarinus collaboratively. © 2016 The Royal Entomological Society.

  18. Lytic Polysaccharide Monooxygenases: The Microbial Power Tool for Lignocellulose Degradation.

    PubMed

    Johansen, Katja Salomon

    2016-11-01

    Lytic polysaccharide monooxygenases (LPMOs) are copper-enzymes that catalyze oxidative cleavage of glycosidic bonds. These enzymes are secreted by many microorganisms to initiate infection and degradation processes. In particular, the concept of fungal degradation of lignocellulose has been revised in the light of this recent finding. LPMOs require a source of electrons for activity, and both enzymatic and plant-derived sources have been identified. Importantly, light-induced electron delivery from light-harvesting pigments can efficiently drive LPMO activity. The possible implications of LPMOs in plant-symbiont and -pathogen interactions are discussed in the context of the very powerful oxidative capacity of these enzymes.

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

    PubMed Central

    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.; Leggio, Leila Lo; Walton, Paul H.

    2016-01-01

    Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes which 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 the first 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

  20. 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.

  1. Pharmacological kynurenine 3-monooxygenase enzyme inhibition significantly reduces neuropathic pain in a rat model.

    PubMed

    Rojewska, Ewelina; Piotrowska, Anna; Makuch, Wioletta; Przewlocka, Barbara; Mika, Joanna

    2016-03-01

    Recent studies have highlighted the involvement of the kynurenine pathway in the pathology of neurodegenerative diseases, but the role of this system in neuropathic pain requires further extensive research. Therefore, the aim of our study was to examine the role of kynurenine 3-monooxygenase (Kmo), an enzyme that is important in this pathway, in a rat model of neuropathy after chronic constriction injury (CCI) to the sciatic nerve. For the first time, we demonstrated that the injury-induced increase in the Kmo mRNA levels in the spinal cord and the dorsal root ganglia (DRG) was reduced by chronic administration of the microglial inhibitor minocycline and that this effect paralleled a decrease in the intensity of neuropathy. Further, minocycline administration alleviated the lipopolysaccharide (LPS)-induced upregulation of Kmo mRNA expression in microglial cell cultures. Moreover, we demonstrated that not only indirect inhibition of Kmo using minocycline but also direct inhibition using Kmo inhibitors (Ro61-6048 and JM6) decreased neuropathic pain intensity on the third and the seventh days after CCI. Chronic Ro61-6048 administration diminished the protein levels of IBA-1, IL-6, IL-1beta and NOS2 in the spinal cord and/or the DRG. Both Kmo inhibitors potentiated the analgesic properties of morphine. In summary, our data suggest that in neuropathic pain model, inhibiting Kmo function significantly reduces pain symptoms and enhances the effectiveness of morphine. The results of our studies show that the kynurenine pathway is an important mediator of neuropathic pain pathology and indicate that Kmo represents a novel pharmacological target for the treatment of neuropathy.

  2. Characterization and Application of Xylene Monooxygenase for Multistep Biocatalysis

    PubMed Central

    Bühler, Bruno; Witholt, Bernard; Hauer, Bernhard; Schmid, Andreas

    2002-01-01

    Xylene monooxygenase of Pseudomonas putida mt-2 catalyzes multistep oxidations of one methyl group of toluene and xylenes. Recombinant Escherichia coli expressing the monooxygenase genes xylM and xylA catalyzes the oxygenation of toluene, pseudocumene, the corresponding alcohols, and the corresponding aldehydes, all by a monooxygenation type of reaction (B. Bühler, A. Schmid, B. Hauer, and B. Witholt, J. Biol. Chem. 275:10085-10092, 2000). Using E. coli expressing xylMA, we investigated the kinetics of this one-enzyme three-step biotransformation. We found that unoxidized substrates like toluene and pseudocumene inhibit the second and third oxygenation steps and that the corresponding alcohols inhibit the third oxygenation step. These inhibitions might promote the energetically more favorable alcohol and aldehyde dehydrogenations in the wild type. Growth of E. coli was strongly affected by low concentrations of pseudocumene and its products. Toxicity and solubility problems were overcome by the use of a two-liquid-phase system with bis(2-ethylhexyl)phthalate as the carrier solvent, allowing high overall substrate and product concentrations. In a fed-batch-based two-liquid-phase process with pseudocumene as the substrate, we observed the consecutive accumulation of aldehyde, acid, and alcohol. Our results indicate that, depending on the reaction conditions, product formation could be directed to one specific product. PMID:11823191

  3. Regulated O2 activation in flavin-dependent monooxygenases.

    PubMed

    Frederick, Rosanne E; Mayfield, Jeffery A; DuBois, Jennifer L

    2011-08-17

    Flavin-dependent monooxygenases (FMOs) are involved in important biosynthetic pathways in diverse organisms, including production of the siderophores used for the import and storage of essential iron in serious pathogens. We have shown that the FMO from Aspergillus fumigatus, an ornithine monooxygenase (Af-OMO), is mechanistically similar to its well-studied distant homologues from mammalian liver. The latter are highly promiscuous in their choice of substrates, while Af-OMO is unusually specific. This presents a puzzle: how do Af-OMO and other FMOs of the biosynthetic classes achieve such specificity? We have discovered substantial enhancement in the rate of O(2) activation in Af-OMO in the presence of L-arginine, which acts as a small molecule regulator. Such protein-level regulation could help explain how this and related biosynthetic FMOs manage to couple O(2) activation and substrate hydroxylation to each other and to the appropriate cellular conditions. Given the essentiality of Fe to Af and the avirulence of the Af-OMO gene knock out, inhibitors of Af-OMO are likely to be drug targets against this medically intractable pathogen.

  4. Regulated O2 Activation in Flavin-Dependent Monooxygenases

    PubMed Central

    Frederick, Rosanne E.; Mayfield, Jeffery A.; DuBois, Jennifer L.

    2012-01-01

    Flavin-dependent monooxygenases (FMOs) are involved in important biosynthetic pathways in diverse organisms, including production of the siderophores used for the import and storage of essential iron in serious pathogens. We have shown that the FMO from Aspergillus fumigatus, an ornithine monooxygenase (Af-OMO), is mechanistically similar to its well-studied distant homologues from mammalian liver. The latter are highly promiscuous in their choice of substrates, while Af-OMO is unusually specific. This presents a puzzle: how do Af-OMO and other FMOs of the biosynthetic classes achieve such specificity? We have discovered substantial enhancement in the rate of O2 activation in Af-OMO in the presence of l-arginine, which acts as a small molecule regulator. Such protein-level regulation could help explain how this and related biosynthetic FMOs manage to couple O2 activation and substrate hydroxylation to each other and to the appropriate cellular conditions. Given the essentiality of Fe to Af and the avirulence of the Af-OMO gene knock out, inhibitors of Af-OMO are likely to be drug targets against this medically intractable pathogen. PMID:21774554

  5. Engineering Styrene Monooxygenase for Biocatalysis: Reductase-Epoxidase Fusion Proteins.

    PubMed

    Heine, Thomas; Tucker, Kathryn; Okonkwo, Nonye; Assefa, Berhanegebriel; Conrad, Catleen; Scholtissek, Anika; Schlömann, Michael; Gassner, George; Tischler, Dirk

    2017-04-01

    The enantioselective epoxidation of styrene and related compounds by two-component styrene monooxygenases (SMOs) has targeted these enzymes for development as biocatalysts. In the present work, we prepare genetically engineered fusion proteins that join the C-terminus of the epoxidase (StyA) to the N-terminus of the reductase (StyB) through a linker peptide and demonstrate their utility as biocatalysts in the synthesis of Tyrain purple and other indigoid dyes. A single-vector expression system offers a simplified platform for transformation and expansion of the catalytic function of styrene monooxygenases, and the resulting fusion proteins are self-regulated and couple efficiently NADH oxidation to styrene epoxidation. We find that the reductase domain proceeds through a sequential ternary-complex mechanism at low FAD concentration and a double-displacement mechanism at higher concentrations of FAD. Single-turnover studies indicate an observed rate constant for FAD-to-FAD hydride transfer of ~8 s(-1). This step is rate limiting in the styrene epoxidation reaction and helps to ensure that flavin reduction and styrene epoxidation reactions proceed without wasteful side reactions. Comparison of the reductase activity of the fusion proteins with the naturally occurring reductase, SMOB, and N-terminally histidine-tagged reductase, NSMOB, suggests that the observed changes in catalytic mechanism are due in part to an increase in flavin-binding affinity associated with the N-terminal extension of the reductase.

  6. 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.

  7. Glyceryl ether monooxygenase resembles aromatic amino acid hydroxylases in metal ion and tetrahydrobiopterin dependence.

    PubMed

    Watschinger, Katrin; Keller, Markus A; Hermetter, Albin; Golderer, Georg; Werner-Felmayer, Gabriele; Werner, Ernst R

    2009-01-01

    Glyceryl ether monooxygenase is a tetrahydrobiopterin-dependent membrane-bound enzyme which catalyses the cleavage of lipid ethers into glycerol and the corresponding aldehyde. Despite many different characterisation and purification attempts, so far no gene and primary sequence have been assigned to this enzyme. The seven other tetrahydrobiopterin-dependent enzymes can be divided in the family of aromatic amino acid hydroxylases - comprising phenylalanine hydroxylase, tyrosine hydroxylase and the two tryptophan hydroxylases - and into the three nitric oxide synthases. We tested the influences of different metal ions and metal ion chelators on glyceryl ether monooxygenase, phenylalanine hydroxylase and nitric oxide synthase activity to elucidate the relationship of glyceryl ether monooxygenase to these two families. 1,10-Phenanthroline, an inhibitor of non-heme iron-dependent enzymes, was able to potently block glyceryl ether monooxygenase as well as phenylalanine hydroxylase, but had no effect on inducible nitric oxide synthase. Two tetrahydrobiopterin analogues, N(5)-methyltetrahydrobiopterin and 4-aminotetrahydrobiopterin, had a similar impact on glyceryl ether monooxygenase activity, as has already been shown for phenylalanine hydroxylase. These observations point to a close analogy of the role of tetrahydrobiopterin in glyceryl ether monooxygenase and in aromatic amino acid hydroxylases and suggest that glyceryl ether monooxygenase may require a non-heme iron for catalysis.

  8. 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.

  9. Lytic polysaccharide monooxygenases disrupt the cellulose fibers structure.

    PubMed

    Villares, Ana; Moreau, Céline; Bennati-Granier, Chloé; Garajova, Sona; Foucat, Loïc; Falourd, Xavier; Saake, Bodo; Berrin, Jean-Guy; Cathala, Bernard

    2017-01-10

    Lytic polysaccharide monooxygenases (LPMOs) are a class of powerful oxidative enzymes that breakdown recalcitrant polysaccharides such as cellulose. Here we investigate the action of LPMOs on cellulose fibers. After enzymatic treatment and dispersion, LPMO-treated fibers show intense fibrillation. Cellulose structure modifications visualized at different scales indicate that LPMO creates nicking points that trigger the disintegration of the cellulose fibrillar structure with rupture of chains and release of elementary nanofibrils. Investigation of LPMO action using solid-state NMR provides direct evidence of modification of accessible and inaccessible surfaces surrounding the crystalline core of the fibrils. The chains breakage likely induces modifications of the cellulose network and weakens fibers cohesion promoting their disruption. Besides the formation of new initiation sites for conventional cellulases, this work provides the first evidence of the direct oxidative action of LPMOs with the mechanical weakening of the cellulose ultrastructure. LPMOs can be viewed as promising biocatalysts for enzymatic modification or degradation of cellulose fibers.

  10. Substrate and inhibitor specificity of kynurenine monooxygenase from Cytophaga hutchinsonii.

    PubMed

    Phillips, Robert S; Anderson, Andrew D; Gentry, Harvey G; Güner, Osman F; Bowen, J Phillip

    2017-04-15

    Kynurenine monooxygenase (KMO) is a potential drug target for treatment of neurodegenerative disorders such as Huntington's and Alzheimer's diseases. We have evaluated substituted kynurenines as substrates or inhibitors of KMO from Cytophaga hutchinsonii. Kynurenines substituted with a halogen at the 5-position are excellent substrates, with values of kcat and kcat/Km comparable to or higher than kynurenine. However, kynurenines substituted in the 3-position are competitive inhibitors, with KI values lower than the Km for kynurenine. Bromination also enhances inhibition, and 3,5-dibromokynurenine is a potent competitive inhibitor with a KI value of 1.5μM. A pharmacophore model of KMO was developed, and predicted that 3,4-dichlorohippuric acid would be an inhibitor. The KI for this compound was found to be 34μM, thus validating the pharmacophore model. We are using these results and our model to design more potent inhibitors of KMO. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Lytic polysaccharide monooxygenases disrupt the cellulose fibers structure

    PubMed Central

    Villares, Ana; Moreau, Céline; Bennati-Granier, Chloé; Garajova, Sona; Foucat, Loïc; Falourd, Xavier; Saake, Bodo; Berrin, Jean-Guy; Cathala, Bernard

    2017-01-01

    Lytic polysaccharide monooxygenases (LPMOs) are a class of powerful oxidative enzymes that breakdown recalcitrant polysaccharides such as cellulose. Here we investigate the action of LPMOs on cellulose fibers. After enzymatic treatment and dispersion, LPMO-treated fibers show intense fibrillation. Cellulose structure modifications visualized at different scales indicate that LPMO creates nicking points that trigger the disintegration of the cellulose fibrillar structure with rupture of chains and release of elementary nanofibrils. Investigation of LPMO action using solid-state NMR provides direct evidence of modification of accessible and inaccessible surfaces surrounding the crystalline core of the fibrils. The chains breakage likely induces modifications of the cellulose network and weakens fibers cohesion promoting their disruption. Besides the formation of new initiation sites for conventional cellulases, this work provides the first evidence of the direct oxidative action of LPMOs with the mechanical weakening of the cellulose ultrastructure. LPMOs can be viewed as promising biocatalysts for enzymatic modification or degradation of cellulose fibers. PMID:28071716

  12. The Oxygen Dilemma: A Severe Challenge for the Application of Monooxygenases?

    PubMed Central

    Holtmann, Dirk

    2016-01-01

    Abstract Monooxygenases are promising catalysts because they in principle enable the organic chemist to perform highly selective oxyfunctionalisation reactions that are otherwise difficult to achieve. For this, monooxygenases require reducing equivalents, to allow reductive activation of molecular oxygen at the enzymes' active sites. However, these reducing equivalents are often delivered to O2 either directly or via a reduced intermediate (uncoupling), yielding hazardous reactive oxygen species and wasting valuable reducing equivalents. The oxygen dilemma arises from monooxygenases' dependency on O2 and the undesired uncoupling reaction. With this contribution we hope to generate a general awareness of the oxygen dilemma and to discuss its nature and some promising solutions. PMID:27194219

  13. Evolutionary history of a specialized P450 propane monooxygenase

    PubMed Central

    Fasan, Rudi; Meharenna, Yergalem T.; Snow, Christopher D.; Poulos, Thomas L.; Arnold, Frances H.

    2008-01-01

    Summary The evolutionary pressures that shaped the specificity and catalytic efficiency of enzymes can only be speculated. While directed evolution experiments show that new functions can be acquired under positive selection with few mutations, the role of negative selection in eliminating undesired activities and achieving high specificity remains unclear. Here we examine intermediates along the ‘lineage’ from a naturally-occurring C12–C20 fatty acid hydroxylase (P450BM3) to a laboratory-evolved P450 propane monooxygenase (P450PMO) having 20 heme domain substitutions compared to P450BM3. Biochemical, crystallographic and computational analyses show that a minimal perturbation of the P450BM3 fold and substrate binding pocket accompanies a significant broadening of enzyme substrate range and the emergence of propane activity. In contrast, refinement of the enzyme catalytic efficiency for propane oxidation (~9,000-fold increase in kcat/Km) involves profound reshaping and partitioning of the substrate access pathway. Remodeling of the substrate recognition mechanisms ultimately results in remarkable narrowing of the substrate profile around propane and enables the acquisition of a basal iodomethane dehalogenase activity as yet unknown in natural alkane monooxygenases. A highly destabilizing L188P substitution in a region of the enzyme that undergoes a large conformational change during catalysis plays an important role in adaptation to the gaseous alkane. This work demonstrates that positive selection alone is sufficient to completely re-specialize the cytochrome P450 for function on a non-native substrate. PMID:18619466

  14. 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.

  15. Structure of nitrilotriacetate monooxygenase component B from Mycobacterium thermoresistibile

    PubMed Central

    Zhang, Y.; Edwards, T. E.; Begley, D. W.; Abramov, A.; Thompkins, K. B.; Ferrell, M.; Guo, W. J.; Phan, I.; Olsen, C.; Napuli, A.; Sankaran, B.; Stacy, R.; Van Voorhis, W. C.; Stewart, L. J.; Myler, P. J.

    2011-01-01

    Mycobacterium tuberculosis belongs to a large family of soil bacteria which can degrade a remarkably broad range of organic compounds and utilize them as carbon, nitrogen and energy sources. It has been proposed that a variety of mycobacteria can subsist on alternative carbon sources during latency within an infected human host, with the help of enzymes such as nitrilotriacetate monooxygenase (NTA-Mo). NTA-Mo is a member of a class of enzymes which consist of two components: A and B. While component A has monooxygenase activity and is responsible for the oxidation of the substrate, component B consumes cofactor to generate reduced flavin mononucleotide, which is required for component A activity. NTA-MoB from M. thermoresistibile, a rare but infectious close relative of M. tuberculosis which can thrive at elevated temperatures, has been expressed, purified and crystallized. The 1.6 Å resolution crystal structure of component B of NTA-Mo presented here is one of the first crystal structures determined from the organism M. thermo­resistibile. The NTA-MoB crystal structure reveals a homodimer with the characteristic split-barrel motif typical of flavin reductases. Surprisingly, NTA-MoB from M. thermoresistibile contains a C-terminal tail that is highly conserved among myco­bacterial orthologs and resides in the active site of the other protomer. Based on the structure, the C-terminal tail may modulate NTA-MoB activity in mycobacteria by blocking the binding of flavins and NADH. PMID:21904057

  16. Osmotic Regulation of a Novel Flavin-Containing Monooxygenase in Primary Cultured Cells from Rainbow Trout (Oncorhynchus mykiss)

    PubMed Central

    Rodríguez-Fuentes, Gabriela; Aparicio-Fabre, Rosaura; Li, Qi; Schlenk, Daniel

    2013-01-01

    A cDNA encoding a hepatic isoform of flavin-containing monooxygenase (hFMO) (EF063736) containing an open reading frame of 1792 base pairs (bp) and encoding 554 amino acids was cloned and sequenced from liver mRNA of rainbow trout (Oncorhynchus mykiss). The genomic sequence of hFMO was also characterized and was 4.379 kilobases, possessing 10 exons and 9 introns (EU519462). Structural analysis of the promoter region showed several cis-acting elements including putative glucocorticoid and osmoregulatory response elements, which have been reported to be functionally related to induction of flavin-containing monooxygenase (FMO) proteins in vertebrates. The amino acid sequence showed 74% identity to a putative FMO gene from fugu (Takifugu rubripes; Q6ZZY9), 52 to 55% to zebrafish (Brachydanio rerio; Q5RGM6, Q5RGM3, Q6TLD2, Q7T1D7) FMO5, and 54 and 50% to human FMO1 (Q01740), FMO3 (P49326), and FMO5 (P49326). Southern blot analysis using a 180-bp fragment of the hFMO cDNA indicated at least seven potential genes. Treatment of primary trout hepatocytes with cortisol and sodium chloride for 24 h enhanced hFMO expression. Expression of hFMO was not detected in untreated or solute-treated primary cultures of gill epithelial cells, suggesting tissue-specific expression of hFMO. Induction of hFMO is consistent with the occurrence of cis-osmoregulatory and glucocorticoid response elements identified in the 5′-upstream sequence, indicating regulation of hFMO in response to hypersaline conditions and the osmoregulatory hormone cortisol. PMID:18372402

  17. Characterization of the peptidylglycine α-amidating monooxygenase (PAM) from the venom ducts of neogastropods, Conus bullatus and Conus geographus

    PubMed Central

    Ul-Hasan, Sabah; Burgess, Daniel M.; Gajewiak, Joanna; Li, Qing; Hu, Hao; Yandell, Mark; Olivera, Baldomero M.; Bandyopadhyay, Pradip K.

    2014-01-01

    Cone snails, genus Conus, are predatory marine snails that use venom to capture their prey. This venom contains a diverse array of peptide toxins, known as conotoxins, which undergo a diverse set of posttranslational modifications. Amidating enzymes modify peptides and proteins containing a C-terminal glycine residue, resulting in loss of the glycine residue and amidation of the preceding residue. A significant fraction of peptides present in the venom of cone snails contain C-terminal amidated residues, which are important for optimizing biological activity. This study describes the characterization of the amidating enzyme, peptidylglycine α-amidating monooxygenase (PAM), present in the venom duct of cone snails, Conus bullatus and Conus geographus. PAM is known to carry out two functions, peptidyl α-hydroxylating monooxygenase (PHM) and peptidylamido-glycolate lyase (PAL). In some animals, such as Drosophila melanogaster, these two functions are present in separate polypeptides, working as individual enzymes. In other animals, such as mammals and in Aplysia californica, PAM activity resides in a single, bifunctional polypeptide. Using specific oligonucleotide primers and reverse transcription-polymerase chain reaction we have identified and cloned from the venom duct cDNA library, a cDNA with 49% homology to PAM from A. californica. We have determined that both the PHM and PAL activities are encoded in one mRNA polynucleotide in both C. bullatus and C. geographus. We have directly demonstrated enzymatic activity catalyzing the conversion of dansyl-YVG-COOH to dansyl-YV-NH2 in cloned cDNA expressed in Drosophila S2 cells. PMID:23994590

  18. Metabolic conditions determining the composition and catalytic activity of cytochrome P-450 monooxygenases in Candida tropicalis.

    PubMed Central

    Sanglard, D; Käppeli, O; Fiechter, A

    1984-01-01

    In the microsomal fraction of Candida tropicalis cells, two distinct monooxygenases were detected, depending on the growth conditions. The distinction of the two monooxygenases was evident from: (i) the absorption maxima in the reduced CO difference spectra of the terminal oxidases (cytochromes P-450 and P-448); (ii) the contents of the monooxygenase components (cytochromes P-450/P-448, NADPH-cytochrome c (P-450) reductase, and cytochrome b5) and (iii) the catalytic activity of the complete system (aliphatic hydroxylation and N-demethylation activity). The occurrence of the respective monooxygenases could be related to the carbon source (n-alkanes or glucose). Oxygen limitation led to a significant increase of cytochrome P-450/P-448 content, independent of the carbon source utilized by the cells. An improved method for the isolation of microsomes enabled us to demonstrate the presence of cytochrome P-448 in glucose-grown cells. PMID:6690424

  19. Comprehensive Spectroscopic, Steady State, and Transient Kinetic Studies of a Representative Siderophore-associated Flavin Monooxygenase*

    PubMed Central

    Mayfield, Jeffery A.; Frederick, Rosanne E.; Streit, Bennett R.; Wencewicz, Timothy A.; Ballou, David P.; DuBois, Jennifer L.

    2010-01-01

    Many siderophores used for the uptake and intracellular storage of essential iron contain hydroxamate chelating groups. Their biosyntheses are typically initiated by hydroxylation of the primary amine side chains of l-ornithine or l-lysine. This reaction is catalyzed by members of a widespread family of FAD-dependent monooxygenases. Here the kinetic mechanism for a representative family member has been extensively characterized by steady state and transient kinetic methods, using heterologously expressed N5-l-ornithine monooxygenase from the pathogenic fungus Aspergillus fumigatus. Spectroscopic data and kinetic analyses suggest a model in which a molecule of hydroxylatable substrate serves as an activator for the reaction of the reduced flavin and O2. The rate acceleration is only ∼5-fold, a mild effect of substrate on formation of the C4a-hydroperoxide that does not influence the overall rate of turnover. The effect is also observed with the bacterial ornithine monooxygenase PvdA. The C4a-hydroperoxide is stabilized in the absence of hydroxylatable substrate by the presence of bound NADP+ (t½ = 33 min, 25 °C, pH 8). NADP+ therefore is a likely regulator of O2 and substrate reactivity in the siderophore-associated monooxygenases. Aside from the activating effect of the hydroxylatable substrate, the siderophore-associated monooxygenases share a kinetic mechanism with the hepatic microsomal flavin monooxygenases and bacterial Baeyer-Villiger monooxygenases, with which they share only moderate sequence homology and from which they are distinguished by their acute substrate specificity. The remarkable specificity of the N5-l-ornithine monooxygenase-catalyzed reaction suggests added means of reaction control beyond those documented in related well characterized flavoenzymes. PMID:20650894

  20. 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

  1. A soluble form of ammonia monooxygenase in Nitrosomonas europaea.

    PubMed

    Gilch, Stefan; Meyer, Ortwin; Schmidt, Ingo

    2009-09-01

    Ammonia monooxygenase (AMO) of Nitrosomonas europaea is a metalloenzyme that catalyzes the oxidation of ammonia to hydroxylamine. This study shows that AMO resides in the cytoplasm of the bacteria in addition to its location in the membrane and is distributed approximately equally in both subcellular fractions. AMO in both fractions catalyzes the oxidation of ammonia and binds [(14)C]acetylene, a mechanism-based inhibitor which specifically interacts with catalytically active AMO. Soluble AMO was purified 12-fold to electrophoretic homogeneity with a yield of 8%. AMO has a molecular mass of approximately 283 kDa with subunits of ca. 27 kDa (alpha-subunit, AmoA), ca. 42 kDa (beta-subunit, AmoB), and ca. 24 kDa (gamma-subunit, cytochrome c(1)) in an alpha(3)beta(3)gamma(3) sub-unit structure. Different from the beta-subunit of membrane-bound AMO, AmoB of soluble AMO possesses an N-terminal signal sequence. AMO contains Cu (9.4+/-0.6 mol per mol AMO), Fe (3.9+/-0.3 mol per mol AMO), and Zn (0.5 to 2.6 mol per mol AMO). Upon reduction the visible absorption spectrum of AMO reveals absorption bands characteristic of cytochrome c. Electron para-magnetic resonance spectroscopy of air-oxidized AMO at 50 K shows a paramagnetic signal originating from Cu(2+) and at 10 K a paramagnetic signal characteristic of heme-Fe.

  2. 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.

  3. Differential microbial transformation of nitrosamines by an inducible propane monooxygenase.

    PubMed

    Homme, Carissa L; Sharp, Jonathan O

    2013-07-02

    The toxicity of N-nitrosamines, their presence in drinking and environmental water supplies, and poorly understood recalcitrance collectively necessitate a better understanding of their potential for bioattenuation. Here, we show that the bacterial strain Rhodococcus jostii RHA1 can biotransform N-nitrosodiethylamine (NDEA), N-nitrosodi-n-propylamine (NDPA), N-nitrosopyrrolidine (NPYR), and possibly N-nitrosomorpholine (NMOR) in addition to N-nitrosodimethylamine (NDMA). Growth of cells on propane as the sole carbon source greatly enhanced degradation rates when contrasted with cells grown on complex organics. Propane-induced rates in order of fastest to slowest were NDMA > NDEA > NDPA > NPYR > NMOR at concentrations <2000 μg/L. Removal rates for linear functional groups scaled inversely with mass and cyclic nitrosamines were more recalcitrant than linear nitrosamines. Controls demonstrated significant NDEA and NDPA losses independent of biomass, suggesting abiotic processes may play a role in attenuation of these two compounds under experimental conditions tested here. In contrast to NDMA, a transition from first to zero order kinetics was not observed for the other nitrosamines included in this study over a concentration range of 20-2000 μg/L. A genetic knockout for the propane monooxygenase enzyme (PrMO) confirmed the role of this enzyme in the biotransformation of NDEA and NPYR. This study furthers our understanding of environmental nitrosamine attenuation by revealing an enzymatic mechanism for the biotransformation of multiple nitrosamines, their relative recalcitrance to transformation, and potential for abiotic loss.

  4. Product Repression of Alkane Monooxygenase Expression in Pseudomonas butanovora

    PubMed Central

    Doughty, D. M.; Sayavedra-Soto, L. A.; Arp, D. J.; Bottomley, P. J.

    2006-01-01

    Physiological and regulatory mechanisms that allow the alkane-oxidizing bacterium Pseudomonas butanovora to consume C2 to C8 alkane substrates via butane monooxygenase (BMO) were examined. Striking differences were observed in response to even- versus odd-chain-length alkanes. Propionate, the downstream product of propane oxidation and of the oxidation of other odd-chain-length alkanes following β-oxidation, was a potent repressor of BMO expression. The transcriptional activity of the BMO promoter was reduced with as little as 10 μM propionate, even in the presence of appropriate inducers. Propionate accumulated stoichiometrically when 1-propanol and propionaldehyde were added to butane- and ethane-grown cells, indicating that propionate catabolism was inactive during growth on even-chain-length alkanes. In contrast, propionate consumption was induced (about 80 nmol propionate consumed · min−1 · mg protein−1) following growth on the odd-chain-length alkanes, propane and pentane. The induction of propionate consumption could be brought on by the addition of propionate or pentanoate to the growth medium. In a reporter strain of P. butanovora in which the BMO promoter controls β-galactosidase expression, only even-chain-length alcohols (C2 to C8) induced β-galactosidase following growth on acetate or butyrate. In contrast, both even- and odd-chain-length alcohols (C3 to C7) were able to induce β-galactosidase following the induction of propionate consumption by propionate or pentanoate. PMID:16547046

  5. 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

  6. The yeast Geotrichum candidum encodes functional lytic polysaccharide monooxygenases.

    PubMed

    Ladevèze, Simon; Haon, Mireille; Villares, Ana; Cathala, Bernard; Grisel, Sacha; Herpoël-Gimbert, Isabelle; Henrissat, Bernard; Berrin, Jean-Guy

    2017-01-01

    Lytic polysaccharide monooxygenases (LPMOs) are a class of powerful oxidative enzymes that have revolutionized our understanding of lignocellulose degradation. Fungal LPMOs of the AA9 family target cellulose and hemicelluloses. AA9 LPMO-coding genes have been identified across a wide range of fungal saprotrophs (Ascomycotina, Basidiomycotina, etc.), but so far they have not been found in more basal lineages. Recent genome analysis of the yeast Geotrichum candidum (Saccharomycotina) revealed the presence of several LPMO genes, which belong to the AA9 family. In this study, three AA9 LPMOs from G. candidum were successfully produced and biochemically characterized. The use of native signal peptides was well suited to ensure correct processing and high recombinant production of GcLPMO9A, GcLPMO9B, and GcLPMO9C in Pichia pastoris. We show that GcLPMO9A and GcLPMO9B were both active on cellulose and xyloglucan, releasing a mixture of soluble C1- and C4-oxidized oligosaccharides from cellulose. All three enzymes disrupted cellulose fibers and significantly improved the saccharification of pretreated lignocellulosic biomass upon addition to a commercial cellulase cocktail. The unique enzymatic arsenal of G. candidum compared to other yeasts could be beneficial for plant cell wall decomposition in a saprophytic or pathogenic context. From a biotechnological point of view, G. candidum LPMOs are promising candidates to further enhance enzyme cocktails used in biorefineries such as consolidated bioprocessing.

  7. Engineering Cyclohexanone Monooxygenase for the Production of Methyl Propanoate.

    PubMed

    van Beek, Hugo L; Romero, Elvira; Fraaije, Marco W

    2017-01-20

    A previous study showed that cyclohexanone monooxygenase from Acinetobacter calcoaceticus (AcCHMO) catalyzes the Baeyer-Villiger oxidation of 2-butanone, yielding ethyl acetate and methyl propanoate as products. Methyl propanoate is of industrial interest as a precursor of acrylic plastic. Here, various residues near the substrate and NADP(+) binding sites in AcCHMO were subjected to saturation mutagenesis to enhance both the activity on 2-butanone and the regioselectivity toward methyl propanoate. The resulting libraries were screened using whole cell biotransformations, and headspace gas chromatography-mass spectrometry was used to identify improved AcCHMO variants. This revealed that the I491A AcCHMO mutant exhibits a significant improvement over the wild type enzyme in the desired regioselectivity using 2-butanone as a substrate (40% vs 26% methyl propanoate, respectively). Another interesting mutant is the T56S AcCHMO mutant, which exhibits a higher conversion yield (92%) and kcat (0.5 s(-1)) than wild type AcCHMO (52% and 0.3 s(-1), respectively). Interestingly, the uncoupling rate for the T56S AcCHMO mutant is also significantly lower than that for the wild type enzyme. The T56S/I491A double mutant combined the beneficial effects of both mutations leading to higher conversion and improved regioselectivity. This study shows that even for a relatively small aliphatic substrate (2-butanone), catalytic efficiency and regioselectivity can be tuned by structure-inspired enzyme engineering.

  8. Diversity and evolution of cytochrome P450 monooxygenases in Oomycetes.

    PubMed

    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

    2015-07-01

    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.

  9. Indigo formation by microorganisms expressing styrene monooxygenase activity.

    PubMed Central

    O'Connor, K E; Dobson, A D; Hartmans, S

    1997-01-01

    The transformation of indole to indigo by microorganisms expressing styrene monooxygenase (SMO) has been studied. Styrene and indole are structurally very similar, and thus we looked at a variety of styrene-degrading strains for indole transformation to indigo. Two strains, Pseudomonas putida S12 and CA-3, gave a blue color on solid media when grown in the presence of indole. Indole induces its own transformation on solid media but is a poor inducer in liquid media. Styrene is the best inducer of indole transformation in both strains. Arginine represses styrene consumption and indigo formation rates in P. putida S12 compared to phenylacetic acid-grown cells, while the opposite effect is seen for P. putida CA-3. Characterization of an SMO- and styrene oxide isomerase (SOI)-negative transposon mutant of P. putida CA-3 and an SOI-negative N-methyl-N'-nitro-N-nitrosoguanidine mutant of P. putida S12 reveals the involvement of both SMO and SOI in indole transformation to indigo. Both strains stoichiometrically produce high-purity indigo from indole. PMID:9361415

  10. 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.

  11. A mechanistic structure-activity relationship for hepatic polysubstrate monooxygenase

    SciTech Connect

    Hollebone, B.R.; Davis, D.; Michelin, N.; Purdy, D. . Chemistry Dept.); Brownlee, L.J. )

    1995-01-01

    The polysubstrate monooxygenase (PSMO) system response to hydrophobic xenobiotics is both intensive in oxidizing power and extensive in enzyme quantity. Under in vitro pseudo-first-order rate conditions the extensive properties become irrelevant, and the intensive rate-determining step depends on chemical structure. Xenobiotics react with PSMO either as inducers (adaptation domain) or as substrates (reaction domain) to produce intended hydroxylation, but accidental oxidations may also occur. Both the induction of intensive oxidizing power in the adaptation domain and the efficiency of reaction in the reaction domain depend on the strength of the weakest C-H bond in the xenobiotic, consistent with either a free radical or an ionic S[sub E]2 reaction process. Only the ionic S[sub E]2 mechanism is sensitive to the electrical charge on the carbon atom of the weakest C-H bond. In this study systematic treatment of simple hydrocarbon structures by adjacent polarizing heteroatoms N, Cl, and O inhibited substrate metabolism in direct proportion to their polarizing power. With this evidence of preferential S[sub E]2 behavior, a QSAR is proposed that allows prediction of four types of disease end points. These arise as combination of conditions of intended or accidental oxidations combined with release of metabolites into cytosolic or lipid media.

  12. Versatile biocatalysis of fungal cytochrome P450 monooxygenases.

    PubMed

    Durairaj, Pradeepraj; Hur, Jae-Seoun; Yun, Hyungdon

    2016-07-18

    Cytochrome P450 (CYP) monooxygenases, the nature's most versatile biological catalysts have unique ability to catalyse regio-, chemo-, and stereospecific oxidation of a wide range of substrates under mild reaction conditions, thereby addressing a significant challenge in chemocatalysis. Though CYP enzymes are ubiquitous in all biological kingdoms, the divergence of CYPs in fungal kingdom is manifold. The CYP enzymes play pivotal roles in various fungal metabolisms starting from housekeeping biochemical reactions, detoxification of chemicals, and adaptation to hostile surroundings. Considering the versatile catalytic potentials, fungal CYPs has gained wide range of attraction among researchers and various remarkable strategies have been accomplished to enhance their biocatalytic properties. Numerous fungal CYPs with multispecialty features have been identified and the number of characterized fungal CYPs is constantly increasing. Literature reveals ample reviews on mammalian, plant and bacterial CYPs, however, modest reports on fungal CYPs urges a comprehensive review highlighting their novel catalytic potentials and functional significances. In this review, we focus on the diversification and functional diversity of fungal CYPs and recapitulate their unique and versatile biocatalytic properties. As such, this review emphasizes the crucial issues of fungal CYP systems, and the factors influencing efficient biocatalysis.

  13. 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

  14. Dioxygen activation in methane monooxygenase: a theoretical study.

    PubMed

    Gherman, Benjamin F; Baik, Mu-Hyun; Lippard, Stephen J; Friesner, Richard A

    2004-03-10

    Using broken-symmetry unrestricted Density Functional Theory, the mechanism of enzymatic dioxygen activation by the hydroxylase component of soluble methane monooxygenase (MMOH) is determined to atomic detail. After a thorough examination of mechanistic alternatives, an optimal pathway was identified. The diiron(II) state H(red) reacts with dioxygen to give a ferromagnetically coupled diiron(II,III) H(superoxo) structure, which undergoes intersystem crossing to the antiferromagnetic surface and affords H(peroxo), a symmetric diiron(III) unit with a nonplanar mu-eta(2):eta(2)-O(2)(2)(-) binding mode. Homolytic cleavage of the O-O bond yields the catalytically competent intermediate Q, which has a di (mu-oxo)diiron(IV) core. A carboxylate shift involving Glu243 is essential to the formation of the symmetric H(peroxo) and Q structures. Both thermodynamic and kinetic features agree well with experimental data, and computed spin-exchange coupling constants are in accord with spectroscopic values. Evidence is presented for pH-independent decay of H(red) and H(peroxo). Key electron-transfer steps that occur in the course of generating Q from H(red) are also detailed and interpreted. In contrast to prior theoretical studies, a requisite large model has been employed, electron spins and couplings have been treated in a quantitative manner, potential energy surfaces have been extensively explored, and quantitative total energies have been determined along the reaction pathway.

  15. Exploring the Structural Basis of Substrate Preferences in Baeyer-Villiger Monooxygenases

    PubMed Central

    Franceschini, Stefano; van Beek, Hugo L.; Pennetta, Alessandra; Martinoli, Christian; Fraaije, Marco W.; Mattevi, Andrea

    2012-01-01

    Steroid monooxygenase (STMO) from Rhodococcus rhodochrous catalyzes the Baeyer-Villiger conversion of progesterone into progesterone acetate using FAD as prosthetic group and NADPH as reducing cofactor. The enzyme shares high sequence similarity with well characterized Baeyer-Villiger monooxygenases, including phenylacetone monooxygenase and cyclohexanone monooxygenase. The comparative biochemical and structural analysis of STMO can be particularly insightful with regard to the understanding of the substrate-specificity properties of Baeyer-Villiger monooxygenases that are emerging as promising tools in biocatalytic applications and as targets for prodrug activation. The crystal structures of STMO in the native, NADP+-bound, and two mutant forms reveal structural details on this microbial steroid-degrading enzyme. The binding of the nicotinamide ring of NADP+ is shifted with respect to the flavin compared with that observed in other monooxygenases of the same class. This finding fully supports the idea that NADP(H) adopts various positions during the catalytic cycle to perform its multiple functions in catalysis. The active site closely resembles that of phenylacetone monooxygenase. This observation led us to discover that STMO is capable of acting also on phenylacetone, which implies an impressive level of substrate promiscuity. The investigation of six mutants that target residues on the surface of the substrate-binding site reveals that enzymatic conversions of both progesterone and phenylacetone are largely insensitive to relatively drastic amino acid changes, with some mutants even displaying enhanced activity on progesterone. These features possibly reflect the fact that these enzymes are continuously evolving to acquire new activities, depending on the emerging availabilities of new compounds in the living environment. PMID:22605340

  16. Phenylalanine 4-monooxygenase and the role of endobiotic metabolism enzymes in xenobiotic biotransformation.

    PubMed

    Steventon, Glyn B; Mitchell, Stephen C

    2009-10-01

    Phenylalanine 4-monooxygenase is the key enzyme in the sulfoxidation of the thioether drug S-carboxymethyl-l-cysteine and its thioether metabolites, S-methyl-l-cysteine, N-acetyl-S-carboxymethyl-l-cysteine and N-acetyl-S-methyl-l-cysteine in humans, and a number of other mammalian species. The kinetics constants of the sulfoxidation reaction (K(m), V(max) and CL(E)) have been investigated in cytosolic fractions derived from rat and human liver, in cytosolic fractions of HepG2 cells and using both human and mouse cDNA expressed phenylalanine 4-monooxygenase. Differences in K(m), V(max) and CL(E) of S-carboxymethyl-l-cysteine have been seen in HepG2 cells and human and mouse cDNA expressed phenylalanine 4-monooxygenase when compared to both rat and human hepatic cytosolic fractions. The association of the genetic polymorphism in the sulfoxidation of S-carboxymethyl-l-cysteine is highlighted with particular reference to this biotransformation reaction as being a biomarker of disease susceptibility in Parkinson's, Alzheimer's and motor neurone diseases and in rheumatoid arthritis. The possible underlying molecular genetics of the sulfoxidation polymorphism is also discussed in relation to the known allelic frequencies of phenylalanine 4-monooxygenase. Finally, the new found role phenylalanine 4-monooxygenase plays in xenobiotic metabolism is discussed.

  17. 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

  18. 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. © 2016 Authors; published by Portland Press Limited.

  19. Targeted Deletion of Kynurenine 3-Monooxygenase in Mice

    PubMed Central

    Giorgini, Flaviano; Huang, Shao-Yi; Sathyasaikumar, Korrapati V.; Notarangelo, Francesca M.; Thomas, Marian A. R.; Tararina, Margarita; Wu, Hui-Qiu; Schwarcz, Robert; Muchowski, Paul J.

    2013-01-01

    Kynurenine 3-monooxygenase (KMO), a pivotal enzyme in the kynurenine pathway (KP) of tryptophan degradation, has been suggested to play a major role in physiological and pathological events involving bioactive KP metabolites. To explore this role in greater detail, we generated mice with a targeted genetic disruption of Kmo and present here the first biochemical and neurochemical characterization of these mutant animals. Kmo−/− mice lacked KMO activity but showed no obvious abnormalities in the activity of four additional KP enzymes tested. As expected, Kmo−/− mice showed substantial reductions in the levels of its enzymatic product, 3-hydroxykynurenine, in liver, brain, and plasma. Compared with wild-type animals, the levels of the downstream metabolite quinolinic acid were also greatly decreased in liver and plasma of the mutant mice but surprisingly were only slightly reduced (by ∼20%) in the brain. The levels of three other KP metabolites: kynurenine, kynurenic acid, and anthranilic acid, were substantially, but differentially, elevated in the liver, brain, and plasma of Kmo−/− mice, whereas the liver and brain content of the major end product of the enzymatic cascade, NAD+, did not differ between Kmo−/− and wild-type animals. When assessed by in vivo microdialysis, extracellular kynurenic acid levels were found to be significantly elevated in the brains of Kmo−/− mice. Taken together, these results provide further evidence that KMO plays a key regulatory role in the KP and indicate that Kmo−/− mice will be useful for studying tissue-specific functions of individual KP metabolites in health and disease. PMID:24189070

  20. Reversal of Physiological Deficits Caused by Diminished Levels of Peptidylglycine α-Amidating Monooxygenase by Dietary Copper

    PubMed Central

    Bousquet-Moore, D.; Ma, X. M.; Nillni, E. A.; Czyzyk, T. A.; Pintar, J. E.; Eipper, B. A.; Mains, R. E.

    2009-01-01

    Amidated peptides are critically involved in many physiological functions. Genetic deletion of peptidylglycine α-amidating monooxygenase (PAM), the only enzyme that can synthesize these peptides, is embryonically lethal. The goal of the present study was the identification of physiological functions impaired by haploinsufficiency of PAM. Regulation of the hypothalamic-pituitary-thyroid axis and body temperature, functions requiring contributions from multiple amidated peptides, were selected for evaluation. Based on serum T4 and pituitary TSH-β mRNA levels, mice heterozygous for PAM (PAM+/−) were euthyroid at baseline. Feedback within the hypothalamic-pituitary-thyroid axis was impaired in PAM+/− mice made hypothyroid using a low iodine/propylthiouracil diet. Despite their normal endocrine response to cold, PAM+/− mice were unable to maintain body temperature as well as wild-type littermates when kept in a 4 C environment. When provided with additional dietary copper, PAM+/− mice maintained body temperature as well as wild-type mice. Pharmacological activation of vasoconstriction or shivering also allowed PAM+/− mice to maintain body temperature. Cold-induced vasoconstriction was deficient in PAM+/− mice. This deficit was eliminated in PAM+/− mice receiving a diet with supplemental copper. These results suggest that dietary deficiency of copper, coupled with genetic deficits in PAM, could result in physiological deficits in humans. PMID:19022883

  1. Marmoset Flavin-Containing Monooxygenase 3 in the Liver Is a Major Benzydamine and Sulindac Sulfide Oxygenase.

    PubMed

    Uehara, Shotaro; Shimizu, Makiko; Uno, Yasuhiro; Inoue, Takashi; Sasaki, Erika; Yamazaki, Hiroshi

    2017-05-01

    Common marmosets (Callithrix jacchus) are potentially primate models for preclinical drug metabolism studies because there are similarities in the molecular characteristics of cytochrome P450 enzymes between this species and humans. However, characterization of non-cytochrome P450 enzymes has not been clarified in marmosets. Here, we report characterization of flavin-containing monooxygenases FMO1-FMO5 identified in marmoset tissues. Marmoset FMO forms shared high amino acid sequence identities (93%-95%) and phylogenetic closeness with human homologous FMO forms. FMO1 and FMO3 mRNA were abundantly expressed in the liver and kidneys among five marmoset tissues examined, where FMO3 protein was detected by immunoblotting. FMO inhibition assays using preheated tissue microsomes indicated that benzydamine N-oxygenation and sulindac sulfide S-oxygenation in the marmoset liver was mainly catalyzed by FMO3, the major hepatic FMO. Marmoset FMO3 protein heterologously expressed in Escherichia coli effectively catalyzed benzydamine N-oxygenation and sulindac sulfide S-oxygenation comparable to marmoset liver microsomes. These results indicate that the FMO3 enzyme expressed in marmoset livers mainly metabolizes benzydamine and sulindac sulfide (typical human FMO substrates), suggesting its importance for FMO-dependent drug metabolism in marmosets. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  2. Suicidal inactivation and labelling of ammonia mono-oxygenase by acetylene.

    PubMed Central

    Hyman, M R; Wood, P M

    1985-01-01

    Acetylene brings about a progressive inactivation of ammonia mono-oxygenase, the ammonia-oxidizing enzyme in Nitrosomonas europaea. High NH4+ ion concentrations were protective. The inactivation followed first-order kinetics, with a rate constant of 1.5 min-1 at saturating concentrations of acetylene. If acetylene was added in the absence of O2, the cells remained active until O2 was re-introduced. A protective effect was also demonstrated with thiourea, a reversible non-competitive inhibitor of ammonia oxidation. Incubation of cells with [14C]acetylene was found to cause labelling of a single membrane polypeptide. This ran on dodecyl sulphate/polyacrylamide-gel electrophoresis with an Mr value of 28 000. It is concluded that acetylene is a suicide substrate for the mono-oxygenase. The labelling experiment provides the first identification of a constituent polypeptide of ammonia mono-oxygenase. Images Fig. 4. PMID:4004794

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

    USDA-ARS?s Scientific Manuscript database

    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

    USDA-ARS?s Scientific Manuscript database

    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. 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

  6. Intermediate P* from soluble methane monooxygenase contains a diferrous cluster.

    PubMed

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

    2013-06-25

    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 Fe(II)Fe(II) state (H(red)). O₂ binds rapidly at a site away from the cluster to form the Fe(II)Fe(II) intermediate O, which converts to an Fe(III)Fe(III)-peroxo intermediate P and finally to the Fe(IV)Fe(IV) 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 H(red) and O is lost. This was interpreted as signaling oxidation of the cluster, but a low level of accumulation of P* prevented further characterization. In this study, three methods for directly detecting and trapping 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 H(red) 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 Fe(II)Fe(II) state. Thus, the

  7. Mutational and crystallographic analysis of l-amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813: Interconversion between oxidase and monooxygenase activities.

    PubMed

    Matsui, Daisuke; Im, Do-Hyun; Sugawara, Asami; Fukuta, Yasuhisa; Fushinobu, Shinya; Isobe, Kimiyasu; Asano, Yasuhisa

    2014-01-01

    In this study, it was shown for the first time that l-amino acid oxidase of Pseudomonas sp. AIU813, renamed as l-amino acid oxidase/monooxygenase (l-AAO/MOG), exhibits l-lysine 2-monooxygenase as well as oxidase activity. l-Lysine oxidase activity of l-AAO/MOG was increased in a p-chloromercuribenzoate (p-CMB) concentration-dependent manner to a final level that was five fold higher than that of the non-treated enzyme. In order to explain the effects of modification by the sulfhydryl reagent, saturation mutagenesis studies were carried out on five cysteine residues, and we succeeded in identifying l-AAO/MOG C254I mutant enzyme, which showed five-times higher specific activity of oxidase activity than that of wild type. The monooxygenase activity shown by the C254I variant was decreased significantly. Moreover, we also determined a high-resolution three-dimensional structure of l-AAO/MOG to provide a structural basis for its biochemical characteristics. The key residue for the activity conversion of l-AAO/MOG, Cys-254, is located near the aromatic cage (Trp-418, Phe-473, and Trp-516). Although the location of Cys-254 indicates that it is not directly involved in the substrate binding, the chemical modification by p-CMB or C254I mutation would have a significant impact on the substrate binding via the side chain of Trp-516. It is suggested that a slight difference of the binding position of a substrate can dictate the activity of this type of enzyme as oxidase or monooxygenase.

  8. Tolerance to Acetaminophen Hepatotoxicity in the Mouse Model of Autoprotection Is Associated with Induction of Flavin-Containing Monooxygenase-3 (FMO3) in Hepatocytes

    PubMed Central

    Rudraiah, Swetha; Rohrer, Philip R.; Gurevich, Igor; Goedken, Michael J.; Rasmussen, Theodore; Hines, Ronald N.; Manautou, José E.

    2014-01-01

    Acetaminophen (APAP) pretreatment with a hepatotoxic dose (400 mg/kg) in mice results in resistance to a second, higher dose (600 mg/kg) of APAP (APAP autoprotection). Recent microarray work by our group showed a drastic induction of liver flavin containing monooxygenase-3 (Fmo3) mRNA expression in our mouse model of APAP autoprotection. The role of liver Fmo3, which detoxifies xenobiotics, in APAP autoprotection is unknown. The purpose of this study was to characterize the gene regulation and protein expression of liver Fmo3 during APAP hepatotoxicity. The functional consequences of Fmo3 induction were also investigated. Plasma and livers were collected from male C57BL/6J mice over a period of 72 h following a single dose of APAP (400 mg/kg) to measure Fmo3 mRNA and protein expression. Although Fmo3 mRNA levels increased significantly following APAP treatment, protein expression changed marginally. In contrast, both Fmo3 mRNA and protein expression were significantly higher in APAP autoprotected livers. Unlike male C57BL/6J mice, female mice have ∼80-times higher constitutive Fmo3 mRNA levels and are highly resistant to APAP hepatotoxicity. Coadministration of APAP with the FMO inhibitor methimazole rendered female mice susceptible to APAP hepatotoxicity, with no changes in susceptibility detected in male mice. Furthermore, a human hepatocyte cell line (HC-04) clone over-expressing human FMO3 showed enhanced resistance to APAP cytotoxicity. Taken together, these findings establish for the first time induction of Fmo3 protein expression and function by xenobiotic treatment. Our results also indicate that Fmo3 expression and function plays a role in protecting the liver from APAP-induced toxicity. Although the mechanism(s) of this protection remains to be elucidated, this work describes a novel protective function for this enzyme. PMID:24973094

  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. TCE degradation by toluene/benzene monooxygenase of Pseudomonas aeruginosa JI104 and Escherichia coli recombinant

    SciTech Connect

    Koizumi, Junichi; Kitayama, Atsushi

    1995-12-31

    Pseudomonas aeruginosa JI104 incorporates more than three degradation pathways for aromatic compounds such as benzene, toluene, and xylene. A dioxygenase and two monooxygenases were cloned in Escherichia coli XL1-Blue. The dioxygenase yielding cis-toluene dihydrodiol and one of the monooxygenases producing o-cresol from toluene did not exhibit conspicuous activity in trichloroethylene (TCE) oxygenation, although DNA sequencing proved that the former enzyme was an isozyme of toluene dioxygenase of the known TCE decomposer P.putida F1. The other toluene/benzene monooxygenase that could generate o-, m-, and p-cresol simultaneously from toluene showed TCE oxygenation activity resulting in TCE decomposition in E. coli. The activity was inhibited competitively by toluene, ethylbenzene, and o- and m-xylene: their inhibition constants were greater than those of propylbenzene and p-xylene. When the E. coli recombinant harboring the monooxygenase was induced by isopropyl {beta}-D-thiogalactopyranoside (IPTG) and incubated in the absence of toluene, TCE degradation activity decreased during incubation, compared to that with toluene. Toluene probably controlled the lifetime of the enzyme.

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

    PubMed

    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.

  13. The discovery of potent and selective kynurenine 3-monooxygenase inhibitors for the treatment of acute pancreatitis.

    PubMed

    Liddle, John; Beaufils, Benjamin; Binnie, Margaret; Bouillot, Anne; Denis, Alexis A; Hann, Michael M; Haslam, Carl P; Holmes, Duncan S; Hutchinson, Jon P; Kranz, Michael; McBride, Andrew; Mirguet, Olivier; Mole, Damian J; Mowat, Christopher G; Pal, Sandeep; Rowland, Paul; Trottet, Lionel; Uings, Iain J; Walker, Ann L; Webster, Scott P

    2017-05-01

    A series of potent, competitive and highly selective kynurenine monooxygenase inhibitors have been discovered via a substrate-based approach for the treatment of acute pancreatitis. The lead compound demonstrated good cellular potency and clear pharmacodynamic activity in vivo. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    PubMed

    Carlin, D A; Bertolani, S J; Siegel, J B

    2015-02-11

    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.

  15. Monooxygenase Levels and Knockdown Resistance (kdr) Allele Frequencies in Anopheles gambiae and Anopheles arabiensis in Kenya

    PubMed Central

    Chen, Hong; Githeko, Andrew K; Githure, John I; Mutunga, James; Zhou, Guofa; Yan, Guiyun

    2013-01-01

    Pyrethroid-treated bed nets and indoor spray are important components of malaria control strategies in Kenya. Information on resistance to pyrethroid insecticides in Anopheles gambiae and An. arabiensis populations is essential to the selection of appropriate insecticides and the management of insecticide resistance. Monooxygenase activity and knockdown resistance (kdr) allele frequency are biochemical and molecular indicators of mosquito resistance to pyrethroids. This study determined baseline information on monooxygenase activity and kdr allele frequency in anopheline mosquitoes in the western region, the Great Rift Valley-central province region, and the coastal region of Kenya. A total of 1990 field-collected individuals, representing 12 An. gambiae and 22 An. arabiensis populations was analyzed. We found significant among-population variation in monooxygenase activity in An. gambiae and An. arabiensis and substantial variability among individuals within populations. Nine out of 12 An. gambiae populations exhibited significantly higher average monooxygenase activity than the susceptible Kisumu reference strain. The kdr alleles (L1014S) were detected in three An. gambiae populations, and one An. arabiensis population in western Kenya, but not in the Rift Valley-central region and the coastal Kenya region. All genotypes with the kdr alleles were heterozygous, and the conservative estimation of kdr allele frequency was below 1% in these four populations. Information on monooxygenase activity and kdr allele frequency reported in this study provided baseline data for monitoring insecticide resistance changes in Kenya during the era when large-scale insecticide-treated bednet and indoor residual spray campaigns were being implemented. PMID:18402140

  16. Cometabolic degradation of chlorinated alkenes by alkene monooxygenase in a propylene-grown Xanthobacter strain.

    PubMed Central

    Ensign, S A; Hyman, M R; Arp, D J

    1992-01-01

    Propylene-grown Xanthobacter cells (strain Py2) degraded several chlorinated alkenes of environmental concern, including trichloroethylene, 1-chloroethylene (vinyl chloride), cis- and trans-1,2-dichloroethylene, 1,3-dichloropropylene, and 2,3-dichloropropylene. 1,1-Dichloroethylene was not degraded efficiently, while tetrachloroethylene was not degraded. The role of alkene monooxygenase in catalyzing chlorinated alkene degradations was established by demonstrating that glucose-grown cells which lack alkene monooxygenase and propylene-grown cells in which alkene monooxygenase was selectively inactivated by propyne were unable to degrade the compounds. C2 and C3 chlorinated alkanes were not oxidized by alkene monooxygenase, but a number of these compounds were inhibitors of propylene and ethylene oxidation, suggesting that they compete for binding to the enzyme. A number of metabolites enhanced the rate of degradation of chlorinated alkenes, including propylene oxide, propionaldehyde, and glucose. Propylene stimulated chlorinated alkene oxidation slightly when present at a low concentration but became inhibitory at higher concentrations. Toxic effects associated with chlorinated alkene oxidations were determined by measuring the propylene oxidation and propylene oxide-dependent O2 uptake rates of cells previously incubated with chlorinated alkenes. Compounds which were substrates for alkene monooxygenase exhibited various levels of toxicity, with 1,1-dichloroethylene and trichloroethylene being the most potent inactivators of propylene oxidation and 1,3- and 2,3-dichloropropylene being the most potent inactivators of propylene oxide-dependent O2 uptake. No toxic effects were seen when cells were incubated with chlorinated alkenes anaerobically, indicating that the product(s) of chlorinated alkene oxidation mediates toxicity. PMID:1444418

  17. Polysome immunoprecipitation of phenylalanine hydroxylase mRNA from rat liver and cloning of its cDNA.

    PubMed Central

    Robson, K J; Chandra, T; MacGillivray, R T; Woo, S L

    1982-01-01

    The mRNA for phenylalanine hydroxylase (phenylalanine 4-monooxygenase, EC 1.14.16.1) has been purified from total rat liver mRNAs, of which it constitutes less than 0.25%, to greater than 10% purity in a single step by specific polysome immunoprecipitation. The purified mRNA was used for synthesis and cloning of its cDNA. Recombinant colonies containing phenylalanine hydroxylase DNA sequences were identified by differential hybridization, hybrid-selected translation, and blot hybridization analysis. The rat cDNA clone was capable of hybridizing with human phenylalanine hydroxylase mRNA, which will permit the isolation of the corresponding human gene for analysis of phenylketonuria, a hereditary disorder in phenylalanine metabolism that causes permanent mental retardation in humans. Images PMID:6750607

  18. Structural and Catalytic Differences between Two FADH2-Dependent Monooxygenases: 2,4,5-TCP 4-Monooxygenase (TftD) from Burkholderia cepacia AC1100 and 2,4,6-TCP 4-Monooxygenase (TcpA) from Cupriavidus necator JMP134

    PubMed Central

    Hayes, Robert P.; Webb, Brian N.; Subramanian, Arun Kumar; Nissen, Mark; Popchock, Andrew; Xun, Luying; Kang, ChulHee

    2012-01-01

    2,4,5-TCP 4-monooxygenase (TftD) and 2,4,6-TCP 4-monooxygenase (TcpA) have been discovered in the biodegradation of 2,4,5-trichlorophenol (2,4,5-TCP) and 2,4,6-trichlorophenol (2,4,6-TCP). TcpA and TftD belong to the reduced flavin adenine dinucleotide (FADH2)-dependent monooxygenases and both use 2,4,6-TCP as a substrate; however, the two enzymes produce different end products. TftD catalyzes a typical monooxygenase reaction, while TcpA catalyzes a typical monooxygenase reaction followed by a hydrolytic dechlorination. We have previously reported the 3D structure of TftD and confirmed the catalytic residue, His289. Here we have determined the crystal structure of TcpA and investigated the apparent differences in specificity and catalysis between these two closely related monooxygenases through structural comparison. Our computational docking results suggest that Ala293 in TcpA (Ile292 in TftD) is possibly responsible for the differences in substrate specificity between the two monooxygenases. We have also identified that Arg101 in TcpA could provide inductive effects/charge stabilization during hydrolytic dechlorination. The collective information provides a fundamental understanding of the catalytic reaction mechanism and the parameters for substrate specificity. The information may provide guidance for designing bioremediation strategies for polychlorophenols, a major group of environmental pollutants. PMID:22949829

  19. Flavin-containing monooxygenase-3: Induction by 3-methylcholanthrene and complex regulation by xenobiotic chemicals in hepatoma cells and mouse liver

    SciTech Connect

    Celius, Trine; Pansoy, Andrea; Matthews, Jason; Okey, Allan B.; Henderson, Marilyn C.; Krueger, Sharon K.; Williams, David E.

    2010-08-15

    Flavin-containing monooxygenases often are thought not to be inducible but we recently demonstrated aryl hydrocarbon receptor (AHR)-dependent induction of FMO mRNAs in mouse liver by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Celius et al., Drug Metab Dispos 36:2499, 2008). We now evaluated FMO induction by other AHR ligands and xenobiotic chemicals in vivo and in mouse Hepa1c1c7 hepatoma cells (Hepa-1). In mouse liver, 3-methylcholanthrene (3MC) induced FMO3 mRNA 8-fold. In Hepa-1 cells, 3MC and benzo[a]pyrene (BaP) induced FMO3 mRNA > 30-fold. Induction by 3MC and BaP was AHR dependent but, surprisingly, the potent AHR agonist, TCDD, did not induce FMO3 mRNA in Hepa-1 cells nor did chromatin immunoprecipitation assays detect recruitment of AHR or ARNT to Fmo3 regulatory elements after exposure to 3MC in liver or in Hepa-1 cells. However, in Hepa-1, 3MC and BaP (but not TCDD) caused recruitment of p53 protein to a p53 response element in the 5'-flanking region of the Fmo3 gene. We tested the possibility that FMO3 induction in Hepa-1 cells might be mediated by Nrf2/anti-oxidant response pathways, but agents known to activate Nrf2 or to induce oxidative stress did not affect FMO3 mRNA levels. The protein synthesis inhibitor, cycloheximide (which causes 'superinduction' of CYP1A1 mRNA in TCDD-treated cells), by itself caused dramatic upregulation (> 300-fold) of FMO3 mRNA in Hepa-1 suggesting that cycloheximide prevents synthesis of a labile protein that suppresses FMO3 expression. Although FMO3 mRNA is highly induced by 3MC or TCDD in mouse liver and in Hepa-1 cells, FMO protein levels and FMO catalytic function showed only modest elevation.

  20. Flavin-containing monooxygenase-3: induction by 3-methylcholanthrene and complex regulation by xenobiotic chemicals in hepatoma cells and mouse liver.

    PubMed

    Celius, Trine; Pansoy, Andrea; Matthews, Jason; Okey, Allan B; Henderson, Marilyn C; Krueger, Sharon K; Williams, David E

    2010-08-15

    Flavin-containing monooxygenases often are thought not to be inducible but we recently demonstrated aryl hydrocarbon receptor (AHR)-dependent induction of FMO mRNAs in mouse liver by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Celius et al., Drug Metab Dispos 36:2499, 2008). We now evaluated FMO induction by other AHR ligands and xenobiotic chemicals in vivo and in mouse Hepa1c1c7 hepatoma cells (Hepa-1). In mouse liver, 3-methylcholanthrene (3MC) induced FMO3 mRNA 8-fold. In Hepa-1 cells, 3MC and benzo[a]pyrene (BaP) induced FMO3 mRNA >30-fold. Induction by 3MC and BaP was AHR dependent but, surprisingly, the potent AHR agonist, TCDD, did not induce FMO3 mRNA in Hepa-1 cells nor did chromatin immunoprecipitation assays detect recruitment of AHR or ARNT to Fmo3 regulatory elements after exposure to 3MC in liver or in Hepa-1 cells. However, in Hepa-1, 3MC and BaP (but not TCDD) caused recruitment of p53 protein to a p53 response element in the 5'-flanking region of the Fmo3 gene. We tested the possibility that FMO3 induction in Hepa-1 cells might be mediated by Nrf2/anti-oxidant response pathways, but agents known to activate Nrf2 or to induce oxidative stress did not affect FMO3 mRNA levels. The protein synthesis inhibitor, cycloheximide (which causes "superinduction" of CYP1A1 mRNA in TCDD-treated cells), by itself caused dramatic upregulation (>300-fold) of FMO3 mRNA in Hepa-1 suggesting that cycloheximide prevents synthesis of a labile protein that suppresses FMO3 expression. Although FMO3 mRNA is highly induced by 3MC or TCDD in mouse liver and in Hepa-1 cells, FMO protein levels and FMO catalytic function showed only modest elevation.

  1. Diversity of the active methanotrophic community in acidic peatlands as assessed by mRNA and SIP-PLFA analyses.

    PubMed

    Chen, Yin; Dumont, Marc G; McNamara, Niall P; Chamberlain, Paul M; Bodrossy, Levente; Stralis-Pavese, Nancy; Murrell, J Colin

    2008-02-01

    The active methanotroph community was investigated for the first time in heather (Calluna)-covered moorlands and Sphagnum/Eriophorum-covered UK peatlands. Direct extraction of mRNA from these soils facilitated detection of expression of methane monooxygenase genes, which revealed that particulate methane monooxygenase and not soluble methane monooxygenase was probably responsible for CH(4) oxidation in situ, because only pmoA transcripts (encoding a subunit of particulate methane monooxygenase) were readily detectable. Differences in methanotroph community structures were observed between the Calluna-covered moorland and Sphagnum/Eriophorum-covered gully habitats. As with many other Sphagnum-covered peatlands, the Sphagnum/Eriophorum-covered gullies were dominated by Methylocystis. Methylocella and Methylocapsa-related species were also present. Methylobacter-related species were found as demonstrated by the use of a pmoA-based diagnostic microarray. In Calluna-covered moorlands, in addition to Methylocella and Methylocystis, a unique group of peat-associated type I methanotrophs (Gammaproteobacteria) and a group of uncultivated type II methanotrophs (Alphaproteobacteria) were also found. The pmoA sequences of the latter were only distantly related to Methylocapsa and also to the RA-14 group of methanotrophs, which are believed to be involved in oxidation of atmospheric concentrations of CH(4). Soil samples were also labelled with (13)CH(4), and subsequent analysis of the (13)C-labelled phospholipid fatty acids (PLFAs) showed that 16:1 omega 7, 18:1 omega 7 and 18:1 omega 9 were the major labelled PLFAs. The presence of (13)C-labelled 18:1 omega 9, which was not a major PLFA of any extant methanotrophs, indicated the presence of novel methanotrophs in this peatland.

  2. Genetic Variant in Flavin-Containing Monooxygenase 3 Alters Lipid Metabolism in Laying Hens in a Diet-Specific Manner

    PubMed Central

    Wang, Jing; Long, Cheng; Zhang, Haijun; Zhang, Yanan; Wang, Hao; Yue, Hongyuan; Wang, Xiaocui; Wu, Shugeng; Qi, Guanghai

    2016-01-01

    Genetic variant T329S in flavin-containing monooxygenase 3 (FMO3) impairs trimethylamine (TMA) metabolism in birds. The TMA metabolism that under complex genetic and dietary regulation, closely linked to cardiovascular disease risk. We determined whether the genetic defects in TMA metabolism may change other metabolic traits in birds, determined whether the genetic effects depend on diets, and to identify genes or gene pathways that underlie the metabolic alteration induced by genetic and diet factors. We used hens genotyped as FMO3 c.984 A>T as well as those with the homozygous normal genotype. For each genotype, hens were provided with either a corn-soybean meal basal diets (SM), which contains lower levels of TMA precursor, or the basal diets supplemented with 21% of rapeseed meal (RM), which contains higher levels of TMA precursor. An integrative analysis of metabolomic and transcriptomic was used to explore the metabolic patterns of FMO3 genetic variant in hens that were fed the two defined diets. In birds that consumed SM diets, the T329S mutation increased levels of plasma TMA and lipids, FMO3 mRNA levels, and the expression of genes involved in long chain polyunsaturated fatty acid biosynthesis. In birds that consumed RM diets, the T329S mutation induced fishy odor syndrome, a repression in LXR pathway and a reciprocal change in lipid metabolism. Variations in TMA and lipid metabolism were linked to the genetic variant in FMO3 in a diet-specific manner, which suggest FMO3 functions in TMA metabolism and lipid homeostasis. LXR pathway and polyunsaturated fatty acid metabolism are two possible mechanisms of FMO3 action in response to dietary TMA precursor. PMID:27877090

  3. Properties of soluble and membrane bound dopamine-beta-monooxygenase from bovine adrenal medulla cross-linked with dimethyl suberimidate.

    PubMed

    Miras-Portugal, M T; Millaruelo, A; Vara, F

    1980-12-10

    Bovine dopamine-beta-monooxygenase from chromaffin granules in its soluble and membrane-bound forms was cross-linked with the bifunctional reagent dimethyl suberimidate, and its structural and kinetic properties were studied. 1. The cross-linking reaction does not affect the activity of soluble dopamine-beta-monooxygenase; it produces a ten percent inactivation in the membrane-bound enzyme, possibly because the linkage to other membrane proteins hinders its activity. 2. The soluble dopamine-beta-monooxygenase reaction mixture was analyzed by sodium dodecyl sulfate gel electrophoresis, showing appreciable amounts of dimer and tetramer, but only small amounts of trimer. In membrane-bound dopamine-beta-monooxygenase, subjected to the same treatment, appreciable amounts of dimer and higher aggregates were found. 3. The kinetic properties of soluble dopamine-beta-monooxygenase after the crosslinking reaction are the same as those of the native enzyme, with a ping-pong kinetic mechanism and the same real Michaelis constants for tyramine and ascorbate: KmT = 0.36 mM and KmA = 0.32 mM. Membrane-bound dopamine-beta-monooxygenase does not present a ping-pong mechanism before or after cross-linking; its real Michaelis constants are slightly modified by the cross-linking reaction: KmT = 0.4 mM and KMA = 0.4 mM.

  4. [A substrate-type induction of liver microsomal monooxygenases by phenobarbital].

    PubMed

    Tsyrlov, I B; Gromova, O A; Rivkind, N B; Vakulin, G M; Liakhovich, V V

    1977-07-01

    A possibility of step-wise induction of microsomal monooxygenases after injection of phenobarbital in the presence of 3-methylcholanthrene-caused induction was studied. It was found that the ratio of the high- and low-spin types of cytochrome, rather than the position of the CO-peak of its reduced form is a criterion for functional specificity of hemoprotein. Induction by phenobarbital appears possible under conditions when the inductor binding to microsomal hemoprotein is lacking, since cytochrome P-488 has no binding sites for phenobarbital. It is assumed that under microsomal monooxygenases induction by phenobarbital activation of genome and subsequent protein synthesis are operated by the substrate rather than by products of its primary metabolism in microsomes.

  5. Discovery, application and protein engineering of Baeyer-Villiger monooxygenases for organic synthesis.

    PubMed

    Balke, Kathleen; Kadow, Maria; Mallin, Hendrik; Sass, Stefan; Bornscheuer, Uwe T

    2012-08-21

    Baeyer-Villiger monooxygenases (BVMOs) are useful enzymes for organic synthesis as they enable the direct and highly regio- and stereoselective oxidation of ketones to esters or lactones simply with molecular oxygen. This contribution covers novel concepts such as searching in protein sequence databases using distinct motifs to discover new Baeyer-Villiger monooxygenases as well as high-throughput assays to facilitate protein engineering in order to improve BVMOs with respect to substrate range, enantioselectivity, thermostability and other properties. Recent examples for the application of BVMOs in synthetic organic synthesis illustrate the broad potential of these biocatalysts. Furthermore, methods to facilitate the more efficient use of BVMOs in organic synthesis by applying e.g. improved cofactor regeneration, substrate feed and in situ product removal or immobilization are covered in this perspective.

  6. tRNA-modifying MiaE protein from Salmonella typhimurium is a nonheme diiron monooxygenase

    PubMed Central

    Mathevon, Carole; Pierrel, Fabien; Oddou, Jean-Louis; Garcia-Serres, Ricardo; Blondin, Geneviève; Latour, Jean-Marc; Ménage, Stéphane; Gambarelli, Serge; Fontecave, Marc; Atta, Mohamed

    2007-01-01

    MiaE catalyzes the posttranscriptional allylic hydroxylation of 2-methylthio-N-6-isopentenyl adenosine in tRNAs. The Salmonella typhimurium enzyme was heterologously expressed in Escherichia coli. The purified enzyme is a monomer with two iron atoms and displays activity in in vitro assays. The type and properties of the iron center were investigated by using a combination of UV-visible absorption, EPR, HYSCORE, and Mössbauer spectroscopies which demonstrated that the MiaE enzyme contains a nonheme dinuclear iron cluster, similar to that found in the hydroxylase component of methane monooxygenase. This is the first example of an enzyme from this important class of diiron monooxygenases to be involved in the hydroxylation of a biological macromolecule and the second example of a redox metalloenzyme participating in tRNA modification. PMID:17679698

  7. Oxygen Activation at the Active Site of a Fungal Lytic Polysaccharide Monooxygenase.

    PubMed

    O'Dell, William B; Agarwal, Pratul K; Meilleur, Flora

    2017-01-16

    Lytic polysaccharide monooxygenases have attracted vast attention owing to their abilities to disrupt glycosidic bonds via oxidation instead of hydrolysis and to enhance enzymatic digestion of recalcitrant substrates including chitin and cellulose. We have determined high-resolution X-ray crystal structures of an enzyme from Neurospora crassa in the resting state and of a copper(II) dioxo intermediate complex formed in the absence of substrate. X-ray crystal structures also revealed "pre-bound" molecular oxygen adjacent to the active site. An examination of protonation states enabled by neutron crystallography and density functional theory calculations identified a role for a conserved histidine in promoting oxygen activation. These results provide a new structural description of oxygen activation by substrate free lytic polysaccharide monooxygenases and provide insights that can be extended to reactivity in the enzyme-substrate complex. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Bacterial degradation of styrene involving a novel flavin adenine dinucleotide-dependent styrene monooxygenase.

    PubMed Central

    Hartmans, S; van der Werf, M J; de Bont, J A

    1990-01-01

    By using styrene as the sole source of carbon and energy in concentrations of 10 to 500 microM, 14 strains of aerobic bacteria and two strains of fungi were isolated from various soil and water samples. In cell extracts of 11 of the bacterial isolates, a novel flavin adenine dinucleotide-requiring styrene monooxygenase activity that oxidized styrene to styrene oxide (phenyl oxirane) was detected. In one bacterial strain (S5), styrene metabolism was studied in more detail. In addition to styrene monooxygenase, cell extracts from strain S5 contained styrene oxide isomerase and phenylacetaldehyde dehydrogenase activities. A pathway for styrene degradation via styrene oxide and phenylacetaldehyde to phenylacetic acid is proposed. PMID:2339888

  9. Improved homology model of cyclohexanone monooxygenase from Acinetobacter calcoaceticus based on multiple templates.

    PubMed

    Bermúdez, Eduardo; Ventura, Oscar N; Eriksson, Leif A; Saenz-Méndez, Patricia

    2014-04-01

    A new homology model of cyclohexanone monooxygenase (CHMO) from Acinetobacter calcoaceticus is derived based on multiple templates, and in particular the crystal structure of CHMO from Rhodococcus sp. The derived model was fully evaluated, showing that the quality of the new structure was improved over previous models. Critically, the nicotinamide cofactor is included in the model for the first time. Analysis of several molecular dynamics snapshots of intermediates in the enzymatic mechanism led to a description of key residues for cofactor binding and intermediate stabilization during the reaction, in particular Arg327 and the well known conserved motif (FxGxxxHxxxW) in Baeyer-Villiger monooxygenases, in excellent agreement with known experimental and computational data.

  10. Biological methane oxidation: regulation, biochemistry, and active site structure of particulate methane monooxygenase.

    PubMed

    Lieberman, Raquel L; Rosenzweig, Amy C

    2004-01-01

    Particulate methane monooxygenase (pMMO) is a three-subunit integral membrane enzyme that catalyzes the oxidation of methane to methanol. Although pMMO is the predominant methane oxidation catalyst in nature, it has proved difficult to isolate, and most questions regarding its molecular structure, active site composition, chemical mechanism, and genetic regulation remain unanswered. Copper ions are believed to play a key role in both pMMO regulation and catalysis, and there is some evidence that the enzyme contains iron as well. A number of research groups have solubilized and purified or partially purified pMMO. These preparations have been characterized by biochemical and biophysical methods. In addition, aspects of methane monooxygenase gene regulation and copper accumulation in methanotrophs have been studied. This review summarizes for the first time the often controversial pMMO literature, focusing on recent progress and highlighting unresolved issues.

  11. The mechanism of methane and dioxygen activation in the catalytic cycle of methane monooxygenase.

    PubMed

    Shteinman, A A

    1995-03-27

    The binuclear structure of the active center of methane monooxygenase plays a determining role in dioxygen activation and in selectivity and specificity of alkane oxidation with this enzyme. A new mechanism is suggested for binding and activation of O2, which involves side-on binding of O2-(2) to iron atoms followed by its conversion to the bis-mu-oxo complex considered as an alternative of ferryl in CH4 activation. This mechanism results in the sequence of the cleavage of the O-O bond of peroxide O/O2-instead of the opposite sequence O2-/O, which takes place in the case of heme monooxygenase cytochrome P-450. Therefore, in this case there is no necessity of the charge relay system [N.B. Gerber and S.G. Sligar, J. Am. Chem. Soc. 114 (1992) 8742] for the transformation of O2 to an active intermediate. The experiment for checking this hypothesis is suggested.

  12. tRNA-modifying MiaE protein from Salmonella typhimurium is a nonheme diiron monooxygenase.

    PubMed

    Mathevon, Carole; Pierrel, Fabien; Oddou, Jean-Louis; Garcia-Serres, Ricardo; Blondin, Geneviève; Latour, Jean-Marc; Ménage, Stéphane; Gambarelli, Serge; Fontecave, Marc; Atta, Mohamed

    2007-08-14

    MiaE catalyzes the posttranscriptional allylic hydroxylation of 2-methylthio-N-6-isopentenyl adenosine in tRNAs. The Salmonella typhimurium enzyme was heterologously expressed in Escherichia coli. The purified enzyme is a monomer with two iron atoms and displays activity in in vitro assays. The type and properties of the iron center were investigated by using a combination of UV-visible absorption, EPR, HYSCORE, and Mössbauer spectroscopies which demonstrated that the MiaE enzyme contains a nonheme dinuclear iron cluster, similar to that found in the hydroxylase component of methane monooxygenase. This is the first example of an enzyme from this important class of diiron monooxygenases to be involved in the hydroxylation of a biological macromolecule and the second example of a redox metalloenzyme participating in tRNA modification.

  13. 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.

  14. Microbial monooxygenase amperometric biosensor for monitoring of Baeyer-Villiger biotransformation.

    PubMed

    Schenkmayerová, Andrea; Bučko, Marek; Gemeiner, Peter; Katrlík, Jaroslav

    2013-12-15

    A whole-cell amperometric biosensor consisting of genetically engineered Escherichia coli immobilised in polyelectrolyte membrane onto a miniaturised oxygen electrode was developed and used for monitoring of biotransformation based on Baeyer-Villiger oxidation. Baeyer-Villiger oxidation is commonly performed using microorganisms overexpressing Baeyer-Villiger monooxygenase enabling the production of enantiopure lactones or esters used in pharmaceutical industry. The biorecognition element, genetically modified E. coli overexpressing either cyclopentanone monooxygenase or cyclohexanone monooxygenase was immobilised in the form of solid polyelectrolyte complex gel membrane made of cellulose sulphate, sodium alginate and poly(methylene-co-guanidine) and attached to the surface of miniaturised oxygen electrode. The time response of the biosensor was 30s, the linear range of the calibration curve (R(2)=0.9993) was 8-130 μM and the sensitivity was 1.8 nA μM(-1) (RSD=5.0%) for substrate of Baeyer-Villiger oxidation (±)-cis-bicyclo[3.2.0]hept-2-en-6-one as analyte. The biosensor sensitivity was assessed for two other commercially available substrates, 4-methylcyclohexanone and 3-methylcyclohexanone. No interferences from ampicillin, citric acid, acetic acid, ethanol, methanol, glucose and products of Baeyer-Villiger oxidation (1R, 5S)-3-oxabicyclo[3.3.0]oct-6-en-2-one and (1S, 5R)-2-oxabicyclo[3.3.0]oct-6-en-3-one were detected. After 1 week of storage at 4°C the biosensor sensitivity was without changes. The biosensor was employed for monitoring of Baeyer-Villiger biotransformation and the results were correlated with gas chromatography. Till now, this is the first described biosensor based on Baeyer-Villiger monooxygenase and the first reported application of biosensor for monitoring of biotransformation based on Baeyer-Villiger oxidation. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. 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.

  16. 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.

  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. Cloning and characterization of the Type I Baeyer-Villiger monooxygenase from Leptospira biflexa.

    PubMed

    Ceccoli, Romina D; Bianchi, Dario A; Fink, Michael J; Mihovilovic, Marko D; Rial, Daniela V

    2017-12-01

    Baeyer-Villiger monooxygenases are recognized by their ability and high selectivity as oxidative biocatalysts for the generation of esters or lactones using ketones as starting materials. These enzymes represent valuable tools for biooxidative syntheses since they can catalyze reactions that otherwise involve strong oxidative reagents. In this work, we present a novel enzyme, the Type I Baeyer-Villiger monooxygenase from Leptospira biflexa. This protein is phylogenetically distant from other well-characterized BVMOs. In order to study this new enzyme, we cloned its gene, expressed it in Escherichia coli and characterized the substrate scope of the Baeyer-Villiger monooxygenase from L. biflexa as a whole-cell biocatalyst. For this purpose, we performed the screening of a collection of ketones with variable structures and sizes, namely acyclic ketones, aromatic ketones, cyclic ketones, and fused ketones. As a result, we observed that this biocatalyst readily oxidized linear- and branched- medium-chain ketones, alkyl levulinates and linear ketones with aromatic substituents with excellent regioselectivity. In addition, this enzyme catalyzed the oxidation of 2-substituted cycloketone derivatives but showed an unusual selection against substituents in positions 3 or 4 of the ring.

  19. Direct inhibitions of the activities of steroidogenic cytochrome P-450 mono-oxygenase systems by anticonvulsants.

    PubMed

    Ohnishi, T; Ichikawa, Y

    1997-01-01

    The effects of anticonvulsants on the activities of cytochromes P-450(17alpha,lyase) (CYP17), P-450arom (CYP19), P-450C21 (CYP21), P-450SCC (CYP11A1), and P-450(11beta) (CYP11B1) mono-oxygenase systems were studied using rat testicular microsomes, human placental microsomes, bovine adrenocortical microsomes, bovine adrenocortical mitochondria and purified cytochrome P-450(11beta). Phenytoin, clonazepam and carbamazepine inhibited the steroidogenesis catalysed by these cytochrome P-450 mono-oxygenase systems and the Ki values for each anticonvulsant were determined. Neither hydantoin nor sodium valproate inhibited the activities of steroidogenic cytochromes P-450. When the activities of cytochromes P-450arom and P-450C21 were measured in the presence of anticonvulsants, the Ki values (0.15 mM) for phenytoin were close to the plasma concentration of phenytoin under therapeutic conditions. Phenytoin, clonazepam and carbamazepine directly inhibited the monooxygenase activities of cytochromes P-450, because they did not affect the activities of NADPH-cytochrome P-450 reductase, NADPH-adrenoferredoxin reductase and adrenoferredoxin.

  20. 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.

  1. Prospecting Biotechnologically-Relevant Monooxygenases from Cold Sediment Metagenomes: An In Silico Approach.

    PubMed

    Musumeci, Matías A; Lozada, Mariana; Rial, Daniela V; Mac Cormack, Walter P; Jansson, Janet K; Sjöling, Sara; Carroll, JoLynn; Dionisi, Hebe M

    2017-04-09

    The goal of this work was to identify sequences encoding monooxygenase biocatalysts with novel features by in silico mining an assembled metagenomic dataset of polar and subpolar marine sediments. The targeted enzyme sequences were Baeyer-Villiger and bacterial cytochrome P450 monooxygenases (CYP153). These enzymes have wide-ranging applications, from the synthesis of steroids, antibiotics, mycotoxins and pheromones to the synthesis of monomers for polymerization and anticancer precursors, due to their extraordinary enantio-, regio-, and chemo- selectivity that are valuable features for organic synthesis. Phylogenetic analyses were used to select the most divergent sequences affiliated to these enzyme families among the 264 putative monooxygenases recovered from the ~14 million protein-coding sequences in the assembled metagenome dataset. Three-dimensional structure modeling and docking analysis suggested features useful in biotechnological applications in five metagenomic sequences, such as wide substrate range, novel substrate specificity or regioselectivity. Further analysis revealed structural features associated with psychrophilic enzymes, such as broader substrate accessibility, larger catalytic pockets or low domain interactions, suggesting that they could be applied in biooxidations at room or low temperatures, saving costs inherent to energy consumption. This work allowed the identification of putative enzyme candidates with promising features from metagenomes, providing a suitable starting point for further developments.

  2. A novel chimera: the "truncated hemoglobin-antibiotic monooxygenase" from Streptomyces avermitilis.

    PubMed

    Bonamore, Alessandra; Attili, Andrea; Arenghi, Fabio; Catacchio, Bruno; Chiancone, Emilia; Morea, Veronica; Boffi, Alberto

    2007-08-15

    Novel chimeric proteins made of a globin domain fused with a "cofactor free" monooxygenase domain have been identified within the Streptomyces avermitilis and Frankia sp. genomes by means of bioinformatics methods. Structure based sequence alignments show that the globin domains of both proteins can be unambiguously assigned to the truncated hemoglobin family, in view of the striking similarity to the truncated hemoglobins from Mycobacterium tuberculosis, Thermobifida fusca and Bacillus subtilis. In turn, the non-heme domains belong to a family of small (about 100 aminoacids) homodimeric proteins annotated as antibiotic biosynthesis monooxygenases, despite the lack of a cofactor (e.g., a metal, a flavin or a heme) necessary for oxygen activation. The chimeric protein from S. avermitilis has been cloned, expressed and characterized. The protein is a stable dimer in solution based on analytical ultracentrifugation experiments. The heme ligand binding properties with oxygen and carbonmonoxide resemble those of other Group II truncated hemoglobins. In addition, an oxygen dependent redox activity has been demonstrated towards easily oxidizable substrates such as menadiol and p-aminophenol. These findings suggest novel functional roles of truncated hemoglobins, which might represent a vast class of multipurpose oxygen activating/scavenging proteins whose catalytic action is mediated by the interaction with cofactor free monooxygenases.

  3. 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

  4. StyA1 and StyA2B from Rhodococcus opacus 1CP: a Multifunctional Styrene Monooxygenase System▿

    PubMed Central

    Tischler, Dirk; Kermer, René; Gröning, Janosch A. D.; Kaschabek, Stefan R.; van Berkel, Willem J. H.; Schlömann, Michael

    2010-01-01

    Two-component flavoprotein monooxygenases are emerging biocatalysts that generally consist of a monooxygenase and a reductase component. Here we show that Rhodococcus opacus 1CP encodes a multifunctional enantioselective flavoprotein monooxygenase system composed of a single styrene monooxygenase (SMO) (StyA1) and another styrene monooxygenase fused to an NADH-flavin oxidoreductase (StyA2B). StyA1 and StyA2B convert styrene and chemical analogues to the corresponding epoxides at the expense of FADH2 provided from StyA2B. The StyA1/StyA2B system presents the highest monooxygenase activity in an equimolar ratio of StyA1 and StyA2B, indicating (transient) protein complex formation. StyA1 is also active when FADH2 is supplied by StyB from Pseudomonas sp. VLB120 or PheA2 from Rhodococcus opacus 1CP. However, in both cases the reductase produces an excess of FADH2, resulting in a high waste of NADH. The epoxidation rate of StyA1 heavily depends on the type of reductase. This supports that the FADH2-induced activation of StyA1 requires interprotein communication. We conclude that the StyA1/StyA2B system represents a novel type of multifunctional flavoprotein monooxygenase. Its unique mechanism of cofactor utilization provides new opportunities for biotechnological applications and is highly relevant from a structural and evolutionary point of view. PMID:20675468

  5. 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

  6. Tolerance to acetaminophen hepatotoxicity in the mouse model of autoprotection is associated with induction of flavin-containing monooxygenase-3 (FMO3) in hepatocytes.

    PubMed

    Rudraiah, Swetha; Rohrer, Philip R; Gurevich, Igor; Goedken, Michael J; Rasmussen, Theodore; Hines, Ronald N; Manautou, José E

    2014-09-01

    Acetaminophen (APAP) pretreatment with a hepatotoxic dose (400 mg/kg) in mice results in resistance to a second, higher dose (600 mg/kg) of APAP (APAP autoprotection). Recent microarray work by our group showed a drastic induction of liver flavin containing monooxygenase-3 (Fmo3) mRNA expression in our mouse model of APAP autoprotection. The role of liver Fmo3, which detoxifies xenobiotics, in APAP autoprotection is unknown. The purpose of this study was to characterize the gene regulation and protein expression of liver Fmo3 during APAP hepatotoxicity. The functional consequences of Fmo3 induction were also investigated. Plasma and livers were collected from male C57BL/6J mice over a period of 72 h following a single dose of APAP (400 mg/kg) to measure Fmo3 mRNA and protein expression. Although Fmo3 mRNA levels increased significantly following APAP treatment, protein expression changed marginally. In contrast, both Fmo3 mRNA and protein expression were significantly higher in APAP autoprotected livers. Unlike male C57BL/6J mice, female mice have ∼80-times higher constitutive Fmo3 mRNA levels and are highly resistant to APAP hepatotoxicity. Coadministration of APAP with the FMO inhibitor methimazole rendered female mice susceptible to APAP hepatotoxicity, with no changes in susceptibility detected in male mice. Furthermore, a human hepatocyte cell line (HC-04) clone over-expressing human FMO3 showed enhanced resistance to APAP cytotoxicity. Taken together, these findings establish for the first time induction of Fmo3 protein expression and function by xenobiotic treatment. Our results also indicate that Fmo3 expression and function plays a role in protecting the liver from APAP-induced toxicity. Although the mechanism(s) of this protection remains to be elucidated, this work describes a novel protective function for this enzyme. © The Author 2014. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions

  7. Flavin-Dependent Redox Transfers by the Two-Component Diketocamphane Monooxygenases of Camphor-Grown Pseudomonas putida NCIMB 10007

    PubMed Central

    Willetts, Andrew; Kelly, David

    2016-01-01

    The progressive titres of key monooxygenases and their requisite native donors of reducing power were used to assess the relative contribution of various camphor plasmid (CAM plasmid)- and chromosome-coded activities to biodegradation of (rac)-camphor at successive stages throughout growth of Pseudomonas putida NCIMB 10007 on the bicylic monoterpenoid. A number of different flavin reductases (FRs) have the potential to supply reduced flavin mononucleotide to both 2,5- and 3,6-diketocamphane monooxygenase, the key isoenzymic two-component monooxygenases that delineate respectively the (+)- and (−)-camphor branches of the convergent degradation pathway. Two different constitutive chromosome-coded ferric reductases able to act as FRs can serve such as role throughout all stages of camphor-dependent growth, whereas Fred, a chromosome-coded inducible FR can only play a potentially significant role in the relatively late stages. Putidaredoxin reductase, an inducible CAM plasmid-coded flavoprotein that serves an established role as a redox intermediate for plasmid-coded cytochrome P450 monooxygenase also has the potential to serve as an important FR for both diketocamphane monooxygenases (DKCMOs) throughout most stages of camphor-dependent growth. PMID:27754389

  8. Antifungal activity of biogenic tellurium nanoparticles against Candida albicans and its effects on squalene monooxygenase gene expression.

    PubMed

    Zare, Bijan; Sepehrizadeh, Zargham; Faramarzi, Mohammad Ali; Soltany-Rezaee-Rad, Mohammad; Rezaie, Sassan; Shahverdi, Ahmad Reza

    2014-01-01

    In this study, we evaluated the antifungal activity of biogenic tellurium nanoparticles (Te NPs) against Candida albicans (ATCC14053). In addition, the effect of these biogenic NPs on squalene monooxygenase activity and the squalene monooxygenase gene (ERG1) expression level was evaluated. Squalene monooxygenase is an important enzyme involved in the synthesis of ergosterol, cholesterol, and phytosterols. Because of the importance of the noted compound, the squalene monooxygenase gene could be considered a good antifungal target. Results showed that biogenic Te NPs had antifungal effect against C. albicans. The minimal fungicidal concentration-minimal inhibitory concentration ratios of the biogenic Te NPs revealed that these NPs exhibited fungicidal effects against the test strain. The results of an enzyme assay using quantitative high-performance liquid chromatography showed squalene accumulation in C. albicans cells because of enzyme inhibition. Real-time PCR analysis showed an increase in the expression of the ERG1 gene in C. albicans cells, which were treated with Te NPs (0.2 mg/mL). It is conclution that Te NPs can inhibit the squalene monooxygenase enzyme, and, as a result, this inhibition phenomenon can cause an increase in the expression level of the ERG1 gene. This is the first report of the anti-Candida effect of biogenic Te NPs and its possible mechanisms.

  9. Preferential inhibition of mouse hepatic coumarin 7-hydroxylase by inhibitors of steroid metabolizing monooxygenases.

    PubMed

    Kojo, A; Honkakoski, P; Järvinen, P; Pelkonen, O; Lang, M

    1989-08-01

    Etomidate, metomidate and metyrapone, all potent inhibitors of steroid metabolizing monooxygenases, inhibit preferentially coumarin 7-hydroxylase (COH) amongst several liver microsomal monooxygenase activities from control and pyrazole-treated D2 mice in vitro. SKF-525A, an inhibitor of phenobarbital-inducible monooxygenase activities has a much weaker effect on COH than the other three drugs, even though COH is a phenobarbital-inducible enzyme. Treatment of mice with eto- and metomidate decreases the microsomal COH also in vivo while the other activities remained unchanged (with the exception of 7-ethoxycoumarin O-deethylase (ECDE) in case of metomidate). Despite of the decrease in COH no parallel decrease in the amount of microsomal P450Coh (P450 isoenzyme highly active in the 7-hydroxylation of coumarin) could be found in dot immuno-binding analysis. These data suggest that among several liver microsomal P450 isoenzymes, metyrapone, eto- and metomidate interact preferentially with the P450Coh and that eto- and metomidate may alter selectively the catalytic properties of P450Coh leading to decreased enzyme activity. Two different Ks-values could be found for all three drug in their binding to microsomal cytochrome(s) P450. Based on substrate binding spectra, potassium ferricyanide treatment does not dissociate the complex between reduced P450 and metomidate and does it only partly for etomidate. Furthermore potassium ferricyanide treatment of microsomes does not increase COH after in vivo treatment of mice with eto- and metomidate. These data further suggest that the complex between P450Coh and eto- and metomidate may be particularly strong and independent from the redox state of the haem iron.

  10. 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

  11. Controlling the Regioselectivity of Baeyer-Villiger Monooxygenases by Mutation of Active-Site Residues.

    PubMed

    Balke, Kathleen; Bäumgen, Marcus; Bornscheuer, Uwe T

    2017-08-17

    Baeyer-Villiger monooxygenase (BVMO)-mediated regiodivergent conversions of asymmetric ketones can lead to the formation of "normal" or "abnormal" lactones. In a previous study, we were able to change the regioselectivity of a BVMO by mutation of the active-site residues to smaller amino acids, which thus created more space. In this study, we demonstrate that this method can also be used for other BVMO/substrate combinations. We investigated the regioselectivity of 2-oxo-Δ(3) -4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase from Pseudomonas putida (OTEMO) for cis-bicyclo[3.2.0]hept-2-en-6-one (1) and trans-dihydrocarvone (2), and we were able to switch the regioselectivity of this enzyme for one of the substrate enantiomers. The OTEMO wild-type enzyme converted (-)-1 into an equal (50:50) mixture of the normal and abnormal products. The F255A/F443V variant produced 90 % of the normal product, whereas the W501V variant formed up to 98 % of the abnormal product. OTEMO F255A exclusively produced the normal lactone from (+)-2, whereas the wild-type enzyme was selective for the production of the abnormal product. The positions of these amino acids were equivalent to those mutated in the cyclohexanone monooxygenases from Arthrobacter sp. and Acinetobacter sp. (CHMOArthro and CHMOAcineto ) to switch their regioselectivity towards (+)-2, which suggests that there are hot spots in the active site of BVMOs that can be targeted with the aim to change the regioselectivity. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Adaptive and Behavioral Changes in Kynurenine 3-Monooxygenase Knockout Mice: Relevance to Psychotic Disorders.

    PubMed

    Erhardt, Sophie; Pocivavsek, Ana; Repici, Mariaelena; Liu, Xi-Cong; Imbeault, Sophie; Maddison, Daniel C; Thomas, Marian A R; Smalley, Joshua L; Larsson, Markus K; Muchowski, Paul J; Giorgini, Flaviano; Schwarcz, Robert

    2016-12-16

    Kynurenine 3-monooxygenase converts kynurenine to 3-hydroxykynurenine, and its inhibition shunts the kynurenine pathway-which is implicated as dysfunctional in various psychiatric disorders-toward enhanced synthesis of kynurenic acid, an antagonist of both α7 nicotinic acetylcholine and N-methyl-D-aspartate receptors. Possibly as a result of reduced kynurenine 3-monooxygenase activity, elevated central nervous system levels of kynurenic acid have been found in patients with psychotic disorders, including schizophrenia. In the present study, we investigated adaptive-and possibly regulatory-changes in mice with a targeted deletion of Kmo (Kmo(-/-)) and characterized the kynurenine 3-monooxygenase-deficient mice using six behavioral assays relevant for the study of schizophrenia. Genome-wide differential gene expression analyses in the cerebral cortex and cerebellum of these mice identified a network of schizophrenia- and psychosis-related genes, with more pronounced alterations in cerebellar tissue. Kynurenic acid levels were also increased in these brain regions in Kmo(-/-) mice, with significantly higher levels in the cerebellum than in the cerebrum. Kmo(-/-) mice exhibited impairments in contextual memory and spent less time than did controls interacting with an unfamiliar mouse in a social interaction paradigm. The mutant animals displayed increased anxiety-like behavior in the elevated plus maze and in a light/dark box. After a D-amphetamine challenge (5 mg/kg, intraperitoneal), Kmo(-/-) mice showed potentiated horizontal activity in the open field paradigm. Taken together, these results demonstrate that the elimination of Kmo in mice is associated with multiple gene and functional alterations that appear to duplicate aspects of the psychopathology of several neuropsychiatric disorders. Copyright © 2017. Published by Elsevier Inc.

  13. Effects of sponge bleaching on ammonia-oxidizing Archaea: distribution and relative expression of ammonia monooxygenase genes associated with the barrel sponge Xestospongia muta.

    PubMed

    López-Legentil, Susanna; Erwin, Patrick M; Pawlik, Joseph R; Song, Bongkeun

    2010-10-01

    Sponge-mediated nitrification is an important process in the nitrogen cycle, however, nothing is known about how nitrification and symbiotic Archaea may be affected by sponge disease and bleaching events. The giant barrel sponge Xestospongia muta is a prominent species on Caribbean reefs that contains cyanobacterial symbionts, the loss of which results in two types of bleaching: cyclic, a recoverable condition; and fatal, a condition associated with the disease-like sponge orange band (SOB) syndrome and sponge death. Terminal restriction fragment length polymorphism (TRFLP) analyses, clone libraries, and relative mRNA quantification of ammonia monooxygenase genes (amoA) were performed using a RNA transcript-based approach to characterize the active ammonia-oxidizing Archaea (AOA) community present in bleached, non-bleached, and SOB tissues of cyclically and fatally bleached sponges. We found that non-bleached and cyclically bleached tissues of X. muta harbored a unique Crenarchaeota community closely related to those reported for other sponges. In contrast, bleached tissue from the most degraded sponge contained a Crenarchaeota community that was more similar to those found in sediment and sand. Although there were no significant differences in amoA expression among the different tissues, amoA expression was higher in the most deteriorated tissues. Results suggest that a shift in the Crenarchaeota community precedes an increase in amoA gene expression in fatally bleached sponges, while cyclic bleaching did not alter the AOA community structure and its amoA gene expression.

  14. Gene structure and spatiotemporal expression profile of tomato genes encoding YUCCA-like flavin monooxygenases: the ToFZY gene family.

    PubMed

    Expósito-Rodríguez, Marino; Borges, Andrés A; Borges-Pérez, Andrés; Pérez, José A

    2011-07-01

    The flavin monooxygenases (FMO) encoded by plant YUCCA genes are thought to catalyze a rate-limiting step in the tryptamine pathway for indole-3-acetic acid biosynthesis. Recent experiments with different plant models have indicate that YUCCA genes play essential roles in growth and development through their contribution to the local pool of free auxin. In this study we have characterized five new genes that encode YUCCA-like FMOs in the tomato genome (ToFZY2 to ToFZY6), including gene structure, conserved motifs and phylogenetic analyses. As a first step towards clarifying the individual functions of ToFZY genes, we have used quantitative real-time RT-PCR to conduct a systematic comparison of the steady-state mRNA levels of 6 ToFZY genes, in 33 samples representing major organs and the entire tomato life cycle. We followed an absolute quantification strategy which allowed us to cross-compare transcript levels among different ToFZY genes in a given spatiotemporal coordinate. Our results indicate that expression of ToFZY genes is temporally and spatially regulated, and that the distinctive expression pattern of each ToFZY gene partially overlaps with other members of the multigenic family. We compare our data with previous results in other plant species and make some predictions about the role of tryptamine pathway in tomato growth and development.

  15. Interaction of the mechanism-based inactivator acetylene with ammonia monooxygenase of Nitrosomonas europaea.

    PubMed

    Gilch, Stefan; Vogel, Manja; Lorenz, Matthias W; Meyer, Ortwin; Schmidt, Ingo

    2009-01-01

    The ammonia monooxygenase (AMO) of Nitrosomonas europaea is a metalloenzyme that catalyses the oxidation of ammonia to hydroxylamine. We have identified histidine 191 of AmoA as the binding site for the oxidized mechanism-based inactivator acetylene. Binding of acetylene changed the molecular mass of His-191 from 155.15 to 197.2 Da (+42.05), providing evidence that acetylene was oxidized to ketene (CH2CO; 42.04 Da) which binds specifically to His-191. It must be assumed that His-191 is part of the acetylene-activating site in AMO or at least directly neighbours this site.

  16. 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

    2015-01-22

    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.

  17. Kinetic characterization of the soluble butane monooxygenase from Thauera butanivorans, formerly 'Pseudomonas butanovora'.

    PubMed

    Cooley, Richard B; Dubbels, Bradley L; Sayavedra-Soto, Luis A; Bottomley, Peter J; Arp, Daniel J

    2009-06-01

    Soluble butane monooxygenase (sBMO), a three-component di-iron monooxygenase complex expressed by the C(2)-C(9) alkane-utilizing bacterium Thauera butanivorans, was kinetically characterized by measuring substrate specificities for C(1)-C(5) alkanes and product inhibition profiles. sBMO has high sequence homology with soluble methane monooxygenase (sMMO) and shares a similar substrate range, including gaseous and liquid alkanes, aromatics, alkenes and halogenated xenobiotics. Results indicated that butane was the preferred substrate (defined by k(cat) : K(m) ratios). Relative rates of oxidation for C(1)-C(5) alkanes differed minimally, implying that substrate specificity is heavily influenced by differences in substrate K(m) values. The low micromolar K(m) for linear C(2)-C(5) alkanes and the millimolar K(m) for methane demonstrate that sBMO is two to three orders of magnitude more specific for physiologically relevant substrates of T. butanivorans. Methanol, the product of methane oxidation and also a substrate itself, was found to have similar K(m) and k(cat) values to those of methane. This inability to kinetically discriminate between the C(1) alkane and C(1) alcohol is observed as a steady-state concentration of methanol during the two-step oxidation of methane to formaldehyde by sBMO. Unlike methanol, alcohols with chain length C(2)-C(5) do not compete effectively with their respective alkane substrates. Results from product inhibition experiments suggest that the geometry of the active site is optimized for linear molecules four to five carbons in length and is influenced by the regulatory protein component B (butane monooxygenase regulatory component; BMOB). The data suggest that alkane oxidation by sBMO is highly specialized for the turnover of C(3)-C(5) alkanes and the release of their respective alcohol products. Additionally, sBMO is particularly efficient at preventing methane oxidation during growth on linear alkanes > or =C(2,) despite its high

  18. Kinetic characterization of the soluble butane monooxygenase from Thauera butanivorans, formerly ‘Pseudomonas butanovora’

    PubMed Central

    Cooley, Richard B.; Dubbels, Bradley L.; Sayavedra-Soto, Luis A.; Bottomley, Peter J.; Arp, Daniel J.

    2009-01-01

    Soluble butane monooxygenase (sBMO), a three-component di-iron monooxygenase complex expressed by the C2–C9 alkane-utilizing bacterium Thauera butanivorans, was kinetically characterized by measuring substrate specificities for C1–C5 alkanes and product inhibition profiles. sBMO has high sequence homology with soluble methane monooxygenase (sMMO) and shares a similar substrate range, including gaseous and liquid alkanes, aromatics, alkenes and halogenated xenobiotics. Results indicated that butane was the preferred substrate (defined by kcat : Km ratios). Relative rates of oxidation for C1–C5 alkanes differed minimally, implying that substrate specificity is heavily influenced by differences in substrate Km values. The low micromolar Km for linear C2–C5 alkanes and the millimolar Km for methane demonstrate that sBMO is two to three orders of magnitude more specific for physiologically relevant substrates of T. butanivorans. Methanol, the product of methane oxidation and also a substrate itself, was found to have similar Km and kcat values to those of methane. This inability to kinetically discriminate between the C1 alkane and C1 alcohol is observed as a steady-state concentration of methanol during the two-step oxidation of methane to formaldehyde by sBMO. Unlike methanol, alcohols with chain length C2–C5 do not compete effectively with their respective alkane substrates. Results from product inhibition experiments suggest that the geometry of the active site is optimized for linear molecules four to five carbons in length and is influenced by the regulatory protein component B (butane monooxygenase regulatory component; BMOB). The data suggest that alkane oxidation by sBMO is highly specialized for the turnover of C3–C5 alkanes and the release of their respective alcohol products. Additionally, sBMO is particularly efficient at preventing methane oxidation during growth on linear alkanes ≥C2, despite its high sequence homology with sMMO. These

  19. Terbinafine resistance conferred by multiple copies of the salicylate 1-monooxygenase gene in Trichophyton rubrum.

    PubMed

    Santos, Hemelin L; Lang, Elza A S; Segato, Fernando; Rossi, Antonio; Martinez-Rossi, Nilce M

    2017-06-02

    Resistance to antifungals is a leading concern in the treatment of human mycoses. We demonstrate that the salA gene, encoding salicylate 1-monooxygenase, is involved in resistance of the dermatophyte Trichophyton rubrum to terbinafine, one of the most effective antifungal drugs against dermatophytes. A strain with multiple copies of salA was constructed and exhibited elevated expression of salA and increased terbinafine resistance. This reflects a mechanism not yet reported in a pathogenic fungus. © The Author 2017. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  20. Fungal secretomics to probe the biological functions of lytic polysaccharide monooxygenases.

    PubMed

    Berrin, Jean-Guy; Rosso, Marie-Noëlle; Abou Hachem, Maher

    2017-08-07

    Enzymatic degradation of plant biomass is of growing interest for the development of a sustainable bio-based industry. Filamentous fungi, which degrade complex and recalcitrant plant polymers, are proficient secretors of enzymes acting on the lignocellulose composite of plant cell walls in addition to starch, the main carbon storage reservoir. In this review, we focus on the identification of lytic polysaccharide monooxygenases (LPMOs) and their redox partners in fungal secretomes to highlight the biological functions of these remarkable enzyme systems and we discuss future trends related to LPMO-potentiated bioconversion. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Oxidation of chlorinated olefins by Escherichia coli transformed with dimethyl sulfide monooxygenase genes or cumene dioxygenase genes.

    PubMed

    Takami, Wako; Yoshida, Takako; Nojiri, Hideaki; Yamane, Hisakazu; Omori, Toshio

    1999-04-01

    In the present work, it was shown that the dimethyl sulfide (DMS) monooxygenase and the cumene dioxygenase catalyzed oxidation of various chlorinated ethenes, propenes, and butenes. The specific activities of these oxygenases were determined for C(2) to C(4) chlorinated olefins, and the oxidation rates ranged from 0.19 to 4.18 nmol.min(-1).mg(-1) of dry cells by the DMS monooxygenase and from 0.19 to 1.29 nmol.min(-1).mg(-1) of dry cells by the cumene dioxygenase. The oxidation products were identified by gas chromatography-mass spectrometry. Most chlorinated olefins were monooxygenated by the DMS monooxygenase to yield chlorinated epoxides. In the case of the cumene dioxygenase, the substrates lacking any chlorine atom on double-bond carbon atoms were dioxygenated, and those with chlorine atoms attaching to double-bond carbon atoms were monooxygenated to yield allyl alcohols.

  2. 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...

  3. 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...

  4. Crystal structure of a Baeyer-Villiger flavin-containing monooxygenase from Staphylococcus aureus MRSA strain MU50.

    PubMed

    Hwang, William C; Xu, Qingping; Wu, Bainan; Godzik, Adam

    2014-08-05

    Flavin-containing Monooxygenase (FMO) catalyzed the oxygenation of broad spectrum of substrates. FMO can also serve as biocatalysts in the Baeyer-Villiger reaction in organic synthesis. Here, we report the high-resolution crystal structure of a Baeyer-Villiger Flavin-containing Monooxygenase (BVFMO) from methicillin- and vancomycin-resistant Staphylococcus aureus strain MU50. The structure of S. aureus FMO should facilitate further development of BVFMO as biocatalysts. A possible role of S. aureus FMO in methicillin and vancomycin resistance is discussed. Proteins 2014. © 2014 Wiley Periodicals, Inc.

  5. 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

  6. Biooxidation of n-butane to 1-butanol by engineered P450 monooxygenase under increased pressure.

    PubMed

    Nebel, Bernd A; Scheps, Daniel; Honda Malca, Sumire; Nestl, Bettina M; Breuer, Michael; Wagner, Hans-Günter; Breitscheidel, Boris; Kratz, Detlef; Hauer, Bernhard

    2014-12-10

    In addition to the traditional 1-butanol production by hydroformylation of gaseous propene and by fermentation of biomass, the cytochrome P450-catalyzed direct terminal oxidation of n-butane into the primary alcohol 1-butanol constitutes an alternative route to provide the high demand of this basic chemical. Moreover the use of n-butane offers an unexploited ubiquitous feed stock available in large quantities. Based on protein engineering of CYP153A from Polaromonas sp. JS666 and the improvement of the native redox system, a highly ω-regioselective (>96%) fusion protein variant (CYP153AP.sp.(G254A)-CPRBM3) for the conversion of n-butane into 1-butanol was developed. Maximum yield of 3.12g/L butanol, of which 2.99g/L comprise for 1-butanol, has been obtained after 20h reaction time. Due to the poor solubility of n-butane in an aqueous system, a high pressure reaction assembly was applied to increase the conversion. After optimization a maximum product content of 4.35g/L 1-butanol from a total amount of 4.53g/L butanol catalyzed by the self-sufficient fusion monooxygenase has been obtained at 15bar pressure. In comparison to the CYP153A wild type the 1-butanol concentration was enhanced fivefold using the engineered monooxygenase whole cell system by using the high-pressure reaction assembly. Copyright © 2014 Elsevier B.V. All rights reserved.

  7. Stabilization of cyclohexanone monooxygenase by a computationally designed disulfide bond spanning only one residue

    PubMed Central

    van Beek, Hugo L.; Wijma, Hein J.; Fromont, Lucie; Janssen, Dick B.; Fraaije, Marco W.

    2014-01-01

    Enzyme stability is an important parameter in biocatalytic applications, and there is a strong need for efficient methods to generate robust enzymes. We investigated whether stabilizing disulfide bonds can be computationally designed based on a model structure. In our approach, unlike in previous disulfide engineering studies, short bonds spanning only a few residues were included. We used cyclohexanone monooxygenase (CHMO), a Baeyer–Villiger monooxygenase (BVMO) from Acinetobacter sp. NCIMB9871 as the target enzyme. This enzyme has been the prototype BVMO for many biocatalytic studies even though it is notoriously labile. After creating a small library of mutant enzymes with introduced cysteine pairs and subsequent screening for improved thermostability, three stabilizing disulfide bonds were identified. The introduced disulfide bonds are all within 12 Å of each other, suggesting this particular region is critical for unfolding. This study shows that stabilizing disulfide bonds do not have to span many residues, as the most stabilizing disulfide bond, L323C–A325C, spans only one residue while it stabilizes the enzyme, as shown by a 6 °C increase in its apparent melting temperature. PMID:24649397

  8. Induced allostery in the directed evolution of an enantioselective Baeyer–Villiger monooxygenase

    PubMed Central

    Wu, Sheng; Acevedo, Juan Pablo; Reetz, Manfred T.

    2010-01-01

    The molecular basis of allosteric effects, known to be caused by an effector docking to an enzyme at a site distal from the binding pocket, has been studied recently by applying directed evolution. Here, we utilize laboratory evolution in a different way, namely to induce allostery by introducing appropriate distal mutations that cause domain movements with concomitant reshaping of the binding pocket in the absence of an effector. To test this concept, the thermostable Baeyer–Villiger monooxygenase, phenylacetone monooxygenase (PAMO), was chosen as the enzyme to be employed in asymmetric Baeyer–Villiger reactions of substrates that are not accepted by the wild type. By using the known X-ray structure of PAMO, a decision was made regarding an appropriate site at which saturation mutagenesis is most likely to generate mutants capable of inducing allostery without any effector compound being present. After screening only 400 transformants, a double mutant was discovered that catalyzes the asymmetric oxidative kinetic resolution of a set of structurally different 2-substituted cyclohexanone derivatives as well as the desymmetrization of three different 4-substituted cyclohexanones, all with high enantioselectivity. Molecular dynamics (MD) simulations and covariance maps unveiled the origin of increased substrate scope as being due to allostery. Large domain movements occur that expose and reshape the binding pocket. This type of focused library production, aimed at inducing significant allosteric effects, is a viable alternative to traditional approaches to “designed” directed evolution that address the binding site directly. PMID:20133612

  9. The hydrogen peroxide reactivity of peptidylglycine monooxygenase supports a Cu(II)-superoxo catalytic intermediate.

    PubMed

    Bauman, Andrew T; Yukl, Erik T; Alkevich, Katsiaryna; McCormack, Ashley L; Blackburn, Ninian J

    2006-02-17

    We have investigated the reaction of peptidylglycine monooxygenase with hydrogen peroxide to determine whether Cu(II)-peroxo is a likely intermediate. When the oxidized enzyme was reacted with the dansyl-YVG substrate and H(2)O(2), the alpha-hydroxyglycine product was formed. The reaction was catalytic and did not require the presence of additional reductant. When (18)O-labeled H(2)O(2) was reacted with peptidylglycine monooxygenase and substrate anaerobically, oxygen in the product was labeled with (18)O and must therefore be derived from H(2)O(2). However, when the reaction was carried out with H (16)(2)O(2) in the presence of (18)O(2), 60% of the product contained the (18)O label. Therefore, the reaction must proceed via an intermediate that can react directly with dioxygen and thus scramble the label. Under strictly anaerobic conditions (in the presence of glucose and glucose oxidase, where no oxygen was released into the medium from nonenzymatic peroxide decomposition), product formation and peroxide consumption were tightly coupled, and the rate of product formation was identical to that measured under aerobic conditions. Peroxide reactivity was eliminated by a mutation at the Cu(H) center, which should not be involved in the peroxide shunt. Our data lend support to recent proposals that Cu(II)-superoxide is the active species.

  10. Lactone-bound structures of cyclohexanone monooxygenase provide insight into the stereochemistry of catalysis.

    PubMed

    Yachnin, Brahm J; McEvoy, Michelle B; MacCuish, Roderick J D; Morley, Krista L; Lau, Peter C K; Berghuis, Albert M

    2014-12-19

    The Baeyer-Villiger monooxygenases (BVMOs) are microbial enzymes that catalyze the synthetically useful Baeyer-Villiger oxidation reaction. The available BVMO crystal structures all lack a substrate or product bound in a position that would determine the substrate specificity and stereospecificity of the enzyme. Here, we report two crystal structures of cyclohexanone monooxygenase (CHMO) with its product, ε-caprolactone, bound: the CHMO(Tight) and CHMO(Loose) structures. The CHMO(Tight) structure represents the enzyme state in which substrate acceptance and stereospecificity is determined, providing a foundation for engineering BVMOs with altered substrate spectra and/or stereospecificity. The CHMO(Loose) structure is the first structure where the product is solvent accessible. This structure represents the enzyme state upon binding and release of the substrate and product. In addition, the role of the invariant Arg329 in chaperoning the substrate/product during the catalytic cycle is highlighted. Overall, these data provide a structural framework for the engineering of BVMOs with altered substrate spectra and/or stereospecificity.

  11. Stabilization of cyclohexanone monooxygenase by a computationally designed disulfide bond spanning only one residue.

    PubMed

    van Beek, Hugo L; Wijma, Hein J; Fromont, Lucie; Janssen, Dick B; Fraaije, Marco W

    2014-01-01

    Enzyme stability is an important parameter in biocatalytic applications, and there is a strong need for efficient methods to generate robust enzymes. We investigated whether stabilizing disulfide bonds can be computationally designed based on a model structure. In our approach, unlike in previous disulfide engineering studies, short bonds spanning only a few residues were included. We used cyclohexanone monooxygenase (CHMO), a Baeyer-Villiger monooxygenase (BVMO) from Acinetobacter sp. NCIMB9871 as the target enzyme. This enzyme has been the prototype BVMO for many biocatalytic studies even though it is notoriously labile. After creating a small library of mutant enzymes with introduced cysteine pairs and subsequent screening for improved thermostability, three stabilizing disulfide bonds were identified. The introduced disulfide bonds are all within 12 Å of each other, suggesting this particular region is critical for unfolding. This study shows that stabilizing disulfide bonds do not have to span many residues, as the most stabilizing disulfide bond, L323C-A325C, spans only one residue while it stabilizes the enzyme, as shown by a 6 °C increase in its apparent melting temperature.

  12. 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.

  13. A family of diiron monooxygenases catalyzing amino acid beta-hydroxylation in antibiotic biosynthesis.

    PubMed

    Makris, Thomas M; Chakrabarti, Mrinmoy; Münck, Eckard; Lipscomb, John D

    2010-08-31

    The biosynthesis of chloramphenicol requires a beta-hydroxylation tailoring reaction of the precursor L-p-aminophenylalanine (L-PAPA). Here, it is shown that this reaction is catalyzed by the enzyme CmlA from an operon containing the genes for biosynthesis of L-PAPA and the nonribosomal peptide synthetase CmlP. EPR, Mössbauer, and optical spectroscopies reveal that CmlA contains an oxo-bridged dinuclear iron cluster, a metal center not previously associated with nonribosomal peptide synthetase chemistry. Single-turnover kinetic studies indicate that CmlA is functional in the diferrous state and that its substrate is L-PAPA covalently bound to CmlP. Analytical studies show that the product is hydroxylated L-PAPA and that O(2) is the oxygen source, demonstrating a monooxygenase reaction. The gene sequence of CmlA shows that it utilizes a lactamase fold, suggesting that the diiron cluster is in a protein environment not previously known to effect monooxygenase reactions. Notably, CmlA homologs are widely distributed in natural product biosynthetic pathways, including a variety of pharmaceutically important beta-hydroxylated antibiotics and cytostatics.

  14. 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.

  15. Mechanism of O2 activation and substrate hydroxylation in noncoupled binuclear copper monooxygenases

    PubMed Central

    Cowley, Ryan E.; Tian, Li; Solomon, Edward I.

    2016-01-01

    Peptidylglycine α-hydroxylating monooxygenase (PHM) and dopamine β-monooxygenase (DβM) are copper-dependent enzymes that are vital for neurotransmitter regulation and hormone biosynthesis. These enzymes feature a unique active site consisting of two spatially separated (by 11 Å in PHM) and magnetically noncoupled copper centers that enables 1e– activation of O2 for hydrogen atom abstraction (HAA) of substrate C–H bonds and subsequent hydroxylation. Although the structures of the resting enzymes are known, details of the hydroxylation mechanism and timing of long-range electron transfer (ET) are not clear. This study presents density-functional calculations of the full reaction coordinate, which demonstrate: (i) the importance of the end-on coordination of superoxide to Cu for HAA along the triplet spin surface; (ii) substrate radical rebound to a CuII hydroperoxide favors the proximal, nonprotonated oxygen; and (iii) long-range ET can only occur at a late step with a large driving force, which serves to inhibit deleterious Fenton chemistry. The large inner-sphere reorganization energy at the ET site is used as a control mechanism to arrest premature ET and dictate the correct timing of ET. PMID:27790986

  16. 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.

  17. Untangling the multiple monooxygenases of Mycobacterium chubuense strain NBB4, a versatile hydrocarbon degrader.

    PubMed

    Coleman, Nicholas V; Yau, Sheree; Wilson, Neil L; Nolan, Laura M; Migocki, Margaret D; Ly, Mai-Anh; Crossett, Ben; Holmes, Andrew J

    2011-06-01

    Mycobacterium strain NBB4 was isolated on ethene as part of a bioprospecting study searching for novel monooxygenase (MO) enzymes of interest to biocatalysis and bioremediation. Previous work indicated that strain NBB4 contained an unprecedented diversity of MO genes, and we hypothesized that each MO type would support growth on a distinct hydrocarbon substrate. Here, we attempted to untangle the relationships between MO types and hydrocarbon substrates. Strain NBB4 was shown to grow on C2 -C4 alkenes and C2 -C16 alkanes. Complete gene clusters encoding six different monooxygenases were recovered from a fosmid library, including homologues of ethene MO (etnABCD), propene MO (pmoABCD), propane MO (smoABCD), butane MO (smoXYB1C1Z), cytochrome P450 (CYP153; fdx-cyp-fdr) and alkB (alkB-rubA1-rubA2). Catabolic enzymes involved in ethene assimilation (EtnA, EtnC, EtnD, EtnE) and alkane assimilation (alcohol and aldehyde dehydrogenases) were identified by proteomics, and we showed for the first time that stress response proteins (catalase/peroxidase, chaperonins) were induced by growth on C2 -C5 alkanes and ethene. Surprisingly, none of the identified MO genes could be specifically associated with oxidation of small alkanes, and thus the nature of the gaseous alkane MO in NBB4 remains mysterious.

  18. Characterization of a Novel Rieske-Type Alkane Monooxygenase System in Pusillimonas sp. Strain T7-7

    PubMed Central

    Li, Ping; Wang, Lei

    2013-01-01

    The cold-tolerant bacterium Pusillimonas sp. strain T7-7 is able to utilize diesel oils (C5 to C30 alkanes) as a sole carbon and energy source. In the present study, bioinformatics, proteomics, and real-time reverse transcriptase PCR approaches were used to identify the alkane hydroxylation system present in this bacterium. This system is composed of a Rieske-type monooxygenase, a ferredoxin, and an NADH-dependent reductase. The function of the monooxygenase, which consists of one large (46.711 kDa) and one small (15.355 kDa) subunit, was further studied using in vitro biochemical analysis and in vivo heterologous functional complementation tests. The purified large subunit of the monooxygenase was able to oxidize alkanes ranging from pentane (C5) to tetracosane (C24) using NADH as a cofactor, with greatest activity on the C15 substrate. The large subunit also showed activity on several alkane derivatives, including nitromethane and methane sulfonic acid, but it did not act on any aromatic hydrocarbons. The optimal reaction condition of the large subunit is pH 7.5 at 30°C. Fe2+ can enhance the activity of the enzyme evidently. This is the first time that an alkane monooxygenase system belonging to the Rieske non-heme iron oxygenase family has been identified in a bacterium. PMID:23417490

  19. Whole genome co-expression analysis of soybean cytochrome P450 genes identifies nodulation-specific P450 monooxygenases

    USDA-ARS?s Scientific Manuscript database

    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...

  20. Controlling the regiospecific oxidation of aromatics via active site engineering of toluene para-monooxygenase of Ralstonia pickettii PKO1.

    PubMed

    Fishman, Ayelet; Tao, Ying; Rui, Lingyun; Wood, Thomas K

    2005-01-07

    A primary goal of protein engineering is to control catalytic activity. Here we show that through mutagenesis of three active site residues, the catalytic activity of a multicomponent monooxygenase is altered so that it hydroxylates all three positions of toluene as well as both positions of naphthalene. Hence, for the first time, an enzyme has been engineered so that its regiospecific oxidation of a substrate can be controlled. Through the A107G mutation in the alpha-subunit of toluene para-monooxygenase, a variant was formed that hydroxylated toluene primarily at the ortho-position while converting naphthalene to 1-naphthol. Conversely, the A107T variant produced >98% p-cresol and p-nitrophenol from toluene and nitrobenzene, respectively, as well as produced 2-naphthol from naphthalene. The mutation I100S/G103S produced a toluene para-monooxygenase variant that formed 75% m-cresol from toluene and 100% m-nitrophenol from nitrobenzene; thus, for the first time a true meta-hydroxylating toluene monooxygenase was created.

  1. Purification and characterization of toluene 2-monooxygenase from Burkholderia cepacia G4.

    PubMed

    Newman, L M; Wackett, L P

    1995-10-31

    Recent in vivo studies indicate that ring monooxygenation is a widespread mechanism by which bacteria metabolize aromatic hydrocarbons and obtain carbon and energy. In this study, toluene 2-monooxygenase from Burkholderia (formerly Pseudomonas) cepacia G4 was purified to homogeneity and found to be a three-component enzyme system. The reconstituted enzyme system oxidized toluene to o-cresol and o-cresol to 3-methylcatechol, an important intermediate for growth of the bacterium on toluene. Steady-state kinetic parameters measured for the water-soluble substrate o-cresol were a Km of 0.8 microM and a Vmax of 131 nmol min-1 (mg of hydroxylase protein)-1. The three protein components were (1) a 40 kDa polypeptide containing one FAD and a [2Fe2S] cluster, (2) a 10.4 kDa polypeptide that contained no identifiable metals or organic cofactors, and (3) a 211 kDa alpha 2 beta 2 gamma 2 component containing five to six iron atoms. The 40 kDa flavo-iron-sulfur protein oxidized NADH and transferred electrons to cytochrome c, dyes, and the alpha 2 beta 2 gamma 2 component. It is analogous to other NADH oxidoreductase components found in a wide range of bacterial mono- and dioxygenases. The 10.4 kDa component, added to the other two components and NADH, increased toluene oxidation rates 10-fold. The alpha 2 beta 2 gamma 2 component was indicated to contain the site for toluene binding and hydroxylation by the following observations: (1) tight binding to a toluene affinity column; (2) oxidation of toluene after reduction of the protein with dithionite and adding O2; (3) H2O2-dependent toluene oxidation and catalase activity; and (4) spectroscopic studies of the iron atoms in the component. The alpha 2 beta 2 gamma 2 component had no significant absorbance in the visible region. EPR spectroscopy yielded a signal at g = 16 upon addition of > 2 equiv of electrons per 2 Fe atoms. Taken with the quantitation of five to six iron atoms, the data suggest that the alpha 2 beta 2 gamma 2

  2. Status of Resistance of Bemisia tabaci (Hemiptera: Aleyrodidae) to Neonicotinoids in Iran and Detoxification by Cytochrome P450-Dependent Monooxygenases.

    PubMed

    Basij, M; Talebi, K; Ghadamyari, M; Hosseininaveh, V; Salami, S A

    2017-02-01

    Nine Bemisia tabaci (Gennadius) populations were collected from different regions of Iran. In all nine populations, only one biotype (B biotype) was detected. Susceptibilities of these populations to imidacloprid and acetamiprid were assayed. The lethal concentration 50 values (LC50) for different populations showed a significant discrepancy in the susceptibility of B. tabaci to imidacloprid (3.76 to 772.06 mg l(-1)) and acetamiprid (4.96 to 865 mg l(-1)). The resistance ratio of the populations ranged from 9.72 to 205.20 for imidacloprid and 6.38 to 174.57 for acetamiprid. The synergistic effects of piperonylbutoxide (PBO) and S,S,S-tributylphosphorotrithioate (DEF) were evaluated for the susceptible (RF) and resistant (JR) populations for the determination of the involvement of cytochrome P450-dependent monooxygenase and carboxylesterase, respectively, in their resistance mechanisms. The results showed that PBO overcame the resistance of the JR population to both imidacloprid and acetamiprid, with synergistic ratios of 72.7 and 106.9, respectively. Carboxylesterase, glutathione S-transferase and cytochrome P450-dependent monooxygenase were studied biochemically, for the purpose of measuring the activity of the metabolizing enzymes in order to determine which enzymes are directly involved in neonicotinoid resistance. There was an increase in the activity of cytochrome P450-dependent monooxygenase up to 17-fold in the resistant JR population (RR = 205.20). The most plausible activity of cytochrome P450-dependent monooxygenase correlated with the resistances of imidacloprid and acetamiprid, and this suggests that cytochrome P450-dependent monooxygenase is the only enzyme system responsible for neonicotinoid resistance in the nine populations of B. tabaci.

  3. Alkyl Formate Ester Synthesis by a Fungal Baeyer-Villiger Monooxygenase.

    PubMed

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

    2017-03-16

    We investigated Baeyer-Villiger monooxygenase (BVMO)-mediated synthesis of alkyl formate esters, which are important flavor and fragrance products. A recombinant fungal BVMO from Aspergillus flavus was found to transform a selection of aliphatic aldehydes into alkyl formates with high regioselectivity. Near complete conversion of 10 mm octanal was achieved within 8 h with a regiomeric excess of ∼80 %. Substrate concentration was found to affect specific activity and regioselectivity of the BVMO, as well as the rate of product autohydrolysis to the primary alcohol. More than 80 % conversion of 50 mm octanal was reached after 72 h (TTN nearly 20 000). Biotransformation on a 200 mL scale under unoptimized conditions gave a space-time yield (STY) of 4.2 g L(-1)  d(-1) (3.4 g L(-1)  d(-1) extracted product).

  4. Distribution and Diversity of Archaeal Ammonia Monooxygenase Genes Associated with Corals▿ †

    PubMed Central

    Beman, J.Michael; Roberts, Kathryn J.; Wegley, Linda; Rohwer, Forest; Francis, Christopher A.

    2007-01-01

    Corals are known to harbor diverse microbial communities of Bacteria and Archaea, yet the ecological role of these microorganisms remains largely unknown. Here we report putative ammonia monooxygenase subunit A (amoA) genes of archaeal origin associated with corals. Multiple DNA samples drawn from nine coral species and four different reef locations were PCR screened for archaeal and bacterial amoA genes, and archaeal amoA gene sequences were obtained from five different species of coral collected in Bocas del Toro, Panama. The 210 coral-associated archaeal amoA sequences recovered in this study were broadly distributed phylogenetically, with most only distantly related to previously reported sequences from coastal/estuarine sediments and oceanic water columns. In contrast, the bacterial amoA gene could not be amplified from any of these samples. These results offer further evidence for the widespread presence of the archaeal amoA gene in marine ecosystems, including coral reefs. PMID:17586663

  5. Dynamics of Alkane Hydroxylation at the Non-Heme Diiron Center in Methane Monooxygenase

    SciTech Connect

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

    2002-03-12

    Semiclassical molecular dynamics simulations have been combined with quantum chemistry calculations to provide detailed modeling of the methane and ethane hydroxylation reactions catalyzed by the hydroxylase enzymes of the soluble methane monooxygenase system. The experimental distribution of enantiomeric alcohols in the reaction of ethanes made chiral by the use of hydrogen isotopes is quantitatively reproduced and explained. The reaction dynamics involve a mixture of concerted and bound radical trajectories, and we characterize each of these reactive channels in detail. Diffusion of the bound radical intermediate at the active site core determines the global rate constant. The results also provide a qualitative rationale for the lack of ring-opened products derived from certain radical clock substrate probes and for the relative rate constants and kinetic isotope effects exhibited by a variety of substrates.

  6. 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.

  7. Xenon and halogenated alkanes track putative substrate binding cavities in the soluble methane monooxygenase hydroxylase.

    PubMed

    Whittington, D A; Rosenzweig, A C; Frederick, C A; Lippard, S J

    2001-03-27

    To investigate the role of protein cavities in facilitating movement of the substrates, methane and dioxygen, in the soluble methane monooxygenase hydroxylase (MMOH), we determined the X-ray structures of MMOH from Methylococcus capsulatus (Bath) cocrystallized with dibromomethane or iodoethane, or by using crystals pressurized with xenon gas. The halogenated alkanes bind in two cavities within the alpha-subunit that extend from one surface of the protein to the buried dinuclear iron active site. Two additional binding sites were located in the beta-subunit. Pressurization of two crystal forms of MMOH with xenon resulted in the identification of six binding sites located exclusively in the alpha-subunit. These results indicate that hydrophobic species bind preferentially in preexisting cavities in MMOH and support the hypothesis that such cavities may play a functional role in sequestering and enhancing the availability of the physiological substrates for reaction at the active site.

  8. Cytochrome P450 monooxygenases involved in anthracene metabolism by the white-rot basidiomycete Phanerochaete chrysosporium.

    PubMed

    Chigu, Nomathemba Loice; Hirosue, Sinji; Nakamura, Chie; Teramoto, Hiroshi; Ichinose, Hirofumi; Wariishi, Hiroyuki

    2010-08-01

    Cytochrome P450 monooxygenases (P450s) involved in anthracene metabolism by the white-rot basidiomycete Phanerochaete chrysosporium were identified by comprehensive screening of both catalytic potentials and transcriptomic profiling. Functional screening of P. chrysosporium P450s (PcCYPs) revealed that 14 PcCYP species catalyze stepwise conversion of anthracene to anthraquinone via intermediate formation of anthrone. Moreover, transcriptomic profiling explored using a complementary DNA microarray system demonstrated that 12 PcCYPs are up-regulated in response to exogenous addition of anthracene. Among the up-regulated PcCYPs, five species showed catalytic activity against anthracene. Based upon both catalytic and transcriptional properties, these five species are most likely to play major roles in anthracene metabolic processes in vivo. Thus, the combination of functional screening and a microarray system may provide a novel strategy for obtaining a thorough understanding of the catalytic functions and biological impacts of PcCYPs.

  9. 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.

  10. Structural basis for substrate targeting and catalysis by fungal polysaccharide monooxygenases

    PubMed Central

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

    2013-01-01

    SUMMARY 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 Å 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

  11. 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

  12. Toluene 4-Monooxygenase and its Complex with Effector Protein T4moD

    SciTech Connect

    Bailey, Lucas J.; Fox, Brian G.

    2012-10-16

    Toluene 4-monooxygenase (T4MO) is a multiprotein diiron enzyme complex that catalyzes the regiospecific oxidation of toluene to p-cresol. Catalytic function requires the presence of a small protein, called the effector protein. Effector protein exerts substantial control on the diiron hydroxylase catalytic cycle through protein-protein interactions. High-resolution crystal structures of the stoichometric hydroxylase and effector protein complex described here reveal how protein-protein interactions and reduction of the diiron center produce an active site configuration poised for reaction with O{sub 2}. Further information from crystal structures of mutated isoforms of the hydroxylase and a peroxo adduct is combined with catalytic results to give a fuller picture of the geometry of the enzyme-substrate complex used for the high fidelity oxidation of hydrocarbon substrates.

  13. Supramolecular control of monooxygenase reactivity in a copper(ii) cryptate.

    PubMed

    Chaloner, L; Khomutovskaya, A; Thomas, F; Ottenwaelder, X

    2016-07-05

    We report a detailed investigation of the formation and self-decomposition of Cu(ii)-hydroperoxo intermediates under the influence of second-coordination-sphere features provided by a cryptand. In solution, an equilibrium between two copper complexes with square-planar and square-pyramidal geometries was identified. Upon addition of H2O2/Et3N, two copper(ii)-hydroperoxo intermediates formed at different rates. Their decomposition via self-oxidation was probed by deuterating select positions on the cryptand. This led to a small kinetic isotope effect of 1.5. Mass spectrometry analysis of the demetallated organic products is consistent with a direct oxygen-atom transfer to a tertiary amine on the cryptand, forming an N-oxide, unlike other models of copper mononuclear monooxygenase enzymes.

  14. Structural and mechanistic basis of differentiated inhibitors of the acute pancreatitis target kynurenine-3-monooxygenase

    NASA Astrophysics Data System (ADS)

    Hutchinson, Jonathan P.; Rowland, Paul; Taylor, Mark R. D.; Christodoulou, Erica M.; Haslam, Carl; Hobbs, Clare I.; Holmes, Duncan S.; Homes, Paul; Liddle, John; Mole, Damian J.; Uings, Iain; Walker, Ann L.; Webster, Scott P.; Mowat, Christopher G.; Chung, Chun-Wa

    2017-06-01

    Kynurenine-3-monooxygenase (KMO) is a key FAD-dependent enzyme of tryptophan metabolism. In animal models, KMO inhibition has shown benefit in neurodegenerative diseases such as Huntington's and Alzheimer's. Most recently it has been identified as a target for acute pancreatitis multiple organ dysfunction syndrome (AP-MODS); a devastating inflammatory condition with a mortality rate in excess of 20%. Here we report and dissect the molecular mechanism of action of three classes of KMO inhibitors with differentiated binding modes and kinetics. Two novel inhibitor classes trap the catalytic flavin in a previously unobserved tilting conformation. This correlates with picomolar affinities, increased residence times and an absence of the peroxide production seen with previous substrate site inhibitors. These structural and mechanistic insights culminated in GSK065(C1) and GSK366(C2), molecules suitable for preclinical evaluation. Moreover, revising the repertoire of flavin dynamics in this enzyme class offers exciting new opportunities for inhibitor design.

  15. 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

  16. Alcaligenes eutrophus JMP134 "2,4-dichlorophenoxyacetate monooxygenase" is an alpha-ketoglutarate-dependent dioxygenase.

    PubMed Central

    Fukumori, F; Hausinger, R P

    1993-01-01

    The Alcaligenes eutrophus JMP134 tfdA gene, encoding the enzyme responsible for the first step in 2,4-dichlorophenoxyacetic acid (2,4-D) biodegradation, was overexpressed in Escherichia coli, and several enzymatic properties of the partially purified gene product were examined. Although the tfdA-encoded enzyme is typically referred to as 2,4-D monooxygenase, we were unable to observe any reductant-dependent activity. Rather, we demonstrate that this enzyme is a ferrous ion-dependent dioxygenase that uses alpha-ketoglutarate as a cosubstrate. The alpha-ketoglutarate is converted to succinate concomitant with 2,4-D conversion to 2,4-dichlorophenol. By using [1-14C]alpha-ketoglutarate, we established that carbon dioxide is the second product derived from alpha-ketoglutarate. Finally, we verified the proposal that glyoxylate is the second product derived from 2,4-D. PMID:8458850

  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. 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

  19. 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

  20. Escherichia coli Overexpressing a Baeyer-Villiger Monooxygenase from Acinetobacter radioresistens Becomes Resistant to Imipenem.

    PubMed

    Minerdi, Daniela; Zgrablic, Ivan; Castrignanò, Silvia; Catucci, Gianluca; Medana, Claudio; Terlizzi, Maria Elena; Gribaudo, Giorgio; Gilardi, Gianfranco; Sadeghi, Sheila J

    2015-10-12

    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.

  1. Cytochrome P450 monooxygenase lipid metabolites are significant second messengers in the resolution of choroidal neovascularization.

    PubMed

    Hasegawa, Eiichi; Inafuku, Saori; Mulki, Lama; Okunuki, Yoko; Yanai, Ryoji; Smith, Kaylee E; Kim, Clifford B; Klokman, Garrett; Bielenberg, Diane R; Puli, Narender; Falck, John R; Husain, Deeba; Miller, Joan W; Edin, Matthew L; Zeldin, Darryl C; Lee, Kin Sing Stephen; Hammock, Bruce D; Schunck, Wolf-Hagen; Connor, Kip M

    2017-09-05

    Age-related macular degeneration (AMD) is the most common cause of blindness for individuals age 50 and above in the developed world. Abnormal growth of choroidal blood vessels, or choroidal neovascularization (CNV), is a hallmark of the neovascular (wet) form of advanced AMD and leads to significant vision loss. A growing body of evidence supports a strong link between neovascular disease and inflammation. Metabolites of long-chain polyunsaturated fatty acids derived from the cytochrome P450 (CYP) monooxygenase pathway serve as vital second messengers that regulate a number of hormones and growth factors involved in inflammation and vascular function. Using transgenic mice with altered CYP lipid biosynthetic pathways in a mouse model of laser-induced CNV, we characterized the role of these lipid metabolites in regulating neovascular disease. We discovered that the CYP-derived lipid metabolites epoxydocosapentaenoic acids (EDPs) and epoxyeicosatetraenoic acids (EEQs) are vital in dampening CNV severity. Specifically, overexpression of the monooxygenase CYP2C8 or genetic ablation or inhibition of the soluble epoxide hydrolase (sEH) enzyme led to increased levels of EDP and EEQ with attenuated CNV development. In contrast, when we promoted the degradation of these CYP-derived metabolites by transgenic overexpression of sEH, the protective effect against CNV was lost. We found that these molecules work in part through their ability to regulate the expression of key leukocyte adhesion molecules, on both leukocytes and endothelial cells, thereby mediating leukocyte recruitment. These results suggest that CYP lipid signaling molecules and their regulators are potential therapeutic targets in neovascular diseases.

  2. The Toluene o-Xylene Monooxygenase Enzymatic Activity for the Biosynthesis of Aromatic Antioxidants.

    PubMed

    Donadio, Giuliana; Sarcinelli, Carmen; 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.

  3. Crystal Structure of Albaflavenone Monooxygenase Containing a Moonlighting Terpene Synthase Active Site*

    PubMed Central

    Zhao, Bin; Lei, Li; Vassylyev, Dmitry G.; Lin, Xin; Cane, David E.; Kelly, Steven L.; Yuan, Hang; Lamb, David C.; Waterman, Michael R.

    2009-01-01

    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 Å) and complex of endogenous substrate (epi-isozizaene) with CYP170A1 (3.3 Å). 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 α-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 α-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. PMID:19858213

  4. Homology modeling and protein engineering of alkane monooxygenase in Burkholderia thailandensis MSMB121: in silico insights.

    PubMed

    Jain, Chakresh Kumar; Gupta, Money; Prasad, Yamuna; Wadhwa, Gulshan; Sharma, Sanjeev Kumar

    2014-07-01

    The degradation of hydrocarbons plays an important role in the eco-balancing of petroleum products, pesticides and other toxic products in the environment. The degradation of hydrocarbons by microbes such as Geobacillus thermodenitrificans, Burkhulderia, Gordonia sp. and Acinetobacter sp. has been studied intensively in the literature. The present study focused on the in silico protein engineering of alkane monooxygenase (ladA)-a protein involved in the alkane degradation pathway. We demonstrated the improvement in substrate binding energy with engineered ladA in Burkholderia thailandensis MSMB121. We identified an ortholog of ladA monooxygenase found in B. thailandensis MSMB121, and showed it to be an enzyme involved in an alkane degradation pathway studied extensively in Geobacillus thermodenitrificans. Homology modeling of the three-dimensional structure of ladA was performed with a crystal structure (protein databank ID: 3B9N) as a template in MODELLER 9v11, and further validated using PROCHECK, VERIFY-3D and WHATIF tools. Specific amino acids were substituted in the region corresponding to amino acids 305-370 of ladA protein, resulting in an enhancement of binding energy in different alkane chain molecules as compared to wild protein structures in the docking experiments. The substrate binding energy with the protein was calculated using Vina (Implemented in VEGAZZ). Molecular dynamics simulations were performed to study the dynamics of different alkane chain molecules inside the binding pockets of wild and mutated ladA. Here, we hypothesize an improvement in binding energies and accessibility of substrates towards engineered ladA enzyme, which could be further facilitated for wet laboratory-based experiments for validation of the alkane degradation pathway in this organism.

  5. Kinetic Mechanism of the Dechlorinating Flavin-dependent Monooxygenase HadA.

    PubMed

    Pimviriyakul, Panu; Thotsaporn, Kittisak; Sucharitakul, Jeerus; Chaiyen, Pimchai

    2017-03-24

    The accumulation of chlorophenols (CPs) in the environment, due to their wide use as agrochemicals, has become a serious environmental problem. These organic halides can be degraded by aerobic microorganisms, where the initial steps of various biodegradation pathways include an oxidative dechlorinating process in which chloride is replaced by a hydroxyl substituent. Harnessing these dechlorinating processes could provide an opportunity for environmental remediation, but detailed catalytic mechanisms for these enzymes are not yet known. To close this gap, we now report transient kinetics and product analysis of the dechlorinating flavin-dependent monooxygenase, HadA, from the aerobic organism Ralstonia pickettii DTP0602, identifying several mechanistic properties that differ from other enzymes in the same class. We first overexpressed and purified HadA to homogeneity. Analyses of the products from single and multiple turnover reactions demonstrated that HadA prefers 4-CP and 2-CP over CPs with multiple substituents. Stopped-flow and rapid-quench flow experiments of HadA with 4-CP show the involvement of specific intermediates (C4a-hydroperoxy-FAD and C4a-hydroxy-FAD) in the reaction, define rate constants and the order of substrate binding, and demonstrate that the hydroxylation step occurs prior to chloride elimination. The data also identify the non-productive and productive paths of the HadA reactions and demonstrate that product formation is the rate-limiting step. This is the first elucidation of the kinetic mechanism of a two-component flavin-dependent monooxygenase that can catalyze oxidative dechlorination of various CPs, and as such it will serve as the basis for future investigation of enzyme variants that will be useful for applications in detoxifying chemicals hazardous to human health.

  6. The Biochemical Mechanism of Auxin Biosynthesis by an Arabidopsis YUCCA Flavin-containing Monooxygenase*

    PubMed Central

    Dai, Xinhua; Mashiguchi, Kiyoshi; Chen, Qingguo; Kasahara, Hiroyuki; Kamiya, Yuji; Ojha, Sunil; DuBois, Jennifer; Ballou, David; Zhao, Yunde

    2013-01-01

    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

  7. Flavin-Dependent Monooxygenases as a Detoxification Mechanism in Insects: New Insights from the Arctiids (Lepidoptera)

    PubMed Central

    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

  8. 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

  9. 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.

  10. 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.

  11. Inactivation of Toluene 2-Monooxygenase in Burkholderia cepacia G4 by Alkynes

    PubMed Central

    Yeager, Chris M.; Bottomley, Peter J.; Arp, Daniel J.; Hyman, Michael R.

    1999-01-01

    High concentrations of acetylene (10 to 50% [vol/vol] gas phase) were required to inhibit the growth of Burkholderia cepacia G4 on toluene, while 1% (vol/vol) (gas phase) propyne or 1-butyne completely inhibited growth. Low concentrations of longer-chain alkynes (C5 to C10) were also effective inhibitors of toluene-dependent growth, and 2- and 3-alkynes were more potent inhibitors than their 1-alkyne counterparts. Exposure of toluene-grown B. cepacia G4 to alkynes resulted in the irreversible loss of toluene- and o-cresol-dependent O2 uptake activities, while acetate- and 3-methylcatechol-dependent O2 uptake activities were unaffected. Toluene-dependent O2 uptake decreased upon the addition of 1-butyne in a concentration- and time-dependent manner. The loss of activity followed first-order kinetics, with apparent rate constants ranging from 0.25 min−1 to 2.45 min−1. Increasing concentrations of toluene afforded protection from the inhibitory effects of 1-butyne. Furthermore, oxygen, supplied as H2O2, was required for inhibition by 1-butyne. These results suggest that alkynes are specific, mechanism-based inactivators of toluene 2-monooxygenase in B. cepacia G4, although the simplest alkyne, acetylene, was relatively ineffective compared to longer alkynes. Alkene analogs of acetylene and propyne—ethylene and propylene—were not inactivators of toluene 2-monooxygenase activity in B. cepacia G4 but were oxidized to their respective epoxides, with apparent Ks and Vmax values of 39.7 μM and 112.3 nmol min−1 mg of protein−1 for ethylene and 32.3 μM and 89.2 nmol min−1 mg of protein−1 for propylene. PMID:9925593

  12. 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.

  13. 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

  14. 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.

  15. 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

    2010-05-03

    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

  16. Enzymatic formation of apo-carotenoids from the xanthophyll carotenoids lutein, zeaxanthin and b-cryptoxanthin by ferret carotene-9, 10-monooxygenase

    USDA-ARS?s Scientific Manuscript database

    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. mRNA stability in mammalian cells.

    PubMed Central

    Ross, J

    1995-01-01

    This review concerns how cytoplasmic mRNA half-lives are regulated and how mRNA decay rates influence gene expression. mRNA stability influences gene expression in virtually all organisms, from bacteria to mammals, and the abundance of a particular mRNA can fluctuate manyfold following a change in the mRNA half-life, without any change in transcription. The processes that regulate mRNA half-lives can, in turn, affect how cells grow, differentiate, and respond to their environment. Three major questions are addressed. Which sequences in mRNAs determine their half-lives? Which enzymes degrade mRNAs? Which (trans-acting) factors regulate mRNA stability, and how do they function? The following specific topics are discussed: techniques for measuring eukaryotic mRNA stability and for calculating decay constants, mRNA decay pathways, mRNases, proteins that bind to sequences shared among many mRNAs [like poly(A)- and AU-rich-binding proteins] and proteins that bind to specific mRNAs (like the c-myc coding-region determinant-binding protein), how environmental factors like hormones and growth factors affect mRNA stability, and how translation and mRNA stability are linked. Some perspectives and predictions for future research directions are summarized at the end. PMID:7565413

  18. A comparison of the substrate and electron-donor specificities of the methane mono-oxygenases from three strains of methane-oxidizing bacteria.

    PubMed Central

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

    1979-01-01

    1. Methane mono-oxygenase from Methylosinus trichosporium has the same broad substrate specificity as the analogous enzyme from Methylococcus capsulatus (Bath); the enzyme from Methylomonas methanica is more specific. 2. Contrary to previous reports, NAD(P)H and not ascorbate is the required electron donor for the enzyme from Methylosinus trichosporium. 3. It is concluded that these three bacteria contain similar methane mono-oxygenases. PMID:106847

  19. Transcriptional regulation of the grape cytochrome P450 monooxygenase gene CYP736B expression in response to Xylella fastidiosa infection

    PubMed Central

    2010-01-01

    Background Plant cytochrome P450 monooxygenases (CYP) mediate synthesis and metabolism of many physiologically important primary and secondary compounds that are related to plant defense against a range of pathogenic microbes and insects. To determine if cytochrome P450 monooxygenases are involved in defense response to Xylella fastidiosa (Xf) infection, we investigated expression and regulatory mechanisms of the cytochrome P450 monooxygenase CYP736B gene in both disease resistant and susceptible grapevines. Results Cloning of genomic DNA and cDNA revealed that the CYP736B gene was composed of two exons and one intron with GT as a donor site and AG as an acceptor site. CYP736B transcript was up-regulated in PD-resistant plants and down-regulated in PD-susceptible plants 6 weeks after Xf inoculation. However, CYP736B expression was very low in stem tissues at all evaluated time points. 5'RACE and 3'RACE sequence analyses revealed that there were three candidate transcription start sites (TSS) in the upstream region and three candidate polyadenylation (PolyA) sites in the downstream region of CYP736B. Usage frequencies of each transcription initiation site and each polyadenylation site varied depending on plant genotype, developmental stage, tissue, and treatment. These results demonstrate that expression of CYP736B is regulated developmentally and in response to Xf infection at both transcriptional and post-transcriptional levels. Multiple transcription start and polyadenylation sites contribute to regulation of CYP736B expression. Conclusions This report provides evidence that the cytochrome P450 monooxygenase CYP736B gene is involved in defense response at a specific stage of Xf infection in grapevines; multiple transcription initiation and polyadenylation sites exist for CYP736B in grapevine; and coordinative and selective use of transcription initiation and polyadenylation sites play an important role in regulation of CYP736B expression during growth

  20. Role of hepatic monooxygenases in generating estrogenic metabolites from methoxychlor and from its identified contaminants.

    PubMed

    Bulger, W H; Feil, V J; Kupfer, D

    1985-01-01

    Previous investigations demonstrated that methoxychlor [1,1,1-trichloro-2,2-bis(4-methoxyphenyl)ethane] contains estrogenic contaminants and that methoxychlor per se is not an estrogen but is a proestrogen being metabolized in vivo into estrogenic products. The present study examined structurally identified methoxychlor contaminants as to their estrogenic or proestrogenic properties. Also, the estrogenic activity of demethylated metabolites of methoxychlor and of one contaminant was determined. To examine these properties, we utilized an assay developed by us that monitors whether a given compound, incubated with isolated rat uteri, can diminish the uterine cytosolic estrogen receptor and elevate the nuclear estrogen receptor and whether metabolic intervention by hepatic microsomal monooxygenase(s) is required by the respective compound for this cellular redistribution of the receptor. Of the 15 compounds examined which constitute with methoxychlor 99.5% of total technical grade methoxychlor, two compounds, 1,1-dichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethene (mono-OH-MDDE) and 1,1,1-trichloro-2-(4-hydroxyphenyl)-2-(4-methoxyphenyl)ethane (mono-OH-methoxychlor), were active per se and two compounds, 1,1-dichloro-2,2-bis(4-methoxyphenyl)ethene (MDDE) and methoxychlor, required metabolic transformation for estrogenic activity to be manifested. Subsequently, it was shown that the mono- and bis-OH metabolites of MDDE and of methoxychlor were active estrogens and that the order of activity, either by the above procedure or in terms of relative binding affinity to rat uterine cytosolic receptor, was as follows: bis-OH-MDDE much greater than bis-OH-methoxychlor greater than mono-OH-MDDE greater than mono-OH-methoxychlor. Following the in vitro observations, the activity of MDDE and bis-OH-MDDE was determined in vivo in immature rats. It appears that both compounds are estrogenic, yielding marked elevation in ornithine decarboxylase (EC 4.1.1.17) levels and moderate

  1. Pseudomonad Cyclopentadecanone Monooxygenase Displaying an Uncommon Spectrum of Baeyer-Villiger Oxidations of Cyclic Ketones†

    PubMed Central

    Iwaki, Hiroaki; Wang, Shaozhao; Grosse, Stephan; Bergeron, Hélène; Nagahashi, Ayako; Lertvorachon, Jittiwud; Yang, Jianzhong; Konishi, Yasuo; Hasegawa, Yoshie; Lau, Peter C. K.

    2006-01-01

    Baeyer-Villiger monooxygenases (BVMOs) are biocatalysts that offer the prospect of high chemo-, regio-, and enantioselectivity in the organic synthesis of lactones or esters from a variety of ketones. In this study, we have cloned, sequenced, and overexpressed in Escherichia coli a new BVMO, cyclopentadecanone monooxygenase (CpdB or CPDMO), originally derived from Pseudomonas sp. strain HI-70. The 601-residue primary structure of CpdB revealed only 29% to 50% sequence identity to those of known BVMOs. A new sequence motif, characterized by a cluster of charged residues, was identified in a subset of BVMO sequences that contain an N-terminal extension of ∼60 to 147 amino acids. The 64-kDa CPDMO enzyme was purified to apparent homogeneity, providing a specific activity of 3.94 μmol/min/mg protein and a 20% yield. CPDMO is monomeric and NADPH dependent and contains ∼1 mol flavin adenine dinucleotide per mole of protein. A deletion mutant suggested the importance of the N-terminal 54 amino acids to CPDMO activity. In addition, a Ser261Ala substitution in a Rossmann fold motif resulted in an improved stability and increased affinity of the enzyme towards NADPH compared to the wild-type enzyme (Km = 8 μM versus Km = 24 μM). Substrate profiling indicated that CPDMO is unusual among known BVMOs in being able to accommodate and oxidize both large and small ring substrates that include C11 to C15 ketones, methyl-substituted C5 and C6 ketones, and bicyclic ketones, such as decalone and β-tetralone. CPDMO has the highest affinity (Km = 5.8 μM) and the highest catalytic efficiency (kcat/Km ratio of 7.2 × 105 M−1 s−1) toward cyclopentadecanone, hence the Cpd designation. A number of whole-cell biotransformations were carried out, and as a result, CPDMO was found to have an excellent enantioselectivity (E > 200) as well as 99% S-selectivity toward 2-methylcyclohexanone for the production of 7-methyl-2-oxepanone, a potentially valuable chiral building block. Although

  2. Identification and treatment of heme depletion attributed to overexpression of a lineage of evolved P450 monooxygenases.

    PubMed

    Michener, Joshua K; Nielsen, Jens; Smolke, Christina D

    2012-11-20

    Recent advances in metabolic engineering have demonstrated that microbial biosynthesis can provide a viable alternative to chemical synthesis for the production of bulk and fine chemicals. Introduction of a new biosynthetic pathway typically requires the expression of multiple heterologous enzymes in the production host, which can impose stress on the host cell and, thereby, limit performance of the pathway. Unfortunately, analysis and treatment of the host stress response can be difficult, because there are many sources of stress that may interact in complex ways. We use a systems biological approach to analyze the stress imposed by expressing different enzyme variants from a lineage of soluble P450 monooxygenases, previously evolved for heterologous activity in Saccharomyces cerevisiae. Our analysis identifies patterns of stress imposed on the host by heterologous enzyme overexpression that are consistent across the evolutionary lineage, ultimately implicating heme depletion as the major stress. We show that the monooxygenase evolution, starting from conditions of either high or low stress, caused the cellular stress to converge to a common level. Overexpression of rate-limiting enzymes in the endogenous heme biosynthetic pathway alleviates the stress imposed by expression of the P450 monooxygenases and increases the enzymatic activity of the final evolved P450 by an additional 2.3-fold. Heme overexpression also increases the total activity of an endogenous cytosolic heme-containing catalase but not a heterologous P450 that is membrane-associated. This work demonstrates the utility of combining systems and synthetic biology to analyze and optimize heterologous enzyme expression.

  3. Regioselective Versatility of Monooxygenase Reactions Catalyzed by CYP2B6 and CYP3A4: Examples with Single Substrates.

    PubMed

    Erratico, Claudio A; Deo, Anand K; Bandiera, Stelvio M

    2015-01-01

    Hepatic microsomal cytochrome P450 (CYP) enzymes have broad and overlapping substrate specificity and catalyze a variety of monooxygenase reactions, including aliphatic and aromatic hydroxylations, N-hydroxylations, oxygenations of heteroatoms (N, S, P and I), alkene and arene epoxidations, dehalogenations, dehydrogenations and N-, O- and S-dealkylations. Individual CYP enzymes typically catalyze the oxidative metabolism of a common substrate in a regioselective and stereoselective manner. In addition, different CYP enzymes often utilize different monooxygenase reactions when oxidizing a common substrate. This review examines various oxidative reactions catalyzed by a CYP enzyme acting on a single substrate. In the first example, 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), a halogenated aromatic environmental contaminant, was oxidatively biotransformed by human CYP2B6. Nine different metabolites of BDE-47 were produced by CYP2B6 via monooxygenase reactions that included aromatic hydroxylation, with and without an NIH-shift, dealkylation and debromination. In the second example, lithocholic acid (3α-hydroxy-5β-cholan-24-oic acid), an endogenous bile acid, served as a substrate for human CYP3A4 and yielded five different metabolites via aliphatic hydroxylation and dehydrogenation reactions.

  4. Total degradation of pentachloroethane by an engineered Alcaligenes strain expressing a modified camphor monooxygenase and a hybrid dioxygenase.

    PubMed

    Iwakiri, Ryo; Yoshihira, Kunichika; Ngadiman; Futagami, Taiki; Goto, Masatoshi; Furukawa, Kensuke

    2004-06-01

    We engineered biphenyl-degrading Alcaligenes sp. strain KF711 for total degradation of pentachloroethane (PCA), which expresses a modified camphor monooxygenase and a hybrid dioxygenase consisting of TodC1 (a large subunit of toluene dioxygenase of Pseudomonas putida F1) and BphA2-BphA3-pbhA4 (a small subunit, ferredoxin and ferredoxin reductase of biphenyl dioxygenase, respectively, in strain KF707). Modified camphor monooxygenase genes (camCAB) were supplied as a plasmid and the todC1 gene was integrated within the chromosomal bph gene cluster by a single crossover recombination. The resultant strain KF711S-3cam dechlorinated PCA to trichloroethene by the action of the modified camphor monooxygenase under anaerobic conditions. The same strain subsequently degraded trichloroethene formed oxidatively by the action of the Tol-Bph hybrid dioxygenase under aerobic conditions. Thus sequential anaerobic and aerobic treatments of the KF711S-3cam resting cells resulted in efficient and total degradation of PCA.

  5. Nutrigenetics of carotenoid metabolism in the chicken: a polymorphism at the β,β-carotene 15,15'-mono-oxygenase 1 (BCMO1) locus affects the response to dietary β-carotene.

    PubMed

    Jlali, Maamer; Graulet, Benoit; Chauveau-Duriot, Béatrice; Godet, Estelle; Praud, Christophe; Nunes, Carlos Simões; Le Bihan-Duval, Elisabeth; Berri, Cécile; Duclos, Michel J

    2014-06-28

    The enzyme β,β-carotene-15,15'-mono-oxygenase 1 (BCMO1) is responsible for the symmetrical cleavage of β-carotene into retinal. We identified a polymorphism in the promoter of the BCMO1 gene, inducing differences in BCMO1 mRNA levels (high in adenines (AA) and low in guanines (GG)) and colour in chicken breast muscle. The present study was designed to test whether this polymorphism could affect the response to dietary β-carotene. Dietary β-carotene supplementation did not change the effects of the genotypes on breast muscle properties: BCMO1 mRNA levels were lower and xanthophyll contents higher in GG than in AA chickens. Lower vitamin E levels in the plasma and duodenum, plasma cholesterol levels and body weight were also observed in GG than in AA chickens. In both genotypes, dietary β-carotene increased vitamin A storage in the liver; however, it reduced numerous parameters such as SCARB1 (scavenger receptor class B type I) in the duodenum, BCMO1 in the liver, vitamin E levels in the plasma and tissues, xanthophyll contents in the pectoralis major muscle and carcass adiposity. However, several diet × genotype interactions were observed. In the GG genotype, dietary β-carotene increased ISX (intestine-specific homeobox) and decreased BCMO1 mRNA levels in the duodenum, decreased xanthophyll concentrations in the duodenum, liver and plasma, and decreased colour index and HDL-cholesterol concentration in the plasma. Retinol accumulation following dietary β-carotene supplementation was observed in the duodenum of AA chickens only. Therefore, the negative feedback control on β-carotene conversion through ISX appears as functional in the duodenum of GG but not of AA chickens. This could result in a higher availability of β-carotene in the duodenum of GG chickens, reducing the uptake of xanthophylls, liposoluble vitamins and cholesterol.

  6. Sequencing and characterization of mixed function monooxygenase genes CYP1A1 and CYP1A2 of Mink (Mustela vison) to facilitate study of dioxin-like compounds

    SciTech Connect

    Zhang Xiaowei; Moore, Jeremy N.; Newsted, John L.; Hecker, Markus Zwiernik, Matthew J.; Jones, Paul D.; Bursian, Steven J.

    2009-02-01

    As part of an ongoing effort to understand aryl hydrocarbon receptor (AhR) mediated toxicity in mink, cDNAs encoding for CYP1A1 and the CYP1A2 mixed function monooxygenases were cloned and characterized. In addition, the effects of selected dibenzofurans on the expression of these genes and the presence of their respective proteins (P4501A) were investigated, and then correlated with the catalytic activities of these proteins as measured by ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-deethylase (MROD) activities. The predicted protein sequences for CYP1A1 and CYP1A2 comprise 517 and 512 amino acid residues, respectively. The phylogenetic analysis of the mink CYP1As with protein sequences of other mammals revealed high sequence homology with sea otter, seals and the dog, with amino acid identities ranging from 89 to 95% for CYP1A1 and 81 to 93% for CYP1A2. Since exposure to both 2,3,7,8-Tetrachlorodibenzofuran (TCDF) and 2,3,4,7,8-Pentachlorodibenzofuran (PeCDF) resulted in dose-dependent increases of CYP1A1 mRNA, CYP1A2 mRNA and CYP1A protein levels an underlying AhR-mediated mechanism is suggested. The up-regulation of CYP1A mRNA in liver was more consistent to the sum adipose TEQ concentration than to the liver TEQ concentration in minks treated with TCDF or PeCDF. The result suggested that the hepatic-sequestered fraction of PeCDF was biologically inactive to the induction of CYP1A1 and CYP1A2.

  7. Safety assessment of dicamba mono-oxygenases that confer dicamba tolerance to various crops.

    PubMed

    Wang, Cunxi; Glenn, Kevin C; Kessenich, Colton; Bell, Erin; Burzio, Luis A; Koch, Michael S; Li, Bin; Silvanovich, Andre

    2016-11-01

    Dicamba tolerant (DT) soybean, cotton and maize were developed through constitutive expression of dicamba mono-oxygenase (DMO) in chloroplasts. DMO expressed in three DT crops exhibit 91.6-97.1% amino acid sequence identity to wild type DMO. All DMO forms maintain the characteristics of Rieske oxygenases that have a history of safe use. Additionally, they are all functionally similar in vivo since the three DT crops are all tolerant to dicamba treatment. None of these DMO sequences were found to have similarity to any known allergens or toxins. Herein, to further understand the safety of these DMO variants, a weight of evidence approach was employed. Each purified DMO protein was found to be completely deactivated in vitro by heating at temperatures 55 °C and above, and all were completely digested within 30 s or 5 min by pepsin and pancreatin, respectively. Mice orally dosed with each of these DMO proteins showed no adverse effects as evidenced by analysis of body weight gain, food consumption and clinical observations. Therefore, the weight of evidence from all these protein safety studies support the conclusion that the various forms of DMO proteins introduced into DT soybean, cotton and maize are safe for food and feed consumption, and the small amino acid sequence differences outside the active site of DMO do not raise any additional safety concerns.

  8. Phenylalanine monooxygenase and the sulfur oxygenation of S-carboxymethyl-L-cysteine in mice.

    PubMed

    Vandenbossche, Evita; Lucas, Christopher; Mistry, Lata; Garfield, Emma; Mitchell, Stephen C; Steventon, Glyn B

    2016-01-01

    1. The extent of sulfoxidation of the drug, S-carboxymethyl-L-cysteine, has been shown to vary between individuals, with this phenomenon being mooted as a biomarker for certain disease states and susceptibilities. Studies in vitro have indicated that the enzyme responsible for this reaction was phenylalanine monooxygenase but to date no in vivo evidence exists to support this assumption. Using the mouse models of mild hyperphenylalaninamia (enu1 PAH variant) and classical phenylketonuria (enu2 PAH variant), the sulfur oxygenation of S-carboxymethyl-L-cysteine has been investigated. 2. Compared to the wild type (wt/wt) mice, both the heterozygous dominant (wt/enu1 and wt/enu2) mice and the homozygous recessive (enu1/enu1 and enu2/enu2) mice were shown to have significantly increased Cmax, AUC(0-180 min) and AUC(0-∞ min) values (15 - to 20-fold higher). These results were primarily attributable to the significantly reduced clearance of S-carboxymethyl-L-cysteine (13 - to 22-fold lower). 3. Only the wild type mice produced measurable quantities of the parent S-oxide metabolites. Those mice possessing one or more allelic variant showed no evidence of blood SCMC (R/S) S-oxides. These observations support the proposition that differences in phenylalanine hydroxylase activity underlie the variation in S-carboxymethyl-L-cysteine sulfoxidation and that no other enzyme is able to undertake this reaction.

  9. Molecular phylogeny, long-term evolution, and functional divergence of flavin-containing monooxygenases.

    PubMed

    Hao, Da Cheng; Chen, Shi Lin; Mu, Jun; Xiao, Pei Gen

    2009-11-01

    Flavin-containing monooxygenases (FMOs) metabolize xenobiotic compounds, many of which are clinically important, as well as endogenous substrates as part of a discrete physiological process. The FMO gene family is conserved and ancient with representatives present in all phyla so far examined. However, there is a lack of information regarding the long-term evolution and functional divergence of these proteins. This study represents the first attempt to characterize the long-term evolution followed by the members of this family. Our analysis shows that there is extensive silent divergence at the nucleotide level suggesting that this family has been subject to strong purifying selection at the protein level. Invertebrate FMOs have a polyphyletic origin. The functional divergence of FMOs 1-5 started before the split between amphibians and mammals. The vertebrate FMO5 is more ancestral than other four FMOs. Moreover, the existence of higher levels of codon bias was detected at the N-terminal ends, which can be ascribed to the critical role played by the FAD binding motif in this region. Finally, critical amino acid residues for FMO functional divergence (type I & II) after gene duplication were detected and characterized.

  10. 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

  11. Flavin-containing monooxygenase, a new clue of pathological proteins in the rotenone model of parkinsonism.

    PubMed

    Li, Boyu; Yuan, Yuhe; Zhang, Wanqing; He, Wenbin; Hu, Jinfeng; Chen, Naihong

    2014-04-30

    Parkinson's disease (PD) is a major age-related neurodegenerative disorder characterized by loss of dopaminergic neurons in the substantia nigra par compacta (SNpc) and accumulation of aggregated alpha-synuclein in brain areas. Rotenone is a neurotoxin that is routinely used to model PD, thus to help us understand the mechanisms of neural death. Flavin-containing monooxygenase (FMO), usually known as an important hepatic microsomal enzyme like cytochrome P450, was found to play a role in the brain recent years. In our study we aimed to find out the role that FMO might play in PD pathology. Thus we successfully generated rotenone model in primary midbrain dopaminergic neurons and identified the apoptosis of neurons caused by rotenone. We found that in rotenone model of Parkinsonism, the expression/protein level of parkin and FMO1 were decreased accompanied by the activation of caspase 3. Blocking FMO activity by FMO inhibitor methimazole directly caused activation of caspase 3, meanwhile parkin protein level was decreased. Our data indicated that FMO, whose dysfunction could be a reason for the apoptosis of dopaminergic neurons in rotenone model, might be a new clue of pathological proteins in rotenone model of parkinsonism. Meanwhile, it was suggested that parkin function was compromised in neuro-pathological states, thereby further adding to the cellular survival stress. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  12. 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.

  13. 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.

  14. 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

  15. Production of four Neurospora crassa lytic polysaccharide monooxygenases in Pichia pastoris monitored by a fluorimetric assay.

    PubMed

    Kittl, Roman; Kracher, Daniel; Burgstaller, Daniel; Haltrich, Dietmar; Ludwig, Roland

    2012-10-26

    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. 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. 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.

  16. 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.

  17. Crystal Structures of Cyclohexanone Monooxygenase Reveal Complex Domain Movements and a Sliding Cofactor

    SciTech Connect

    Mirza, I.; Yachnin, B; Wang, S; Grosse, S; Bergeron, H; Imura, A; Iwaki, H; Hasegawa, Y; Lau, P; Berghuis, A

    2009-01-01

    Cyclohexanone monooxygenase (CHMO) is a flavoprotein that carries out the archetypical Baeyer-Villiger oxidation of a variety of cyclic ketones into lactones. Using NADPH and O{sub 2} as cosubstrates, the enzyme inserts one atom of oxygen into the substrate in a complex catalytic mechanism that involves the formation of a flavin-peroxide and Criegee intermediate. We present here the atomic structures of CHMO from an environmental Rhodococcus strain bound with FAD and NADP+ in two distinct states, to resolutions of 2.3 and 2.2 {angstrom}. The two conformations reveal domain shifts around multiple linkers and loop movements, involving conserved arginine 329 and tryptophan 492, which effect a translation of the nicotinamide resulting in a sliding cofactor. Consequently, the cofactor is ideally situated and subsequently repositioned during the catalytic cycle to first reduce the flavin and later stabilize formation of the Criegee intermediate. Concurrent movements of a loop adjacent to the active site demonstrate how this protein can effect large changes in the size and shape of the substrate binding pocket to accommodate a diverse range of substrates. Finally, the previously identified BVMO signature sequence is highlighted for its role in coordinating domain movements. Taken together, these structures provide mechanistic insights into CHMO-catalyzed Baeyer-Villiger oxidation.

  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.

  19. 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.

  20. Emerging Roles of Flavin Monooxygenase 3 (FMO3) in Cholesterol Metabolism and Atherosclerosis

    PubMed Central

    Schugar, Rebecca C.; Brown, J. Mark

    2015-01-01

    Purpose of Review Atherosclerosis and associated cardiovascular disease (CVD) still remain the largest cause of mortality worldwide. Several recent studies have discovered that metabolism of common nutrients by gut microbes can produce a proatherogenic metabolite called trimethylamine-N-oxide (TMAO). The goal of this review is to discuss emerging evidence that the hepatic enzyme that generates TMAO, flavin monooxygenase 3 (FMO3), plays a regulatory role in maintaining whole body cholesterol balance and atherosclerosis development. Recent Findings Several independent studies have recently uncovered a link between either FMO3 itself or its enzymatic product TMAO with atherosclerosis and hepatic insulin resistance. These recent studies show that inhibition of FMO3 stimulates macrophage reverse cholesterol transport (RCT) and protects against atherosclerosis in mice. Summary A growing body of work demonstrates that nutrients present in high fat foods (phosphatidylcholine, choline, and L-carnitine) can be metabolized by the gut microbial enzymes to generate trimethylamine (TMA), which is then further metabolized by the host enzyme FMO3 to produce proatherogenic TMAO. Here we discuss emerging evidence that the TMAO producing enzyme FMO3 is centrally involved in the pathogenesis of atherosclerosis by regulating cholesterol metabolism and insulin resistance, and how these new insights provide exciting new avenues for CVD therapies. PMID:26218418

  1. 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

  2. Characterization of maize cytochrome P450 monooxygenases induced in response to safeners and bacterial pathogens.

    PubMed

    Persans, M W; Wang, J; Schuler, M A

    2001-02-01

    Plants use a diverse array of cytochrome P450 monooxygenases in their biosynthetic and detoxification pathways. To determine the extent to which various maize P450s are induced in response to chemical inducers, such as naphthalic anhydride (NA), triasulfuron (T), phenobarbital, and bacterial pathogens (Erwinia stuartii, Acidovorax avenae), we have analyzed the response patterns of seven P450 transcripts after treatment of seedlings with these inducers. Each of these P450 transcripts has distinct developmental, tissue-specific, and chemical cues regulating their expression even when they encode P450s within the same biosynthetic pathway. Most notably, the CYP71C1 and CYP71C3 transcripts, encoding P450s in the DIMBOA biosynthetic pathway, are induced to the same level in response to wounding and NA treatment of younger seedlings and differentially in response to NA/T treatment of younger seedlings and NA and NA/T treatment of older seedlings. NA and T induce expression of both CYP92A1 and CYP72A5 transcripts in older seedling shoots, whereas phenobarbital induces CYP92A1 expression in older seedling shoots and highly induces CYP72A5 expression in young and older seedling roots. Expressed sequence tag (EST) 6c06b11 transcripts, encoding an undefined P450 activity, are highly induced in seedling shoots infected with bacterial pathogens.

  3. Characterization of Maize Cytochrome P450 Monooxygenases Induced in Response to Safeners and Bacterial Pathogens1

    PubMed Central

    Persans, Michael W.; Wang, Jian; Schuler, Mary A.

    2001-01-01

    Plants use a diverse array of cytochrome P450 monooxygenases in their biosynthetic and detoxification pathways. To determine the extent to which various maize P450s are induced in response to chemical inducers, such as naphthalic anhydride (NA), triasulfuron (T), phenobarbital, and bacterial pathogens (Erwinia stuartii, Acidovorax avenae), we have analyzed the response patterns of seven P450 transcripts after treatment of seedlings with these inducers. Each of these P450 transcripts has distinct developmental, tissue-specific, and chemical cues regulating their expression even when they encode P450s within the same biosynthetic pathway. Most notably, the CYP71C1 and CYP71C3 transcripts, encoding P450s in the DIMBOA biosynthetic pathway, are induced to the same level in response to wounding and NA treatment of younger seedlings and differentially in response to NA/T treatment of younger seedlings and NA and NA/T treatment of older seedlings. NA and T induce expression of both CYP92A1 and CYP72A5 transcripts in older seedling shoots, whereas phenobarbital induces CYP92A1 expression in older seedling shoots and highly induces CYP72A5 expression in young and older seedling roots. Expressed sequence tag (EST) 6c06b11 transcripts, encoding an undefined P450 activity, are highly induced in seedling shoots infected with bacterial pathogens. PMID:11161067

  4. 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

  5. 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

  6. Soluble methane monooxygenase component B gene probe for identification of methanotrophs that rapidly degrade trichloroethylene.

    PubMed Central

    Tsien, H C; Hanson, R S

    1992-01-01

    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. Fragments produced by digestion of DNA with rare cutting restriction endonucleases were separated by pulsed-field gel electrophoresis and transferred to Zeta-Probe membrane (Bio-Rad) for Southern blot analysis. Samples were also analyzed for the presence of soluble MMO by Western blot analysis and the ability to degrade TCE. The physiological groups of methanotrophs in each sample were determined by hybridizing cells with fluorescence-labelled signature probes. 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 Bath also produces a soluble MMO. Images PMID:1349468

  7. Prediction and analysis of the modular structure of cytochrome P450 monooxygenases

    PubMed Central

    2010-01-01

    Background Cytochrome P450 monooxygenases (CYPs) form a vast and diverse family of highly variable sequences. They catalyze a wide variety of oxidative reactions and are therefore of great relevance in drug development and biotechnological applications. Despite their differences in sequence and substrate specificity, the structures of CYPs are highly similar. Although being in research focus for years, factors mediating selectivity and activity remain vague. Description This systematic comparison of CYPs based on the Cytochrome P450 Engineering Database (CYPED) involved sequence and structure analysis of more than 8000 sequences. 31 structures have been applied to generate a reliable structure-based HMM profile in order to predict structurally conserved regions. Therefore, it was possible to automatically transfer these modules on CYP sequences without any secondary structure information, to analyze substrate interacting residues and to compare interaction sites with redox partners. Conclusions Functionally relevant structural sites of CYPs were predicted. Regions involved in substrate binding were analyzed in all sequences among the CYPED. For all CYPs that require a reductase, two reductase interaction sites were identified and classified according to their length. The newly gained insights promise an improvement of engineered enzyme properties for potential biotechnological application. The annotated sequences are accessible on the current version of the CYPED. The prediction tool can be applied to any CYP sequence via the web interface at http://www.cyped.uni-stuttgart.de/cgi-bin/strpred/dosecpred.pl. PMID:20950472

  8. Catalase improves saccharification of lignocellulose by reducing lytic polysaccharide monooxygenase-associated enzyme inactivation.

    PubMed

    Scott, Brian R; Huang, Hong Zhi; Frickman, Jesper; Halvorsen, Rune; Johansen, Katja S

    2016-03-01

    Efficient enzymatic saccharification of plant cell wall material is key to industrial processing of agricultural and forestry waste such as straw and wood chips into fuels and chemicals. Saccharification assays were performed on steam-pretreated wheat straw under ambient and O2-deprived environments and in the absence and presence of a lytic polysaccharide monooxygenase (LPMO) and catalase. A kinetic model was used to calculate catalytic rate and first-order inactivation rate constants of the cellulases from reaction progress curves. The addition of a LPMO significantly (P < 0.01, Student's T test) enhanced the rate of glucose release from 2.8 to 6.9 h(-1) under ambient O2 conditions. However, this also significantly (P < 0.01, Student's T test) increased the rate of inactivation of the enzyme mixture, thereby reducing the performance half-life from 65 to 35 h. Decreasing O2 levels or, strikingly, the addition of catalase significantly reduced (P < 0.01, Student's T test) enzyme inactivation and, as a consequence, higher efficiency of the cellulolytic enzyme cocktail was achieved. Oxidative inactivation of commercial cellulase mixtures is a significant factor influencing the overall saccharification efficiency and the addition of catalase can be used to protect these mixtures from inactivation.

  9. Signaling Mediated by the Cytosolic Domain of Peptidylglycine α-Amidating Monooxygenase

    PubMed Central

    Alam, M. Rashidul; Steveson, Tami C.; Johnson, Richard C.; Bäck, Nils; Abraham, Benjamin; Mains, Richard E.; Eipper, Betty A.

    2001-01-01

    The luminal domains of membrane peptidylglycine α-amidating monooxygenase (PAM) are essential for peptide α-amidation, and the cytosolic domain (CD) is essential for trafficking. Overexpression of membrane PAM in corticotrope tumor cells reorganizes the actin cytoskeleton, shifts endogenous adrenocorticotropic hormone (ACTH) from mature granules localized at the tips of processes to the TGN region, and blocks regulated secretion. PAM-CD interactor proteins include a protein kinase that phosphorylates PAM (P-CIP2) and Kalirin, a Rho family GDP/GTP exchange factor. We engineered a PAM protein unable to interact with either P-CIP2 or Kalirin (PAM-1/K919R), along with PAM proteins able to interact with Kalirin but not with P-CIP2. AtT-20 cells expressing PAM-1/K919R produce fully active membrane enzyme but still exhibit regulated secretion, with ACTH-containing granules localized to process tips. Immunoelectron microscopy demonstrates accumulation of PAM and ACTH in tubular structures at the trans side of the Golgi in AtT-20 cells expressing PAM-1 but not in AtT-20 cells expressing PAM-1/K919R. The ability of PAM to interact with P-CIP2 is critical to its ability to block exit from the Golgi and affect regulated secretion. Consistent with this, mutation of its P-CIP2 phosphorylation site alters the ability of PAM to affect regulated secretion. PMID:11251076

  10. 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

  11. 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.

  12. 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. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Crystal structures of cyclohexanone monooxygenase reveal complex domain movements and a sliding cofactor.

    PubMed

    Mirza, I Ahmad; Yachnin, Brahm J; Wang, Shaozhao; Grosse, Stephan; Bergeron, Hélène; Imura, Akihiro; Iwaki, Hiroaki; Hasegawa, Yoshie; Lau, Peter C K; Berghuis, Albert M

    2009-07-01

    Cyclohexanone monooxygenase (CHMO) is a flavoprotein that carries out the archetypical Baeyer-Villiger oxidation of a variety of cyclic ketones into lactones. Using NADPH and O(2) as cosubstrates, the enzyme inserts one atom of oxygen into the substrate in a complex catalytic mechanism that involves the formation of a flavin-peroxide and Criegee intermediate. We present here the atomic structures of CHMO from an environmental Rhodococcus strain bound with FAD and NADP(+) in two distinct states, to resolutions of 2.3 and 2.2 A. The two conformations reveal domain shifts around multiple linkers and loop movements, involving conserved arginine 329 and tryptophan 492, which effect a translation of the nicotinamide resulting in a sliding cofactor. Consequently, the cofactor is ideally situated and subsequently repositioned during the catalytic cycle to first reduce the flavin and later stabilize formation of the Criegee intermediate. Concurrent movements of a loop adjacent to the active site demonstrate how this protein can effect large changes in the size and shape of the substrate binding pocket to accommodate a diverse range of substrates. Finally, the previously identified BVMO signature sequence is highlighted for its role in coordinating domain movements. Taken together, these structures provide mechanistic insights into CHMO-catalyzed Baeyer-Villiger oxidation.

  14. Switching the Regioselectivity of a Cyclohexanone Monooxygenase toward (+)-trans-Dihydrocarvone by Rational Protein Design.

    PubMed

    Balke, Kathleen; Schmidt, Sandy; Genz, Maika; Bornscheuer, Uwe T

    2016-01-15

    The regioselectivity of the Baeyer-Villiger monooxygenase-catalyzed oxidation is governed mostly by electronic effects leading to the migration of the higher substituted residue. However, in some cases, substrate binding occurs in a way that the less substituted residue lies in an antiperiplanar orientation to the peroxy bond in the Criegee intermediate yielding in the formation of the "abnormal" lactone product. We are the first to demonstrate a complete switch in the regioselectivity of the BVMO from Arthrobacter sp. (CHMOArthro) as exemplified for (+)-trans-dihydrocarvone by redesigning the active site of the enzyme. In the designed triple mutant, the substrate binds in an inverted orientation leading to a ratio of 99:1 in favor of the normal lactone instead of exclusive formation of the abnormal lactone in case of the wild type enzyme. In order to validate our computational study, the beneficial mutations were successfully transferred to the CHMO from Acinetobacter sp. (CHMOAcineto), again yielding in a complete switch of regioselectivity.

  15. Absence of kynurenine 3-monooxygenase reduces mortality of acute viral myocarditis in mice.

    PubMed

    Kubo, Hisako; Hoshi, Masato; Mouri, Akihiro; Tashita, Chieko; Yamamoto, Yasuko; Nabeshima, Toshitaka; Saito, Kuniaki

    2017-01-01

    Infection of the encephalomyocarditis virus (EMCV) in mice is an established model for viral myocarditis. Previously, we have demonstrated that indoleamine 2,3-dioxygenase (IDO), an L-tryptophan - kynurenine pathway (KP) enzyme, affects acute viral myocarditis. However, the roles of KP metabolites in EMCV infection remain unclear. Kynurenine 3-monooxygenase (KMO) is one of the key regulatory enzymes, which metabolizes kynurenine to 3-hydroxykynurenine in the KP. Therefore, we examined the role of KMO in acute viral infection by comparing between KMO(-/-) mice and KMO(+/+) mice. KMO deficiency resulted in suppressed mortality after EMCV infection. The number of infiltrating cells and F4/80(+) cells in KMO(-/-) mice was suppressed compared with those in KMO(+/+) mice. KMO(-/-) mice showed significantly increased levels of serum KP metabolites, and induction of KMO expression upon EMCV infection was involved in its effect on mortality through EMCV suppression. Furthermore, KMO(-/-) mice showed significantly suppression of CCL2, CCL3 and CCL4 on day 2 and CXCL1 on day 4 after infection. These results suggest that increased KP metabolites reduced chemokine production, resulting in suppressed mortality upon KMO knockdown in EMCV infection. KP metabolites may thus provide an effective strategy for treating acute viral myocarditis. Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  16. Microsomal monooxygenase as a multienzyme system: the role of P450-P450 interactions

    PubMed Central

    Davydov, Dmitri R.

    2011-01-01

    Introduction There is increasing evidence of physical interactions (association) among cytochromes P450 in the membranes of the endoplasmic reticulum. Functional consequences of these interactions are often underestimated. Areas covered This article provides a comprehensive overview of available experimental material regarding P450-P450 interactions. Special emphasis is given to the interactions between different P450 species and to the functional consequences of homo- and heterooligomerization. Expert opinion Recent advances provide conclusive evidence for a substantial degree of P450 oligomerization in membranes. Interactions between different P450 species resulting in the formation of mixed oligomers with altered activity and substrate specificity have been demonstrated clearly. There are important indications that oligomerization of cytochromes P450 impedes electron flow to a fraction of the P450 population, which render some P450 species non-functional. Functional consequences of P450-P450 interactions make the integrated properties of the microsomal monooxygenase remarkably different from a simple summation of the properties of the individual P450 species. This complexity compromises the predictive power of the current in vitro models of drug metabolism and warrants an urgent need for development of new model systems that consider the interactions of multiple P450 species. PMID:21395496

  17. Identification of selectivity determinants in CYP monooxygenases by modelling and systematic analysis of sequence and structure.

    PubMed

    Seifert, Alexander; Pleiss, Jurgen

    2012-02-01

    Cytochrome P450 monooxygenases (CYPs) form a large, ubiquitous enzyme family and are of great interest in red and white biotechnology. To investigate the effect of protein structure on selectivity, the binding of substrate molecules near to the active site was modelled by molecular dynamics simulations. From a comprehensive and systematic comparison of more than 6300 CYP sequences and 31 structures using the Cytochrome P450 Engineering Database (CYPED), residues were identified which are predicted to point close to the heme centre and thus restrict accessibility for substrates. As a result, sequence-structure-function relationships are described that can be used to predict selectivity-determining positions from CYP sequences and structures. Based on this analysis, a minimal library consisting of bacterial CYP102A1 (P450(BM3)) and 24 variants was constructed. All variants were functionally expressed in E. coli, and the library was screened with four terpene substrates. Only 3 variants showed no activity towards all 4 terpenes, while 11 variants demonstrated either a strong shift or improved regio- or stereoselectivity during oxidation of at least one substrate as compared to CYP102A1 wild type. The minimal library also contains variants that show interesting side products which are not generated by the wild type enzyme. By two additional rounds of molecular modelling, diversification, and screening, the selectivity of one of these variants for a new product was optimised with a minimal screening effort. We propose this as a generic approach for other CYP substrates.

  18. 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

  19. 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.

  20. 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.

  1. 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

  2. Monooxygenase-mediated 1,2-dichloroethane degradation by Pseudomonas sp. strain DCA1

    SciTech Connect

    Hage, J.C.; Hartmans, S.

    1999-06-01

    A bacterial strain, designated Pseudomonas sp. strain DCA1, was isolated from a 1,2-dichloroethane (DCA)-degrading biofilm. Strain DCA1 utilizes DCA as the sole carbon and energy source and does not require additional organic nutrients, such as vitamins, for optimal growth. The affinity of strain DCA1 for DCA is very high, with a K{sub m} value below the detection limit of 0.5 {micro}M. Instead of a hydrolytic dehalogenation, as in other DCA utilizers, the first step in DCA degradation in strain DCA1 is an oxidation reaction. Oxygen and NAD(P)H are required for this initial step. Propene was converted to 1,2-epoxypropane by DCA-grown cells and competitively inhibited DCA degradation. The authors concluded that a monooxygenase is responsible for the first step in DCA degradation in strain DCA1. Oxidation of DCA probably results in the formation of the unstable intermediate 1,2-dichloroethanol, which spontaneously releases chloride, yielding chloroacetaldehyde. The DCA degradation pathway is strain DCA1 proceeds from chloroacetaldehyde via chloroacetic acid and presumably glycolic acid, which is similar to degradation routes observed in other DCA-utilizing bacteria.

  3. Structural and Functional Characterization of a Lytic Polysaccharide Monooxygenase with Broad Substrate Specificity*

    PubMed Central

    Borisova, Anna S.; Isaksen, Trine; Dimarogona, Maria; Kognole, Abhishek A.; Mathiesen, Geir; Várnai, Anikó; Røhr, Åsmund K.; Payne, Christina M.; Sørlie, Morten; Sandgren, Mats; Eijsink, Vincent G. H.

    2015-01-01

    The recently discovered lytic polysaccharide monooxygenases (LPMOs) carry out oxidative cleavage of polysaccharides and are of major importance for efficient processing of biomass. NcLPMO9C from Neurospora crassa acts both on cellulose and on non-cellulose β-glucans, including cellodextrins and xyloglucan. The crystal structure of the catalytic domain of NcLPMO9C revealed an extended, highly polar substrate-binding surface well suited to interact with a variety of sugar substrates. The ability of NcLPMO9C to act on soluble substrates was exploited to study enzyme-substrate interactions. EPR studies demonstrated that the Cu2+ center environment is altered upon substrate binding, whereas isothermal titration calorimetry studies revealed binding affinities in the low micromolar range for polymeric substrates that are due in part to the presence of a carbohydrate-binding module (CBM1). Importantly, the novel structure of NcLPMO9C enabled a comparative study, revealing that the oxidative regioselectivity of LPMO9s (C1, C4, or both) correlates with distinct structural features of the copper coordination sphere. In strictly C1-oxidizing LPMO9s, access to the solvent-facing axial coordination position is restricted by a conserved tyrosine residue, whereas access to this same position seems unrestricted in C4-oxidizing LPMO9s. LPMO9s known to produce a mixture of C1- and C4-oxidized products show an intermediate situation. PMID:26178376

  4. Oxygenation of Organoboronic Acids by a Nonheme Iron(II) Complex: Mimicking Boronic Acid Monooxygenase Activity.

    PubMed

    Chatterjee, Sayanti; Paine, Tapan Kanti

    2015-10-19

    Phenolic compounds are important intermediates in the bacterial biodegradation of aromatic compounds in the soil. An Arthrobacter sp. strain has been shown to exhibit boronic acid monooxygenase activity through the conversion of different substituted phenylboronic acids to the corresponding phenols using dioxygen. While a number of methods have been reported to cleave the C-B bonds of organoboronic acids, there is no report on biomimetic iron complex exhibiting this activity using dioxygen as the oxidant. In that direction, we have investigated the reactivity of a nucleophilic iron-oxygen oxidant, generated upon oxidative decarboxylation of an iron(II)-benzilate complex [(Tp(Ph2))Fe(II)(benzilate)] (Tp(Ph2) = hydrotris(3,5-diphenyl-pyrazol-1-yl)borate), toward organoboronic acids. The oxidant converts different aryl/alkylboronic acids to the corresponding oxygenated products with the incorporation of one oxygen atom from dioxygen. This method represents an efficient protocol for the oxygenation of boronic acids with dioxygen as the terminal oxidant.

  5. 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

  6. Mechanisms of reduced flavin transfer in the two-component flavin-dependent monooxygenases.

    PubMed

    Sucharitakul, Jeerus; Tinikul, Ruchanok; Chaiyen, Pimchai

    2014-08-01

    Two-component flavin-dependent enzymes are abundant in nature and are involved in a wide variety of biological reactions. These enzymes consist of a reductase which generates a reduced flavin and a monooxygenase that utilizes the reduced flavin as a substrate for monooxygenation. As reduced flavin is unstable and can be oxidized by oxygen, these enzymes must have a means to efficiently coordinate the transfer of the reduced flavin such that auto-oxidation can be minimized. Various types of experiments and methodologies have been used to probe the mode of reduced flavin transfer. Results from many systems have indicated that the transfer can be achieved by free diffusion and that the presence of one component has no influence on the kinetics of the other component. Contradicting results indicating that the transfer of the reduced flavin may be achieved via protein-protein mediation also exist. Regardless of the mode of reduced flavin transfer, these enzymes have a means to control their overall kinetics such that the reaction rate is slow when the demand for oxygenation is not high.

  7. Kinetic and spectroscopic characterization of the putative monooxygenase domain of human MICAL-1.

    PubMed

    Zucchini, Daniela; Caprini, Gianluca; Pasterkamp, R Jeroen; Tedeschi, Gabriella; Vanoni, Maria A

    2011-11-01

    MICALs form a conserved multidomain protein family essential for cytoskeletal rearrangements. To complement structural information available, we produced the FAD-containing monooxygenase-like domain of human MICAL-1 (MICAL-MO) in forms differing for the presence and location of a His-tag, which only influences the protein yields. The K(m) for NADPH of the NADPH oxidase reaction is sensitive to ionic strength and type of ions. The apparent k(cat) (pH 7) is limited by enzyme reduction by NADPH, which occurs without detectable intermediates, as established by anaerobic rapid reaction experiments. The sensitivity to ionic strength and type of ions and the pH dependence of the steady-state kinetic parameters extend MICAL-MO similarity with enzymes of the p-hydroxybenzoate hydroxylase class at the functional level. The reaction is also sensitive to solvent viscosity, providing a tool to monitor the conformational changes predicted to occur during turnover. Finally, it was confirmed that MICAL-MO promotes actin depolymerization, and it was shown that F-actin, but not G-actin, stimulates NADPH oxidation by increasing k(cat) and k(cat)/K(NADPH) (≈5 and ≈200-fold, respectively) with an apparent K(m) for actin of 4.7μM, under conditions that stabilize F-actin. The time-course of NADPH oxidation shows substrate recycling, indicating the possible reversibility of MICAL effect. Copyright © 2011 Elsevier Inc. All rights reserved.

  8. Subclade of Flavin-Monooxygenases Involved in Aliphatic Glucosinolate Biosynthesis1[W

    PubMed Central

    Li, Jing; Hansen, Bjarne Gram; Ober, James A.; Kliebenstein, Daniel J.; Halkier, Barbara Ann

    2008-01-01

    Glucosinolates (GSLs) are amino acid-derived secondary metabolites with diverse biological activities dependent on chemical modifications of the side chain. We previously identified the flavin-monooxygenase FMOGS-OX1 as an enzyme in the biosynthesis of aliphatic GSLs in Arabidopsis (Arabidopsis thaliana) that catalyzes the S-oxygenation of methylthioalkyl to methylsulfinylalkyl GSLs. Here, we report the fine mapping of a quantitative trait locus for the S-oxygenating activity in Arabidopsis. In this region, there are three FMOs that, together with FMOGS-OX1 and a fifth FMO, form what appears to be a crucifer-specific subclade. We report the identification of these four uncharacterized FMOs, designated FMOGS-OX2 to FMOGS-OX5. Biochemical characterization of the recombinant protein combined with the analysis of GSL content in knockout mutants and overexpression lines show that FMOGS-OX2, FMOGS-OX3, and FMOGS-OX4 have broad substrate specificity and catalyze the conversion from methylthioalkyl GSL to the corresponding methylsulfinylalkyl GSL independent of chain length. In contrast, FMOGS-OX5 shows substrate specificity toward the long-chain 8-methylthiooctyl GSL. Identification of the FMOGS-OX subclade will generate better understanding of the evolution of biosynthetic activities and specificities in secondary metabolism and provides an important tool for breeding plants with improved cancer prevention characteristics as provided by the methylsulfinylalkyl GSL. PMID:18799661

  9. Electron paramagnetic studies of the copper and iron containing soluble ammonia monooxygenase from Nitrosomonas europaea.

    PubMed

    Gilch, Stefan; Meyer, Ortwin; Schmidt, Ingo

    2010-08-01

    Soluble ammonia monooxygenase (AMO) from Nitrosomonas europaea was purified to homogeneity and metals in the active sites of the enzyme (Cu, Fe) were analyzed by electron paramagnetic resonance (EPR) spectroscopy. EPR spectra were obtained for a type 2 Cu(II) site with g(parallel) = 2.24, A(parallel) = 18.4 mT and g(perpendicular) = 2.057 as well as for heme and non heme iron present in purified soluble AMO from N. europaea. A second type 2 Cu(II) EPR signal with g(parallel) = 2.29, A(parallel) = 16.1 mT and g(perpendicular) = 2.03 appeared in the spectrum of the ferricyanide oxidized enzyme and was attributed to oxidation of cuprous sites. Comparison of EPR-detectable Cu(2+) with total copper determined by inductively coupled plasma-mass spectrometry (ICP-MS) suggests that there are six paramagnetic Cu(2+) and three diamagnetic Cu(1+) per heterotrimeric soluble AMO (two paramagnetic and one diamagnetic Cu per alphabetagamma-protomer). A trigonal EPR signal at g = 6.01, caused by a high-spin iron, indicative for cytochrome bound iron, and a rhombic signal at g = 4.31, characteristic of specifically bound Fe(3+) was detectable. The binding of nitric oxide in the presence of reductant resulted in a ferrous S = 3/2 signal, characteristic of a ferrous nitrosyl complex. Inactivation of soluble AMO with acetylene did neither diminish the ferrous signal nor the intensity of the Cu(2+)-EPR signal.

  10. A small lytic polysaccharide monooxygenase from Streptomyces griseus targeting α- and β-chitin.

    PubMed

    Nakagawa, Yuko S; Kudo, Madoka; Loose, Jennifer S M; Ishikawa, Takahiro; Totani, Kazuhide; Eijsink, Vincent G H; Vaaje-Kolstad, Gustav

    2015-03-01

    The lytic polysaccharide monooxygenases (LPMOs) have received considerable attention subsequent to their discovery because of their ability to boost the enzymatic conversion of recalcitrant polysaccharides. In the present study, we describe the enzymatic properties of SgLPMO10F, a small (15 kDa) auxilliary activity (AA) family 10 LPMO from Streptomyces griseus belonging to a clade of the phylogenetic tree without any characterized representative. The protein was expressed using a Brevibacillus-based expression system that had not been used previously for LPMO expression and that also ensures correct processing of the N-terminus crucial for LPMO activity. The enzyme was active towards both α- and β-chitin and showed stronger binding and a greater release of soluble oxidized products for the latter allomorph. In chitinase synergy assays, however, SgLPMO10F worked slightly better for α-chitin, increasing chitin solubilization yields by up to 30-fold and 20-fold for α- and β-chitin, respectively. Synergy experiments with various chitinases showed that the addition of SgLPMO10F leads to a substantial increase in the (GlcNAc)2 :GlcNAc product ratio, in reactions with α-chitin only. This underpins the structural differences between the substrates and also shows that, on α-chitin, SgLPMO10F affects the binding mode and/or degree of processivity of the chitinases tested. Variation in the only exposed aromatic residue in the substrate-binding surface of LPMO10s has previously been linked to preferential binding for α-chitin (exposed Trp) or β-chitin (exposed Tyr). Mutation of this residue, Tyr56, in SgLPMO10F to Trp had no detectable effect on substrate-binding preferences but, in synergy experiments, the mutant appeared to be more efficient on α-chitin.

  11. Activity-Based Protein Profiling of Ammonia Monooxygenase in Nitrosomonas europaea

    PubMed Central

    Bennett, Kristen; Sadler, Natalie C.; Wright, Aaron T.; Yeager, Chris

    2016-01-01

    Nitrosomonas europaea is an aerobic nitrifying bacterium that oxidizes ammonia (NH3) to nitrite (NO2−) through the sequential activities of ammonia monooxygenase (AMO) and hydroxylamine dehydrogenase (HAO). Many alkynes are mechanism-based inactivators of AMO, and here we describe an activity-based protein profiling method for this enzyme using 1,7-octadiyne (17OD) as a probe. Inactivation of NH4+-dependent O2 uptake by N. europaea by 17OD was time- and concentration-dependent. The effects of 17OD were specific for ammonia-oxidizing activity, and de novo protein synthesis was required to reestablish this activity after cells were exposed to 17OD. Cells were reacted with Alexa Fluor 647 azide using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) (click) reaction, solubilized, and analyzed by SDS-PAGE and infrared (IR) scanning. A fluorescent 28-kDa polypeptide was observed for cells previously exposed to 17OD but not for cells treated with either allylthiourea or acetylene prior to exposure to 17OD or for cells not previously exposed to 17OD. The fluorescent polypeptide was membrane associated and aggregated when heated with β-mercaptoethanol and SDS. The fluorescent polypeptide was also detected in cells pretreated with other diynes, but not in cells pretreated with structural homologs containing a single ethynyl functional group. The membrane fraction from 17OD-treated cells was conjugated with biotin-azide and solubilized in SDS. Streptavidin affinity-purified polypeptides were on-bead trypsin-digested, and amino acid sequences of the peptide fragments were determined by liquid chromatography-mass spectrometry (LC-MS) analysis. Peptide fragments from AmoA were the predominant peptides detected in 17OD-treated samples. In-gel digestion and matrix-assisted laser desorption ionization–tandem time of flight (MALDI-TOF/TOF) analyses also confirmed that the fluorescent 28-kDa polypeptide was AmoA. PMID:26826234

  12. Analyzing Activities of Lytic Polysaccharide Monooxygenases by Liquid Chromatography and Mass Spectrometry.

    PubMed

    Westereng, Bjørge; Arntzen, Magnus Ø; Agger, Jane Wittrup; Vaaje-Kolstad, Gustav; Eijsink, Vincent G H

    2017-01-01

    Lytic polysaccharide monooxygenases perform oxidative cleavage of glycosidic bonds in various polysaccharides. The majority of LMPOs studied so far possess activity on either cellulose or chitin and analysis of these activities is therefore the main focus of this review. Notably, however, the number of LPMOs that are active on other polysaccharides is increasing. The products generated by LPMOs from cellulose are either oxidized in the downstream end (at C1) or upstream end (at C4), or at both ends. These modifications only result in small structural changes, which makes both chromatographic separation and product identification by mass spectrometry challenging. The changes in physicochemical properties that are associated with oxidation need to be considered when choosing analytical approaches. C1 oxidation leads to a sugar that is no longer reducing but instead has an acidic functionality, whereas C4 oxidation leads to products that are inherently labile at high and low pH and that exist in a keto-gemdiol equilibrium that is strongly shifted toward the gemdiol in aqueous solutions. Partial degradation of C4-oxidized products leads to the formation of native products, which could explain why some authors claim to have observed glycoside hydrolase activity for LPMOs. Notably, apparent glycoside hydrolase activity may also be due to small amounts of contaminating glycoside hydrolases since these normally have much higher catalytic rates than LPMOs. The low catalytic turnover rates of LPMOs necessitate the use of sensitive product detection methods, which limits the analytical possibilities considerably. Modern liquid chromatography and mass spectrometry have become essential tools for evaluating LPMO activity, and this chapter provides an overview of available methods together with a few novel tools. The methods described constitute a suite of techniques for analyzing oxidized carbohydrate products, which can be applied to LPMOs as well as other carbohydrate

  13. Flavin-containing monooxygenase S-oxygenation of a series of thioureas and thiones

    SciTech Connect

    Henderson, Marilyn C.; Siddens, Lisbeth K.; Krueger, Sharon K.; Stevens, J. Fred; Kedzie, Karen; Fang, Wenkui K.; Heidelbaugh, Todd; Nguyen, Phong; Chow, Ken; Garst, Michael; Gil, Daniel; Williams, David E.

    2014-07-15

    Mammalian flavin-containing monooxygenase (FMO) is active towards many drugs with a heteroatom having the properties of a soft nucleophile. Thiocarbamides and thiones are S-oxygenated to the sulfenic acid which can either react with glutathione and initiate a redox-cycle or be oxygenated a second time to the unstable sulfinic acid. In this study, we utilized LC–MS/MS to demonstrate that the oxygenation by hFMO of the thioureas under test terminated at the sulfenic acid. With thiones, hFMO catalyzed the second reaction and the sulfinic acid rapidly lost sulfite to form the corresponding imidazole. Thioureas are often pulmonary toxicants in mammals and, as previously reported by our laboratory, are excellent substrates for hFMO2. This isoform is expressed at high levels in the lung of most mammals, including non-human primates. Genotyping to date indicates that individuals of African (up to 49%) or Hispanic (2–7%) ancestry have at least one allele for functional hFMO2 in lung, but not Caucasians nor Asians. In this study the major metabolite formed by hFMO2 with thioureas from Allergan, Inc. was the sulfenic acid that reacted with glutathione. The majority of thiones were poor substrates for hFMO3, the major form in adult human liver. However, hFMO1, the major isoform expressed in infant and neonatal liver and adult kidney and intestine, readily S-oxygenated thiones under test, with K{sub m}s ranging from 7 to 160 μM and turnover numbers of 30–40 min{sup −1}. The product formed was identified by LC–MS/MS as the imidazole. The activities of the mouse and human FMO1 and FMO3 orthologs were in good agreement with the exception of some thiones for which activity was much greater with hFMO1 than mFMO1.

  14. Plant activation of aromatic amines mediated by cytochromes P450 and flavin-containing monooxygenases.

    PubMed

    Chiapella, C; Radovan, R D; Moreno, J A; Casares, L; Barbé, J; Llagostera, M

    2000-10-31

    To know the mechanisms involved in the activation of promutagenic aromatic amines mediated by plants, we used Persea americana S117 system (S117) for the activation of 2-aminofluorene (2-AF) and m-phenylenediamine (m-PDA) in Ames assays. In these assays, the effect of the diphenylene iodonium (DPI), an inhibitor of flavin-containing monooxygenases (FMOs), of the 1-aminobenzotriazole (1-ABT), an inhibitor of cytochromes P450 (cyt-P450s) and of the methimazole, a high-affinity substrate for FMOs, was studied. The efficacy of both inhibitors and of the methimazole was verified to find that they did partially inhibit the mutagenesis of both aromatic amines, activated with rat liver S9. Similarly, both inhibitors and methimazole did produce a significant decrease in 2-AF and m-PDA mutagenesis, when the activation system was S117, indicating that, similar to what occurs in mammalian systems, plant FMOs and cyt-P450s can metabolize aromatic amines to mutagenic product(s). However, the affinity of both FMOs and cyt-P450s of plant for 2-AF and m-PDA was different. Data obtained indicate that the activities of plant FMOs must be the main enzymatic system of m-PDA activation while, in 2-AF activation, plant cyt-P450s have the most relevant activities. In addition, peroxidases of the S117 system must contribute to 2-AF activation and some isoforms of FMOs and/or cyt-P450s of the S117 system, uninhibited by the inhibitors used, must be the responsible for a partial activation of m-PDA.

  15. The Catalytic Copper of Peptidylglycine alpha-Hydroxylating Monooxygenase also Plays a Critical Structural Role

    SciTech Connect

    Siebert,X.; Eipper, B.; Mains, R.; Prigge, S.; Blackburn, N.; Amzel, L.

    2005-01-01

    Many bioactive peptides require amidation of their carboxy terminus to exhibit full biological activity. Peptidylglycine alpha-Hydroxylating Monooxygenase (PHM; EC 1.14.17.3), the enzyme that catalyzes the first of the two steps of this reaction, is composed of two domains, each of which binds one copper atom (CuH and CuM). The CuM site includes Met314 and two His residues as ligands. Mutation of Met314 to Ile inactivates PHM, but has only a minimal effect on the EXAFS spectrum of the oxidized enzyme, implying that it contributes only marginally to stabilization of the CuM site. To characterize the role of Met314 as a CuM ligand, we determined the structure of the M314I-PHM mutant. Since the mutant protein failed to crystallize in the conditions of the original wild-type protein, this structure determination required finding a new crystal form. The M314I-PHM mutant structure confirms that the mutation does not abolish CuM binding to the enzyme, but causes other structural perturbations that affect the overall stability of the enzyme and the integrity of the CuH site. To eliminate possible effects of crystal contacts, we redetermined the structure of wt-PHM in the M314I-PHM crystal form and showed that it does not differ from the structure of wt-PHM in the original crystals. M314I-PHM was also shown to be less stable than wt-PHM by Differential Scanning Calorimetry (DSC). Both structural and calorimetric studies point to a structural role for the CuM site, in addition to its established catalytic role.

  16. Molecular analysis of the pmo (particulate methane monooxygenase) operons from two type II methanotrophs.

    PubMed

    Gilbert, B; McDonald, I R; Finch, R; Stafford, G P; Nielsen, A K; Murrell, J C

    2000-03-01

    The particulate methane monooxygenase gene clusters, pmoCAB, from two representative type II methanotrophs of the alpha-Proteobacteria, Methylosinus trichosporium OB3b and Methylocystis sp. strain M, have been cloned and sequenced. Primer extension experiments revealed that the pmo cluster is probably transcribed from a single transcriptional start site located 300 bp upstream of the start of the first gene, pmoC, for Methylocystis sp. strain M. Immediately upstream of the putative start site, consensus sequences for sigma(70) promoters were identified, suggesting that these pmo genes are recognized by sigma(70) and negatively regulated under low-copper conditions. The pmo genes were cloned in several overlapping fragments, since parts of these genes appeared to be toxic to the Escherichia coli host. Methanotrophs contain two virtually identical copies of pmo genes, and it was necessary to use Southern blotting and probing with pmo gene fragments in order to differentiate between the two pmoCAB clusters in both methanotrophs. The complete DNA sequence of one copy of pmo genes from each organism is reported here. The gene sequences are 84% similar to each other and 75% similar to that of a type I methanotroph of the gamma-Proteobacteria, Methylococcus capsulatus Bath. The derived proteins PmoC and PmoA are predicted to be highly hydrophobic and consist mainly of transmembrane-spanning regions, whereas PmoB has only two putative transmembrane-spanning helices. Hybridization experiments showed that there are two copies of pmoC in both M. trichosporium OB3b and Methylocystis sp. strain M, and not three copies as found in M. capsulatus Bath.

  17. Proteomic and targeted qPCR analyses of subsurface microbial communities for presence of methane monooxygenase

    SciTech Connect

    Andrzej J. Paszczynski; Ravindra Paidisetti; Andrew K. Johnson; Ronald L. Crawford; Frederick S. Colwell; Tonia Green; Mark Delwiche; Hope Lee; Deborah Newby; Eoin L. Brodie; Mark Conrad

    2011-11-01

    The Test Area North (TAN) site at the Idaho National Laboratory near Idaho Falls, ID, USA, sits over a trichloroethylene (TCE) contaminant plume in the Snake River Plain fractured basalt aquifer. Past observations have provided evidence that TCE at TAN is being transformed by biological natural attenuation that may be primarily due to co-metabolism in aerobic portions of the plume by methanotrophs. TCE co-metabolism by methanotrophs is the result of the broad substrate specificity of microbial methane monooxygenase which permits non-specific oxidation of TCE in addition to the primary substrate, methane. Arrays of experimental approaches have been utilized to understand the biogeochemical processes driving intrinsic TCE co-metabolism at TAN. In this study, aerobic methanotrophs were enumerated by qPCR using primers targeting conserved regions of the genes pmoA and mmoX encoding subunits of the particulate MMO (pMMO) and soluble MMO (sMMO) enzymes, respectively, as well as the gene mxa encoding the downstream enzyme methanol dehydrogenase. Identification of proteins in planktonic and biofilm samples from TAN was determined using reverse phase ultraperformance liquid chromatography (UPLC) coupled with a quadrupole-time-of-flight (QToF) mass spectrometer to separate and sequence peptides from trypsin digests of the protein extracts. Detection of MMO in unenriched water samples from TAN provides direct evidence of intrinsic methane oxidation and TCE co-metabolic potential of the indigenous microbial population. Mass spectrometry is also well suited for distinguishing which form of MMO is expressed in situ either soluble or particulate. Using this method, pMMO proteins were found to be abundant in samples collected from wells within and adjacent to the TCE plume at TAN.

  18. Inactivation of the particulate methane monooxygenase (pMMO) in Methylococcus capsulatus (Bath) by acetylene.

    PubMed

    Pham, Minh D; Lin, Ya-Ping; Van Vuong, Quan; Nagababu, Penumaka; Chang, Brian T-A; Ng, Kok Yaoh; Chen, Chein-Hung; Han, Chau-Chung; Chen, Chung-Hsuan; Li, Mai Suan; Yu, Steve S-F; Chan, Sunney I

    2015-12-01

    Acetylene (HCCH) has a long history as a mechanism-based enzyme inhibitor and is considered an active-site probe of the particulate methane monooxygenase (pMMO). Here, we report how HCCH inactivates pMMO in Methylococcus capsulatus (Bath) by using high-resolution mass spectrometry and computational simulation. High-resolution MALDI-TOF MS of intact pMMO complexes has allowed us to confirm that the enzyme oxidizes HCCH to the ketene (C2H2O) intermediate, which then forms an acetylation adduct with the transmembrane PmoC subunit. LC-MS/MS analysis of the peptides derived from in-gel proteolytic digestion of the protein subunit identifies K196 of PmoC as the site of acetylation. No evidence is obtained for chemical modification of the PmoA or PmoB subunit. The inactivation of pMMO by a single adduct in the transmembrane PmoC domain is intriguing given the complexity of the structural fold of this large membrane-protein complex as well as the complicated roles played by the various metal cofactors in the enzyme catalysis. Computational studies suggest that the entry of hydrophobic substrates to, and migration of products from, the catalytic site of pMMO are controlled tightly within the transmembrane domain. Support of these conclusions is provided by parallel experiments with two related alkynes: propyne (CH3CCH) and trifluoropropyne (CF3CCH). Finally, we discuss the implication of these findings to the location of the catalytic site in pMMO.

  19. Characterization of alternate reductant binding and electron transfer in the dopamine. beta. -monooxygenase reaction

    SciTech Connect

    Stewart, L.C.; Klinman, J.P.

    1987-08-25

    The steady-state limiting kinetic parameters V/sub max/, V/K/sub DA/, and V/K/sub O/sub 2//, together with deuterium isotope effects on these parameters, have been determined for the dopamine ..beta..-monooxygenase (D..beta..M) reaction in the presence of structurally distinct reductants. The results show the one-electron reductant ferrocyanide to be nearly as kinetically competent as the presumed in vivo reductant ascrobate. Further, a reductant system of ferricyanide plus substrate dopamine yields steady-state kinetic parameters and isotope effects very similar to those measured solely in the presence of ferrocyanide, indicating a role for catecholamine in the rapid recycling of oxidized ferrocyanide. Use of substrate dopamine as the sole reductant is found to lead to a highly unusual kinetic independence of oxygen concentration, as well as significantly reduced values of V/sub max/ and V/K/sub DA/, and the authors conclude that dopamine reduces enzymic copper in a rate-limiting step that is 40-fold slower than with ascorbate. The near-identical kinetic parameters measured in the presence of either ascorbate or ferrocyanide, together with markedly reduced rates with dopamine, are interpreted in terms of a binding site for reductant that is physically distinct from the substrate binding site. This view is supported by molecular modeling, which reveals ascorbate and ferrocyanide to possess an unexpected similarity in potential sites for interaction with enzymic residues. With regard to electron flux, identical values of V/K/sub O/sub 2// have been measured with (2,2-/sup 2/H/sub 2/)dopamine as substrate both in the presence and in the absence of added ascorbate. This key result unambiguously rules out an entry of electrons to enzyme forms leading from the enzyme-dopamine complex to enzyme-bound product and, hence, reaction mechanisms involving a reductive activation of the putative Cu(II)-OOH prior to substrate hydroxylation.

  20. Ascorbate depletion as a consequence of product recycling during dopamine. beta. -monooxygenase catalyzed selenoxidation

    SciTech Connect

    May, S.W.; Herman, H.H.; Roberts, S.F.; Ciccarello, M.C.

    1987-03-24

    The competence of dopamine ..beta..-monooxygenase (DBM) to process selenide substrates was investigated, in anticipation that the expected selenoxide products would exhibit unique reactivity and redox properties. The prototypical selenide phenyl 2-aminoethyl selenide (PAESe) was synthesized and shown to be a substrate for DBM with the characteristic e/O/sub 2/ ratio of 2:1 for monooxygenation. The kinetic parameters for oxygenation of PAESe were found to be similar to those for the DBM-catalyzed sulfoxidation of the cognate sulfide phenyl 2-aminoethyl sulfide, and selenoxidation was stimulated by fumarate in a manner similar to other well-characterized DBM monooxygenation reactions. Identification of phenyl 2-aminoethyl selenoxide (PAESeO) as the enzymatic product was accomplished by the demonstration of coincident elution of authentic PAESeO with the enzymatic product in three significantly different HPLC systems. PAESeO was found to oxidize ascorbic acid with the concomitant and stoichiometric reduction of PAESeO back to the selenide, PAESe. As a consequence of this nonenzymatic reaction, ascorbate-supported DBM turnover was prematurely terminated under standard assay conditions due to depletion of reduced ascorbate. The kinetics of the redox reaction between PAESeO and ascorbate were investigated with a spectrophotometric assay of ascorbate at 300 nm, and a second-order rate constant of 3.4 M/sup -1/ s/sup -1/ was determined at pH 5.0, 25/sup 0/C. Spectrophotometric assay of cytochrome c (cyt c) reduction at 550 nm during the oxidation of ascorbate by PAESeO demonstrated that no cyt c trappable semidehydroascorbate was produced in this nonenzymatic reaction.

  1. Integration of bacterial lytic polysaccharide monooxygenases into designer cellulosomes promotes enhanced cellulose degradation

    PubMed Central

    Arfi, Yonathan; Shamshoum, Melina; Rogachev, Ilana; Peleg, Yoav; Bayer, Edward A.

    2014-01-01

    Efficient conversion of cellulose into soluble sugars is a key technological bottleneck limiting efficient production of plant-derived biofuels and chemicals. In nature, the process is achieved by the action of a wide range of cellulases and associated enzymes. In aerobic microrganisms, cellulases are secreted as free enzymes. Alternatively, in certain anaerobic microbes, cellulases are assembled into large multienzymes complexes, termed “cellulosomes,” which allow for efficient hydrolysis of cellulose. Recently, it has been shown that enzymes classified as lytic polysaccharide monooxygenases (LPMOs) were able to strongly enhance the activity of cellulases. However, LPMOs are exclusively found in aerobic organisms and, thus, cannot benefit from the advantages offered by the cellulosomal system. In this study, we designed several dockerin-fused LPMOs based on enzymes from the bacterium Thermobifida fusca. The resulting chimeras exhibited activity levels on microcrystalline cellulose similar to that of the wild-type enzymes. The dockerin moieties of the chimeras were demonstrated to be functional and to specifically bind to their corresponding cohesin partner. The chimeric LPMOs were able to self-assemble in designer cellulosomes alongside an endo- and an exo-cellulase also converted to the cellulosomal mode. The resulting complexes showed a 1.7-fold increase in the release of soluble sugars from cellulose, compared with the free enzymes, and a 2.6-fold enhancement compared with free cellulases without LPMO enhancement. These results highlight the feasibility of the conversion of LPMOs to the cellulosomal mode, and that these enzymes can benefit from the proximity effects generated by the cellulosome architecture. PMID:24927597

  2. Structural and functional characterization of a conserved pair of bacterial cellulose-oxidizing lytic polysaccharide monooxygenases

    PubMed Central

    Forsberg, Zarah; Mackenzie, Alasdair K.; Sørlie, Morten; Røhr, Åsmund K.; Helland, Ronny; Arvai, Andrew S.; Vaaje-Kolstad, Gustav; Eijsink, Vincent G. H.

    2014-01-01

    For decades, the enzymatic conversion of cellulose was thought to rely on the synergistic action of hydrolytic enzymes, but recent work has shown that lytic polysaccharide monooxygenases (LPMOs) are important contributors to this process. We describe the structural and functional characterization of two functionally coupled cellulose-active LPMOs belonging to auxiliary activity family 10 (AA10) that commonly occur in cellulolytic bacteria. One of these LPMOs cleaves glycosidic bonds by oxidation of the C1 carbon, whereas the other can oxidize both C1 and C4. We thus demonstrate that C4 oxidation is not confined to fungal AA9-type LPMOs. X-ray crystallographic structures were obtained for the enzyme pair from Streptomyces coelicolor, solved at 1.3 Å (ScLPMO10B) and 1.5 Å (CelS2 or ScLPMO10C) resolution. Structural comparisons revealed differences in active site architecture that could relate to the ability to oxidize C4 (and that also seem to apply to AA9-type LPMOs). Despite variation in active site architecture, the two enzymes exhibited similar affinities for Cu2+ (12–31 nM), redox potentials (242 and 251 mV), and electron paramagnetic resonance spectra, with only the latter clearly different from those of chitin-active AA10-type LPMOs. We conclude that substrate specificity depends not on copper site architecture, but rather on variation in substrate binding and orientation. During cellulose degradation, the members of this LPMO pair act in synergy, indicating different functional roles and providing a rationale for the abundance of these enzymes in biomass-degrading organisms. PMID:24912171

  3. Bioinspired copper(I) complexes that exhibit monooxygenase and catechol dioxygenase activity.

    PubMed

    Arnold, Aline; Metzinger, Ramona; Limberg, Christian

    2015-01-12

    New tripodal ligand L2 featuring three different pyridyl/imidazolyl-based N-donor units at a bridgehead C atom, from which one of the imidazolyl units is separated by a phenylene linker, was synthesized and investigated with regards to copper(I) complexation. The resulting complex [(L2)Cu]OTf (2(OTf)), the known complex [(L1)Cu]OTf (1(OTf); L1 differs from L2 in that it lacks the phenylene spacer) and [(L3)Cu]OTf (3(OTf)), prepared from a known chiral, tripodal, N-donor ligand featuring pyridyl, pyrazolyl, and imidazolyl donors, were tested as catalysts for the oxidation of sodium 2,4-di-tert-butylphenolate (NaDTBP) with O2. Indeed, they mediated NaDTBP oxidation to give mainly the corresponding catecholate and quinone (Q). None of the complexes 1(OTf), 2(OTf), and 3(OTf) is superior to the others, as yields were comparable and, if the presence of protons is guaranteed by concomitant addition of the phenol DTBP, the oxidation can also be performed catalytically. For all complexes stoichiometric oxidations under certain conditions (concentrated solutions, high NaDTBP content) were found to also generate products typical for metal-mediated intradiol cleavage of the catecholate with O2. As shown representatively for 1(OTf) this dioxygenation sets in at a later stage of the reaction. Initially a copper species responsible for the monooxygenation must form from 1(OTf)/NaDTBP/O2, and only thereafter is the copper species responsible for dioxygenation formed and consumes Q as substrate. Hence, under these circumstances complexes 1(OTf)-3(OTf) show both monooxygenase and catechol dioxygenase activity.

  4. Two Novel Flavin-Containing Monooxygenases Involved in Biosynthesis of Aliphatic Glucosinolates

    PubMed Central

    Kong, Wenwen; Li, Jing; Yu, Qingyue; Cang, Wei; Xu, Rui; Wang, Yang; Ji, Wei

    2016-01-01

    Glucosinolates, a class of secondary metabolites from cruciferous plants, are derived from amino acids and have diverse biological activities, such as in biotic defense, depending on their side chain modification. The first structural modification step in the synthesis of aliphatic (methionine-derived) glucosinolates—S-oxygenation of methylthioalkyl glucosinolates to methylsulfinylalkyl glucosinolates—was found to be catalyzed by five flavin-containing monooxygenases (FMOs), FMOGS-OX1-5. Here, we report two additional FMOGS-OX enzymes, FMOGS-OX6, and FMOGS-OX7, encoded by At1g12130 and At1g12160, respectively. The overexpression of both FMOGS-OX6 and FMOGS-OX7 decreased the ratio of methylthioalkyl glucosinolates to the sum of methylthioalkyl and methylsulfinylalkyl glucosinolates, suggesting that the introduction of the two genes converted methylthioalkyl glucosinolates into methylsulfinylalkyl glucosinolates. Analysis of expression pattern revealed that the spatial expression of the two genes is quite similar and partially overlapped with the other FMOGS-OX genes, which are primarily expressed in vascular tissue. We further analyzed the responsive expression pattern of all the seven FMOGS-OX genes to exogenous treatment with abscisic acid, 1-aminocyclopropane-1-carboxylic acid (ACC), jasmonic acid (JA), salicylic acid, indole-3-acetic acid (IAA), and low and high temperatures. Although these genes showed same tendency toward the changing stimulus, the sensitivity of each gene was quite different. The variety in spatial expression among the FMOGS-OX genes while responding to environmental stimulus indicated a complex and finely tuned regulation of glucosinolates modifications. Identification of these two novel FMOGS-OX enzymes will enhance the understanding of glucosinolates modifications and the importance of evolution of these duplicated genes. PMID:27621741

  5. The kynurenine pathway is activated in human obesity and shifted toward kynurenine monooxygenase activation.

    PubMed

    Favennec, Marie; Hennart, Benjamin; Caiazzo, Robert; Leloire, Audrey; Yengo, Loïc; Verbanck, Marie; Arredouani, Abdelilah; Marre, Michel; Pigeyre, Marie; Bessede, Alban; Guillemin, Gilles J; Chinetti, Giulia; Staels, Bart; Pattou, François; Balkau, Beverley; Allorge, Delphine; Froguel, Philippe; Poulain-Godefroy, Odile

    2015-10-01

    This study characterized the kynurenine pathway (KP) in human obesity by evaluating circulating levels of kynurenines and the expression of KP enzymes in adipose tissue. Tryptophan and KP metabolite levels were measured in serum of individuals from the D.E.S.I.R. cohort (case-cohort study: 212 diabetic, 836 randomly sampled) and in women with obesity, diabetic or normoglycemic, from the ABOS cohort (n = 100). KP enzyme gene expressions were analyzed in omental and subcutaneous adipose tissue of women from the ABOS cohort, in human primary adipocytes and in monocyte-derived macrophages. In the D.E.S.I.R. cohort, kynurenine levels were positively associated with body mass index (BMI) (P = 4.68 × 10(-19) ) and with a higher HOMA2-IR insulin resistance index (P = 6.23 × 10(-4) ). The levels of kynurenine, kynurenic acid, and quinolinic acid were associated with higher BMI (P < 0.05). The expression of several KP enzyme genes (indoleamine 2,3-dioxygenase 1 [IDO1], kynureninase [KYNU], kynurenine 3-monooxygenase [KMO], and kynurenine aminotransferase III [CCBL2]) was increased in the omental adipose tissue of women with obesity compared to lean (P < 0.05), and their expression was induced by proinflammatory cytokines in human primary adipocytes (P < 0.05), except for KMO that is not expressed in these cells. The expressions of IDO1, KYNU, KMO, and CCBL2 were higher in proinflammatory than in anti-inflammatory macrophages (P < 0.05). In the context of obesity, the presence of macrophages in adipose tissue may contribute to diverting KP toward KMO activation. © 2015 The Obesity Society.

  6. Gating effects of component B on oxygen activation by the methane monooxygenase hydroxylase component.

    PubMed

    Liu, Y; Nesheim, J C; Lee, S K; Lipscomb, J D

    1995-10-20

    Component B (MMOB) of the soluble methane monooxygenase (MMO) system accelerates the initial velocity of methane oxidation by up to 150-fold by an unknown mechanism. The active site of MMO contains a diferric, hydroxo-bridged diiron cluster located on the hydroxylase component (MMOH). This cluster is reduced by the NAD(P)H-coupled reductase component to the diferrous state, which then reacts with O2 to yield two reaction cycle intermediates sequentially termed compounds P and Q. The rate of compound P formation is shown here to be independent of O2 concentration, suggesting that an MMOH-O2 complex (compound O) is (congruent to irreversibly) formed before compound P. Compound Q is capable of reacting with hydrocarbons to yield the MMOH-product complex, compound T. It is shown here that MMOB accelerates catalysis by increasing congruent to 1000-fold the rate of O2 association and reaction with diferrous MMOH leading to compound P. Modeling of the single turnover reaction in the presence of substoichiometric MMOB suggests that MMOB also accelerates the compound P to Q conversion by congruent to 40-fold. Due to this O2-gating effect of MMOB, either compound Q or T becomes the dominant species during turnover, depending upon the substrate concentration and type. Because these are the species that either react with substrate (Q) or release product (T), their buildup maximizes the turnover rate. This is the first direct role in catalysis to be recognized for MMOB and represents a novel method for oxygenase regulation.

  7. Component interactions in the soluble methane monooxygenase system from Methylococcus capsulatus (Bath).

    PubMed

    Gassner, G T; Lippard, S J

    1999-09-28

    The soluble methane monooxygenase system of Methylococcus capsulatus (Bath) includes three protein components: a 251-kDa non-heme dinuclear iron hydroxylase (MMOH), a 39-kDa iron-sulfur- and FAD-containing reductase (MMOR), and a 16-kDa regulatory protein (MMOB). The thermodynamic stability and kinetics of formation of complexes between oxidized MMOH and MMOB or MMOR were measured by isothermal titration calorimetry and stopped-flow fluorescence spectroscopy at temperatures ranging from 3.3 to 45 degrees C. The results, in conjunction with data from equilibrium analytical ultracentrifugation studies of MMOR and MMOB, indicate that free MMOR and MMOB exist as monomers in solution and bind MMOH with 2:1 stoichiometry. The role of component interactions in the catalytic mechanism of sMMO was investigated through simultaneous measurement of oxidase and hydroxylase activities as a function of varied protein component concentrations during steady-state turnover. The partitioning of oxidase and hydroxylase activities of sMMO is highly dependent on both the MMOR concentration and the nature of the organic substrate. In particular, NADH oxidation is significantly uncoupled from methane hydroxylation at MMOR concentrations exceeding 20% of the hydroxylase concentration but remains tightly coupled to propylene epoxidation at MMOR concentrations ranging up to the MMOH concentration. The steady-state kinetic data were fit to numerical simulations of models that include both the oxidase activities of free MMOR and of MMOH/MMOR complexes and the hydroxylase activity of MMOH/MMOB complexes. The data were well described by a model in which MMOR and MMOB bind noncompetitively at distinct interacting sites on the hydroxylase. MMOB manifests its regulatory effects by differentially accelerating intermolecular electron transfer from MMOR to MMOH containing bound substrate and product in a manner consistent with its activating and inhibitory effects on the hydroxylase.

  8. Hydroxylation mechanism of methane and its derivatives over designed methane monooxygenase model with peroxo dizinc core.

    PubMed

    Li, Cai-Qin; Yang, Hua-Qing; Xu, Jian; Hu, Chang-Wei

    2012-05-21

    The peroxo dizinc Zn(2)O(2) complex Q coordinated by imidazole and carboxylate groups for each Zn center has been designed to model the hydroxylase component of methane monooxygenase (MMO) enzyme, on the basis of the experimentally available structure information of enzyme with divalent zinc ion and the MMO with Fe(2)O(2) core. The reaction mechanism for the hydroxylation of methane and its derivatives catalyzed by Q has been investigated at the B3LYP*/cc-pVTZ, Lanl2tz level in protein solution environment. These hydroxylation reactions proceed via a radical-rebound mechanism, with the rate-determining step of the C-H bond cleavage. This radical-rebound reaction mechanism is analogous to the experimentally available MMOs with diamond Fe(2)O(2) core accompanied by a coordinate number of six for the hydroxylation of methane. The rate constants for the hydroxylation of substrates catalyzed by Q increase along CH(4) < CH(3)F < CH(3)CN ≈ CH(3)NO(2) < CH(3)CH(3). Both the activation strain ΔE(≠)(strain) and the stabilizing interaction ΔE(≠)(int) jointly affect the activation energy ΔE(≠). For the C-H cleavage of substrate CH(3)X, with the decrease of steric shielding for the substituted CH(3)X (X = F > H > CH(3) > NO(2) > CN) attacking the O center in Q, the activation strain ΔE(≠)(strain) decreases, whereas the stabilizing interaction ΔE(≠)(int) increases. It is predicted that the MMO with peroxo dizinc Zn(2)O(2) core should be a promising catalyst for the hydroxylation of methane and its derivatives.

  9. Evaluating cytochrome P450 in birds by monooxygenases and immunohistochemistry: possible nonlethal assessment by skin immunohistochemistry

    USGS Publications Warehouse

    Melancon, M.J.; Kutay, A.L.; Woodin, Bruce R.; Stegeman, John J.

    2000-01-01

    Six month old Lesser Scaup and nestling Tree Swallows were injected intraperitoneally with beta-naphthoflavone (BNF) or vehicle. Nestling Tree Swallows were also collected from five sites with differing levels of contaminants. Liver samples were taken and stored at -80C until microsome preparation and monooxygenase (MO) assay. Skin and heart samples were placed in buffered formalin until immunohistochemical (IMHC) analysis for cytochrome P4501A (CYP1A). Scaup treated with BNF at 20 or 100 mg/kg body weight showed approximately 20- to 65-fold increases in four MOs. Responses of two of the four MOs were as high at 20 mg/kg as at 100mg/kg. There was no IMHC response in the vehicle-injected ducks, while in skin the IMHC response was the same for both dose levels of BNF and in heart there was response in two of four samples at 20 mg/kg and in all five samples at 100mg/kg. Tree Swallows injected with BNF at 100, but not at 20 mg/kg showed significant increases (ca.5-fold) in two MO activities. There was no IMHC response in control swallows. In skin and heart there were IMHC responses in one of five swallows at 20 mg/kg and four of five swallows at 100mg/kg. There was poor correlation between individual skin IMHC responses and MO activities and PCB concentrations in 47 field-collected Tree Swallow samples, but 14 of the 16 skin samples with positive IMHC responses were from the location with the highest MO activities and PCB concentrations. Although present data do not allow construction of significant dose response curves, the responses in skin make it well worth continuing study on this potential nonlethal technique for biomonitoring contaminant exposure of birds.

  10. pH dependence on functional activity of human and mouse flavin-containing monooxygenase 5.

    PubMed

    Motika, Meike S; Zhang, Jun; Ralph, Erik C; Dwyer, Mary A; Cashman, John R

    2012-04-01

    Flavin-containing monooxygenase (FMO) 5 belongs to a family of enzymes that catalyze the oxygenation of nucleophilic N- and S-containing compounds. The FMO enzyme family consists of five forms (FMOs1-5) that share about 50-60% sequence identity to each other. A comparison of FMOs showed that the pH-dependence profile for functional activity of FMO5 differed significantly from that of other FMO enzymes. The objective of this study was to examine the pH-dependence of FMO5 to gain insight into the mechanism of action of FMO5. Recombinant mouse and human FMO5 (mFMO5 and hFMO5, respectively) were expressed as maltose-binding fusion proteins from Escherichia coli, purified with affinity chromatography, and examined for their N-oxygenation functional activity at different pH values. hFMO5 showed a broader range and greater functional activity from pH 6 to 11 compared to mFMO5. mFMO5 lost almost all functional activity at pH 6, while hFMO5 maintained almost normal enzyme activity. In order to identify the amino acid residues involved in the effects of pH on hFMO5 and mFMO5 functional enzyme activity, pH-studies in the range of pH 6-9 were done with chimeras of recombinant mouse and human FMO5 and variants of both. Results of these studies and molecular modeling showed that residues responsible for the differences in the pH profile between mFMO5 and hFMO5 were located at positions 227 and 228 of the enzyme. Further variants were made to investigate the role of these amino acids. The results of this study may help to explain the mechanism of FMO function. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Fungal Cytochrome P450 Monooxygenases: Their Distribution, Structure, Functions, Family Expansion, and Evolutionary Origin

    PubMed Central

    Chen, Wanping; Lee, Mi-Kyung; Jefcoate, Colin; Kim, Sun-Chang; Chen, Fusheng; Yu, Jae-Hyuk

    2014-01-01

    Cytochrome P450 (CYP) monooxygenase superfamily contributes a broad array of biological functions in living organisms. In fungi, CYPs play diverse and pivotal roles in versatile metabolism and fungal adaptation to specific ecological niches. In this report, CYPomes in the 47 genomes of fungi belong to the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota have been studied. The comparison of fungal CYPomes suggests that generally fungi possess abundant CYPs belonging to a variety of families with the two global families CYP51 and CYP61, indicating individuation of CYPomes during the evolution of fungi. Fungal CYPs show highly conserved characteristic motifs, but very low overall sequence similarities. The characteristic motifs of fungal CYPs are distinguishable from those of CYPs in animals, plants, and especially archaea and bacteria. The four representative motifs contribute to the general function of CYPs. Fungal CYP51s and CYP61s can be used as the models for the substrate recognition sites analysis. The CYP proteins are clustered into 15 clades and the phylogenetic analyses suggest that the wide variety of fungal CYPs has mainly arisen from gene duplication. Two large duplication events might have been associated with the booming of Ascomycota and Basidiomycota. In addition, horizontal gene transfer also contributes to the diversification of fungal CYPs. Finally, a possible evolutionary scenario for fungal CYPs along with fungal divergences is proposed. Our results provide the fundamental information for a better understanding of CYP distribution, structure and function, and new insights into the evolutionary events of fungal CYPs along with the evolution of fungi. PMID:24966179

  12. Biomaterials for mRNA Delivery

    PubMed Central

    Islam, Mohammad Ariful; Reesor, Emma K. G.; Xu, Yingjie; Zope, Harshal R.; Zetter, Bruce R.; Shi, Jinjun

    2015-01-01

    Messenger RNA (mRNA) has recently emerged with remarkable potential as an effective alternative to DNA-based therapies because of several unique advantages. mRNA does not require nuclear entry for transfection activity and has a negligible chance of integrating into the host genome which excludes the possibility of potentially detrimental genomic alternations. Chemical modification of mRNA has further enhanced its stability and decreased its activation of innate immune responses. Additionally, mRNA has been found to have rapid expression and predictable kinetics. Nevertheless, the ubiquitous application of mRNA remains challenging given its unfavorable attributes, such as large size, negative charge and susceptibility to enzymatic degradation. Further refinement of mRNA delivery modalities is therefore essential for its development as a therapeutic tool. This review provides an exclusive overview of current state-of-the-art biomaterials and nanotechnology platforms for mRNA delivery, and discusses future prospects to bring these exciting technologies into clinical practice. PMID:26280625

  13. Occurrence of a barbiturate-inducible catalytically self-sufficient 119,000 dalton cytochrome P-450 monooxygenase in bacilli.

    PubMed

    Fulco, A J; Ruettinger, R T

    1987-05-04

    In a recent publication (Narhi, L.O. and Fulco, A.J.[1986] J. Biol. Chem. 261, 7160-7169) we described the characterization of a catalytically self-sufficient 119,000 Dalton cytochrome P-450 fatty acid monooxygenase (P-450BM-3) induced by barbiturates in Bacillus megaterium ATCC 14581. We have now examined cell-free preparations from 12 distinct strains of B. megaterium and from one or two strains each of B. alvei, B. brevis, B. cereus, B. licheniformis, B. macerans, B. pumilis and B. subtilis for the presence of this inducible enzyme. Using Western blot analyses in combination with assays for fatty acid hydroxylase activity and cytochrome P-450, we were able to show that 11 of the 12 B. megaterium strains contained not only a strongly pentobarbital-inducible fatty acid monooxygenase identical to or polymorphic with P-450BM-3 but also significant levels of two smaller P-450 cytochromes that were the same as or similar to cytochromes P-450BM-1 and P-450BM-2 originally found in ATCC 14581. Unlike the 119,000 Dalton P-450, however, the two smaller P-450s were generally easily detectable in cultures grown to stationary phase in the absence of barbiturates and, with some exceptions, were not strongly induced by pentobarbital. None of the non-megaterium species of Bacillus tested exhibited significant levels of either fatty acid monooxygenase activity or cytochrome P-450. The one strain of B. megaterium that lacked inducible P-450BM-3 was also negative for BM-1 and BM-2. However, this strain (ATCC 13368) did contain a small but significant level of another P-450 cytochrome that others have identified as the oxygenase component of a steroid 15-beta-hydroxylase system. Our evidence suggests that the BM series of P-450 cytochromes is encoded by chromosomal (rather than by plasmid) DNA.

  14. The effect of disulfide bond introduction and related Cys/Ser mutations on the stability of a cyclohexanone monooxygenase.

    PubMed

    Schmidt, Sandy; Genz, Maika; Balke, Kathleen; Bornscheuer, Uwe T

    2015-11-20

    Baeyer-Villiger monooxygenases (BVMO) belong to the class B of flavin-dependent monooxygenases (type I BVMOs) and catalyze the oxidation of (cyclic) ketones into esters and lactones. The prototype BVMO is the cyclohexanone monooxygenase (CHMO) from Acinetobacter sp. NCIMB 9871. This enzyme shows an impressive substrate scope with a high chemo-, regio- and/or enantioselectivity. BVMO reactions are often difficult, if not impossible to achieve by chemical approaches and this makes these enzymes thus highly desired candidates for industrial applications. Unfortunately, the industrial use is hampered by several factors related to the lack of stability of these biocatalysts. Thus, the aim of this study was to improve the CHMO's long-term stability, one of the most relevant parameter for biocatalytic processes, and additionally its stability against oxidation. We used an easy computational method for the prediction of stabilizing disulfide bonds in the CHMO-scaffold. The three most promising predicted disulfide pairs were created and biochemically characterized. The most oxidatively stable variant (Y411C-A463C) retained nearly 60% activity after incubation with 25 mM H2O2 whereas the wild type retained only 16%. In addition, one extra disulfide pair (T415C-A463C) was created and tested for increased stability. The melting temperature (Tm) of this variant was increased by 5°C with simultaneous improved long-term stability. After verification by ABD-F labeling that this mutant does not form a disulfide bond, single and double Cys/Ser mutants were prepared and investigated. Subsequent analysis revealed that the T415C single point variant is the most stable variant with a 30-fold increased long-term stability (33% residual activity after 24h incubation at 25°C) showcasing a great achievement for practical applications.

  15. Independent Recruitment of a Flavin-Dependent Monooxygenase for Safe Accumulation of Sequestered Pyrrolizidine Alkaloids in Grasshoppers and Moths

    PubMed Central

    Wang, Linzhu; Beuerle, Till; Timbilla, James; Ober, Dietrich

    2012-01-01

    Several insect lineages have developed diverse strategies to sequester toxic pyrrolizidine alkaloids from food-plants for their own defense. Here, we show that in two highly divergent insect taxa, the hemimetabolous grasshoppers and the holometabolous butterflies, an almost identical strategy evolved independently for safe accumulation of pyrrolizidine alkaloids. This strategy involves a pyrrolizidine alkaloid N-oxygenase that transfers the pyrrolizidine alkaloids to their respective N-oxide, enabling the insects to avoid high concentrations of toxic pyrrolizidine alkaloids in the hemolymph. We have identified a pyrrolizidine alkaloid N-oxygenase, which is a flavin-dependent monooxygenase, of the grasshopper Zonocerus variegatus. After heterologous expression in E. coli, this enzyme shows high specificity for pyrrolizidine alkaloids of various structural types and for the tropane alkaloid atropine as substrates, a property that has been described previously for a pyrrolizidine alkaloid N-oxygenase of the arctiid moth Grammia geneura. Phylogenetic analyses of insect flavin-dependent monooxygenase sequences suggest that independent gene duplication events preceded the establishment of this specific enzyme in the lineages of the grasshoppers and of arctiid moths. Two further flavin-dependent monooxygenase sequences have been identified from Z. variegatus sharing amino acid identities of approximately 78% to the pyrrolizidine alkaloid N-oxygenase. After heterologous expression, both enzymes are also able to catalyze the N-oxygenation of pyrrolizidine alkaloids, albeit with a 400-fold lower specific activity. With respect to the high sequence identity between the three Z. variegatus sequences this ability to N-oxygenize pyrrolizidine alkaloids is interpreted as a relict of a former bifunctional ancestor gene of which one of the gene copies optimized this activity for the specific adaptation to pyrrolizidine alkaloid containing food plants. PMID:22363737

  16. Competition between Metals for Binding to Methanobactin Enables Expression of Soluble Methane Monooxygenase in the Presence of Copper

    PubMed Central

    Kalidass, Bhagyalakshmi; Ul-Haque, Muhammad Farhan; Baral, Bipin S.; DiSpirito, Alan A.

    2014-01-01

    It is well known that copper is a key factor regulating expression of the two forms of methane monooxygenase found in proteobacterial methanotrophs. Of these forms, the cytoplasmic, or soluble, methane monooxygenase (sMMO) is expressed only at low copper concentrations. The membrane-bound, or particulate, methane monooxygenase (pMMO) is constitutively expressed with respect to copper, and such expression increases with increasing copper. Recent findings have shown that copper uptake is mediated by a modified polypeptide, or chalkophore, termed methanobactin. Although methanobactin has high specificity for copper, it can bind other metals, e.g., gold. Here we show that in Methylosinus trichosporium OB3b, sMMO is expressed and active in the presence of copper if gold is also simultaneously present. Such expression appears to be due to gold binding to methanobactin produced by M. trichosporium OB3b, thereby limiting copper uptake. Such expression and activity, however, was significantly reduced if methanobactin preloaded with copper was also added. Further, quantitative reverse transcriptase PCR (RT-qPCR) of transcripts of genes encoding polypeptides of both forms of MMO and SDS-PAGE results indicate that both sMMO and pMMO can be expressed when copper and gold are present, as gold effectively competes with copper for binding to methanobactin. Such findings suggest that under certain geochemical conditions, both forms of MMO may be expressed and active in situ. Finally, these findings also suggest strategies whereby field sites can be manipulated to enhance sMMO expression, i.e., through the addition of a metal that can compete with copper for binding to methanobactin. PMID:25416758

  17. Independent recruitment of a flavin-dependent monooxygenase for safe accumulation of sequestered pyrrolizidine alkaloids in grasshoppers and moths.

    PubMed

    Wang, Linzhu; Beuerle, Till; Timbilla, James; Ober, Dietrich

    2012-01-01

    Several insect lineages have developed diverse strategies to sequester toxic pyrrolizidine alkaloids from food-plants for their own defense. Here, we show that in two highly divergent insect taxa, the hemimetabolous grasshoppers and the holometabolous butterflies, an almost identical strategy evolved independently for safe accumulation of pyrrolizidine alkaloids. This strategy involves a pyrrolizidine alkaloid N-oxygenase that transfers the pyrrolizidine alkaloids to their respective N-oxide, enabling the insects to avoid high concentrations of toxic pyrrolizidine alkaloids in the hemolymph. We have identified a pyrrolizidine alkaloid N-oxygenase, which is a flavin-dependent monooxygenase, of the grasshopper Zonocerus variegatus. After heterologous expression in E. coli, this enzyme shows high specificity for pyrrolizidine alkaloids of various structural types and for the tropane alkaloid atropine as substrates, a property that has been described previously for a pyrrolizidine alkaloid N-oxygenase of the arctiid moth Grammia geneura. Phylogenetic analyses of insect flavin-dependent monooxygenase sequences suggest that independent gene duplication events preceded the establishment of this specific enzyme in the lineages of the grasshoppers and of arctiid moths. Two further flavin-dependent monooxygenase sequences have been identified from Z. variegatus sharing amino acid identities of approximately 78% to the pyrrolizidine alkaloid N-oxygenase. After heterologous expression, both enzymes are also able to catalyze the N-oxygenation of pyrrolizidine alkaloids, albeit with a 400-fold lower specific activity. With respect to the high sequence identity between the three Z. variegatus sequences this ability to N-oxygenize pyrrolizidine alkaloids is interpreted as a relict of a former bifunctional ancestor gene of which one of the gene copies optimized this activity for the specific adaptation to pyrrolizidine alkaloid containing food plants.

  18. Inactivation of tyrosine 3-monooxygenase by acetone precipitation and its restoration by incubation with a sulfhydryl agent and iron.

    PubMed

    Okuno, S; Fujisawa, H

    1981-04-14

    The acetone precipitation of a partially purified tyrosine 3-monooxygenase (L-tyrosine, tetrahydropteridine: oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2) resulted in the complete loss of enzymatic activity. The enzymatic activity was restored by incubation with iron and dithiothreitol. The restoration of the activity was a pH-, temperature- and time-dependent reaction. Since cobalt, nickel, copper, zinc, manganese, cadmium, magnesium calcium and barium ions were all ineffective in restoring activity, iron ion appeared to be specifically required in the restoration of the enzyme activity. Dithiothreitol could be partially replaced in the restoration step by glutathione, 2-mercaptoethanol or cysteine.

  19. Aliphatic and chlorinated alkenes and epoxides as inducers of alkene monooxygenase and epoxidase activities in Xanthobacter strain Py2

    SciTech Connect

    Ensign, S.A.

    1996-01-01

    Xanthobacter strain Py2 is one of a number of bacteria capable of aerobic growth using aliphatic alkenes as the sole carbon and energy source. The oxidation of alkenes in initiated by an alkene monooxygenase which catalyzes the CO{sub 2} and reductant-dependent oxidation of the alkenes to their corresponding epoxides. In this study, the induction of the alkene-oxidizing enzymes of Xantheobacter strain Py2 by the physiological substrates propylene and proplyene oxide is characterized. A variety of aliphatic and chlorinated alkenes and epoxides are shown to serve as effective inducers of the system. 21 refs., 6 figs., 2 tabs.

  20. Comparison of the peptide map and functional properties of monooxygenases induced by 3-methylcholanthrene and. beta. -naphthoflavone

    SciTech Connect

    Chasovnikova, O.B.; Mishin, V.M.; Tsyrlov, I.B.

    1987-02-20

    The similarity of the catalytic, spectral, electrophoretic, and immunochemical properties of microsomal cytochromes P-448 (molecular weight 56,000), synthesized de novo after administration of 3-methylcholanthrene and ..beta..-naphthoflavone to rats, was demonstrated. The identity of the peptide maps of the microsomal and isolated cytochrome P-448 is evidence of adequacy of the method of limited proteolysis for establishing the homogeneity and comparing the structure of the microsomal hemoproteins. The data obtained substantiate the approach for the study of the similarity and differences in the structure and enzymatic activity of various forms of monooxygenases without their preliminary isolation from the microsomal membrane.

  1. Development of a Series of Kynurenine 3-Monooxygenase Inhibitors Leading to a Clinical Candidate for the Treatment of Acute Pancreatitis.

    PubMed

    Walker, Ann L; Ancellin, Nicolas; Beaufils, Benjamin; Bergeal, Marylise; Binnie, Margaret; Bouillot, Anne; Clapham, David; Denis, Alexis; Haslam, Carl P; Holmes, Duncan S; Hutchinson, Jonathan P; Liddle, John; McBride, Andrew; Mirguet, Olivier; Mowat, Christopher G; Rowland, Paul; Tiberghien, Nathalie; Trottet, Lionel; Uings, Iain; Webster, Scott P; Zheng, Xiaozhong; Mole, Damian J

    2017-04-27

    Recently, we reported a novel role for KMO in the pathogenesis of acute pancreatitis (AP). A number of inhibitors of kynurenine 3-monooxygenase (KMO) have previously been described as potential treatments for neurodegenerative conditions and particularly for Huntington's disease. However, the inhibitors reported to date have insufficient aqueous solubility relative to their cellular potency to be compatible with the intravenous (iv) dosing route required in AP. We have identified and optimized a novel series of high affinity KMO inhibitors with favorable physicochemical properties. The leading example is exquisitely selective, has low clearance in two species, prevents lung and kidney damage in a rat model of acute pancreatitis, and is progressing into preclinical development.

  2. H2-driven biotransformation of n-octane to 1-octanol by a recombinant Pseudomonas putida strain co-synthesizing an O2-tolerant hydrogenase and a P450 monooxygenase.

    PubMed

    Lonsdale, Thomas H; Lauterbach, Lars; Honda Malca, Sumire; Nestl, Bettina M; Hauer, Bernhard; Lenz, Oliver

    2015-11-21

    An in vivo biotransformation system is presented that affords the hydroxylation of n-octane to 1-octanol on the basis of NADH-dependent CYP153A monooxygenase and NAD(+)-reducing hydrogenase heterologously synthesized in a bacterial host. The hydrogenase sustains H2-driven NADH cofactor regeneration even in the presence of O2, the co-substrate of monooxygenase.

  3. Regulation of cytoplasmic mRNA decay

    PubMed Central

    Schoenberg, Daniel R.; Maquat, Lynne E.

    2012-01-01

    Discoveries made over the past 20 years highlight the importance of mRNA decay as a means to modulate gene expression and thereby protein production. Up until recently, studies focused largely on identifying cis-acting sequences that serve as mRNA stability or instability elements, the proteins that bind these elements, how the process of translation influences mRNA decay, and the ribonucleases that catalyze decay. Now, current studies have begun to elucidate how the decay process is regulated. This review examines our current understanding of how mammalian-cell mRNA decay is controlled by different signaling pathways and lays out a framework for future research. PMID:22392217

  4. Disorders in barrier protein mRNA expression and placenta secretory activity under the influence of polychlorinated biphenyls in vitro.

    PubMed

    Wojciechowska, A; Mlynarczuk, J; Kotwica, J

    2017-02-01

    Pregnancy disorders are often correlated with the presence of organic pollutants in the tissues of living bodies. The aim of this study was to investigate the effects (over 24 and 48 hours) of polychlorinated biphenyls (PCBs) 153, 126, and 77 at doses of 1, 10, and 100 ng/mL on barrier function and secretory activity in cow placentome sections collected during the second trimester of pregnancy. None of the PCBs affected the viability of the sections (P > 0.05). Polychlorinated biphenyl 153 decreased (P < 0.05) connexin 26 (Cx 26) mRNA expression, and all three PCBs reduced (P < 0.05) Cx 43 mRNA expression. Cx 32 mRNA expression showed a downward trend (P > 0.05) under the influence of PCBs 126 and 77. Moreover, PCBs 153 and 126 increased keratin 8 (KRT8) mRNA expression, whereas all PCBs decreased (P < 0.05) placenta specific protein 1 (PLAC-1) mRNA expression without changing (P > 0.05) hypoxia inducible factor 1α (HIF1α) mRNA expression. Concomitantly, PCBs 153 and 126 stimulated (P < 0.05) cyclooxygenase 2 (COX-2) mRNA expression, all PCBs increased (P < 0.05) prostaglandin E2 synthase (PGES) mRNA expression, and PCBs 126 and 77 increased prostaglandin E2 (PGE2) secretion. All three PCBs decreased (P < 0.05) prostaglandin F2α synthase (PGFS) mRNA expression and prostaglandin F2α (PGF2α) secretion. In addition, all three PCBs increased (P < 0.05) neurophysin I/oxytocin (NP-I/OT) mRNA expression and OT secretion but did not affect peptidyl-glycine-α-amidating monooxygenase (PGA) mRNA expression (P > 0.05). Moreover, the PCBs increased (P < 0.05) estradiol (E2) secretion, whereas progesterone (P4) secretion remained unchanged (P > 0.05). These changes could affect trophoblast invasion and uterine contractility and thus impact the course of gestation and/or fetal development in the cow.

  5. Cytoplasmic mRNA turnover and ageing

    PubMed Central

    Borbolis, Fivos; Syntichaki, Popi

    2015-01-01

    Messenger RNA (mRNA) turnover that determines the lifetime of cytoplasmic mRNAs is a means to control gene expression under both normal and stress conditions, whereas its impact on ageing and age-related disorders has just become evident. Gene expression control is achieved at the level of the mRNA clearance as well as mRNA stability and accessibility to other molecules. All these processes are regulated by cis-acting motifs and trans-acting factors that determine the rates of translation and degradation of transcripts. Specific messenger RNA granules that harbor the mRNA decay machinery or various factors, involved in translational repression and transient storage of mRNAs, are also part of the mRNA fate regulation. Their assembly and function can be modulated to promote stress resistance to adverse conditions and over time affect the ageing process and the lifespan of the organism. Here, we provide insights into the complex relationships of ageing modulators and mRNA turnover mechanisms. PMID:26432921

  6. Activity-Based Protein Profiling of Ammonia Monooxygenase in Nitrosomonas europaea.

    PubMed

    Bennett, Kristen; Sadler, Natalie C; Wright, Aaron T; Yeager, Chris; Hyman, Michael R

    2016-04-01

    Nitrosomonas europaea is an aerobic nitrifying bacterium that oxidizes ammonia (NH3) to nitrite (NO2 (-)) through the sequential activities of ammonia monooxygenase (AMO) and hydroxylamine dehydrogenase (HAO). Many alkynes are mechanism-based inactivators of AMO, and here we describe an activity-based protein profiling method for this enzyme using 1,7-octadiyne (17OD) as a probe. Inactivation of NH4 (+)-dependent O2 uptake by N. europaea by 17OD was time- and concentration-dependent. The effects of 17OD were specific for ammonia-oxidizing activity, andde novoprotein synthesis was required to reestablish this activity after cells were exposed to 17OD. Cells were reacted with Alexa Fluor 647 azide using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) (click) reaction, solubilized, and analyzed by SDS-PAGE and infrared (IR) scanning. A fluorescent 28-kDa polypeptide was observed for cells previously exposed to 17OD but not for cells treated with either allylthiourea or acetylene prior to exposure to 17OD or for cells not previously exposed to 17OD. The fluorescent polypeptide was membrane associated and aggregated when heated with β-mercaptoethanol and SDS. The fluorescent polypeptide was also detected in cells pretreated with other diynes, but not in cells pretreated with structural homologs containing a single ethynyl functional group. The membrane fraction from 17OD-treated cells was conjugated with biotin-azide and solubilized in SDS. Streptavidin affinity-purified polypeptides were on-bead trypsin-digested, and amino acid sequences of the peptide fragments were determined by liquid chromatography-mass spectrometry (LC-MS) analysis. Peptide fragments from AmoA were the predominant peptides detected in 17OD-treated samples. In-gel digestion and matrix-assisted laser desorption ionization-tandem time of flight (MALDI-TOF/TOF) analyses also confirmed that the fluorescent 28-kDa polypeptide was AmoA. Copyright © 2016, American Society for Microbiology. All

  7. Oxidative Metabolism of Seleno-L-Methionine to L-Methionine Selenoxide by Flavin- Containing Monooxygenases

    PubMed Central

    Krause, Renee J.; Glocke, Steven C.; Sicuri, Anna Rita; Ripp, Sharon L.; Elfarra, Adnan A

    2008-01-01

    The roles of flavin-containing monooxygenases (FMOs) in the oxidation of seleno-L-methionine (SeMet) to L-methionine selenoxide (MetSeO) were investigated using cDNA-expressed human FMOs, purified rat liver FMOs, and rat liver microsomes. MetSeO and the N-2,4-dinitrophenyl-derivatives of SeMet and MetSeO were synthesized and characterized by 1H-NMR and ESI/MS. These reference compounds were then used to develop a sensitive HPLC assay to monitor SeMet oxidation to MetSeO. Formation of MetSeO in rat liver microsomes was time-, protein concentration-, SeMet concentration-, and NADPH-dependent. The microsomal activity exhibited a SeMet Km value (mean ±S.D.; n=4) of 0.91 ± 0.29 mM and a Vmax value of 44 ± 8.0 nmol MetSeO/mg protein/min. Inclusion of 1-benzylimidazole, superoxide dismutase or deferoxamine caused no inhibition of the rat liver microsomal activity. Because these results suggested the involvement of FMOs in the oxidation of SeMet in rat liver microsomes, formation of MetSeO was also examined using cDNA-expressed human and purified rat FMOs. The results showed that both rat and human FMO1 and FMO3 but not FMO5 can catalyze the reaction. The SeMet kinetic constants were obtained with purified rat liver FMO3 (Km = 0.11 mM, Vmax = 280 nmol/mg protein/min) and rat liver FMO1 (Km = 7.8 mM, Vmax = 1200 nmol/mg protein/min). Because SeMet has anti-cancer, chemopreventive, and toxic properties, the kinetic results suggest FMO3 is likely to play a role in the biological activities of SeMet at low exposure conditions. PMID:17173378

  8. Activity-Based Protein Profiling of Ammonia Monooxygenase in Nitrosomonas europaea

    SciTech Connect

    Bennett, Kristen; Sadler, Natalie C.; Wright, Aaron T.; Yeager, Chris; Hyman, Michael R.; Löffler, F. E.

    2016-01-29

    Nitrosomonas europaeais an aerobic nitrifying bacterium that oxidizes ammonia (NH3) to nitrite (NO2) through the sequential activities of ammonia monooxygenase (AMO) and hydroxylamine dehydrogenase (HAO). Many alkynes are mechanism-based inactivators of AMO, and here we describe an activity-based protein profiling method for this enzyme using 1,7-octadiyne (17OD) as a probe. Inactivation of NH4+-dependent O2uptake byN. europaeaby 17OD was time- and concentration-dependent. The effects of 17OD were specific for ammonia-oxidizing activity, andde novoprotein synthesis was required to reestablish this activity after cells were exposed to 17OD. Cells were reacted with Alexa Fluor 647 azide using a copper-catalyzed azide-alkyne cycloaddition (CuAAC) (click) reaction, solubilized, and analyzed by SDS-PAGE and infrared (IR) scanning. A fluorescent 28-kDa polypeptide was observed for cells previously exposed to 17OD but not for cells treated with either allylthiourea or acetylene prior to exposure to 17OD or for cells not previously exposed to 17OD. The fluorescent polypeptide was membrane associated and aggregated when heated with β-mercaptoethanol and SDS. The fluorescent polypeptide was also detected in cells pretreated with other diynes, but not in cells pretreated with structural homologs containing a single ethynyl functional group. The membrane fraction from 17OD-treated cells was conjugated with biotin-azide and solubilized in SDS. Streptavidin affinity-purified polypeptides were on-bead trypsin-digested, and amino acid sequences of the peptide fragments were determined by liquid chromatography-mass spectrometry (LC-MS) analysis. Peptide fragments from AmoA were the predominant peptides detected in 17OD-treated samples. In-gel digestion and matrix

  9. Differential Reactivity between Two Copper Sites in Peptidylglycine r-Hydroxylating Monooxygenase

    SciTech Connect

    E Chufan; S Prigge; X Siebert; B Eipper; R Mains; L Amzel

    2011-12-31

    Peptidylglycine {alpha}-hydroxylating monooxygenase (PHM) catalyzes the stereospecific hydroxylation of the C{alpha} of C-terminal glycine-extended peptides and proteins, the first step in the activation of many peptide hormones, growth factors, and neurotransmitters. The crystal structure of the enzyme revealed two nonequivalent Cu sites (Cu{sub M} and Cu{sub H}) separated by {approx}11 {angstrom}. In the resting state of the enzyme, Cu{sub M} is coordinated in a distorted tetrahedral geometry by one methionine, two histidines, and a water molecule. The coordination site of the water molecule is the position where external ligands bind. The Cu{sub H} has a planar T-shaped geometry with three histidines residues and a vacant position that could potentially be occupied by a fourth ligand. Although the catalytic mechanism of PHM and the role of the metals are still being debated, Cu{sub M} is identified as the metal involved in catalysis, while Cu{sub H} is associated with electron transfer. To further probe the role of the metals, we studied how small molecules such as nitrite (NO{sub 2}{sup -}), azide (N{sub 3}{sup -}), and carbon monoxide (CO) interact with the PHM copper ions. The crystal structure of an oxidized nitrite-soaked PHMcc, obtained by soaking for 20 h in mother liquor supplemented with 300 mM NaNO{sub 2}, shows that nitrite anion coordinates Cu{sub M} in an asymmetric bidentate fashion. Surprisingly, nitrite does not bind Cu{sub H}, despite the high concentration used in the experiments (nitrite/protein > 1000). Similarly, azide and carbon monoxide coordinate Cu{sub M} but not Cu{sub H} in the PHMcc crystal structures obtained by cocrystallization with 40 mM NaN{sub 3} and by soaking CO under 3 atm of pressure for 30 min. This lack of reactivity at the Cu{sub H} is also observed in the reduced form of the enzyme: CO binds Cu{sub M} but not Cu{sub H} in the structure of PHMcc obtained by exposure of a crystal to 3 atm CO for 15 min in the presence of 5

  10. Mechanistic studies of cyclohexanone monooxygenase: chemical properties of intermediates involved in catalysis.

    PubMed

    Sheng, D; Ballou, D P; Massey, V

    2001-09-18

    Cyclohexanone monooxygenase (CHMO), a bacterial flavoenzyme, carries out an oxygen insertion reaction on cyclohexanone to form a seven-membered cyclic product, epsilon-caprolactone. The reaction catalyzed involves the four-electron reduction of O2 at the expense of a two-electron oxidation of NADPH and a two-electron oxidation of cyclohexanone to form epsilon-caprolactone. Previous studies suggested the participation of either a flavin C4a-hydroperoxide or a flavin C4a-peroxide intermediate during the enzymatic catalysis [Ryerson, C. C., Ballou, D. P., and Walsh, C. (1982) Biochemistry 21, 2644-2655]. However, there was no kinetic or spectral evidence to distinguish between these two possibilities. In the present work we used double-mixing stopped-flow techniques to show that the C4a-flavin-oxygen adduct, which is formed rapidly from the reaction of oxygen with reduced enzyme in the presence of NADP, can exist in two states. When the reaction is carried out at pH 7.2, the first intermediate is a flavin C4a-peroxide with maximum absorbance at 366 nm; this intermediate becomes protonated at about 3 s(-1) to form what is believed to be the flavin C4a-hydroperoxide with maximum absorbance at 383 nm. These two intermediates can be interconverted by altering the pH, with a pK(a) of 8.4. Thus, at pH 9.0 the flavin C4a-peroxide persists mainly in the deprotonated form. Further kinetic studies also demonstrated that only the flavin C4a-peroxide intermediate could oxygenate the substrate, cyclohexanone. The requirement in catalysis of the deprotonated flavin C4a-peroxide, a nucleophile, is consistent with a Baeyer-Villiger rearrangement mechanism for the enzymatic oxygenation of cyclohexanone. In the course of these studies, the Kd for cyclohexanone to the C4a-peroxyflavin form of CHMO was determined to be approximately 1 microM. The rate-determining step in catalysis was shown to be the release of NADP from the oxidized enzyme.

  11. Induction of Flavin-Containing Monooxygenase in Mice by Oral Administration of Phellinus baumii (Agaricomycetes) Extract.

    PubMed

    Sainkhuu, Batbayar; Park, Beom Su; Kim, Ha Won

    2016-01-01

    Phellinus baumii is a yellow mushroom long used in alternative medicine in Korea and other central Asian countries. To identify genes affected by a single or 7-day oral administration of a water extract of Ph. Baumii, mouse liver tissue was analyzed using microarrays. The results showed that 8 and 23 genes were upregulated and 3 and 11 genes downregulated more than 3-fold by single and multiple oral administrations of 100 mg/kg PBE, respectively. Among the upregulated genes, the expression of 3 flavin-containing monooxygenase (Fmo) family genes, Fmo2-4, was upregulated in a concentration-dependent manner. The microarray analysis also showed that single and multiple administrations of PBE increased Fmo3 expression in the mouse liver by 5.1- and 17.6-fold, respectively. To validate the Fmo expression microarray data, polymerase chain reaction was used to confirm the induction of Fmo subclass genes. Mice were orally administered Ph. Baumii extract (PBE), Ph. Baumii water, or Ph. Baumii β-glucan fraction (PBG) for 7 days, and induction of the expression of the Fmo subclasses in the liver, lung, and kidney was investigated. Fmo2, Fmo3, and Fmo4 expression was induced by both PBE and PBG in the lung, liver, and kidney, respectively. However, no induction of Fmo1 and Fmo5 was detected. To investigate the metabolic acceleration of xenobiotic by PBE, carbendazim was orally administered to mice and its clearance from the blood analyzed. High-performance liquid chromatography analysis showed accelerated clearance of serum carbendazim by oral administration of PBE for 7 days, as evidenced by the reduced peak plasma concentration, time to reach the peak plasma concentration, and area under the curve values. Moreover, PBE increased the carbendazim clearance rate at the higher concentration. These data indicate that oral administration of PBE resulted in modulation of gene expression: PBE was responsible for the induction of Fmo2, Fmo3, and Fmo4 expression. PBE also accelerated

  12. Structure and Mechanism of Styrene Monooxygenase Reductase: New Insight into the FAD–Transfer Reaction†

    PubMed Central

    Morrison, Eliot; Kantz, Auric; Gassner, George T.; Sazinsky, Matthew H.

    2013-01-01

    The two–component flavoprotein styrene monooxygenase (SMO) from Pseudomonas putida S12 catalyzes the NADH– and FAD–dependent epoxidation of styrene to styrene oxide. In this study we investigate the mechanism of flavin reduction and transfer from the reductase (SMOB) to epoxidase (NSMOA) component and report our findings in light of the 2.2–Å crystal structure of SMOB. Upon rapidly mixing with NADH, SMOB forms an NADH→FADox charge–transfer intermediate and catalyzes a hydride–transfer reaction from NADH to FAD, with a rate constant of 49.1 ± 1.4 s−1, in a step that is coupled to the rapid dissociation of NAD+. Electrochemical and equilibrium–binding studies indicate that NSMOA binds FADhq ~13–times more tightly than SMOB, which supports a vectoral transfer of FADhq from the reductase to the epoxidase. After binding to NSMOA, FADhq rapidly reacts with molecular oxygen to form a stable C(4a)–hydroperoxide intermediate. The half–life of apoSMOB generated in the FAD–transfer reaction is increased ~21–fold, supporting the model of a protein–protein interaction between apoSMOB and NSMOA with the peroxide intermediate. The mechanisms of FAD–dissociation and transport from SMOB to NSMOA were probed by monitoring the competitive reduction of cytochrome c in the presence and absence of pyridine nucleotides. Based on these studies, we propose a model in which reduced FAD binds to SMOB in equilibrium between an unreactive, sequestered state (S–state) and more reactive, transfer state (T–state). Dissociation of NAD+ after the hydride transfer–reaction transiently populates the T–state, promoting the transfer of FADhq to NSMOA. The binding of pyridine nucleotides to SMOB–FADhq shifts the FADhq–binding equilibrium from the T–state to the S–state. Additionally, the 2.2–Å crystal structure of SMOB–FADox reported in this work is discussed in light of the pyridine nucleotide–gated flavin–transfer and electron

  13. Key amino acid residues in the regulation of soluble methane monooxygenase catalysis by component B.

    PubMed

    Brazeau, Brian J; Lipscomb, John D

    2003-05-20

    The regulatory component MMOB of soluble methane monooxygenase (sMMO) has been hypothesized to control access of substrates into the active site of the hydroxylase component (MMOH) through formation of a size specific channel or region of increased structural flexibility tuned to methane and O(2). Accordingly, a decrease in the size of four MMOB residues (N107G/S109A/S110A/T111A, the Quad mutant) was shown to accelerate the reaction of substrates larger than methane with the reactive MMOH intermediate Q [Wallar, B. J., and Lipscomb, J. D. (2001) Biochemistry 40, 2220-2233]. Here, this hypothesis is tested by construction of single and double mutations involving the residues of the Quad mutant. It is shown that mutations of residues that extend into the core structure of MMOB alter many aspects of the MMOH catalyzed reaction but do not mimic the effects of the Quad mutant. In contrast, the MMOB residues that are thought to form part of the interface in the MMOH-MMOB complex increase active site accessibility as observed for the Quad mutant. In particular, the mutant T111A mimics most of the effects of the Quad mutant; thus, Thr111 is proposed to most directly control access. Unexpectedly, mutation of Thr111 to the larger Tyr greatly increases the rate constant for the reaction of larger substrates such as ethane, furan, and nitrobenzene with Q while decreasing the rate constant for the reaction with methane. Other steps in the cycle are dramatically slowed, the regiospecificity for nitrobenzene oxidation is altered, and 10-fold more T111Y than wild-type MMOB is required to maximize the rate of turnover. Thus, T111Y appears to make a more extensive change in local interface structure that allows hydrocarbons at least as large as ethane to bind and react with Q similarly. As a result, the bond cleavage rates for methane, ethane, and their deuterated analogues are shown for the first time to correlate with bond strength in accord with a mechanism in which C-H bond

  14. Kinetics and activation thermodynamics of methane monooxygenase compound Q formation and reaction with substrates.

    PubMed

    Brazeau, B J; Lipscomb, J D

    2000-11-07

    The transient kinetics of formation and decay of the reaction cycle intermediates of the Methylosinus trichosporium OB3b methane monooxygenase (MMO) catalytic cycle are studied as a function of temperature and substrate type and deuteration. Kinetic evidence is presented for the existence of three intermediates termed compounds O, P, and P forming after the addition of O(2) to diferrous MMO hydroxylase (H(r)) and before the formation of the reactive intermediate compound Q. The Arrhenius plots for these reactions are linear and independent of substrate concentration and type, showing that substrate does not participate directly in the oxygen activation phase of the catalytic cycle. Analysis of the transient kinetic data revealed only small changes relative to the weak optical spectrum of H(r) for any of these intermediates. In contrast, large changes in the 430 nm spectral region are associated with the formation of Q. The decay reaction of Q exhibits an apparent first-order concentration dependence for all substrates tested, and the observed rate constant depends on the substrate type. The kinetics of the decay reaction of Q yield a nonlinear Arrhenius plot when methane is the substrate, and the rates in both segments of the plot increase linearly with methane concentration. Together these observations suggest that at least two reactions with a methane concentration dependence, and perhaps two methane molecules, are involved in the decay process. When CD(4) is used as the substrate, a large isotope effect and a linear Arrhenius plot are observed. Analogous plots for all other MMO substrates tested (e.g., ethane) are linear, and no isotope effect for deuterated analogues is observed. This demonstrates that a step other than C-H bond breaking is rate limiting for alternative MMO substrates. A two step Q decay mechanism is proposed that provides an explanation for the lack of an isotope effect for alternative MMO substrates and the fact that rate of oxidation of

  15. Recombinant heteromeric phenylalanine monooxygenase and the oxygenation of carbon and sulfur substrates.

    PubMed

    Boonyapiwat, Boontarika; Mitchell, Stephen C; Steventon, Glyn B

    2011-04-01

    The aim of this investigation was to provide in-vitro enzyme kinetic data to support the hypothesis that the in-vivo heterozygous dominant phenotype for phenylalanine monooxygenase (hPAH) was responsible for the S-oxidation polymorphism in the metabolism of S-carboxymethyl-l-cysteine reported in humans. Using a dual-vector expression strategy for the co-production of wild-type and mutant human hPAH subunits we report for the first time the kinetic parameters (K(m) , V(max) , CL(E) ) for the C-oxidation of l-phenylalanine and the S-oxidation of S-carboxymethyl-l-cysteine in homomeric wild-type, heteromeric mutant and homomeric mutant hPAH proteins in vitro. A PRO(TM) dual-vector bacterial expression system was used to produce the required hPAH proteins. Enzyme activity was determined by HPLC with fluorescence detection. The heteromeric hPAH proteins (I65T, R68S, R158Q, I174T, R261Q, V338M, R408W and Y414C) all showed significantly decreased V(max) and CL(E) values when compared to the homomeric wild-type hPAH enzyme. For both substrates, all calculated K(m) values were significantly higher than homomeric wild-type hPAH enzyme, with the exception of I65T, R68S and Y414C heteromeric hPAH proteins employing l-phenylalanine as substrate. The net outcome for the heteromeric mutant hPAH proteins was a decrease significantly more dramatic for S-carboxymethyl-l-cysteine S-oxidation (1.0-18.8% of homomeric wild-type hPAH activity) when compared to l-phenylalanine C-oxidation (25.9-52.9% of homomeric wild-type hPAH activity) as a substrate. Heteromeric hPAH enzyme may be related to the variation in S-carboxymethyl-l-cysteine S-oxidation capacity observed in humans. © 2011 The Authors. JPP © 2011 Royal Pharmaceutical Society.

  16. Methane monooxygenase component B mutants alter the kinetics of steps throughout the catalytic cycle.

    PubMed

    Wallar, B J; Lipscomb, J D

    2001-02-20

    Component interactions play important roles in the regulation of catalysis by methane monooxygenase (MMO). The binding of component B (MMOB) to the hydroxylase component (MMOH) has been shown in previous studies to cause structural changes in MMOH that result in altered thermodynamic and kinetic properties during the reduction and oxygen binding steps of the catalytic cycle. Here, specific amino acid residues of MMOB that play important roles in the interconversion of several intermediates of the MMO cycle have been identified. Both of the histidine residues in Methylosinus trichosporium OB3b MMOB (H5 and H33) were chemically modified by diethylpyrocarbonate (DEPC). Although the DEPC--MMOB species exhibited only minor changes relative to unmodified MMOB in steady-state MMO turnover, large decreases in the formation rate constants of the reaction cycle intermediates, compound P and compound Q, were observed. The site specific mutants H5A, H33A, and H5A/H33A were made and characterized. H5A and wild type MMOB elicited similar steady-state and transient kinetics, although the mutant caused a slightly lower rate constant for Q formation. Conversely, H33A exhibited a >50-fold decrease in the P formation rate constant, which resulted in slower formation of Q. The kinetics of the double mutant (H5A/H33A) were similar to those of H33A, suggesting that the highly conserved residue, H33, has the most significant effect on the efficient progress of the cycle. Ongoing NMR investigations of residues perturbed by formation of the MMOH-MMOB complex suggested construction of the MMOB N107G/S109A/S110A/T111A quadruple mutant. This mutant was found to elicit a nearly 2-fold increase in specific activity for steady-state MMO turnover of large substrates such as furan and nitrobenzene but caused no similar increase for the physiological substrate, methane. While the quadruple mutant did not have a significant effect on P and Q formation, it caused an almost 3-fold increase in the

  17. Roles of the methane monooxygenase reductase component in the regulation of catalysis.

    PubMed

    Liu, Y; Nesheim, J C; Paulsen, K E; Stankovich, M T; Lipscomb, J D

    1997-04-29

    The reductase component (MMOR) of the soluble methane monooxygenase isolated from Methylosinus trichosporium OB3b catalyzes transfer of 2e- from NADH to the hydroxylase component (MMOH) where oxygen activation and substrate oxidation occur. It is shown here that MMOR can also exert regulatory effects on catalysis by binding to MMOH or to the binary complex of MMOH and component B (MMOB), another regulatory protein. MMOR alters the oxidation-reduction potentials of the dinuclear iron cluster at the active site of MMOH. Although little change is observed in the potential for the first electron transfer to the cluster (E(1)0' = 76 mV), the E(2)0' potential value for the second electron transfer is increased from 21 to 125 mV. This shift provides a larger driving force for electron transfer from MMOR and favors transfer of two rather than one electron as required by catalysis. Similar positive shifts in potential are observed even in the presence of MMOB which has been shown to cause a 132 mV negative shift in the midpoint potential of MMOH in the absence of MMOR. MMOR is also shown to decrease the rate of reaction between the fully reduced MMOH-MMOB and O2 approximately 20-fold at 4 degrees C. However, the time course of the key catalytic cycle intermediate that can react with substates, compound Q, is unaffected. This implies a compensating faster decay of one or more of the intermediates that occur between diferrous MMOH and compound Q in the reaction cycle, thereby limiting potential nonproductive autodecay of these intermediates. Accordingly, an increase in single turnover product yield is observed in the presence of MMOR. Interestingly, MMOR can cause the redox potential increases, changes in rates, and the increase in product yield when present at only 10% of the concentration of MMOH active sites. Substrate binding is shown to induce negligible changes in the redox potentials. Two alternative regulatory schemes are presented based on (i) thermodynamic coupling

  18. Crystallographic Analysis of Active Site Contributions to Regiospecificity in the Diiron Enzyme Toluene 4-Monooxygenase

    SciTech Connect

    Bailey, Lucas J.; Acheson, Justin F.; McCoy, Jason G.; Elsen, Nathaniel L.; Phillips, Jr., George N.; Fox, Brian G.

    2014-10-02

    Crystal structures of toluene 4-monooxygenase hydroxylase in complex with reaction products and effector protein reveal active site interactions leading to regiospecificity. Complexes with phenolic products yield an asymmetric {mu}-phenoxo-bridged diiron center and a shift of diiron ligand E231 into a hydrogen bonding position with conserved T201. In contrast, complexes with inhibitors p-NH{sub 2}-benzoate and p-Br-benzoate showed a {mu}-1,1 coordination of carboxylate oxygen between the iron atoms and only a partial shift in the position of E231. Among active site residues, F176 trapped the aromatic ring of products against a surface of the active site cavity formed by G103, E104 and A107, while F196 positioned the aromatic ring against this surface via a {pi}-stacking interaction. The proximity of G103 and F176 to the para substituent of the substrate aromatic ring and the structure of G103L T4moHD suggest how changes in regiospecificity arise from mutations at G103. Although effector protein binding produced significant shifts in the positions of residues along the outer portion of the active site (T201, N202, and Q228) and in some iron ligands (E231 and E197), surprisingly minor shifts (<1 {angstrom}) were produced in F176, F196, and other interior residues of the active site. Likewise, products bound to the diiron center in either the presence or absence of effector protein did not significantly shift the position of the interior residues, suggesting that positioning of the cognate substrates will not be strongly influenced by effector protein binding. Thus, changes in product distributions in the absence of the effector protein are proposed to arise from differences in rates of chemical steps of the reaction relative to motion of substrates within the active site channel of the uncomplexed, less efficient enzyme, while structural changes in diiron ligand geometry associated with cycling between diferrous and diferric states are discussed for their potential

  19. Evolution of substrate specificity in bacterial AA10 lytic polysaccharide monooxygenases.

    PubMed

    Book, Adam J; Yennamalli, Ragothaman M; Takasuka, Taichi E; Currie, Cameron R; Phillips, George N; Fox, Brian G

    2014-01-01

    Understanding the diversity of lignocellulose-degrading enzymes in nature will provide insights for the improvement of cellulolytic enzyme cocktails used in the biofuels industry. Two families of enzymes, fungal AA9 and bacterial AA10, have recently been characterized as crystalline cellulose or chitin-cleaving lytic polysaccharide monooxygenases (LPMOs). Here we analyze the sequences, structures, and evolution of LPMOs to understand the factors that may influence substrate specificity both within and between these enzyme families. Comparative analysis of sequences, solved structures, and homology models from AA9 and AA10 LPMO families demonstrated that, although these two LPMO families are highly conserved, structurally they have minimal sequence similarity outside the active site residues. Phylogenetic analysis of the AA10 family identified clades with putative chitinolytic and cellulolytic activities. Estimation of the rate of synonymous versus non-synonymous substitutions (dN/dS) within two major AA10 subclades showed distinct selective pressures between putative cellulolytic genes (subclade A) and CBP21-like chitinolytic genes (subclade D). Estimation of site-specific selection demonstrated that changes in the active sites were strongly negatively selected in all subclades. Furthermore, all codons in the subclade D had dN/dS values of less than 0.7, whereas codons in the cellulolytic subclade had dN/dS values of greater than 1.5. Positively selected codons were enriched at sites localized on the surface of the protein adjacent to the active site. The structural similarity but absence of significant sequence similarity between AA9 and AA10 families suggests that these enzyme families share an ancient ancestral protein. Combined analysis of amino acid sites under Darwinian selection and structural homology modeling identified a subclade of AA10 with diversifying selection at different surfaces, potentially used for cellulose-binding and protein

  20. Fusion proteins of an enoate reductase and a Baeyer-Villiger monooxygenase facilitate the synthesis of chiral lactones.

    PubMed

    Peters, Christin; Rudroff, Florian; Mihovilovic, Marko D; T Bornscheuer, Uwe

    2017-01-01

    Nature uses the advantages of fusion proteins for multi-step reactions to facilitate the metabolism in cells as the conversion of substrates through intermediates to the final product can take place more rapidly and with less side-product formation. In a similar fashion, also for enzyme cascade reactions, the fusion of biocatalysts involved can be advantageous. In the present study, we investigated fusion of an alcohol dehydrogenase (ADH), an enoate reductase (ERED) and a Baeyer-Villiger monooxygenase (BVMO) to enable the synthesis of (chiral) lactones starting from unsaturated alcohols as substrates. The domain order and various linkers were studied to find optimal conditions with respect to expression levels and enzymatic activities. Best results were achieved for the ERED xenobiotic reductase B (XenB) from Pseudomonas putida and the cyclohexanone monooxygenase (CHMO) from Acinetobacter sp., whereas none of the ADHs studied could be fused successfully. This fusion protein together with separately supplied ADH resulted in similar reaction rates in in vivo biocatalysis reactions. After 1.5 h we could detect 40% more dihydrocarvone lactone in in vivo reactions with the fusion protein and ADH then with the single enzymes.

  1. Directed evolution of phenylacetone monooxygenase as an active catalyst for the Baeyer-Villiger conversion of cyclohexanone to caprolactone.

    PubMed

    Parra, Loreto P; Acevedo, Juan P; Reetz, Manfred T

    2015-07-01

    Phenylacetone monooxygenase (PAMO) is an exceptionally robust Baeyer-Villiger monooxygenase, which makes it ideal for potential industrial applications. However, its substrate scope is limited, unreactive cyclohexanone being a prominent example. Such a limitation is unfortunate, because this particular transformation in an ecologically viable manner would be highly desirable, the lactone and the respective lactam being of considerable interest as monomers in polymer science. We have applied directed evolution in search of an active mutant for this valuable C-C activating reaction. Using iterative saturation mutagenesis (ISM), several active mutants were evolved, with only a minimal trade-off in terms of stability. The best mutants allow for quantitative conversion of 2 mM cyclohexanone within 1 h reaction time. In order to circumvent the NADP(+) regeneration problem, whole E. coli resting cells were successfully applied. Molecular dynamics simulations and induced fit docking throw light on the origin of enhanced PAMO activity. The PAMO mutants constitute ideal starting points for future directed evolution optimization necessary for an industrial process. © 2015 Wiley Periodicals, Inc.

  2. Cloning, Expression, Characterization, and Biocatalytic Investigation of the 4-Hydroxyacetophenone Monooxygenase from Pseudomonas putida JD1▿ †

    PubMed Central

    Rehdorf, Jessica; Zimmer, Christian L.; Bornscheuer, Uwe T.

    2009-01-01

    While the number of available recombinant Baeyer-Villiger monooxygenases (BVMOs) has grown significantly over the last few years, there is still the demand for other BVMOs to expand the biocatalytic diversity. Most BVMOs that have been described are dedicated to convert efficiently cyclohexanone and related cyclic aliphatic ketones. To cover a broader range of substrate types and enantio- and/or regioselectivities, new BVMOs have to be discovered. The gene encoding a BVMO identified in Pseudomonas putida JD1 converting aromatic ketones (HAPMO; 4-hydroxyacetophenone monooxygenase) was amplified from genomic DNA using SiteFinding-PCR, cloned, and functionally expressed in Escherichia coli. Furthermore, four other open reading frames could be identified clustered around this HAPMO. It has been suggested that these proteins, including the HAPMO, might be involved in the degradation of 4-hydroxyacetophenone. Substrate specificity studies revealed that a large variety of other arylaliphatic ketones are also converted via Baeyer-Villiger oxidation into the corresponding esters, with preferences for para-substitutions at the aromatic ring. In addition, oxidation of aldehydes and some heteroaromatic compounds was observed. Cycloketones and open-chain ketones were not or poorly accepted, respectively. It was also found that this enzyme oxidizes aromatic ketones such as 3-phenyl-2-butanone with excellent enantioselectivity (E ≫100). PMID:19251889

  3. The substrate-bound crystal structure of a Baeyer-Villiger monooxygenase exhibits a Criegee-like conformation.

    PubMed

    Yachnin, Brahm J; Sprules, Tara; McEvoy, Michelle B; Lau, Peter C K; Berghuis, Albert M

    2012-05-09

    The Baeyer-Villiger monooxygenases (BVMOs) are a family of bacterial flavoproteins that catalyze the synthetically useful Baeyer-Villiger oxidation reaction. This involves the conversion of ketones into esters or cyclic ketones into lactones by introducing an oxygen atom adjacent to the carbonyl group. The BVMOs offer exquisite regio- and enantiospecificity while acting on a wide range of substrates. They use only NADPH and oxygen as cosubstrates, and produce only NADP(+) and water as byproducts, making them environmentally attractive for industrial purposes. Here, we report the first crystal structure of a BVMO, cyclohexanone monooxygenase (CHMO) from Rhodococcus sp. HI-31 in complex with its substrate, cyclohexanone, as well as NADP(+) and FAD, to 2.4 Å resolution. This structure shows a drastic rotation of the NADP(+) cofactor in comparison to previously reported NADP(+)-bound structures, as the nicotinamide moiety is no longer positioned above the flavin ring. Instead, the substrate, cyclohexanone, is found at this location, in an appropriate position for the formation of the Criegee intermediate. The rotation of NADP(+) permits the substrate to gain access to the reactive flavin peroxyanion intermediate while preventing it from diffusing out of the active site. The structure thus reveals the conformation of the enzyme during the key catalytic step. CHMO is proposed to undergo a series of conformational changes to gradually move the substrate from the solvent, via binding in a solvent excluded pocket that dictates the enzyme's chemospecificity, to a location above the flavin-peroxide adduct where catalysis occurs.

  4. The Substrate-Bound Crystal Structure of a Baeyer–Villiger Monooxygenase Exhibits a Criegee-like Conformation

    PubMed Central

    2012-01-01

    The Baeyer–Villiger monooxygenases (BVMOs) are a family of bacterial flavoproteins that catalyze the synthetically useful Baeyer–Villiger oxidation reaction. This involves the conversion of ketones into esters or cyclic ketones into lactones by introducing an oxygen atom adjacent to the carbonyl group. The BVMOs offer exquisite regio- and enantiospecificity while acting on a wide range of substrates. They use only NADPH and oxygen as cosubstrates, and produce only NADP+ and water as byproducts, making them environmentally attractive for industrial purposes. Here, we report the first crystal structure of a BVMO, cyclohexanone monooxygenase (CHMO) from Rhodococcus sp. HI-31 in complex with its substrate, cyclohexanone, as well as NADP+ and FAD, to 2.4 Å resolution. This structure shows a drastic rotation of the NADP+ cofactor in comparison to previously reported NADP+-bound structures, as the nicotinamide moiety is no longer positioned above the flavin ring. Instead, the substrate, cyclohexanone, is found at this location, in an appropriate position for the formation of the Criegee intermediate. The rotation of NADP+ permits the substrate to gain access to the reactive flavin peroxyanion intermediate while preventing it from diffusing out of the active site. The structure thus reveals the conformation of the enzyme during the key catalytic step. CHMO is proposed to undergo a series of conformational changes to gradually move the substrate from the solvent, via binding in a solvent excluded pocket that dictates the enzyme’s chemospecificity, to a location above the flavin–peroxide adduct where catalysis occurs. PMID:22506764

  5. Factors affecting the relative importance of amine oxidases and monooxygenases in the in vivo metabolism of xenobiotic amines in humans.

    PubMed

    Strolin Benedetti, M; Tipton, K F; Whomsley, R; Baltes, E

    2007-01-01

    The monooxygenases and the amine oxidases (AOs) are the major enzyme systems involved in vivo in the oxidative metabolism of xenobiotic amines in humans. With the exception of the inhibition of the metabolism of tyramine ingested by subjects taking inhibitors of MAO-A or of both MAO-A and -B, which has been extensively investigated, the involvement of the monoamine oxidases in xenobiotic amine metabolism (drugs in particular) has been largely neglected. Furthermore, with the exception of amlodipine, there have been essentially no studies on the metabolism of drug amines by amine oxidases such as SSAOs and PAOs in humans. In contrast, monooxygenases (CYP isoenzymes, and to a lesser extent, FMOs) have been extensively investigated in terms of their involvement in xenobiotic metabolism. It is possible that the contribution of AOs to the overall metabolism of xenobiotic amines in humans has been underestimated, or erroneously estimated, as most investigations of drug metabolism have been performed using in vitro test systems optimized for CYP activity, such as liver microsomes, and most investigations of drug metabolism in vivo in humans have identified only the final, stable metabolites.

  6. Continuous testing system for Baeyer-Villiger biooxidation using recombinant Escherichia coli expressing cyclohexanone monooxygenase encapsulated in polyelectrolyte complex capsules.

    PubMed

    Bučko, Marek; Schenkmayerová, Andrea; Gemeiner, Peter; Vikartovská, Alica; Mihovilovič, Marko D; Lacík, Igor

    2011-08-10

    An original strategy for universal laboratory testing of Baeyer-Villiger monooxygenases based on continuous packed-bed minireactor connected with flow calorimeter and integrated with bubble-free oxygenation is reported. Model enantioselective Baeyer-Villiger biooxidations of rac-bicyclo[3.2.0]hept-2-en-6-one to corresponding lactones (1R,5S)-3-oxabicyclo-[3.3.0]oct-6-en-3-one and (1S,5R)-2-oxabicyclo-[3.3.0]oct-6-en-3-one as important chiral synthons for the synthesis of bioactive compounds were performed in the minireactor equipped with a column packed with encapsulated recombinant cells Escherichia coli overexpressing cyclohexanone monooxygenase. The cells were encapsulated in polyelectrolyte complex capsules formed by reaction of oppositely charged polymers utilizing highly reproducible and controlled encapsulation process. Encapsulated cells tested in minireactor exhibited high operational stability with 4 complete substrate conversions to products and 6 conversions above 80% within 14 repeated consecutive biooxidation tests. Moreover, encapsulated cells showed high enzyme stability during 91 days of storage with substrate conversions above 80% up to 60 days of storage. Furthermore, usable thermometric signal of Baeyer-Villiger biooxidation obtained by flow calorimetry using encapsulated cells was utilized for preparatory kinetic study in order to guarantee sub-inhibitory initial substrate concentration for biooxidation tests.

  7. Monooxygenase activity and contaminant burdens of pipping heron embryos in Virginia, the Great Lakes and San Francisco Bay

    USGS Publications Warehouse

    Rattner, B.A.; Melancon, M.J.; Custer, T.W.; Hothem, R.L.; King, K.A.; LeCaptain, L.J.; Spann, J.W.

    1991-01-01

    Black-crowned night-heron (Nvcticorax nvcticorax) pipping embryos were studied from undisturbed (Chincoteague National Wildl ife Refuge, VA) and industrialized (Cat Island, Green Bay WI, and Bair and W. Marin Islands, San Francisco Bay, CA) locations. Hepatic aryl hydrocarbon hydroxylase (AHH) , ethoxyresorufin-O-dealkylase, (EROD), benzyloxyROD (BROD), pentoxyROD (PROD) and ethoxycoumarinOD (ECOD) activities and burdens of organochlorines (embryo + yolk sac - liver) were quantified. AHH, BROD, ECOD and EROD were induced up to 100-fold (P<.O5) in embryos from Cat Island compared to the other sites. Greatest burdens of total PCBs and p,p?DDE were detected in Cat Island embryos. Monooxygenase activities (AHH, BROD, ECOD and EROD) and PCB concentrations were significantly correlated (r=O.50 to 0.72). These and other data indicate that monooxygenases may be rapid and inexpensive biomarkers of exposure to some PCB congeners. Current efforts include determination of PCB congeners and other contaminants in these embryos, additional characterization of the induced P-450 isozymes, and expanding the study to include heron embryos and nestlings at other estuaries.

  8. Isolation and initial characterization of a novel type of Baeyer-Villiger monooxygenase activity from a marine microorganism.

    PubMed

    Willetts, Andrew; Joint, Ian; Gilbert, Jack A; Trimble, William; Mühling, Martin

    2012-07-01

    A novel type of Baeyer-Villiger monooxygenase (BVMO) has been found in a marine strain of Stenotrophomonas maltophila strain PML168 that was isolated from a temperate intertidal zone. The enzyme is able to use NADH as the source of reducing power necessary to accept the atom of diatomic oxygen not incorporated into the oxyfunctionalized substrate. Growth studies have establish that the enzyme is inducible, appears to serve a catabolic role, and is specifically induced by one or more unidentified components of seawater as well as various anthropogenic xenobiotic compounds. A blast search of the primary sequence of the enzyme, recovered from the genomic sequence of the isolate, has placed this atypical BVMO in the context of the several hundred known members of the flavoprotein monooxygenase superfamily. A particular feature of this BVMO lies in its truncated C-terminal domain, which results in a relatively small protein (357 amino acids; 38.4 kDa). In addition, metagenomic screening has been conducted on DNA recovered from an extensive range of marine environmental samples to gauge the relative abundance and distribution of similar enzymes within the global marine microbial community. Although low, abundance was detected in samples from many marine provinces, confirming the potential for biodiscovery in marine microorganisms.

  9. A comparative study on the activity of fungal lytic polysaccharide monooxygenases for the depolymerization of cellulose in soybean spent flakes.

    PubMed

    Pierce, Brian C; Agger, Jane Wittrup; Zhang, Zhenghong; Wichmann, Jesper; Meyer, Anne S

    2017-09-08

    Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes capable of the oxidative breakdown of polysaccharides. They are of industrial interest due to their ability to enhance the enzymatic depolymerization of recalcitrant substrates by glycoside hydrolases. In this paper, twenty-four lytic polysaccharide monooxygenases (LPMOs) expressed in Trichoderma reesei were evaluated for their ability to oxidize the complex polysaccharides in soybean spent flakes, an abundant and industrially relevant substrate. TrCel61A, a soy-polysaccharide-active AA9 LPMO from T. reesei, was used as a benchmark in this evaluation. In total, seven LPMOs demonstrated activity on pretreated soy spent flakes, with the products from enzymatic treatments evaluated using mass spectrometry and high performance anion exchange chromatography. The hydrolytic boosting effect of the top-performing enzymes was evaluated in combination with endoglucanase and beta-glucosidase. Two enzymes (TrCel61A and Aspte6) showed the ability to release more than 36% of the pretreated soy spent flake glucose - a greater than 75% increase over the same treatment without LPMO addition. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. Interactions between the HIV-1 Unspliced mRNA and Host mRNA Decay Machineries

    PubMed Central

    Toro-Ascuy, Daniela; Rojas-Araya, Bárbara; Valiente-Echeverría, Fernando; Soto-Rifo, Ricardo

    2016-01-01

    The human immunodeficiency virus type-1 (HIV-1) unspliced transcript is used both as mRNA for the synthesis of structural proteins and as the packaged genome. Given the presence of retained introns and instability AU-rich sequences, this viral transcript is normally retained and degraded in the nucleus of host cells unless the viral protein REV is present. As such, the stability of the HIV-1 unspliced mRNA must be particularly controlled in the nucleus and the cytoplasm in order to ensure proper levels of this viral mRNA for translation and viral particle formation. During its journey, the HIV-1 unspliced mRNA assembles into highly specific messenger ribonucleoproteins (mRNPs) containing many different host proteins, amongst which are well-known regulators of cytoplasmic mRNA decay pathways such as up-frameshift suppressor 1 homolog (UPF1), Staufen double-stranded RNA binding protein 1/2 (STAU1/2), or components of miRNA-induced silencing complex (miRISC) and processing bodies (PBs). More recently, the HIV-1 unspliced mRNA was shown to contain N6-methyladenosine (m6A), allowing the recruitment of YTH N6-methyladenosine RNA binding protein 2 (YTHDF2), an m6A reader host protein involved in mRNA decay. Interestingly, these host proteins involved in mRNA decay were shown to play positive roles in viral gene expression and viral particle assembly, suggesting that HIV-1 interacts with mRNA decay components to successfully accomplish viral replication. This review summarizes the state of the art in terms of the interactions between HIV-1 unspliced mRNA and components of different host mRNA decay machineries. PMID:27886048

  11. Interactions between the HIV-1 Unspliced mRNA and Host mRNA Decay Machineries.

    PubMed

    Toro-Ascuy, Daniela; Rojas-Araya, Bárbara; Valiente-Echeverría, Fernando; Soto-Rifo, Ricardo

    2016-11-23

    The human immunodeficiency virus type-1 (HIV-1) unspliced transcript is used both as mRNA for the synthesis of structural proteins and as the packaged genome. Given the presence of retained introns and instability AU-rich sequences, this viral transcript is normally retained and degraded in the nucleus of host cells unless the viral protein REV is present. As such, the stability of the HIV-1 unspliced mRNA must be particularly controlled in the nucleus and the cytoplasm in order to ensure proper levels of this viral mRNA for translation and viral particle formation. During its journey, the HIV-1 unspliced mRNA assembles into highly specific messenger ribonucleoproteins (mRNPs) containing many different host proteins, amongst which are well-known regulators of cytoplasmic mRNA decay pathways such as up-frameshift suppressor 1 homolog (UPF1), Staufen double-stranded RNA binding protein 1/2 (STAU1/2), or components of miRNA-induced silencing complex (miRISC) and processing bodies (PBs). More recently, the HIV-1 unspliced mRNA was shown to contain N⁶-methyladenosine (m⁶A), allowing the recruitment of YTH N⁶-methyladenosine RNA binding protein 2 (YTHDF2), an m⁶A reader host protein involved in mRNA decay. Interestingly, these host proteins involved in mRNA decay were shown to play positive roles in viral gene expression and viral particle assembly, suggesting that HIV-1 interacts with mRNA decay components to successfully accomplish viral replication. This review summarizes the state of the art in terms of the interactions between HIV-1 unspliced mRNA and components of different host mRNA decay machineries.

  12. EXPRESSION OF BRANCHIAL FLAVIN-CONTAINING MONOOXYGENASE IS DIRECTLY CORRELATED WITH SALINITY-INDUCED ALDICARB TOXICITY IN THE EURYHALINE FISH (ORYZIAS LATIPES). (R826109)

    EPA Science Inventory

    Abstract

    Earlier studies in our laboratory have demonstrated a reduction of flavin-containing monooxygenase (FMO) activity when salt-water adapted euryhaline fish were transferred to water of less salinity. Since FMOs have been shown to be responsible for the bioact...

  13. EXPRESSION OF BRANCHIAL FLAVIN-CONTAINING MONOOXYGENASE IS DIRECTLY CORRELATED WITH SALINITY-INDUCED ALDICARB TOXICITY IN THE EURYHALINE FISH (ORYZIAS LATIPES). (R826109)

    EPA Science Inventory

    Abstract

    Earlier studies in our laboratory have demonstrated a reduction of flavin-containing monooxygenase (FMO) activity when salt-water adapted euryhaline fish were transferred to water of less salinity. Since FMOs have been shown to be responsible for the bioact...

  14. Cloning, sequencing, and analysis of a gene cluster from Chelatobacter heintzii ATCC 29600 encoding nitrilotriacetate monooxygenase and NADH:flavin mononucleotide oxidoreductase.

    PubMed Central

    Xu, Y; Mortimer, M W; Fisher, T S; Kahn, M L; Brockman, F J; Xun, L

    1997-01-01

    Nitrilotriacetate (NTA) is an important chelating agent in detergents and has also been used extensively in processing radionuclides. In Chelatobacter heintzii ATCC 29600, biodegradation of NTA is initiated by NTA monooxygenase that oxidizes NTA to iminodiacetate and glyoxylate. The NTA monooxygenase activity requires two component proteins, component A and component B, but the function of each component is unclear. We have cloned and sequenced a gene cluster encoding components A and B (nmoA and nmoB) and two additional open reading frames, nmoR and nmoT, downstream of nmoA. Based on sequence similarities, nmoR and nmoT probably encode a regulatory protein and a transposase, respectively. The NmoA sequence was similar to a monooxygenase that uses reduced flavin mononucleotide (FMNH2) as reductant; NmoB was similar to an NADH:flavin mononucleotide (FMN) oxidoreductase. On the basis of this information, we tested the function of each component. Purified component B was shown to be an NADH:FMN oxidoreductase, and its activity could be separated from that of component A. When the Photobacterium fischeri NADH:FMN oxidoreductase was substituted for component B in the complete reaction, NTA was oxidized, showing that the substrate specificity of the reaction resides in component A. Component A is therefore an NTA monooxygenase that uses FMNH2 and O2 to oxidize NTA, and component B is an NADH:FMN oxidoreductase that provides FMNH2 for NTA oxidation. PMID:9023192

  15. Binary function of mRNA.

    PubMed

    Kloc, Malgorzata; Foreman, Victor; Reddy, Sriyutha A

    2011-11-01

    Since the discovery of messenger RNA (mRNA) over half a century ago, the assumption has always been that the only function of mRNA is to make a protein. However, recent studies of prokaryotic and eukaryotic organisms unexpectedly show that some mRNAs may be functionally binary and have additional structural functions that are unrelated to their translation product. These findings imply that some of the phenotypic features of cells and organisms can also be binary, that is, they depend both on the function of a protein and the independent structural function of its mRNA. In this review, we will discuss this concept within the framework of multifunctional RNA molecules and the RNA World Hypothesis. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  16. Self-amplifying mRNA vaccines.

    PubMed

    Brito, Luis A; Kommareddy, Sushma; Maione, Domenico; Uematsu, Yasushi; Giovani, Cinzia; Berlanda Scorza, Francesco; Otten, Gillis R; Yu, Dong; Mandl, Christian W; Mason, Peter W; Dormitzer, Philip R; Ulmer, Jeffrey B; Geall, Andrew J

    2015-01-01

    This chapter provides a brief introduction to nucleic acid-based vaccines and recent research in developing self-amplifying mRNA vaccines. These vaccines promise the flexibility of plasmid DNA vaccines with enhanced immunogenicity and safety. The key to realizing the full potential of these vaccines is efficient delivery of nucleic acid to the cytoplasm of a cell, where it can amplify and express the encoded antigenic protein. The hydrophilicity and strong net negative charge of RNA impedes cellular uptake. To overcome this limitation, electrostatic complexation with cationic lipids or polymers and physical delivery using electroporation or ballistic particles to improve cellular uptake has been evaluated. This chapter highlights the rapid progress made in using nonviral delivery systems for RNA-based vaccines. Initial preclinical testing of self-amplifying mRNA vaccines has shown nonviral delivery to be capable of producing potent and robust innate and adaptive immune responses in small animals and nonhuman primates. Historically, the prospect of developing mRNA vaccines was uncertain due to concerns of mRNA instability and the feasibility of large-scale manufacturing. Today, these issues are no longer perceived as barriers in the widespread implementation of the technology. Currently, nonamplifying mRNA vaccines are under investigation in human clinical trials and can be produced at a sufficient quantity and quality to meet regulatory requirements. If the encouraging preclinical data with self-amplifying mRNA vaccines are matched by equivalently positive immunogenicity, potency, and tolerability in human trials, this platform could establish nucleic acid vaccines as a versatile new tool for human immunization.

  17. Identification of the Lomofungin Biosynthesis Gene Cluster and Associated Flavin-Dependent Monooxygenase Gene in Streptomyces lomondensis S015

    PubMed Central

    Zhang, Chunxiao; Sheng, Chaolan; Wang, Wei; Hu, Hongbo; Peng, Huasong; Zhang, Xuehong

    2015-01-01

    Streptomyces lomondensis S015 synthesizes the broad-spectrum phenazine antibiotic lomofungin. Whole genome sequencing of this strain revealed a genomic locus consisting of 23 open reading frames that includes the core phenazine biosynthesis gene cluster lphzGFEDCB. lomo10, encoding a putative flavin-dependent monooxygenase, was also identified in this locus. Inactivation of lomo10 by in-frame partial deletion resulted in the biosynthesis of a new phenazine metabolite, 1-carbomethoxy-6-formyl-4,9-dihydroxy-phenazine, along with the absence of lomofungin. This result suggests that lomo10 is responsible for the hydroxylation of lomofungin at its C-7 position. This is the first description of a phenazine hydroxylation gene in Streptomyces, and the results of this study lay the foundation for further investigation of phenazine metabolite biosynthesis in Streptomyces. PMID:26305803

  18. Hydroxylation of Longiborneol by a Clm2-Encoded CYP450 Monooxygenase to Produce Culmorin in Fusarium graminearum.

    PubMed

    Bahadoor, Adilah; Schneiderman, Danielle; Gemmill, Larissa; Bosnich, Whynn; Blackwell, Barbara; Melanson, Jeremy E; McRae, Garnet; Harris, Linda J

    2016-01-22

    A second structural gene required for culmorin biosynthesis in the plant pathogen Fusarium graminearum is described. Clm2 encodes a regio- and stereoselective cytochrome P450 monooxygenase for C-11 of longiborneol (1). Clm2 gene disruptants were grown in liquid culture and assessed for culmorin production via HPLC-evaporative light scattering detection. The analysis indicated a complete loss of culmorin (2) from the liquid culture of the ΔClm2 mutants. Culmorin production resumed in a ΔClm2 complementation experiment. A detailed analysis of the secondary metabolites extracted from the large-scale liquid culture of disruptant ΔClm2D20 revealed five new natural products: 3-hydroxylongiborneol (3), 5-hydroxylongiborneol (4), 12-hydroxylongiborneol (5), 15-hydroxylongiborneol (6), and 11-epi-acetylculmorin (7). The structures of the new compounds were elucidated by a combination of HRMS, 1D and 2D NMR, and X-ray crystallography.

  19. Localization of human flavin-containing monooxygenase genes FMO2 and FMO5 to chromosome 1q

    SciTech Connect

    McCombie, R.R.; Shephard, E.A.; Dolphin, C.T.

    1996-06-15

    The human flavin-containing monooxygenase (FMO) gene family comprises at least five distinct members (FMO1 to FMO5) that code for enzymes responsible for the oxidation of a wide variety of soft nucleophilic substrates, including drugs and environmental pollutants. Three of these genes (FMO1, FMO3, and FMO4) have previously been localized to human chromosome 1q, raising the possibility that the entire gene family is clustered in this chromosomal region. Analysis by polymerase chain reaction of DNA isolated from a panel of human-rodent somatic cell hybrids demonstrates that the two remaining identified members of the FMO gene family, FMO2 and FMO5, also are located on chromosome 1q. 19 refs., 1 fig., 1 tab.

  20. Oxidative cleavage and hydrolytic boosting of cellulose in soybean spent flakes by Trichoderma reesei Cel61A lytic polysaccharide monooxygenase.

    PubMed

    Pierce, Brian C; Agger, Jane Wittrup; Wichmann, Jesper; Meyer, Anne S

    2017-03-01

    The auxiliary activity family 9 (AA9) copper-dependent lytic polysaccharide monooxygenase (LPMO) from Trichoderma reesei (EG4; TrCel61A) was investigated for its ability to oxidize the complex polysaccharides from soybean. The substrate specificity of the enzyme was assessed against a variety of substrates, including both soy spent flake, a by-product of the soy food industry, and soy spent flake pretreated with sodium hydroxide. Products from enzymatic treatments were analyzed using mass spectrometry and high performance anion exchange chromatography. We demonstrate that TrCel61A is capable of oxidizing cellulose from both pretreated soy spent flake and phosphoric acid swollen cellulose, oxidizing at both the C1 and C4 positions. In addition, we show that the oxidative activity of TrCel61A displays a synergistic effect capable of boosting endoglucanase activity, and thereby substrate depolymerization of soy cellulose, by 27%.

  1. Oxidation of trichloroethylene, 1,1-dichloroethylene, and chloroform by toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1

    SciTech Connect

    Chauhan, S.; Wood, T.K.; Barbieri, P.

    1998-08-01

    Toluene/o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1, which oxidizes toluene and o-xylene, was examined for its ability to degrade the environmental pollutants trichloroethylene (TCE), 1,1-dichloroethylene (1,1-DCE), cis-1,2-DCE, trans-1,2-DCE, chloroform, dichloromethane, phenol, 2,4-dichlorophenol, 2,4,5-trichlorophenol, 2,4,6-trichlorophenol, 2,3,5,6-tetrachlorophenol, and 2,3,4,5,6-pentachlorophenol. Escherichia coli JM109 that expressed ToMO from genes on plasmid pBZ1260 under control of the lac promoter degraded TCE, 1,1-DCE, and chloroform at initial rates of 3.1, 3.6, and 1.6 nmol, respectively. Stoichiometric amounts of chloride release were seen, indicating mineralization. Thus, the substrate range of ToMO is extended to include aliphatic chlorinated compounds.

  2. Fungal cellulose degradation by oxidative enzymes: from dysfunctional GH61 family to powerful lytic polysaccharide monooxygenase family

    PubMed Central

    Powlowski, Justin; Tsang, Adrian

    2014-01-01

    Our understanding of fungal cellulose degradation has shifted dramatically in the past few years with the characterization of a new class of secreted enzymes, the lytic polysaccharide monooxygenases (LPMO). After a period of intense research covering structural, biochemical, theoretical and evolutionary aspects, we have a picture of them as wedge-like copper-dependent metalloenzymes that on reduction generate a radical copper-oxyl species, which cleaves mainly crystalline cellulose. The main biological function lies in the synergism of fungal LPMOs with canonical hydrolytic cellulases in achieving efficient cellulose degradation. Their important role in cellulose degradation is highlighted by the wide distribution and often numerous occurrences in the genomes of almost all plant cell-wall degrading fungi. In this review, we provide an overview of the latest achievements in LPMO research and consider the open questions and challenges that undoubtedly will continue to stimulate interest in this new and exciting group of enzymes. PMID:25217478

  3. Transcript Analysis of Multiple Copies of amo (Encoding Ammonia Monooxygenase) and hao (Encoding Hydroxylamine Oxidoreductase) in Nitrosomonas europaea

    PubMed Central

    Hommes, Norman G.; Sayavedra-Soto, Luis A.; Arp, Daniel J.

    2001-01-01

    The genes encoding ammonia monooxygenase (amoCAB), hydroxylamine oxidoreductase (hao), and the c-type cytochrome c-554 (hcy) are present in multiple copies in the genome of Nitrosomonas europaea. The upstream regions of the two copies of amoC, the three copies of hao, and one copy of hcy were cloned and sequenced. Primer extension reactions were done to identify transcription start sites for these genes, as well as for amoA. Putative ς70 promoter sequences were found associated with all but one of the mapped transcription start sites. Primer extensions were done with amoC primers using RNA harvested from cells incubated with and without ammonium. The experiments suggested that N. europaea cells may be able to use different promoters in the presence and absence of ammonium. PMID:11208810

  4. Characterization of a tryptophan 2-monooxygenase gene from Puccinia graminis f. sp. tritici involved in auxin biosynthesis and rust pathogenicity.

    PubMed

    Yin, Chuntao; Park, Jeong-Jin; Gang, David R; Hulbert, Scot H

    2014-03-01

    The plant hormone indole-3-acetic acid (IAA) is best known as a regulator of plant growth and development but its production can also affect plant-microbe interactions. Microorganisms, including numerous plant-associated bacteria and several fungi, are also capable of producing IAA. The stem rust fungus Puccinia graminis f. sp. tritici induced wheat plants to accumulate auxin in infected leaf tissue. A gene (Pgt-IaaM) encoding a putative tryptophan 2-monooxygenase, which makes the auxin precursor indole-3-acetamide (IAM), was identified in the P. graminis f. sp. tritici genome and found to be expressed in haustoria cells in infected plant tissue. Transient silencing of the gene in infected wheat plants indicated that it was required for full pathogenicity. Expression of Pgt-IaaM in Arabidopsis caused a typical auxin expression phenotype and promoted susceptibility to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000.

  5. Simultaneous biocatalyst production and Baeyer-Villiger oxidation for bioconversion of cyclohexanone by recombinant Escherichia coli expressing cyclohexanone monooxygenase.

    PubMed

    Lee, Won-Heong; Park, Yong-Cheol; Lee, Dae-Hee; Park, Kyungmoon; Seo, Jin-Ho

    2005-01-01

    Cyclohexanone monooxygenase (CHMO) catalyzing Baeyer-Villiger oxidation converts cyclic ketones into optically pure lactones, which have been used as building blocks in organic synthesis. A recombinant Escherichia coli BL21(DE3)/pMM4 expressing CHMO originated from Acinetobacter sp. NCIB 9871 was used to produce epsilon-caprolactone through a simultaneous biocatalyst production and Baeyer-Villiger oxidation (SPO) process. A fed-batch process was designed to obtain high cell density for improving production of epsilon-caprolactone. The fed-batch SPO process gave the best results, 10.2 g/L of epsilon-caprolactone and 0.34 g/(L.h) of productivity, corresponding to a 10.5- and 3.4-fold enhancement compared with those of the batch SPO, respectively.

  6. Enhanced production of ε-caprolactone by coexpression of bacterial hemoglobin gene in recombinant Escherichia coli expressing cyclohexanone monooxygenase gene.

    PubMed

    Lee, Won-Heong; Park, Eun-Hee; Kim, Myoung-Dong

    2014-12-28

    Baeyer-Villiger (BV) oxidation of cyclohexanone to epsilon-caprolactone in a microbial system expressing cyclohexanone monooxygenase (CHMO) can be influenced by not only the efficient regeneration of NADPH but also a sufficient supply of oxygen. In this study, the bacterial hemoglobin gene from Vitreoscilla stercoraria (vhb) was introduced into the recombinant Escherichia coli expressing CHMO to investigate the effects of an oxygen-carrying protein on microbial BV oxidation of cyclohexanone. Coexpression of Vhb allowed the recombinant E. coli strain to produce a maximum epsilon-caprolactone concentration of 15.7 g/l in a fed-batch BV oxidation of cyclohexanone, which corresponded to a 43% improvement compared with the control strain expressing CHMO only under the same conditions.

  7. Copper-dioxygen complex mediated C-H bond oxygenation: relevance for particulate methane monooxygenase (pMMO).

    PubMed

    Himes, Richard A; Karlin, Kenneth D

    2009-02-01

    Particulate methane monooxygenase (pMMO), an integral membrane protein found in methanotrophic bacteria, catalyzes the oxidation of methane to methanol. Expression and greater activity of the enzyme in the presence of copper ion suggest that pMMO is a cuprous metalloenzyme. Recent advances - especially the first crystal structures of pMMO - have energized the field, but the nature of the active site(s) and the mechanism of methane oxidation remain poorly understood-yet hotly contested. Herein the authors briefly review the current understanding of the pMMO metal sites and discuss advances in small molecule Cu-O(2) chemistry that may contribute to an understanding of copper-ion mediated hydrocarbon oxidation chemistry.

  8. Lytic polysaccharide monooxygenases: a crystallographer’s view on a new class of biomass-degrading enzymes

    PubMed Central

    Frandsen, Kristian E. H.; Lo Leggio, Leila

    2016-01-01

    Lytic polysaccharide monooxygenases (LPMOs) are a new class of microbial copper enzymes involved in the degradation of recalcitrant polysaccharides. They have only been discovered and characterized in the last 5–10 years and have stimulated strong interest both in biotechnology and in bioinorganic chemistry. In biotechnology, the hope is that these enzymes will finally help to make enzymatic biomass conversion, especially of lignocellulosic plant waste, economically attractive. Here, the role of LPMOs is likely to be in attacking bonds that are not accessible to other enzymes. LPMOs have attracted enormous interest since their discovery. The emphasis in this review is on the past and present contribution of crystallographic studies as a guide to functional understanding, with a final look towards the future. PMID:27840684

  9. Crystallographic insights into a cobalt (III) sepulchrate based alternative cofactor system of P450 BM3 monooxygenase.

    PubMed

    Panneerselvam, Saravanan; Shehzad, Aamir; Mueller-Dieckmann, Jochen; Wilmanns, Matthias; Bocola, Marco; Davari, Mehdi D; Schwaneberg, Ulrich

    2017-07-22

    P450 BM3 is a multi-domain heme-containing soluble bacterial monooxygenase. P450 BM3 and variants are known to oxidize structurally diverse substrates. Crystal structures of individual domains of P450 BM3 are available. However, the spatial organization of the full-length protein is unknown. In this study, crystal structures of the P450 BM3 M7 heme domain variant with and without cobalt (III) sepulchrate are reported. Cobalt (III) sepulchrate acts as an electron shuttle in an alternative cofactor system employing zinc dust as the electron source. The crystal structure shows a binding site for the mediator cobalt (III) sepulchrate at the entrance of the substrate access channel. The mediator occupies an unusual position which is far from the active site and distinct from the binding of the natural redox partner (FAD/NADPH binding domain). Copyright © 2017 Elsevier B.V. All rights reserved.

  10. A chicory cytochrome P450 mono-oxygenase CYP71AV8 for the oxidation of (+)-valencene.

    PubMed

    Cankar, Katarina; van Houwelingen, Adèle; Bosch, Dirk; Sonke, Theo; Bouwmeester, Harro; Beekwilder, Jules

    2011-01-03

    Chicory (Cichorium intybus L.), which is known to have a variety of terpene-hydroxylating activities, was screened for a P450 mono-oxygenase to convert (+)-valencene to (+)-nootkatone. A novel P450 cDNA was identified in a chicory root EST library. Co-expression of the enzyme with a valencene synthase in yeast, led to formation of trans-nootkatol, cis-nootkatol and (+)-nootkatone. The novel enzyme was also found to catalyse a three step conversion of germacrene A to germacra-1(10),4,11(13)-trien-12-oic acid, indicating its involvement in chicory sesquiterpene lactone biosynthesis. Likewise, amorpha-4,11-diene was converted to artemisinic acid. Surprisingly, the chicory P450 has a different regio-specificity on (+)-valencene compared to germacrene A and amorpha-4,11-diene.

  11. Characterization of in vitro metabolites of methylenedioxypyrovalerone (MDPV): An N-oxide metabolite formation mediated by flavin monooxygenase.

    PubMed

    Kim, In Sook; Rehman, Shaheed Ur; Choi, Min Sun; Jang, Moonhee; Yang, Wonkyung; Kim, Eunmi; Yoo, Hye Hyun

    2016-11-30

    Methylenedioxypyrovalerone (MDPV) has emerged in recent years as a recreational substance with psychostimulant properties. In this study, in vitro metabolites of MDPV were characterized based on liquid chromatography/quadrupole-time-of-flight mass spectrometry (LC/QTOF MS). MDPV was incubated with human liver microsomes, human recombinant cDNA-expressed cytochrome P450 enzymes and flavin monooxygenase (FMO). MDPV was metabolized to yield eight metabolites (M1-M8) with major metabolic reactions such as demethylenation and oxidation. Among them, M6 was assigned as an N-oxide metabolite. FMO was found to be a principal enzyme responsible for the formation of M6; FMO1 and FMO3 were the main enzymes involved in N-oxidation of MDPV. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Effects of the neem tree compounds azadirachtin, salannin, nimbin, and 6-desacetylnimbin on ecdysone 20-monooxygenase activity.

    PubMed

    Mitchell, M J; Smith, S L; Johnson, S; Morgan, E D

    1997-01-01

    The effects of azadirachtin, salannin, nimbin, and 6-desacetylnimbin 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 radiolabeled ecdysone and increasing concentrations (from 1 x 10(-8) to 1 x 10(-3) M) of the four compounds isolated from seed kernels of the neem tree, Azadirachta indica. All four neem tree compounds were found to inhibit, in a dose-dependent fashion, the E-20-M activity in 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 2 x 10(-5) to 1 x 10(-3).

  13. Sensitivity of mRNA Translation.

    PubMed

    Poker, Gilad; Margaliot, Michael; Tuller, Tamir

    2015-08-04

    Using the dynamic mean-field approximation of the totally asymmetric simple exclusion process (TASEP), we investigate the effect of small changes in the initiation, elongation, and termination rates along the mRNA strand on the steady-state protein translation rate. We show that the sensitivity of mRNA translation is equal to the sensitivity of the maximal eigenvalue of a symmetric, nonnegative, tridiagonal, and irreducible matrix. This leads to new analytical results as well as efficient numerical schemes that are applicable for large-scale models. Our results show that in the usual endogenous case, when initiation is more rate-limiting than elongation, the sensitivity of the translation rate to small mutations rapidly increases towards the 5' end of the ORF. When the initiation rate is high, as may be the case for highly expressed and/or heterologous optimized genes, the maximal sensitivity is with respect to the elongation rates at the middle of the mRNA strand. We also show that the maximal possible effect of a small increase/decrease in any of the rates along the mRNA is an increase/decrease of the same magnitude in the translation rate. These results are in agreement with previous molecular evolutionary and synthetic biology experimental studies.

  14. Sensitivity of mRNA Translation

    PubMed Central

    Poker, Gilad; Margaliot, Michael; Tuller, Tamir

    2015-01-01

    Using the dynamic mean-field approximation of the totally asymmetric simple exclusion process (TASEP), we investigate the effect of small changes in the initiation, elongation, and termination rates along the mRNA strand on the steady-state protein translation rate. We show that the sensitivity of mRNA translation is equal to the sensitivity of the maximal eigenvalue of a symmetric, nonnegative, tridiagonal, and irreducible matrix. This leads to new analytical results as well as efficient numerical schemes that are applicable for large-scale models. Our results show that in the usual endogenous case, when initiation is more rate-limiting than elongation, the sensitivity of the translation rate to small mutations rapidly increases towards the 5′ end of the ORF. When the initiation rate is high, as may be the case for highly expressed and/or heterologous optimized genes, the maximal sensitivity is with respect to the elongation rates at the middle of the mRNA strand. We also show that the maximal possible effect of a small increase/decrease in any of the rates along the mRNA is an increase/decrease of the same magnitude in the translation rate. These results are in agreement with previous molecular evolutionary and synthetic biology experimental studies. PMID:26238363

  15. Identification of the Monooxygenase Gene Clusters Responsible for the Regioselective Oxidation of Phenol to Hydroquinone in Mycobacteria▿

    PubMed Central

    Furuya, Toshiki; Hirose, Satomi; Osanai, Hisashi; Semba, Hisashi; Kino, Kuniki

    2011-01-01

    Mycobacterium goodii strain 12523 is an actinomycete that is able to oxidize phenol regioselectively at the para position to produce hydroquinone. In this study, we investigated the genes responsible for this unique regioselective oxidation. On the basis of the fact that the oxidation activity of M. goodii strain 12523 toward phenol is induced in the presence of acetone, we first identified acetone-induced proteins in this microorganism by two-dimensional electrophoretic analysis. The N-terminal amino acid sequence of one of these acetone-induced proteins shares 100% identity with that of the protein encoded by the open reading frame Msmeg_1971 in Mycobacterium smegmatis strain mc2155, whose genome sequence has been determined. Since Msmeg_1971, Msmeg_1972, Msmeg_1973, and Msmeg_1974 constitute a putative binuclear iron monooxygenase gene cluster, we cloned this gene cluster of M. smegmatis strain mc2155 and its homologous gene cluster found in M. goodii strain 12523. Sequence analysis of these binuclear iron monooxygenase gene clusters revealed the presence of four genes designated mimABCD, which encode an oxygenase large subunit, a reductase, an oxygenase small subunit, and a coupling protein, respectively. When the mimA gene (Msmeg_1971) of M. smegmatis strain mc2155, which was also found to be able to oxidize phenol to hydroquinone, was deleted, this mutant lost the oxidation ability. This ability was restored by introduction of the mimA gene of M. smegmatis strain mc2155 or of M. goodii strain 12523 into this mutant. Interestingly, we found that these gene clusters also play essential roles in propane and acetone metabolism in these mycobacteria. PMID:21183637

  16. Mechanism of the 6-Hydroxy-3-succinoyl-pyridine 3-Monooxygenase Flavoprotein from Pseudomonas putida S16*

    PubMed Central

    Yu, Hao; Hausinger, Robert P.; Tang, Hong-Zhi; Xu, Ping

    2014-01-01

    6-Hydroxy-3-succinoyl-pyridine (HSP) 3-monooxygenase (HspB), a flavoprotein essential to the pyrrolidine pathway of nicotine degradation, catalyzes pyridine-ring β-hydroxylation, resulting in carbon-carbon cleavage and production of 2,5-dihydroxypyridine. Here, we generated His6-tagged HspB in Escherichia coli, characterized the properties of the recombinant enzyme, and investigated its mechanism of catalysis. In contrast to conclusions reported previously, the second product of the HspB reaction was shown to be succinate, with isotope labeling experiments providing direct evidence that the newly introduced oxygen atom of succinate is derived from H2O. Phylogenetic analysis reveals that HspB is the most closely related to two p-nitrophenol 4-monooxygenases, and the experimental results exhibit that p-nitrophenol is a substrate of HspB. The reduction of HspB (with maxima at 375 and 460 nm, and a shoulder at 485 nm) by NADH was followed by stopped-flow spectroscopy, and the rate constant for reduction was shown to be stimulated by HSP. Reduced HspB reacts with oxygen to form a C(4a)-(hydro)peroxyflavin intermediate with an absorbance maximum at ∼400 nm within the first few milliseconds before converting to the oxidized flavoenzyme species. The formed C(4a)-hydroperoxyflavin intermediate reacts with HSP to form an intermediate that hydrolyzes to the products 2,5-dihydroxypyridine and succinate. The investigation on the catalytic mechanism of a flavoprotein pyridine-ring β-position hydroxylase provides useful information for the biosynthesis of pyridine derivatives. PMID:25172510

  17. Mutations of Toluene-4-Monooxygenase That Alter Regiospecificity of Indole Oxidation and Lead to Production of Novel Indigoid Pigments

    PubMed Central

    McClay, Kevin; Boss, Corinne; Keresztes, Ivan; Steffan, Robert J.

    2005-01-01

    Broad-substrate-range monooygenase enzymes, including toluene-4-monooxygenase (T4MO), can catalyze the oxidation of indole. The indole oxidation products can then condense to form the industrially important dye indigo. Site-directed mutagenesis of T4MO resulted in the creation of T4MO isoforms with altered pigment production phenotypes. High-pressure liquid chromatography, thin-layer chromatography, and nuclear magnetic resonance analysis of the indole oxidation products generated by the mutant T4MO isoforms revealed that the phenotypic differences were primarily due to changes in the regiospecificity of indole oxidation. Most of the mutations described in this study changed the ratio of the primary indole oxidation products formed (indoxyl, 2-oxindole, and isatin), but some mutations, particularly those involving amino acid G103 of tmoA, allowed for the formation of additional products, including 7-hydroxyindole and novel indigoid pigments. For example, mutant G103L converted 17% of added indole to 7-hydroxyindole and 29% to indigoid pigments including indigo and indirubin and two other structurally related pigments. The double mutant G103L:A107G converted 47% of indole to 7-hydroxyindole, but no detectable indigoid pigments were formed, similar to the product distribution observed with the toluene-2-monooxygenase (T2MO) of Burkholderia cepacia G4. These results demonstrate that modification of the tmoA active site can change the products produced by the enzyme and lead to the production of novel pigments and other indole oxidation products with potential commercial and medicinal utility. PMID:16151140

  18. Spectroscopic and computational insight into the activation of O2 by the mononuclear Cu center in polysaccharide monooxygenases.

    PubMed

    Kjaergaard, Christian H; Qayyum, Munzarin F; Wong, Shaun D; Xu, Feng; Hemsworth, Glyn R; Walton, Daniel J; Young, Nigel A; Davies, Gideon J; Walton, Paul H; Johansen, Katja Salomon; Hodgson, Keith O; Hedman, Britt; Solomon, Edward I

    2014-06-17

    Strategies for O2 activation by copper enzymes were recently expanded to include mononuclear Cu sites, with the discovery of the copper-dependent polysaccharide monooxygenases, also classified as auxiliary-activity enzymes 9-11 (AA9-11). These enzymes are finding considerable use in industrial biofuel production. Crystal structures of polysaccharide monooxygenases have emerged, but experimental studies are yet to determine the solution structure of the Cu site and how this relates to reactivity. From X-ray absorption near edge structure and extended X-ray absorption fine structure spectroscopies, we observed a change from four-coordinate Cu(II) to three-coordinate Cu(I) of the active site in solution, where three protein-derived nitrogen ligands coordinate the Cu in both redox states, and a labile hydroxide ligand is lost upon reduction. The spectroscopic data allowed for density functional theory calculations of an enzyme active site model, where the optimized Cu(I) and (II) structures were consistent with the experimental data. The O2 reactivity of the Cu(I) site was probed by EPR and stopped-flow absorption spectroscopies, and a rapid one-electron reduction of O2 and regeneration of the resting Cu(II) enzyme were observed. This reactivity was evaluated computationally, and by calibration to Cu-superoxide model complexes, formation of an end-on Cu-AA9-superoxide species was found to be thermodynamically favored. We discuss how this thermodynamically difficult one-electron reduction of O2 is enabled by the unique protein structure where two nitrogen ligands from His1 dictate formation of a T-shaped Cu(I) site, which provides an open coordination position for strong O2 binding with very little reorganization energy.

  19. Development of a plant viral-vector-based gene expression assay for the screening of yeast cytochrome p450 monooxygenases.

    PubMed

    Hanley, Kathleen; Nguyen, Long V; Khan, Faizah; Pogue, Gregory P; Vojdani, Fakhrieh; Panda, Sanjay; Pinot, Franck; Oriedo, Vincent B; Rasochova, Lada; Subramanian, Mani; Miller, Barbara; White, Earl L

    2003-02-01

    Development of a gene discovery tool for heterologously expressed cytochrome P450 monooxygenases has been inherently difficult. The activity assays are labor-intensive and not amenable to parallel screening. Additionally, biochemical confirmation requires coexpression of a homologous P450 reductase or complementary heterologous activity. Plant virus gene expression systems have been utilized for a diverse group of organisms. In this study we describe a method using an RNA vector expression system to phenotypically screen for cytochrome P450-dependent fatty acid omega-hydroxylase activity. Yarrowia lipolytica CYP52 gene family members involved in n-alkane assimilation were amplified from genomic DNA, cloned into a plant virus gene expression vector, and used as a model system for determining heterologous expression. Plants infected with virus vectors expressing the yeast CYP52 genes (YlALK1-YlALK7) showed a distinct necrotic lesion phenotype on inoculated plant leaves. No phenotype was detected on negative control constructs. YlALK3-, YlALK5-, and YlALK7-inoculated plants all catalyzed the terminal hydroxylation of lauric acid as confirmed using thin-layer and gas chromatography/mass spectrometry methods. The plant-based cytochrome P450 phenotypic screen was tested on an n-alkane-induced Yarrowia lipolytica plant virus expression library. A subset of 1,025 random library clones, including YlALK1-YlALK7 constructs, were tested on plants. All YlALK gene constructs scored positive in the randomized screen. Following nucleotide sequencing of the clones that scored positive using a phenotypic screen, approximately 5% were deemed appropriate for further biochemical analysis. This report illustrates the utility of a plant-based system for expression of heterologous cytochrome P450 monooxygenases and for the assignment of gene function.

  20. Methanobactin from Methylocystis sp. strain SB2 affects gene expression and methane monooxygenase activity in Methylosinus trichosporium OB3b.

    PubMed

    Farhan Ul-Haque, Muhammad; Kalidass, Bhagyalakshmi; Vorobev, Alexey; Baral, Bipin S; DiSpirito, Alan A; Semrau, Jeremy D

    2015-04-01

    Methanotrophs can express a cytoplasmic (soluble) methane monooxygenase (sMMO) or membrane-bound (particulate) methane monooxygenase (pMMO). Expression of these MMOs is strongly regulated by the availability of copper. Many methanotrophs have been found to synthesize a novel compound, methanobactin (Mb), that is responsible for the uptake of copper, and methanobactin produced by Methylosinus trichosporium OB3b plays a key role in controlling expression of MMO genes in this strain. As all known forms of methanobactin are structurally similar, it was hypothesized that methanobactin from one methanotroph may alter gene expression in another. When Methylosinus trichosporium OB3b was grown in the presence of 1 μM CuCl2, expression of mmoX, encoding a subunit of the hydroxylase component of sMMO, was very low. mmoX expression increased, however, when methanobactin from Methylocystis sp. strain SB2 (SB2-Mb) was added, as did whole-cell sMMO activity, but there was no significant change in the amount of copper associated with M. trichosporium OB3b. If M. trichosporium OB3b was grown in the absence of CuCl2, the mmoX expression level was high but decreased by several orders of magnitude if copper prebound to SB2-Mb (Cu-SB2-Mb) was added, and biomass-associated copper was increased. Exposure of Methylosinus trichosporium OB3b to SB2-Mb had no effect on expression of mbnA, encoding the polypeptide precursor of methanobactin in either the presence or absence of CuCl2. mbnA expression, however, was reduced when Cu-SB2-Mb was added in both the absence and presence of CuCl2. These data suggest that methanobactin acts as a general signaling molecule in methanotrophs and that methanobactin "piracy" may be commonplace.

  1. Catalytic activity of the two-component flavin-dependent monooxygenase from Pseudomonas aeruginosa toward cinnamic acid derivatives.

    PubMed

    Furuya, Toshiki; Kino, Kuniki

    2014-02-01

    4-Hydroxyphenylacetate 3-hydroxylases (HPAHs) of the two-component flavin-dependent monooxygenase family are attractive enzymes that possess the catalytic potential to synthesize valuable ortho-diphenol compounds from simple monophenol compounds. In this study, we investigated the catalytic activity of HPAH from Pseudomonas aeruginosa strain PAO1 toward cinnamic acid derivatives. We prepared Escherichia coli cells expressing the hpaB gene encoding the monooxygenase component and the hpaC gene encoding the oxidoreductase component. E. coli cells expressing HpaBC exhibited no or very low oxidation activity toward cinnamic acid, o-coumaric acid, and m-coumaric acid, whereas they rapidly oxidized p-coumaric acid to caffeic acid. Interestingly, after p-coumaric acid was almost completely consumed, the resulting caffeic acid was further oxidized to 3,4,5-trihydroxycinnamic acid. In addition, HpaBC exhibited oxidation activity toward 3-(4-hydroxyphenyl)propanoic acid, ferulic acid, and coniferaldehyde to produce the corresponding ortho-diphenols. We also investigated a flask-scale production of caffeic acid from p-coumaric acid as the model reaction for HpaBC-catalyzed syntheses of hydroxycinnamic acids. Since the initial concentrations of the substrate p-coumaric acid higher than 40 mM markedly inhibited its HpaBC-catalyzed oxidation, the reaction was carried out by repeatedly adding 20 mM of this substrate to the reaction mixture. Furthermore, by using the HpaBC whole-cell catalyst in the presence of glycerol, our experimental setup achieved the high-yield production of caffeic acid, i.e., 56.6 mM (10.2 g/L) within 24 h. These catalytic activities of HpaBC will provide an easy and environment-friendly synthetic approach to hydroxycinnamic acids.

  2. Mechanism of Cytoplasmic mRNA Translation

    PubMed Central

    2015-01-01

    Protein synthesis is a fundamental process in gene expression that depends upon the abundance and accessibility of the mRNA transcript as well as the activity of many protein and RNA-protein complexes. Here we focus on the intricate mechanics of mRNA translation in the cytoplasm of higher plants. This chapter includes an inventory of the plant translational apparatus and a detailed review of the translational processes of initiation, elongation, and termination. The majority of mechanistic studies of cytoplasmic translation have been carried out in yeast and mammalian systems. The factors and mechanisms of translation are for the most part conserved across eukaryotes; however, some distinctions are known to exist in plants. A comprehensive understanding of the complex translational apparatus and its regulation in plants is warranted, as the modulation of protein production is critical to development, environmental plasticity and biomass yield in diverse ecosystems and agricultural settings. PMID:26019692

  3. Staufen-mediated mRNA decay

    PubMed Central

    Park, Eonyoung; Maquat, Lynne E.

    2013-01-01

    Staufen1 (STAU1)-mediated mRNA decay (SMD) is an mRNA degradation process in mammalian cells that is mediated by the binding of STAU1 to a STAU1-binding site (SBS) within the 3'-untranslated region (3'UTR) of target mRNAs. During SMD, STAU1, a double-stranded (ds) RNA-binding protein, recognizes dsRNA structures formed either by intramolecular base-pairing of 3'UTR sequences or by intermolecular base-pairing of 3'UTR sequences with a long noncoding RNA (lncRNA) via partially complementary Alu elements. Recently, STAU2, a paralog of STAU1, has also been reported to mediate SMD. Both STAU1 and STAU2 interact directly with the ATP-dependent RNA helicase UPF1, a key SMD factor, enhancing its helicase activity to promote effective SMD. Moreover, STAU1 and STAU2 form homodimeric and heterodimeric interactions via domain-swapping. Since both SMD and the mechanistically related nonsense-mediated mRNA decay (NMD) employ UPF1, SMD and NMD are competitive pathways. Competition contributes to cellular differentiation processes, such as myogenesis and adipogenesis, placing SMD at the heart of various physiologically important mechanisms. PMID:23681777

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

    PubMed

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

    2012-11-01

    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.

  5. 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

  6. 1,4-Dioxane-degrading consortia can be enriched from uncontaminated soils: prevalence of Mycobacterium and soluble di-iron monooxygenase genes.

    PubMed

    He, Ya; Mathieu, Jacques; da Silva, Marcio L B; Li, Mengyan; Alvarez, Pedro J J

    2017-10-06

    Two bacterial consortia were enriched from uncontaminated soil by virtue of their ability to grow on 1,4-dioxane (dioxane) as a sole carbon and energy source. Their specific dioxane degradation rates at 30°C, pH = 7 (i.e. 5.7 to 7.1 g-dioxane per g-protein per day) were comparable to those of two dioxane-metabolizing archetypes: Pseudonocardia dioxanivoransCB1190 and Mycobacterium dioxanotrophicusPH-06. Based on 16S rRNA sequencing, Mycobacterium was the dominant genus. Acetylene inhibition tests suggest that dioxane degradation was mediated by monooxygenases. However, qPCR analyses targeting the tetrahydrofuran/dioxane monooxygenase gene (thmA/dxmA) (which is, to date, the only sequenced dioxane monooxygenase gene) were negative, indicating that other (as yet unknown) catabolic gene(s) were responsible. DNA sequence analyses also showed threefold to sevenfold enrichment of group 5 and group 6 soluble di-iron monooxygenase (SDIMO) genes relative to the original soil samples. Whereas biodegradation of trace levels of dioxane is a common challenge at contaminated sites, both consortia degraded dioxane at low initial concentrations (300 μg l(-1) ) below detectable levels (5 μg l(-1) ) in bioaugmented microcosms prepared with impacted groundwater. Overall, this work shows that dioxane-degrading bacteria (and the associated natural attenuation potential) exist even in some uncontaminated soils, and may be enriched to broaden bioaugmentation options for sites experiencing insufficient dioxane catabolic capacity. © 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  7. Characterization of 4-Hydroxyphenylacetate 3-Hydroxylase (HpaB) of Escherichia coli as a Reduced Flavin Adenine Dinucleotide-Utilizing Monooxygenase

    PubMed Central

    Xun, Luying; Sandvik, Erik R.

    2000-01-01

    4-Hydroxyphenylacetate 3-hydroxylase (HpaB and HpaC) of Escherichia coli W has been reported as a two-component flavin adenine dinucleotide (FAD)-dependent monooxygenase that attacks a broad spectrum of phenolic compounds. However, the function of each component in catalysis is unclear. The large component (HpaB) was demonstrated here to be a reduced FAD (FADH2)-utilizing monooxygenase. When an E. coli flavin reductase (Fre) having no apparent homology with HpaC was used to generate FADH2 in vitro, HpaB was able to use FADH2 and O2 for the oxidation of 4-hydroxyphenylacetate. HpaB also used chemically produced FADH2 for 4-hydroxyphenylacetate oxidation, further demonstrating that HpaB is an FADH2-utilizing monooxygenase. FADH2 generated by Fre was rapidly oxidized by O2 to form H2O2 in the absence of HpaB. When HpaB was included in the reaction mixture without 4-hydroxyphenylacetate, HpaB bound FADH2 and transitorily protected it from rapid autoxidation by O2. When 4-hydroxyphenylacetate was also present, HpaB effectively competed with O2 for FADH2 utilization, leading to 4-hydroxyphenylacetate oxidation. With sufficient amounts of HpaB in the reaction mixture, FADH2 produced by Fre was mainly used by HpaB for the oxidation of 4-hydroxyphenylacetate. At low HpaB concentrations, most FADH2 was autoxidized by O2, causing uncoupling. However, the coupling of the two enzymes' activities was increased by lowering FAD concentrations in the reaction mixture. A database search revealed that HpaB had sequence similarities to several proteins and gene products involved in biosynthesis and biodegradation in both bacteria and archaea. This is the first report of an FADH2-utilizing monooxygenase that uses FADH2 as a substrate rather than as a cofactor. PMID:10653707

  8. Comparative analysis of the conventional and novel pmo (particulate methane monooxygenase) operons from methylocystis strain SC2.

    PubMed

    Ricke, Peter; Erkel, Christoph; Kube, Michael; Reinhardt, Richard; Liesack, Werner

    2004-05-01

    In addition to the conventional pmoA gene (pmoA1) encoding the active site polypeptide of particulate methane monooxygenase, a novel pmoA gene copy (pmoA2) is widely distributed among type II methanotrophs (methane-oxidizing bacteria [MOB]) (M. Tchawa Yimga, P. F. Dunfield, P. Ricke, J. Heyer, and W. Liesack, Appl. Environ. Microbiol. 69:5593-5602, 2003). Here we report that the pmoA1 and pmoA2 gene copies in the type II MOB Methylocystis strain SC2 are each part of a complete pmoCAB gene cluster (pmoCAB1, pmoCAB2). A bacterial artificial chromosome (BAC) library of strain SC2 genomic DNA was constructed, and BAC clones carrying either pmoCAB1 or pmoCAB2 were identified. Comparative sequence analysis showed that these two gene clusters exhibit low levels of identity at both the DNA level (67.4 to 70.9%) and the derived protein level (59.3 to 65.6%). In contrast, the secondary structures predicted for PmoCAB1 and PmoCAB2, as well as the derived transmembrane-spanning regions, are nearly identical. This suggests that PmoCAB2 is, like PmoCAB1, a highly hydrophobic, membrane-associated protein. A total of 190 of the 203 amino acid residues representing a highly conserved consensus sequence of the currently known PmoCAB1 and AmoCAB sequence types could be identified in PmoCAB2. The amoCAB gene cluster encodes ammonia monooxygenase and is evolutionarily related to pmoCAB. Analysis of a set of amino acid residues that allowed differentiation between conventional PmoA and AmoA provided further support for the hypothesis that pmoCAB2 encodes a functional equivalent of PmoCAB1. In experiments in which we used 5' rapid amplification of cDNA ends we identified transcriptional start sites 320 and 177 bp upstream of pmoC1 and pmoC2, respectively. Immediately upstream of the transcriptional start sites of both pmoCAB1 and pmoCAB2, sequence motifs similar to Escherichia coli sigma(70) promoters were identified.

  9. Evidence for Involvement of Copper Ions and Redox State in Regulation of Butane Monooxygenase in Pseudomonas butanovora▿

    PubMed Central

    Doughty, D. M.; Kurth, E. G.; Sayavedra-Soto, L. A.; Arp, D. J.; Bottomley, P. J.

    2008-01-01

    Pseudomonas butanovora possesses an alcohol-inducible alkane monooxygenase, butane monooxygenase (BMO), that initiates growth on C2-C9 alkanes. A lacZ transcriptional reporter strain, P. butanovora bmoX::lacZ, in which the BMO promoter controls the expression of β-galactosidase activity, was used to show that 1-butanol induced the BMO promoter in the presence or absence of O2 when lactate-grown, BMO-repressed cells were washed free of lactate and incubated in NH4Cl-KNa phosphate buffer. In contrast, when lactate-grown cells of the reporter strain were incubated in phosphate buffer containing the mineral salts of standard growth medium, 1-butanol-dependent induction was significantly repressed at low O2 (1 to 2% [vol/vol]) and totally repressed under anoxic conditions. The repressive effect of the mineral salts was traced to its copper content. In cells exposed to 1% (vol/vol) O2, CuSO4 (0.5 μM) repressed 1-butanol-dependent induction of β-galactosidase activity. Under oxic conditions (20% O2 [vol/vol]), significantly higher concentrations of CuSO4 (2 μM) were required for almost complete repression of induction in lactate-grown cells. A combination of the Cu2+ reducing agent Na ascorbate (100 μM) and CuSO4 (0.5 μM) repressed the induction of β-galactosidase activity under oxic conditions to the same extent that 0.5 μM CuSO4 alone repressed it under anoxic conditions. Under oxic conditions, 2 μM CuSO4 repressed induction of the BMO promoter less effectively in butyrate-grown cells of the bmoX::lacZ strain and of an R8-bmoX::lacZ mutant reporter strain with a putative BMO regulator, BmoR, inactivated. Under anoxic conditions, CuSO4 repression remained highly effective, regardless of the growth substrate, in both BmoR-positive and -negative reporter strains. PMID:18281403

  10. Comparative Analysis of the Conventional and Novel pmo (Particulate Methane Monooxygenase) Operons from Methylocystis Strain SC2

    PubMed Central

    Ricke, Peter; Erkel, Christoph; Kube, Michael; Reinhardt, Richard; Liesack, Werner

    2004-01-01

    In addition to the conventional pmoA gene (pmoA1) encoding the active site polypeptide of particulate methane monooxygenase, a novel pmoA gene copy (pmoA2) is widely distributed among type II methanotrophs (methane-oxidizing bacteria [MOB]) (M. Tchawa Yimga, P. F. Dunfield, P. Ricke, J. Heyer, and W. Liesack, Appl. Environ. Microbiol. 69:5593-5602, 2003). Here we report that the pmoA1 and pmoA2 gene copies in the type II MOB Methylocystis strain SC2 are each part of a complete pmoCAB gene cluster (pmoCAB1, pmoCAB2). A bacterial artificial chromosome (BAC) library of strain SC2 genomic DNA was constructed, and BAC clones carrying either pmoCAB1 or pmoCAB2 were identified. Comparative sequence analysis showed that these two gene clusters exhibit low levels of identity at both the DNA level (67.4 to 70.9%) and the derived protein level (59.3 to 65.6%). In contrast, the secondary structures predicted for PmoCAB1 and PmoCAB2, as well as the derived transmembrane-spanning regions, are nearly identical. This suggests that PmoCAB2 is, like PmoCAB1, a highly hydrophobic, membrane-associated protein. A total of 190 of the 203 amino acid residues representing a highly conserved consensus sequence of the currently known PmoCAB1 and AmoCAB sequence types could be identified in PmoCAB2. The amoCAB gene cluster encodes ammonia monooxygenase and is evolutionarily related to pmoCAB. Analysis of a set of amino acid residues that allowed differentiation between conventional PmoA and AmoA provided further support for the hypothesis that pmoCAB2 encodes a functional equivalent of PmoCAB1. In experiments in which we used 5′ rapid amplification of cDNA ends we identified transcriptional start sites 320 and 177 bp upstream of pmoC1 and pmoC2, respectively. Immediately upstream of the transcriptional start sites of both pmoCAB1 and pmoCAB2, sequence motifs similar to Escherichia coli σ70 promoters were identified. PMID:15128567

  11. Recombinant expression and purification of the 2,5-diketocamphane 1,2-monooxygenase from the camphor metabolizing Pseudomonas putida strain NCIMB 10007

    PubMed Central

    2011-01-01

    Three different Baeyer-Villiger monooxygenases (BVMOs) were reported to be involved in the camphor metabolism by Pseudomonas putida NCIMB 10007. During (+)-camphor degradation, 2,5-diketocamphane is formed serving as substrate for the 2,5-diketocamphane 1,2-monooxygenase. This enzyme is encoded on the CAM plasmid and depends on the cofactors FMN and NADH and hence belongs to the group of type II BVMOs. We have cloned and recombinantly expressed the oxygenating subunit of the 2,5-diketocamphane 1,2-monooxygenase (2,5-DKCMO) in E. coli followed by His-tag-based affinity purification. A range of compounds representing different BVMO substrate classes were then investigated, but only bicyclic ketones were converted by 2,5-DKCMO used as crude cell extract or after purification. Interestingly, also (-)-camphor was oxidized, but conversion was about 3-fold lower compared to (+)-camphor. Moreover, activity of purified 2,5-DKCMO was observed in the absence of an NADH-dehydrogenase subunit. PMID:21906366

  12. An Unprecedented NADPH Domain Conformation in Lysine Monooxygenase NbtG Provides Insights into Uncoupling of Oxygen Consumption from Substrate Hydroxylation

    DOE PAGES

    Binda, Claudia; Robinson, Reeder M.; Martin del Campo, Julia S.; ...

    2015-03-23

    N-hydroxylating monooxygenases (NMOs) are involved in the biosynthesis of iron-chelating hydroxamate-containing siderophores that play a role in microbial virulence. These flavoenzymes catalyze the NADPH- and oxygen-dependent hydroxylation of amines, such as those found on the side chains of lysine and ornithine. In this work we report the biochemical and structural characterization of Nocardia farcinica Lys monooxygenase (NbtG), which has similar biochemical properties to mycobacterial homologs. NbtG is also active on D-Lys although it binds L-Lys with a higher affinity. Differently from the ornithine monooxygenases PvdA, SidA and KtzI, NbtG can use both NADH and NADPH and is highly uncoupled, producingmore » more superoxide and hydrogen peroxide than hydroxylated Lys. The crystal structure of NbtG solved at 2.4 Å resolution revealed an unexpected protein conformation with a 30° rotation of the NAD(P)H domain with respect to the FAD domain that precludes binding of the nicotinamide cofactor. This “occluded” structure may explain the biochemical properties of NbtG, specifically with regard to the substantial uncoupling and limited stabilization of the C4a-hydroperoxyflavin intermediate. We discuss the biological implications of these findings.« less

  13. Cloning and expression of three ladA-type alkane monooxygenase genes from an extremely thermophilic alkane-degrading bacterium Geobacillus thermoleovorans B23.

    PubMed

    Boonmak, Chanita; Takahashi, Yasunori; Morikawa, Masaaki

    2014-05-01

    An extremely thermophilic bacterium, Geobacillus thermoleovorans B23, is capable of degrading a broad range of alkanes (with carbon chain lengths ranging between C11 and C32) at 70 °C. Whole-genome sequence analysis revealed that unlike most alkane-degrading bacteria, strain B23 does not possess an alkB-type alkane monooxygenase gene. Instead, it possesses a cluster of three ladA-type genes, ladAαB23, ladAβB23, and ladB B23, on its chromosome, whose protein products share significant amino acid sequence identities, 49.8, 34.4, and 22.7 %, respectively, with that of ladA alkane monooxygenase gene found on a plasmid of Geobacillus thermodetrificans NG 80-2. Each of the three genes, ladAαB23, ladAβB23, and ladB B23, was heterologously expressed individually in an alkB1 deletion mutant strain, Pseudomonas fluorescens KOB2Δ1. It was found that all three genes were functional in P. fluorescens KOB2Δ1, and partially restored alkane degradation activity. In this study, we suggest that G. thermoleovorans B23 utilizes multiple LadA-type alkane monooxygenases for the degradation of a broad range of alkanes.

  14. An Unprecedented NADPH Domain Conformation in Lysine Monooxygenase NbtG Provides Insights into Uncoupling of Oxygen Consumption from Substrate Hydroxylation*

    PubMed Central

    Binda, Claudia; Robinson, Reeder M.; Martin del Campo, Julia S.; Keul, Nicholas D.; Rodriguez, Pedro J.; Robinson, Howard H.; Mattevi, Andrea; Sobrado, Pablo

    2015-01-01

    N-Hydroxylating monooxygenases are involved in the biosynthesis of iron-chelating hydroxamate-containing siderophores that play a role in microbial virulence. These flavoenzymes catalyze the NADPH- and oxygen-dependent hydroxylation of amines such as those found on the side chains of lysine and ornithine. In this work we report the biochemical and structural characterization of Nocardia farcinica Lys monooxygenase (NbtG), which has similar biochemical properties to mycobacterial homologs. NbtG is also active on d-Lys, although it binds l-Lys with a higher affinity. Differently from the ornithine monooxygenases PvdA, SidA, and KtzI, NbtG can use both NADH and NADPH and is highly uncoupled, producing more superoxide and hydrogen peroxide than hydroxylated Lys. The crystal structure of NbtG solved at 2.4 Å resolution revealed an unexpected protein conformation with a 30° rotation of the NAD(P)H domain with respect to the flavin adenine dinucleotide (FAD) domain that precludes binding of the nicotinamide cofactor. This “occluded” structure may explain the biochemical properties of NbtG, specifically with regard to the substantial uncoupling and limited stabilization of the C4a-hydroperoxyflavin intermediate. Biological implications of these findings are discussed. PMID:25802330

  15. Identification of bacterial carotenoid cleavage dioxygenase homologs that cleave the interphenyl α,β double bond of stilbene derivatives via a monooxygenase reaction

    PubMed Central

    Marasco, Erin K.; Schmidt-Dannert, Claudia

    2013-01-01

    Carotenoid cleavage oxygenases (CCOs, also referred to as carotenoid cleavage dioxygenases (CCDs) in the literature) are a new class of non-heme iron-type enzymes that oxidatively cleave double bonds in the conjugated carbon chain of carotenoids. The oxidative cleavage mechanism of these enzymes is not clear and both monooxygenase and dioxygenase mechanisms have been proposed for different carotenoid cleavage enzymes. CCOs have been described from plants, animals, fungi and cyanobacteria but little is known about their distribution and activities in bacteria other than cyanobacteria. We surveyed bacterial genome sequences for CCO homologs and report the characterization of CCO homologs identified in Novosphingobium aromaticivorans DSM 12444 (NOV1 and NOV2) and in Bradyrhizobium sp. (BRA-J and BRA-S). In vitro and in vivo assays with carotenoid and stilbene compounds were used to investigate cleavage activities of the recombinant enzymes. The NOV enzymes cleaved the interphenyl α-β double bond of stilbenes with an oxygen functional group at the 4’ carbon (e.g. resveratrol, piceatannol, and rhaponticin) to the corresponding aldehyde products. Carotenoids and apocarotenoids were not substrates for these enzymes. The two homologous enzymes from Bradyrhizobium sp. did not possess carotenoid or stilbene cleavage oxygenase activities, but showed activity with farnesol. To investigate whether oxidative cleavage of stilbenes proceeds via a monooxygenase or dioxygenase reaction, oxygen labeling studies were conducted with NOV2. Our labeling studies show that double-bond cleavage of stilbenes occurs via a monooxygenase reaction mechanism. PMID:18478524

  16. An improved protocol with a highly degenerate primer targeting copper-containing membrane-bound monooxygenase genes for community analysis of methane- and ammonia-oxidizing bacteria.

    PubMed

    Wang, Jian-Gong; Xia, Fei; Zeleke, Jemaneh; Zou, Bin; Rhee, Sung-Keun; Quan, Zhe-Xue

    2017-03-01

    The copper-containing membrane-bound monooxygenase (CuMMO) family comprises key enzymes for methane or ammonia oxidation: particulate methane monooxygenase (PMMO) and ammonia monooxygenase (AMO). To comprehensively amplify CuMMO genes, a two-step PCR strategy was developed using a newly designed tagged highly degenerate primer (THDP; degeneracy = 4608). Designated THDP-PCR, the technique consists of primary CuMMO gene-specific PCR followed by secondary PCR with a tag as a single primer. No significant bias in THDP-PCR amplification was found using various combinations of template mixtures of pmoA and amoA genes, which encode key subunits of the pMMO and AMO enzymes, respectively, from different microbes. THDP-PCR was successfully applied to nine different environmental samples and revealed relatively high contents of complete ammonia oxidation (Comammox)-related bacteria and a novel group of the CuMMO family. The levels of freshwater cluster methanotrophs obtained by THDP-PCR were much higher than those obtained by conventional methanotroph-specific PCR. The THDP-PCR strategy developed in this study can be extended to other functional gene-based community analyses, particularly when the target gene sequences lack regions of high consensus for primer design. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  17. Post-translational activation of human phenylalanine 4-monooxygenase from an endobiotic to a xenobiotic enzyme by reactive oxygen and reactive nitrogen species.

    PubMed

    Antypa, A; Rebello, C; Biernacka, A; Krajewski, K; Cassam, J; Mitchell, S C; Steventon, G B

    2010-05-01

    An investigation into the post-translational activation of cDNA-expressed human phenylalanine 4-monooxygenase and human hepatic cytosolic fraction phenylalanine 4-monooxygenase activity with respect to both endobiotic metabolism and xenobiotic metabolism revealed that the reactive oxygen species (hydrogen peroxide and hydroxyl radical) and reactive nitrogen species (nitric oxide and peroxynitrite) could elicit the post-translational activation of the enzyme with respect to both of these biotransformation reactions. In virtually all instances, the K(m) values were decreased and the V(max) values were increased; the only exceptions observed being with hydrogen peroxide and L-phenylalanine. These effects were shown to occur at activator concentrations known to exist in physiological situations and, hence, suggest that reactive oxygen and reactive nitrogen species may cause, and may be involved with, the post-translational activation of phenylalanine 4-monooxygenase within the human body. This mechanism, in response to free-radical bursts, may enable the enzyme to expand its substrate range and to process certain xenobiotics as and when required.

  18. An Unprecedented NADPH Domain Conformation in Lysine Monooxygenase NbtG Provides Insights into Uncoupling of Oxygen Consumption from Substrate Hydroxylation

    SciTech Connect

    Binda, Claudia; Robinson, Reeder M.; Martin del Campo, Julia S.; Keul, Nicholas D.; Rodriguez, Pedro J.; Robinson, Howard H.; Mattevi, Andrea; Sobrado, Pablo

    2015-03-23

    N-hydroxylating monooxygenases (NMOs) are involved in the biosynthesis of iron-chelating hydroxamate-containing siderophores that play a role in microbial virulence. These flavoenzymes catalyze the NADPH- and oxygen-dependent hydroxylation of amines, such as those found on the side chains of lysine and ornithine. In this work we report the biochemical and structural characterization of Nocardia farcinica Lys monooxygenase (NbtG), which has similar biochemical properties to mycobacterial homologs. NbtG is also active on D-Lys although it binds L-Lys with a higher affinity. Differently from the ornithine monooxygenases PvdA, SidA and KtzI, NbtG can use both NADH and NADPH and is highly uncoupled, producing more superoxide and hydrogen peroxide than hydroxylated Lys. The crystal structure of NbtG solved at 2.4 Å resolution revealed an unexpected protein conformation with a 30° rotation of the NAD(P)H domain with respect to the FAD domain that precludes binding of the nicotinamide cofactor. This “occluded” structure may explain the biochemical properties of NbtG, specifically with regard to the substantial uncoupling and limited stabilization of the C4a-hydroperoxyflavin intermediate. We discuss the biological implications of these findings.

  19. Expression of aryl hydrocarbon receptor 1 (AHR1), AHR1 nuclear translocator 1 (ARNT1) and CYP1 family monooxygenase mRNAs and their activity in chicken ovarian follicles following in vitro exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD).

    PubMed

    Antos, Piotr A; Błachuta, Małgorzata; Hrabia, Anna; Grzegorzewska, Agnieszka K; Sechman, Andrzej

    2015-09-02

    The aim of this in vitro study was to determine the effect of TCDD and luteinizing hormone (LH) on mRNA expression of aryl hydrocarbon receptor 1 (AHR1), AHR1 nuclear translocator 1 (ARNT1), and the CYP1 family monooxygenases (CYP1A4, CYP1A5, CYP1B1), and to assess the basal and TCDD-induced activity of these enzymes in chicken ovarian follicles. White (WF) and yellowish (YF) prehierarchical follicles and fragments of the theca (TL) and granulosa (GL) layers of the 3 largest preovulatory follicles (F3-F1) were exposed to TCDD (10nM), ovine LH (oLH; 10ng/mL) or a combination of TCDD (10nM) and oLH (10ng/mL), and increasing doses of TCDD (0.01-100nM). AHR1 and ARNT1 mRNA transcripts were found in all examined follicles. The effect of TCDD and oLH on AHR1 and ARNT1 mRNA expression depended on the maturational state of the follicle. CYP1A4 was predominantly expressed in the GL of the F3-F1 follicles; in comparison with the WF, a higher level of CYP1A5 mRNA was found both in the GL and TL of F3-F1 follicles. Alternatively, the highest level of CYP1B1 mRNA was noticed in the WF follicles. In different developmental stages of the follicle TCDD and oLH induced a different CYP1 isoform. TCDD increased EROD and MROD activities in all the investigated ovarian follicles. In conclusion, AHR1 and ARNT1 mRNA expression indicate that the chicken ovary is a target tissue for dioxin and dioxin-like compounds. The expression of CYP1-family genes and TCDD-inducible EROD and MROD activities in ovarian follicles suggest the possibility of xenobiotic detoxification in the chicken ovary. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  20. Local Auxin Biosynthesis Mediated by a YUCCA Flavin Monooxygenase Regulates Haustorium Development in the Parasitic Plant Phtheirospermum japonicum[OPEN

    PubMed Central

    Takebayashi, Yumiko; Kasahara, Hiroyuki; Wafula, Eric; dePamphilis, Claude W.; Namba, Shigetou

    2016-01-01

    Parasitic plants in the Orobanchaceae cause serious agricultural problems worldwide. Parasitic plants develop a multicellular infectious organ called a haustorium after recognition of host-released signals. To understand the molecular events associated with host signal perception and haustorium development, we identified differentially regulated genes expressed during early haustorium development in the facultative parasite Phtheirospermum japonicum using a de novo assembled transcriptome and a customized microarray. Among the genes that were upregulated during early haustorium development, we identified YUC3, which encodes a functional YUCCA (YUC) flavin monooxygenase involved in auxin biosynthesis. YUC3 was specifically expressed in the epidermal cells around the host contact site at an early time point in haustorium formation. The spatio-temporal expression patterns of YUC3 coincided with those of the auxin response marker DR5, suggesting generation of auxin response maxima at the haustorium apex. Roots transformed with YUC3 knockdown constructs formed haustoria less frequently than nontransgenic roots. Moreover, ectopic expression of YUC3 at the root epidermal cells induced the formation of haustorium-like structures in transgenic P. japonicum roots. Our results suggest that expression of the auxin biosynthesis gene YUC3 at the epidermal cells near the contact site plays a pivotal role in haustorium formation in the root parasitic plant P. japonicum. PMID:27385817

  1. Kinetic and spectroscopic characterization of intermediates and component interactions in reactions of methane monooxygenase from methylococcus capsulatus (Bath)

    SciTech Connect

    Liu, K.E.; Valentine, A.M.; Salifoglou, A.; Lippard, S.J.; Wang, D.; Huynh, B.H.; Edmondson, D.E.

    1995-10-18

    We describe mechanistic studies of the soluble methane monooxygenase (sMMO) enzyme system from Methylococcus capsulatus (Bath). Interactions among the three sMMO components, the hydroxylase (H), reductase (R), and protein B (B), were investigated by monitoring conversion of nitrobenzene to nitrophenol under both single turnover and catalytic conditions. During catalytic turnover, hydroxylation occurs to afford 3-nitrophenol (43%) and 4-nitrophenol (57%), whereas hydroxylation takes place exclusively (> 95%) to give 4-nitrophenol under single turnover conditions in the absence of reductase. Protein B exerts a strong influence on single turnover reactions of nitrobenzene, with optimal rate constants and yields obtained by using 1.5-2 equiv of protein R per equivalent of hydroxylase. The temperature dependence of these kinetic values was determined. Changes in dioxygen concentration and pH, as well as exchange of solvent accessible protons with D{sub 2}O, did not significantly affect the rate constants for either of these processes, the implications of which for the kinetic mechanism are discussed. From the present and related evidence, structures for H{sub peroxo} and Q are proposed. 54 refs., 11 figs., 4 tabs.

  2. Two Cytochrome P450 Monooxygenases Catalyze Early Hydroxylation Steps in the Potato Steroid Glycoalkaloid Biosynthetic Pathway1[OPEN

    PubMed Central

    Nakayasu, Masaru; Ohyama, Kiyoshi; Saito, Kazuki

    2016-01-01

    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

  3. Monooxygenase activity of black-crowned night-heron (BCNH) nestlings in Virginia, the Great Lakes and San Francisco Bay

    USGS Publications Warehouse

    Rattner, B.A.; Melancon, M.J.; Custer, T.W.; Hothem, R.L.; King, K.A.; LeCaptain, L.J.; Spann, J.W.

    1990-01-01

    To evaluate cytochrome P-450 related parameters as biomarkers of pollutant exposure, rates of arylhydrocarbon hydroxylase (AHH), ethoxyresorufin-O-deethylase (EROD), benzyloxyROD (BROD), pentoxyROD (PROD) and ethoxycoumarinOD (ECOD) were studied in 10-day-old BCNHs (Nycticorax nycticorax). Nestlings were collected from Chincoteague National Wildlife Refuge, VA ('controls') and from polluted sites including. Cat Island, Green Bay, WI, and Bair and West Marin Islands, San Francisco Bay, CA. Livers were frozen (-70.C) for monooxygenase assays and SDS-PAGE. Microsomal AHH and BROD activities were greater (P2 standard deviations from the control mean (induced up to 3-fold). EROD, PROD and ECOD did not differ among sites. Absence of an EROD response with AHH and BROD induction in BCNHs is different than responses in other species. The association of pollutant burdens with P-450 parameters is being studied. These biomarkers may serve as a rapid screen of exposure in a national contaminant biomonitoring program and other assessment activities.

  4. Phylogenetic diversity of archaea and the archaeal ammonia monooxygenase gene in uranium mining-impacted locations in Bulgaria.

    PubMed

    Radeva, Galina; Kenarova, Anelia; Bachvarova, Velina; Flemming, Katrin; Popov, Ivan; Vassilev, Dimitar; Selenska-Pobell, Sonja

    2014-01-01

    Uranium mining and milling activities adversely affect the microbial populations of impacted sites. The negative effects of uranium on soil bacteria and fungi are well studied, but little is known about the effects of radionuclides and heavy metals on archaea. The composition and diversity of archaeal communities inhabiting the waste pile of the Sliven uranium mine and the soil of the Buhovo uranium mine were investigated using 16S rRNA gene retrieval. A total of 355 archaeal clones were selected, and their 16S rDNA inserts were analysed by restriction fragment length polymorphism (RFLP) discriminating 14 different RFLP types. All evaluated archaeal 16S rRNA gene sequences belong to the 1.1b/Nitrososphaera cluster of Crenarchaeota. The composition of the archaeal community is distinct for each site of interest and dependent on environmental characteristics, including pollution levels. Since the members of 1.1b/Nitrososphaera cluster have been implicated in the nitrogen cycle, the archaeal communities from these sites were probed for the presence of the ammonia monooxygenase gene (amoA). Our data indicate that amoA gene sequences are distributed in a similar manner as in Crenarchaeota, suggesting that archaeal nitrification processes in uranium mining-impacted locations are under the control of the same key factors controlling archaeal diversity.

  5. Quantum mechanical calculations suggest that lytic polysaccharide monooxygenases use a copper-oxyl, oxygen-rebound mechanism.

    PubMed

    Kim, Seonah; Ståhlberg, Jerry; Sandgren, Mats; Paton, Robert S; Beckham, Gregg T

    2014-01-07

    Lytic polysaccharide monooxygenases (LPMOs) exhibit a mononuclear copper-containing active site and use dioxygen and a reducing agent to oxidatively cleave glycosidic linkages in polysaccharides. LPMOs represent a unique paradigm in carbohydrate turnover and exhibit synergy with hydrolytic enzymes in biomass depolymerization. To date, several features of copper binding to LPMOs have been elucidated, but the identity of the reactive oxygen species and the key steps in the oxidative mechanism have not been elucidated. Here, density functional theory calculations are used with an enzyme active site model to identify the reactive oxygen species and compare two hypothesized reaction pathways in LPMOs for hydrogen abstraction and polysaccharide hydroxylation; namely, a mechanism that employs a η(1)-superoxo intermediate, which abstracts a substrate hydrogen and a hydroperoxo species is responsible for substrate hydroxylation, and a mechanism wherein a copper-oxyl radical abstracts a hydrogen and subsequently hydroxylates the substrate via an oxygen-rebound mechanism. The results predict that oxygen binds end-on (η(1)) to copper, and that a copper-oxyl-mediated, oxygen-rebound mechanism is energetically preferred. The N-terminal histidine methylation is also examined, which is thought to modify the structure and reactivity of the enzyme. Density functional theory calculations suggest that this posttranslational modification has only a minor effect on the LPMO active site structure or reactivity for the examined steps. Overall, this study suggests the steps in the LPMO mechanism for oxidative cleavage of glycosidic bonds.

  6. Quantum mechanical calculations suggest that lytic polysaccharide monooxygenases use a copper-oxyl, oxygen-rebound mechanism

    PubMed Central

    Kim, Seonah; Ståhlberg, Jerry; Sandgren, Mats; Paton, Robert S.; Beckham, Gregg T.

    2014-01-01

    Lytic polysaccharide monooxygenases (LPMOs) exhibit a mononuclear copper-containing active site and use dioxygen and a reducing agent to oxidatively cleave glycosidic linkages in polysaccharides. LPMOs represent a unique paradigm in carbohydrate turnover and exhibit synergy with hydrolytic enzymes in biomass depolymerization. To date, several features of copper binding to LPMOs have been elucidated, but the identity of the reactive oxygen species and the key steps in the oxidative mechanism have not been elucidated. Here, density functional theory calculations are used with an enzyme active site model to identify the reactive oxygen species and compare two hypothesized reaction pathways in LPMOs for hydrogen abstraction and polysaccharide hydroxylation; namely, a mechanism that employs a η1-superoxo intermediate, which abstracts a substrate hydrogen and a hydroperoxo species is responsible for substrate hydroxylation, and a mechanism wherein a copper-oxyl radical abstracts a hydrogen and subsequently hydroxylates the substrate via an oxygen-rebound mechanism. The results predict that oxygen binds end-on (η1) to copper, and that a copper-oxyl–mediated, oxygen-rebound mechanism is energetically preferred. The N-terminal histidine methylation is also examined, which is thought to modify the structure and reactivity of the enzyme. Density functional theory calculations suggest that this posttranslational modification has only a minor effect on the LPMO active site structure or reactivity for the examined steps. Overall, this study suggests the steps in the LPMO mechanism for oxidative cleavage of glycosidic bonds. PMID:24344312

  7. Identification of dimethylamine monooxygenase in marine bacteria reveals a metabolic bottleneck in the methylated amine degradation pathway.

    PubMed

    Lidbury, Ian; Mausz, Michaela A; Scanlan, David J; Chen, Yin

    2017-07-01

    Methylated amines (MAs) are ubiquitous in the marine environment and their subsequent flux into the atmosphere can result in the formation of aerosols and ultimately cloud condensation nuclei. Therefore, these compounds have a potentially important role in climate regulation. Using Ruegeria pomeroyi as a model, we identified the genes encoding dimethylamine (DMA) monooxygenase (dmmABC) and demonstrate that this enzyme degrades DMA to monomethylamine (MMA). Although only dmmABC are required for enzyme activity in recombinant Escherichia coli, we found that an additional gene, dmmD, was required for the growth of R. pomeroyi on MAs. The dmmDABC genes are absent from the genomes of multiple marine bacteria, including all representatives of the cosmopolitan SAR11 clade. Consequently, the abundance of dmmDABC in marine metagenomes was substantially lower than the genes required for other metabolic steps of the MA degradation pathway. Thus, there is a genetic and potential metabolic bottleneck in the marine MA degradation pathway. Our data provide an explanation for the observation that DMA-derived secondary organic aerosols (SOAs) are among the most abundant SOAs detected in fine marine particles over the North and Tropical Atlantic Ocean.

  8. The Cercospora nicotianae gene encoding dual O-methyltransferase and FAD-dependent monooxygenase domains mediates cercosporin toxin biosynthesis.

    PubMed

    Dekkers, Katherine L; You, Bang-Jau; Gowda, Vivek S; Liao, Hui-Ling; Lee, Miin-Huey; Bau, Huey-Jiunn; Ueng, Peter P; Chung, Kuang-Ren

    2007-05-01

    Cercosporin, a photo-activated, non-host-selective phytotoxin produced by many species of the plant pathogenic fungus Cercospora, causes peroxidation of plant cell membranes by generating reactive oxygen species and is an important virulence determinant. Here we report a new gene, CTB3 that is involved in cercosporin biosynthesis in Cercospora nicotianae. CTB3 is adjacent to a previously identified CTB1 encoding a polyketide synthase which is also required for cercosporin production. CTB3 contains a putative O-methyltransferase domain in the N-terminus and a putative flavin adenine dinucleotide (FAD)-dependent monooxygenase domain in the C-terminus. The N-terminal amino acid sequence also is similar to that of the transcription enhancer AFLS (formerly AFLJ) involved in aflatoxin biosynthesis. Expression of CTB3 was differentially regulated by light, medium, nitrogen and carbon sources and pH. Disruption of the N- or C-terminus of CTB3 yielded mutants that failed to accumulate the CTB3 transcript and cercosporin. The Deltactb3 disruptants produced a yellow pigment that is not toxic to tobacco suspension cells. Production of cercosporin in a Deltactb3 null mutant was fully restored when transformed with a functional CTB3 clone or when paired with a Deltactb1-null mutant (defective in polyketide synthase) by cross feeding of the biosynthetic intermediates. Pathogenicity assays using detached tobacco leaves revealed that the Deltactb3 disruptants drastically reduced lesion formation.

  9. Coenzyme Q Biosynthesis: Evidence for a Substrate Access Channel in the FAD-Dependent Monooxygenase Coq6

    PubMed Central

    Ismail, Alexandre; Leroux, Vincent; Smadja, Myriam; Gonzalez, Lucie; Lombard, Murielle; Pierrel, Fabien; Mellot-Draznieks, Caroline; Fontecave, Marc

    2016-01-01

    Coq6 is an enzyme involved in the biosynthesis of coenzyme Q, a polyisoprenylated benzoquinone lipid essential to the function of the mitochondrial respiratory chain. In the yeast Saccharomyces cerevisiae, this putative flavin-dependent monooxygenase is proposed to hydroxylate the benzene ring of coenzyme Q (ubiquinone) precursor at position C5. We show here through biochemical studies that Coq6 is a flavoprotein using FAD as a cofactor. Homology models of the Coq6-FAD complex are constructed and studied through molecular dynamics and substrate docking calculations of 3-hexaprenyl-4-hydroxyphenol (4-HP6), a bulky hydrophobic model substrate. We identify a putative access channel for Coq6 in a wild type model and propose in silico mutations positioned at its entrance capable of partially (G248R and L382E single mutations) or completely (a G248R-L382E double-mutation) blocking access to the channel for the substrate. Further in vivo assays support the computational predictions, thus explaining the decreased activities or inactivation of the mutated enzymes. This work provides the first detailed structural information of an important and highly conserved enzyme of ubiquinone biosynthesis. PMID:26808124

  10. Loss-of-function mutation in carotenoid 15,15'-monooxygenase identified in a patient with hypercarotenemia and hypovitaminosis A.

    PubMed

    Lindqvist, Annika; Sharvill, John; Sharvill, Denis E; Andersson, Stefan

    2007-11-01

    The enzyme carotenoid 15,15'-monooxygenase (CMO1) catalyzes the first step in the conversion of dietary provitamin A carotenoids to vitamin A in the small intestine. Plant carotenoids are an important dietary source of vitamin A (retinol) and the sole source of vitamin A for vegetarians. Vitamin A is essential for normal embryonic development as well as normal physiological functions in children and adults. Here, we describe one heterozygous T170M missense mutation in the CMO1 gene in a subject with hypercarotenemia and mild hypovitaminosis A. The replacement of a highly conserved threonine with methionine results in a 90% reduction in enzyme activity when analyzed in vitro using purified recombinant enzymes. The Michaelis-Menten constant (K(m)) for the mutated enzyme is normal. Ample amounts of carotenoids are present in plasma of persons consuming a normal Western diet, suggesting that the enzyme is saturated with substrate under normal conditions. Therefore, we propose that haploinsufficiency of the CMO1 enzyme may cause symptoms of hypercarotenemia and hypovitaminosis A in individuals consuming a carotenoid-containing and vitamin A-deficient diet.

  11. Biocatalytic Conversion of Avermectin to 4"-Oxo-Avermectin: Heterologous Expression of the ema1 Cytochrome P450 Monooxygenase

    PubMed Central

    Molnár, István; Hill, D. Steven; Zirkle, Ross; Hammer, Philip E.; Gross, Frank; Buckel, Thomas G.; Jungmann, Volker; Pachlatko, Johannes Paul; Ligon, James M.

    2005-01-01

    The cytochrome P450 monooxygenase Ema1 from Streptomyces tubercidicus R-922 and its homologs from closely related Streptomyces strains are able to catalyze the regioselective oxidation of avermectin into 4"-oxo-avermectin, a key intermediate in the manufacture of the agriculturally important insecticide emamectin benzoate (V. Jungmann, I. Molnár, P. E. Hammer, D. S. Hill, R. Zirkle, T. G. Buckel, D. Buckel, J. M. Ligon, and J. P. Pachlatko, Appl. Environ. Microbiol. 71:6968-6976, 2005). The gene for Ema1 has been expressed in Streptomyces lividans, Streptomyces avermitilis, and solvent-tolerant Pseudomonas putida strains using different promoters and vectors to provide biocatalytically competent cells. Replacing the extremely rare TTA codon with the more frequent CTG codon to encode Leu4 in Ema1 increased the biocatalytic activities of S. lividans strains producing this enzyme. Ferredoxins and ferredoxin reductases were also cloned from Streptomyces coelicolor and biocatalytic Streptomyces strains and tested in ema1 coexpression systems to optimize the electron transport towards Ema1. PMID:16269733

  12. The TMAO-Producing Enzyme Flavin-Containing Monooxygenase 3 Regulates Obesity and the Beiging of White Adipose Tissue.

    PubMed

    Schugar, Rebecca C; Shih, Diana M; Warrier, Manya; Helsley, Robert N; Burrows, Amy; Ferguson, Daniel; Brown, Amanda L; Gromovsky, Anthony D; Heine, Markus; Chatterjee, Arunachal; Li, Lin; Li, Xinmin S; Wang, Zeneng; Willard, Belinda; Meng, YongHong; Kim, Hanjun; Che, Nam; Pan, Calvin; Lee, Richard G; Crooke, Rosanne M; Graham, Mark J; Morton, Richard E; Langefeld, Carl D; Das, Swapan K; Rudel, Lawrence L; Zein, Nizar; McCullough, Arthur J; Dasarathy, Srinivasan; Tang, W H Wilson; Erokwu, Bernadette O; Flask, Chris A; Laakso, Markku; Civelek, Mete; Naga Prasad, Sathyamangla V; Heeren, Joerg; Lusis, Aldons J; Hazen, Stanley L; Brown, J Mark

    2017-06-20

    Emerging evidence suggests that microbes resident in the human intestine represent a key environmental factor contributing to obesity-associated disorders. Here, we demonstrate that the gut microbiota-initiated trimethylamine N-oxide (TMAO)-generating pathway is linked to obesity and energy metabolism. In multiple clinical cohorts, systemic levels of TMAO were observed to strongly associate with type 2 diabetes. In addition, circulating TMAO levels were associated with obesity traits in the different inbred strains represented in the Hybrid Mouse Diversity Panel. Further, antisense oligonucleotide-mediated knockdown or genetic deletion of the TMAO-producing enzyme flavin-containing monooxygenase 3 (FMO3) conferred protection against obesity in mice. Complimentary mouse and human studies indicate a negative regulatory role for FMO3 in the beiging of white adipose tissue. Collectively, our studies reveal a link between the TMAO-producing enzyme FMO3 and obesity and the beiging of white adipose tissue. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  13. Xenobiotics in gametes of Lake Michigan lake trout (Salvelinus namaycush) induce hepatic monooxygenase activity in their offspring

    SciTech Connect

    Binder, R.L.; Lech, J.J.

    1984-12-01

    Eggs spawned from Lake Michigan lake trout contain a number of xenobiotic compounds, including polychlorinated biphenyls (PCBs). To assess whether this contamination is sufficient to induce hepatic cytochrome P-450-dependent monooxygenase (MO) activity during early development, the hepatic MO systems of laboratory-cultured offspring of Lake Michigan, Green Bay, and Marquette Hatchery lake trout were compared. Additionally, the induction of hepatic cytochrome P-450 systems in developing lake trout by the commercial PCB mixture, Aroclor 1254 (A1254), was characterized. During late embryonic development and at the swim-up stage, the hepatic MO systems of the feral lake trout offspring appeared induced, based on levels of aryl hydrocarbon hydroxylase (AHH) activity that were 3.5- to 8.6-fold higher than the hatchery control levels. Furthermore, at the swim-up stage the feral trout offspring resembled A1254-treated hatchery fry with regard to the degree of inhibition of hepatic AHH activity by alpha-naphthoflavone, and the presence of an inducible Mr . 58,000 polypeptide in hepatic microsomes. The levels of aminopyrine N-demethylase activity, which was relatively unresponsive to inducers, were moderately lower in the Lake Michigan and Green Bay swim-up fry compared to the hatchery control levels. After 7 months of posthatching laboratory culture, when residues of xenobiotics present at fertilization were greatly diluted by growth, the hepatic MO systems of the Lake Michigan and hatchery trout offspring appeared essentially indistinguishable with regard to a number of parameters.

  14. Tertiary amines related to brompheniramine: preferred conformations for N-oxygenation by the hog liver flavin-containing monooxygenase.

    PubMed

    Cashman, J R; Celestial, J R; Leach, A; Newdoll, J; Park, S B

    1993-08-01

    The metabolism of racemic, (D)- and (L)-brompheniramine, a widely used antihistamine, was studied with microsomes and with highly purified flavin-containing monooxygenase (FMO) from hog liver. In addition, a number of other similar tertiary amines were evaluated as substrates for FMO activity from hog liver and the kinetic constants obtained were compared with brompheniramine. Although some N-demethylation was observed, the major metabolite of brompheniramine and the other tertiary amines examined in hog liver microsomes was the metabolite containing an aliphatic nitrogen N-oxide. Brompheniramine was extensively N-oxygenated by the highly purified FMO from hog liver. N-Oxygenation of brompheniramine in both microsomes and with highly purified FMO from hog liver was enantioselective. The Km for N-oxygenation of (D)-brompheniramine was markedly lower than the Km for (L)-brompheniramine. (E)- and (Z)-zimeldine are less conformationally flexible model compounds of brompheniramine, and these compounds were also examined and were found to be stereoselectively N-oxygenated by the highly purified FMO from hog liver. The similarities and differences in Km and Vmax values were evaluated in terms of possible conformations of the substrates determined by SYBYL molecular mechanics calculations. Distance map data indicated that FMO preferentially accommodated selected conformations of tertiary amines. Thus, (D)-brompheniramine and (Z)-zimeldine presumably have the aliphatic tertiary amine nitrogen atom and aromatic ring center at a defined distance and geometry and were more efficiently N-oxygenated than their respective isomers.

  15. Conformational analysis of putative regulatory subunit D of the toluene/o-xylene-monooxygenase complex from Pseudomonas stutzeri OX1

    PubMed Central

    Scognamiglio, Roberta; Notomista, Eugenio; Barbieri, Paola; Pucci, Piero; Piaz, Fabrizio Dal; Tramontano, Anna; Di Donato, Alberto

    2001-01-01

    A gene cluster isolated from Pseudomonas stutzeri OX1 genomic DNA and containing six ORFs codes for toluene/o-xylene-monooxygenase. The putative regulatory D subunit was expressed in Escherichia coli and purified. Its protein sequence was verified by mass spectrometry mapping and found to be identical to the sequence predicted on the basis of the DNA sequence. The surface topology of subunit D in solution was probed by limited proteolysis carried out under strictly controlled conditions using several proteases as proteolytic probes. The same experiments were carried out on the homologous P2 component of the multicomponent phenol hydroxylase from Pseudomonas putida CF600. The proteolytic fragments released from both proteins in their native state were analyzed by electrospray mass spectrometry, and the preferential cleavage sites were assessed. The results indicated that despite the relatively high similarity between the sequences of the two proteins, some differences in the distribution of preferential proteolytic cleavages were detected, and a much higher conformational flexibility of subunit D was inferred. Moreover, automatic modeling of subunit D was attempted, based on the known three-dimensional structure of P2. Our results indicate that, at least in this case, standard modeling procedures based on automatic alignment on the structure of P2 fail to produce a model consistent with limited proteolysis experimental data. Thus, it is our opinion that reliable techniques such as limited proteolysis can be employed to test three-dimensional models and highlight problems in automatic model building. PMID:11344317

  16. Differences in metabolism of the marine biotoxin okadaic acid by human and rat cytochrome P450 monooxygenases.

    PubMed

    Kolrep, Franziska; Hessel, Stefanie; These, Anja; Ehlers, Anke; Rein, Kathleen; Lampen, Alfonso

    2016-08-01

    The ingestion of seafood contaminated with the marine biotoxin okadaic acid (OA) can lead to diarrhetic shellfish poisoning with symptoms like nausea, vomiting and abdominal cramps. Both rat and the human hepatic cytochrome P450 monooxygenases (CYP) metabolize OA. However, liver cell toxicity of metabolized OA is mainly unclear. The aim of our study was to detect the cellular effects in HepG2 cells exposed to OA in the presence of recombinant CYP enzymes of both rat and human for the investigation of species differences. The results should be set in correlation with a CYP-specific metabolite pattern. Comparative metabolite profiles of OA after incubation in rat and human recombinant CYP enzymes were established by using LC-MS/MS technique. Results demonstrated that metabolism of OA to oxygenated metabolites correlates with detoxification which was mainly catalyzed by human CYP3A4 and CYP3A5. Detoxification by rat Cyp3a1 was lower compared to human CYP3A enzymes and activation of OA by Cyp3a2 was observed, coincident with minor overall conversion capacity of OA. By contrast human and rat CYP1A2 seem to activate OA into cytotoxic intermediates. In conclusion, different mechanisms of OA metabolism may occur in the liver. At low OA doses, the human liver is likely well protected against cytotoxic OA, but for high shellfish consumers a potential risk cannot be excluded.

  17. Local Auxin Biosynthesis Mediated by a YUCCA Flavin Monooxygenase Regulates Haustorium Development in the Parasitic Plant Phtheirospermum japonicum.

    PubMed

    Ishida, Juliane K; Wakatake, Takanori; Yoshida, Satoko; Takebayashi, Yumiko; Kasahara, Hiroyuki; Wafula, Eric; dePamphilis, Claude W; Namba, Shigetou; Shirasu, Ken

    2016-08-01

    Parasitic plants in the Orobanchaceae cause serious agricultural problems worldwide. Parasitic plants develop a multicellular infectious organ called a haustorium after recognition of host-released signals. To understand the molecular events associated with host signal perception and haustorium development, we identified differentially regulated genes expressed during early haustorium development in the facultative parasite Phtheirospermum japonicum using a de novo assembled transcriptome and a customized microarray. Among the genes that were upregulated during early haustorium development, we identified YUC3, which encodes a functional YUCCA (YUC) flavin monooxygenase involved in auxin biosynthesis. YUC3 was specifically expressed in the epidermal cells around the host contact site at an early time point in haustorium formation. The spatio-temporal expression patterns of YUC3 coincided with those of the auxin response marker DR5, suggesting generation of auxin response maxima at the haustorium apex. Roots transformed with YUC3 knockdown constructs formed haustoria less frequently than nontransgenic roots. Moreover, ectopic expression of YUC3 at the root epidermal cells induced the formation of haustorium-like structures in transgenic P. japonicum roots. Our results suggest that expression of the auxin biosynthesis gene YUC3 at the epidermal cells near the contact site plays a pivotal role in haustorium formation in the root parasitic plant P. japonicum.

  18. Phylogenetic Diversity of Archaea and the Archaeal Ammonia Monooxygenase Gene in Uranium Mining-Impacted Locations in Bulgaria

    PubMed Central

    Radeva, Galina; Kenarova, Anelia; Bachvarova, Velina; Popov, Ivan; Selenska-Pobell, Sonja

    2014-01-01

    Uranium mining and milling activities adversely affect the microbial populations of impacted sites. The negative effects of uranium on soil bacteria and fungi are well studied, but little is known about the effects of radionuclides and heavy metals on archaea. The composition and diversity of archaeal communities inhabiting the waste pile of the Sliven uranium mine and the soil of the Buhovo uranium mine were investigated using 16S rRNA gene retrieval. A total of 355 archaeal clones were selected, and their 16S rDNA inserts were analysed by restriction fragment length polymorphism (RFLP) discriminating 14 different RFLP types. All evaluated archaeal 16S rRNA gene sequences belong to the 1.1b/Nitrososphaera cluster of Crenarchaeota. The composition of the archaeal community is distinct for each site of interest and dependent on environmental characteristics, including pollution levels. Since the members of 1.1b/Nitrososphaera cluster have been implicated in the nitrogen cycle, the archaeal communities from these sites were probed for the presence of the ammonia monooxygenase gene (amoA). Our data indicate that amoA gene sequences are distributed in a similar manner as in Crenarchaeota, suggesting that archaeal nitrification processes in uranium mining-impacted locations are under the control of the same key factors controlling archaeal diversity. PMID:24711725

  19. Modeling trichloroethylene degradation by a recombinant pseudomonad expressing toluene ortho-monooxygenase in a fixed-film bioreactor

    SciTech Connect

    Sun, A.K.; Hong, J.; Wood, T.K.

    1998-07-05

    Burkholderia cepacia PR1{sub 23}(TOM{sub 23C}), expressing constitutively the TCE-degrading enzyme toluene ortho-monooxygenase (Tom), was immobilized on SIRAN{trademark} glass beads in a biofilter for the degradation and mineralization of gas-phase trichloroethylene (TCE). To interpret the experimental results, a mathematical model has been developed which includes axial dispersion, convection, film mass-transfer, and biodegradation coupled with deactivation of the TCE-degrading enzyme. Parameters used for numerical simulation were determined from either independent experiments or values reported in the literature. The model was compared with the experimental data, and there was good agreement between the predicted and measured TCE breakthrough curves. The simulations indicated that TCE degradation in the biofilter was not limited by mass transfer of TCE or oxygen from the gas phase to the liquid/biofilm phase (biodegradation limits), and predicts that improving the specific TCE degradation rates of bacteria will not significantly enhance long-term biofilter performance. The most important factors for prolonging the performance of biofilter are increasing the amount of active biomass and the transformation capacity enhancing resistance to TCE metabolism.

  20. A novel flavin-containing monooxygenase from Methylophaga sp strain SK1 and its indigo synthesis in Escherichia coli.

    PubMed

    Choi, Hack Sun; Kim, Jin Kwon; Cho, Eun Hee; Kim, Yong Chul; Kim, Jae Il; Kim, Si Wouk

    2003-07-11

    We cloned a gene from Methylophaga sp. strain SK1. This gene was responsible for producing, the blue pigment, indigo. The complete open reading frame was 1371 bp long, which encodes a protein of 456 amino acids. The molecular mass of the encoded protein was 105 kDa, consisting of homodimer of 54 kDa with an isoelectric point of 5.14. The deduced amino acid sequence from the gene showed approximately 30% identities with flavin-containing monooxygenases (FMOs) of human (FMO1-FMO5), Arabidopsis, and yeast. It contained three characteristic sequence motifs of FMOs: FAD binding domain, FMO-identifying sequence motif, and NADPH binding domain. In addition, the biochemical properties such as substrate specificities and absorption spectra were similar to the eukaryotic FMO families. Thus, we assigned the enzyme to be a bacterial FMO. The recombinant Escherichia coli expressing the bacterial FMO produced up to 160 mg of indigo per liter in the tryptophan medium after 12h cultivation. This suggests that the recombinant strain has a potential to be applied in microbial indigo production.

  1. Cholesterol-dependent degradation of squalene monooxygenase, a control point in cholesterol synthesis beyond HMG-CoA reductase.

    PubMed

    Gill, Saloni; Stevenson, Julian; Kristiana, Ika; Brown, Andrew J

    2011-03-02

    Exquisite control of cholesterol synthesis is crucial for maintaining homeostasis of this vital yet potentially toxic lipid. Squalene monooxygenase (SM) catalyzes the first oxygenation step in cholesterol synthesis, acting on squalene before cyclization into the basic steroid structure. Using model cell systems, we found that cholesterol caused the accumulation of the substrate squalene, suggesting that SM may serve as a flux-controlling enzyme beyond 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR, considered as rate limiting). Cholesterol accelerated the proteasomal degradation of SM which required the N-terminal domain, partially conserved in vertebrates but not in lower organisms. Unlike HMGR, SM degradation is not mediated by Insig, 24,25-dihydrolanosterol, or side-chain oxysterols, but rather by cholesterol itself. Importantly, SM's N-terminal domain conferred cholesterol-regulated turnover on heterologous fusion proteins. Furthermore, proteasomal inhibition almost totally eliminated squalene accumulation, highlighting the importance of this degradation mechanism for the control of SM and suggesting this as a possible control point in cholesterol synthesis. Copyright © 2011 Elsevier Inc. All rights reserved.

  2. 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.

  3. Genomic and transcriptomic insights into the cytochrome P450 monooxygenase gene repertoire in the rice pest brown planthopper, Nilaparvata lugens.

    PubMed

    Lao, Shu-Hua; Huang, Xiao-Hui; Huang, Hai-Jian; Liu, Cheng-Wen; Zhang, Chuan-Xi; Bao, Yan-Yuan

    2015-11-01

    The cytochrome P450 monooxygenase (P450) gene family is one of the most abundant eukaryotic gene families that encode detoxification enzymes. In this study, we identified an abundance of P450 gene repertoire through genome- and transcriptome-wide analysis in the brown planthopper (Nilaparvata lugens), the most destructive rice pest in Asia. Detailed gene information including the exon-intron organization, size, transcription orientation and distribution in the genome revealed that many P450 loci were closely situated on the same scaffold, indicating frequent occurrence of gene duplications. Insecticide-response expression profiling revealed that imidacloprid significantly increased NlCYP6CS1v2, NLCYP4CE1v2, NlCYP4DE1, NlCYP417A1v2 and NlCYP439A1 expression; while triazophos and deltamethrin notably enhanced NlCYP303A1 expression. Expression analysis at the developmental stage showed the egg-, nymph-, male- and female-specific expression patterns of N. lugens P450 genes. These novel findings will be helpful for clarifying the P450 functions in physiological processes including development, reproduction and insecticide resistance in this insect species. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Molecular evolutionary dynamics of cytochrome P450 monooxygenases across kingdoms: Special focus on mycobacterial P450s

    PubMed Central

    Parvez, Mohammad; Qhanya, Lehlohonolo Benedict; Mthakathi, Ntsane Trevor; Kgosiemang, Ipeleng Kopano Rosinah; Bamal, Hans Denis; Pagadala, Nataraj Sekhar; Xie, Ting; Yang, Haoran; Chen, Hengye; Theron, Chrispian William; Monyaki, Richie; Raselemane, Seiso Caiphus; Salewe, Vuyani; Mongale, Bogadi Lorato; Matowane, Retshedisitswe Godfrey; Abdalla, Sara Mohamed Hasaan; Booi, Wool Isaac; van Wyk, Mari; Olivier, Dedré; Boucher, Charlotte E.; Nelson, David R.; Tuszynski, Jack A.; Blackburn, Jonathan Michael; Yu, Jae-Hyuk; Mashele, Samson Sitheni; Chen, Wanping; Syed, Khajamohiddin

    2016-01-01

    Since the initial identification of cytochrome P450 monooxygenases (CYPs/P450s), great progress has been made in understanding their structure-function relationship, diversity and application in producing compounds beneficial to humans. However, the molecular evolution of P450s in terms of their dynamics both at protein and DNA levels and functional conservation across kingdoms still needs investigation. In this study, we analyzed 17 598 P450s belonging to 113 P450 families (bacteria −42; fungi −19; plant −28; animal −22; plant and animal −1 and common P450 family −1) and found highly conserved and rapidly evolving P450 families. Results suggested that bacterial P450s, particularly P450s belonging to mycobacteria, are highly conserved both at protein and DNA levels. Mycobacteria possess the highest P450 diversity percentage compared to other microbes and have a high coverage of P450s (≥1%) in their genomes, as found in fungi and plants. Phylogenetic and functional analyses revealed the functional conservation of P450s despite belonging to different biological kingdoms, suggesting the adherence of P450s to their innate function such as their involvement in either generation or oxidation of steroids and structurally related molecules, fatty acids and terpenoids. This study’s results offer new understanding of the dynamic structural nature of P450s. PMID:27616185

  5. Sekiguchi Lesion Gene Encodes a Cytochrome P450 Monooxygenase That Catalyzes Conversion of Tryptamine to Serotonin in Rice*

    PubMed Central

    Fujiwara, Tadashi; Maisonneuve, Sylvie; Isshiki, Masayuki; Mizutani, Masaharu; Chen, Letian; Wong, Hann Ling; Kawasaki, Tsutomu; Shimamoto, Ko

    2010-01-01

    Serotonin is a well known neurotransmitter in mammals and plays an important role in various mental functions in humans. In plants, the serotonin biosynthesis pathway and its function are not well understood. The rice sekiguchi lesion (sl) mutants accumulate tryptamine, a candidate substrate for serotonin biosynthesis. We isolated the SL gene by map-based cloning and found that it encodes CYP71P1 in a cytochrome P450 monooxygenase family. A recombinant SL protein exhibited tryptamine 5-hydroxylase enzyme activity and catalyzed the conversion of tryptamine to serotonin. This pathway is novel and has not been reported in mammals. Expression of SL was induced by the N-acetylchitooligosaccharide (chitin) elicitor and by infection with Magnaporthe grisea, a causal agent for rice blast disease. Exogenously applied serotonin induced defense gene expression and cell death in rice suspension cultures and increased resistance to rice blast infection in plants. We also found that serotonin-induced defense gene expression is mediated by the RacGTPase pathway and by the Gα subunit of the heterotrimeric G protein. These results suggest that serotonin plays an important role in rice innate immunity. PMID:20150424

  6. Sekiguchi lesion gene encodes a cytochrome P450 monooxygenase that catalyzes conversion of tryptamine to serotonin in rice.

    PubMed

    Fujiwara, Tadashi; Maisonneuve, Sylvie; Isshiki, Masayuki; Mizutani, Masaharu; Chen, Letian; Wong, Hann Ling; Kawasaki, Tsutomu; Shimamoto, Ko

    2010-04-09

    Serotonin is a well known neurotransmitter in mammals and plays an important role in various mental functions in humans. In plants, the serotonin biosynthesis pathway and its function are not well understood. The rice sekiguchi lesion (sl) mutants accumulate tryptamine, a candidate substrate for serotonin biosynthesis. We isolated the SL gene by map-based cloning and found that it encodes CYP71P1 in a cytochrome P450 monooxygenase family. A recombinant SL protein exhibited tryptamine 5-hydroxylase enzyme activity and catalyzed the conversion of tryptamine to serotonin. This pathway is novel and has not been reported in mammals. Expression of SL was induced by the N-acetylchitooligosaccharide (chitin) elicitor and by infection with Magnaporthe grisea, a causal agent for rice blast disease. Exogenously applied serotonin induced defense gene expression and cell death in rice suspension cultures and increased resistance to rice blast infection in plants. We also found that serotonin-induced defense gene expression is mediated by the RacGTPase pathway and by the G alpha subunit of the heterotrimeric G protein. These results suggest that serotonin plays an important role in rice innate immunity.

  7. Isolation of Homogeneous Polysaccharide Monooxygenases from Fungal Sources and Investigation of Their Synergism with Cellulases when Acting on Cellulose.

    PubMed

    Bulakhov, A G; Gusakov, A V; Chekushina, A V; Satrutdinov, A D; Koshelev, A V; Matys, V Yu; Sinitsyn, A P

    2016-05-01

    Lytic polysaccharide monooxygenases (PMO) discovered several years ago are enzymes classified as oxidoreductases. In nature, they participate in microbial degradation of cellulose together with cellulases that belong to the hydrolytic type of enzymes (class of hydrolases). Three PMO from ascomycetes - Thielavia terrestris, Trichoderma reesei, and Myceliophthora thermophila - were isolated and purified to homogeneous state using various types of chromatography. The first two enzymes are recombinant proteins heterologously expressed by the Penicillium verruculosum fungus, while the third is a native PMO secreted by M. thermophila. When acting on microcrystalline cellulose, all these PMOs displayed synergism with the cellulase complex of the P. verruculosum fungus. Replacing 10% of cellulases (by protein concentration) with PMO in the presence of 6.25 mM gallic acid or 2.5 µM of cellobiose dehydrogenase from M. thermophila, used as electron donors for PMO, resulted in the 17-31% increase in the yield of reducing sugars after 24-48 h of the enzymatic reaction.

  8. Overexpression of human kynurenine-3-monooxygenase protects against 3-hydroxykynurenine-mediated apoptosis through bidirectional nonlinear feedback.

    PubMed

    Wilson, K; Auer, M; Binnie, M; Zheng, X; Pham, N T; Iredale, J P; Webster, S P; Mole, D J

    2016-04-14

    Kynurenine 3-monooxygenase (KMO) is a critical regulator of inflammation. The preferred KMO substrate, kynurenine, is converted to 3-hydroxykynurenine (3HK), and this product exhibits cytotoxicity through mechanisms that culminate in apoptosis. Here, we report that overexpression of human KMO with orthotopic localisation to mitochondria creates a metabolic environment during which the cell exhibits increased tolerance for exogenous 3HK-mediated cellular injury. Using the selective KMO inhibitor Ro61-8048, we show that KMO enzyme function is essential for cellular protection. Pan-caspase inhibition with Z-VAD-FMK confirmed apoptosis as the mode of cell death. By defining expression of pathway components upstream and downstream of KMO, we observed alterations in other key kynurenine pathway components, particularly tryptophan-2,3-dioxygenase upregulation, through bidirectional nonlinear feedback. KMO overexpression also increased expression of inducible nitric oxide synthase (iNOS). These changes in gene expression are functionally relevant, because siRNA knockdown of the pathway components kynureninase and quinolinate phosphoribosyl transferase caused cells to revert to a state of susceptibility to 3HK-mediated apoptosis. In summary, KMO overexpression, and importantly KMO activity, have metabolic repercussions that fundamentally affect resistance to cell stress.

  9. Targeted deletion of kynurenine 3-monooxygenase in mice: a new tool for studying kynurenine pathway metabolism in periphery and brain.

    PubMed

    Giorgini, Flaviano; Huang, Shao-Yi; Sathyasaikumar, Korrapati V; Notarangelo, Francesca M; Thomas, Marian A R; Tararina, Margarita; Wu, Hui-Qiu; Schwarcz, Robert; Muchowski, Paul J

    2013-12-20

    Kynurenine 3-monooxygenase (KMO), a pivotal enzyme in the kynurenine pathway (KP) of tryptophan degradation, has been suggested to play a major role in physiological and pathological events involving bioactive KP metabolites. To explore this role in greater detail, we generated mice with a targeted genetic disruption of Kmo and present here the first biochemical and neurochemical characterization of these mutant animals. Kmo(-/-) mice lacked KMO activity but showed no obvious abnormalities in the activity of four additional KP enzymes tested. As expected, Kmo(-/-) mice showed substantial reductions in the levels of its enzymatic product, 3-hydroxykynurenine, in liver, brain, and plasma. Compared with wild-type animals, the levels of the downstream metabolite quinolinic acid were also greatly decreased in liver and plasma of the mutant mice but surprisingly were only slightly reduced (by ∼20%) in the brain. The levels of three other KP metabolites: kynurenine, kynurenic acid, and anthranilic acid, were substantially, but differentially, elevated in the liver, brain, and plasma of Kmo(-/-) mice, whereas the liver and brain content of the major end product of the enzymatic cascade, NAD(+), did not differ between Kmo(-/-) and wild-type animals. When assessed by in vivo microdialysis, extracellular kynurenic acid levels were found to be significantly elevated in the brains of Kmo(-/-) mice. Taken together, these results provide further evidence that KMO plays a key regulatory role in the KP and indicate that Kmo(-/-) mice will be useful for studying tissue-specific functions of individual KP metabolites in health and disease.

  10. Overexpression of human kynurenine-3-monooxygenase protects against 3-hydroxykynurenine-mediated apoptosis through bidirectional nonlinear feedback

    PubMed Central

    Wilson, K; Auer, M; Binnie, M; Zheng, X; Pham, N T; Iredale, J P; Webster, S P; Mole, D J

    2016-01-01

    Kynurenine 3-monooxygenase (KMO) is a critical regulator of inflammation. The preferred KMO substrate, kynurenine, is converted to 3-hydroxykynurenine (3HK), and this product exhibits cytotoxicity through mechanisms that culminate in apoptosis. Here, we report that overexpression of human KMO with orthotopic localisation to mitochondria creates a metabolic environment during which the cell exhibits increased tolerance for exogenous 3HK-mediated cellular injury. Using the selective KMO inhibitor Ro61-8048, we show that KMO enzyme function is essential for cellular protection. Pan-caspase inhibition with Z-VAD-FMK confirmed apoptosis as the mode of cell death. By defining expression of pathway components upstream and downstream of KMO, we observed alterations in other key kynurenine pathway components, particularly tryptophan-2,3-dioxygenase upregulation, through bidirectional nonlinear feedback. KMO overexpression also increased expression of inducible nitric oxide synthase (iNOS). These changes in gene expression are functionally relevant, because siRNA knockdown of the pathway components kynureninase and quinolinate phosphoribosyl transferase caused cells to revert to a state of susceptibility to 3HK-mediated apoptosis. In summary, KMO overexpression, and importantly KMO activity, have metabolic repercussions that fundamentally affect resistance to cell stress. PMID:27077813

  11. Prognostic significance of kynurenine 3-monooxygenase and effects on proliferation, migration, and invasion of human hepatocellular carcinoma

    PubMed Central

    Jin, Haojie; Zhang, Yurong; You, Haiyan; Tao, Xuemei; Wang, Cun; Jin, Guangzhi; Wang, Ning; Ruan, Haoyu; Gu, Dishui; Huo, Xisong; Cong, Wenming; Qin, Wenxin

    2015-01-01

    Kynurenine 3-monooxygenase (KMO) is a pivotal enzyme in the kynurenine pathway of tryptophan degradation and plays a critical role in Huntington’s and Alzheimer’s diseases. This study aimed to examine the expression of KMO in human hepatocellular carcinoma (HCC) and investigate the relationship between its expression and prognosis of HCC patients. We first analyzed KMO expression in 120 paired HCC samples (HCC tissues vs matched adjacent non-cancerous liver tissues), and 205 clinical HCC specimens using immunohistochemistry (IHC). Kaplan-Meier survival and Cox regression analyses were executed to evaluate the prognosis of HCC. The results of IHC analysis showed that KMO expression was significantly higher in HCC tissues than that in normal liver tissues (all p < 0.05). Survival and recurrence analyses showed that KMO was an independent prognostic factor for overall survival (OS) and time to recurrence (TTR) (both p<0.01). And in vitro studies revealed that KMO positively regulated proliferation, migration, and invasion of HCC cells. These results suggest that KMO exhibits tumor-promoting effects towards HCC and it may serve as a novel prognostic marker in HCC. PMID:26099564

  12. Prognostic significance of kynurenine 3-monooxygenase and effects on proliferation, migration, and invasion of human hepatocellular carcinoma.

    PubMed

    Jin, Haojie; Zhang, Yurong; You, Haiyan; Tao, Xuemei; Wang, Cun; Jin, Guangzhi; Wang, Ning; Ruan, Haoyu; Gu, Dishui; Huo, Xisong; Cong, Wenming; Qin, Wenxin

    2015-06-23

    Kynurenine 3-monooxygenase (KMO) is a pivotal enzyme in the kynurenine pathway of tryptophan degradation and plays a critical role in Huntington's and Alzheimer's diseases. This study aimed to examine the expression of KMO in human hepatocellular carcinoma (HCC) and investigate the relationship between its expression and prognosis of HCC patients. We first analyzed KMO expression in 120 paired HCC samples (HCC tissues vs matched adjacent non-cancerous liver tissues), and 205 clinical HCC specimens using immunohistochemistry (IHC). Kaplan-Meier survival and Cox regression analyses were executed to evaluate the prognosis of HCC. The results of IHC analysis showed that KMO expression was significantly higher in HCC tissues than that in normal liver tissues (all p < 0.05). Survival and recurrence analyses showed that KMO was an independent prognostic factor for overall survival (OS) and time to recurrence (TTR) (both p<0.01). And in vitro studies revealed that KMO positively regulated proliferation, migration, and invasion of HCC cells. These results suggest that KMO exhibits tumor-promoting effects towards HCC and it may serve as a novel prognostic marker in HCC.

  13. Oxidative biodegradation of 4-chlorophenol by using recombinant monooxygenase cloned and overexpressed from Arthrobacter chlorophenolicus A6.

    PubMed

    Kang, Christina; Yang, Jun Won; Cho, Wooyoun; Kwak, Seonyeong; Park, Sungyoon; Lim, Yejee; Choe, Jae Wan; Kim, Han S

    2017-09-01

    In this study, cphC-I and cphB, encoding a putative two-component flavin-diffusible monooxygenase (TC-FDM) complex, were cloned from Arthrobacter chlorophenolicus A6. The corresponding enzymes were overexpressed to assess the feasibility of their utilization for the oxidative decomposition of 4-chlorophenol (4-CP). Soluble CphC-I was produced at a high level (∼50%), and subsequently purified. Since CphB was expressed in an insoluble form, a flavin reductase, Fre, cloned from Escherichia coli was used as an alternative reductase. CphC-I utilized cofactor FADH2, which was reduced by Fre for the hydroxylation of 4-CP. This recombinant enzyme complex exhibited a higher specific activity for the oxidation of 4-CP (45.34U/mg-protein) than that exhibited by CphC-I contained in cells (0.18U/mg-protein). The Michaelis-Menten kinetic parameters were determined as: vmax=223.3μM·min(-1), KM=249.4μM, and kcat/KM=0.052min(-1)·μM(-1). These results could be useful for the development of a new biochemical remediation technique based on enzymatic agents catalyzing the degradation of phenolic contaminants. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. The Regulatory Domain of Squalene Monooxygenase Contains a Re-entrant Loop and Senses Cholesterol via a Conformational Change*

    PubMed Central

    Howe, Vicky; Chua, Ngee Kiat; Stevenson, Julian; Brown, Andrew J.

    2015-01-01

    Squalene monooxygenase (SM) is an important control point in cholesterol synthesis beyond 3-hydroxy-3-methylglutaryl-CoA reductase. Although it is known to associate with the endoplasmic reticulum, its topology has not been determined. We have elucidated the membrane topology of the sterol-responsive domain of SM comprising the first 100 amino acids fused to GFP (SM N100-GFP) by determining the accessibility of 16 introduced cysteines to the cysteine-reactive, membrane-impermeable reagent PEG-maleimide. We have identified a region integrally associated with the endoplasmic reticulum membrane that is likely to interact with cholesterol or respond to cholesterol-induced membrane effects. By comparing cysteine accessibility with and without cholesterol treatment, we further present evidence to suggest that cholesterol induces a conformational change in SM N100-GFP. This change is likely to lead to its targeted degradation by the ubiquitin-proteasome system because degradation is blunted by treatment with the chemical chaperone glycerol, which retains SM N100-GFP in its native conformation. Furthermore, degradation can be disrupted by insertion of two N-terminal myc tags, implicating the N terminus in this process. Together, this information provides new molecular insights into the regulation of this critical control point in cholesterol synthesis. PMID:26434806

  15. Oxidative demethylation in monooxygenase model systems. Competing pathways for binuclear and helical multinuclear copper(I) complexes

    SciTech Connect

    Gelling, O.J.; Feringa, B.L. )

    1990-10-10

    The ligand 2,6-bis(N-(2-pyridylethyl)formimidoyl)-1-methoxybenzene (2,6-BPB-1-OCH{sub 3}) (4) reacts with Cu-(CH{sub 3}CN){sub 4}BF{sub 4} to form novel binuclear copper(I) complexes (Cu{sub 2}(2,6-BPB-1-OCH{sub 3})(BF{sub 4}){sub 2}(CH{sub 3}CN){sub 4}) (11) and (Cu{sub 2}(2,6-BPB-1-OCH{sub 3})(BF{sub 4}){sub 2}(CH{sub 2}Cl{sub 2}){sub 0.5}) (14), or the helical polynuclear copper(I) complex (Cu(2,6-BPB-1-OCH{sub 3})(BF{sub 4})){sub n} (16). The complexes mimic certain monooxygenases as they rapidly take up O{sub 2} followed by demethylation of the anisole moiety (up to 95% yield). {sup 18}O experiments are provided that show competing aryl-oxygen ({ge}60%) and alkyl-oxygen (20%) bond cleavage pathways. Introduction of a p-methoxy substituent in the arene moiety of complex 11 decreases the oxygenation rate and led to an unprecedented O{sub 2} induced arene-OCH{sub 3}-OCD{sub 3} exchange at 20{degree}C in CD{sub 3}OD. A mechanistic rational is given.

  16. Crystal Structure and Computational Characterization of the Lytic Polysaccharide Monooxygenase GH61D from the Basidiomycota Fungus Phanerochaete chrysosporium*

    PubMed Central

    Wu, Miao; Beckham, Gregg T.; Larsson, Anna M.; Ishida, Takuya; Kim, Seonah; Payne, Christina M.; Himmel, Michael E.; Crowley, Michael F.; Horn, Svein J.; Westereng, Bjørge; Igarashi, Kiyohiko; Samejima, Masahiro; Ståhlberg, Jerry; Eijsink, Vincent G. H.; Sandgren, Mats

    2013-01-01

    Carbohydrate structures are modified and degraded in the biosphere by a myriad of mostly hydrolytic enzymes. Recently, lytic polysaccharide mono-oxygenases (LPMOs) were discovered as a new class of enzymes for cleavage of recalcitrant polysaccharides that instead employ an oxidative mechanism. LPMOs employ copper as the catalytic metal and are dependent on oxygen and reducing agents for activity. LPMOs are found in many fungi and bacteria, but to date no basidiomycete LPMO has been structurally characterized. Here we present the three-dimensional crystal structure of the basidiomycete Phanerochaete chrysosporium GH61D LPMO, and, for the first time, measure the product distribution of LPMO action on a lignocellulosic substrate. The structure reveals a copper-bound active site common to LPMOs, a collection of aromatic and polar residues near the binding surface that may be responsible for regio-selectivity, and substantial differences in loop structures near the binding face compared with other LPMO structures. The activity assays indicate that this LPMO primarily produces aldonic acids. Last, molecular simulations reveal conformational changes, including the binding of several regions to the cellulose surface, leading to alignment of three tyrosine residues on the binding face of the enzyme with individual cellulose chains, similar to what has been observed for family 1 carbohydrate-binding modules. A calculated potential energy surface for surface translation indicates that P. chrysosporium GH61D exhibits energy wells whose spacing seems adapted to the spacing of cellobiose units along a cellulose chain. PMID:23525113

  17. Pilot-scale production of (S)-styrene oxide from styrene by recombinant Escherichia coli synthesizing styrene monooxygenase.

    PubMed

    Panke, Sven; Held, Martin; Wubbolts, Marcel G; Witholt, Bernard; Schmid, Andreas

    2002-10-05

    Recombinant Escherichia coli JM101(pSPZ10) cells produce the styrene monooxygenase of Pseudomonas sp. strain VLB120, which catalyzes the oxidation of styrene to (S)-styrene oxide at an enantiomeric excess larger than 99%. This biocatalyst was used to produce 388 g of styrene oxide in a two-liquid phase 30-L fed-batch bioconversion. The average overall volumetric activity was 170 U per liter over a period of more than 10 h, equivalent to mass transfer rates of 10.2 mmoles per liter per hour at a phase ratio of 0.5. At this transfer rate, the biotransformation system appeared to be substrate mass-transfer limited. The reactor had an estimated power input in the order of 5 W. L(-1), which is close to values typically obtained with commercially operating units. The product could be easily purified by fractional distillation to a purity in excess of 97%. The process illustrates the feasibility of recombinant whole cell biotransformations in two-liquid phase systems with toxic substrates and products. Copyright 2002 Wiley Periodicals, Inc. Biotechnol Bioeng 80: 33-41, 2002.

  18. Intermediate Q from soluble methane monooxygenase hydroxylates the mechanistic substrate probe norcarane: evidence for a stepwise reaction.

    PubMed

    Brazeau, B J; Austin, R N; Tarr, C; Groves, J T; Lipscomb, J D

    2001-12-05

    Norcarane is a valuable mechanistic probe for enzyme-catalyzed hydrocarbon oxidation reactions because different products or product distributions result from concerted, radical, and cation based reactions. Soluble methane monooxygenase (sMMO) from Methylosinus trichosporium OB3b catalyzes the oxidation of norcarane to afford 3-hydroxymethylcyclohexene and 3-cycloheptenol, compounds characteristic of radical and cationic intermediates, respectively, in addition to 2- and 3-norcaranols. Past single turnover transient kinetic studies have identified several optically distinct intermediates from the catalytic cycle of the hydroxylase component of sMMO. Thus, the reaction between norcarane and key reaction intermediates can be directly monitored. The presence of norcarane increases the rate of decay of only one intermediate, the high-valent bis-mu-oxo Fe(IV)(2) cluster-containing species compound Q, showing that it is responsible for the majority of the oxidation chemistry. The observation of products from both radical and cationic intermediates from norcarane oxidation catalyzed by sMMO is consistent with a mechanism in which an initial substrate radical intermediate is formed by hydrogen atom abstraction. This intermediate then undergoes either oxygen rebound, intramolecular rearrangement followed by oxygen rebound, or loss of a second electron to yield a cationic intermediate to which OH(-) is transferred. The estimated lower limit of 20 ps for the lifetime of the putative radical intermediate is in accord with values determined from previous studies of sterically hindered sMMO probes.

  19. Suitability of recombinant Escherichia coli and Pseudomonas putida strains for selective biotransformation of m-nitrotoluene by xylene monooxygenase.

    PubMed

    Meyer, Daniel; Witholt, Bernard; Schmid, Andreas

    2005-11-01

    Escherichia coli JM101(pSPZ3), containing xylene monooxygenase (XMO) from Pseudomonas putida mt-2, catalyzes specific oxidations and reductions of m-nitrotoluene and derivatives thereof. In addition to reactions catalyzed by XMO, we focused on biotransformations by native enzymes of the E. coli host and their effect on overall biocatalyst performance. While m-nitrotoluene was consecutively oxygenated to m-nitrobenzyl alcohol, m-nitrobenzaldehyde, and m-nitrobenzoic acid by XMO, the oxidation was counteracted by an alcohol dehydrogenase(s) from the E. coli host, which reduced m-nitrobenzaldehyde to m-nitrobenzyl alcohol. Furthermore, the enzymatic background of the host reduced the nitro groups of the reactants resulting in the formation of aromatic amines, which were shown to effectively inhibit XMO in a reversible fashion. Host-intrinsic oxidoreductases and their reaction products had a major effect on the activity of XMO during biocatalysis of m-nitrotoluene. P. putida DOT-T1E and P. putida PpS81 were compared to E. coli JM101 as alternative hosts for XMO. These promising strains contained an additional dehydrogenase that oxidized m-nitrobenzaldehyde to the corresponding acid but catalyzed the formation of XMO-inhibiting aromatic amines at a significantly lower level than E. coli JM101.

  20. Identification of structural determinants of NAD(P)H selectivity and lysine binding in lysine N(6)-monooxygenase.

    PubMed

    Abdelwahab, Heba; Robinson, Reeder; Rodriguez, Pedro; Adly, Camelia; El-Sohaimy, Sohby; Sobrado, Pablo

    2016-09-15

    l-lysine (l-Lys) N(6)-monooxygenase (NbtG), from Nocardia farcinica, is a flavin-dependent enzyme that catalyzes the hydroxylation of l-Lys in the presence of oxygen and NAD(P)H in the biosynthetic pathway of the siderophore nocobactin. NbtG displays only a 3-fold preference for NADPH over NADH, different from well-characterized related enzymes, which are highly selective for NADPH. The structure of NbtG with bound NAD(P)(+) or l-Lys is currently not available. Herein, we present a mutagenesis study targeting M239, R301, and E216. These amino acids are conserved and located in either the NAD(P)H binding domain or the l-Lys binding pocket. M239R resulted in high production of hydrogen peroxide and little hydroxylation with no change in coenzyme selectivity. R301A caused a 300-fold decrease on kcat/Km value with NADPH but no change with NADH. E216Q increased the Km value for l-Lys by 30-fold with very little change on the kcat value or in the binding of NAD(P)H. These results suggest that R301 plays a major role in NADPH selectivity by interacting with the 2'-phosphate of the adenine-ribose moiety of NADPH, while E216 plays a role in l-Lys binding.

  1. Cello-oligosaccharide oxidation reveals differences between two lytic polysaccharide monooxygenases (family GH61) from Podospora anserina.

    PubMed

    Bey, Mathieu; Zhou, Simeng; Poidevin, Laetitia; Henrissat, Bernard; Coutinho, Pedro M; Berrin, Jean-Guy; Sigoillot, Jean-Claude

    2013-01-01

    The genome of the coprophilic ascomycete Podospora anserina encodes 33 different genes encoding copper-dependent lytic polysaccharide monooxygenases (LPMOs) from glycoside hydrolase family 61 (GH61). In this study, two of these enzymes (P. anserina GH61A [PaGH61A] and PaGH61B), which both harbored a family 1 carbohydrate binding module, were successfully produced in Pichia pastoris. Synergistic cooperation between PaGH61A or PaGH61B with the cellobiose dehydrogenase (CDH) of Pycnoporus cinnabarinus on cellulose resulted in the formation of oxidized and nonoxidized cello-oligosaccharides. A striking difference between PaGH61A and PaGH61B was observed through the identification of the products, among which were doubly and triply oxidized cellodextrins, which were released only by the combination of PaGH61B with CDH. The mass spectrometry fragmentation patterns of these oxidized products could be consistent with oxidation at the C-6 position with a geminal diol group. The different properties of PaGH61A and PaGH61B and their effect on the interaction with CDH are discussed in regard to the proposed in vivo function of the CDH/GH61 enzyme system in oxidative cellulose hydrolysis.

  2. Cello-Oligosaccharide Oxidation Reveals Differences between Two Lytic Polysaccharide Monooxygenases (Family GH61) from Podospora anserina

    PubMed Central

    Bey, Mathieu; Zhou, Simeng; Poidevin, Laetitia; Henrissat, Bernard; Coutinho, Pedro M.; Sigoillot, Jean-Claude

    2013-01-01

    The genome of the coprophilic ascomycete Podospora anserina encodes 33 different genes encoding copper-dependent lytic polysaccharide monooxygenases (LPMOs) from glycoside hydrolase family 61 (GH61). In this study, two of these enzymes (P. anserina GH61A [PaGH61A] and PaGH61B), which both harbored a family 1 carbohydrate binding module, were successfully produced in Pichia pastoris. Synergistic cooperation between PaGH61A or PaGH61B with the cellobiose dehydrogenase (CDH) of Pycnoporus cinnabarinus on cellulose resulted in the formation of oxidized and nonoxidized cello-oligosaccharides. A striking difference between PaGH61A and PaGH61B was observed through the identification of the products, among which were doubly and triply oxidized cellodextrins, which were released only by the combination of PaGH61B with CDH. The mass spectrometry fragmentation patterns of these oxidized products could be consistent with oxidation at the C-6 position with a geminal diol group. The different properties of PaGH61A and PaGH61B and their effect on the interaction with CDH are discussed in regard to the proposed in vivo function of the CDH/GH61 enzyme system in oxidative cellulose hydrolysis. PMID:23124232

  3. Stopped-flow Fourier transform infrared spectroscopy of nitromethane oxidation by the diiron(IV) intermediate of methane monooxygenase.

    PubMed

    Muthusamy, Mylrajan; Ambundo, Edna A; George, Simon J; Lippard, Stephen J; Thorneley, Roger N F

    2003-09-17

    The hydroxylase component (MMOH) of soluble methane monooxygenase from Methylococcus capsulatus (Bath) was reduced to the diiron(II) form and then allowed to react with dioxygen to generate the diiron(IV) intermediate Q in the first phase of a double-mixing stopped-flow experiment. CD3NO2 was then introduced in the second phase of the experiment, which was carried out in D2O at 25 degrees C. The kinetics of the reaction of the substrate with Q were monitored by stopped-flow Fourier transform infrared spectroscopy, observing the disappearance of the asymmetric NO2 bending vibration at 1548 cm-1. The data were fit to a single-exponential function, which yielded a kobs of 0.45 +/- 0.07 s-1. This result is in quantitative agreement with a kobs of 0.39 +/- 0.01 s-1 obtained by observing the disappearance of Q by double-mixing stopped-flow optical spectroscopy at its absorption maximum of 420 nm. These results provide for the first time direct monitoring of the hydroxylation of a methane-derived substrate in the MMOH reaction pathway and demonstrate that Q decay occurs concomitantly with substrate consumption.

  4. Sparteine monooxygenase in brain and liver: Identified by the dopamine uptake blocker ( sup 3 H)GBR-12935

    SciTech Connect

    Kalow, W.; Tyndale, R.F.; Niznik, H.B.; Inaba, T. )

    1990-02-26

    P450IID6 (human sparteine monooxygenase) metabolizes many drugs including neuroleptics, antidepressants, and beta-blockers. The P450IID6 exists in human, bovine, rat and canine brains, but in very low quantities causing methodological difficulties in its assessment. Work with ({sup 3}H)GBR-12935; 1-(2-(diphenylmethoxy) ethyl)-4-(3-phenyl propyl) piperazine has shown that it binds a neuronal/hepatic protein with high affinity ({approximately}7nM) and a rank order of inhibitory potency suggesting that the binding protein is cytochrome P450IID6. The binding was used to predict that d-amphetamine and methamphetamine would interact with P450IID6. Inhibition studies indicated that these compounds were competitive inhibitors of P450IID6. Haloperidol (HAL) and it's metabolite hydroxy-haloperidol (RHAL) are both competitive inhibitors of P450IID6 activity and were found to inhibit ({sup 3}H)GBR-12935 binding. K{sub i} values of twelve compounds (known to interact with the DA transporter or P450IID6) for ({sup 3}H)GRB-12935 binding and P450IID6 activity. The techniques are now available for measurements of cytochrome P450IID6 in healthy and diseased brain/liver tissue using radio-receptor binding assay techniques with ({sup 3}H)GBR-12935.

  5. Inhibition of the Flavin-Dependent Monooxygenase Siderophore A (SidA) Blocks Siderophore Biosynthesis and Aspergillus fumigatus Growth.

    PubMed

    Martín Del Campo, Julia S; Vogelaar, Nancy; Tolani, Karishma; Kizjakina, Karina; Harich, Kim; Sobrado, Pablo

    2016-11-18

    Aspergillus fumigatus is an opportunistic fungal pathogen and the most common causative agent of fatal invasive mycoses. The flavin-dependent monooxygenase siderophore A (SidA) catalyzes the oxygen and NADPH dependent hydroxylation of l-ornithine (l-Orn) to N(5)-l-hydroxyornithine in the biosynthetic pathway of hydroxamate-containing siderophores in A. fumigatus. Deletion of the gene that codes for SidA has shown that it is essential in establishing infection in mice models. Here, a fluorescence polarization high-throughput assay was used to screen a 2320 compound library for inhibitors of SidA. Celastrol, a natural quinone methide, was identified as a noncompetitive inhibitor of SidA with a MIC value of 2 μM. Docking experiments suggest that celastrol binds across the NADPH and l-Orn pocket. Celastrol prevents A. fumigatus growth in blood agar. The addition of purified ferric-siderophore abolished the inhibitory effect of celastrol. Thus, celastrol inhibits A. fumigatus growth by blocking siderophore biosynthesis through SidA inhibiton.

  6. Mutation of the Glucosinolate Biosynthesis Enzyme Cytochrome P450 83A1 Monooxygenase Increases Camalexin Accumulation and Powdery Mildew Resistance.

    PubMed

    Liu, Simu; Bartnikas, Lisa M; Volko, Sigrid M; Ausubel, Frederick M; Tang, Dingzhong

    2016-01-01

    Small secondary metabolites, including glucosinolates and the major phytoalexin camalexin, play important roles in immunity in Arabidopsis thaliana. We isolated an Arabidopsis mutant with increased resistance to the powdery mildew fungus Golovinomyces cichoracearum and identified a mutation in the gene encoding cytochrome P450 83A1 monooxygenase (CYP83A1), which functions in glucosinolate biosynthesis. The cyp83a1-3 mutant exhibited enhanced defense responses to G. cichoracearum and double mutant analysis showed that this enhanced resistance requires NPR1, EDS1, and PAD4, but not SID2 or EDS5. In cyp83a1-3 mutants, the expression of genes related to camalexin synthesis increased upon G. cichoracearum infection. Significantly, the cyp83a1-3 mutant also accumulated higher levels of camalexin. Decreasing camalexin levels by mutation of the camalexin synthetase gene PAD3 or the camalexin synthesis regulator AtWRKY33 compromised the powdery mildew resistance in these mutants. Consistent with these observations, overexpression of PAD3 increased camalexin levels and enhanced resistance to G. cichoracearum. Taken together, our data indicate that accumulation of higher levels of camalexin contributes to increased resistance to powdery mildew.

  7. Effect of topical application of clotrimazole to rats on epidermal and hepatic monooxygenase activities and cytochrome P-450.

    PubMed

    Merk, H F; Khan, W A; Kuhn, C; Bickers, D R; Mukhtar, H

    1989-01-01

    Clotrimazole, an N-substituted imidazole, is a widely used topical agent for the treatment of superficial fungal infections. In this study, the effect of application of clotrimazole to the skin of neonatal rats on the induction response of the cytochrome P-450-dependent monooxygenase system in epidermis and liver has been examined. A single topical application of clotrimazole (10 mg/100 g) to rats resulted in a 53% increase in hepatic cytochrome P-450 content. Clotrimazole treatment also resulted in significant induction of epidermal 7-ethoxycoumarin-O-deethylase activity. Hepatic p-nitrophenol hydroxylase, an enzyme, catalyzed principally by the ethanol inducible cytochrome P-450 isozyme, was also significantly induced (58%) by topically applied clotrimazole. This enzyme activity was undetectable in epidermal microsomes. Further characterization of the cytochrome P-450 isozymes induced in liver by clotrimazole treatment was based on monoclonal antibodies (MAbs) raised against purified rat liver cytochrome P-450 isozymes induced by phenobarbital (MAb 2-66-3) and ethanol (MAb 1-98-1). Hepatic microsomes prepared from clotrimazole-treated rats showed significant immunoreactivity on Western blot with both the MAbs whereas no reactivity occurred in epidermal microsomes. Our data indicate that topical application of clotrimazole to rats results in the induction of selected cytochrome P-450 isozyme(s) in liver and epidermis which may have implications for the therapeutic use of this compound.

  8. Messenger RNA (mRNA) nanoparticle tumour vaccination

    NASA Astrophysics Data System (ADS)

    Phua, Kyle K. L.; Nair, Smita K.; Leong, Kam W.

    2014-06-01

    Use of mRNA-based vaccines for tumour immunotherapy has gained increasing attention in recent years. A growing number of studies applying nanomedicine concepts to mRNA tumour vaccination show that the mRNA delivered in nanoparticle format can generate a more robust immune response. Advances in the past decade have deepened our understanding of gene delivery barriers, mRNA's biological stability and immunological properties, and support the notion for engineering innovations tailored towards a more efficient mRNA nanoparticle vaccine delivery system. In this review we will first examine the suitability of mRNA for engineering manipulations, followed by discussion of a model framework that highlights the barriers to a robust anti-tumour immunity mediated by mRNA encapsulated in nanoparticles. Finally, by consolidating existing literature on mRNA nanoparticle tumour vaccination within the context of this framework, we aim to identify bottlenecks that can be addressed by future nanoengineering research.

  9. Alternative polyadenylation of mRNA precursors

    PubMed Central

    Tian, Bin; Manley, James L.

    2017-01-01

    Alternative polyadenylation (APA) is an RNA-processing mechanism that generates distinct 3′ termini on mRNAs and other RNA polymerase II transcripts. It is widespread across all eukaryotic species and is recognized as a major mechanism of gene regulation. APA exhibits tissue specificity and is important for cell proliferation and differentiation. In this Review, we discuss the roles of APA in diverse cellular processes, including mRNA metabolism, protein diversification and protein localization, and more generally in gene regulation. We also discuss the molecular mechanisms underlying APA, such as variation in the concentration of core processing factors and RNA-binding proteins, as well as transcription-based regulation. PMID:27677860

  10. Sequences controlling histone H4 mRNA abundance.

    PubMed Central

    Capasso, O; Bleecker, G C; Heintz, N

    1987-01-01

    The post-transcriptional regulation of histone mRNA abundance is manifest both by accumulation of histone mRNA during the S phase, and by the rapid degradation of mature histone mRNA following the inhibition of DNA synthesis. We have constructed a comprehensive series of substitution mutants within a human H4 histone gene, introduced them into the mouse L cell genome, and analyzed their effects on the post-transcriptional control of the H4 mRNA. Our results demonstrate that most of the H4 mRNA is dispensable for proper regulation of histone mRNA abundance. However, recognition of the 3' terminus of the mature H4 mRNA is critically important for regulating its cytoplasmic half-life. Thus, this region of the mRNA functions both in the nucleus as a signal for proper processing of the mRNA terminus, and in the cytoplasm as an essential element in the control of mRNA stability. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:3608993

  11. Coupled Evolution of Transcription and mRNA Degradation

    PubMed Central

    Dori-Bachash, Mally; Shema, Efrat; Tirosh, Itay

    2011-01-01

    mRNA levels are determined by the balance between transcription and mRNA degradation, and while transcription has been extensively studied, very little is known regarding the regulation of mRNA degradation and its coordination with transcription. Here we examine the evolution of mRNA degradation rates between two closely related yeast species. Surprisingly, we find that around half of the evolutionary changes in mRNA degradation were coupled to transcriptional changes that exert opposite effects on mRNA levels. Analysis of mRNA degradation rates in an interspecific hybrid further suggests that opposite evolutionary changes in transcription and in mRNA degradation are mechanistically coupled and were generated by the same individual mutations. Coupled changes are associated with divergence of two complexes that were previously implicated both in transcription and in mRNA degradation (Rpb4/7 and Ccr4-Not), as well as with sequence divergence of transcription factor binding motifs. These results suggest that an opposite coupling between the regulation of transcription and that of mRNA degradation has shaped the evolution of gene regulation in yeast. PMID:21811398

  12. The Two-Component Monooxygenase MeaXY Initiates the Downstream Pathway of Chloroacetanilide Herbicide Catabolism in Sphingomonads

    PubMed Central

    Cheng, Minggen; Meng, Qiang; Yang, Youjian; Chu, Cuiwei; Chen, Qing; Li, Yi; Cheng, Dan; Hong, Qing; He, Jian

    2017-01-01

    ABSTRACT Due to the extensive use of chloroacetanilide herbicides over the past 60 years, bacteria have evolved catabolic pathways to mineralize these compounds. In the upstream catabolic pathway, chloroacetanilide herbicides are transformed into the two common metabolites 2-methyl-6-ethylaniline (MEA) and 2,6-diethylaniline (DEA) through N-dealkylation and amide hydrolysis. The pathway downstream of MEA is initiated by the hydroxylation of aromatic rings, followed by its conversion to a substrate for ring cleavage after several steps. Most of the key genes in the pathway have been identified. However, the genes involved in the initial hydroxylation step of MEA are still unknown. As a special aniline derivative, MEA cannot be transformed by the aniline dioxygenases that have been characterized. Sphingobium baderi DE-13 can completely degrade MEA and use it as a sole carbon source for growth. In this work, an MEA degradation-deficient mutant of S. baderi DE-13 was isolated. MEA catabolism genes were predicted through comparative genomic analysis. The results of genetic complementation and heterologous expression demonstrated that the products of meaX and meaY are responsible for the initial step of MEA degradation in S. baderi DE-13. MeaXY is a two-component flavoprotein monooxygenase system that catalyzes the hydroxylation of MEA and DEA using NADH and flavin mononucleotide (FMN) as cofactors. Nuclear magnetic resonance (NMR) analysis confirmed that MeaXY hydroxylates MEA and DEA at the para-position. Transcription of meaX was enhanced remarkably upon induction of MEA or DEA in S. baderi DE-13. Additionally, meaX and meaY were highly conserved among other MEA-degrading sphingomonads. This study fills a gap in our knowledge of the biochemical pathway that carries out mineralization of chloroacetanilide herbicides in sphingomonads. IMPORTANCE Much attention has been paid to the environmental fate of chloroacetanilide herbicides used for the past 60 years. Microbial

  13. The Two-Component Monooxygenase MeaXY Initiates the Downstream Pathway of Chloroacetanilide Herbicide Catabolism in Sphingomonads.

    PubMed

    Cheng, Minggen; Meng, Qiang; Yang, Youjian; Chu, Cuiwei; Chen, Qing; Li, Yi; Cheng, Dan; Hong, Qing; Yan, Xin; He, Jian

    2017-04-01

    Due to the extensive use of chloroacetanilide herbicides over the past 60 years, bacteria have evolved catabolic pathways to mineralize these compounds. In the upstream catabolic pathway, chloroacetanilide herbicides are transformed into the two common metabolites 2-methyl-6-ethylaniline (MEA) and 2,6-diethylaniline (DEA) through N-dealkylation and amide hydrolysis. The pathway downstream of MEA is initiated by the hydroxylation of aromatic rings, followed by its conversion to a substrate for ring cleavage after several steps. Most of the key genes in the pathway have been identified. However, the genes involved in the initial hydroxylation step of MEA are still unknown. As a special aniline derivative, MEA cannot be transformed by the aniline dioxygenases that have been characterized. Sphingobium baderi DE-13 can completely degrade MEA and use it as a sole carbon source for growth. In this work, an MEA degradation-deficient mutant of S. baderi DE-13 was isolated. MEA catabolism genes were predicted through comparative genomic analysis. The results of genetic complementation and heterologous expression demonstrated that the products of meaX and meaY are responsible for the initial step of MEA degradation in S. baderi DE-13. MeaXY is a two-component flavoprotein monooxygenase system that catalyzes the hydroxylation of MEA and DEA using NADH and flavin mononucleotide (FMN) as cofactors. Nuclear magnetic resonance (NMR) analysis confirmed that MeaXY hydroxylates MEA and DEA at the para-position. Transcription of meaX was enhanced remarkably upon induction of MEA or DEA in S. baderi DE-13. Additionally, meaX and meaY were highly conserved among other MEA-degrading sphingomonads. This study fills a gap in our knowledge of the biochemical pathway that carries out mineralization of chloroacetanilide herbicides in sphingomonads.IMPORTANCE Much attention has been paid to the environmental fate of chloroacetanilide herbicides used for the past 60 years. Microbial degradation

  14. Altering toluene 4-monooxygenase by active-site engineering for the synthesis of 3-methoxycatechol, methoxyhydroquinone, and methylhydroquinone.

    PubMed

    Tao, Ying; Fishman, Ayelet; Bentley, William E; Wood, Thomas K

    2004-07-01

    Wild-type toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 oxidizes toluene to p-cresol (96%) and oxidizes benzene sequentially to phenol, to catechol, and to 1,2,3-trihydroxybenzene. In this study T4MO was found to oxidize o-cresol to 3-methylcatechol (91%) and methylhydroquinone (9%), to oxidize m-cresol and p-cresol to 4-methylcatechol (100%), and to oxidize o-methoxyphenol to 4-methoxyresorcinol (87%), 3-methoxycatechol (11%), and methoxyhydroquinone (2%). Apparent Vmax values of 6.6 +/- 0.9 to 10.7 +/- 0.1 nmol/min/ mg of protein were obtained for o-, m-, and p-cresol oxidation by wild-type T4MO, which are comparable to the toluene oxidation rate (15.1 +/- 0.8 nmol/min/mg of protein). After these new reactions were discovered, saturation mutagenesis was performed near the diiron catalytic center at positions I100, G103, and A107 of the alpha subunit of the hydroxylase (TmoA) based on directed evolution of the related toluene o-monooxygenase of Burkholderia cepacia G4 (K. A. Canada, S. Iwashita, H. Shim, and T. K. Wood, J. Bacteriol. 184:344-349, 2002) and a previously reported T4MO G103L regiospecific mutant (K. H. Mitchell, J. M. Studts, and B. G. Fox, Biochemistry 41:3176-3188, 2002). By using o-cresol and o-methoxyphenol as model substrates, regiospecific mutants of T4MO were created; for example, TmoA variant G103A/A107S produced 3-methylcatechol (98%) from o-cresol twofold faster and produced 3-methoxycatechol (82%) from 1 mM o-methoxyphenol seven times faster than the wild-type T4MO (1.5 +/- 0.2 versus 0.21 +/- 0.01 nmol/min/mg of protein). Variant I100L produced 3-methoxycatechol from o-methoxyphenol four times faster than wild-type T4MO, and G103S/A107T produced methylhydroquinone (92%) from o-cresol fourfold faster than wild-type T4MO and there was 10 times more in terms of the percentage of the product. Variant G103S produced 40-fold more methoxyhydroquinone from o-methoxyphenol than the wild-type enzyme produced (80 versus 2%) and

  15. Rational Reprogramming of the R2 Subunit of Escherichia coli Ribonucleotide Reductase into a Self-Hydroxylating Monooxygenase

    SciTech Connect

    Baldwin, J.; Voegtli, W.C.; Khidekel, N.; Moënne-Loccoz, P.; Krebs, C.; Ley, B.A.; Huynh, B.H.; Loehr, T.M.; Rosenzweig, A.C.; Bollinger, Jr., J.M.

    2010-03-05

    The outcome of O{sub 2} activation at the diiron(II) cluster in the R2 subunit of Escherichia coli (class I) ribonucleotide reductase has been rationally altered from the normal tyrosyl radical (Y122) production to self-hydroxylation of a phenylalanine side-chain by two amino acid substitutions that leave intact the (histidine){sub 2}-(carboxylate){sub 4} ligand set characteristic of the diiron-carboxylate family. Iron ligand Asp (D) 84 was replaced with Glu (E), the amino acid found in the cognate position of the structurally similar diiron-carboxylate protein, methane monooxygenase hydroxylase (MMOH). We previously showed that this substitution allows accumulation of a {mu}-1,2-peroxodiiron(III) intermediate, which does not accumulate in the wild-type (wt) protein and is probably a structural homologue of intermediate P (H{sub peroxo}) in O{sub 2} activation by MMOH. In addition, the near-surface residue Trp (W) 48 was replaced with Phe (F), blocking transfer of the 'extra' electron that occurs in wt R2 during formation of the formally Fe(III)Fe(IV) cluster X. Decay of the {mu}-1,2-peroxodiiron(III) complex in R2-W48F/D84E gives an initial brown product, which contains very little Y122 and which converts very slowly (t{sub 1/2} {approx} 7 h) upon incubation at 0 C to an intensely purple final product. X-ray crystallographic analysis of the purple product indicates that F208 has undergone {epsilon}-hydroxylation and the resulting phenol has shifted significantly to become a ligand to Fe2 of the diiron cluster. Resonance Raman (RR) spectra of the purple product generated with {sup 16}O{sub 2} or {sub 18}O{sub 2} show appropriate isotopic sensitivity in bands assigned to O-phenyl and Fe-O-phenyl vibrational modes, confirming that the oxygen of the Fe(III)-phenolate species is derived from O{sub 2}. Chemical analysis, experiments involving interception of the hydroxylating intermediate with exogenous reductant, and Moessbauer and EXAFS characterization of the brown

  16. Adaptor Protein-1 Complex Affects the Endocytic Trafficking and Function of Peptidylglycine α-Amidating Monooxygenase, a Luminal Cuproenzyme.

    PubMed

    Bonnemaison, Mathilde L; Bäck, Nils; Duffy, Megan E; Ralle, Martina; Mains, Richard E; Eipper, Betty A

    2015-08-28

    The adaptor protein-1 complex (AP-1), which transports cargo between the trans-Golgi network and endosomes, plays a role in the trafficking of Atp7a, a copper-transporting P-type ATPase, and peptidylglycine α-amidating monooxygenase (PAM), a copper-dependent membrane enzyme. Lack of any of the four AP-1 subunits impairs function, and patients with MEDNIK syndrome, a rare genetic disorder caused by lack of expression of the σ1A subunit, exhibit clinical and biochemical signs of impaired copper homeostasis. To explore the role of AP-1 in copper homeostasis in neuroendocrine cells, we used corticotrope tumor cells in which AP-1 function was diminished by reducing expression of its μ1A subunit. Copper levels were unchanged when AP-1 function was impaired, but cellular levels of Atp7a declined slightly. The ability of PAM to function was assessed by monitoring 18-kDa fragment-NH2 production from proopiomelanocortin. Reduced AP-1 function made 18-kDa fragment amidation more sensitive to inhibition by bathocuproine disulfonate, a cell-impermeant Cu(I) chelator. The endocytic trafficking of PAM was altered, and PAM-1 accumulated on the cell surface when AP-1 levels were reduced. Reduced AP-1 function increased the Atp7a presence in early/recycling endosomes but did not alter the ability of copper to stimulate its appearance on the plasma membrane. Co-immunoprecipitation of a small fraction of PAM and Atp7a supports the suggestion that copper can be transferred directly from Atp7a to PAM, a process that can occur only when both proteins are present in the same subcellular compartment. Altered luminal cuproenzyme function may contribute to deficits observed when the AP-1 function is compromised.

  17. Structural and Functional Analysis of a Lytic Polysaccharide Monooxygenase Important for Efficient Utilization of Chitin in Cellvibrio japonicus.

    PubMed

    Forsberg, Zarah; Nelson, Cassandra E; Dalhus, Bjørn; Mekasha, Sophanit; Loose, Jennifer S M; Crouch, Lucy I; Røhr, Åsmund K; Gardner, Jeffrey G; Eijsink, Vincent G H; Vaaje-Kolstad, Gustav

    2016-04-01

    Cellvibrio japonicusis a Gram-negative soil bacterium that is primarily known for its ability to degrade plant cell wall polysaccharides through utilization of an extensive repertoire of carbohydrate-active enzymes. Several putative chitin-degrading enzymes are also found among these carbohydrate-active enzymes, such as chitinases, chitobiases, and lytic polysaccharide monooxygenases (LPMOs). In this study, we have characterized the chitin-active LPMO,CjLPMO10A, a tri-modular enzyme containing a catalytic family AA10 LPMO module, a family 5 chitin-binding module, and a C-terminal unclassified module of unknown function. Characterization of the latter module revealed tight and specific binding to chitin, thereby unraveling a new family of chitin-binding modules (classified as CBM73). X-ray crystallographic elucidation of theCjLPMO10A catalytic module revealed that the active site of the enzyme combines structural features previously only observed in either cellulose or chitin-active LPMO10s. Analysis of the copper-binding site by EPR showed a signal signature more similar to those observed for cellulose-cleaving LPMOs. The full-length LPMO shows no activity toward cellulose but is able to bind and cleave both α- and β-chitin. Removal of the chitin-binding modules reduced LPMO activity toward α-chitin compared with the full-length enzyme. Interestingly, the full-length enzyme and the individual catalytic LPMO module boosted the activity of an endochitinase equally well, also yielding similar amounts of oxidized products. Finally, gene deletion studies show thatCjLPMO10A is needed byC. japonicusto obtain efficient growth on both purified chitin and crab shell particles.

  18. Phenylalanine 4-monooxygenase and the S-oxidation of S-carboxymethyl-L-cysteine by human cytosolic fractions.

    PubMed

    Boonyapiwat, Boontarika; Forbes, Ben; Mitchell, Stephen; Steventon, Glyn B

    2008-01-01

    The purpose of this investigation was to reaction phenotype the identity of the cytosolic enzyme responsible for the S-oxidation of S-carboxymethyl-L-cysteine (SCMC) in female human hepatic cytosolic fractions. The identity of this enzyme in the female Wistar rat hepatic cytosolic fraction was found to be phenylalanine 4-monooxygenase (PAH). In pooled female human hepatic cytosolic fractions the calculated K(m) and V(max) for substrate (SCMC) activated PAH was 16.22 +/- 11.31 mM and 0.87 +/- 0.41 nmoles x min(-1) mg(-1). The experimental data modelled to the Michaelis-Menten equation with noncompetitive substrate inhibition. When the cytosolic fractions were activated with lysophophatidylcholine the V(max) increased to 52.31 +/- 11.72 nmoles x min(-1) mg(-1) but the K(m) remained unchanged at 16.53 +/- 2.32 mM. A linear correlation was seen in the production of Tyr and SCMC R/S S-oxide in 20 individual female hepatic cytosolic fractions for both substrate and lysophosphatidylcholine activated PAH (r(s) > 0.96). Inhibitor studies found that the specific chemical and antibody inhibitors of PAH reduced the production of Tyr and SCMC R/S S-oxide in these in vitro PAH assays. An investigation of the mechanism of interaction of SCMC with PAH indicated that the drug was a competitive inhibitor of the aromatic C-oxidation of Phe with a calculated K(i) of 17.23 +/- 4.15 mM. The requirement of BH4 as cofactor and the lack of effect of the specific tyrosine hydroxylase, tryptophan hydroxylase and nitric oxide synthase inhibitors on the S-oxidation of SCMC all indicate that PAH was the enzyme responsible for this biotransformation reaction in human hepatic cytosolic fractions.

  19. 4-Toluene sulfonate methyl-monooxygenase from Comamonas testosteroni T-2: purification and some properties of the oxygenase component.

    PubMed Central

    Locher, H H; Leisinger, T; Cook, A M

    1991-01-01

    Comamonas testosteroni T-2 synthesizes an inducible enzyme system that oxygenates 4-toluene sulfontate (TS) to 4-sulfobenzyl alcohol when grown in TS-salts medium. We purified this TS methyl-monooxygenase system (TSMOS) and found it to consist of two components. A monomeric, iron-sulfur flavoprotein (component B), which has been shown to act as a reductase in the 4-sulfobenzoate dioxygenase system of this organism (H. H. Locher, T. Leisinger, and A. M. Cook, Biochem. J. 274:833-842, 1991), carried electrons from NADH to component M, an oxygenase. This oxygenase had the UV-visible spectral characteristics of an iron-sulfur protein. Mrs of about 152,000 for the native oxygenase and of 43,000 under denaturing conditions indicated a homotri- or homotetrameric enzyme, whose N-terminal amino acids and amino acid composition were determined. The activity of the purified enzyme was enhanced about fivefold by the addition of Fe2+. In the presence of O2 and NADH, components B and M together catalyzed the stoichiometric transformation of TS or p-toluate to the corresponding alcohol. The reaction was confirmed as oxygenation of the methyl group by observation of an oxygen atom from 18O2 in carboxybenzyl alcohol. The substrate range of TSMOS included carboxylated analogs of TS (p- and m-toluates and 4-ethylbenzoate), whereas p-xylene, toluene, and p-cresol were not substrates. TSMOS also catalyzed demethylation; 4-methoxybenzoate was transformed to 4-hydroxybenzoate and formaldehyde. Images PMID:2050632

  20. Heterogeneity in the Histidine-brace Copper Coordination Sphere in Auxiliary Activity Family 10 (AA10) Lytic Polysaccharide Monooxygenases.

    PubMed

    Chaplin, Amanda K; Wilson, Michael T; Hough, Michael A; Svistunenko, Dimitri A; Hemsworth, Glyn R; Walton, Paul H; Vijgenboom, Erik; Worrall, Jonathan A R

    2016-06-10

    Copper-dependent lytic polysaccharide monooxygenases (LPMOs) are enzymes that oxidatively deconstruct polysaccharides. The active site copper in LPMOs is coordinated by a histidine-brace. This utilizes the amino group and side chain of the N-terminal His residue with the side chain of a second His residue to create a T-shaped arrangement of nitrogen ligands. We report a structural, kinetic, and thermodynamic appraisal of copper binding to the histidine-brace in an auxiliary activity family 10 (AA10) LPMO from Streptomyces lividans (SliLPMO10E). Unexpectedly, we discovered the existence of two apo-SliLPMO10E species in solution that can each bind copper at a single site with distinct kinetic and thermodynamic (exothermic and endothermic) properties. The experimental EPR spectrum of copper-bound SliLPMO10E requires the simulation of two different line shapes, implying two different copper-bound species, indicative of three and two nitrogen ligands coordinating the copper. Amino group coordination was probed through the creation of an N-terminal extension variant (SliLPMO10E-Ext). The kinetics and thermodynamics of copper binding to SliLPMO10E-Ext are in accord with copper binding to one of the apo-forms in the wild-type protein, suggesting that amino group coordination is absent in the two-nitrogen coordinate form of SliLPMO10E. Copper binding to SliLPMO10B was also investigated, and again it revealed the presence of two apo-forms with kinetics and stoichiometry of copper binding identical to that of SliLPMO10E. Our findings highlight that heterogeneity exists in the active site copper coordination sphere of LPMOs that may have implications for the mechanism of loading copper in the cell.

  1. TESTING THE SPECIFICITY OF PRIMERS TO ENVIRONMENTAL AMMONIA MONOOXYGENASE (AMOA) GENES IN GROUNDWATER TREATED WITH UREA TO PROMOTE CALCITE PRECIPITATION

    SciTech Connect

    Stephanie Freeman; David Reed; Yoshiko Fujita

    2006-12-01

    The diversity of bacterial ammonia monooxygenase (amoA) genes in DNA isolated from microorganisms in groundwater was characterized by amplification of amoA DNA using polymerase chain reaction (PCR), Restriction Fragment Length Polymorphism (RFLP) analysis, and sequencing. The amoA gene is characteristic of ammonia oxidizing bacteria (AOB). The DNA extracts were acquired from an experiment where dilute molasses and urea were sequentially introduced into a well in the Eastern Snake River Plain Aquifer (ESRPA) in Idaho to examine whether such amendments could stimulate enhanced ureolytic activity. The hydrolysis of urea into ammonium and carbonate serves as the basis for a potential remediation technique for trace metals and radionuclide contaminants that co-precipitate in calcite. The ammonium ion resulting from ureolysis can promote the growth of AOB. The goal of this work was to investigate the effectiveness of primers designed for quantitative PCR of environmental amoA genes and to evaluate the effect of the molasses and urea amendments upon the population diversity of groundwater AOB. PCR primers designed to target a portion of the amoA gene were used to amplify amoA gene sequences in the groundwater DNA extracts. Following PCR, amplified gene products were cloned and the clones were characterized by RFLP, a DNA restriction technique that can distinguish different DNA sequences, to gauge the initial diversity. Clones exhibiting unique RFLP patterns were subjected to DNA sequencing. Initial sequencing results suggest that the primers were successful at specific detection of amoA sequences and the RFLP analyses indicated that the diversity of detected amoA sequences in the ESRPA decreased with the additions of molasses and urea.

  2. The oxidation of hydrophobic aromatic substrates using a variant of the P450 monooxygenase CYP101B1.

    PubMed

    Sarkar, Md Raihan; Lee, Joel H Z; Bell, Stephen Graham

    2017-09-03

    The cytochrome P450 monooxygenase CYP101B1, from a Novosphingobium bacterium is able to bind and oxidise aromatic substrates but at a lower activity and efficiency compared to norisoprenoids and monoterpenoid esters. Histidine 85 of CYP101B1 aligns with tyrosine 96 of CYP101A1, which in this enzyme forms the only hydrophilic interaction with its substrate, camphor. The histidine residue of CYP101B1 was modified to a phenylalanine with the aim of improving the activity of the enzyme for hydrophobic substrates. The H85F mutant lowered the binding affinity and activity of the enzyme for β-ionone and altered the oxidation selectivity. This variant also showed enhanced affinity and activity towards alkylbenzenes, styrenes and methylnaphthalenes. For example the product formation rate of acenaphthene oxidation was improved 6-fold to 245 nmol.nmol-CYP-1.min-1. Certain disubstituted naphthalenes and substrates such as phenylcyclohexane, and biphenyls, were oxidised with lower activity by the H85F variant. Variants at H85 (A and G) designed to introduce additional space in the active site to accommodate these larger substrates did not engender improvements in the oxidation activity. As the H85F mutant of CYP101B1 improved the oxidation of hydrophobic substrates this residue is likely to be in the substrate binding pocket or the access channel of the enzyme. The side chain of the histidine may interact with the carbonyl groups of the favoured norisoprenoid substrates of CYP101B1. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Possible Peroxo State of the Dicopper Site of Particulate Methane Monooxygenase from Combined Quantum Mechanics and Molecular Mechanics Calculations.

    PubMed

    Itoyama, Shuhei; Doitomi, Kazuki; Kamachi, Takashi; Shiota, Yoshihito; Yoshizawa, Kazunari

    2016-03-21

    Enzymatic methane hydroxylation is proposed to efficiently occur at the dinuclear copper site of particulate methane monooxygenase (pMMO), which is an integral membrane metalloenzyme in methanotrophic bacteria. The resting state and a possible peroxo state of the dicopper active site of pMMO are discussed by using combined quantum mechanics and molecular mechanics calculations on the basis of reported X-ray crystal structures of the resting state of pMMO by Rosenzweig and co-workers. The dicopper site has a unique structure, in which one copper is coordinated by two histidine imidazoles and another is chelated by a histidine imidazole and primary amine of an N-terminal histidine. The resting state of the dicopper site is assignable to the mixed-valent Cu(I)Cu(II) state from a computed Cu-Cu distance of 2.62 Å from calculations at the B3LYP-D/TZVP level of theory. A μ-η(2):η(2)-peroxo-Cu(II)2 structure similar to those of hemocyanin and tyrosinase is reasonably obtained by using the resting state structure and dioxygen. Computed Cu-Cu and O-O distances are 3.63 and 1.46 Å, respectively, in the open-shell singlet state. Structural features of the dicopper peroxo species of pMMO are compared with those of hemocyanin and tyrosinase and synthetic dicopper model compounds. Optical features of the μ-η(2):η(2)-peroxo-Cu(II)2 state are calculated and analyzed with TD-DFT calculations.

  4. Structural characterization by EXAFS spectroscopy of the binuclear iron center in protein A of methane monooxygenase from methylococcus capsulatus (bath)

    SciTech Connect

    Ericson, A.; Hedman, B.; Hodgson, K.O.; Green, J.; Dalton, H.; Bentsen, J.G.; Beer, R.H.; Lippard, S.J.

    1988-03-30

    Soluble methane monooxygenase (MMO) from Methylococcus capsulatus (Bath) activates dioxygen for incorporation into a remarkable variety of substrates including methane, which is required for bacterial growth (eq 1). MMO is a three component CH/sub 4/ + NADH + H/sup +/ + O/sub 2/ /sup MMO/ ..-->.. CH/sub 3/OH + NAD/sup +/ + H/sub 2/O (1) enzyme. Protein A (M/sub r/ 210,000), believed to be the oxygenase component, contains two iron atoms. Protein B (M/sub r/ 15,700) serves a regulatory function and lacks prosthetic groups, while protein C, the reductase component of the enzyme, is an iron-sulfur flavoprotein (M/sub r/ 42,000) responsible for electron transfer from NADH to protein A. Recently, a binuclear iron center was postulated to occur in protein A based on the finding that one-electron reduction gives rise to electron spin resonance (ESR) signals (g 1.95, 1.88, 1.78) very similar to those observed for the binuclear mixed-valence Fe/sub 2/(III,II) centers in semimet hemerythrin (Hr) and purple acid phosphatase (PAP). In conjunction with model studies, extended X-ray absorption fine structure (EXAFS) spectroscopy has proven to be a powerful method for identifying bridged binuclear iron centers in Hr, ribonucleotide reductase (RR), and PAP. Here the authors report iron K-edge EXAFS results on semireduced protein A of MMO which support the occurrence of a binuclear iron center (Fe-Fe distance, 3.41 A), with no short ..mu..-oxo bridge.

  5. Optimization of trichloroethylene degradation using soluble methane monooxygenase of Methylosinus trichosporium OB3b expressed in recombinant bacteria

    SciTech Connect

    Jahng, D.; Kim, C.S.; Wood, T.K.; Hanson, R.S.

    1996-08-05

    By complementing cell-free extracts of Pseudomonas putida F1/pSMMO20 with purified soluble methane monooxygenase (sMMO) components of Methylosinus trichospoirium OB3b, the low cloned-gene sMMO activity in the recombinant strain was found to be due to incomplete activity of the hydroxylase component. To address this incomplete activity, additional sMMO-expressing strains were formed by transferring mmo-containing pSMMO20 and pSMMO50 into various bacterial species including pseudomonads and {alpha}-2 subdivision strains such as methanotrophs, methylotrophs, Agrobacterium tumefaciens A114, and Rhizobium meliloti 102F34 (11 new strains screened); sMMO activity was detected in the last two strains. To increase plasmid segregational stability, the hok/sok locus originally from Escherichia coli plasmid R1 was inserted downstream of the mmo locus of pSMMO20 (resulting in pSMMO40) and found to enhance plasmid stability in P. putida F1 and R. meliloti 102F34 (first report of hok/sok in Rhizobium). To further increase sMMO activity, a modified Whittenbury minimal medium was selected from various minimal and complex media based on trichloroethylene (TCE) degradation and growth rates and was improved by removing the sMMO-inhibiting metal ions [Cu(II), Ni(II), and Zn(II)] and chloramphenicol from the medium and by supplementing with an iron source (3.6 {micro}M of ferrous ammonium sulfate). Using chemostat-grown P. putida F1/pSMMO40, it was found that sMMO activity was higher for cells grown at higher dilution rates. These optimization efforts resulted in a twofold increase in the extent of TCE degradation and more consistent sMMO activity.

  6. Isolation and characterization of two YUCCA flavin monooxygenase genes from cultivated strawberry (Fragaria × ananassa Duch.).

    PubMed

    Liu, Hong; Ying, Yang-Yang; Zhang, Ling; Gao, Qing-Hua; Li, Jing; Zhang, Zhen; Fang, Jing-Gui; Duan, Ke

    2012-08-01

    In strawberry (Fragaria × ananassa Duch.), auxin has been recognized as the main signal molecule coordinating the growth and initiation of ripening of fruits. The molecular mechanism regulating auxin biosynthesis in strawberry remains unknown. This project reports two YUCCA flavin monooxygenase genes FaYUC1-2 isolated from cultivated strawberry. FaYUC1 and FaYUC2 are most homologous to AtYUC6 and AtYUC4, respectively. Significant expression of FaYUC1-2 is found in vegetative meristems and reproductive organs, with overlapping but distinct patterns. During fruit development, both transcripts of FaYUC1 and FaYUC2 in achenes reach a peak around large green fruit (G2) stage, but the sudden rise in FaYUC2 transcript level is much steeper and begins earlier than that in FaYUC1. FaYUC2 is also obviously expressed in the receptacles from green fruits, hinting another auxin source for receptacle development, other than achenes. FaYUC1 over-expression Arabidopsis exhibits typical auxin hyper-accumulation phenotype in many aspects, such as the narrow and downward curled leaves, strong apical dominance, short and hairy root. It is also severely sterile, due to the disruption of floral meristems initiation and floral organs development. Transgenic analysis indicates that strawberry YUC gene may hold conserved role in auxin biosynthesis like their homologs in other plants. Integrated with the spatiotemporal expression features, these results led us to propose that FaYUC1-2 may involve in many developmental processes including flower and fruit development in strawberry. This paper is the first report of isolation and characterization of strawberry auxin biosynthesis genes. And their conserved functions in auxin biosynthesis were confirmed after ectopic expression.

  7. Disulfide bridges as essential elements for the thermostability of lytic polysaccharide monooxygenase LPMO10C from Streptomyces coelicolor.

    PubMed

    Tanghe, Magali; Danneels, Barbara; Last, Matthias; Beerens, Koen; Stals, Ingeborg; Desmet, Tom

    2017-05-01

    Lytic polysaccharide monooxygenases (LPMOs) are crucial components of cellulase mixtures but their stability has not yet been studied in detail, let alone been engineered for industrial applications. In this work, we have evaluated the importance of disulfide bridges for the thermodynamic stability of Streptomyces coelicolor LPMO10C. Interestingly, this enzyme was found to retain 34% of its activity after 2-h incubation at 80°C while its apparent melting temperature (Tm) is only 51°C. When its three disulfide bridges were broken, however, irreversible unfolding occurred and no residual activity could be detected after a similar heat treatment. Based on these findings, additional disulfide bridges were introduced, as predicted by computational tools (MOdelling of DIsulfide bridges in Proteins (MODiP) and Disulfide by Design (DbD)) and using the most flexible positions in the structure as target sites. Four out of 16 variants displayed an improvement in Tm, ranging from 2 to 9°C. Combining the positive mutations yielded additional improvements (up to 19°C) but aberrant unfolding patterns became apparent in some cases, resulting in a diminished capacity for heat resistance. Nonetheless, the best variant, a combination of A143C-P183C and S73C-A115C, displayed a 12°C increase in Tm and was able to retain and was able to retain no less than 60% of its activity after heat treatment. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  8. Expression and purification of the recombinant subunits of toluene/o-xylene monooxygenase and reconstitution of the active complex.

    PubMed

    Cafaro, Valeria; Scognamiglio, Roberta; Viggiani, Ambra; Izzo, Viviana; Passaro, Irene; Notomista, Eugenio; Piaz, Fabrizio Dal; Amoresano, Angela; Casbarra, Annarita; Pucci, Piero; Di Donato, Alberto

    2002-11-01

    This paper describes the cloning of the genes coding for each component of the complex of toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1, their expression, purification and characterization. Moreover, the reconstitution of the active complex from the recombinant subunits has been obtained, and the functional role of each component in the electron transfer from the electron donor to molecular oxygen has been determined. The coexpression of subunits B, E and A leads to the formation of a subcomplex, named H, with a quaternary structure (BEA)2, endowed with hydroxylase activity. Tomo F component is an NADH oxidoreductase. The purified enzyme contains about 1 mol of FAD, 2 mol of iron, and 2 mol of acid labile sulfide per mol of protein, as expected for the presence of one [2Fe-2S] cluster, and exhibits a typical flavodoxin absorption spectrum. Interestingly, the sequence of the protein does not correspond to that previously predicted on the basis of DNA sequence. We have shown that this depends on minor errors in the gene sequence that we have corrected. C component is a Rieske-type ferredoxin, whose iron and acid labile sulfide content is in agreement with the presence of one [2Fe-2S] cluster. The cluster is very sensitive to oxygen damage. Mixtures of the subcomplex H and of the subunits F, C and D are able to oxidize p-cresol into 4-methylcathecol, thus demonstrating the full functionality of the recombinant subunits as purified. Finally, experimental evidence is reported which strongly support a model for the electron transfer. Subunit F is the first member of an electron transport chain which transfers electrons from NADH to C, which tunnels them to H subcomplex, and eventually to molecular oxygen.

  9. The detection and phylogenetic analysis of the alkane 1-monooxygenase gene of members of the genus Rhodococcus.

    PubMed

    Táncsics, András; Benedek, Tibor; Szoboszlay, Sándor; Veres, Péter G; Farkas, Milán; Máthé, István; Márialigeti, Károly; Kukolya, József; Lányi, Szabolcs; Kriszt, Balázs

    2015-02-01

    Naturally occurring and anthropogenic petroleum hydrocarbons are potential carbon sources for many bacteria. The AlkB-related alkane hydroxylases, which are integral membrane non-heme iron enzymes, play a key role in the microbial degradation of many of these hydrocarbons. Several members of the genus Rhodococcus are well-known alkane degraders and are known to harbor multiple alkB genes encoding for different alkane 1-monooxygenases. In the present study, 48 Rhodococcus strains, representing 35 species of the genus, were investigated to find out whether there was a dominant type of alkB gene widespread among species of the genus that could be used as a phylogenetic marker. Phylogenetic analysis of rhodococcal alkB gene sequences indicated that a certain type of alkB gene was present in almost every member of the genus Rhodococcus. These alkB genes were common in a unique nucleotide sequence stretch absent from other types of rhodococcal alkB genes that encoded a conserved amino acid motif: WLG(I/V/L)D(G/D)GL. The sequence identity of the targeted alkB gene in Rhodococcus ranged from 78.5 to 99.2% and showed higher nucleotide sequence variation at the inter-species level compared to the 16S rRNA gene (93.9-99.8%). The results indicated that the alkB gene type investigated might be applicable for: (i) differentiating closely related Rhodococcus species, (ii) properly assigning environmental isolates to existing Rhodococcus species, and finally (iii) assessing whether a new Rhodococcus isolate represents a novel species of the genus. Copyright © 2014 Elsevier GmbH. All rights reserved.

  10. The metabolomics of (+/-)-arecoline 1-oxide in the mouse and its formation by human flavin-containing monooxygenases.

    PubMed

    Giri, Sarbani; Krausz, Kristopher W; Idle, Jeffrey R; Gonzalez, Frank J

    2007-02-15

    The alkaloid arecoline is a main constituent of areca nuts that are chewed by approximately 600 million persons worldwide. A principal metabolite of arecoline is arecoline 1-oxide whose metabolism has been poorly studied. To redress this, synthetic (+/-)-arecoline 1-oxide was administered to mice (20mg/kg p.o.) and a metabolomic study performed on 0-12h urine using ultra-performance liquid chromatography-coupled time-of-flight mass spectrometry (UPLC-TOFMS) with multivariate data analysis. A total of 16 mass/retention time pairs yielded 13 metabolites of (+/-)-arecoline 1-oxide, most of them novel. Identity of metabolites was confirmed by tandem mass spectrometry. The principal pathways of metabolism of (+/-)-arecoline 1-oxide were mercapturic acid formation, with catabolism to mercaptan and methylmercaptan metabolites, apparent CC double-bond reduction, carboxylic acid reduction to the aldehyde (a novel pathway in mammals), N-oxide reduction, and de-esterification. Relative percentages of metabolites were determined directly from the metabolomic data. Approximately, 50% of the urinary metabolites corresponded to unchanged (+/-)-arecoline 1-oxide, 25% to other N-oxide metabolites, while approximately, 30% corresponded to mercapturic acids or their metabolites. Many metabolites, principally mercapturic acids and their derivatives, were excreted as diastereomers that could be resolved by UPLC-TOFMS. Arecoline was converted to arecoline 1-oxide in vitro by human flavin-containing monooxygenases FMO1 (K(M): 13.6+/-4.9muM; V(MAX): 0.114+/-0.01nmolmin(-1)microg(-1) protein) and FMO3 (K(M): 44.5+/-8.0microM; V(MAX): 0.014+/-0.001nmolmin(-1)microg(-1) protein), but not by FMO5 or any of 11 human cytochromes P450. This report underscores the power of metabolomics in drug metabolite mining.

  11. Haploinsufficiency in peptidylglycine α-amidating monooxygenase leads to altered synaptic transmission in the amygdala and impaired emotional responses

    PubMed Central

    Gaier, ED; Rodriguiz, RM; Ma, XM; Sivaramakrishnan, S; Bousquet-Moore, D; Wetsel, WC; Eipper, BA

    2010-01-01

    The mammalian amygdala expresses various neuropeptides whose signaling has been implicated in emotionality. Many neuropeptides require amidation for full activation by peptidylglycine α-amidating monooxygenase (PAM), a transmembrane vesicular cuproenzyme and regulator of the secretory pathway. Mice heterozygous for the Pam gene (PAM+/−) exhibit physiological and behavioral abnormalities related to specific peptidergic pathways. In the present study, we evaluated emotionality and examined molecular and cellular responses that characterize neurophysiological differences in the PAM+/− amygdala. PAM+/− mice presented with anxiety-like behaviors in the zero maze that were alleviated by diazepam. PAM+/− animals were deficient in short- and long-term contextual and cued fear conditioning and required higher shock intensities to establish fear-potentiated startle than their wildtype littermates. Immunohistochemical analysis of the amygdala revealed PAM expression in pyramidal neurons and local interneurons that synthesize γ-aminobutyric acid (GABA). We performed whole-cell recordings of pyramidal neurons in the PAM+/− amygdala to elucidate neurophysiological correlates of the fear behavioral phenotypes. Consistent with these observations, thalamic afferent synapses in the PAM+/− lateral nucleus were deficient in long-term potentiation. This deficit was apparent in the absence and presence of the GABAA receptor antagonist picrotoxin and was abolished when both GABAA and GABAB receptors were blocked. Both evoked and spontaneous excitatory signals were enhanced in the PAM+/− lateral nucleus. Phasic GABAergic signaling was also augmented in the PAM+/− amygdala, and this difference was comprised of activity-independent and activity-dependent components. These physiological findings represent perturbations in the PAM+/− amygdala that may underlie the aberrant emotional responses in the intact animal. PMID:20943906

  12. Adaptor Protein-1 Complex Affects the Endocytic Trafficking and Function of Peptidylglycine α-Amidating Monooxygenase, a Luminal Cuproenzyme*

    PubMed Central

    Bonnemaison, Mathilde L.; Bäck, Nils; Duffy, Megan E.; Ralle, Martina; Mains, Richard E.; Eipper, Betty A.

    2015-01-01

    The adaptor protein-1 complex (AP-1), which transports cargo between the trans-Golgi network and endosomes, plays a role in the trafficking of Atp7a, a copper-transporting P-type ATPase, and peptidylglycine α-amidating monooxygenase (PAM), a copper-dependent membrane enzyme. Lack of any of the four AP-1 subunits impairs function, and patients with MEDNIK syndrome, a rare genetic disorder caused by lack of expression of the σ1A subunit, exhibit clinical and biochemical signs of impaired copper homeostasis. To explore the role of AP-1 in copper homeostasis in neuroendocrine cells, we used corticotrope tumor cells in which AP-1 function was diminished by reducing expression of its μ1A subunit. Copper levels were unchanged when AP-1 function was impaired, but cellular levels of Atp7a declined slightly. The ability of PAM to function was assessed by monitoring 18-kDa fragment-NH2 production from proopiomelanocortin. Reduced AP-1 function made 18-kDa fragment amidation more sensitive to inhibition by bathocuproine disulfonate, a cell-impermeant Cu(I) chelator. The endocytic trafficking of PAM was altered, and PAM-1 accumulated on the cell surface when AP-1 levels were reduced. Reduced AP-1 function increased the Atp7a presence in early/recycling endosomes but did not alter the ability of copper to stimulate its appearance on the plasma membrane. Co-immunoprecipitation of a small fraction of PAM and Atp7a supports the suggestion that copper can be transferred directly from Atp7a to PAM, a process that can occur only when both proteins are present in the same subcellular compartment. Altered luminal cuproenzyme function may contribute to deficits observed when the AP-1 function is compromised. PMID:26170456

  13. An Investigation into the Prediction of in Vivo Clearance for a Range of Flavin-containing Monooxygenase Substrates.

    PubMed

    Jones, Barry C; Srivastava, Abhishek; Colclough, Nicola; Wilson, Joanne; Reddy, Venkatesh Pilla; Amberntsson, Sara; Li, Danxi

    2017-10-01

    Flavin-containing monooxygenases (FMO) are metabolic enzymes mediating the oxygenation of nucleophilic atoms such as nitrogen, sulfur, phosphorus, and selenium. These enzymes share similar properties to the cytochrome P450 system but can be differentiated through heat inactivation and selective substrate inhibition by methimazole. This study investigated 10 compounds with varying degrees of FMO involvement to determine the nature of the correlation between human in vitro and in vivo unbound intrinsic clearance. To confirm and quantify the extent of FMO involvement six of the compounds were investigated in human liver microsomal (HLM) in vitro assays using heat inactivation and methimazole substrate inhibition. Under these conditions FMO contribution varied from 21% (imipramine) to 96% (itopride). Human hepatocyte and HLM intrinsic clearance (CLint) data were scaled using standard methods to determine the predicted unbound intrinsic clearance (predicted CLint u) for each compound. This was compared with observed unbound intrinsic clearance (observed CLint u) values back calculated from human pharmacokinetic studies. A good correlation was observed between the predicted and observed CLint u using hepatocytes (R(2) = 0.69), with 8 of the 10 compounds investigated within or close to a factor of 2. For HLM the in vitro-in vivo correlation was maintained (R(2) = 0.84) but the accuracy was reduced with only 3 out of 10 compounds falling within, or close to, twofold. This study demonstrates that human hepatocytes and HLM can be used with standard scaling approaches to predict the human in vivo clearance for FMO substrates. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  14. A substrate-specific cytochrome P450 monooxygenase, CYP6AB11, from the polyphagous navel orangeworm (Amyelois transitella).

    PubMed

    Niu, Guodong; Rupasinghe, Sanjeewa G; Zangerl, Arthur R; Siegel, Joel P; Schuler, Mary A; Berenbaum, May R

    2011-04-01

    The navel orangeworm Amyelois transitella (Walker) (Lepidoptera: Pyralidae) is a serious pest of many tree crops in California orchards, including almonds, pistachios, walnuts and figs. To understand the molecular mechanisms underlying detoxification of phytochemicals, insecticides and mycotoxins by this species, full-length CYP6AB11 cDNA was isolated from larval midguts using RACE PCR. Phylogenetic analysis of this insect cytochrome P450 monooxygenase established its evolutionary relationship to a P450 that selectively metabolizes imperatorin (a linear furanocoumarin) and myristicin (a natural methylenedioxyphenyl compound) in another lepidopteran species. Metabolic assays conducted with baculovirus-expressed P450 protein, P450 reductase and cytochrome b(5) on 16 compounds, including phytochemicals, mycotoxins, and synthetic pesticides, indicated that CYP6AB11 efficiently metabolizes imperatorin (0.88 pmol/min/pmol P450) and slowly metabolizes piperonyl butoxide (0.11 pmol/min/pmol P450). LC-MS analysis indicated that the imperatorin metabolite is an epoxide generated by oxidation of the double bond in its extended isoprenyl side chain. Predictive structures for CYP6AB11 suggested that its catalytic site contains a doughnut-like constriction over the heme that excludes aromatic rings on substrates and allows only their extended side chains to access the catalytic site. CYP6AB11 can also metabolize the principal insecticide synergist piperonyl butoxide (PBO), a synthetic methylenedioxyphenyl compound, albeit slowly, which raises the possibility that resistance may evolve in this species after exposure to synergists under field conditions. Copyright © 2011 Elsevier Ltd. All rights reserved.

  15. Effect of repeated ether anesthesias on the mono-oxygenase system of rat liver S-9 fraction.

    PubMed

    Paolini, M; Bauer, C; Corsi, C; Tonelli, F; Hrelia, P; Bronzetti, G; Forti, G C

    1984-01-01

    This study was designed to investigate the effect of ether anesthesia in rats, before i.p. injections to induce the mono-oxygenase enzyme system, on biochemical properties of liver S9 fractions. Aminopyrine N-demethylase and rho-nitroanisole O-demethylase activity levels, their stability, and lipid peroxidation were determined in S9 fractions after etherization (about 1 min in ether vapor chamber daily for 3 consecutive days, before i.p. injections of Na-phenobarbital and beta-naphthoflavone) and compared with controls receiving the same injections without etherization. The activities were slightly (but not significatively) enhanced after this treatment, but stability was markedly and significatively greater after 1 h of incubation in the conditions of the liver microsomal assay (+ 14.8% and + 74.7%, respectively); lipid peroxidation was strongly and significatively depressed (-76.0%). Etherization sufficient to kill the animals on the 4th day resulted in equally active but less stable S9 fraction enzymes. Dimethylnitrosamine (as a standard premutagen) was assayed with the D7 strain of Saccharomyces cerevisiae using S9 fractions obtained from both anesthetized and nonanesthetized rats. According to biochemical data, results obtained with S9 from partially anesthetized rats were comparable with the conventional ones (S9 from nonanesthetized rats). On the contrary, the use of more prolonged ether anesthesia, including one on the day the animals are killed, gives S9 fraction significantly less effective. We conclude that if brief etherizations are used, for i.p. injections only, the S9 fractions obtained are entirely satisfactory and the procedures involved in production are simplified; the additional animal treatment (etherization) must be specified.

  16. Relative contribution of monooxygenase and esterase to pyrethroid resistance in Triatoma infestans (Hemiptera: Reduviidae) from Argentina and Bolivia.

    PubMed

    Orihuela, Pablo L Santo; Vassena, Claudia V; Zerba, Eduardo N; Picollo, Maria I

    2008-03-01

    Recently, high resistance to pyrethroid insecticides has been associated with ineffective field treatments against Triatoma infestans (Klug) (Hemiptera: Reduviidae) in northern Argentina. Samples were collected from two areas in Argentina (Salta and La Rioja) and one are in Bolivia (Yacuiba), and they were subjected to toxicological and biochemical assays. All populations were resistant to deltamethrin, but they showed different profiles to nonpyrethroid insecticides. The Salta population showed high resistance ratios (RRs) to deltamethrin and only slight differences in the susceptibility to fenitrothion and fipronil compared with the reference strain. Otherwise, the La Rioja population showed a lower RR to deltamethrin and no resistance to fenitrothion or fipronil. Finally, the Yacuiba population had high a RR to deltamethrin, but it was susceptibility to fenitrothion and fipronil. In several cases, deltamethrin-resistant populations had higher susceptibility to bendiocarb than the reference strain. Measured activity of P450 monooxygenase in individual insects (based on ethoxycoumarine-O-deethylase), tended to be higher in the deltamethrin-resistant populations, but the differences were not statistically significant. Activity of specific esterases determined by the hydrolysis of 7-coumaryl permethrate demonstrated an increase in the percentage of insects with higher esterase activity in the Salta and La Rioja populations. Unexpectedly, the Yacuiba population showed lower pyrethroid esterase activity than the reference strain. The different pyrethroid resistance patterns found in T. infestans from three geographical regions within Argentina and in Bolivia suggests that enzyme-based pyrethroid resistance in this species has multiple origins. Nevertheless, because nerve insensitivity (related to the presence of the kdr gene) is also an important mechanism related to pyrethroid resistance, further studies on the kdr gene should be carried to clarify the relative

  17. Heterogeneity in the Histidine-brace Copper Coordination Sphere in Auxiliary Activity Family 10 (AA10) Lytic Polysaccharide Monooxygenases*

    PubMed Central

    Chaplin, Amanda K.; Wilson, Michael T.; Hough, Michael A.; Svistunenko, Dimitri A.; Hemsworth, Glyn R.; Walton, Paul H.; Vijgenboom, Erik; Worrall, Jonathan A. R.

    2016-01-01

    Copper-dependent lytic polysaccharide monooxygenases (LPMOs) are enzymes that oxidatively deconstruct polysaccharides. The active site copper in LPMOs is coordinated by a histidine-brace. This utilizes the amino group and side chain of the N-terminal His residue with the side chain of a second His residue to create a T-shaped arrangement of nitrogen ligands. We report a structural, kinetic, and thermodynamic appraisal of copper binding to the histidine-brace in an auxiliary activity family 10 (AA10) LPMO from Streptomyces lividans (SliLPMO10E). Unexpectedly, we discovered the existence of two apo-SliLPMO10E species in solution that can each bind copper at a single site with distinct kinetic and thermodynamic (exothermic and endothermic) properties. The experimental EPR spectrum of copper-bound SliLPMO10E requires the simulation of two different line shapes, implying two different copper-bound species, indicative of three and two nitrogen ligands coordinating the copper. Amino group coordination was probed through the creation of an N-terminal extension variant (SliLPMO10E-Ext). The kinetics and thermodynamics of copper binding to SliLPMO10E-Ext are in accord with copper binding to one of the apo-forms in the wild-type protein, suggesting that amino group coordination is absent in the two-nitrogen coordinate form of SliLPMO10E. Copper binding to SliLPMO10B was also investigated, and again it revealed the presence of two apo-forms with kinetics and stoichiometry of copper binding identical to that of SliLPMO10E. Our findings highlight that heterogeneity exists in the active site copper coordination sphere of LPMOs that may have implications for the mechanism of loading copper in the cell. PMID:27129229

  18. Stereospecificity and physiology of co-oxidative production of chemicals by methanotrophic bacteria. [Methane monooxygenase:a2

    SciTech Connect

    Kelley, R.L.; Hoefer, D.E.; Conrad, J.R.; Srivastava, V.J.; Akin, C.

    1991-01-01

    Methanotrophic bacteria can use methane as a sole source of carbon and energy. The enzyme responsible for the oxidation of methane in these organisms, methane monooxygenase, is also able to biotransform a number of substrates into industrially important chemicals. One such example is the oxidation of propylene to propylene oxide. Several strains of mesophilic and thermophilic methanotrophs have been tested for their ability to produce propylene oxide with formate, methanol, and methane as the co-oxidative substrates. Cultures in the late stationary phase were found to be more productive than exponentially growing cultures. Methane did not inhibit propylene epoxidation on low-density cultures; in fact, the yields of propylene oxide were greater when both methane and propylene were present than when only propylene was present. In higher-density cultures, however, methane did appear to inhibit oxidation of propylene. The effects of several culture parameters such as pH, temperature, and concentration of micronutrients on propylene oxide production ad steroespecificity were determined. Propylene oxide production was proportional to the amount of cell loading up to 14 g/L. Unwashed cells produced more propylene oxide than washed cells. The long- and short-term inhibitory effects of propylene oxide on the methanotrophic strains were also investigated. A tolerance of up to 1 M propylene oxide was observed, and the maximum inhibitory effect was seen within 30 minute. The steroespecificity for propylene oxide production and oxidation of 3-methylcyclohexene was determined for several strains. Methylosinus trichosporium (OB3b), particularly a cell-free extract of this strain, had the greatest steroespecificity. 13 refs., 12 figs., 3 tabs.

  19. Biochemical evidence for the tyrosine involvement in cationic intermediate stabilization in mouse β-carotene 15, 15'-monooxygenase

    PubMed Central

    2009-01-01

    Background β-carotene 15,15'-monooxygenase (BCMO1) catalyzes the crucial first step in vitamin A biosynthesis in animals. We wished to explore the possibility that a carbocation intermediate is formed during the cleavage reaction of BCMO1, as is seen for many isoprenoid biosynthesis enzymes, and to determine which residues in the substrate binding cleft are necessary for catalytic and substrate binding activity. To test this hypothesis, we replaced substrate cleft aromatic and acidic residues by site-directed mutagenesis. Enzymatic activity was measured in vitro using His-tag purified proteins and in vivo in a β-carotene-accumulating E. coli system. Results Our assays show that mutation of either Y235 or Y326 to leucine (no cation-π stabilization) significantly impairs the catalytic activity of the enzyme. Moreover, mutation of Y326 to glutamine (predicted to destabilize a putative carbocation) almost eliminates activity (9.3% of wt activity). However, replacement of these same tyrosines with phenylalanine or tryptophan does not significantly impair activity, indicating that aromaticity at these residues is crucial. Mutations of two other aromatic residues in the binding cleft of BCMO1, F51 and W454, to either another aromatic residue or to leucine do not influence the catalytic activity of the enzyme. Our ab initio model of BCMO1 with β-carotene mounted supports a mechanism involving cation-π stabilization by Y235 and Y326. Conclusions Our data are consistent with the formation of a substrate carbocation intermediate and cation-π stabilization of this intermediate by two aromatic residues in the substrate-binding cleft of BCMO1. PMID:20003456

  20. Cooperation between MEF2 and PPARγ in human intestinal β,β-carotene 15,15'-monooxygenase gene expression

    PubMed Central

    Gong, Xiaoming; Tsai, Shu-Whei; Yan, Bingfang; Rubin, Lewis P

    2006-01-01

    Background Vitamin A and its derivatives, the retinoids, are essential for normal embryonic development and maintenance of cell differentiation. β, β-carotene 15,15'-monooxygenase 1 (BCMO1) catalyzes the central cleavage of β-carotene to all-trans retinal and is the key enzyme in the intestinal metabolism of carotenes to vitamin A. However, human and various rodent species show markedly different efficiencies in intestinal BCMO1-mediated carotene to retinoid conversion. The aim of this study is to identify potentially human-specific regulatory control mechanisms of BCMO1 gene expression. Results We identified and functionally characterized the human BCMO1 promoter sequence and determined the transcriptional regulation of the BCMO1 gene in a BCMO1 expressing human intestinal cell line, TC-7. Several functional transcription factor-binding sites were identified in the human promoter that are absent in the mouse BCMO1 promoter. We demonstrate that the proximal promoter sequence, nt -190 to +35, confers basal transcriptional activity of the human BCMO1 gene. Site-directed mutagenesis of the myocyte enhancer factor 2 (MEF2) and peroxisome proliferator-activated receptor (PPAR) binding elements resulted in decreased basal promoter activity. Mutation of both promoter elements abrogated the expression of intestinal cell BCMO1. Electrophoretic mobility shift and supershift assays and transcription factor co-expression in TC-7 cells showed MEF2C and PPARγ bind to their respective DNA elements and synergistically transactivate BCMO1 expression. Conclusion We demonstrate that human intestinal cell BCMO1 expression is dependent on the functional cooperation between PPARγ and MEF2 isoforms. The findings suggest that the interaction between MEF2 and PPAR factors may provide a molecular basis for interspecies differences in the transcriptional regulation of the BCMO1 gene. PMID:16504037

  1. The in vitro metabolism of benzo[a]pyrene by polychlorinated and polybrominated biphenyl induced rat hepatic microsomal monooxygenases.

    PubMed

    Haake, J M; Merrill, J C; Safe, S

    1985-09-01

    The metabolism of benzo[a]pyrene by halogenated biphenyl-induced rat hepatic microsomal monooxygenases was determined using a high pressure liquid chromatographic assay system. Incubation of benzo[a]pyrene with microsomes from rats pretreated with phenobarbitone or phenobarbitone-type inducers (2,2',4,4',5,5'-hexachlorobiphenyl, 2,2',4,4',6,6'-hexachlorobiphenyl, 2,2',5,5'-tetrachlorobiphenyl, 2,2',4,4',5,5'-hexabromobiphenyl, and 2,2',5,5'-tetrabromobiphenyl) resulted in increased overall metabolism of the hydrocarbon (less than fourfold) into phenolic, quinone, and diol metabolites, with the most striking increase observed in the formation of 4,5-dihydro-4,5-dihydroxybenzo[a]pyrene. In contrast, the metabolism of benzo[a]pyrene by microsomes from rats induced with 3-methylcholanthrene or 3,3',4,4'-tetrachlorobiphenyl resulted in a greater than 10-fold increase in overall benzo[a]pyrene metabolism, with the largest increases observed in the formation of the trans-7,8- and -9,10-dihydrodiol metabolites of benzo[a]pyrene. However, in comparison to control and phenobarbitone-induced microsomes, the oxidative conversion of benzo[a]pyrene by microsomes induced with 3-methylcholanthrene and 3,3',4,4'-tetrachlorobiphenyl into the 6,12-quinone was substantially inhibited. Previous reports have shown that the commercial halogenated biphenyl mixtures, fireMaster BP-6, and Aroclor 1254 are mixed-type inducers and that microsomes from rats pretreated with these mixtures markedly enhance the overall metabolism of benzo[a]pyrene. Not surprisingly, the metabolism of benzo[a]pyrene by microsomes from rats pretreated with the mixed-type inducers, 2,3,3',4,4'-penta-,2,3,3',4,4',5-hexa-, and 2',3,3',4,4',5-hexa- chlorobiphenyl was also increased and the metabolic profile was similar to that observed with fireMaster BP-6 and Aroclor 1254 induced microsomes.

  2. CYP709B3, a cytochrome P450 monooxygenase gene involved in salt tolerance in Arabidopsis thaliana

    PubMed Central

    2013-01-01

    Background Within the Arabidopsis genome, there are 272 cytochrome P450 monooxygenase (P450) genes. However, the biological functions of the majority of these P450s remain unknown. The CYP709B family of P450s includes three gene members, CYP709B1, CYP709B2 and CYP709B3, which have high amino acid sequence similarity and lack reports elucidating biological functions. Results We identified T-DNA insertion-based null mutants of the CYP709B subfamily of genes. No obvious morphological phenotypes were exhibited under normal growth conditions. When the responses to ABA and salt stress were studied in these mutants, only the cyp709b3 mutant showed sensitivity to ABA and salt during germination. Under moderate salt treatment (150 mM NaCl), cyp709b3 showed a higher percentage of damaged seedlings, indicating a lower tolerance to salt stress. CYP709B3 was highly expressed in all analyzed tissues and especially high in seedlings and leaves. In contrast, CYP709B1 and CYP709B2 were highly expressed in siliques, but were at very low levels in other tissues. Under salt stress condition, CYP709B3 gene expression was induced after 24 hr and remained at high expression level. Expression of the wild type CYP709B3 gene in the cyp709b3 mutant fully complemented the salt intolerant phenotype. Furthermore, metabolite profiling analysis revealed some differences between wild type and cyp709b3 mutant plants, supporting the salt intolerance phenotype of the cyp709b3 mutant. Conclusions These results suggest that CYP709B3 plays a role in ABA and salt stress response and provides evidence to support the functions of cytochrome P450 enzymes in plant stress response. PMID:24164720

  3. Purification a