Cytochrome P450 2C8 and flavin-containing monooxygenases are involved in the metabolism of tazarotenic acid in humans.

PubMed

Attar, Mayssa; Dong, Dahai; Ling, Kah-Hiing John; Tang-Liu, Diane D-S

2003-04-01

Upon oral administration, tazarotene is rapidly converted to tazarotenic acid by esterases. The main circulating agent, tazarotenic acid is subsequently oxidized to the inactive sulfoxide metabolite. Therefore, alterations in the metabolic clearance of tazarotenic acid may have significant effects on its systemic exposure. The objective of this study was to identify the human liver microsomal enzymes responsible for the in vitro metabolism of tazarotenic acid. Tazarotenic acid was incubated with 1 mg/ml pooled human liver microsomes, in 100 mM potassium phosphate buffer (pH 7.4), at 37 degrees C, over a period of 30 min. The microsomal enzymes that may be involved in tazarotenic acid metabolism were identified through incubation with microsomes containing cDNA-expressed human microsomal isozymes. Chemical inhibition studies were then conducted to confirm the identity of the enzymes potentially involved in tazarotenic acid metabolism. Reversed-phase high performance liquid chromatography was used to quantify the sulfoxide metabolite, the major metabolite of tazarotenic acid. Upon incubation of tazarotenic acid with microsomes expressing CYP2C8, flavin-containing monooxygenase 1 (FMO1), or FMO3, marked formation of the sulfoxide metabolite was observed. The involvement of these isozymes in tazarotenic acid metabolism was further confirmed by inhibition of metabolite formation in pooled human liver microsomes by specific inhibitors of CYP2C8 or FMO. In conclusion, the in vitro metabolism of tazarotenic acid to its sulfoxide metabolite in human liver microsomes is mediated by CYP2C8 and FMO.

Species Differences in the Oxidative Desulfurization of a Thiouracil-Based Irreversible Myeloperoxidase Inactivator by Flavin-Containing Monooxygenase Enzymes.

PubMed

Eng, Heather; Sharma, Raman; Wolford, Angela; Di, Li; Ruggeri, Roger B; Buckbinder, Leonard; Conn, Edward L; Dalvie, Deepak K; Kalgutkar, Amit S

2016-08-01

N1-Substituted-6-arylthiouracils, represented by compound 1 [6-(2,4-dimethoxyphenyl)-1-(2-hydroxyethyl)-2-thioxo-2,3-dihydropyrimidin-4(1H)-one], are a novel class of selective irreversible inhibitors of human myeloperoxidase. The present account is a summary of our in vitro studies on the facile oxidative desulfurization in compound 1 to a cyclic ether metabolite M1 [5-(2,4-dimethoxyphenyl)-2,3-dihydro-7H-oxazolo[3,2-a]pyrimidin-7-one] in NADPH-supplemented rats (t1/2 [half-life = mean ± S.D.] = 8.6 ± 0.4 minutes) and dog liver microsomes (t1/2 = 11.2 ± 0.4 minutes), but not in human liver microsomes (t1/2 > 120 minutes). The in vitro metabolic instability also manifested in moderate-to-high plasma clearances of the parent compound in rats and dogs with significant concentrations of M1 detected in circulation. Mild heat deactivation of liver microsomes or coincubation with the flavin-containing monooxygenase (FMO) inhibitor imipramine significantly diminished M1 formation. In contrast, oxidative metabolism of compound 1 to M1 was not inhibited by the pan cytochrome P450 inactivator 1-aminobenzotriazole. Incubations with recombinant FMO isoforms (FMO1, FMO3, and FMO5) revealed that FMO1 principally catalyzed the conversion of compound 1 to M1. FMO1 is not expressed in adult human liver, which rationalizes the species difference in oxidative desulfurization. Oxidation by FMO1 followed Michaelis-Menten kinetics with Michaelis-Menten constant, maximum rate of oxidative desulfurization, and intrinsic clearance values of 209 μM, 20.4 nmol/min/mg protein, and 82.7 μl/min/mg protein, respectively. Addition of excess glutathione essentially eliminated the conversion of compound 1 to M1 in NADPH-supplemented rat and dog liver microsomes, which suggests that the initial FMO1-mediated S-oxygenation of compound 1 yields a sulfenic acid intermediate capable of redox cycling to the parent compound in a glutathione-dependent fashion or undergoing further oxidation to a more

Targeted sequencing identifies genetic polymorphisms of flavin-containing monooxygenase genes contributing to susceptibility of nicotine dependence in European American and African American.

PubMed

Zhang, Tian-Xiao; Saccone, Nancy L; Bierut, Laura J; Rice, John P

2017-04-01

Smoking is a leading cause of preventable death. Early studies based on samples of twins have linked the lifetime smoking practices to genetic predisposition. The flavin-containing monooxygenase (FMO) protein family consists of a group of enzymes that metabolize drugs and xenobiotics. Both FMO1 and FMO3 were potentially susceptible genes for nicotine metabolism process. In this study, we investigated the potential of FMO genes to confer risk of nicotine dependence via deep targeted sequencing in 2,820 study subjects comprising 1,583 nicotine dependents and 1,237 controls from European American and African American. Specifically, we focused on the two genomic segments including FMO1 , FMO3 , and pseudo gene FMO6P , and aimed to investigate the potential association between FMO genes and nicotine dependence. Both common and low-frequency/rare variants were analyzed using different algorithms. The potential functional significance of SNPs with association signal was investigated with relevant bioinformatics tools. We identified different clusters of significant common variants in European (with most significant SNP rs6674596, p  =   .0004, OR = 0.67, MAF_EA = 0.14, FMO1 ) and African Americans (with the most significant SNP rs6608453, p  =   .001, OR = 0.64, MAF_AA = 0.1, FMO6P ). No significant signals were identified through haplotype-based analyses. Gene network investigation indicated that both FMO1 and FMO3 have a strong relation with a variety of genes belonging to CYP gene families (with combined score greater than 0.9). Most of the significant variants identified were SNPs located within intron regions or with unknown functional significance, indicating a need for future work to understand the underlying functional significance of these signals. Our findings indicated significant association between FMO genes and nicotine dependence. Replications of our findings in other ethnic groups were needed in the future. Most of the significant variants

OnpA, an Unusual Flavin-Dependent Monooxygenase Containing a Cytochrome b5 Domain

PubMed Central

Xiao, Yi; Liu, Ting-Ting; Dai, Hui; Zhang, Jun-Jie; Liu, Hong; Tang, Huiru; Leak, David J.

2012-01-01

ortho-Nitrophenol 2-monooxygenase (EC 1.14.13.31) from Alcaligenes sp. strain NyZ215 catalyzes monooxygenation of ortho-nitrophenol to form catechol via ortho-benzoquinone. Sequence analysis of this onpA-encoded enzyme revealed that it contained a flavin-binding monooxygenase domain and a heme-binding cytochrome b5 domain. OnpA was purified to homogeneity as a His-tagged protein and was considered a monomer, as determined by gel filtration. FAD and heme were identified by high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry (HPLC-MS) as cofactors in this enzyme, and quantitative analysis indicated that 1 mol of the purified recombinant OnpA contained 0.66 mol of FAD and 0.20 mol of heme. However, the enzyme activity of OnpA was increased by 60% and 450% after addition of FAD and hemin, respectively, suggesting that the optimal stoichiometry was 1:1:1. In addition, site-directed mutagenesis experiments confirmed that two highly conserved histidines located in the cytochrome b5 domain were associated with binding of the heme, and the cytochrome b5 domain was involved in the OnpA activity. These results indicate that OnpA is an unusual FAD-dependent monooxygenase containing a fused cytochrome b5 domain that is essential for its activity. Therefore, we here demonstrate a link between cytochrome b5 and flavin-dependent monooxygenases. PMID:22267507

The Flavin-Containing Monooxygenase 3 Gene and Essential Hypertension: The Joint Effect of Polymorphism E158K and Cigarette Smoking on Disease Susceptibility

PubMed Central

Bushueva, Olga; Solodilova, Maria; Churnosov, Mikhail; Ivanov, Vladimir; Polonikov, Alexey

2014-01-01

Gene encoding flavin-containing monooxygenase 3 (FMO3), a microsomal antioxidant defense enzyme, has been suggested to contribute to essential hypertension (EH). The present study was designed to investigate whether common functional polymorphism E158K (rs2266782) of the FMO3 gene is associated with EH susceptibility in a Russian population. A total of 2 995 unrelated subjects from Kursk (1 362 EH patients and 843 healthy controls) and Belgorod (357 EH patients and 422 population controls) regions of Central Russia were recruited for this study. DNA samples from all study participants were genotyped for the FMO3 gene polymorphism through PCR followed by RFLP analysis. We found that the polymorphism E158K is associated with increased risk of essential hypertension in both discovery population from Kursk region (OR 1.36 95% CI 1.09–1.69, P = 0.01) and replication population from Belgorod region (OR 1.54 95% CI 1.07–1.89, P = 0.02) after adjustment for gender and age using logistic regression analysis. Further analysis showed that the increased hypertension risk in carriers of genotype 158KK gene occurred in cigarette smokers, whereas nonsmoker carriers of this genotype did not show the disease risk. This is the first study reporting the association of the FMO3 gene polymorphism and the risk of essential hypertension. PMID:25243081

The participation of human hepatic P450 isoforms, flavin-containing monooxygenases and aldehyde oxidase in the biotransformation of the insecticide fenthion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Leoni, Claudia; Buratti, Franca M.; Testai, Emanuela

Although fenthion (FEN) is widely used as a broad spectrum insecticide on various crops in many countries, very scant data are available on its biotransformation in humans. In this study the in vitro human hepatic FEN biotransformation was characterized, identifying the relative contributions of cytochrome P450 (CYPs) and/or flavin-containing monooxygenase (FMOs) by using single c-DNA expressed human enzymes, human liver microsomes and cytosol and CYP/FMO-specific inhibitors. Two major metabolites, FEN-sulfoxide and FEN-oxon (FOX), are formed by some CYPs although at very different levels, depending on the relative CYP hepatic content. Formation of further oxidation products and the reduction of FEN-sulfoxidemore » back to FEN by the cytosolic aldehyde oxidase enzyme were ruled out. Comparing intrinsic clearance values, FOX formation seemed to be favored and at low FEN concentrations CYP2B6 and 1A2 are mainly involved in its formation. At higher levels, a more widespread CYP involvement was evident, as in the case of FEN-sulfoxide, although a higher efficiency of CYP2C family was suggested. Hepatic FMOs were able to catalyze only sulfoxide formation, but at low FEN concentrations hepatic FEN sulfoxidation is predominantly P450-driven. Indeed, the contribution of the hepatic isoforms FMO{sub 3} and FMO{sub 5} was generally negligible, although at high FEN concentrations FMO's showed activities comparable to the active CYPs, accounting for up to 30% of total sulfoxidation. Recombinant FMO{sub 1} showed the highest efficiency with respect to CYPs and the other FMOs, but it is not expressed in the adult human liver. This suggests that FMO{sub 1}-catalysed sulfoxidation may represent the major extra-hepatic pathway of FEN biotransformation.« less

Quantification of Flavin-containing Monooxygenases 1, 3, and 5 in Human Liver Microsomes by UPLC-MRM-Based Targeted Quantitative Proteomics and Its Application to the Study of Ontogeny.

PubMed

Chen, Yao; Zane, Nicole R; Thakker, Dhiren R; Wang, Michael Zhuo

2016-07-01

Flavin-containing monooxygenases (FMOs) have a significant role in the metabolism of small molecule pharmaceuticals. Among the five human FMOs, FMO1, FMO3, and FMO5 are the most relevant to hepatic drug metabolism. Although age-dependent hepatic protein expression, based on immunoquantification, has been reported previously for FMO1 and FMO3, there is very little information on hepatic FMO5 protein expression. To overcome the limitations of immunoquantification, an ultra-performance liquid chromatography (UPLC)-multiple reaction monitoring (MRM)-based targeted quantitative proteomic method was developed and optimized for the quantification of FMO1, FMO3, and FMO5 in human liver microsomes (HLM). A post-in silico product ion screening process was incorporated to verify LC-MRM detection of potential signature peptides before their synthesis. The developed method was validated by correlating marker substrate activity and protein expression in a panel of adult individual donor HLM (age 39-67 years). The mean (range) protein expression of FMO3 and FMO5 was 46 (26-65) pmol/mg HLM protein and 27 (11.5-49) pmol/mg HLM protein, respectively. To demonstrate quantification of FMO1, a panel of fetal individual donor HLM (gestational age 14-20 weeks) was analyzed. The mean (range) FMO1 protein expression was 7.0 (4.9-9.7) pmol/mg HLM protein. Furthermore, the ontogenetic protein expression of FMO5 was evaluated in fetal, pediatric, and adult HLM. The quantification of FMO proteins also was compared using two different calibration standards, recombinant proteins versus synthetic signature peptides, to assess the ratio between holoprotein versus total protein. In conclusion, a UPLC-MRM-based targeted quantitative proteomic method has been developed for the quantification of FMO enzymes in HLM. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

Quantification of Flavin-containing Monooxygenases 1, 3, and 5 in Human Liver Microsomes by UPLC-MRM-Based Targeted Quantitative Proteomics and Its Application to the Study of Ontogeny

PubMed Central

Chen, Yao; Zane, Nicole R.; Thakker, Dhiren R.

2016-01-01

Flavin-containing monooxygenases (FMOs) have a significant role in the metabolism of small molecule pharmaceuticals. Among the five human FMOs, FMO1, FMO3, and FMO5 are the most relevant to hepatic drug metabolism. Although age-dependent hepatic protein expression, based on immunoquantification, has been reported previously for FMO1 and FMO3, there is very little information on hepatic FMO5 protein expression. To overcome the limitations of immunoquantification, an ultra-performance liquid chromatography (UPLC)-multiple reaction monitoring (MRM)-based targeted quantitative proteomic method was developed and optimized for the quantification of FMO1, FMO3, and FMO5 in human liver microsomes (HLM). A post-in silico product ion screening process was incorporated to verify LC-MRM detection of potential signature peptides before their synthesis. The developed method was validated by correlating marker substrate activity and protein expression in a panel of adult individual donor HLM (age 39–67 years). The mean (range) protein expression of FMO3 and FMO5 was 46 (26–65) pmol/mg HLM protein and 27 (11.5–49) pmol/mg HLM protein, respectively. To demonstrate quantification of FMO1, a panel of fetal individual donor HLM (gestational age 14–20 weeks) was analyzed. The mean (range) FMO1 protein expression was 7.0 (4.9–9.7) pmol/mg HLM protein. Furthermore, the ontogenetic protein expression of FMO5 was evaluated in fetal, pediatric, and adult HLM. The quantification of FMO proteins also was compared using two different calibration standards, recombinant proteins versus synthetic signature peptides, to assess the ratio between holoprotein versus total protein. In conclusion, a UPLC-MRM-based targeted quantitative proteomic method has been developed for the quantification of FMO enzymes in HLM. PMID:26839369

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

Predicting the Metabolic Sites by Flavin-Containing Monooxygenase on Drug Molecules Using SVM Classification on Computed Quantum Mechanics and Circular Fingerprints Molecular Descriptors

PubMed Central

Fu, Chien-wei; Lin, Thy-Hou

2017-01-01

As an important enzyme in Phase I drug metabolism, the flavin-containing monooxygenase (FMO) also metabolizes some xenobiotics with soft nucleophiles. The site of metabolism (SOM) on a molecule is the site where the metabolic reaction is exerted by an enzyme. Accurate prediction of SOMs on drug molecules will assist the search for drug leads during the optimization process. Here, some quantum mechanics features such as the condensed Fukui function and attributes from circular fingerprints (called Molprint2D) are computed and classified using the support vector machine (SVM) for predicting some potential SOMs on a series of drugs that can be metabolized by FMO enzymes. The condensed Fukui function fA− representing the nucleophilicity of central atom A and the attributes from circular fingerprints accounting the influence of neighbors on the central atom. The total number of FMO substrates and non-substrates collected in the study is 85 and they are equally divided into the training and test sets with each carrying roughly the same number of potential SOMs. However, only N-oxidation and S-oxidation features were considered in the prediction since the available C-oxidation data was scarce. In the training process, the LibSVM package of WEKA package and the option of 10-fold cross validation are employed. The prediction performance on the test set evaluated by accuracy, Matthews correlation coefficient and area under ROC curve computed are 0.829, 0.659, and 0.877 respectively. This work reveals that the SVM model built can accurately predict the potential SOMs for drug molecules that are metabolizable by the FMO enzymes. PMID:28072829

Human plasma metabolic profiles of benzydamine, a flavin-containing monooxygenase probe substrate, simulated with pharmacokinetic data from control and humanized-liver mice.

PubMed

Yamazaki-Nishioka, Miho; Shimizu, Makiko; Suemizu, Hiroshi; Nishiwaki, Megumi; Mitsui, Marina; Yamazaki, Hiroshi

2018-02-01

1. Benzydamine is used clinically as a nonsteroidal anti-inflammatory drug in oral rinses and is employed in preclinical research as a flavin-containing monooxygenase (FMO) probe substrate. In this study, plasma concentrations of benzydamine and its primary N-oxide and N-demethylated metabolites were investigated in control TK-NOG mice, in humanized-liver mice, and in mice whose liver cells had been ablated with ganciclovir. 2. Following oral administration of benzydamine (10 mg/kg) in humanized-liver TK-NOG mice, plasma concentrations of benzydamine N-oxide were slightly higher than those of demethyl benzydamine. In contrast, in control and ganciclovir-treated TK-NOG mice, concentrations of demethyl benzydamine were slightly higher than those of benzydamine N-oxide. 3. Simulations of human plasma concentrations of benzydamine and its N-oxide were achieved using simplified physiologically based pharmacokinetic models based on data from control TK-NOG mice and from reported benzydamine concentrations after low-dose administration in humans. Estimated clearance rates based on data from humanized-liver and ganciclovir-treated TK-NOG mice were two orders magnitude high. 4. The pharmacokinetic profiles of benzydamine were different for control and humanized-liver TK-NOG mice. Humanized-liver mice are generally accepted human models; however, drug oxidation in mouse kidney might need to be considered when probe substrates undergo FMO-dependent drug oxidation in mouse liver and kidney.

Formation of a quinoneimine intermediate of 4-fluoro-N-methylaniline by FMO1: carbon oxidation plus defluorination.

PubMed

Driscoll, James P; Aliagas, Ignacio; Harris, Jennifer J; Halladay, Jason S; Khatib-Shahidi, Sheerin; Deese, Alan; Segraves, Nathaniel; Khojasteh-Bakht, S Cyrus

2010-05-17

Here, we report on the mechanism by which flavin-containing monooxygenase 1 (FMO1) mediates the formation of a reactive intermediate of 4-fluoro-N-methylaniline. FMO1 catalyzed a carbon oxidation reaction coupled with defluorination that led to the formation of 4-N-methylaminophenol, which was a reaction first reported by Boersma et al. (Boersma et al. (1993) Drug Metab. Dispos. 21 , 218 - 230). We propose that a labile 1-fluoro-4-(methylimino)cyclohexa-2,5-dienol intermediate was formed leading to an electrophilic quinoneimine intermediate. The identification of N-acetylcysteine adducts by LC-MS/MS and NMR further supports the formation of a quinoneimine intermediate. Incubations containing stable labeled oxygen (H(2)(18)O or (18)O(2)) and ab initio calculations were performed to support the proposed reaction mechanism.

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

PubMed Central

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

2014-01-01

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

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

PubMed

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

2012-06-01

N-Hydroxylating monooxygenases (NMOs) are essential for pathogenesis in fungi and bacteria. NMOs catalyze the hydroxylation of sine and ornithine in the biosynthesis of hydroxamate-containing siderophores. Inhibition of kynurenine monooxygenase (KMO), which catalyzes the conversion of kynurenine to 3-hydroxykynurenine, alleviates neurodegenerative disorders such as Huntington's and Alzheimer's diseases and brain infections caused by the parasite Trypanosoma brucei. These enzymes are examples of flavin-dependent monooxygenases, which are validated drug targets. Here, we describe the development and optimization of a fluorescence polarization assay to identify potential inhibitors of flavin-dependent monooxygenases. Fluorescently labeled ADP molecules were synthesized and tested. An ADP-TAMRA chromophore bound to KMO with a K(d) value of 0.60 ± 0.05 μM and to the NMOs from Aspergillus fumigatus and Mycobacterium smegmatis with K(d) values of 2.1 ± 0.2 and 4.0 ± 0.2 μM, respectively. The assay was tested in competitive binding experiments with substrates and products of KMO and an NMO. Furthermore, we show that this assay can be used to identify inhibitors of NMOs. A Z' factor of 0.77 was calculated, and we show that the assay exhibits good tolerance to temperature, incubation time, and dimethyl sulfoxide concentration. Copyright © 2012 Elsevier Inc. All rights reserved.

How pH Modulates the Reactivity and Selectivity of a Siderophore-Associated Flavin Monooxygenase

PubMed Central

2015-01-01

Flavin-containing monooxygenases (FMOs) catalyze the oxygenation of diverse organic molecules using O2, NADPH, and the flavin adenine dinucleotide (FAD) cofactor. The fungal FMO SidA initiates peptidic siderophore biosynthesis via the highly selective hydroxylation of l-ornithine, while the related amino acid l-lysine is a potent effector of reaction uncoupling to generate H2O2. We hypothesized that protonation states could critically influence both substrate-selective hydroxylation and H2O2 release, and therefore undertook a study of SidA’s pH-dependent reaction kinetics. Consistent with other FMOs that stabilize a C4a-OO(H) intermediate, SidA’s reductive half reaction is pH independent. The rate constant for the formation of the reactive C4a-OO(H) intermediate from reduced SidA and O2 is likewise independent of pH. However, the rate constants for C4a-OO(H) reactions, either to eliminate H2O2 or to hydroxylate l-Orn, were strongly pH-dependent and influenced by the nature of the bound amino acid. Solvent kinetic isotope effects of 6.6 ± 0.3 and 1.9 ± 0.2 were measured for the C4a-OOH/H2O2 conversion in the presence and absence of l-Lys, respectively. A model is proposed in which l-Lys accelerates H2O2 release via an acid–base mechanism and where side-chain position determines whether H2O2 or the hydroxylation product is observed. PMID:24490904

Development of a physiologically based pharmacokinetic model to predict the effects of flavin-containing monooxygenase 3 (FMO3) polymorphisms on itopride exposure.

PubMed

Zhou, Wangda; Humphries, Helen; Neuhoff, Sibylle; Gardner, Iain; Masson, Eric; Al-Huniti, Nidal; Zhou, Diansong

2017-09-01

Itopride, a substrate of FMO3, has been used for the symptomatic treatment of various gastrointestinal disorders. Physiologically based pharmacokinetic (PBPK) modeling was applied to evaluate the impact of FMO3 polymorphism on itopride pharmacokinetics (PK). The Asian populations within the Simcyp simulator were updated to incorporate information on the frequency, activity and abundance of FMO3 enzyme with different phenotypes. A meta-analysis of relative enzyme activities suggested that FMO3 activity in subjects with homozygous Glu158Lys and Glu308Gly mutations (Lys158 and Gly308) in both alleles is ~47% lower than those carrying two wild-type FMO3 alleles. Individuals with homozygous Lys158 and Gly308 mutations account for about 5% of the total population in Asian populations. A CL int of 9 μl/min/pmol was optimised for itopride via a retrograde approach as human liver microsomal results would under-predict its clearance by ~7.9-fold. The developed itopride PBPK model was first verified with three additional clinical studies in Korean and Japanese subjects resulting in a predicted clearance of 52 to 69 l/h, which was comparable to those observed (55 to 88 l/h). The model was then applied to predict plasma concentration-time profiles of itopride in Chinese subjects with wild type or homozygous Lys158 and Gly308 FMO3 genotypes. The ratios of predicted to observed AUC of itopride in subjects with each genotype were 1.23 and 0.94, respectively. In addition, the results also suggested that for FMO3 metabolised drugs with a safety margin of 2 or more, proactive genotyping FMO3 to exclude subjects with homozygous Lys158/Gly308 alleles may not be necessary. Copyright © 2017 John Wiley & Sons, Ltd.

Benzydamine N-oxidation as an index reaction reflecting FMO activity in human liver microsomes and impact of FMO3 polymorphisms on enzyme activity

PubMed Central

Störmer, Elke; Roots, Ivar; Brockmöller, Jürgen

2000-01-01

Aims The role of flavin containing monooxygenases (FMO) on the disposition of many drugs has been insufficiently explored. In vitro and in vivo tests are required to study FMO activity in humans. Benzydamine (BZD) N-oxidation was evaluated as an index reaction for FMO as was the impact of genetic polymorphisms of FMO3 on activity. Methods BZD was incubated with human liver microsomes (HLM) and recombinant enzymes. Human liver samples were genotyped using PCR-RFLP. Results BZD N-oxide formation rates in HLM followed Michaelis-Menten kinetics (mean Km = 64.0 μm, mean Vmax = 6.9 nmol mg−1 protein min−1; n = 35). N-benzylimidazole, a nonspecific CYP inhibitor, and various CYP isoform selective inhibitors did not affect BZD N-oxidation. In contrast, formation of BZD N-oxide was almost abolished by heat treatment of microsomes in the absence of NADPH and strongly inhibited by methimazole, a competitive FMO inhibitor. Recombinant FMO3 and FMO1 (which is not expressed in human liver), but not FMO5, showed BZD N-oxidase activity. Respective Km values for FMO3 and FMO1 were 40.4 μm and 23.6 μm, and respective Vmax values for FMO3 and FMO1 were 29.1 and 40.8 nmol mg−1 protein min−1. Human liver samples (n = 35) were analysed for six known FMO3 polymorphisms. The variants I66M, P135L and E305X were not detected. Samples homozygous for the K158 variant showed significantly reduced vmax values (median 2.7 nmol mg−1 protein min−1) compared to the carriers of at least one wild type allele (median 6.2 nmol mg−1 protein min−1) (P<0.05, Mann–Whitney- U-test). The V257M and E308G substitutions had no effect on enzyme activity. Conclusions BZD N-oxidation in human liver is mainly catalysed by FMO3 and enzyme activity is affected by FMO3 genotype. BZD may be used as a model substrate for human liver FMO3 activity in vitro and may be further developed as an in vivo probe reflecting FMO3 activity. PMID:11136294

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

Salicylic Acid–Independent ENHANCED DISEASE SUSCEPTIBILITY1 Signaling in Arabidopsis Immunity and Cell Death Is Regulated by the Monooxygenase FMO1 and the Nudix Hydrolase NUDT7[W

PubMed Central

Bartsch, Michael; Gobbato, Enrico; Bednarek, Pawel; Debey, Svenja; Schultze, Joachim L.; Bautor, Jaqueline; Parker, Jane E.

2006-01-01

Arabidopsis thaliana ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) controls defense activation and programmed cell death conditioned by intracellular Toll-related immune receptors that recognize specific pathogen effectors. EDS1 is also needed for basal resistance to invasive pathogens by restricting the progression of disease. In both responses, EDS1, assisted by its interacting partner, PHYTOALEXIN-DEFICIENT4 (PAD4), regulates accumulation of the phenolic defense molecule salicylic acid (SA) and other as yet unidentified signal intermediates. An Arabidopsis whole genome microarray experiment was designed to identify genes whose expression depends on EDS1 and PAD4, irrespective of local SA accumulation, and potential candidates of an SA-independent branch of EDS1 defense were found. We define two new immune regulators through analysis of corresponding Arabidopsis loss-of-function insertion mutants. FLAVIN-DEPENDENT MONOOXYGENASE1 (FMO1) positively regulates the EDS1 pathway, and one member (NUDT7) of a family of cytosolic Nudix hydrolases exerts negative control of EDS1 signaling. Analysis of fmo1 and nudt7 mutants alone or in combination with sid2-1, a mutation that severely depletes pathogen-induced SA production, points to SA-independent functions of FMO1 and NUDT7 in EDS1-conditioned disease resistance and cell death. We find instead that SA antagonizes initiation of cell death and stunting of growth in nudt7 mutants. PMID:16531493

Diagnosis and management of trimethylaminuria (FMO3 deficiency) in children.

PubMed

Chalmers, R A; Bain, M D; Michelakakis, H; Zschocke, J; Iles, R A

2006-02-01

Persistent trimethylaminuria in children is caused by autosomal recessively inherited impairment of hepatic trimethylamine (TMA) oxidation due to deficiency of flavin monooxygenase 3 (FMO3) secondary to mutations in the FMO3 gene. Trimethylaminuria or 'fish odour syndrome' is due to excessive excretion into body fluids and breath of TMA derived from the enterobacterial metabolism of dietary precursors. The disorder is present from birth but becomes apparent as foods containing high amounts of choline or of trimethylamine N-oxide (TMAO) from marine (sea or saltwater) fish are introduced into the diet. In our experience, trimethylaminuria (FMO3 deficiency) in children is rare. We have compared the dynamics and diagnostic efficacy of choline loading with marine fish meals in six children with trimethylaminuria. Loading with a marine fish meal provides a simple and acceptable method for confirmation of diagnosis of suspected trimethylaminuria in children, with the effects being cleared more quickly than with a choline load test. However, oral loading with choline bitartrate allows estimation of residual oxidative capacity in vivo and is a useful adjunct to molecular studies. Patients homozygous for the 'common' P153L mutation in the FMO3 gene showed virtual complete lack of residual TMA N-oxidative capacity, consistent with a nonfunctional or absent FMO3 enzyme, whereas a patient with the M82T mutation showed some residual oxidative capacity. A patient compound heterozygous for two novel mutations, G193E and R483T, showed considerable residual N-oxidative capacity. A further patient, heterozygous for two novel sequence variations in the FMO3 gene, consistently showed malodour and elevated urinary TMA/TMAO ratios under basal conditions but a negative response to both choline and marine fish meal loading. Comparison of the effects of administration of antibiotics (metronidazole, amoxicillin, neomycin) on gut bacterial production of trimethylamine from choline showed they

Identification of human cytochrome P450 and flavin-containing monooxygenase enzymes involved in the metabolism of lorcaserin, a novel selective human 5-hydroxytryptamine 2C agonist.

PubMed

Usmani, Khawja A; Chen, Weichao G; Sadeque, Abu J M

2012-04-01

Lorcaserin, a selective serotonin 5-hydroxytryptamine 2C receptor agonist, is being developed for weight management. The oxidative metabolism of lorcaserin, mediated by recombinant human cytochrome P450 (P450) and flavin-containing monooxygenase (FMO) enzymes, was examined in vitro to identify the enzymes involved in the generation of its primary oxidative metabolites, N-hydroxylorcaserin, 7-hydroxylorcaserin, 5-hydroxylorcaserin, and 1-hydroxylorcaserin. Human CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, CYP3A4, and FMO1 are major enzymes involved in N-hydroxylorcaserin; CYP2D6 and CYP3A4 are enzymes involved in 7-hydroxylorcaserin; CYP1A1, CYP1A2, CYP2D6, and CYP3A4 are enzymes involved in 5-hydroxylorcaserin; and CYP3A4 is an enzyme involved in 1-hydroxylorcaserin formation. In 16 individual human liver microsomal preparations (HLM), formation of N-hydroxylorcaserin was correlated with CYP2B6, 7-hydroxylorcaserin was correlated with CYP2D6, 5-hydroxylorcaserin was correlated with CYP1A2 and CYP3A4, and 1-hydroxylorcaserin was correlated with CYP3A4 activity at 10.0 μM lorcaserin. No correlation was observed for N-hydroxylorcaserin with any P450 marker substrate activity at 1.0 μM lorcaserin. N-Hydroxylorcaserin formation was not inhibited by CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, and CYP3A4 inhibitors at the highest concentration tested. Furafylline, quinidine, and ketoconazole, selective inhibitors of CYP1A2, CYP2D6, and CYP3A4, respectively, inhibited 5-hydroxylorcaserin (IC(50) = 1.914 μM), 7-hydroxylorcaserin (IC(50) = 0.213 μM), and 1-hydroxylorcaserin formation (IC(50) = 0.281 μM), respectively. N-Hydroxylorcaserin showed low and high K(m) components in HLM and 7-hydroxylorcaserin showed lower K(m) than 5-hydroxylorcaserin and 1-hydroxylorcaserin in HLM. The highest intrinsic clearance was observed for N-hydroxylorcaserin, followed by 7-hydroxylorcaserin, 5-hydroxylorcaserin, and 1-hydroxylorcaserin in HLM. Multiple human P450 and FMO enzymes catalyze

Effect of two-linked mutations of the FMO3 gene on itopride metabolism in Chinese healthy volunteers.

PubMed

Zhou, Li-Ping; Tan, Zhi-Rong; Chen, Hao; Guo, Dong; Chen, Yao; Huang, Wei-Hua; Wang, Lian-Sheng; Zhang, Guo-Gang

2014-11-01

Itopride is an effective gastroprokinetic agent mainly used for the treatment of functional dyspepsia. Flavin-containing monooxygenase 3 (FMO3) has been confirmed to be the key enzyme involved in the main itopride metabolic pathway. We investigated whether the FMO3 genotypes can affect itopride metabolism in Chinese healthy volunteers. Twelve healthy volunteers who had been genotyped for FMO3 gene were selected to participate in our study. Volunteers were given 50 mg itopride orally and then blood samples were collected from 0 to 24 h. The plasma concentrations of itopride and itopride N-oxide were determined by HPLC-MS/MS method. Itopride and itopride N-oxide both exhibit FMO3 genotype-dependent pharmacokinetic profiles. The area under the plasma concentration-time curve (AUC) of itopride increased by 127.82 ± 41.99 % (P < 0.001) and the AUC of itopride N-oxide decreased by 30.30 ± 25.70 % (P < 0.05) in homozygous FMO3 hhdd subjects (n = 6) compared with the HHDD group (n = 6). The CL/F value was lower in the hhdd group than that in the HHDD group (36.60 ± 7.06 vs. 80.20 ± 15.34 L/h, P < 0.001). But no significant differences in t1/2 value and tmax of itopride and itopride N-oxide were observed between these two genotypes. The FMO3 allele can significantly affect the metabolism of itopride. The pharmacokinetic parameters of both itopride and itopride N-oxide were significantly different between these two genotypes.

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

Kinetic Mechanism of the Dechlorinating Flavin-dependent Monooxygenase HadA*

PubMed Central

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

2017-01-01

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. PMID:28159841

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.

Arg279 is the key regulator of coenzyme selectivity in the flavin-dependent ornithine monooxygenase SidA.

PubMed

Robinson, Reeder; Franceschini, Stefano; Fedkenheuer, Michael; Rodriguez, Pedro J; Ellerbrock, Jacob; Romero, Elvira; Echandi, Maria Paulina; Martin Del Campo, Julia S; Sobrado, Pablo

2014-04-01

Siderophore A (SidA) is a flavin-dependent monooxygenase that catalyzes the NAD(P)H- and oxygen-dependent hydroxylation of ornithine in the biosynthesis of siderophores in Aspergillus fumigatus and is essential for virulence. SidA can utilize both NADPH or NADH for activity; however, the enzyme is selective for NADPH. Structural analysis shows that R279 interacts with the 2'-phosphate of NADPH. To probe the role of electrostatic interactions in coenzyme selectivity, R279 was mutated to both an alanine and a glutamate. The mutant proteins were active but highly uncoupled, oxidizing NADPH and producing hydrogen peroxide instead of hydroxylated ornithine. For wtSidA, the catalytic efficiency was 6-fold higher with NADPH as compared to NADH. For the R279A mutant the catalytic efficiency was the same with both coenyzmes, while for the R279E mutant the catalytic efficiency was 5-fold higher with NADH. The effects are mainly due to an increase in the KD values, as no major changes on the kcat or flavin reduction values were observed. Thus, the absence of a positive charge leads to no coenzyme selectivity while introduction of a negative charge leads to preference for NADH. Flavin fluorescence studies suggest altered interaction between the flavin and NADP⁺ in the mutant enzymes. The effects are caused by different binding modes of the coenzyme upon removal of the positive charge at position 279, as no major conformational changes were observed in the structure for R279A. The results indicate that the positive charge at position 279 is critical for tight binding of NADPH and efficient hydroxylation. Copyright © 2014 Elsevier B.V. All rights reserved.

Characterization of a flavin reductase from a thermophilic dibenzothiophene-desulfurizing bacterium, Bacillus subtilis WU-S2B.

PubMed

Takahashi, Shusuke; Furuya, Toshiki; Ishii, Yoshitaka; Kino, Kuniki; Kirimura, Kohtaro

2009-01-01

Bacillus subtilis WU-S2B is a thermophilic dibenzothiophene (DBT)-desulfurizing bacterium and produces a flavin reductase (Frb) that couples with DBT and DBT sulfone monooxygenases. The recombinant Frb was purified from Escherichia coli cells expressing the frb gene and was characterized. The purified Frb exhibited high stability over wide temperature and pH ranges of 20-55 degrees C and 2-12, respectively. Frb contained FMN and exhibited both flavin reductase and nitroreductase activities.

Kynurenine 3-monooxygenase from Pseudomonas fluorescens: substrate-like inhibitors both stimulate flavin reduction and stabilize the flavin-peroxo intermediate yet result in the production of hydrogen peroxide.

PubMed

Crozier-Reabe, Karen R; Phillips, Robert S; Moran, Graham R

2008-11-25

Kynurenine 3-monooxygenase (KMO) is a flavin-dependent hydroxylase that catalyzes the conversion of l-kynurenine (l-Kyn) to 3-hydroxykynurenine (3OHKyn) in the pathway for tryptophan catabolism. KMO inhibition has been widely suggested as an early treatment for stroke and other neurological disorders that involve ischemia. We have investigated the reductive and the oxidative half-reactions of a stable form of KMO from Pseudomonas fluorescens (KMO). The binding of l-Kyn by the enzyme is relatively slow and involves at least two reversible steps. The rate constant for reduction of the flavin cofactor by NADPH increases by a factor of approximately 2.5 x 10(3) when l-Kyn is bound. The rate of reduction of the KMO.l-Kyn complex is 160 s(-1), and the K(d) for the NADPH complex is 200 microM with charge-transfer absorption bands for the KMO(RED).l-Kyn.NADP(+) complex accumulating after reduction. The reduction potential of KMO is -188 mV and is unresponsive to the addition of l-Kyn or other inhibitory ligands. KMO inhibitors whose structures are reminiscent of l-Kyn such as m-nitrobenzoylalanine and benzoylalanine also stimulate reduction of flavin by NADPH and, in the presence of dioxygen, result in the stoichiometric liberation of hydrogen peroxide, diminishing the perceived therapeutic potential of inhibitors of this type. In the presence of the native substrate, the oxidative half-reaction exhibits triphasic absorbance data. A spectrum consistent with that of a peroxyflavin species accumulates and then decays to yield the oxidized enzyme. This species then undergoes minor spectral changes that, based on flavin difference spectra defined in the presence of 3OHKyn, can be correlated with product release. The oxidative half-reaction observed in the presence of saturating benzoylalanine or m-nitrobenzoylalanine also shows the accumulation of a peroxyflavin species that then decays to yield hydrogen peroxide without hydroxylation.

The involvement of flavin-containing monooxygenase but not CYP3A4 in metabolism of itopride hydrochloride, a gastroprokinetic agent: comparison with cisapride and mosapride citrate.

PubMed

Mushiroda, T; Douya, R; Takahara, E; Nagata, O

2000-10-01

The goals of the present study were to identify the enzyme responsible for metabolism of itopride hydrochloride (itopride) and to evaluate the likelihood of drug interaction involving itopride. In human liver microsomes, the involvement of flavin-containing monooxygenase in N-oxygenation, the major metabolic pathway of itopride, was indicated by the following results: inhibition by methimazole and thiourea, heat inactivation, and protection against heat inactivation by NADPH. When the effects of ketoconazole on the metabolism of itopride, cisapride, and mosapride citrate (mosapride) were examined using human liver microsomes, ketoconazole strongly inhibited the formation of the primary metabolites of cisapride and mosapride, but not itopride. Other cytochrome P450 (CYP) 3A4 inhibitors, cimetidine, erythromycin, and clarithromycin, also inhibited the metabolism of cisapride and mosapride. In an in vivo study, itopride (30 mg/kg), cisapride (1.5 mg/kg), or mosapride (3 mg/kg) was orally administered to male rats with or without oral pretreatment with ketoconazole (120 mg/kg) twice daily for 2 days. The ketoconazole pretreatment significantly increased the area under the serum concentration curve and the maximum serum concentration of cisapride and mosapride but had no significant effect on the pharmacokinetics of itopride. In addition, itopride did not inhibit five specific CYP-mediated reactions of human liver microsomes. These results suggest that itopride is unlikely to alter the pharmacokinetics of other concomitantly administered drugs.

A Novel Aerobic Degradation Pathway for Thiobencarb Is Initiated by the TmoAB Two-Component Flavin Mononucleotide-Dependent Monooxygenase System in Acidovorax sp. Strain T1

PubMed Central

Chu, Cui-Wei; Liu, Bin; Li, Na; Yao, Shi-Gang; Cheng, Dan; Zhao, Jia-Dong; Qiu, Ji-Guo; Yan, Xin; He, Jian

2017-01-01

ABSTRACT Thiobencarb is a thiocarbamate herbicide used in rice paddies worldwide. Microbial degradation plays a crucial role in the dissipation of thiobencarb in the environment. However, the physiological and genetic mechanisms underlying thiobencarb degradation remain unknown. In this study, a novel thiobencarb degradation pathway was proposed in Acidovorax sp. strain T1. Thiobencarb was oxidized and cleaved at the C—S bond, generating diethylcarbamothioic S-acid and 4-chlorobenzaldehyde (4CDA). 4CDA was then oxidized to 4-chlorobenzoic acid (4CBA) and hydrolytically dechlorinated to 4-hydroxybenzoic acid (4HBA). The identification of catabolic genes suggested further hydroxylation to protocatechuic acid (PCA) and finally degradation through the protocatechuate 4,5-dioxygenase pathway. A novel two-component monooxygenase system identified in the strain, TmoAB, was responsible for the initial catabolic reaction. TmoA shared 28 to 32% identity with the oxygenase components of pyrimidine monooxygenase from Agrobacterium fabrum, alkanesulfonate monooxygenase from Pseudomonas savastanoi, and dibenzothiophene monooxygenase from Rhodococcus sp. TmoB shared 25 to 37% identity with reported flavin reductases and oxidized NADH but not NADPH. TmoAB is a flavin mononucleotide (FMN)-dependent monooxygenase and catalyzed the C—S bond cleavage of thiobencarb. Introduction of tmoAB into cells of the thiobencarb degradation-deficient mutant T1m restored its ability to degrade and utilize thiobencarb. A dehydrogenase gene, tmoC, was located 7,129 bp downstream of tmoAB, and its transcription was clearly induced by thiobencarb. The purified TmoC catalyzed the dehydrogenation of 4CDA to 4CBA using NAD+ as a cofactor. A gene cluster responsible for the complete 4CBA metabolic pathway was also cloned, and its involvement in thiobencarb degradation was preliminarily verified by transcriptional analysis. IMPORTANCE Microbial degradation is the main factor in thiobencarb dissipation

A Novel Aerobic Degradation Pathway for Thiobencarb Is Initiated by the TmoAB Two-Component Flavin Mononucleotide-Dependent Monooxygenase System in Acidovorax sp. Strain T1.

PubMed

Chu, Cui-Wei; Liu, Bin; Li, Na; Yao, Shi-Gang; Cheng, Dan; Zhao, Jia-Dong; Qiu, Ji-Guo; Yan, Xin; He, Qin; He, Jian

2017-12-01

Thiobencarb is a thiocarbamate herbicide used in rice paddies worldwide. Microbial degradation plays a crucial role in the dissipation of thiobencarb in the environment. However, the physiological and genetic mechanisms underlying thiobencarb degradation remain unknown. In this study, a novel thiobencarb degradation pathway was proposed in Acidovorax sp. strain T1. Thiobencarb was oxidized and cleaved at the C-S bond, generating diethylcarbamothioic S -acid and 4-chlorobenzaldehyde (4CDA). 4CDA was then oxidized to 4-chlorobenzoic acid (4CBA) and hydrolytically dechlorinated to 4-hydroxybenzoic acid (4HBA). The identification of catabolic genes suggested further hydroxylation to protocatechuic acid (PCA) and finally degradation through the protocatechuate 4,5-dioxygenase pathway. A novel two-component monooxygenase system identified in the strain, TmoAB, was responsible for the initial catabolic reaction. TmoA shared 28 to 32% identity with the oxygenase components of pyrimidine monooxygenase from Agrobacterium fabrum , alkanesulfonate monooxygenase from Pseudomonas savastanoi , and dibenzothiophene monooxygenase from Rhodococcus sp. TmoB shared 25 to 37% identity with reported flavin reductases and oxidized NADH but not NADPH. TmoAB is a flavin mononucleotide (FMN)-dependent monooxygenase and catalyzed the C-S bond cleavage of thiobencarb. Introduction of tmoAB into cells of the thiobencarb degradation-deficient mutant T1m restored its ability to degrade and utilize thiobencarb. A dehydrogenase gene, tmoC , was located 7,129 bp downstream of tmoAB , and its transcription was clearly induced by thiobencarb. The purified TmoC catalyzed the dehydrogenation of 4CDA to 4CBA using NAD + as a cofactor. A gene cluster responsible for the complete 4CBA metabolic pathway was also cloned, and its involvement in thiobencarb degradation was preliminarily verified by transcriptional analysis. IMPORTANCE Microbial degradation is the main factor in thiobencarb dissipation in soil

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

Transient trimethylaminuria related to menstruation

PubMed Central

Shimizu, Makiko; Cashman, John R; Yamazaki, Hiroshi

2007-01-01

Background Trimethylaminuria, or fish odor syndrome, includes a transient or mild malodor caused by an excessive amount of malodorous trimethylamine as a result of body secretions. Herein, we describe data to support the proposal that menses can be an additional factor causing transient trimethylaminuria in self-reported subjects suffering from malodor and even in healthy women harboring functionally active flavin-containing monooxygenase 3 (FMO3). Methods FMO3 metabolic capacity (conversion of trimethylamine to trimethylamine N-oxide) was defined as the urinary ratio of trimethylamine N-oxide to total trimethylamine. Results Self-reported Case (A) that was homozygous for inactive Arg500stop FMO3, showed decreased metabolic capacity of FMO3 (i.e., ~10% the unaffected metabolic capacity) during 120 days of observation. For Case (B) that was homozygous for common [Glu158Lys; Glu308Gly] FMO3 polymorphisms, metabolic capacity of FMO3 was almost ~90%, except for a few days surrounding menstruation showing < 40% metabolic capacity. In comparison, three healthy control subjects that harbored heterozygous polymorphisms for [Glu158Lys; Glu308Gly] FMO3 or homozygous for wild FMO3 showed normal (> 90%) metabolic capacity, however, on days around menstruation the FMO3 metabolic capacity was decreased to ~60–70%. Conclusion Together, these results indicate that abnormal FMO3 capacity is caused by menstruation particularly in the presence, in homozygous form, of mild genetic variants such as [Glu158Lys; Glu308Gly] that cause a reduced FMO3 function. PMID:17257434

Camphor Pathway Redux: Functional Recombinant Expression of 2,5- and 3,6-Diketocamphane Monooxygenases of Pseudomonas putida ATCC 17453 with Their Cognate Flavin Reductase Catalyzing Baeyer-Villiger Reactions

PubMed Central

Iwaki, Hiroaki; Grosse, Stephan; Bergeron, Hélène; Leisch, Hannes; Morley, Krista; Hasegawa, Yoshie

2013-01-01

Whereas the biochemical properties of the monooxygenase components that catalyze the oxidation of 2,5-diketocamphane and 3,6-diketocamphane (2,5-DKCMO and 3,6-DKCMO, respectively) in the initial catabolic steps of (+) and (−) isomeric forms of camphor (CAM) metabolism in Pseudomonas putida ATCC 17453 are relatively well characterized, the actual identity of the flavin reductase (Fred) component that provides the reduced flavin to the oxygenases has hitherto been ill defined. In this study, a 37-kDa Fred was purified from a camphor-induced culture of P. putida ATCC 17453 and this facilitated cloning and characterization of the requisite protein. The active Fred is a homodimer with a subunit molecular weight of 18,000 that uses NADH as an electron donor (Km = 32 μM), and it catalyzes the reduction of flavin mononucleotide (FMN) (Km = 3.6 μM; kcat = 283 s−1) in preference to flavin adenine dinucleotide (FAD) (Km = 19 μM; kcat = 128 s−1). Sequence determination of ∼40 kb of the CAM degradation plasmid revealed the locations of two isofunctional 2,5-DKCMO genes (camE25–1 for 2,5-DKCMO-1 and camE25–2 for 2,5-DKCMO-2) as well as that of a 3,6-DKCMO-encoding gene (camE36). In addition, by pulsed-field gel electrophoresis, the CAM plasmid was established to be linear and ∼533 kb in length. To enable functional assessment of the two-component monooxygenase system in Baeyer-Villiger oxidations, recombinant plasmids expressing Fred in tandem with the respective 2,5-DKCMO- and 3,6-DKCMO-encoding genes in Escherichia coli were constructed. Comparative substrate profiling of the isofunctional 2,5-DCKMOs did not yield obvious differences in Baeyer-Villiger biooxidations, but they are distinct from 3,6-DKCMO in the stereoselective oxygenations with various mono- and bicyclic ketone substrates. PMID:23524667

Sequential enzymatic epoxidation involved in polyether lasalocid biosynthesis.

PubMed

Minami, Atsushi; Shimaya, Mayu; Suzuki, Gaku; Migita, Akira; Shinde, Sandip S; Sato, Kyohei; Watanabe, Kenji; Tamura, Tomohiro; Oguri, Hiroki; Oikawa, Hideaki

2012-05-02

Enantioselective epoxidation followed by regioselective epoxide opening reaction are the key processes in construction of the polyether skeleton. Recent genetic analysis of ionophore polyether biosynthetic gene clusters suggested that flavin-containing monooxygenases (FMOs) could be involved in the oxidation steps. In vivo and in vitro analyses of Lsd18, an FMO involved in the biosynthesis of polyether lasalocid, using simple olefin or truncated diene of a putative substrate as substrate mimics demonstrated that enantioselective epoxidation affords natural type mono- or bis-epoxide in a stepwise manner. These findings allow us to figure out enzymatic polyether construction in lasalocid biosynthesis. © 2012 American Chemical Society

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.

Spliceostatin hemiketal biosynthesis in Burkholderia spp. is catalyzed by an iron/α-ketoglutarate–dependent dioxygenase

PubMed Central

Eustáquio, Alessandra S.; Janso, Jeffrey E.; Ratnayake, Anokha S.; O’Donnell, Christopher J.; Koehn, Frank E.

2014-01-01

Spliceostatins are potent spliceosome inhibitors biosynthesized by a hybrid nonribosomal peptide synthetase−polyketide synthase (NRPS−PKS) system of the trans-acyl transferase (AT) type. Burkholderia sp. FERM BP-3421 produces hemiketal spliceostatins, such as FR901464, as well as analogs containing a terminal carboxylic acid. We provide genetic and biochemical evidence for hemiketal biosynthesis by oxidative decarboxylation rather than the previously hypothesized Baeyer–Villiger oxidative release postulated to be catalyzed by a flavin-dependent monooxygenase (FMO) activity internal to the last module of the PKS. Inactivation of Fe(II)/α-ketoglutarate–dependent dioxygenase gene fr9P led to loss of hemiketal congeners, whereas the mutant was still able to produce all major carboxylic acid-type compounds. FMO mutants, on the other hand, produced both hemiketal and carboxylic acid analogs containing an exocyclic methylene instead of an epoxide, indicating that the FMO is involved in epoxidation rather than Baeyer–Villiger oxidation. Moreover, recombinant Fr9P enzyme was shown to catalyze hydroxylation to form β-hydroxy acids, which upon decarboxylation led to hemiketal FR901464. Finally, a third oxygenase activity encoded in the biosynthetic gene cluster, the cytochrome P450 monooxygenase Fr9R, was assigned as a 4-hydroxylase based on gene inactivation results. Identification and deletion of the gene involved in hemiketal formation allowed us to generate a strain—the dioxygenase fr9P− mutant—that accumulates only the carboxylic acid-type spliceostatins, which are as potent as the hemiketal analogs, when derivatized to increase cell permeability, but are chemically more stable. PMID:25097259

Role of Valine 464 in the Flavin Oxidation Reaction Catalyzed by Choline Oxidase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Finnegan, Steffan; Agniswamy, Johnson; Weber, Irene T.

2010-11-03

The oxidation of reduced flavin cofactors by oxygen is a very important reaction that is central to the chemical versatility of hundreds of flavoproteins classified as monooxygenases and oxidases. These enzymes are characterized by bimolecular rate constants {ge} 10{sup 5} M{sup -1} s{sup -1} and produce water and hydrogen peroxide, respectively. A hydrophobic cavity close to the reactive flavin C(4a) atom has been previously identified in the 3D structure of monooxygenases but not in flavoprotein oxidases. In the present study, we have investigated by X-ray crystallography, mutagenesis, steady-state, and rapid reaction approaches the role of Val464, which is <6 {angstrom}more » from the flavin C(4a) atom in choline oxidase. The 3D structure of the Val464Ala enzyme was essentially identical to that of the wild-type enzyme as shown by X-ray crystallography. Time-resolved anaerobic substrate reduction of the enzymes showed that replacement of Val464 with alanine or threonine did not affect the reductive half-reaction. Steady-state and rapid kinetics as well as enzyme-monitored turnovers indicated that the oxidative half-reaction in the Ala464 and Thr464 enzymes was decreased by 50-fold with respect to the wild-type enzyme. We propose that the side chain of Val464 in choline oxidase provides a nonpolar site that is required to guide oxygen in proximity of the C(4a) atom of the flavin, where it will subsequently react via electrostatic catalysis. Visual analysis of available structures suggests that analogous nonpolar sites are likely present in most flavoprotein oxidases. Mechanistic considerations provide rationalization for the differences between sites in monooxygenases and oxidases.« less

Flavin-mediated dual oxidation controls an enzymatic Favorskii-type rearrangement

PubMed Central

Louie, Gordon; Noel, Joseph P.; Baran, Phil S.; Palfey, Bruce; Moore, Bradley S.

2013-01-01

Flavoproteins catalyze a diversity of fundamental redox reactions and are one of the most studied enzyme families1,2. As monooxygenases, they are universally thought to control oxygenation by means of a peroxyflavin species that transfers a single atom of molecular oxygen to an organic substrate1,3,4. Here we report that the bacterial flavoenzyme EncM5,6 catalyzes the peroxyflavin-independent oxygenation-dehydrogenation dual oxidation of a highly reactive poly(β-carbonyl). The crystal structure of EncM with bound substrate mimics coupled with isotope labeling studies reveal previously unknown flavin redox biochemistry. We show that EncM maintains an unanticipated stable flavin oxygenating species, proposed to be a flavin-N5-oxide, to promote substrate oxidation and trigger a rare Favorskii-type rearrangement that is central to the biosynthesis of the antibiotic enterocin. This work provides new insight into the fine-tuning of the flavin cofactor in offsetting the innate reactivity of a polyketide substrate to direct its efficient electrocyclization. PMID:24162851

Purification, Characterization, and Overexpression of Flavin Reductase Involved in Dibenzothiophene Desulfurization by Rhodococcus erythropolis D-1

PubMed Central

Matsubara, Toshiyuki; Ohshiro, Takashi; Nishina, Yoshihiro; Izumi, Yoshikazu

2001-01-01

The dibenzothiophene (DBT)-desulfurizing bacterium, Rhodococcus erythropolis D-1, removes sulfur from DBT to form 2-hydroxybiphenyl using four enzymes, DszC, DszA, DszB, and flavin reductase. In this study, we purified and characterized the flavin reductase from R. erythropolis D-1 grown in a medium containing DBT as the sole source of sulfur. It is conceivable that the enzyme is essential for two monooxygenase (DszC and DszA) reactions in vivo. The purified flavin reductase contains no chromogenic cofactors and was found to have a molecular mass of 86 kDa and four identical 22-kDa subunits. The enzyme catalyzed NADH-dependent reduction of flavin mononucleotide (FMN), and the Km values for NADH and FMN were 208 and 10.8 μM, respectively. Flavin adenine dinucleotide was a poor substrate, and NADPH was inert. The enzyme did not catalyze reduction of any nitroaromatic compound. The optimal temperature and optimal pH for enzyme activity were 35°C and 6.0, respectively, and the enzyme retained 30% of its activity after heat treatment at 80°C for 30 min. The N-terminal amino acid sequence of the purified flavin reductase was identical to that of DszD of R. erythropolis IGTS8 (K. A. Gray, O. S. Pogrebinsky, G. T. Mrachko, L. Xi, D. J. Monticello, and C. H. Squires, Nat. Biotechnol. 14:1705–1709, 1996). The flavin reductase gene was amplified with primers designed by using dszD of R. erythropolis IGTS8, and the enzyme was overexpressed in Escherichia coli. The specific activity in crude extracts of the overexpressed strain was about 275-fold that of the wild-type strain. PMID:11229908

Role of Ser-257 in the sliding mechanism of NADP(H) in the reaction catalyzed by the Aspergillus fumigatus flavin-dependent ornithine N5-monooxygenase SidA.

PubMed

Shirey, Carolyn; Badieyan, Somayesadat; Sobrado, Pablo

2013-11-08

SidA (siderophore A) is a flavin-dependent N-hydroxylating monooxygenase that is essential for virulence in Aspergillus fumigatus. SidA catalyzes the NADPH- and oxygen-dependent formation of N(5)-hydroxyornithine. In this reaction, NADPH reduces the flavin, and the resulting NADP(+) is the last product to be released. The presence of NADP(+) is essential for activity, as it is required for stabilization of the C4a-hydroperoxyflavin, which is the hydroxylating species. As part of our efforts to determine the molecular details of the role of NADP(H) in catalysis, we targeted Ser-257 for site-directed mutagenesis and performed extensive characterization of the S257A enzyme. Using a combination of steady-state and stopped-flow kinetic experiments, substrate analogs, and primary kinetic isotope effects, we show that the interaction between Ser-257 and NADP(H) is essential for stabilization of the C4a-hydroperoxyflavin. Molecular dynamics simulation results suggest that Ser-257 functions as a pivot point, allowing the nicotinamide of NADP(+) to slide into position for stabilization of the C4a-hydroperoxyflavin.

Identification of human drug-metabolizing enzymes involved in the metabolism of SNI-2011.

PubMed

Washio, T; Arisawa, H; Kohsaka, K; Yasuda, H

2001-11-01

In vitro studies were conducted to identify human drug-metabolizing enzymes involved in the metabolism of SNI-2011 ((+/-)-cis-2-methylspiro [1,3-oxathiolane-5,3'-quinuclidine] monohydrochloride hemihydrate, cevimeline hydrochloride hydrate). When 14C-SNI-2011 was incubated with human liver microsomes, SNI-2011 trans-sulfoxide and cis-sulfoxide were detected as major metabolites. These oxidations required NADPH, and were markedly inhibited by SKF-525A, indicating that cytochrome P450 (CYP) was involved. In a chemical inhibition study, metabolism of SNI-2011 in liver microsomes was inhibited (35-65%) by CYP3A4 inhibitors (ketoconazole and troleandomycin) and CYP2D6 inhibitors (quinidine and chlorpromazine). Furthermore, using microsomes containing cDNA-expressed CYPs, it was found that high rates of sulfoxidation activities were observed with CYP2D6 and CYP3A4. On the other hand, when 14C-SNI-2011 was incubated with human kidney microsomes, SNI-2011 N-oxide was identified as a major metabolite. This N-oxidation required NADPH, and was completely inhibited by thiourea, indicating that flavin-containing monooxygenase (FMO) was involved. In addition, microsomes containing cDNA-expressed FMO1, a major isoform in human kidney, mainly catalyzed N-oxidation of SNI-2011, but microsomes containing FMO3, a major isoform in adult human liver, did not. These results suggest that SNI-2011 is mainly catalyzed to sulfoxides and N-oxide by CYP2D6/3A4 in liver and FMOI in kidney, respectively.

Effects of salinity on the toxicity and biotransformation of L-selenomethionine in Japanese medaka (Oryzias latipes) embryos: mechanisms of oxidative stress.

PubMed

Lavado, Ramon; Shi, Dalin; Schlenk, Daniel

2012-02-01

Previous studies in mammals have shown that organoselenium depletes the cellular antioxidant, glutathione (GSH) due to activation of organoselenides to organoselenoxides by flavin-containing monooxygenases (FMO). Since FMO tends to be induced in euryhaline fish exposed to hypersaline conditions, the developmental toxicity of salinity and organoselenium was examined in the euryhaline fish Japanese medaka (Oryzias latipes). FMO activity, GSH, and selenium concentrations in Japanese medaka embryos were measured following a 24-h exposure to 0.05 mM L-selenomethionine (SeMet) under different saline conditions: freshwater (<0.5 dS/m), 4.2, 6.7, and 16.8 dS/m. Concentrations of GSH and the hatch-out ratio of the SeMet-treated embryos decreased in a salinity dependent manner. While SeMet treatment led to accumulation within embryos, selenium concentrations were unaltered by salinity treatment. Compared to freshwater-exposed embryos, microsomes from embryos at 6.7 and 16.8 dS/m had enhanced oxidation of SeMet to the selenoxide (10- and 14.3-fold, respectively), which correlated with GSH depletion. The results show that increased SeMet oxidation by hypersaline conditions with subsequent GSH depletion may play an important role in the developmental toxicity of selenomethionine. Copyright © 2011 Elsevier B.V. All rights reserved.

Flavins contained in yeast extract are exploited for anodic electron transfer by Lactococcus lactis.

PubMed

Masuda, Masaki; Freguia, Stefano; Wang, Yung-Fu; Tsujimura, Seiya; Kano, Kenji

2010-06-01

Cyclic voltammograms of yeast extract-containing medium exhibit a clear redox peak around -0.4V vs. Ag|AgCl. Fermentative bacterium Lactococcus lactis was hereby shown to exploit this redox compound for extracellular electron transfer towards a graphite anode using glucose as an electron donor. High performance liquid chromatography revealed that this may be a flavin-type compound. The ability of L. lactis to exploit exogenous flavins for anodic glucose oxidation was confirmed by tests where flavin-type compounds were supplied to the bacterium in well defined media. Based on its mid-point potential, riboflavin can be regarded as a near-optimal mediator for microbially catalyzed anodic electron transfer. Riboflavin derivative flavin mononucleotide (FMN) was also exploited by L. lactis as a redox shuttle, unlike flavin adenine dinucleotide (FAD), possibly due to the absence of a specific transporter for the latter. The use of yeast extract in microbial fuel cell media is herein discouraged based on the related unwanted artificial addition of redox mediators which may distort experimental results. Copyright 2009 Elsevier B.V. All rights reserved.

Structural Basis for Inhibitor-Induced Hydrogen Peroxide Production by Kynurenine 3-Monooxygenase.

PubMed

Kim, Hyun Tae; Na, Byeong Kwan; Chung, Jiwoung; Kim, Sulhee; Kwon, Sool Ki; Cha, Hyunju; Son, Jonghyeon; Cho, Joong Myung; Hwang, Kwang Yeon

2018-04-19

Kynurenine 3-monooxygenase (KMO) inhibitors have been developed for the treatment of neurodegenerative disorders. The mechanisms of flavin reduction and hydrogen peroxide production by KMO inhibitors are unknown. Herein, we report the structure of human KMO and crystal structures of Saccharomyces cerevisiae (sc) and Pseudomonas fluorescens (pf) KMO with Ro 61-8048. Proton transfer in the hydrogen bond network triggers flavin reduction in p-hydroxybenzoate hydroxylase, but the mechanism triggering flavin reduction in KMO is different. Conformational changes via π-π interactions between the loop above the flavin and substrate or non-substrate effectors lead to disorder of the C-terminal α helix in scKMO and shifts of domain III in pfKMO, stimulating flavin reduction. Interestingly, Ro 61-8048 has two different binding modes. It acts as a competitive inhibitor in scKMO and as a non-substrate effector in pfKMO. These findings provide understanding of the catalytic cycle of KMO and insight for structure-based drug design of KMO inhibitors. Copyright © 2018 Elsevier Ltd. All rights reserved.

Constraints on the evolution of a doublesex target gene arising from doublesex’s pleiotropic deployment

PubMed Central

Luo, Shengzhan D.; Baker, Bruce S.

2015-01-01

“Regulatory evolution,” that is, changes in a gene’s expression pattern through changes at its regulatory sequence, rather than changes at the coding sequence of the gene or changes of the upstream transcription factors, has been increasingly recognized as a pervasive evolution mechanism. Many somatic sexually dimorphic features of Drosophila melanogaster are the results of gene expression regulated by the doublesex (dsx) gene, which encodes sex-specific transcription factors (DSXF in females and DSXM in males). Rapid changes in such sexually dimorphic features are likely a result of changes at the regulatory sequence of the target genes. We focused on the Flavin-containing monooxygenase-2 (Fmo-2) gene, a likely direct dsx target, to elucidate how sexually dimorphic expression and its evolution are brought about. We found that dsx is deployed to regulate the Fmo-2 transcription both in the midgut and in fat body cells of the spermatheca (a female-specific tissue), through a canonical DSX-binding site in the Fmo-2 regulatory sequence. In the melanogaster group, Fmo-2 transcription in the midgut has evolved rapidly, in contrast to the conserved spermathecal transcription. We identified two cis-regulatory modules (CRM-p and CRM-d) that direct sexually monomorphic or dimorphic Fmo-2 transcription, respectively, in the midguts of these species. Changes of Fmo-2 transcription in the midgut from sexually dimorphic to sexually monomorphic in some species are caused by the loss of CRM-d function, but not the loss of the canonical DSX-binding site. Thus, conferring transcriptional regulation on a CRM level allows the regulation to evolve rapidly in one tissue while evading evolutionary constraints posed by other tissues. PMID:25675536

Analysis of changes in hepatic gene expression in a murine model of tolerance to acetaminophen hepatotoxicity (autoprotection)

DOE Office of Scientific and Technical Information (OSTI.GOV)

O'Connor, Meeghan A., E-mail: meeghan.oconnor@boehringer-ingelheim.com; Boehringer Ingelheim Pharmaceuticals Inc., 900 Ridgebury Road, Ridgefield, CT 06877-0368; Koza-Taylor, Petra, E-mail: petra.h.koza-taylor@pfizer.com

Pretreatment of mice with a low hepatotoxic dose of acetaminophen (APAP) results in resistance to a subsequent, higher dose of APAP. This mouse model, termed APAP autoprotection was used here to identify differentially expressed genes and cellular pathways that could contribute to this development of resistance to hepatotoxicity. Male C57BL/6J mice were pretreated with APAP (400 mg/kg) and then challenged 48 h later with 600 mg APAP/kg. Livers were obtained 4 or 24 h later and total hepatic RNA was isolated and hybridized to Affymetrix Mouse Genome MU430{sub 2} GeneChip. Statistically significant genes were determined and gene expression changes weremore » also interrogated using the Causal Reasoning Engine (CRE). Extensive literature review narrowed our focus to methionine adenosyl transferase-1 alpha (MAT1A), nuclear factor (erythroid-derived 2)-like 2 (Nrf2), flavin-containing monooxygenase 3 (Fmo3) and galectin-3 (Lgals3). Down-regulation of MAT1A could lead to decreases in S-adenosylmethionine (SAMe), which is known to protect against APAP toxicity. Nrf2 activation is expected to play a role in protective adaptation. Up-regulation of Lgals3, one of the genes supporting the Nrf2 hypothesis, can lead to suppression of apoptosis and reduced mitochondrial dysfunction. Fmo3 induction suggests the involvement of an enzyme not known to metabolize APAP in the development of tolerance to APAP toxicity. Subsequent quantitative RT-PCR and immunochemical analysis confirmed the differential expression of some of these genes in the APAP autoprotection model. In conclusion, our genomics strategy identified cellular pathways that might further explain the molecular basis for APAP autoprotection. - Highlights: • Differential expression of genes in mice resistant to acetaminophen hepatotoxicity. • Increased gene expression of Flavin-containing monooxygenase 3 and Galectin-3. • Decrease in MAT1A expression and compensatory hepatocellular regeneration. • Two distinct

In vitro metabolism of an estrogen-related receptor γ modulator, GSK5182, by human liver microsomes and recombinant cytochrome P450s.

PubMed

Joo, Jeongmin; Wu, Zhexue; Lee, Boram; Shon, Jong Cheol; Lee, Taeho; Lee, In-Kyu; Sim, Taebo; Kim, Kyung-Hee; Kim, Nam Doo; Kim, Seong Heon; Liu, Kwang-Hyeon

2015-04-01

GSK5182 (4-[(Z)-1-[4-(2-dimethylaminoethyloxy)phenyl]-hydroxy-2-phenylpent-1-enyl]phenol) is a specific inverse agonist for estrogen-related receptor γ, a member of the orphan nuclear receptor family that has important functions in development and homeostasis. This study was performed to elucidate the metabolites of GSK5182 and to characterize the enzymes involved in its metabolism. Incubation of human liver microsomes with GSK5182 in the presence of NADPH resulted in the formation of three metabolites, M1, M2 and M3. M1 and M3 were identified as N-desmethyl-GSK5182 and GSK5182 N-oxide, respectively, on the basis of liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis. M2 was suggested to be hydroxy-GSK5182 through interpretation of its MS/MS fragmentation pattern. In addition, the specific cytochrome P450 (P450) and flavin-containing monooxygenase (FMO) isoforms responsible for GSK5182 oxidation to the three metabolites were identified using a combination of correlation analysis, chemical inhibition in human liver microsomes and metabolism by expressed recombinant P450 and FMO isoforms. GSK5182 N-demethylation and hydroxylation is mainly mediated by CYP3A4, whereas FMO1 and FMO3 contribute to the formation of GSK5182 N-oxide from GSK5182. The present data will be useful for understanding the pharmacokinetics and drug interactions of GSK5182 in vivo. Copyright © 2014 John Wiley & Sons, Ltd.

Biochemical establishment and characterization of EncM's flavin-N5-oxide cofactor

PubMed Central

Teufel, Robin; Stull, Frederick; Meehan, Michael J.; Michaudel, Quentin; Dorrestein, Pieter C.; Palfey, Bruce; Moore, Bradley S.

2016-01-01

The ubiquitous flavin-dependent monooxygenases commonly catalyze oxygenation reactions by means of a transient C4a-peroxyflavin. A recent study, however, suggested an unprecedented flavin-oxygenating species - proposed as the flavin-N5-oxide (FlN5[O]) - as key to an oxidative Favorskii-type rearrangement in the biosynthesis of the bacterial polyketide antibiotic enterocin. This stable superoxidized flavin is covalently tethered to the enzyme EncM and converted into FADH2 (Flred) during substrate turnover. Subsequent reaction of Flred with molecular oxygen restores the postulated FlN5[O] via an unknown pathway. Here we provide direct evidence for the FlN5[O] species via isotope labeling, proteolytic digestion, and high-resolution tandem mass spectrometry of EncM. We propose that formation of this species occurs by hydrogen-transfer from Flred to molecular oxygen, allowing radical coupling of the formed protonated superoxide and anionic flavin semiquinone at N5, before elimination of water affords the FlN5[O] cofactor. Further biochemical and spectroscopic investigations reveal important features of the FlN5[O] species and the EncM catalytic mechanism. We speculate that flavin-N5-oxides may be intermediates or catalytically active species in other flavoproteins that form the anionic semiquinone and promote access of oxygen to N5. PMID:26067765

Kinetic mechanism and quaternary structure of Aminobacter aminovorans NADH:flavin oxidoreductase: an unusual flavin reductase with bound flavin.

PubMed

Russell, Thomas R; Demeler, Borries; Tu, Shiao-Chun

2004-02-17

The homodimeric NADH:flavin oxidoreductase from Aminobacter aminovorans is an NADH-specific flavin reductase herein designated FRD(Aa). FRD(Aa) was characterized with respect to purification yields, thermal stability, isoelectric point, molar absorption coefficient, and effects of phosphate buffer strength and pH on activity. Evidence from this work favors the classification of FRD(Aa) as a flavin cofactor-utilizing class I flavin reductase. The isolated native FRD(Aa) contained about 0.5 bound riboflavin-5'-phosphate (FMN) per enzyme monomer, but one bound flavin cofactor per monomer was obtainable in the presence of excess FMN or riboflavin. In addition, FRD(Aa) holoenzyme also utilized FMN, riboflavin, or FAD as a substrate. Steady-state kinetic results of substrate titrations, dead-end inhibition by AMP and lumichrome, and product inhibition by NAD(+) indicated an ordered sequential mechanism with NADH as the first binding substrate and reduced FMN as the first leaving product. This is contrary to the ping-pong mechanism shown by other class I flavin reductases. The FMN bound to the native FRD(Aa) can be fully reduced by NADH and subsequently reoxidized by oxygen. No NADH binding was detected using 90 microM FRD(Aa) apoenzyme and 300 microM NADH. All results favor the interpretation that the bound FMN was a cofactor rather than a substrate. It is highly unusual that a flavin reductase using a sequential mechanism would require a flavin cofactor to facilitate redox exchange between NADH and a flavin substrate. FRD(Aa) exhibited a monomer-dimer equilibrium with a K(d) of 2.7 microM. Similarities and differences between FRD(Aa) and certain flavin reductases are discussed.

Aspergillus fumigatus SidA is a highly specific ornithine hydroxylase with bound flavin cofactor.

PubMed

Chocklett, Samuel W; Sobrado, Pablo

2010-08-10

Ferrichrome is a hydroxamate-containing siderophore produced by the pathogenic fungus Aspergillus fumigatus under iron-limiting conditions. This siderophore contains N(5)-hydroxylated l-ornithines essential for iron binding. A. fumigatus siderophore A (Af SidA) catalyzes the flavin- and NADPH-dependent hydroxylation of l-ornithine in ferrichrome biosynthesis. Af SidA was recombinantly expressed and purified as a soluble tetramer and is the first member of this class of flavin monooxygenases to be isolated with a bound flavin cofactor. The enzyme showed typical saturation kinetics with respect to l-ornithine while substrate inhibition was observed at high concentrations of NADPH and NADH. Increasing amounts of hydrogen peroxide were measured as a function of reduced nicotinamide coenzyme concentration, indicating that inhibition was caused by increased uncoupling. Af SidA is highly specific for its amino acid substrate, only hydroxylating l-ornithine. An 8-fold preference in the catalytic efficiency was determined for NADPH compared to NADH. In the absence of substrate, Af SidA can be reduced by NADPH, and a C4a-(hydro)peroxyflavin intermediate is observed. The decay of this intermediate is accelerated by l-ornithine binding. This intermediate was only stabilized by NADPH and not by NADH, suggesting a role for NADP(+) in the stabilization of intermediates in the reaction of Af SidA. NADP(+) is a competitive inhibitor with respect to NADPH, demonstrating that Af SidA forms a ternary complex with NADP(+) and l-ornithine during catalysis. The data suggest that Af SidA likely proceeds by a sequential kinetic mechanism.

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. © 2016 American Society of Plant Biologists. All rights reserved.

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.

Chloramphenicol Biosynthesis: The Structure of CmlS, a Flavin-Dependent Halogenase Shwing a Covalent Flavin-Aspartate Bond

DOE Office of Scientific and Technical Information (OSTI.GOV)

Podzelinska, K.; Latimer, R; Bhattacharya, A

2010-01-01

Chloramphenicol is a halogenated natural product bearing an unusual dichloroacetyl moiety that is critical for its antibiotic activity. The operon for chloramphenicol biosynthesis in Streptomyces venezuelae encodes the chloramphenicol halogenase CmlS, which belongs to the large and diverse family of flavin-dependent halogenases (FDH's). CmlS was previously shown to be essential for the formation of the dichloroacetyl group. Here we report the X-ray crystal structure of CmlS determined at 2.2 {angstrom} resolution, revealing a flavin monooxygenase domain shared by all FDHs, but also a unique 'winged-helix' C-terminal domain that creates a T-shaped tunnel leading to the halogenation active site. Intriguingly, themore » C-terminal tail of this domain blocks access to the halogenation active site, suggesting a structurally dynamic role during catalysis. The halogenation active site is notably nonpolar and shares nearly identical residues with Chondromyces crocatus tyrosyl halogenase (CndH), including the conserved Lys (K71) that forms the reactive chloramine intermediate. The exception is Y350, which could be used to stabilize enolate formation during substrate halogenation. The strictly conserved residue E44, located near the isoalloxazine ring of the bound flavin adenine dinucleotide (FAD) cofactor, is optimally positioned to function as a remote general acid, through a water-mediated proton relay, which could accelerate the reaction of the chloramine intermediate during substrate halogenation, or the oxidation of chloride by the FAD(C4{alpha})-OOH intermediate. Strikingly, the 8{alpha} carbon of the FAD cofactor is observed to be covalently attached to D277 of CmlS, a residue that is highly conserved in the FDH family. In addition to representing a new type of flavin modification, this has intriguing implications for the mechanism of FDHs. Based on the crystal structure and in analogy to known halogenases, we propose a reaction mechanism for CmlS.« less

Molecular Analysis of a Novel Methanesulfonic Acid Monooxygenase from the Methylotroph Methylosulfonomonas methylovora

PubMed Central

de Marco, Paolo; Moradas-Ferreira, Pedro; Higgins, Timothy P.; McDonald, Ian; Kenna, Elizabeth M.; Murrell, J. Colin

1999-01-01

Methylosulfonomonas methylovora M2 is an unusual gram-negative methylotrophic bacterium that can grow on methanesulfonic acid (MSA) as the sole source of carbon and energy. Oxidation of MSA by this bacterium is carried out by a multicomponent MSA monooxygenase (MSAMO). Cloning and sequencing of a 7.5-kbp SphI fragment of chromosomal DNA revealed four tightly linked genes encoding this novel monooxygenase. Analysis of the deduced MSAMO polypeptide sequences indicated that the enzyme contains a two-component hydroxylase of the mononuclear-iron-center type. The large subunit of the hydroxylase, MsmA (48 kDa), contains a typical Rieske-type [2Fe–2S] center with an unusual iron-binding motif and, together with the small subunit of the hydroxylase, MsmB (20 kDa), showed a high degree of identity with a number of dioxygenase enzymes. However, the other components of the MSAMO, MsmC, the ferredoxin component, and MsmD, the reductase, more closely resemble those found in other classes of oxygenases. MsmC has a high degree of identity to ferredoxins from toluene and methane monooxygenases, which are enzymes characterized by possessing hydroxylases containing μ-oxo bridge binuclear iron centers. MsmD is a reductase of 38 kDa with a typical chloroplast-like [2Fe–2S] center and conserved flavin adenine dinucleotide- and NAD-binding motifs and is similar to a number of mono- and dioxygenase reductase components. Preliminary analysis of the genes encoding MSAMO from a marine MSA-degrading bacterium, Marinosulfonomonas methylotropha, revealed the presence of msm genes highly related to those found in Methylosulfonomonas, suggesting that MSAMO is a novel type of oxygenase that may be conserved in all MSA-utilizing bacteria. PMID:10094704

Structure and Ligand Binding Properties of the Epoxidase Component of Styrene Monooxygenase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ukaegbu, Uchechi E.; Kantz, Auric; Beaton, Michelle

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. Atmore » 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.« less

Consecutive emamectin benzoate and deltamethrin treatments affect the expressions and activities of detoxification enzymes in the rainbow trout (Oncorhynchus mykiss).

PubMed

Cárcamo, Juan Guillermo; Aguilar, Marcelo N; Carreño, Constanza F; Vera, Tamara; Arias-Darraz, Luis; Figueroa, Jaime E; Romero, Alex P; Alvarez, Marco; Yañez, Alejandro J

2017-01-01

Rainbow trout (Oncorhynchus mykiss) subjected to three consecutive, alternating treatments with emamectin benzoate (EMB) and deltamethrin (DM) during outbreaks of Caligus rogercresseyi in a farm located in southern Chile (Hornopiren, Chiloé), were studied to determine the effects of these treatments on the protein and enzymatic activity levels of cytochrome P450 1A (CYP1A), flavin-containing monooxygenase (FMO) and glutathione S-transferase (GST) in different tissues. Consecutive and alternating EMB/DM treatments resulted in a 10-fold increase and 3-fold decrease of CYP1A protein levels in the intestine and gills, respectively. Notably, CYP1A activity levels decreased in most of the analyzed tissues. FMO protein and activity levels markedly increased in the kidney and the intestine. GST was up-regulated in all tissues, either as protein or enzyme activity. When comparing consecutive EMB/DM treatments against previous studies of EMB treatment alone, CYP1A activity levels were similarly diminished, except in muscle. Likewise, FMO activity levels were increased in most of the analyzed tissues, particularly in the muscle, kidney, and intestine. The increases observed for GST were essentially unchanged between consecutive EMB/DM and EMB only treatments. These results indicate that consecutive EMB/DM treatments in rainbow trout induce the expression and activity of FMO and GST enzymes and decrease CYP1A activity. These altered activities of detoxification enzymes could generate imbalances in metabolic processes, synthesis, degradation of hormones and complications associated with drug interactions. It is especially important when analyzing possible effects of consecutive antiparasitic treatments on withholding periods and salmon farming yields. Copyright © 2016 Elsevier Inc. All rights reserved.

Purification and Characterization of EDTA Monooxygenase from the EDTA-Degrading Bacterium BNC1

PubMed Central

Payne, Jason W.; Bolton, Harvey; Campbell, James A.; Xun, Luying

1998-01-01

The synthetic chelating agent EDTA can mobilize radionuclides and heavy metals in the environment. Biodegradation of EDTA should reduce this mobilization. Although several bacteria have been reported to mineralize EDTA, little is known about the biochemistry of EDTA degradation. Understanding the biochemistry will facilitate the removal of EDTA from the environment. EDTA-degrading activities were detected in cell extracts of bacterium BNC1 when flavin mononucleotide (FMN), NADH, and O2 were present. The degradative enzyme system was separated into two different enzymes, EDTA monooxygenase and an FMN reductase. EDTA monooxygenase oxidized EDTA to glyoxylate and ethylenediaminetriacetate (ED3A), with the coconsumption of FMNH2 and O2. The FMN reductase provided EDTA monooxygenase with FMNH2 by reducing FMN with NADH. The FMN reductase was successfully substituted in the assay mixture by other FMN reductases. EDTA monooxygenase was purified to greater than 95% homogeneity and had a single polypeptide with a molecular weight of 45,000. The enzyme oxidized both EDTA complexed with various metal ions and uncomplexed EDTA. The optimal conditions for activity were pH 7.8 and 35°C. Kms were 34.1 μM for uncomplexed EDTA and 8.5 μM for MgEDTA2−; this difference in Km indicates that the enzyme has greater affinity for MgEDTA2−. The enzyme also catalyzed the release of glyoxylate from nitrilotriacetate and diethylenetriaminepentaacetate. EDTA monooxygenase belongs to a small group of FMNH2-utilizing monooxygenases that attack carbon-nitrogen, carbon-sulfur, and carbon-carbon double bonds. PMID:9683478

Survey of Human Oxidoreductases and Cytochrome P450 Enzymes Involved in the Metabolism of Xenobiotic and Natural Chemicals

PubMed Central

2015-01-01

Analyzing the literature resources used in our previous reports, we calculated the fractions of the oxidoreductase enzymes FMO (microsomal flavin-containing monooxygenase), AKR (aldo-keto reductase), MAO (monoamine oxidase), and cytochrome P450 participating in metabolic reactions. The calculations show that the fractions of P450s involved in the metabolism of all chemicals (general chemicals, natural, and physiological compounds, and drugs) are rather consistent in the findings that >90% of enzymatic reactions are catalyzed by P450s. Regarding drug metabolism, three-fourths of the human P450 reactions can be accounted for by a set of five P450s: 1A2, 2C9, 2C19, 2D6, and 3A4, and the largest fraction of the P450 reactions is catalyzed by P450 3A enzymes. P450 3A4 participation in metabolic reactions of drugs varied from 13% for general chemicals to 27% for drugs. PMID:25485457

Genome-wide association identifies genetic variants associated with lentiform nucleus volume in N = 1345 young and elderly subjects.

PubMed

Hibar, Derrek P; Stein, Jason L; Ryles, April B; Kohannim, Omid; Jahanshad, Neda; Medland, Sarah E; Hansell, Narelle K; McMahon, Katie L; de Zubicaray, Greig I; Montgomery, Grant W; Martin, Nicholas G; Wright, Margaret J; Saykin, Andrew J; Jack, Clifford R; Weiner, Michael W; Toga, Arthur W; Thompson, Paul M

2013-06-01

Deficits in lentiform nucleus volume and morphometry are implicated in a number of genetically influenced disorders, including Parkinson's disease, schizophrenia, and ADHD. Here we performed genome-wide searches to discover common genetic variants associated with differences in lentiform nucleus volume in human populations. We assessed structural MRI scans of the brain in two large genotyped samples: the Alzheimer's Disease Neuroimaging Initiative (ADNI; N = 706) and the Queensland Twin Imaging Study (QTIM; N = 639). Statistics of association from each cohort were combined meta-analytically using a fixed-effects model to boost power and to reduce the prevalence of false positive findings. We identified a number of associations in and around the flavin-containing monooxygenase (FMO) gene cluster. The most highly associated SNP, rs1795240, was located in the FMO3 gene; after meta-analysis, it showed genome-wide significant evidence of association with lentiform nucleus volume (P MA  = 4.79 × 10(-8)). This commonly-carried genetic variant accounted for 2.68 % and 0.84 % of the trait variability in the ADNI and QTIM samples, respectively, even though the QTIM sample was on average 50 years younger. Pathway enrichment analysis revealed significant contributions of this gene to the cytochrome P450 pathway, which is involved in metabolizing numerous therapeutic drugs for pain, seizures, mania, depression, anxiety, and psychosis. The genetic variants we identified provide replicated, genome-wide significant evidence for the FMO gene cluster's involvement in lentiform nucleus volume differences in human populations.

NHR-49/HNF4 integrates regulation of fatty acid metabolism with a protective transcriptional response to oxidative stress and fasting.

PubMed

Goh, Grace Y S; Winter, Johnathan J; Bhanshali, Forum; Doering, Kelsie R S; Lai, Regina; Lee, Kayoung; Veal, Elizabeth A; Taubert, Stefan

2018-06-01

Endogenous and exogenous stresses elicit transcriptional responses that limit damage and promote cell/organismal survival. Like its mammalian counterparts, hepatocyte nuclear factor 4 (HNF4) and peroxisome proliferator-activated receptor α (PPARα), Caenorhabditis elegans NHR-49 is a well-established regulator of lipid metabolism. Here, we reveal that NHR-49 is essential to activate a transcriptional response common to organic peroxide and fasting, which includes the pro-longevity gene fmo-2/flavin-containing monooxygenase. These NHR-49-dependent, stress-responsive genes are also upregulated in long-lived glp-1/notch receptor mutants, with two of them making critical contributions to the oxidative stress resistance of wild-type and long-lived glp-1 mutants worms. Similar to its role in lipid metabolism, NHR-49 requires the mediator subunit mdt-15 to promote stress-induced gene expression. However, NHR-49 acts independently from the transcription factor hlh-30/TFEB that also promotes fmo-2 expression. We show that activation of the p38 MAPK, PMK-1, which is important for adaptation to a variety of stresses, is also important for peroxide-induced expression of a subset of NHR-49-dependent genes that includes fmo-2. However, organic peroxide increases NHR-49 protein levels, by a posttranscriptional mechanism that does not require PMK-1 activation. Together, these findings establish a new role for the HNF4/PPARα-related NHR-49 as a stress-activated regulator of cytoprotective gene expression. © 2018 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Effects of salinity acclimation on the expression and activity of Phase I enzymes (CYP450 and FMOs) in coho salmon (Oncorhynchus kisutch)

PubMed Central

Lavado, Ramon; Aparicio-Fabre, Rosaura; Schlenk, Daniel

2013-01-01

Phase I biotransformation enzymes are critically important in the disposition of xenobiotics within biota and are regulated by multiple environmental cues, particularly in anadromous fish species. Given the importance of these enzyme systems in xenobiotic/endogenous chemical bioactivation and detoxification, the current study was designed to better characterize the expression of Phase I biotransformation enzymes in coho salmon (Oncorhynchus kisutch) and the effects of salinity acclimation on those enzymes. Livers, gills and olfactory tissues were collected from coho salmon (Oncorhynchus kisutch) after they had undergone acclimation from freshwater to various salinity regimes of seawater (8, 16 and 32 g/L). Using immunoblot techniques coupled with testosterone hydroxylase catalytic activities, 4 orthologs of cytochrome P450 (CYP1A, CYP2K1, CYP2M1 and CYP3A27) were measured in each tissue. Also the expression of 2 transcripts of flavin-containing monooxygenases (FMO A and B) and associated activities were measured. With the exception of CYP1A, which was down-regulated in liver, protein expression of the other 3 enzymes was induced at higher salinity, with the greatest increase observed in CYP2M1 from olfactory tissues. In liver and gills, 6 - and 16 -hydroxylation of testosterone was also significantly increased after hypersaline acclimation. Similarly, FMO A was up-regulated in all 3 tissues in a salinity-dependent pattern, whereas FMO B mRNA was down-regulated. FMO-catalyzed benzydamine N-oxygenase and methyl p-tolyl sulfoxidation were significantly induced in liver and gills by hypersalinity, but was either unchanged or not detected in olfactory tissues. These data demonstrate thatenvironmental conditions may significantly alter the toxicity of environmental chemicals in salmon during freshwater/saltwater acclimation. PMID:23925894

Effects of FMO3 Polymorphisms on Pharmacokinetics of Sulindac in Chinese Healthy Male Volunteers

PubMed Central

Tang, Yong-Jun; Hu, Kai; Liu, Zhi; Chen, Yao; Ouyang, Dong-Sheng; Zhou, Hong-Hao

2017-01-01

Sulindac is a nonsteroidal anti-inflammatory drug, which is clinically used for the ailments of various inflammations. This study investigated the allele frequencies of FMO3 E158K and E308G and evaluated the influences of these two genetic polymorphisms on the pharmacokinetics of sulindac and its metabolites in Chinese healthy male volunteers. Eight FMO3 wild-type (FMO3 HHDD) subjects and seven FMO3 homozygotes E158K and E308G mutant (FMO3 hhdd) subjects were recruited from 247 healthy male volunteers genotyped by PCR-RFLP method. The plasma concentrations of sulindac, sulindac sulfide, and sulindac sulfone were determined by UPLC, while the pharmacokinetic parameters of the two different FMO3 genotypes were compared with each other. The frequencies of FMO3 E158K and E308G were 20.3% and 20.1%, respectively, which were in line with Hardy-Weinberg equilibrium (D′ = 0.977, r2 = 0.944). The mean values of Cmax, AUC0–24, and AUC0–∞ of sulindac were significantly higher in FMO3 hhdd group than those of FMO3 HHDD group (P < 0.05), while the pharmacokinetic parameters except Tmax of sulindac sulfide and sulindac sulfone showed no statistical difference between the two groups. The two FMO3 mutants were in close linkage disequilibrium and might play an important role in the pharmacokinetics of sulindac in Chinese healthy male volunteers. PMID:28331852

Pericyclic Reactions: FMO Approach-Abstract of Issue 9904M

NASA Astrophysics Data System (ADS)

Lee, Albert W. M.; So, C. T.; Chan, C. L.; Wu, Y. K.

1999-05-01

Pericyclic Reactions: FMO Approach is a program for Macintosh computers in which the frontier molecular orbital approaches to electrocyclic and cycloaddition reactions are animated. The bonding or antibonding interactions of the frontier molecular orbital(s) determine whether the reactions are thermally or photochemically allowed or forbidden. Pericyclic reactions that involve a redistribution of bonding and nonbonding electrons in a cyclic, concerted manner are an important class of organic reactions. Since the publications of the Woodward-Hoffmann rules on the conservation of orbital symmetry (1) and the frontier molecular orbital theory (FMO) by Fukui first described in the late 1960s (2), the underlying principles of these processes at the molecular level have become fully understood. Many modern organic chemistry textbooks include pericyclic reactions as a major topic. They are usually covered in detail in a typical introductory organic chemistry course. In the Classroom Between the two fundamental approaches to pericyclic reactions, the FMO approach has gained some popularity at the undergraduate teaching level. It is simpler and can be based on a pictorial approach. A detailed understanding of molecular orbital theories and symmetry is not required. Screen from Pericyclic Reactions: FMO Approach When learning the mechanisms of organic reactions, our students have often expressed a wish that they could see how the electrons "jump" and the orbitals "move" in the microscopic world. Pericyclic Reactions: FMO Approach has partially fulfilled the students' request. With its color 3-D graphics and animation, Pericyclic Reactions: FMO Approach can greatly enhance the teaching and learning of the FMO approach to pericyclic reactions. The stereochemical outcomes of these highly stereospecific reactions can be seen clearly as the reaction process is animated on the computer screen. Based on the

Reversible Oxidation of a Conserved Methionine in the Nuclear Export Sequence Determines Subcellular Distribution and Activity of the Fungal Nitrate Regulator NirA.

PubMed

Gallmetzer, Andreas; Silvestrini, Lucia; Schinko, Thorsten; Gesslbauer, Bernd; Hortschansky, Peter; Dattenböck, Christoph; Muro-Pastor, María Isabel; Kungl, Andreas; Brakhage, Axel A; Scazzocchio, Claudio; Strauss, Joseph

2015-07-01

The assimilation of nitrate, a most important soil nitrogen source, is tightly regulated in microorganisms and plants. In Aspergillus nidulans, during the transcriptional activation process of nitrate assimilatory genes, the interaction between the pathway-specific transcription factor NirA and the exportin KapK/CRM1 is disrupted, and this leads to rapid nuclear accumulation and transcriptional activity of NirA. In this work by mass spectrometry, we found that in the absence of nitrate, when NirA is inactive and predominantly cytosolic, methionine 169 in the nuclear export sequence (NES) is oxidized to methionine sulfoxide (Metox169). This oxidation depends on FmoB, a flavin-containing monooxygenase which in vitro uses methionine and cysteine, but not glutathione, as oxidation substrates. The function of FmoB cannot be replaced by alternative Fmo proteins present in A. nidulans. Exposure of A. nidulans cells to nitrate led to rapid reduction of NirA-Metox169 to Met169; this reduction being independent from thioredoxin and classical methionine sulfoxide reductases. Replacement of Met169 by isoleucine, a sterically similar but not oxidizable residue, led to partial loss of NirA activity and insensitivity to FmoB-mediated nuclear export. In contrast, replacement of Met169 by alanine transformed the protein into a permanently nuclear and active transcription factor. Co-immunoprecipitation analysis of NirA-KapK interactions and subcellular localization studies of NirA mutants lacking different parts of the protein provided evidence that Met169 oxidation leads to a change in NirA conformation. Based on these results we propose that in the presence of nitrate the activation domain is exposed, but the NES is masked by a central portion of the protein (termed nitrate responsive domain, NiRD), thus restricting active NirA molecules to the nucleus. In the absence of nitrate, Met169 in the NES is oxidized by an FmoB-dependent process leading to loss of protection by the Ni

Regulated methionine oxidation by monooxygenases

PubMed Central

Manta, Bruno; Gladyshev, Vadim N.

2017-01-01

Protein function can be regulated via post-translational modifications by numerous enzymatic and non-enzymatic mechanisms, including oxidation of cysteine and methionine residues. Redox-dependent regulatory mechanisms have been identified for nearly every cellular process, but the major paradigm has been that cellular components are oxidized (damaged) by reactive oxygen species (ROS) in a relatively unspecific way, and then reduced (repaired) by designated reductases. While this scheme may work with cysteine, it cannot be ascribed to other residues, such as methionine, whose reaction with ROS is too slow to be biologically relevant. However, methionine is clearly oxidized in vivo and enzymes for its stereoselective reduction are present in all three domains of life. Here, we revisit the chemistry and biology of methionine oxidation, with emphasis on its generation by enzymes from the monooxygenase family. Particular attention is placed on MICALs, a recently discovered family of proteins that harbor an unusual flavin-monooxygenase domain with an NADPH-dependent methionine sulfoxidase activity. Based on the structural and kinetic information we provide a rational framework to explain MICAL mechanism, inhibition, and regulation. Methionine residues that are targeted by MICALs are reduced back by methionine sulfoxide reductases, suggesting that reversible methionine oxidation may be a general mechanism analogous to the regulation by phosphorylation by kinases/phosphatases. The identification of new enzymes that catalyze the oxidation of methionine will open a new area of research at the forefront of redox signaling. PMID:28229915

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.

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

PubMed

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

2017-06-12

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.

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

PubMed Central

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

2017-01-01

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. PMID:28604669

Structural basis of kynurenine 3-monooxygenase inhibition.

PubMed

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

2013-04-18

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

The 1.6 Å Crystal Structure of Pyranose Dehydrogenase from Agaricus meleagris Rationalizes Substrate Specificity and Reveals a Flavin Intermediate

PubMed Central

Wongnate, Thanyaporn; Sucharitakul, Jeerus; Krondorfer, Iris; Sygmund, Christoph; Haltrich, Dietmar; Chaiyen, Pimchai; Peterbauer, Clemens K.; Divne, Christina

2013-01-01

Pyranose dehydrogenases (PDHs) are extracellular flavin-dependent oxidoreductases secreted by litter-decomposing fungi with a role in natural recycling of plant matter. All major monosaccharides in lignocellulose are oxidized by PDH at comparable yields and efficiencies. Oxidation takes place as single-oxidation or sequential double-oxidation reactions of the carbohydrates, resulting in sugar derivatives oxidized primarily at C2, C3 or C2/3 with the concomitant reduction of the flavin. A suitable electron acceptor then reoxidizes the reduced flavin. Whereas oxygen is a poor electron acceptor for PDH, several alternative acceptors, e.g., quinone compounds, naturally present during lignocellulose degradation, can be used. We have determined the 1.6-Å crystal structure of PDH from Agaricus meleagris. Interestingly, the flavin ring in PDH is modified by a covalent mono- or di-atomic species at the C(4a) position. Under normal conditions, PDH is not oxidized by oxygen; however, the related enzyme pyranose 2-oxidase (P2O) activates oxygen by a mechanism that proceeds via a covalent flavin C(4a)-hydroperoxide intermediate. Although the flavin C(4a) adduct is common in monooxygenases, it is unusual for flavoprotein oxidases, and it has been proposed that formation of the intermediate would be unfavorable in these oxidases. Thus, the flavin adduct in PDH not only shows that the adduct can be favorably accommodated in the active site, but also provides important details regarding the structural, spatial and physicochemical requirements for formation of this flavin intermediate in related oxidases. Extensive in silico modeling of carbohydrates in the PDH active site allowed us to rationalize the previously reported patterns of substrate specificity and regioselectivity. To evaluate the regioselectivity of D-glucose oxidation, reduction experiments were performed using fluorinated glucose. PDH was rapidly reduced by 3-fluorinated glucose, which has the C2 position accessible

FAD C(4a)-hydroxide stabilized in a naturally fused styrene monooxygenase

PubMed Central

Schlömann, Michael; van Berkel, Willem J.H.; Gassner, George T.

2013-01-01

StyA2B represents a new class of styrene monooxygenases that integrates flavin-reductase and styrene-epoxidase activities into a single polypeptide. This naturally-occurring fusion protein offers new avenues for studying and engineering biotechnologically relevant enantioselective biochemical epoxidation reactions. Stopped-flow kinetic studies of StyA2B reported here identify reaction intermediates similar to those reported for the separate reductase and epoxidase components of related two-component systems. Our studies identify substrate epoxidation and elimination of water from the FAD C(4a)-hydroxide as rate-limiting steps in the styrene epoxidation reaction. Efforts directed at accelerating these reaction steps are expected to greatly increase catalytic efficiency and the value of StyA2B as biocatalyst. PMID:24157359

Camphor revisited: studies of 2,5-diketocamphane 1,2-monooxygenase from Pseudomonas putida ATCC 17453.

PubMed Central

Taylor, D G; Trudgill, P W

1986-01-01

The oxygenating component of 2,5-diketocamphane 1,2-monooxygenase from Pseudomonas putida ATCC 17453 was purified to homogeneity by a combination of ammonium sulfate fractionation and chromatography on DEAE-cellulose and polyanion SI-17 columns. It had an Mr of 78,000, bound one molecule of nonautooxidizable flavin mononucleotide (FMN), consisted of two subunits of equal molecular weight, and existed in two electrophoretically distinguishable active forms. The oxygenating complex was constructed from equimolecular amounts of an NADH oxidase, which could be purified separately (Mr, 36,000), and the oxygenating component. Most of the NADH oxidase dissociated from the oxygenating component during purification, although traces remained, to give the final preparation of the oxygenating component significant oxygenase activity. FMN did not dissociate significantly from the oxygenating component during purification, but it was not covalently bound and could be removed under a variety of conditions. Binding between the two proteins that made up the active complex was fairly weak and freely reversible. It probably occurred through the FMN which was strongly bound to the oxygenating component and for which the NADH had a weak binding site. Iron was not present at a significant level in the oxygenating component, and in common with other characterized Baeyer Villiger monooxygenases, 2,5-diketocamphane 1,2-monooxygenase was found to be a simple flavoprotein. Images PMID:3944058

Camphor revisited: studies of 2,5-diketocamphane 1,2-monooxygenase from Pseudomonas putida ATCC 17453.

PubMed

Taylor, D G; Trudgill, P W

1986-02-01

The oxygenating component of 2,5-diketocamphane 1,2-monooxygenase from Pseudomonas putida ATCC 17453 was purified to homogeneity by a combination of ammonium sulfate fractionation and chromatography on DEAE-cellulose and polyanion SI-17 columns. It had an Mr of 78,000, bound one molecule of nonautooxidizable flavin mononucleotide (FMN), consisted of two subunits of equal molecular weight, and existed in two electrophoretically distinguishable active forms. The oxygenating complex was constructed from equimolecular amounts of an NADH oxidase, which could be purified separately (Mr, 36,000), and the oxygenating component. Most of the NADH oxidase dissociated from the oxygenating component during purification, although traces remained, to give the final preparation of the oxygenating component significant oxygenase activity. FMN did not dissociate significantly from the oxygenating component during purification, but it was not covalently bound and could be removed under a variety of conditions. Binding between the two proteins that made up the active complex was fairly weak and freely reversible. It probably occurred through the FMN which was strongly bound to the oxygenating component and for which the NADH had a weak binding site. Iron was not present at a significant level in the oxygenating component, and in common with other characterized Baeyer Villiger monooxygenases, 2,5-diketocamphane 1,2-monooxygenase was found to be a simple flavoprotein.

Antioxidant-related gene polymorphisms associated with the cardio-ankle vascular index in young Russians.

PubMed

Sorokin, Alexander V; Kotani, Kazuhiko; Bushueva, Olga Y; Polonikov, Alexey V

2016-04-01

The cardio-ankle vascular index is a measure of arterial stiffness, whereas oxidative stress underlies arterial pathology. This study aimed to investigate the association between the cardio-ankle vascular index and antioxidant-related gene polymorphisms in young Russians. A total of 89 patients (mean age, 21.6 years) were examined by the cardio-ankle vascular index and for 15 gene polymorphisms related to antioxidant enzymes including FMO3 (flavin-containing monooxygenase 3), GPX1 (glutathione peroxidase 1), and GPX4 (glutathione peroxidase 4). A higher cardio-ankle vascular index level was detected in carriers with the KK-genotype of FMO3 polymorphism rs2266782 than in those without (mean levels: 6.2 versus 5.6, respectively, p<0.05). Similarly, a higher cardio-ankle vascular index level was seen in carriers with the CC-genotype of GPX4 polymorphism rs713041 than in those without (6.0 versus 5.5, respectively, p<0.05). We did not observe significant associations between the cardio-ankle vascular index levels and the other gene polymorphisms. Although carriers with the LL-genotype of GPX1 polymorphism rs1050450 showed a higher diastolic blood pressure level than those without, the polymorphism did not affect the cardio-ankle vascular index level. This study showed a significant association between rs2266782 and rs713041 polymorphisms and arterial stiffness, as measured by the cardio-ankle vascular index, in young Russians. The pathways utilised by antioxidant enzymes may be responsible for early arterial stiffening in the Russian population.

Initial reaction(s) in biotransformation of CL-20 is catalyzed by salicylate 1-monooxygenase from Pseudomonas sp. strain ATCC 29352.

PubMed

Bhushan, Bharat; Halasz, Annamaria; Spain, Jim C; Hawari, Jalal

2004-07-01

CL-20 (2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane) (C(6)H(6)N(12)O(12)), a future-generation high-energy explosive, is biodegradable by Pseudomonas sp. strain FA1 and Agrobacterium sp. strain JS71; however, the nature of the enzyme(s) involved in the process was not understood. In the present study, salicylate 1-monooxygenase, a flavin adenine dinucleotide (FAD)-containing purified enzyme from Pseudomonas sp. strain ATCC 29352, biotransformed CL-20 at rates of 0.256 +/- 0.011 and 0.043 +/- 0.003 nmol min(-1) mg of protein(-1) under anaerobic and aerobic conditions, respectively. The disappearance of CL-20 was accompanied by the release of nitrite ions. Using liquid chromatography/mass spectrometry in the negative electrospray ionization mode, we detected a metabolite with a deprotonated mass ion [M - H](-) at 345 Da, corresponding to an empirical formula of C(6)H(6)N(10)O(8), produced as a result of two sequential N denitration steps on the CL- 20 molecule. We also detected two isomeric metabolites with [M - H](-) at 381 Da corresponding to an empirical formula of C(6)H(10)N(10)O(10). The latter was a hydrated product of the metabolite C(6)H(6)N(10)O(8) with addition of two H(2)O molecules, as confirmed by tests using (18)O-labeled water. The product stoichiometry showed that each reacted CL-20 molecule produced about 1.7 nitrite ions, 3.2 molecules of nitrous oxide, 1.5 molecules of formic acid, and 0.6 ammonium ion. Diphenyliodonium-mediated inhibition of salicylate 1-monooxygenase and a comparative study between native, deflavo, and reconstituted enzyme(s) showed that FAD site of the enzyme was involved in the biotransformation of CL-20 catalyzed by salicylate 1-monooxygenase. The data suggested that salicylate 1-monooxygenase catalyzed two oxygen-sensitive single-electron transfer steps necessary to release two nitrite ions from CL-20 and that this was followed by the secondary decomposition of this energetic chemical.

Association between microbiota-dependent metabolite trimethylamine-N-oxide and type 2 diabetes.

PubMed

Shan, Zhilei; Sun, Taoping; Huang, Hao; Chen, Sijing; Chen, Liangkai; Luo, Cheng; Yang, Wei; Yang, Xuefeng; Yao, Ping; Cheng, Jinquan; Hu, Frank B; Liu, Liegang

2017-09-01

Background: The association of trimethylamine- N -oxide (TMAO), a microbiota-dependent metabolite from dietary choline and carnitine, with type 2 diabetes was inconsistent. Objective: We evaluated the association of plasma TMAO with newly diagnosed type 2 diabetes and the potential modification of TMAO-generating enzyme flavin monooxygenase 3 (FMO3) polymorphisms. Design: This was an age- and sex-matched case-control study of 2694 participants: 1346 newly diagnosed cases of type 2 diabetes and 1348 controls. Concentrations of plasma TMAO were measured, and FMO3 E158K polymorphisms (rs2266782) were genotyped. Results: Medians (IQRs) of plasma TMAO concentration were 1.47 μmol/L (0.81-2.20 μmol/L) for controls and 1.77 μmol/L (1.09-2.80 μmol/L) for type 2 diabetes cases. From the lowest to the highest quartiles of plasma TMAO, the multivariable adjusted ORs of type 2 diabetes were 1.00 (reference), 1.38 (95% CI: 1.08, 1.77), 1.64 (95% CI: 1.28, 2.09), and 2.55 (95% CI: 1.99, 3.28) ( P -trend < 0.001); each SD of ln-transformed plasma TMAO was associated with a 38% (95% CI: 26%, 51%) increment in ORs of type 2 diabetes. The FMO3 rs2266782 polymorphism was not associated with type 2 diabetes. The positive association between plasma TMAO and type 2 diabetes was consistent in each rs2266782 genotype group, and no significant interaction was observed ( P = 0.093). Conclusions: Our results suggested that higher plasma TMAO was associated with increased odds of newly diagnosed type 2 diabetes and that this association was not modified by the FMO3 rs2266782 polymorphism. This study was registered at clinicaltrials.gov as NCT03130894. © 2017 American Society for Nutrition.

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

Quantification of riboflavin, flavin mononucleotide, and flavin adenine dinucleotide in mammalian model cells by CE with LED-induced fluorescence detection.

PubMed

Hühner, Jens; Ingles-Prieto, Álvaro; Neusüß, Christian; Lämmerhofer, Michael; Janovjak, Harald

2015-02-01

Cultured mammalian cells essential are model systems in basic biology research, production platforms of proteins for medical use, and testbeds in synthetic biology. Flavin cofactors, in particular flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), are critical for cellular redox reactions and sense light in naturally occurring photoreceptors and optogenetic tools. Here, we quantified flavin contents of commonly used mammalian cell lines. We first compared three procedures for extraction of free and noncovalently protein-bound flavins and verified extraction using fluorescence spectroscopy. For separation, two CE methods with different BGEs were established, and detection was performed by LED-induced fluorescence with limit of detections (LODs 0.5-3.8 nM). We found that riboflavin (RF), FMN, and FAD contents varied significantly between cell lines. RF (3.1-14 amol/cell) and FAD (2.2-17.0 amol/cell) were the predominant flavins, while FMN (0.46-3.4 amol/cell) was found at markedly lower levels. Observed flavin contents agree with those previously extracted from mammalian tissues, yet reduced forms of RF were detected that were not described previously. Quantification of flavins in mammalian cell lines will allow a better understanding of cellular redox reactions and optogenetic tools. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Initial Reaction(s) in Biotransformation of CL-20 Is Catalyzed by Salicylate 1-Monooxygenase from Pseudomonas sp. Strain ATCC 29352

PubMed Central

Bhushan, Bharat; Halasz, Annamaria; Spain, Jim C.; Hawari, Jalal

2004-01-01

CL-20 (2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane) (C6H6N12O12), a future-generation high-energy explosive, is biodegradable by Pseudomonas sp. strain FA1 and Agrobacterium sp. strain JS71; however, the nature of the enzyme(s) involved in the process was not understood. In the present study, salicylate 1-monooxygenase, a flavin adenine dinucleotide (FAD)-containing purified enzyme from Pseudomonas sp. strain ATCC 29352, biotransformed CL-20 at rates of 0.256 ± 0.011 and 0.043 ± 0.003 nmol min−1 mg of protein−1 under anaerobic and aerobic conditions, respectively. The disappearance of CL-20 was accompanied by the release of nitrite ions. Using liquid chromatography/mass spectrometry in the negative electrospray ionization mode, we detected a metabolite with a deprotonated mass ion [M − H]− at 345 Da, corresponding to an empirical formula of C6H6N10O8, produced as a result of two sequential N denitration steps on the CL- 20 molecule. We also detected two isomeric metabolites with [M − H]− at 381 Da corresponding to an empirical formula of C6H10N10O10. The latter was a hydrated product of the metabolite C6H6N10O8 with addition of two H2O molecules, as confirmed by tests using 18O-labeled water. The product stoichiometry showed that each reacted CL-20 molecule produced about 1.7 nitrite ions, 3.2 molecules of nitrous oxide, 1.5 molecules of formic acid, and 0.6 ammonium ion. Diphenyliodonium-mediated inhibition of salicylate 1-monooxygenase and a comparative study between native, deflavo, and reconstituted enzyme(s) showed that FAD site of the enzyme was involved in the biotransformation of CL-20 catalyzed by salicylate 1-monooxygenase. The data suggested that salicylate 1-monooxygenase catalyzed two oxygen-sensitive single-electron transfer steps necessary to release two nitrite ions from CL-20 and that this was followed by the secondary decomposition of this energetic chemical. PMID:15240281

Quantum transport in the FMO photosynthetic light-harvesting complex.

PubMed

Karafyllidis, Ioannis G

2017-06-01

The very high light-harvesting efficiency of natural photosynthetic systems in conjunction with recent experiments, which showed quantum-coherent energy transfer in photosynthetic complexes, raised questions regarding the presence of non-trivial quantum effects in photosynthesis. Grover quantum search, quantum walks, and entanglement have been investigated as possible effects that lead to this efficiency. Here we explain the near-unit photosynthetic efficiency without invoking non-trivial quantum effects. Instead, we use non-equilibrium Green's functions, a mesoscopic method used to study transport in nano-conductors to compute the transmission function of the Fenna-Matthews-Olson (FMO) complex using an experimentally derived exciton Hamiltonian. The chlorosome antenna and the reaction center play the role of input and output contacts, connected to the FMO complex. We show that there are two channels for which the transmission is almost unity. Our analysis also revealed a dephasing-driven regulation mechanism that maintains the efficiency in the presence of varying dephasing potentials.

Catalytically important flavin linked through a phosphoester bond in a eukaryotic fumarate reductase.

PubMed

Serebryakova, Marina V; Bertsova, Yulia V; Sokolov, Svyatoslav S; Kolesnikov, Alexander A; Baykov, Alexander A; Bogachev, Alexander V

2018-06-01

One of the three domains of kinetoplastid NADH:fumarate oxidoreductase (FRD) is homologous to bacterial flavin transferase that catalyzes transfer of FMN residue from FAD to threonine in flavoproteins. Leptomonas pyrrhocoris FRD produced in yeast cells, which lack flavin transferase gene in their proteome, reduces fumarate in the presence of NADH and contains an FMN residue covalently linked to a Ser9 residue. The conserved flavinylation motif of FRD, D 3 (g/s)x(s/t)(s/g)AS 9 , is similar to the Dxx(s/t)gAT motif recognized by flavin transferase in prokaryotic proteins. Ser9 replacement abolished the flavinylation and fumarate reductase activity of FRD. These findings suggest that the flavinylation is important for the activity of FRD and that this post-translational modification is carried out by the own flavin transferase domain. Copyright © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Flavin-N5 Covalent Intermediate in a Nonredox Dehalogenation Reaction Catalyzed by an Atypical Flavoenzyme.

PubMed

Dai, Yumin; Kizjakina, Karina; Campbell, Ashley C; Korasick, David A; Tanner, John J; Sobrado, Pablo

2018-01-04

The flavin-dependent enzyme 2-haloacrylate hydratase (2-HAH) catalyzes the conversion of 2-chloroacrylate, a major component in the manufacture of acrylic polymers, to pyruvate. The enzyme was expressed in Escherichia coli, purified, and characterized. 2-HAH was shown to be monomeric in solution and contained a non-covalent, yet tightly bound, flavin adenine dinucleotide (FAD). Although the catalyzed reaction was redox-neutral, 2-HAH was active only in the reduced state. A covalent flavin-substrate intermediate, consistent with the flavin-acrylate iminium ion, was trapped with cyanoborohydride and characterized by mass spectrometry. Small-angle X-ray scattering was consistent with 2-HAH belonging to the succinate dehydrogenase/fumarate reductase family of flavoproteins. These studies establish 2-HAH as a novel noncanonical flavoenzyme. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

2016-01-01

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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Multidomain flavin-dependent sulfhydryl oxidases.

PubMed

Coppock, Donald L; Thorpe, Colin

2006-01-01

Eukaryotic flavin-dependent sulfhydryl oxidases catalyze oxidative protein folding with the generation of disulfides and the reduction of oxygen to hydrogen peroxide. This review deals principally with the Quiescinsulfhydryl oxidases (QSOX) that are found in multiple forms in multicellular organisms and singly in a number of protozoan parasites. QSOX is an ancient fusion of thioredoxin domains and an FAD-binding module, ERV1/ALR. Interdomain disulfide exchanges transmit reducing equivalents from substrates to the flavin cofactor and thence to molecular oxygen. The in vitro substrate specificity of avian QSOX1 and the likely substrates of QSOXs in vivo are discussed. The location of QSOX immunoreactivity and mRNA expression levels in human cells and tissues is reviewed. Generally, there is a marked association of QSOX1 expression with cell types that have a high secretory load of disulfide-containing peptides and proteins. The abundance of sulfhydryl oxidases in the islets of Langerhans suggests that oxidative protein folding may directly contribute to the oxidative stress believed to be a factor in the progression to type II diabetes. Finally, the structure and mechanism of QSOX proteins is compared to their smaller stand-alone cousins: yeast ERV1p and ERV2p, the mammalian augmenter of liver regeneration (ALR), and the viral ALR homologs.

Active site residues critical for flavin binding and 5,6-dimethylbenzimidazole biosynthesis in the flavin destructase enzyme BluB.

PubMed

Yu, Ta-Yi; Mok, Kenny C; Kennedy, Kristopher J; Valton, Julien; Anderson, Karen S; Walker, Graham C; Taga, Michiko E

2012-06-01

The "flavin destructase" enzyme BluB catalyzes the unprecedented conversion of flavin mononucleotide (FMN) to 5,6-dimethylbenzimidazole (DMB), a component of vitamin B(12). Because of its unusual chemistry, the mechanism of this transformation has remained elusive. This study reports the identification of 12 mutant forms of BluB that have severely reduced catalytic function, though most retain the ability to bind flavin. The "flavin destructase" BluB is an unusual enzyme that fragments the flavin cofactor FMNH(2) in the presence of oxygen to produce 5,6-dimethylbenzimidazole (DMB), the lower axial ligand of vitamin B(12) (cobalamin). Despite the similarities in sequence and structure between BluB and the nitroreductase and flavin oxidoreductase enzyme families, BluB is the only enzyme known to fragment a flavin isoalloxazine ring. To explore the catalytic residues involved in this unusual reaction, mutants of BluB impaired in DMB biosynthesis were identified in a genetic screen in the bacterium Sinorhizobium meliloti. Of the 16 unique point mutations identified in the screen, the majority were located in conserved residues in the active site or in the unique "lid" domain proposed to shield the active site from solvent. Steady-state enzyme assays of 12 purified mutant proteins showed a significant reduction in DMB synthesis in all of the mutants, with eight completely defective in DMB production. Ten of these mutants have weaker binding affinities for both oxidized and reduced FMN, though only two have a significant effect on complex stability. These results implicate several conserved residues in BluB's unique ability to fragment FMNH(2) and demonstrate the sensitivity of BluB's active site to structural perturbations. This work lays the foundation for mechanistic studies of this enzyme and further advances our understanding of the structure-function relationship of BluB. Copyright © 2012 The Protein Society.

Pipecolic Acid Orchestrates Plant Systemic Acquired Resistance and Defense Priming via Salicylic Acid-Dependent and -Independent Pathways

PubMed Central

Bernsdorff, Friederike; Döring, Anne-Christin; Gruner, Katrin; Schuck, Stefan; Bräutigam, Andrea; Zeier, Jürgen

2016-01-01

We investigated the relationships of the two immune-regulatory plant metabolites, salicylic acid (SA) and pipecolic acid (Pip), in the establishment of plant systemic acquired resistance (SAR), SAR-associated defense priming, and basal immunity. Using SA-deficient sid2, Pip-deficient ald1, and sid2 ald1 plants deficient in both SA and Pip, we show that SA and Pip act both independently from each other and synergistically in Arabidopsis thaliana basal immunity to Pseudomonas syringae. Transcriptome analyses reveal that SAR establishment in Arabidopsis is characterized by a strong transcriptional response systemically induced in the foliage that prepares plants for future pathogen attack by preactivating multiple stages of defense signaling and that SA accumulation upon SAR activation leads to the downregulation of photosynthesis and attenuated jasmonate responses systemically within the plant. Whereas systemic Pip elevations are indispensable for SAR and necessary for virtually the whole transcriptional SAR response, a moderate but significant SA-independent component of SAR activation and SAR gene expression is revealed. During SAR, Pip orchestrates SA-dependent and SA-independent priming of pathogen responses in a FLAVIN-DEPENDENT-MONOOXYGENASE1 (FMO1)-dependent manner. We conclude that a Pip/FMO1 signaling module acts as an indispensable switch for the activation of SAR and associated defense priming events and that SA amplifies Pip-triggered responses to different degrees in the distal tissue of SAR-activated plants. PMID:26672068

Role of Free Radicals and Biotransformation in Trichloronitrobenzene-Induced Nephrotoxicity In Vitro

PubMed Central

Rankin, Gary O.; Tyree, Connor; Pope, Deborah; Tate, Jordan; Racine, Christopher; Anestis, Dianne K.; Brown, Kathleen C.; Dial, Mason; Valentovic, Monica A.

2017-01-01

This study determined the comparative nephrotoxic potential of four trichloronitrobenzenes (TCNBs) (2,3,4-; 2,4,5-; 2,4,6-; and 3,4,5-TCNB) and explored the effects of antioxidants and biotransformation inhibitors on TCNB-induced cytotoxicity in isolated renal cortical cells (IRCC) from male Fischer 344 rats. IRCC were incubated with a TCNB up to 1.0 mM for 15–120 min. Pretreatment with an antioxidant or cytochrome P450 (CYP), flavin monooxygenase (FMO), or peroxidase inhibitor was used in some experiments. Among the four TCNBs, the order of decreasing nephrotoxic potential was approximately 3,4,5- > 2,4,6- > 2,3,4- > 2,4,5-TCNB. The four TCNBs exhibited a similar profile of attenuation of cytotoxicity in response to antioxidant pretreatments. 2,3,4- and 3,4,5-TCNB cytotoxicity was attenuated by most of the biotransformation inhibitors tested, 2,4,5-TCNB cytotoxicity was only inhibited by isoniazid (CYP 2E1 inhibitor), and 2,4,6-TCNB-induced cytotoxicity was inhibited by one CYP inhibitor, one FMO inhibitor, and one peroxidase inhibitor. All of the CYP specific inhibitors tested offered some attenuation of 3,4,5-TCNB cytotoxicity. These results indicate that 3,4,5-TCNB is the most potent nephrotoxicant, free radicals play a role in the TCNB cytotoxicity, and the role of biotransformation in TCNB nephrotoxicity in vitro is variable and dependent on the position of the chloro groups. PMID:28561793

Role of Free Radicals and Biotransformation in Trichloronitrobenzene-Induced Nephrotoxicity In Vitro.

PubMed

Rankin, Gary O; Tyree, Connor; Pope, Deborah; Tate, Jordan; Racine, Christopher; Anestis, Dianne K; Brown, Kathleen C; Dial, Mason; Valentovic, Monica A

2017-05-31

This study determined the comparative nephrotoxic potential of four trichloronitrobenzenes (TCNBs) (2,3,4-; 2,4,5-; 2,4,6-; and 3,4,5-TCNB) and explored the effects of antioxidants and biotransformation inhibitors on TCNB-induced cytotoxicity in isolated renal cortical cells (IRCC) from male Fischer 344 rats. IRCC were incubated with a TCNB up to 1.0 mM for 15-120 min. Pretreatment with an antioxidant or cytochrome P450 (CYP), flavin monooxygenase (FMO), or peroxidase inhibitor was used in some experiments. Among the four TCNBs, the order of decreasing nephrotoxic potential was approximately 3,4,5- > 2,4,6- > 2,3,4- > 2,4,5-TCNB. The four TCNBs exhibited a similar profile of attenuation of cytotoxicity in response to antioxidant pretreatments. 2,3,4- and 3,4,5-TCNB cytotoxicity was attenuated by most of the biotransformation inhibitors tested, 2,4,5-TCNB cytotoxicity was only inhibited by isoniazid (CYP 2E1 inhibitor), and 2,4,6-TCNB-induced cytotoxicity was inhibited by one CYP inhibitor, one FMO inhibitor, and one peroxidase inhibitor. All of the CYP specific inhibitors tested offered some attenuation of 3,4,5-TCNB cytotoxicity. These results indicate that 3,4,5-TCNB is the most potent nephrotoxicant, free radicals play a role in the TCNB cytotoxicity, and the role of biotransformation in TCNB nephrotoxicity in vitro is variable and dependent on the position of the chloro groups.

Pipecolic Acid Orchestrates Plant Systemic Acquired Resistance and Defense Priming via Salicylic Acid-Dependent and -Independent Pathways.

PubMed

Bernsdorff, Friederike; Döring, Anne-Christin; Gruner, Katrin; Schuck, Stefan; Bräutigam, Andrea; Zeier, Jürgen

2016-01-01

We investigated the relationships of the two immune-regulatory plant metabolites, salicylic acid (SA) and pipecolic acid (Pip), in the establishment of plant systemic acquired resistance (SAR), SAR-associated defense priming, and basal immunity. Using SA-deficient sid2, Pip-deficient ald1, and sid2 ald1 plants deficient in both SA and Pip, we show that SA and Pip act both independently from each other and synergistically in Arabidopsis thaliana basal immunity to Pseudomonas syringae. Transcriptome analyses reveal that SAR establishment in Arabidopsis is characterized by a strong transcriptional response systemically induced in the foliage that prepares plants for future pathogen attack by preactivating multiple stages of defense signaling and that SA accumulation upon SAR activation leads to the downregulation of photosynthesis and attenuated jasmonate responses systemically within the plant. Whereas systemic Pip elevations are indispensable for SAR and necessary for virtually the whole transcriptional SAR response, a moderate but significant SA-independent component of SAR activation and SAR gene expression is revealed. During SAR, Pip orchestrates SA-dependent and SA-independent priming of pathogen responses in a FLAVIN-DEPENDENT-MONOOXYGENASE1 (FMO1)-dependent manner. We conclude that a Pip/FMO1 signaling module acts as an indispensable switch for the activation of SAR and associated defense priming events and that SA amplifies Pip-triggered responses to different degrees in the distal tissue of SAR-activated plants. © 2016 American Society of Plant Biologists. All rights reserved.

Large-scale FMO-MP3 calculations on the surface proteins of influenza virus, hemagglutinin (HA) and neuraminidase (NA)

NASA Astrophysics Data System (ADS)

Mochizuki, Yuji; Yamashita, Katsumi; Fukuzawa, Kaori; Takematsu, Kazutomo; Watanabe, Hirofumi; Taguchi, Naoki; Okiyama, Yoshio; Tsuboi, Misako; Nakano, Tatsuya; Tanaka, Shigenori

2010-06-01

Two proteins on the influenza virus surface have been well known. One is hemagglutinin (HA) associated with the infection to cells. The fragment molecular orbital (FMO) calculations were performed on a complex consisting of HA trimer and two Fab-fragments at the third-order Møller-Plesset perturbation (MP3) level. The numbers of residues and 6-31G basis functions were 2351 and 201276, and thus a massively parallel-vector computer was utilized to accelerate the processing. This FMO-MP3 job was completed in 5.8 h with 1024 processors. Another protein is neuraminidase (NA) involved in the escape from infected cells. The FMO-MP3 calculation was also applied to analyze the interactions between oseltamivir and surrounding residues in pharmacophore.

Ultrafast Excited-state Deactivation of Flavins Bound to Dodecin*

PubMed Central

Staudt, Heike; Oesterhelt, Dieter; Grininger, Martin; Wachtveitl, Josef

2012-01-01

Dodecins, a group of flavin-binding proteins with a dodecameric quaternary structure, are able to incorporate two flavins within each of their six identical binding pockets building an aromatic tetrade with two tryptophan residues. Dodecin from the archaeal Halobacterium salinarum is a riboflavin storage device. We demonstrate that unwanted side reactions induced by reactive riboflavin species and degradation of riboflavin are avoided by ultrafast depopulation of the reactive excited state of riboflavin. Intriguingly, in this process, the staggered riboflavin dimers do not interact in ground and photoexcited states. Rather, within the tetrade assembly, each riboflavin is kept under the control of the respective adjacent tryptophan, which suggests that the stacked arrangement is a matter of optimizing the flavin load. We further identify an electron transfer in combination with a proton transfer as a central element of the effective excited state depopulation mechanism. Structural and functional comparisons of the archaeal dodecin with bacterial homologs reveal diverging evolution. Bacterial dodecins bind the flavin FMN instead of riboflavin and exhibit a clearly different binding pocket design with inverse incorporations of flavin dimers. The different adoption of flavin changes photochemical properties, making bacterial dodecin a comparably less efficient quencher of flavins. This supports a functional role different for bacterial and archaeal dodecins. PMID:22451648

FLOOZY of petunia is a flavin mono-oxygenase-like protein required for the specification of leaf and flower architecture

PubMed Central

Tobeña-Santamaria, Rafael; Bliek, Mattijs; Ljung, Karin; Sandberg, Göran; Mol, Joseph N.M.; Souer, Erik; Koes, Ronald

2002-01-01

The mechanisms that determine the relative positions of floral organs, and thereby their numbers, is a poorly understood aspect of flower development. We isolated a petunia mutant, floozy (fzy), in which the formation of floral organ primordia in the outermost three floral whorls and one of the two bracts at the base of the flower is blocked at an early stage. In addition, fzy mutants fail to generate secondary veins in leaves and bracts and display a decreased apical dominance in the inflorescence. FZY encodes an enzyme with homology to flavin mono-oxygenases and appears to be the ortholog of YUCCA genes of Arabidopsis. FZY is expressed in young leafs and bracts and in developing flowers. In young floral meristems FZY is expressed in the center of the meristem dome and, later, expression becomes localized on the flanks of the initiating petal and stamen primordia and at several sites in maturing anthers and carpels. These findings indicate that FZY is involved in synthesizing a signaling compound that is required for floral organ initiation and specification of the vascularization pattern in leaves. Although fzy mutants contain normal auxin levels, ectopic expression of FZY results in excessive auxin accumulation, suggesting that the signaling compound is auxin. PMID:11914280

Cloning of a Gene Cluster Involved in the Catabolism of p-Nitrophenol by Arthrobacter sp. Strain JS443 and Characterization of the p-Nitrophenol Monooxygenase▿

PubMed Central

Perry, Lynda L.; Zylstra, Gerben J.

2007-01-01

The npd gene cluster, which encodes the enzymes of a p-nitrophenol catabolic pathway from Arthrobacter sp. strain JS443, was cloned and sequenced. Three genes, npdB, npdA1, and npdA2, were independently expressed in Escherichia coli in order to confirm the identities of their gene products. NpdA2 is a p-nitrophenol monooxygenase belonging to the two-component flavin-diffusible monooxygenase family of reduced flavin-dependent monooxygenases. NpdA1 is an NADH-dependent flavin reductase, and NpdB is a hydroxyquinol 1,2-dioxygenase. The npd gene cluster also includes a putative maleylacetate reductase gene, npdC. In an in vitro assay containing NpdA2, an E. coli lysate transforms p-nitrophenol stoichiometrically to hydroquinone and hydroxyquinol. It was concluded that the p-nitrophenol catabolic pathway in JS443 most likely begins with a two-step transformation of p-nitrophenol to hydroxy-1,4-benzoquinone, catalyzed by NpdA2. Hydroxy-1,4-benzoquinone is reduced to hydroxyquinol, which is degraded through the hydroxyquinol ortho cleavage pathway. The hydroquinone detected in vitro is a dead-end product most likely resulting from chemical or enzymatic reduction of the hypothetical intermediate 1,4-benzoquinone. NpdA2 hydroxylates a broad range of chloro- and nitro-substituted phenols, resorcinols, and catechols. Only p-nitro- or p-chloro-substituted phenols are hydroxylated twice. Other substrates are hydroxylated once, always at a position para to a hydroxyl group. PMID:17720792

Camphor revisited: involvement of a unique monooxygenase in metabolism of 2-oxo-delta 3-4,5,5-trimethylcyclopentenylacetic acid by Pseudomonas putida.

PubMed Central

Ougham, H J; Taylor, D G; Trudgill, P W

1983-01-01

Previously, Pseudomonas putida was shown to degrade (+)-camphor, and cleavage of the first ring of the bicyclic structure involved two monooxygenases (a hydroxylase and a ring oxygen-inserting enzyme), a dehydrogenase, and spontaneous cleavage of an unstable oxygenation product (lactone). Cleavage of the second ring was not demonstrated but was assumed also to occur by ring oxygen insertion, since the predicted oxygenation product was extracted from whole-cell incubation systems. Our investigation established that metabolism of the first ring cleavage intermediate, 2-oxo-delta 3-4,5,5-trimethylcyclopentenylacetic acid, occurred through the sequential action of two inducible enzymes, a coenzyme A ester synthetase and an oxygenase. The oxygenase was purified to homogeneity and had a molecular weight of 106,000. This enzyme carried a single molecule of flavin adenine dinucleotide and consisted of two identical subunits. Iron was not present at a significant level. The oxygenase was specific for NADPH as the electron donor and absolutely specific for the coenzyme A ester of 2-oxo-delta 3-4,5,5-trimethylcyclopentenylacetic acid as the substrate. The reaction stoichiometry was compatible with this enzyme being a monooxygenase, and a mass spectral analysis of the methyl ester of the product confirmed the insertion of a single oxygen atom. The enzyme appeared to be analogous to, although distinct from. 2,5-diketocamphane 1,2-monooxygenase in catalyzing a "biological Baeyer-Villiger" reaction with the formation of a lactone. Structural analogy suggested that this lactone, like the first, was also unstable and susceptible to spontaneous ring opening, although this was not experimentally established. Images PMID:6848481

Camphor revisited: involvement of a unique monooxygenase in metabolism of 2-oxo-delta 3-4,5,5-trimethylcyclopentenylacetic acid by Pseudomonas putida.

PubMed

Ougham, H J; Taylor, D G; Trudgill, P W

1983-01-01

Previously, Pseudomonas putida was shown to degrade (+)-camphor, and cleavage of the first ring of the bicyclic structure involved two monooxygenases (a hydroxylase and a ring oxygen-inserting enzyme), a dehydrogenase, and spontaneous cleavage of an unstable oxygenation product (lactone). Cleavage of the second ring was not demonstrated but was assumed also to occur by ring oxygen insertion, since the predicted oxygenation product was extracted from whole-cell incubation systems. Our investigation established that metabolism of the first ring cleavage intermediate, 2-oxo-delta 3-4,5,5-trimethylcyclopentenylacetic acid, occurred through the sequential action of two inducible enzymes, a coenzyme A ester synthetase and an oxygenase. The oxygenase was purified to homogeneity and had a molecular weight of 106,000. This enzyme carried a single molecule of flavin adenine dinucleotide and consisted of two identical subunits. Iron was not present at a significant level. The oxygenase was specific for NADPH as the electron donor and absolutely specific for the coenzyme A ester of 2-oxo-delta 3-4,5,5-trimethylcyclopentenylacetic acid as the substrate. The reaction stoichiometry was compatible with this enzyme being a monooxygenase, and a mass spectral analysis of the methyl ester of the product confirmed the insertion of a single oxygen atom. The enzyme appeared to be analogous to, although distinct from. 2,5-diketocamphane 1,2-monooxygenase in catalyzing a "biological Baeyer-Villiger" reaction with the formation of a lactone. Structural analogy suggested that this lactone, like the first, was also unstable and susceptible to spontaneous ring opening, although this was not experimentally established.

Chain mapping approach of Hamiltonian for FMO complex using associated, generalized and exceptional Jacobi polynomials

NASA Astrophysics Data System (ADS)

Mahdian, M.; Arjmandi, M. B.; Marahem, F.

2016-06-01

The excitation energy transfer (EET) in photosynthesis complex has been widely investigated in recent years. However, one of the main problems is simulation of this complex under realistic condition. In this paper by using the associated, generalized and exceptional Jacobi polynomials, firstly, we introduce the spectral density of Fenna-Matthews-Olson (FMO) complex. Afterward, we obtain a map that transforms the Hamiltonian of FMO complex as an open quantum system to a one-dimensional chain of oscillatory modes with only nearest neighbor interaction in which the system is coupled only to first mode of chain. The frequency and coupling strength of each mode can be analytically obtained from recurrence coefficient of mentioned orthogonal polynomials.

Flavins in Coastal Marine Sediments: New Perspectives on Diagenetic Electron Transfer

NASA Astrophysics Data System (ADS)

Monteverde, D.; Berelson, W.; Baronas, J. J.; Sanudo-Wilhelmy, S. A.

2016-02-01

Coastal marine sediments play a critical role in the global cycling of metals and nutrients, many of which undergo diagenetic alteration. Central to these transformations are redox reactions where electron-rich organic matter is oxidized via transfer to terminal electron acceptors (NO3-, MnOx, FeOx, SO42-). The flavins (flavin adenine dinucleotide [FAD], flavin mononucleotide [FMN], and riboflavin [B2]) are microbially synthesized organic coenzymes that perform both single and double electron transfer and are known to mediate reduction of insoluble metal oxides. Culture experiments have found high rates of flavin excretion in metal-reducing Shewanella and Geobacter species, however environmental measurements of these highly labile molecules have not been previously reported. Here we present porewater measurements of FAD, FMN, and B2 from San Pedro Basin. This California Borderland basin is silled, suboxic, 900 m deep, and experiences high sedimentation. Flavin concentrations ranged from pico- (FAD: 0- 60 pM; B2: 40 - 90 pM) to nanomolar (FMN: 0.4 - 1.2 nM). The concentration cascade of FMN>B2>FAD fits well within culture experiments. Interestingly, profiles of all three flavins show a near linear increase with depth from 0-30 cm and a relatively steady concentration from 30-45 cm, supporting likely in situ production. Additionally, the flavins showed a negative correlation with dissolved Fe (R2 = 0.7 for FMN, 0.8 for FAD, and 0.9 for B2), which decreased linearly with depth from 160µM to 65µM. We discuss hypothesized mechanisms controlling flavin concentrations based on a suite of sediment geochemical parameters (dissolved Fe, Mn, TCO2, δ13C, NH3, DOM, and SO42-) as well as implications for microbial redox syntrophy. These data provide a critical link between the extensive culture-based mechanistic understanding of flavin function and the sedimentary environment. Furthermore, these results demonstrate that flavins likely serve as a significant electron transfer

The Structure of the Flavoprotein Tryptophan-2-Monooxygenase, a Key Enzyme in the Formation of Galls in Plants†

PubMed Central

Gaweska, Helena M.; Taylor, Alexander B.; Hart, P. John; Fitzpatrick, Paul F.

2013-01-01

The flavoprotein tryptophan 2-monooxygenase catalyzes the oxidative decarboxylation of tryptophan to yield indole-3-acetamide. This is the initial step in the biosynthesis of the plant growth hormone indole-acetic-acid by bacterial pathogens that cause crown gall and related diseases. The structure of the enzyme from Pseudomonas savastanoi has been determined by X-ray diffraction methods to a resolution of 1.95 Å. The overall structure of the protein shows that it has the same fold as the monoamine oxidase family of flavoproteins, with the greatest similarities to the L-amino acid oxidases. The location of bound indole-3-acetamide in the active site enables identification of residues responsible for substrate binding and specificity. Two residues in the enzyme are conserved in all members of the monoamine oxidase family, Lys365 and Trp466. The K365M mutation decreases the kcat and kcat/KTrp values by 60,000 and 2 million-fold, respectively. The deuterium kinetic isotope effect increases to 3.2, consistent with carbon-hydrogen bond cleavage becoming rate-limiting in the mutant enzyme. The W466F mutation decreases the kcat value less than 2-fold and the kcat/KTrp value only 5-fold, while the W466M mutation results in enzyme lacking flavin and detectable activity. This is consistent with a role for Trp466 in maintaining the structure of the flavin binding site in the more conserved FAD domain. PMID:23521653

A biomimetic redox flow battery based on flavin mononucleotide

PubMed Central

Orita, Akihiro; Verde, Michael G.; Sakai, Masanori; Meng, Ying Shirley

2016-01-01

The versatility in design of redox flow batteries makes them apt to efficiently store energy in large-scale applications at low cost. The discovery of inexpensive organic electroactive materials for use in aqueous flow battery electrolytes is highly attractive, but is thus far limited. Here we report on a flow battery using an aqueous electrolyte based on the sodium salt of flavin mononucleotide. Flavins are highly versatile electroactive molecules, which catalyse a multitude of redox reactions in biological systems. We use nicotinamide (vitamin B3) as a hydrotropic agent to enhance the water solubility of flavin mononucleotide. A redox flow battery using flavin mononucleotide negative and ferrocyanide positive electrolytes in strong base shows stable cycling performance, with over 99% capacity retention over the course of 100 cycles. We hypothesize that this is enabled due to the oxidized and reduced forms of FMN-Na being stabilized by resonance structures. PMID:27767026

A biomimetic redox flow battery based on flavin mononucleotide

NASA Astrophysics Data System (ADS)

Orita, Akihiro; Verde, Michael G.; Sakai, Masanori; Meng, Ying Shirley

2016-10-01

The versatility in design of redox flow batteries makes them apt to efficiently store energy in large-scale applications at low cost. The discovery of inexpensive organic electroactive materials for use in aqueous flow battery electrolytes is highly attractive, but is thus far limited. Here we report on a flow battery using an aqueous electrolyte based on the sodium salt of flavin mononucleotide. Flavins are highly versatile electroactive molecules, which catalyse a multitude of redox reactions in biological systems. We use nicotinamide (vitamin B3) as a hydrotropic agent to enhance the water solubility of flavin mononucleotide. A redox flow battery using flavin mononucleotide negative and ferrocyanide positive electrolytes in strong base shows stable cycling performance, with over 99% capacity retention over the course of 100 cycles. We hypothesize that this is enabled due to the oxidized and reduced forms of FMN-Na being stabilized by resonance structures.

A biomimetic redox flow battery based on flavin mononucleotide.

PubMed

Orita, Akihiro; Verde, Michael G; Sakai, Masanori; Meng, Ying Shirley

2016-10-21

The versatility in design of redox flow batteries makes them apt to efficiently store energy in large-scale applications at low cost. The discovery of inexpensive organic electroactive materials for use in aqueous flow battery electrolytes is highly attractive, but is thus far limited. Here we report on a flow battery using an aqueous electrolyte based on the sodium salt of flavin mononucleotide. Flavins are highly versatile electroactive molecules, which catalyse a multitude of redox reactions in biological systems. We use nicotinamide (vitamin B3) as a hydrotropic agent to enhance the water solubility of flavin mononucleotide. A redox flow battery using flavin mononucleotide negative and ferrocyanide positive electrolytes in strong base shows stable cycling performance, with over 99% capacity retention over the course of 100 cycles. We hypothesize that this is enabled due to the oxidized and reduced forms of FMN-Na being stabilized by resonance structures.

Time-resolved fluorescence spectroscopic study of flavin fluorescence in purified enzymes of bioluminescent bacteria

NASA Astrophysics Data System (ADS)

Vetrova, Elena; Kudryasheva, N.; Cheng, K.

2006-10-01

Time-resolved fluorescence intensity and anisotropy decay measurements have been used to study the environment and rotational mobility of endogenous flavin in two purified enzymes of bioluminescent bacteria, Luciferase from Photobacterium leiognathi and NAD(P)H:FMN-oxidoreductase from Vibrio fischeri. We compared the time-resolved fluorescence parameters, intensity decay lifetimes, rotational correlation times, and their fractional contribution, of the endogeneous flavin fluorescence in each of the two enzymes in the presence or absence of quinones of different structures and redox potentials. The endogeneous flavin exhibited multi-exponential decay characteristics as compared to a single decay lifetime of around 5 ns for free flavin, suggesting a complex and heterogeneous environment of flavin bound to the enzyme. In addition, a significant increase in the rotational correlation time and a certain degree of ordering of the molecule were observed for endogenous flavin when compared to a single and fast rotational correlation time of 150 ps of free flavin. Quinone significantly altered both the lifetime and rotational characteristics of endogenous flavin suggesting specific interactions of quinones to the endogeneous flavin in the bacterial enzyme.

Hepatic microsomal metabolism of montelukast, a potent leukotriene D4 receptor antagonist, in humans.

PubMed

Chiba, M; Xu, X; Nishime, J A; Balani, S K; Lin, J H

1997-09-01

Montelukast (L-706,631, MK-0476, SINGULAIR), a potent and selective leukotriene D4 (CysLT1) receptor antagonist, is currently under development for the treatment of asthma. In vitro studies were conducted using human liver microsomes to evaluate: 1) the difference in the metabolic kinetics of montelukast between adult and pediatric subjects; 2) the relative contribution of flavin-containing monooxygenase and cytochrome P450 (P450) to the sulfoxidation; and 3) the P450 isoforms responsible for montelukast oxidation. No statistically significant difference was observed in the in vitro kinetics for acyl glucuronidation and oxidative metabolism between the two age groups. Results from studies on heat inactivation of flavin-containing monooxygenase and immunochemical inhibition by an anti-rat NADPH P450 reductase antibody on montelukast oxidation indicated that all oxidative metabolism of montelukast-including diastereomeric sulfoxidations, as well as 21- and methyl-hydroxylations-are catalyzed exclusively by P450. Five in vitro approaches have been used to identify the P450 isoforms responsible for the human liver microsomal oxidation of montelukast. The experimental results consistently indicated that CYP3A4 catalyzes sulfoxidation and 21-hydroxylation, whereas CYP2C9 selectively mediates methyl-hydroxylation.

Dual role of the carboxyl-terminal region of pig liver L-kynurenine 3-monooxygenase: mitochondrial-targeting signal and enzymatic activity.

PubMed

Hirai, Kumiko; Kuroyanagi, Hidehito; Tatebayashi, Yoshitaka; Hayashi, Yoshitaka; Hirabayashi-Takahashi, Kanako; Saito, Kuniaki; Haga, Seiich; Uemura, Tomihiko; Izumi, Susumu

2010-12-01

l-kynurenine 3-monooxygenase (KMO) is an NAD(P)H-dependent flavin monooxygenase that catalyses the hydroxylation of l-kynurenine to 3-hydroxykynurenine, and is localized as an oligomer in the mitochondrial outer membrane. In the human brain, KMO may play an important role in the formation of two neurotoxins, 3-hydroxykynurenine and quinolinic acid, both of which provoke severe neurodegenerative diseases. In mosquitos, it plays a role in the formation both of eye pigment and of an exflagellation-inducing factor (xanthurenic acid). Here, we present evidence that the C-terminal region of pig liver KMO plays a dual role. First, it is required for the enzymatic activity. Second, it functions as a mitochondrial targeting signal as seen in monoamine oxidase B (MAO B) or outer membrane cytochrome b(5). The first role was shown by the comparison of the enzymatic activity of two mutants (C-terminally FLAG-tagged KMO and carboxyl-terminal truncation form, KMOΔC50) with that of the wild-type enzyme expressed in COS-7 cells. The second role was demonstrated with fluorescence microscopy by the comparison of the intracellular localization of the wild-type, three carboxyl-terminal truncated forms (ΔC20, ΔC30 and ΔC50), C-terminally FLAG-tagged wild-type and a mutant KMO, where two arginine residues, Arg461-Arg462, were replaced with Ser residues.

Covalent Binding of Flavins to RnfG and RnfD in the Rnf Complex from Vibrio cholerae

PubMed Central

Backiel, Julianne; Juárez, Oscar; Zagorevski, Dmitri V.; Wang, Zhenyu; Nilges, Mark J.; Barquera, Blanca

2009-01-01

Enzymes of the Rnf family are believed to be bacterial redox-driven ion pumps, coupling an oxidoreduction process to the translocation of Na+ across the cell membrane. Here we show for the first time that Rnf is a flavoprotein, with FMN covalently bound to threonine-175 in RnfG and a second flavin bound to threonine-187 in RnfD. Rnf subunits D and G are homologous to subunits B and C of Na+-NQR, respectively. Each of these Na+-NQR subunits includes a conserved S(T)GAT motif, with FMN covalently bound to the final threonine. RnfD and RnfG both contain the same motif, suggesting that they bind flavins in a similar way. In order to investigate this, the genes for RnfD and RnfG from Vibrio cholerae were cloned and expressed individually in that organism. In both cases the produced protein fluoresced under UV illumination on an SDS gel, further indicating the presence of flavin. However, analysis of the mutants RnfG-T175L, RnfD-T278L, and RnfD-T187V showed that RnfG-T175 and RnfD-T187 are the likely flavin ligands. This indicates that, in the case of RnfD, the flavin is bound, not to the SGAT sequence but to the final residues of a TMAT sequence, a novel variant of the flavin binding motif. In the case of RnfG, flavin analysis, followed by MALDI-TOF-TOF mass spectrometry, showed that an FMN is covalently attached to threonine-175, the final threonine of the S(T)GAT sequence. Studies by visible, EPR, and ENDOR spectroscopy showed that, upon partial reduction, the isolated RnfG produces a neutral semiquinone intermediate. The semiquinone species disappeared upon full reduction and was not observed in the denatured protein. A topological analysis combining reporter protein fusion and computer predictions indicated that the flavins in RnfG and RnfD are localized in the periplasmic space. In contrast, in NqrC and NqrB the flavins are located in a cytoplasmic loop. This topological analysis suggests that there may be mechanistic differences between the Rnf and Na

Enhancing Bidirectional Electron Transfer of Shewanella oneidensis by a Synthetic Flavin Pathway.

PubMed

Yang, Yun; Ding, Yuanzhao; Hu, Yidan; Cao, Bin; Rice, Scott A; Kjelleberg, Staffan; Song, Hao

2015-07-17

Flavins regulate the rate and direction of extracellular electron transfer (EET) in Shewanella oneidensis. However, low concentration of endogenously secreted flavins by the wild-type S. oneidensis MR-1 limits its EET efficiency in bioelectrochemical systems (BES). Herein, a synthetic flavin biosynthesis pathway from Bacillus subtilis was heterologously expressed in S. oneidensis MR-1, resulting in ∼25.7 times' increase in secreted flavin concentration. This synthetic flavin module enabled enhanced bidirectional EET rate of MR-1, in which its maximum power output in microbial fuel cells increased ∼13.2 times (from 16.4 to 233.0 mW/m(2)), and the inward current increased ∼15.5 times (from 15.5 to 255.3 μA/cm(2)).

Effect of xenon on the excited states of phototropic receptor flavin in corn seedlings

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vierstra, R.D.; Poff, K.L.; Walker, E.B.

1981-05-01

The chemically inert, water-soluble heavy atom gas, xenon, at millimolar concentrations specifically quenches the triplet excited state of flavin in solution without quenching the flavin singlet excited state. The preferential quenching of the flavin triplet over the singlet excited state by Xe has been established by showing that the flavin triplet-sensitized photooxidation of NADH is inhibited while the fluorescence intensity and lifetime of flavin are not affected by Xe. No significant inhibition of phototropism and geotropism by Xe was observed, suggesting that a flavin singlet state is more likely involved than the triplet state in the primary photoprocess of phototropismmore » in corn.« less

Pharmacokinetics and metabolism of the novel muscarinic receptor agonist SNI-2011 in rats and dogs.

PubMed

Washio, Takuo; Kohsaka, Kazuhiro; Arisawa, Hirohiko; Masunaga, Hiroaki

2003-01-01

In this study, the pharmacokinetics of SNI-2011 ((+/-)-cis-2-methylspiro[1,3-oxathiolane-5,3'-quinuclidine]monohydrochloride hemihydrate, cevimeline, CAS 153504-70-2), a novel muscarinic acetylcholine receptor agonist developed for the treatment of Sjögren's syndrome, in rats and dogs were determined following intravenous or oral administration using liquid chromatography/mass spectrometry (LC/MS). The in vitro metabolism of SNI-2011 was also evaluated with rat and dog liver microsomes. After oral administration, plasma concentrations of SNI-2011 reached to Cmax within 1 h in both species, suggesting that SNI-2011 was quickly absorbed, and then decreased with a t1/2 of 0.4-1.1 h. The bioavailability was approximately 50% and 30% in rats and dogs, respectively. Major metabolites in plasma were both S- and N-oxidized metabolites in rats and only N-oxidized metabolite in dogs, indicating that a large species difference was observed in the metabolism of SNI-2011. Sex difference was also observed in the pharmacokinetics of SNI-2011 in rats, but not in dogs. In the in vitro study, chemical inhibition and pH-dependent studies revealed that the sulf-oxidation and N-oxidation of SNI-2011 were mediated by cytochrome P450 (CYP) and flavin-containing monooxygenase (FMO), respectively, in both species. In addition, CYP2D and CYP3A were mainly responsible for the sulfoxidation in rat liver microsomes.

Nrf2-Mediated Antioxidant Defense and Peroxiredoxin 6 Are Linked to Biosynthesis of Palmitic Acid Ester of 9-Hydroxystearic Acid.

PubMed

Kuda, Ondrej; Brezinova, Marie; Silhavy, Jan; Landa, Vladimir; Zidek, Vaclav; Dodia, Chandra; Kreuchwig, Franziska; Vrbacky, Marek; Balas, Laurence; Durand, Thierry; Hübner, Norbert; Fisher, Aron B; Kopecky, Jan; Pravenec, Michal

2018-06-01

Fatty acid esters of hydroxy fatty acids (FAHFAs) are lipid mediators with promising antidiabetic and anti-inflammatory properties that are formed in white adipose tissue (WAT) via de novo lipogenesis, but their biosynthetic enzymes are unknown. Using a combination of lipidomics in WAT, quantitative trait locus mapping, and correlation analyses in rat BXH/HXB recombinant inbred strains, as well as response to oxidative stress in murine models, we elucidated the potential pathway of biosynthesis of several FAHFAs. Comprehensive analysis of WAT samples identified ∼160 regioisomers, documenting the complexity of this lipid class. The linkage analysis highlighted several members of the nuclear factor, erythroid 2 like 2 ( Nrf2 )-mediated antioxidant defense system ( Prdx6, Mgst1, Mgst3 ), lipid-handling proteins ( Cd36, Scd6, Acnat1, Acnat2, Baat ), and the family of flavin containing monooxygenases ( Fmo ) as the positional candidate genes. Transgenic expression of Nrf2 and deletion of Prdx6 genes resulted in reduction of palmitic acid ester of 9-hydroxystearic acid (9-PAHSA) and 11-PAHSA levels, while oxidative stress induced by an inhibitor of glutathione synthesis increased PAHSA levels nonspecifically. Our results indicate that the synthesis of FAHFAs via carbohydrate-responsive element-binding protein-driven de novo lipogenesis depends on the adaptive antioxidant system and suggest that FAHFAs may link activity of this system with insulin sensitivity in peripheral tissues. © 2018 by the American Diabetes Association.

Identification of the intermediates of in vivo oxidation of 1 ,4-dioxane by monooxygenase-containing bacteria.

PubMed

Mahendra, Shaily; Petzold, Christopher J; Baidoo, Edward E; Keasling, Jay D; Alvarez-Cohen, Lisa

2007-11-01

1,4-dioxane is a probable human carcinogen and an emerging water contaminant. Monooxygenase-expressing bacteria have been shown to degrade dioxane via growth-supporting as well as cometabolic mechanisms. In this study, the intermediates of dioxane degradation by monooxygenase-expressing bacteria were determined by triple quadrupole-mass spectrometry and Fourier transform ion cyclotron resonance-mass spectrometry. The major intermediates were identified as 2-hydroxyethoxyacetic acid (HEAA), ethylene glycol, glycolate, and oxalate. Studies with uniformly labeled 14C dioxane showed that over 50% of the dioxane was mineralized to CO2 by CB1190, while 5% became biomass-associated after 48 h. Volatile organic acids and non-volatiles, respectively, accounted for 20 and 11% of the radiolabeled carbon. Although strains cometabolizing dioxane exhibited limited transformation capacities, nearly half of the initial dioxane was recovered as CO2. On the basis of these analytical results, we propose a pathway for dioxane oxidation by monooxygenase-expressing cells in which dioxane is first converted to 2-hydroxy-1,4-dioxane, which is spontaneously oxidized to HEAA. During a second monooxygenation step, HEAA is further hydroxylated, resulting in a mixture of dihydroxyethoxyacetic acids with a hydroxyl group at the ortho or para position. After cleavage of the second ether bond, small organic molecules such as ethylene glycol, glycolate, glyoxalate, and oxalate are progressively formed, which are then mineralized to CO2 via common cellular metabolic pathways. Bioremediation of dioxane via this pathway is not expected to cause an accumulation of toxic compounds in the environment.

Flavin-catalyzed redox tailoring reactions in natural product biosynthesis.

PubMed

Teufel, Robin

2017-10-15

Natural products are distinct and often highly complex organic molecules that constitute not only an important drug source, but have also pushed the field of organic chemistry by providing intricate targets for total synthesis. How the astonishing structural diversity of natural products is enzymatically generated in biosynthetic pathways remains a challenging research area, which requires detailed and sophisticated approaches to elucidate the underlying catalytic mechanisms. Commonly, the diversification of precursor molecules into distinct natural products relies on the action of pathway-specific tailoring enzymes that catalyze, e.g., acylations, glycosylations, or redox reactions. This review highlights a selection of tailoring enzymes that employ riboflavin (vitamin B2)-derived cofactors (FAD and FMN) to facilitate unusual redox catalysis and steer the formation of complex natural product pharmacophores. Remarkably, several such recently reported flavin-dependent tailoring enzymes expand the classical paradigms of flavin biochemistry leading, e.g., to the discovery of the flavin-N5-oxide - a novel flavin redox state and oxygenating species. Copyright © 2017 Elsevier Inc. All rights reserved.

Crystal Structures of SgcE6 and SgcC, the Two-Component Monooxygenase That Catalyzes Hydroxylation of a Carrier ProteinTethered Substrate during the Biosynthesis of the Enediyne Antitumor Antibiotic C-1027 in Streptomyces globisporus

DOE PAGES

Chang, Chin -Yuan; Lohman, Jeremy; Cao, Hongnan; ...

2016-08-25

C-1027 is a chromoprotein enediyne antitumor antibiotic produced by Streptomyces globisporus. In the last step of biosynthesis of the (S)-3-chloro-5-hydroxy-beta-tyrosine moiety of the C-1027 enediyne chromophore, SgcE6 and SgcC compose a two-component monooxygenase that hydroxylates the C-5 position of (S)-3-chloro-beta-tyrosine. This two-component monooxygenase is remarkable for two reasons. (i) SgcE6 specifically reacts with FAD and NADH, and (ii) SgcC is active with only the peptidyl carrier protein (PCP)-tethered substrate. To address the molecular details of substrate specificity, we determined the crystal structures of SgcE6 and SgcC at 1.66 and 2.63 A resolution, respectively. SgcE6 shares a similar β-barrel fold withmore » the class I HpaC-like flavin reductases. A flexible loop near the active site of SgcE6 plays a role in FAD binding, likely by providing sufficient space to accommodate the AMP moiety of FAD, when compared to that of FMN-utilizing homologues. SgcC shows structural similarity to a few, other known FADH 2-dependent monooxygenases and sheds light on some biochemically but not structurally characterized homologues. In conclusion, the crystal structures reported here provide insights into substrate specificity, and comparison with homologues provides a catalytic mechanism of the two-component, FADH 2-dependent monooxygenase (SgcE6 and SgcC) that catalyzes the hydroxylation of a PCP-tethered substrate.« less

Flavins in Marine Sediments: A Potentially Ubiquitous Intermediary In Microbial Electron Transfer

NASA Astrophysics Data System (ADS)

Monteverde, D.; Sylvan, J. B.; Suffridge, C.; Berelson, W.; Sanudo-Wilhelmy, S. A.; Baronas, J. J.

2016-12-01

The flavins (riboflavin, flavin mononucleotide [FMN], flavin adenine dinucleotide [FA­­D]) are a class of organic compounds synthesized by organisms to assist in redox reactions. They represent the largest class of required coenzymes, rivaled only by iron in the number of unique enzymes they bind. In addition to internal use, cultured metal-reducing organisms such as Shewanella and Geobacter have been known to release flavins into the extracellular pool to aid in external electron transfer. So called "electron shuttles" can allow organisms to overcome unfavorable geochemical zonation by transferring electrons onto a relatively distant insoluble acceptor. Despite the extensive culture work, flavins have not been systematically measured in the environment. Here we present the first set of flavin profiles from the water column and pore waters of a marine environment. Samples were taken from San Pedro Basin, a 900 meter deep, silled basin, with high seasonal inputs of organic carbon, low bottom water oxygen concentrations, and laminated sediments - making it ideal to explore variations in sediment geochemical zonations. Dissolved flavin concentrations in the water column and pore waters collected from two cores were preconcentrated via solid phase extraction and measured via LC/MS. Flavin profiles are compared to a suite of geochemical parameters as well as sediment microbial 16s rRNA data. Preliminary results show that FMN is typically an order of magnitude higher concentration than riboflavin (800-300pM versus 100-50pM). Porewater concentrations were elevated over water column values for all analytes (ranging from 100-2000pM) and displayed an increasing trend with depth in both cores. This increasing trend correlated with a decrease in dissolved Fe (ranging from 160 µM in surface sediments to 65 µM at 40 cm) and shifts in microbial diversity from sediments shallower than 5 cm depth dominated by Delta- and Gammaproteobacteria to subsurface sediments dominated by

Human Augmenter of Liver Regeneration; probing the catalytic mechanism of a flavin-dependent sulfhydryl oxidase†

PubMed Central

Schaefer-Ramadan, Stephanie; Gannon, Shawn A.; Thorpe, Colin

2013-01-01

Augmenter of liver regeneration is a member of the ERV family of small flavin-dependent sulfhydryl oxidases that contain a redox-active CxxC disulfide bond in redox communication with the isoalloxazine ring of bound FAD. These enzymes catalyze the oxidation of thiol substrates with the reduction of molecular oxygen to hydrogen peroxide. This work studies the catalytic mechanism of the short, cytokine, form of augmenter of liver regeneration (sfALR) using model thiol substrates of the enzyme. The redox potential of the proximal disulfide in sfALR was found to be approximately 57 mV more reducing than the flavin chromophore, in agreement with titration experiments. Rapid reaction studies show that dithiothreitol (DTT) generates a transient mixed disulfide intermediate with sfALR signaled by a weak charge-transfer interaction between the thiolate of C145 and the oxidized flavin. The subsequent transfer of reducing equivalents to the flavin ring is relatively slow, with a limiting apparent rate constant of 12.4 s−1. However, reoxidation of the reduced flavin by molecular oxygen is even slower (2.3 s−1 at air saturation), and thus largely limits turnover at 5 mM DTT. The nature of the charge-transfer complexes observed with DTT was explored using a range of simple monothiols to mimic the initial nucleophilic attack on the proximal disulfide. While β–mercaptoethanol is a very poor substrate of sfALR (~ 0.3 min−1 at 100 mM thiol), it rapidly generates a mixed disulfide intermediate allowing the thiolate of C145 to form a strong charge-transfer complex with the flavin. Unlike the other monothiols tested, glutathione is unable to form charge-transfer complexes and is an undetectable substrate of the oxidase. These data are rationalized on the basis of the stringent steric requirements for thiol-disulfide exchange reactions. The inability of the relatively bulky glutathione to attain the in-line geometry required for efficient disulfide exchange in sfALR may be

Evidence for Formation of a Radical-Mediated Flavin-N5 Covalent Intermediate.

PubMed

Dai, Yumin; Valentino, Hannah R; Sobrado, Pablo

2018-05-18

The redox-neutral reaction catalyzed by 2-haloacrylate hydratase (2-HAH) leads to the conversion of 2-chloroacrylate to pyruvate. Previous mechanistic studies demonstrated formation of a flavin-iminium ion as an important intermediate in the 2-HAH catalytic cycle. Time-resolved flavin absorbance studies were performed in this study and the data showed that the enzyme is capable of stabilizing both anionic and neutral flavin semiquinone species. The presence of a radical scavenger decreases the activity in a concentration-dependent manner. These data are consistent with the flavin iminium intermediate occurring via radical recombination. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The light-harvesting antenna of Chlorobium tepidum: interactions between the FMO protein and the major chlorosome protein CsmA studied by surface plasmon resonance.

PubMed

Pedersen, Marie Østergaard; Borch, Jonas; Højrup, Peter; Cox, Raymond P; Miller, Mette

2006-09-01

Green sulfur bacteria possess two external light-harvesting antenna systems, the chlorosome and the FMO protein, which participate in a sequential energy transfer to the reaction centers embedded in the cytoplasmic membrane. However, little is known about the physical interaction between these two antenna systems. We have studied the interaction between the major chlorosome protein, CsmA, and the FMO protein in Chlorobium tepidum using surface plasmon resonance (SPR). Our results show an interaction between the FMO protein and an immobilized synthetic peptide corresponding to 17 amino acids at the C terminal of CsmA. This interaction is dependent on the presence of a motif comprising six amino acids that are highly conserved in all the currently available CsmA protein sequences.

Synthesis of rigidified flavin-guanidinium ion conjugates and investigation of their photocatalytic properties.

PubMed

Schmaderer, Harald; Bhuyan, Mouchumi; König, Burkhard

2009-05-28

Flavin chromophores can mediate redox reactions upon irradiation by blue light. In an attempt to increase their catalytic efficacy, flavin derivatives bearing a guanidinium ion as oxoanion binding site were prepared. Chromophore and substrate binding site are linked by a rigid Kemp's acid structure. The molecular structure of the new flavins was confirmed by an X-ray structure analysis and their photocatalytic activity was investigated in benzyl ester cleavage, nitroarene reduction and a Diels-Alder reaction. The modified flavins photocatalyze the reactions, but the introduced substrate binding site does not enhance their performance.

Semiquantitative FMO Analysis of Substituent Effect on the Reaction of Permanganate Ion with Unsymmetrical Alkenes.

PubMed

Ogino, Toshio; Watanabe, Toru; Matsuura, Masato; Watanabe, Chikara; Ozaki, Hidetoshi

1998-04-17

The substituent effects on the reactions of permanganate ion with unsymmetrical alkenes are analyzed on the assumption of a concerted (3 + 2) cycloaddition model by using an equation obtained by approximation based on the FMO theory in which development and localization of the frontier molecular orbitals at the reaction sites with progress of the reaction are considered. The Hammett plots are successfully reproduced with the newly obtained rate data for the reactions of trans-chalcone and its derivatives and the data for methyl cinnamates, cinnamate ions, and alkyl vinyl ethers taken from the literature using FMO energies and orbital coefficients calculated by the PM3 method. It was indicated that a factor introduced to the basic equation in order to estimate the extent of localization of the molecular orbitals at the transition state is closely related to the position of the transition state along the reaction path.

UbiX is a flavin prenyltransferase required for bacterial ubiquinone biosynthesis

PubMed Central

White, Mark D.; Payne, Karl A.P.; Fisher, Karl; Marshall, Stephen A.; Parker, David; Rattray, Nicholas J.W.; Trivedi, Drupad K.; Goodacre, Royston; Rigby, Stephen E.J.; Scrutton, Nigel S.; Hay, Sam; Leys, David

2016-01-01

Ubiquinone, or coenzyme Q, is a ubiquitous lipid-soluble redox cofactor that is an essential component of electron transfer chains1. Eleven genes have been implicated in bacterial ubiquinone biosynthesis, including ubiX and ubiD, which are responsible for decarboxylation of the 3-octaprenyl-4-hydroxybenzoate precursor2. Despite structural and biochemical characterization of UbiX as an FMN-binding protein, no decarboxylase activity has been detected3–4. We report here that UbiX produces a novel flavin-derived cofactor required for the decarboxylase activity of UbiD5. UbiX acts as a flavin prenyltransferase, linking a dimethylallyl moiety to the flavin N5 and C6 atoms. This adds a fourth non-aromatic ring to the flavin isoalloxazine group. In contrast to other prenyltransferases6–7, UbiX is metal-independent and requires dimethylallyl-monophosphate as substrate. Kinetic crystallography reveals that the prenyl transferase mechanism of UbiX resembles that of the terpene synthases8. The active site environment is dominated by π-systems, which assist phosphate-C1’ bond breakage following FMN reduction, leading to formation of the N5-C1’ bond. UbiX then acts as a chaperone for adduct reorientation, via transient carbocation species, leading ultimately to formation of the dimethylallyl C3’-C6 bond. The study establishes the mechanism for formation of a new flavin-derived cofactor, extending both flavin and terpenoid biochemical repertoire. PMID:26083743

Structure and biocatalytic scope of thermophilic flavin-dependent halogenase and flavin reductase enzymes.

PubMed

Menon, Binuraj R K; Latham, Jonathan; Dunstan, Mark S; Brandenburger, Eileen; Klemstein, Ulrike; Leys, David; Karthikeyan, Chinnan; Greaney, Michael F; Shepherd, Sarah A; Micklefield, Jason

2016-10-04

Flavin-dependent halogenase (Fl-Hal) enzymes have been shown to halogenate a range of synthetic as well as natural aromatic compounds. The exquisite regioselectively of Fl-Hal enzymes can provide halogenated building blocks which are inaccessible using standard halogenation chemistries. Consequently, Fl-Hal are potentially useful biocatalysts for the chemoenzymatic synthesis of pharmaceuticals and other valuable products, which are derived from haloaromatic precursors. However, the application of Fl-Hal enzymes, in vitro, has been hampered by their poor catalytic activity and lack of stability. To overcome these issues, we identified a thermophilic tryptophan halogenase (Th-Hal), which has significantly improved catalytic activity and stability, compared with other Fl-Hal characterised to date. When used in combination with a thermostable flavin reductase, Th-Hal can efficiently halogenate a number of aromatic substrates. X-ray crystal structures of Th-Hal, and the reductase partner (Th-Fre), provide insights into the factors that contribute to enzyme stability, which could guide the discovery and engineering of more robust and productive halogenase biocatalysts.

Terbinafine Resistance Mediated by Salicylate 1-Monooxygenase in Aspergillus nidulans

PubMed Central

Graminha, Marcia A. S.; Rocha, Eleusa M. F.; Prade, Rolf A.; Martinez-Rossi, Nilce M.

2004-01-01

Resistance to antifungal agents is a recurring and growing problem among patients with systemic fungal infections. UV-induced Aspergillus nidulans mutants resistant to terbinafine have been identified, and we report here the characterization of one such gene. A sib-selected, 6.6-kb genomic DNA fragment encodes a salicylate 1-monooxygenase (salA), and a fatty acid synthase subunit (fasC) confers terbinafine resistance upon transformation of a sensitive strain. Subfragments carrying salA but not fasC confer terbinafine resistance. salA is present as a single-copy gene on chromosome VI and encodes a protein of 473 amino acids that is homologous to salicylate 1-monooxygenase, a well-characterized naphthalene-degrading enzyme in bacteria. salA transcript accumulation analysis showed terbinafine-dependent induction in the wild type and the UV-induced mutant Terb7, as well as overexpression in a strain containing the salA subgenomic DNA fragment, probably due to the multicopy effect caused by the transformation event. Additional naphthalene degradation enzyme-coding genes are present in fungal genomes, suggesting that resistance could follow degradation of the naphthalene ring contained in terbinafine. PMID:15328121

Flavins as Covalent Catalysts: New Mechanisms Emerge.

PubMed

Piano, Valentina; Palfey, Bruce A; Mattevi, Andrea

2017-06-01

With approximately 1% of proteins being flavoproteins, flavins are at the heart of a plethora of redox reactions in all areas of biology. Thanks to a series of fascinating recent discoveries, in addition to redox chemistry, covalent catalysis is now being recognized more frequently as a common strategy in flavoenzymes, with unprecedented mechanisms becoming apparent. Thus, noncanonical covalent reactions by flavins are emerging as a new pervasive concept in basic enzymology and biochemistry. These diverse enzymes are engaged in most biological processes, positioning the knowledge being gained from these new mechanisms to be translated into drugs that function through covalent mechanisms. Copyright © 2017 Elsevier Ltd. All rights reserved.

Trichomonas vaginalis Flavin Reductase 1 and its Role in Metronidazole Resistance

PubMed Central

Leitsch, David; Janssen, Brian D.; Kolarich, Daniel; Johnson, Patricia J.; Duchêne, Michael

2015-01-01

Summary The enzyme flavin reductase 1 (FR1) from Trichomonas vaginalis, formerly known as NADPH oxidase, was isolated and identified. Flavin reductase is part of the antioxidative defense in T. vaginalis and indirectly reduces molecular oxygen to hydrogen peroxide via free flavins. Importantly, a reduced or absent flavin reductase activity has been reported in metronidazole-resistant T. vaginalis, resulting in elevated intracellular oxygen levels and futile cycling of metronidazole. Interestingly, FR1 has no close homologue in any other sequenced genome, but seven full-length and three truncated isoforms exist in the T. vaginalis genome. However, out of these, only FR1 has an affinity for flavins, i.e. FMN, FAD, and riboflavin, which is high enough to be of physiological relevance. Although there are no relevant changes in the gene sequence or any alterations of the predicted FR1-mRNA structure in any of the strains studied, FR1 is not expressed in highly metronidazole-resistant strains. Transfection of a metronidazole-resistant clinical isolate (B7268), which does not express any detectable amounts of FR, with a plasmid bearing a functional FR1 gene nearly completely restored metronidazole sensitivity. Our results indicate that FR1 has a significant role in the emergence of metronidazole resistance in T. vaginalis. PMID:24256032

A major integral protein of the plant plasma membrane binds flavin.

PubMed

Lorenz, Astrid; Kaldenhoff, Ralf; Hertel, Rainer

2003-05-01

Abundant flavin binding sites have been found in membranes of plants and fungi. With flavin mononucleotide-agarose affinity columns, riboflavin-binding activity from microsomes of Cucurbita pepoL. hypocotyls was purified and identified as a specific PIP1-homologous protein of the aquaporin family. Sequences such as gi|2149955 in Phaseolus vulgaris, PIP1b of Arabidopsis thaliana, and NtAQP1 of tobacco are closely related. The identification as a riboflavin-binding protein was confirmed by binding tests with an extract of Escherichia coli cells expressing the tobacco NtAQP1 as well as leaves of transgenic tobacco plants that overexpress NtAQP1 or were inhibited in PIP1 expression by antisense constructs. When binding was assayed in the presence of dithionite, the reduced flavin formed a relatively stable association with the protein. Upon dilution under oxidizing conditions, the adduct was resolved, and free flavin reappeared with a half time of about 30 min. Such an association can also be induced photochemically, with oxidized flavin by blue light at 450 nm, in the presence of an electron donor. Several criteria, localization in the plasma membrane, high abundance, affinity to roseoflavin, and photochemistry, argue for a role of the riboflavin-binding protein PIP1 as a photoreceptor.

Mechanism of Rifampicin Inactivation in Nocardia farcinica

PubMed Central

Abdelwahab, Heba; Martin Del Campo, Julia S.; Dai, Yumin; Adly, Camelia; El-Sohaimy, Sohby; Sobrado, Pablo

2016-01-01

A novel mechanism of rifampicin (Rif) resistance has recently been reported in Nocardia farcinica. This new mechanism involves the activity of rifampicin monooxygenase (RifMO), a flavin-dependent monooxygenase that catalyzes the hydroxylation of Rif, which is the first step in the degradation pathway. Recombinant RifMO was overexpressed and purified for biochemical analysis. Kinetic characterization revealed that Rif binding is necessary for effective FAD reduction. RifMO exhibits only a 3-fold coenzyme preference for NADPH over NADH. RifMO catalyzes the incorporation of a single oxygen atom forming an unstable intermediate that eventually is converted to 2′-N-hydroxy-4-oxo-Rif. Stable C4a-hydroperoxyflavin was not detected by rapid kinetics methods, which is consistent with only 30% of the activated oxygen leading to product formation. These findings represent the first reported detailed biochemical characterization of a flavin-monooxygenase involved in antibiotic resistance. PMID:27706151

Hepatic biotransformation pathways and ruminal metabolic stability of the novel anthelmintic monepantel in sheep and cattle.

PubMed

Ballent, M; Virkel, G; Maté, L; Viviani, P; Lanusse, C; Lifschitz, A

2016-10-01

Monepantel (MNP) is a new amino-acetonitrile derivative anthelmintic drug used for the treatment of gastrointestinal (GI) nematodes in sheep. The present work investigated the main enzymatic pathways involved in the hepatic biotransformation of MNP in sheep and cattle. The metabolic stability in ruminal fluid of both the parent drug and its main metabolite (monepantel sulphone, MNPSO2 ) was characterized as well. Additionally, the relative distribution of both anthelmintic molecules between the fluid and particulate phases of the ruminal content was studied. Liver microsomal fractions from six (6) rams and five (5) steers were incubated with a 40 μm of MNP. Heat pretreatment (50 °C for 2 min) of liver microsomes was performed for inactivation of the flavin-monooxygenase (FMO) system. Additionally, MNP was incubated in the presence of 4, 40, and 80 μm of methimazole (MTZ), a FMO inhibitor, or equimolar concentrations of piperonyl butoxide (PBx), a well-known general cytochrome P450 (CYP) inhibitor. In both ruminant species, MNPSO2 was the main metabolite detected after MNP incubation with liver microsomes. The conversion rate of MNP into MNPSO2 was fivefold higher (P < 0.05) in sheep (0.15 ± 0.08 nmol/min·mg) compared to cattle. In sheep, the relative involvement of both FMO and CYP systems (FMO/CYP) was 36/64. Virtually, only the CYP system appeared to be involved in the production of MNPSO2 in cattle liver. Methimazole significantly reduced (41 to 79%) the rate of MNPSO2 production in sheep liver microsomes whereas it did not inhibit MNP oxidation in cattle liver microsomes. On the other hand, PBx inhibited the production of MNPSO2 in liver microsomes of both sheep (58 to 98%, in a dose-dependent manner) and cattle (almost 100%, independently of the PBx concentration added). The incubation of MNP and MNPSO2 with ruminal contents of both species showed a high chemical stability without evident metabolism and/or degradation as well as an extensive

Inactivation of peptidylglycine α-hydroxylating monooxygenase by cinnamic acid analogs

PubMed Central

McIntyre, Neil R.; Lowe, Edward W.; Battistini, Matthew R.; Leahy, James W.; Merkler, David J.

2016-01-01

Peptidylglycine α-amidating monooxygenase (PAM) is a bifunctional enzyme that catalyzes the final reaction in the maturation of α-amidated peptide hormones. Peptidylglycine α-hydroxylating monooxygenase (PHM) is the PAM domain responsible for the copper-, ascorbate- and O2-dependent hydroxylation of a glycine-extended peptide. Peptidylamidoglycolate lyase is the PAM domain responsible for the Zn(II)-dependent dealkylation of the α-hydroxyglycine-containing precursor to the final α-amidated peptide. We report herein that cinnamic acid and cinnamic acid analogs are inhibitors or inactivators of PHM. The inactivation chemistry exhibited by the cinnamates exhibits all the attributes of a suicide-substrate. However, we find no evidence for the formation of an irreversible linkage between cinnamate and PHM in the inactivated enzyme. Our data support the reversible formation of a Michael adduct between an active site nucleophile and cinnamate that leads to inactive enzyme. Our data are of significance given that cinnamates are found in foods, perfumes, cosmetics and pharmaceuticals. PMID:26024288

Flavins secreted by roots of iron-deficient Beta vulgaris enable mining of ferric oxide via reductive mechanisms.

PubMed

Sisó-Terraza, Patricia; Rios, Juan J; Abadía, Javier; Abadía, Anunciación; Álvarez-Fernández, Ana

2016-01-01

Iron (Fe) is abundant in soils but generally poorly soluble. Plants, with the exception of Graminaceae, take up Fe using an Fe(III)-chelate reductase coupled to an Fe(II) transporter. Whether or not nongraminaceous species can convert scarcely soluble Fe(III) forms into soluble Fe forms has deserved little attention so far. We have used Beta vulgaris, one among the many species whose roots secrete flavins upon Fe deficiency, to study whether or not flavins are involved in Fe acquisition. Flavins secreted by Fe-deficient plants were removed from the nutrient solution, and plants were compared with Fe-sufficient plants and Fe-deficient plants without flavin removal. Solubilization of a scarcely soluble Fe(III)-oxide was assessed in the presence or absence of flavins, NADH (nicotinamide adenine dinucleotide, reduced form) or plant roots, and an Fe(II) trapping agent. The removal of flavins from the nutrient solution aggravated the Fe deficiency-induced leaf chlorosis. Flavins were able to dissolve an Fe(III)-oxide in the presence of NADH. The addition of extracellular flavins enabled roots of Fe-deficient plants to reductively dissolve an Fe(III)-oxide. We concluded that root-secretion of flavins improves Fe nutrition in B. vulgaris. Flavins allow B. vulgaris roots to mine Fe from Fe(III)-oxides via reductive mechanisms. © 2015 CSIC New Phytologist © 2015 New Phytologist Trust.

Thermodynamic and electron paramagnetic resonance characterization of flavin in succinate dehydrogenase.

PubMed

Ohnishi, T; King, T E; Salerno, J C; Blum, H; Bowyer, J R; Maida, T

1981-06-10

Thermodynamic parameters of succinate dehydrogenase flavin were determined potentiometrically from the analysis of free radical signal levels as a function of the oxidation-reduction potential. Midpoint redox potentials of consecutive 1-electron transfer steps are -127 and -31 mV at pH 7.0. This corresponds to a stability constant of intermediate stability, 2.5 x 10(-2), which suggests flavin itself may be a converter from n = 2 to n = 1 electron transfer steps. The pK values of the free radical (FlH . in equilibrium Fl . -) and the fully reduced form (FlH2 in equilibrium FlH-) were estimated as 8.0 +/- 0.2 and 7.7 +/- 0.2, respectively. Succinate dehydrogenase flavosemiquinone elicits an EPR spectrum at g = 2.00 with a peak to peak width of 1.2 mT even in the protonated form, suggesting the delocalization in the unpaired electron density. A close proximity of succinate dehydrogenase flavin and iron-sulfur cluster S-1 was demonstrated based on the enhancement of flavin spin relaxation by Center S-1.

Cloning, Baeyer-Villiger Biooxidations, and Structures of the Camphor Pathway 2-Oxo-Δ3-4,5,5-Trimethylcyclopentenylacetyl-Coenzyme A Monooxygenase of Pseudomonas putida ATCC 17453

PubMed Central

Leisch, Hannes; Shi, Rong; Grosse, Stephan; Morley, Krista; Bergeron, Hélène; Cygler, Miroslaw; Iwaki, Hiroaki; Hasegawa, Yoshie

2012-01-01

A dimeric Baeyer-Villiger monooxygenase (BVMO) catalyzing the lactonization of 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-coenzyme A (CoA), a key intermediate in the metabolism of camphor by Pseudomonas putida ATCC 17453, had been initially characterized in 1983 by Ougham and coworkers (H. J. Ougham, D. G. Taylor, and P. W. Trudgill, J. Bacteriol. 153:140–152, 1983). Here we cloned and overexpressed the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) in Escherichia coli and determined its three-dimensional structure with bound flavin adenine dinucleotide (FAD) at a 1.95-Å resolution as well as with bound FAD and NADP+ at a 2.0-Å resolution. OTEMO represents the first homodimeric type 1 BVMO structure bound to FAD/NADP+. A comparison of several crystal forms of OTEMO bound to FAD and NADP+ revealed a conformational plasticity of several loop regions, some of which have been implicated in contributing to the substrate specificity profile of structurally related BVMOs. Substrate specificity studies confirmed that the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetic acid coenzyme A ester is preferred over the free acid. However, the catalytic efficiency (kcat/Km) favors 2-n-hexyl cyclopentanone (4.3 × 105 M−1 s−1) as a substrate, although its affinity (Km = 32 μM) was lower than that of the CoA-activated substrate (Km = 18 μM). In whole-cell biotransformation experiments, OTEMO showed a unique enantiocomplementarity to the action of the prototypical cyclohexanone monooxygenase (CHMO) and appeared to be particularly useful for the oxidation of 4-substituted cyclohexanones. Overall, this work extends our understanding of the molecular structure and mechanistic complexity of the type 1 family of BVMOs and expands the catalytic repertoire of one of its original members. PMID:22267661

Dissipative particle dynamics (DPD) simulations with fragment molecular orbital (FMO) based effective parameters for 1-Palmitoyl-2-oleoyl phosphatidyl choline (POPC) membrane

NASA Astrophysics Data System (ADS)

Doi, Hideo; Okuwaki, Koji; Mochizuki, Yuji; Ozawa, Taku; Yasuoka, Kenji

2017-09-01

In dissipative particle dynamics (DPD) simulations, it is necessary to use the so-called χ parameter set that express the effective interactions between particles. Recently, we have developed a new scheme to evaluate the χ parameters in a non-empirical way through a series of fragment molecular orbital (FMO) calculations. As a challenging test, we have performed the DPD simulations using the FMO-based χ parameters for a mixture of 1-Palmitoyl-2-oleoyl phosphatidyl choline (POPC) and water. The structures of both membrane and vesicle were formed successfully. The calculated structural parameters of membrane were in good agreement with experimental results.

Excitonic Energy Landscape of the Y16F Mutant of the Chlorobium tepidum Fenna-Matthews-Olson (FMO) Complex: High Resolution Spectroscopic and Modeling Studies.

PubMed

Khmelnitskiy, Anton; Saer, Rafael G; Blankenship, Robert E; Jankowiak, Ryszard

2018-04-12

We report high-resolution (low-temperature) absorption, emission, and nonresonant/resonant hole-burned (HB) spectra and results of excitonic calculations using a non-Markovian reduced density matrix theory (with an improved algorithm for parameter optimization in heterogeneous samples) obtained for the Y16F mutant of the Fenna-Matthews-Olson (FMO) trimer from the green sulfur bacterium Chlorobium tepidum. We show that the Y16F mutant is a mixture of FMO complexes with three independent low-energy traps (located near 817, 821, and 826 nm), in agreement with measured composite emission and HB spectra. Two of these traps belong to mutated FMO subpopulations characterized by significantly modified low-energy excitonic states. Hamiltonians for the two major subpopulations (Sub 821 and Sub 817 ) provide new insight into extensive changes induced by the single-point mutation in the vicinity of BChl 3 (where tyrosine Y16 was replaced with phenylalanine F16). The average decay time(s) from the higher exciton state(s) in the Y16F mutant depends on frequency and occurs on a picosecond time scale.

Time- and spectrally resolved characteristics of flavin fluorescence in U87MG cancer cells in culture

NASA Astrophysics Data System (ADS)

Horilova, Julia; Cunderlikova, Beata; Marcek Chorvatova, Alzbeta

2015-05-01

Early detection of cancer is crucial for the successful diagnostics of its presence and its subsequent treatment. To improve cancer detection, we tested the progressive multimodal optical imaging of U87MG cells in culture. A combination of steady-state spectroscopic methods with the time-resolved approach provides a new insight into the native metabolism when focused on endogenous tissue fluorescence. In this contribution, we evaluated the metabolic state of living U87MG cancer cells in culture by means of endogenous flavin fluorescence. Confocal microscopy and time-resolved fluorescence imaging were employed to gather spectrally and time-resolved images of the flavin fluorescence. We observed that flavin fluorescence in U87MG cells was predominantly localized outside the cell nucleus in mitochondria, while exhibiting a spectral maximum under 500 nm and fluorescence lifetimes under 1.4 ns, suggesting the presence of bound flavins. In some cells, flavin fluorescence was also detected inside the cell nuclei in the nucleoli, exhibiting longer fluorescence lifetimes and a red-shifted spectral maximum, pointing to the presence of free flavin. Extra-nuclear flavin fluorescence was diminished by 2-deoxyglucose, but failed to increase with 2,4-dinitrophenol, the uncoupler of oxidative phosphorylation, indicating that the cells use glycolysis, rather than oxidative phosphorylation for functioning. These gathered data are the first step toward monitoring the metabolic state of U87MG cancer cells.

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

Metabolism of MRX-I, a novel antibacterial oxazolidinone, in humans: the oxidative ring opening of 2,3-Dihydropyridin-4-one catalyzed by non-P450 enzymes.

PubMed

Meng, Jian; Zhong, Dafang; Li, Liang; Yuan, Zhengyu; Yuan, Hong; Xie, Cen; Zhou, Jialan; Li, Chen; Gordeev, Mikhail Fedorovich; Liu, Jinqian; Chen, Xiaoyan

2015-05-01

MRX-I is an analog of linezolid containing a 2,3-dihydropyridin-4-one (DHPO) ring rather than a morpholine ring. Our objectives were to characterize the major metabolic pathways of MRX-I in humans and clarify the mechanism underlying the oxidative ring opening of DHPO. After an oral dose of MRX-I (600 mg), nine metabolites were identified in humans. The principal metabolic pathway proposed involved the DHPO ring opening, generating the main metabolites in the plasma and urine: the hydroxyethyl amino propionic acid metabolite MRX445-1 and the carboxymethyl amino propionic acid metabolite MRX459. An in vitro phenotyping study demonstrated that multiple non-cytochrome P450 enzymes are involved in the formation of MRX445-1 and MRX459, including flavin-containing monooxygenase 5, short-chain dehydrogenase/reductase, aldehyde ketone reductase, and aldehyde dehydrogenase (ALDH). H2 (18)O experiments revealed that two (18)O atoms are incorporated into MRX445-1, one in the carboxyethyl group and the other in the hydroxyl group, and three (18)O atoms are incorporated into MRX459, two in the carboxymethyl group and one in the hydroxyl group. Based on these results, the mechanism proposed for the DHPO ring opening involves the metabolism of MRX-I via FMO5-mediated Baeyer-Villiger oxidation to an enol lactone, hydrolysis to an enol, and enol-aldehyde tautomerism to an aldehyde. The aldehyde is reduced by short-chain dehydrogenase/reductase, aldehyde ketone reductase, ALDH to MRX445-1, or oxidized by ALDH to MRX459. Our study suggests that few clinical adverse drug-drug interactions should be anticipated between MRX-I and cytochrome P450 inhibitors or inducers. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Comparative study of flavins binding with human serum albumin: a fluorometric, thermodynamic, and molecular dynamics approach.

PubMed

Sengupta, Abhigyan; Sasikala, Wilbee D; Mukherjee, Arnab; Hazra, Partha

2012-06-04

Flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) are derivatives of riboflavin (RF), a water-soluble vitamin, more commonly known as vitamin B(2). Flavins have attracted special attention in the last few years because of the recent discovery of a large number of flavoproteins. In this work, these flavins are used as extrinsic fluorescence markers for probing the microheterogeneous environment of a well-known transport protein, human serum albumin (HSA). Steady-state and time-resolved fluorescence experiments confirm that both FMN and FAD bind to the Sudlow's site-1 (SS1) binding pocket of HSA, where Trp214 resides. In the case of RF, a fraction of RF molecules binds at the SS1, whereas the major fraction of RF molecules remains unbound or surface bound to the protein. Moreover, flavin(s)-HSA interactions are monitored with the help of isothermal titration calorimetry, which provides free energy, enthalpy, and entropy changes of binding along with the binding constants. The molecular picture of binding interaction between flavins and HSA is well explored by docking and molecular dynamics studies. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of the gene encoding the major NAD(P)H-flavin oxidoreductase of the bioluminescent bacterium Vibrio fischeri ATCC 7744.

PubMed Central

Zenno, S; Saigo, K; Kanoh, H; Inouye, S

1994-01-01

The gene encoding the major NAD(P)H-flavin oxidoreductase (flavin reductase) of the luminous bacterium Vibrio fischeri ATCC 7744 was isolated by using synthetic oligonucleotide probes corresponding to the N-terminal amino acid sequence of the enzyme. Nucleotide sequence analysis suggested that the major flavin reductase of V. fischeri consisted of 218 amino acids and had a calculated molecular weight of 24,562. Cloned flavin reductase expressed in Escherichia coli was purified virtually to homogeneity, and its basic biochemical properties were examined. As in the major flavin reductase in crude extracts of V. fischeri, cloned flavin reductase showed broad substrate specificity and served well as a catalyst to supply reduced flavin mononucleotide (FMNH2) to the bioluminescence reaction. The major flavin reductase of V. fischeri not only showed significant similarity in amino acid sequence to oxygen-insensitive NAD(P)H nitroreductases of Salmonella typhimurium, Enterobacter cloacae, and E. coli but also was associated with a low level of nitroreductase activity. The major flavin reductase of V. fischeri and the nitroreductases of members of the family Enterobacteriaceae would thus appear closely related in evolution and form a novel protein family. Images PMID:8206830

Reconstitution of active mycobacterial binuclear iron monooxygenase complex in Escherichia coli.

PubMed

Furuya, Toshiki; Hayashi, Mika; Kino, Kuniki

2013-10-01

Bacterial binuclear iron monooxygenases play numerous physiological roles in oxidative metabolism. Monooxygenases of this type found in actinomycetes also catalyze various useful reactions and have attracted much attention as oxidation biocatalysts. However, difficulties in expressing these multicomponent monooxygenases in heterologous hosts, particularly in Escherichia coli, have hampered the development of engineered oxidation biocatalysts. Here, we describe a strategy to functionally express the mycobacterial binuclear iron monooxygenase MimABCD in Escherichia coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of the mimABCD gene expression in E. coli revealed that the oxygenase components MimA and MimC were insoluble. Furthermore, although the reductase MimB was expressed at a low level in the soluble fraction of E. coli cells, a band corresponding to the coupling protein MimD was not evident. This situation rendered the transformed E. coli cells inactive. We found that the following factors are important for functional expression of MimABCD in E. coli: coexpression of the specific chaperonin MimG, which caused MimA and MimC to be soluble in E. coli cells, and the optimization of the mimD nucleotide sequence, which led to efficient expression of this gene product. These two remedies enabled this multicomponent monooxygenase to be actively expressed in E. coli. The strategy described here should be generally applicable to the E. coli expression of other actinomycetous binuclear iron monooxygenases and related enzymes and will accelerate the development of engineered oxidation biocatalysts for industrial processes.

Sub-millitesla magnetic field effects on the recombination reaction of flavin and ascorbic acid radicals

NASA Astrophysics Data System (ADS)

Evans, Emrys W.; Kattnig, Daniel R.; Henbest, Kevin B.; Hore, P. J.; Mackenzie, Stuart R.; Timmel, Christiane R.

2016-08-01

Even though the interaction of a <1 mT magnetic field with an electron spin is less than a millionth of the thermal energy at room temperature (kBT), it still can have a profound effect on the quantum yields of radical pair reactions. We present a study of the effects of sub-millitesla magnetic fields on the photoreaction of flavin mononucleotide with ascorbic acid. Direct control of the reaction pathway is achieved by varying the rate of electron transfer from ascorbic acid to the photo-excited flavin. At pH 7.0, we verify the theoretical prediction that, apart from a sign change, the form of the magnetic field effect is independent of the initial spin configuration of the radical pair. The data agree well with model calculations based on a Green's function approach that allows multinuclear spin systems to be treated including the diffusive motion of the radicals, their spin-selective recombination reactions, and the effects of the inter-radical exchange interaction. The protonation states of the radicals are uniquely determined from the form of the magnetic field-dependence. At pH 3.0, the effects of two chemically distinct radical pair complexes combine to produce a pronounced response to ˜500 μT magnetic fields. These findings are relevant to the magnetic responses of cryptochromes (flavin-containing proteins proposed as magnetoreceptors in birds) and may aid the evaluation of effects of weak magnetic fields on other biologically relevant electron transfer processes.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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,more » 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.« less

Vibrational and vibronic coherences in the dynamics of the FMO complex

NASA Astrophysics Data System (ADS)

Liu, Xiaomeng; Kühn, Oliver

2016-12-01

The coupled exciton-vibrational dynamics of a seven site Frenkel exciton model of the Fenna-Matthews-Olson (FMO) complex is investigated using a Quantum Master Equation approach. Thereby, one vibrational mode per monomer is treated explicitly as being part of the relevant system. Emphasis is put on the comparison of this model with that of a purely excitonic relevant system. Further, the effects of two different approximations to the exciton-vibrational basis are investigated, namely the one- and two-particle description. Analysis of the vibronic and vibrational density matrix in the site basis points to the importance of on- and inter-site coherences for the exciton transfer. Here, one- and two-particle approximations give rise to qualitatively different results.

RNA-sequencing quantification of hepatic ontogeny of phase-I enzymes in mice.

PubMed

Peng, Lai; Cui, Julia Y; Yoo, Byunggil; Gunewardena, Sumedha S; Lu, Hong; Klaassen, Curtis D; Zhong, Xiao-Bo

2013-12-01

Phase-I drug metabolizing enzymes catalyze reactions of hydrolysis, reduction, and oxidation of drugs and play a critical role in drug metabolism. However, the functions of most phase-I enzymes are not mature at birth, which markedly affects drug metabolism in newborns. Therefore, characterization of the expression profiles of phase-I enzymes and the underlying regulatory mechanisms during liver maturation is needed for better estimation of using drugs in pediatric patients. The mouse is an animal model widely used for studying the mechanisms in the regulation of developmental expression of phase-I genes. Therefore, we applied RNA sequencing to provide a "true quantification" of the mRNA expression of phase-I genes in the mouse liver during development. Liver samples of male C57BL/6 mice at 12 different ages from prenatal to adulthood were used for defining the ontogenic mRNA profiles of phase-I families, including hydrolysis: carboxylesterase (Ces), paraoxonase (Pon), and epoxide hydrolase (Ephx); reduction: aldo-keto reductase (Akr), quinone oxidoreductase (Nqo), and dihydropyrimidine dehydrogenase (Dpyd); and oxidation: alcohol dehydrogenase (Adh), aldehyde dehydrogenase (Aldh), flavin monooxygenases (Fmo), molybdenum hydroxylase (Aox and Xdh), cytochrome P450 (P450), and cytochrome P450 oxidoreductase (Por). Two rapidly increasing stages of total phase-I gene expression after birth reflect functional transition of the liver during development. Diverse expression patterns were identified, and some large gene families contained the mRNA of genes that are enriched at different stages of development. Our study reveals the mRNA abundance of phase-I genes in the mouse liver during development and provides a valuable foundation for mechanistic studies in the future.

H2O2 Production in Species of the Lactobacillus acidophilus Group: a Central Role for a Novel NADH-Dependent Flavin Reductase

PubMed Central

Hertzberger, Rosanne; Arents, Jos; Dekker, Henk L.; Pridmore, R. David; Gysler, Christof; Kleerebezem, Michiel

2014-01-01

Hydrogen peroxide production is a well-known trait of many bacterial species associated with the human body. In the presence of oxygen, the probiotic lactic acid bacterium Lactobacillus johnsonii NCC 533 excretes up to 1 mM H2O2, inducing growth stagnation and cell death. Disruption of genes commonly assumed to be involved in H2O2 production (e.g., pyruvate oxidase, NADH oxidase, and lactate oxidase) did not affect this. Here we describe the purification of a novel NADH-dependent flavin reductase encoded by two highly similar genes (LJ_0548 and LJ_0549) that are conserved in lactobacilli belonging to the Lactobacillus acidophilus group. The genes are predicted to encode two 20-kDa proteins containing flavin mononucleotide (FMN) reductase conserved domains. Reductase activity requires FMN, flavin adenine dinucleotide (FAD), or riboflavin and is specific for NADH and not NADPH. The Km for FMN is 30 ± 8 μM, in accordance with its proposed in vivo role in H2O2 production. Deletion of the encoding genes in L. johnsonii led to a 40-fold reduction of hydrogen peroxide formation. H2O2 production in this mutant could only be restored by in trans complementation of both genes. Our work identifies a novel, conserved NADH-dependent flavin reductase that is prominently involved in H2O2 production in L. johnsonii. PMID:24487531

Convenient microtiter plate-based, oxygen-independent activity assays for flavin-dependent oxidoreductases based on different redox dyes

PubMed Central

Brugger, Dagmar; Krondorfer, Iris; Zahma, Kawah; Stoisser, Thomas; Bolivar, Juan M; Nidetzky, Bernd; Peterbauer, Clemens K; Haltrich, Dietmar

2014-01-01

Flavin-dependent oxidoreductases are increasingly recognized as important biocatalysts for various industrial applications. In order to identify novel activities and to improve these enzymes in engineering approaches, suitable screening methods are necessary. We developed novel microtiter-plate-based assays for flavin-dependent oxidases and dehydrogenases using redox dyes as electron acceptors for these enzymes. 2,6-dichlorophenol-indophenol, methylene green, and thionine show absorption changes between their oxidized and reduced forms in the visible range, making it easy to judge visually changes in activity. A sample set of enzymes containing both flavoprotein oxidases and dehydrogenases – pyranose 2-oxidase, pyranose dehydrogenase, cellobiose dehydrogenase, d-amino acid oxidase, and l-lactate oxidase – was selected. Assays for these enzymes are based on a direct enzymatic reduction of the redox dyes and not on the coupled detection of a reaction product as in the frequently used assays based on hydrogen peroxide formation. The different flavoproteins show low Michaelis constants with these electron acceptor substrates, and therefore these dyes need to be added in only low concentrations to assure substrate saturation. In conclusion, these electron acceptors are useful in selective, reliable and cheap MTP-based screening assays for a range of flavin-dependent oxidoreductases, and offer a robust method for library screening, which could find applications in enzyme engineering programs. PMID:24376171

Alternative Pyrimidine Biosynthesis Protein ApbE Is a Flavin Transferase Catalyzing Covalent Attachment of FMN to a Threonine Residue in Bacterial Flavoproteins*

PubMed Central

Bertsova, Yulia V.; Fadeeva, Maria S.; Kostyrko, Vitaly A.; Serebryakova, Marina V.; Baykov, Alexander A.; Bogachev, Alexander V.

2013-01-01

Na+-translocating NADH:quinone oxidoreductase (Na+-NQR) contains two flavin residues as redox-active prosthetic groups attached by a phosphoester bond to threonine residues in subunits NqrB and NqrC. We demonstrate here that flavinylation of truncated Vibrio harveyi NqrC at Thr-229 in Escherichia coli cells requires the presence of a co-expressed Vibrio apbE gene. The apbE genes cluster with genes for Na+-NQR and other FMN-binding flavoproteins in bacterial genomes and encode proteins with previously unknown function. Experiments with isolated NqrC and ApbE proteins confirmed that ApbE is the only protein factor required for NqrC flavinylation and also indicated that the reaction is Mg2+-dependent and proceeds with FAD but not FMN. Inactivation of the apbE gene in Klebsiella pneumoniae, wherein the nqr operon and apbE are well separated in the chromosome, resulted in a complete loss of the quinone reductase activity of Na+-NQR, consistent with its dependence on covalently bound flavin. Our data thus identify ApbE as a novel modifying enzyme, flavin transferase. PMID:23558683

Sub-millitesla magnetic field effects on the recombination reaction of flavin and ascorbic acid radicals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Evans, Emrys W.; Henbest, Kevin B.; Timmel, Christiane R., E-mail: christiane.timmel@chem.ox.ac.uk, E-mail: stuart.mackenzie@chem.ox.ac.uk

Even though the interaction of a <1 mT magnetic field with an electron spin is less than a millionth of the thermal energy at room temperature (k{sub B}T), it still can have a profound effect on the quantum yields of radical pair reactions. We present a study of the effects of sub-millitesla magnetic fields on the photoreaction of flavin mononucleotide with ascorbic acid. Direct control of the reaction pathway is achieved by varying the rate of electron transfer from ascorbic acid to the photo-excited flavin. At pH 7.0, we verify the theoretical prediction that, apart from a sign change, themore » form of the magnetic field effect is independent of the initial spin configuration of the radical pair. The data agree well with model calculations based on a Green’s function approach that allows multinuclear spin systems to be treated including the diffusive motion of the radicals, their spin-selective recombination reactions, and the effects of the inter-radical exchange interaction. The protonation states of the radicals are uniquely determined from the form of the magnetic field-dependence. At pH 3.0, the effects of two chemically distinct radical pair complexes combine to produce a pronounced response to ∼500 μT magnetic fields. These findings are relevant to the magnetic responses of cryptochromes (flavin-containing proteins proposed as magnetoreceptors in birds) and may aid the evaluation of effects of weak magnetic fields on other biologically relevant electron transfer processes.« less

Outer membrane cytochromes/flavin interactions in Shewanella spp.—A molecular perspective

DOE PAGES

Babanova, Sofia; Matanovic, Ivana; Cornejo, Jose; ...

2017-05-31

Extracellular electron transfer (EET) is intrinsically associated with the core phenomena of energy harvesting/energy conversion in natural ecosystems and biotechnology applications. But, the mechanisms associated with EET are complex and involve molecular interactions that take place at the “bionano interface” where biotic/abiotic interactions are usually explored. Our work provides molecular perspective on the electron transfer mechanism(s) employed by Shewanella oneidensis MR-1. Molecular docking simulations were used to explain the interfacial relationships between two outer-membrane cytochromes (OMC) OmcA and MtrC and riboflavin (RF) and flavin mononucleotide (FMN), respectively. OMC-flavin interactions were analyzed by studying the electrostatic potential, the hydrophilic/hydrophobic surface properties,more » and the van der Waals surface of the OMC proteins. As a result, it was proposed that the interactions between flavins and OMCs are based on geometrical recognition event. The possible docking positions of RF and FMN to OmcA and MtrC were also shown.« less

N-terminus determines activity and specificity of styrene monooxygenase reductases.

PubMed

Heine, Thomas; Scholtissek, Anika; Westphal, Adrie H; van Berkel, Willem J H; Tischler, Dirk

2017-12-01

Styrene monooxygenases (SMOs) are two-enzyme systems that catalyze the enantioselective epoxidation of styrene to (S)-styrene oxide. The FADH 2 co-substrate of the epoxidase component (StyA) is supplied by an NADH-dependent flavin reductase (StyB). The genome of Rhodococcus opacus 1CP encodes two SMO systems. One system, which we define as E1-type, displays homology to the SMO from Pseudomonas taiwanensis VLB120. The other system, originally reported as a fused system (RoStyA2B), is defined as E2-type. Here we found that E1-type RoStyB is inhibited by FMN, while RoStyA2B is known to be active with FMN. To rationalize the observed specificity of RoStyB for FAD, we generated an artificial reductase, designated as RoStyBart, in which the first 22 amino acid residues of RoStyB were joined to the reductase part of RoStyA2B, while the oxygenase part (A2) was removed. RoStyBart mainly purified as apo-protein and mimicked RoStyB in being inhibited by FMN. Pre-incubation with FAD yielded a turnover number at 30°C of 133.9±3.5s -1 , one of the highest rates observed for StyB reductases. RoStyBart holo-enzyme switches to a ping-pong mechanism and fluorescence analysis indicated for unproductive binding of FMN to the second (co-substrate) binding site. In summary, it is shown for the first time that optimization of the N-termini of StyB reductases allows the evolution of their activity and specificity. Copyright © 2017 Elsevier B.V. All rights reserved.

Genetic Control of Biosynthesis and Transport of Riboflavin and Flavin Nucleotides and Construction of Robust Biotechnological Producers†

PubMed Central

Abbas, Charles A.; Sibirny, Andriy A.

2011-01-01

Summary: Riboflavin [7,8-dimethyl-10-(1′-d-ribityl)isoalloxazine, vitamin B2] is an obligatory component of human and animal diets, as it serves as the precursor of flavin coenzymes, flavin mononucleotide, and flavin adenine dinucleotide, which are involved in oxidative metabolism and other processes. Commercially produced riboflavin is used in agriculture, medicine, and the food industry. Riboflavin synthesis starts from GTP and ribulose-5-phosphate and proceeds through pyrimidine and pteridine intermediates. Flavin nucleotides are synthesized in two consecutive reactions from riboflavin. Some microorganisms and all animal cells are capable of riboflavin uptake, whereas many microorganisms have distinct systems for riboflavin excretion to the medium. Regulation of riboflavin synthesis in bacteria occurs by repression at the transcriptional level by flavin mononucleotide, which binds to nascent noncoding mRNA and blocks further transcription (named the riboswitch). In flavinogenic molds, riboflavin overproduction starts at the stationary phase and is accompanied by derepression of enzymes involved in riboflavin synthesis, sporulation, and mycelial lysis. In flavinogenic yeasts, transcriptional repression of riboflavin synthesis is exerted by iron ions and not by flavins. The putative transcription factor encoded by SEF1 is somehow involved in this regulation. Most commercial riboflavin is currently produced or was produced earlier by microbial synthesis using special selected strains of Bacillus subtilis, Ashbya gossypii, and Candida famata. Whereas earlier RF overproducers were isolated by classical selection, current producers of riboflavin and flavin nucleotides have been developed using modern approaches of metabolic engineering that involve overexpression of structural and regulatory genes of the RF biosynthetic pathway as well as genes involved in the overproduction of the purine precursor of riboflavin, GTP. PMID:21646432

Genetic control of biosynthesis and transport of riboflavin and flavin nucleotides and construction of robust biotechnological producers.

PubMed

Abbas, Charles A; Sibirny, Andriy A

2011-06-01

Riboflavin [7,8-dimethyl-10-(1'-d-ribityl)isoalloxazine, vitamin B₂] is an obligatory component of human and animal diets, as it serves as the precursor of flavin coenzymes, flavin mononucleotide, and flavin adenine dinucleotide, which are involved in oxidative metabolism and other processes. Commercially produced riboflavin is used in agriculture, medicine, and the food industry. Riboflavin synthesis starts from GTP and ribulose-5-phosphate and proceeds through pyrimidine and pteridine intermediates. Flavin nucleotides are synthesized in two consecutive reactions from riboflavin. Some microorganisms and all animal cells are capable of riboflavin uptake, whereas many microorganisms have distinct systems for riboflavin excretion to the medium. Regulation of riboflavin synthesis in bacteria occurs by repression at the transcriptional level by flavin mononucleotide, which binds to nascent noncoding mRNA and blocks further transcription (named the riboswitch). In flavinogenic molds, riboflavin overproduction starts at the stationary phase and is accompanied by derepression of enzymes involved in riboflavin synthesis, sporulation, and mycelial lysis. In flavinogenic yeasts, transcriptional repression of riboflavin synthesis is exerted by iron ions and not by flavins. The putative transcription factor encoded by SEF1 is somehow involved in this regulation. Most commercial riboflavin is currently produced or was produced earlier by microbial synthesis using special selected strains of Bacillus subtilis, Ashbya gossypii, and Candida famata. Whereas earlier RF overproducers were isolated by classical selection, current producers of riboflavin and flavin nucleotides have been developed using modern approaches of metabolic engineering that involve overexpression of structural and regulatory genes of the RF biosynthetic pathway as well as genes involved in the overproduction of the purine precursor of riboflavin, GTP.

Aminobacter aminovorans NADH:flavin oxidoreductase His140: a highly conserved residue critical for NADH binding and utilization.

PubMed

Russell, Thomas R; Tu, Shiao-Chun

2004-10-12

Homodimeric FRD(Aa) Class I is an NADH:flavin oxidoreductase from Aminobacter aminovorans. It is unusual because it contains an FMN cofactor but utilizes a sequential-ordered kinetic mechanism. Because little is known about NADH-specific flavin reductases in general and FRD(Aa) in particular, this study aimed to further explore FRD(Aa) by identifying the functionalities of a key residue. A sequence alignment of FRD(Aa) with several known and hypothetical flavoproteins in the same subfamily reveals within the flavin reductase active-site domain a conserved GDH motif, which is believed to be responsible for the enzyme and NADH interaction. Mutation of the His140 in this GDH motif to alanine reduced FRD(Aa) activity to <3%. An ultrafiltration assay and fluorescence quenching demonstrated that H140A FRD(Aa) binds FMN in the same 1:1 stoichiometric ratio as the wild-type enzyme, but with slightly weakened affinity (K(d) = 0.9 microM). Anaerobic stopped-flow studies were carried out using both the native and mutated FRD(Aa). Similar to the native enzyme, H140A FRD(Aa) was also able to reduce the FMN cofactor by NADH although much less efficiently. Kinetic analysis of anaerobic reduction measurements indicated that the His140 residue of FRD(Aa) was essential to NADH binding, as well as important for the reduction of the FMN cofactor. For the native enzyme, the cofactor reduction was followed by at least one slower step in the catalytic pathway.

Flavin-Induced Oligomerization in Escherichia coli Adaptive Response Protein AidB

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hamill, Michael J.; Jost, Marco; Wong, Cintyu

2011-11-21

The process known as 'adaptive response' allows Escherichia coli to respond to small doses of DNA-methylating agents by upregulating the expression of four proteins. While the role of three of these proteins in mitigating DNA damage is well understood, the function of AidB is less clear. Although AidB is a flavoprotein, no catalytic role has been established for the bound cofactor. Here we investigate the possibility that flavin plays a structural role in the assembly of the AidB tetramer. We report the generation and biophysical characterization of deflavinated AidB and of an AidB mutant that has greatly reduced affinity formore » flavin adenine dinucleotide (FAD). Using fluorescence quenching and analytical ultracentrifugation, we find that apo AidB has a high affinity for FAD, as indicated by an apparent dissociation constant of 402.1 {+-} 35.1 nM, and that binding of substoichiometric amounts of FAD triggers a transition in the AidB oligomeric state. In particular, deflavinated AidB is dimeric, whereas the addition of FAD yields a tetramer. We further investigate the dimerization and tetramerization interfaces of AidB by determining a 2.8 {angstrom} resolution crystal structure in space group P3{sub 2} that contains three intact tetramers in the asymmetric unit. Taken together, our findings provide strong evidence that FAD plays a structural role in the formation of tetrameric AidB.« less

Purification and sequence analysis of 4-methyl-5-nitrocatechol oxygenase from Burkholderia sp. strain DNT.

PubMed Central

Haigler, B E; Suen, W C; Spain, J C

1996-01-01

4-Methyl-5-nitrocatechol (MNC) is an intermediate in the degradation of 2,4-dinitrotoluene by Burkholderia sp. strain DNT. In the presence of NADPH and oxygen, MNC monooxygenase catalyzes the removal of the nitro group from MNC to form 2-hydroxy-5-methylquinone. The gene (dntB) encoding MNC monooxygenase has been previously cloned and characterized. In order to examine the properties of MNC monooxygenase and to compare it with other enzymes, we sequenced the gene encoding the MNC monooxygenase and purified the enzyme from strain DNT. dntB was localized within a 2.2-kb ApaI DNA fragment. Sequence analysis of this fragment revealed an open reading frame of 1,644 bp with an N-terminal amino acid sequence identical to that of purified MNC monooxygenase from strain DNT. Comparison of the derived amino acid sequences with those of other genes showed that DntB contains the highly conserved ADP and flavin adenine dinucleotide (FAD) binding motifs characteristic of flavoprotein hydroxylases. MNC monooxygenase was purified to homogeneity from strain DNT by anion exchange and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein with a molecular weight of 60,200, which is consistent with the size determined from the gene sequence. The native molecular weight determined by gel filtration was 65,000, which indicates that the native enzyme is a monomer. It used either NADH or NADPH as electron donors, and NADPH was the preferred cofactor. The purified enzyme contained 1 mol of FAD per mol of protein, which is also consistent with the detection of an FAD binding motif in the amino acid sequence of DntB. MNC monooxygenase has a narrow substrate specificity. MNC and 4-nitrocatechol are good substrates whereas 3-methyl-4-nitrophenol, 3-methyl-4-nitrocatechol, 4-nitrophenol, 3-nitrophenol, and 4-chlorocatechol were not. These studies suggest that MNC monooxygenase is a flavoprotein that shares some properties with

Mechanistic insights into energy conservation by flavin-based electron bifurcation.

PubMed

Lubner, Carolyn E; Jennings, David P; Mulder, David W; Schut, Gerrit J; Zadvornyy, Oleg A; Hoben, John P; Tokmina-Lukaszewska, Monika; Berry, Luke; Nguyen, Diep M; Lipscomb, Gina L; Bothner, Brian; Jones, Anne K; Miller, Anne-Frances; King, Paul W; Adams, Michael W W; Peters, John W

2017-06-01

The recently realized biochemical phenomenon of energy conservation through electron bifurcation provides biology with an elegant means to maximize utilization of metabolic energy. The mechanism of coordinated coupling of exergonic and endergonic oxidation-reduction reactions by a single enzyme complex has been elucidated through optical and paramagnetic spectroscopic studies revealing unprecedented features. Pairs of electrons are bifurcated over more than 1 volt of electrochemical potential by generating a low-potential, highly energetic, unstable flavin semiquinone and directing electron flow to an iron-sulfur cluster with a highly negative potential to overcome the barrier of the endergonic half reaction. The unprecedented range of thermodynamic driving force that is generated by flavin-based electron bifurcation accounts for unique chemical reactions that are catalyzed by these enzymes.

Effective use of heterologous hosts for characterization of biosynthetic enzymes allows production of natural products and promotes new natural product discovery.

PubMed

Watanabe, Kenji

2014-01-01

In the past few years, there has been impressive progress in elucidating the mechanism of biosynthesis of various natural products accomplished through the use of genetic, molecular biological and biochemical techniques. Here, we present a comprehensive overview of the current results from our studies on fungal natural product biosynthetic enzymes, including nonribosomal peptide synthetase and polyketide synthase-nonribosomal peptide synthetase hybrid synthetase, as well as auxiliary enzymes, such as methyltransferases and oxygenases. Specifically, biosynthesis of the following compounds is described in detail: (i) Sch210972, potentially involving a Diels-Alder reaction that may be catalyzed by CghA, a functionally unknown protein identified by targeted gene disruption in the wild type fungus; (ii) chaetoglobosin A, formed via multi-step oxidations catalyzed by three redox enzymes, one flavin-containing monooxygenase and two cytochrome P450 oxygenases as characterized by in vivo biotransformation of relevant intermediates in our engineered Saccharomyces cerevisiae; (iii) (-)-ditryptophenaline, formed by a cytochrome P450, revealing the dimerization mechanism for the biosynthesis of diketopiperazine alkaloids; (iv) pseurotins, whose variations in the C- and O-methylations and the degree of oxidation are introduced combinatorially by multiple redox enzymes; and (v) spirotryprostatins, whose spiro-carbon moiety is formed by a flavin-containing monooxygenase or a cytochrome P450 as determined by heterologous de novo production of the biosynthetic intermediates and final products in Aspergillus niger. We close our discussion by summarizing some of the key techniques that have facilitated the discovery of new natural products, production of their analogs and identification of biosynthetic mechanisms in our study.

Biosynthetic machinery of ionophore polyether lasalocid: enzymatic construction of polyether skeleton.

PubMed

Minami, Atsushi; Oguri, Hiroki; Watanabe, Kenji; Oikawa, Hideaki

2013-08-01

Diversity of natural polycyclic polyethers originated from very simple yet versatile strategy consisting of epoxidation of linear polyene followed by epoxide opening cascade. To understand two-step enzymatic transformations at molecular basis, a flavin containing monooxygenase (EPX) Lsd18 and an epoxide hydrolase (EH) Lsd19 were selected as model enzymes for extensive investigation on substrate specificity, catalytic mechanism, cofactor requirement and crystal structure. This pioneering study on prototypical lasalocid EPX and EH provides insight into detailed mechanism of ionophore polyether assembly machinery and clarified remaining issues for polyether biosynthesis. Copyright © 2013 Elsevier Ltd. All rights reserved.

Mechanistic insights into energy conservation by flavin-based electron bifurcation

DOE PAGES

Lubner, Carolyn E.; Jennings, David P.; Mulder, David W.; ...

2017-04-10

The recently realized biochemical phenomenon of energy conservation through electron bifurcation provides biology with an elegant means to maximize utilization of metabolic energy. The mechanism of coordinated coupling of exergonic and endergonic oxidation-reduction reactions by a single enzyme complex has been elucidated through optical and paramagnetic spectroscopic studies revealing unprecedented features. Pairs of electrons are bifurcated over more than 1 volt of electrochemical potential by generating a low-potential, highly energetic, unstable flavin semiquinone and directing electron flow to an iron-sulfur cluster with a highly negative potential to overcome the barrier of the endergonic half reaction. As a result, the unprecedentedmore » range of thermodynamic driving force that is generated by flavin-based electron bifurcation accounts for unique chemical reactions that are catalyzed by these enzymes.« less

Mechanistic insights into energy conservation by flavin-based electron bifurcation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lubner, Carolyn E.; Jennings, David P.; Mulder, David W.

The recently realized biochemical phenomenon of energy conservation through electron bifurcation provides biology with an elegant means to maximize utilization of metabolic energy. The mechanism of coordinated coupling of exergonic and endergonic oxidation-reduction reactions by a single enzyme complex has been elucidated through optical and paramagnetic spectroscopic studies revealing unprecedented features. Pairs of electrons are bifurcated over more than 1 volt of electrochemical potential by generating a low-potential, highly energetic, unstable flavin semiquinone and directing electron flow to an iron-sulfur cluster with a highly negative potential to overcome the barrier of the endergonic half reaction. As a result, the unprecedentedmore » range of thermodynamic driving force that is generated by flavin-based electron bifurcation accounts for unique chemical reactions that are catalyzed by these enzymes.« less

The Role of Biotransformation and Oxidative Stress in 3,5-Dichloroaniline (3,5-DCA) Induced Nephrotoxicity in Isolated Renal Cortical Cells from Male Fischer 344 Rats

PubMed Central

Racine, Christopher R.; Ferguson, Travis; Preston, Debbie; Ward, Dakota; Ball, John; Anestis, Dianne; Valentovic, Monica; Rankin, Gary O.

2016-01-01

Among the mono- and dichloroanilines, 3,5-Dichloroaniline (3,5-DCA) is the most potent nephrotoxicant in vivo and in vitro. However, the role of renal biotransformation in 3,5-DCA induced nephrotoxicity is unknown. The current study was designed to determine the in vitro nephrotoxic potential of 3,5-DCA in isolated renal cortical cells (IRCC) obtained from male Fischer 344 rats, and the role of renal bioactivation and oxidative stress in 3,5-DCA nephrotoxicity. IRCC (~4 million cells/ml) from male rats were exposed to 3,5-DCA (0-1.0 mM) for up to 120 min. In IRCC, 3,5-DCA was cytotoxic at 1.0 mM by 60 min as evidenced by the increased release of lactate dehydrogenase (LDH), but 120 min was required for 3,5-DCA 0.5 mM to increase LDH release. In subsequent studies, IRCC were exposed to a pretreatment (antioxidant or enzyme inhibitor) prior to exposure to 3,5-DCA (1.0 mM) for 90 min. Cytotoxicity induced by 3,5-DCA was attenuated by pretreatment with inhibitors of flavin-containing monooxygenase (FMO; methimazole, N-octylamine), cytochrome P450 (CYP; piperonyl butoxide, metyrapone), or peroxidase (indomethacin, mercaptosuccinate) enzymes. Use of more selective CYP inhibitors suggested that the CYP 2C family contributed to 3,5-DCA bioactivation. Antioxidants (glutathione, N-acetyl-L-cysteine, α-tocopherol, ascorbate, pyruvate) also attenuated 3,5-DCA nephrotoxicity, but oxidized glutathione levels and the oxidized/reduced glutathione ratios were not increased. These results indicate that 3,5-DCA may be activated via several renal enzyme systems to toxic metabolites, and that free radicals, but not oxidative stress, contribute to 3,5-DCA induced nephrotoxicity in vitro. PMID:26808022

Structural basis for cellobiose dehydrogenase action during oxidative cellulose degradation

NASA Astrophysics Data System (ADS)

Tan, Tien-Chye; Kracher, Daniel; Gandini, Rosaria; Sygmund, Christoph; Kittl, Roman; Haltrich, Dietmar; Hällberg, B. Martin; Ludwig, Roland; Divne, Christina

2015-07-01

A new paradigm for cellulose depolymerization by fungi focuses on an oxidative mechanism involving cellobiose dehydrogenases (CDH) and copper-dependent lytic polysaccharide monooxygenases (LPMO); however, mechanistic studies have been hampered by the lack of structural information regarding CDH. CDH contains a haem-binding cytochrome (CYT) connected via a flexible linker to a flavin-dependent dehydrogenase (DH). Electrons are generated from cellobiose oxidation catalysed by DH and shuttled via CYT to LPMO. Here we present structural analyses that provide a comprehensive picture of CDH conformers, which govern the electron transfer between redox centres. Using structure-based site-directed mutagenesis, rapid kinetics analysis and molecular docking, we demonstrate that flavin-to-haem interdomain electron transfer (IET) is enabled by a haem propionate group and that rapid IET requires a closed CDH state in which the propionate is tightly enfolded by DH. Following haem reduction, CYT reduces LPMO to initiate oxygen activation at the copper centre and subsequent cellulose depolymerization.

Synthesis of 10-Ethyl Flavin: A Multistep Synthesis Organic Chemistry Laboratory Experiment for Upper-Division Undergraduate Students

ERIC Educational Resources Information Center

Sichula, Vincent A.

2015-01-01

A multistep synthesis of 10-ethyl flavin was developed as an organic chemistry laboratory experiment for upper-division undergraduate students. Students synthesize 10-ethyl flavin as a bright yellow solid via a five-step sequence. The experiment introduces students to various hands-on experimental organic synthetic techniques, such as column…

The P450 Monooxygenase BcABA1 Is Essential for Abscisic Acid Biosynthesis in Botrytis cinerea

PubMed Central

Siewers, Verena; Smedsgaard, Jørn; Tudzynski, Paul

2004-01-01

The phytopathogenic ascomycete Botrytis cinerea is known to produce abscisic acid (ABA), which is thought to be involved in host-pathogen interaction. Biochemical analyses had previously shown that, in contrast to higher plants, the fungal ABA biosynthesis probably does not proceed via carotenoids but involves direct cyclization of farnesyl diphosphate and subsequent oxidation steps. We present here evidence that this “direct” pathway is indeed the only one used by an ABA-overproducing strain of B. cinerea. Targeted inactivation of the gene bccpr1 encoding a cytochrome P450 oxidoreductase reduced the ABA production significantly, proving the involvement of P450 monooxygenases in the pathway. Expression analysis of 28 different putative P450 monooxygenase genes revealed two that were induced under ABA biosynthesis conditions. Targeted inactivation showed that one of these, bcaba1, is essential for ABA biosynthesis: ΔBcaba1 mutants contained no residual ABA. Thus, bcaba1 represents the first identified fungal ABA biosynthetic gene. PMID:15240257

An extended N-H bond, driven by a conserved second-order interaction, orients the flavin N5 orbital in cholesterol oxidase

NASA Astrophysics Data System (ADS)

Golden, Emily; Yu, Li-Juan; Meilleur, Flora; Blakeley, Matthew P.; Duff, Anthony P.; Karton, Amir; Vrielink, Alice

2017-01-01

The protein microenvironment surrounding the flavin cofactor in flavoenzymes is key to the efficiency and diversity of reactions catalysed by this class of enzymes. X-ray diffraction structures of oxidoreductase flavoenzymes have revealed recurrent features which facilitate catalysis, such as a hydrogen bond between a main chain nitrogen atom and the flavin redox center (N5). A neutron diffraction study of cholesterol oxidase has revealed an unusual elongated main chain nitrogen to hydrogen bond distance positioning the hydrogen atom towards the flavin N5 reactive center. Investigation of the structural features which could cause such an unusual occurrence revealed a positively charged lysine side chain, conserved in other flavin mediated oxidoreductases, in a second shell away from the FAD cofactor acting to polarize the peptide bond through interaction with the carbonyl oxygen atom. Double-hybrid density functional theory calculations confirm that this electrostatic arrangement affects the N-H bond length in the region of the flavin reactive center. We propose a novel second-order partial-charge interaction network which enables the correct orientation of the hydride receiving orbital of N5. The implications of these observations for flavin mediated redox chemistry are discussed.

Comparative Activity-Based Flavin-Dependent Oxidase Profiling.

PubMed

Krysiak, Joanna; Breinbauer, Rolf

2017-01-01

Activity-based protein profiling (ABPP) has become a powerful chemoproteomic technology allowing for the dissection of complex ligand-protein interactions in their native cellular environment. One of the biggest challenges for ABPP is the extension of the proteome coverage. In this chapter a new ABPP strategy dedicated to monoamine oxidases (MAO) is presented. These enzymes are representative examples of flavin-dependent oxidases, playing a crucial role in the regulation of nervous system signaling.

Heterologous expression and purification of kynurenine-3-monooxygenase from Pseudomonas fluorescens strain 17400.

PubMed

Crozier, Karen R; Moran, Graham R

2007-02-01

Kynurenine 3-monooxygenase (KMO) is an NADPH-dependent flavoprotein hydroxylase that catalyzes the conversion of l-Kynurenine (L-Kyn) to 3-hydroxykynurenine (3OHKyn). The reaction is central to the tryptophan degradative pathway and takes place within microglial cells defining cellular concentrations of the N-methyl-d-aspatate (NMDA) receptor agonist quinolinate and antagonist kynurenate. The influence over the cellular concentrations of these NMDA receptor effectors makes KMO an attractive target for the treatment of ischemic stroke. Pseudomonas fluorescens str 17400, expresses five activities of tryptophan catabolism including that of KMO. The KMO gene from P. fluorescens was cloned into the pET-17b plasmid using incorporated NdeI and XhoI restriction sites. This construct yielded PfKMO to 20% of total cell protein after 12h of expression at 22 degrees C without induction by isopropyl-beta-thiogalactopyranoside (IPTG). The enzyme could be readily purified using ammonium sulfate fractionation and ion exchange chromatography, resulting in pure KMO with a turnover number of 5.0 s(-1). PfKMO activity was dependent on the reduction state of the enzyme. Preparation and storage benefited from the presence of a reductant such as dithiothreitol or beta-mercaptoethanol. The loss of activity was found to be directly related to the oxidation of thiols as measured by dinitrothiobenzoate assay. Steady-state assays monitoring the consumption of dioxygen were used to measure apparent kinetic parameters and ligand perturbation of flavin fluorescence was used to determine a Kd value for both L-Kyn and the inhibitor m-nitrobenzoylalanine. PfKMO is offered as prototypical bacterial form of the enzyme to serve as a viable platform on which to base future KMO studies.

Redox-dependent substrate-cofactor interactions in the Michaelis-complex of a flavin-dependent oxidoreductase

NASA Astrophysics Data System (ADS)

Werther, Tobias; Wahlefeld, Stefan; Salewski, Johannes; Kuhlmann, Uwe; Zebger, Ingo; Hildebrandt, Peter; Dobbek, Holger

2017-07-01

How an enzyme activates its substrate for turnover is fundamental for catalysis but incompletely understood on a structural level. With redox enzymes one typically analyses structures of enzyme-substrate complexes in the unreactive oxidation state of the cofactor, assuming that the interaction between enzyme and substrate is independent of the cofactors oxidation state. Here, we investigate the Michaelis complex of the flavoenzyme xenobiotic reductase A with the reactive reduced cofactor bound to its substrates by X-ray crystallography and resonance Raman spectroscopy and compare it to the non-reactive oxidized Michaelis complex mimics. We find that substrates bind in different orientations to the oxidized and reduced flavin, in both cases flattening its structure. But only authentic Michaelis complexes display an unexpected rich vibrational band pattern uncovering a strong donor-acceptor complex between reduced flavin and substrate. This interaction likely activates the catalytic ground state of the reduced flavin, accelerating the reaction within a compressed cofactor-substrate complex.

Redox-dependent substrate-cofactor interactions in the Michaelis-complex of a flavin-dependent oxidoreductase

PubMed Central

Werther, Tobias; Wahlefeld, Stefan; Salewski, Johannes; Kuhlmann, Uwe; Zebger, Ingo; Hildebrandt, Peter; Dobbek, Holger

2017-01-01

How an enzyme activates its substrate for turnover is fundamental for catalysis but incompletely understood on a structural level. With redox enzymes one typically analyses structures of enzyme–substrate complexes in the unreactive oxidation state of the cofactor, assuming that the interaction between enzyme and substrate is independent of the cofactors oxidation state. Here, we investigate the Michaelis complex of the flavoenzyme xenobiotic reductase A with the reactive reduced cofactor bound to its substrates by X-ray crystallography and resonance Raman spectroscopy and compare it to the non-reactive oxidized Michaelis complex mimics. We find that substrates bind in different orientations to the oxidized and reduced flavin, in both cases flattening its structure. But only authentic Michaelis complexes display an unexpected rich vibrational band pattern uncovering a strong donor–acceptor complex between reduced flavin and substrate. This interaction likely activates the catalytic ground state of the reduced flavin, accelerating the reaction within a compressed cofactor–substrate complex.

Coq6 Is Responsible for the C4-deamination Reaction in Coenzyme Q Biosynthesis in Saccharomyces cerevisiae*

PubMed Central

Ozeir, Mohammad; Pelosi, Ludovic; Ismail, Alexandre; Mellot-Draznieks, Caroline; Fontecave, Marc; Pierrel, Fabien

2015-01-01

The yeast Saccharomyces cerevisiae is able to use para-aminobenzoic acid (pABA) in addition to 4-hydroxybenzoic acid as a precursor of coenzyme Q, a redox lipid essential to the function of the mitochondrial respiratory chain. The biosynthesis of coenzyme Q from pABA requires a deamination reaction at position C4 of the benzene ring to substitute the amino group with an hydroxyl group. We show here that the FAD-dependent monooxygenase Coq6, which is known to hydroxylate position C5, also deaminates position C4 in a reaction implicating molecular oxygen, as demonstrated with labeling experiments. We identify mutations in Coq6 that abrogate the C4-deamination activity, whereas preserving the C5-hydroxylation activity. Several results support that the deletion of Coq9 impacts Coq6, thus explaining the C4-deamination defect observed in Δcoq9 cells. The vast majority of flavin monooxygenases catalyze hydroxylation reactions on a single position of their substrate. Coq6 is thus a rare example of a flavin monooxygenase that is able to act on two different carbon atoms of its C4-aminated substrate, allowing its deamination and ultimately its conversion into coenzyme Q by the other proteins constituting the coenzyme Q biosynthetic pathway. PMID:26260787

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

2017-07-12

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 O 2 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.

Biotransformation and bioactivation reactions - 2015 literature highlights.

PubMed

Baillie, Thomas A; Dalvie, Deepak; Rietjens, Ivonne M C M; Cyrus Khojasteh, S

2016-05-01

Since 1972, Drug Metabolism Reviews has been recognized as one of the principal resources for researchers in pharmacological, pharmaceutical and toxicological fields to keep abreast of advances in drug metabolism science in academia and the pharmaceutical industry. With a distinguished list of authors and editors, the journal covers topics ranging from relatively mature fields, such as cytochrome P450 enzymes, to a variety of emerging fields. We hope to continue this tradition with the current compendium of mini-reviews that highlight novel biotransformation processes that were published during the past year. Each review begins with a summary of the article followed by our comments on novel aspects of the research and their biological implications. This collection of highlights is not intended to be exhaustive, but rather to be illustrative of recent research that provides new insights or approaches that advance the field of drug metabolism. Abbreviations NAPQI N-acetyl-p-benzoquinoneimine ALDH aldehyde dehydrogenase AO aldehyde oxidase AKR aldo-keto reductase CES carboxylesterase CSB cystathionine β-synthase CSE cystathionine γ-lyase P450 cytochrome P450 DHPO 2,3-dihydropyridin-4-one ESI electrospray FMO flavin monooxygenase GSH glutathione GSSG glutathione disulfide ICPMS inductively coupled plasma mass spectrometry i.p. intraperitoneal MDR multidrug-resistant NNAL 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol NNK 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone oaTOF orthogonal acceleration time-of-flight PBK physiologically based kinetic PCP pentachlorophenol SDR short-chain dehydrogenase/reductase SULT sulfotransferase TB tuberculosis.

Cytochrome P450 monooxygenases: perspectives for synthetic application.

PubMed

Urlacher, Vlada B; Eiben, Sabine

2006-07-01

Cytochrome P450 monooxygenases are versatile biocatalysts that introduce oxygen into a vast range of molecules. These enzymes catalyze diverse reactions in a regio- and stereoselective manner, and their properties have been used for drug development, bioremediation and the synthesis of fine chemicals and other useful compounds. However, the potential of P450 monooxygenases has not been fully exploited; there are some drawbacks limiting the broader implementation of these catalysts for commercial needs. Protein engineering has produced P450 enzymes with widely altered substrate specificities, substantially increased activity and higher stability. Furthermore, electrochemical and enzymatic approaches for the replacement or regeneration of NAD(P)H have been developed, enabling the more cost-effective use of P450 enzymes. In this review, we focus on the aspects relevant to the synthetic applications of P450 enzymes and their optimization for commercial needs.

Investigation of the enzymology and pharmacology of novel substrates and inhibitors of dopamine beta-monooxygenase

DOE Office of Scientific and Technical Information (OSTI.GOV)

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 inmore » 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.« less

The Structure of the Antibiotic Deactivating, N-hydroxylating Rifampicin Monooxygenase*

PubMed Central

Liu, Li-Kai; Abdelwahab, Heba; Martin Del Campo, Julia S.; Mehra-Chaudhary, Ritcha; Sobrado, Pablo; Tanner, John J.

2016-01-01

Rifampicin monooxygenase (RIFMO) catalyzes the N-hydroxylation of the natural product antibiotic rifampicin (RIF) to 2′-N-hydroxy-4-oxo-rifampicin, a metabolite with much lower antimicrobial activity. RIFMO shares moderate sequence similarity with well characterized flavoprotein monooxygenases, but the protein has not been isolated and characterized at the molecular level. Herein, we report crystal structures of RIFMO from Nocardia farcinica, the determination of the oligomeric state in solution with small angle x-ray scattering, and the spectrophotometric characterization of substrate binding. The structure identifies RIFMO as a class A flavoprotein monooxygenase and is similar in fold and quaternary structure to MtmOIV and OxyS, which are enzymes in the mithramycin and oxytetracycline biosynthetic pathways, respectively. RIFMO is distinguished from other class A flavoprotein monooxygenases by its unique middle domain, which is involved in binding RIF. Small angle x-ray scattering analysis shows that RIFMO dimerizes via the FAD-binding domain to form a bell-shaped homodimer in solution with a maximal dimension of 110 Å. RIF binding was monitored using absorbance at 525 nm to determine a dissociation constant of 13 μm. Steady-state oxygen consumption assays show that NADPH efficiently reduces the FAD only when RIF is present, implying that RIF binds before NADPH in the catalytic scheme. The 1.8 Å resolution structure of RIFMO complexed with RIF represents the precatalytic conformation that occurs before formation of the ternary E-RIF-NADPH complex. The RIF naphthoquinone blocks access to the FAD N5 atom, implying that large conformational changes are required for NADPH to reduce the FAD. A model for these conformational changes is proposed. PMID:27557658

Chromophore-Dependent Intramolecular Exciton-Vibrational Coupling in the FMO Complex: Quantification and Importance for Exciton Dynamics.

PubMed

Padula, Daniele; Lee, Myeong H; Claridge, Kirsten; Troisi, Alessandro

2017-11-02

In this paper, we adopt an approach suitable for monitoring the time evolution of the intramolecular contribution to the spectral density of a set of identical chromophores embedded in their respective environments. We apply the proposed method to the Fenna-Matthews-Olson (FMO) complex, with the objective to quantify the differences among site-dependent spectral densities and the impact of such differences on the exciton dynamics of the system. Our approach takes advantage of the vertical gradient approximation to reduce the computational demands of the normal modes analysis. We show that the region of the spectral density that is believed to strongly influence the exciton dynamics changes significantly in the timescale of tens of nanoseconds. We then studied the impact of the intramolecular vibrations on the exciton dynamics by considering a model of FMO in a vibronic basis and neglecting the interaction with the environment to isolate the role of the intramolecular exciton-vibration coupling. In agreement with the assumptions in the literature, we demonstrate that high frequency modes at energy much larger than the excitonic energy splitting have negligible influence on exciton dynamics despite the large exciton-vibration coupling. We also find that the impact of including the site-dependent spectral densities on exciton dynamics is not very significant, indicating that it may be acceptable to apply the same spectral density on all sites. However, care needs to be taken for the description of the exciton-vibrational coupling in the low frequency part of intramolecular modes because exciton dynamics is more susceptible to low frequency modes despite their small Huang-Rhys factors.

Atomic-Resolution Structure of an N(5) Flavin Adduct in D-Arginine Dehydrogenase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fu, Guoxing; Yuan, Hongling; Wang, Siming

2011-09-06

D-Arginine dehydrogenase (DADH) catalyzes the flavin-dependent oxidative deamination of D-arginine and other D-amino acids to the corresponding imino acids. The 1.07 {angstrom} atomic-resolution structure of DADH crystallized with D-leucine unexpectedly revealed a covalent N(5) flavin adduct, instead of the expected iminoleucine product in the active site. This acyl adduct has been successfully reproduced by photoreduction of DADH in the presence of 4-methyl-2-oxopentanoic acid (ketoleucine). The iminoleucine may be released readily because of weak interactions in the binding site, in contrast to iminoarginine, converted to ketoleucine, which reacts with activated FAD to form the covalently linked acyl adduct.

Blue light induced reactive oxygen species from flavin mononucleotide and flavin adenine dinucleotide on lethality of HeLa cells.

PubMed

Yang, Ming-Yeh; Chang, Chih-Jui; Chen, Liang-Yü

2017-08-01

Photodynamic therapy (PDT) is a safe and non-invasive treatment for cancers and microbial infections. Various photosensitizers and light sources have been developed for clinical cancer therapies. Flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) are the cofactor of enzymes and are used as photosensitizers in this study. Targeting hypoxia and light-triggering reactive oxygen species (ROS) are experimental strategies for poisoning tumor cells in vitro. HeLa cells are committed to apoptosis when treated with FMN or FAD and exposed to visible blue light (the maximum emitted wavelength of blue light is 462nm). Under blue light irradiation at 3.744J/cm 2 (=0.52mW/cm 2 irradiated for 2h), the minimal lethal dose is 3.125μM and the median lethal doses (LD 50 ) for FMN and FAD are 6.5μM and 7.2μM, respectively. Individual exposure to visible blue light irradiation or riboflavin photosensitizers does not produce cytotoxicity and no side effects are observed in this study. The western blotting results also show that an intrinsic apoptosis pathway is activated by the ROS during photolysis of riboflavin analogues. Blue light triggers the cytotoxicity of riboflavins on HeLa cells in vitro. Based on these results, this is a feasible and efficient of PDT with an intrinsic photosensitizer for cancer research. Copyright © 2017 Elsevier B.V. All rights reserved.

How can EPR spectroscopy help to unravel molecular mechanisms of flavin-dependent photoreceptors?

PubMed

Nohr, Daniel; Rodriguez, Ryan; Weber, Stefan; Schleicher, Erik

2015-01-01

Electron paramagnetic resonance (EPR) spectroscopy is a well-established spectroscopic method for the examination of paramagnetic molecules. Proteins can contain paramagnetic moieties in form of stable cofactors, transiently formed intermediates, or spin labels artificially introduced to cysteine sites. The focus of this review is to evaluate potential scopes of application of EPR to the emerging field of optogenetics. The main objective for EPR spectroscopy in this context is to unravel the complex mechanisms of light-active proteins, from their primary photoreaction to downstream signal transduction. An overview of recent results from the family of flavin-containing, blue-light dependent photoreceptors is given. In detail, mechanistic similarities and differences are condensed from the three classes of flavoproteins, the cryptochromes, LOV (Light-oxygen-voltage), and BLUF (blue-light using FAD) domains. Additionally, a concept that includes spin-labeled proteins and examination using modern pulsed EPR is introduced, which allows for a precise mapping of light-induced conformational changes.

How can EPR spectroscopy help to unravel molecular mechanisms of flavin-dependent photoreceptors?

PubMed Central

Nohr, Daniel; Rodriguez, Ryan; Weber, Stefan; Schleicher, Erik

2015-01-01

Electron paramagnetic resonance (EPR) spectroscopy is a well-established spectroscopic method for the examination of paramagnetic molecules. Proteins can contain paramagnetic moieties in form of stable cofactors, transiently formed intermediates, or spin labels artificially introduced to cysteine sites. The focus of this review is to evaluate potential scopes of application of EPR to the emerging field of optogenetics. The main objective for EPR spectroscopy in this context is to unravel the complex mechanisms of light-active proteins, from their primary photoreaction to downstream signal transduction. An overview of recent results from the family of flavin-containing, blue-light dependent photoreceptors is given. In detail, mechanistic similarities and differences are condensed from the three classes of flavoproteins, the cryptochromes, LOV (Light-oxygen-voltage), and BLUF (blue-light using FAD) domains. Additionally, a concept that includes spin-labeled proteins and examination using modern pulsed EPR is introduced, which allows for a precise mapping of light-induced conformational changes. PMID:26389123

On-line focusing of flavin derivatives using Dynamic pH junction-sweeping capillary electrophoresis with laser-induced fluorescence detection.

PubMed

Britz-McKibbin, Philip; Otsuka, Koji; Terabe, Shigeru

2002-08-01

Simple yet effective methods to enhance concentration sensitivity is needed for capillary electrophoresis (CE) to become a practical method to analyze trace levels of analytes in real samples. In this report, the development of a novel on-line preconcentration technique combining dynamic pH junction and sweeping modes of focusing is applied to the sensitive and selective analysis of three flavin derivatives: riboflavin, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). Picomolar (pM) detectability of flavins by CE with laser-induced fluorescence (LIF) detection is demonstrated through effective focusing of large sample volumes (up to 22% capillary length) using a dual pH junction-sweeping focusing mode. This results in greater than a 1,200-fold improvement in sensitivity relative to conventional injection methods, giving a limit of detection (S/N = 3) of approximately 4.0 pM for FAD and FMN. Flavin focusing is examined in terms of analyte mobility dependence on buffer pH, borate complexation and SDS interaction. Dynamic pH junction-sweeping extends on-line focusing to both neutral (hydrophobic) and weakly acidic (hydrophilic) species and is considered useful in cases when either conventional sweeping or dynamic pH junction techniques used alone are less effective for certain classes of analytes. Enhanced focusing performance by this hyphenated method was demonstrated by greater than a 4-fold reduction in flavin bandwidth, as compared to either sweeping or dynamic pH junction, reflected by analyte detector bandwidths <0.20 cm. Novel on-line focusing strategies are required to improve sensitivity in CE, which may be applied toward more effective biochemical analysis methods for diverse types of analytes.

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

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

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

PubMed Central

Ensign, S A

1996-01-01

The inducible nature of the alkene oxidation system of Xanthobacter strain Py2 has been investigated. Cultures grown with glucose as the carbon source did not contain detectable levels of alkene monooxygenase or epoxidase, two key enzymes of alkene and epoxide metabolism. Upon addition of propylene to glucose-grown cultures, alkene monooxygenase and epoxidase activities increased and after an 11-h induction period reached levels of specific activity comparable to those in propylene-grown cells. Addition of chloramphenicol or rifampin prevented the increase in the enzyme activities. Comparison of the banding patterns of proteins present in cell extracts revealed that polypeptides with molecular masses of 43, 53, and 57 kDa accumulate in propylene-grown but not glucose-grown cells. Pulse-labeling of glucose-grown cells with [35S]methionine and [35S]cysteine revealed that the 43-, 53-, and 57-kDa proteins, as well as two additional polypeptides with molecular masses of 12 and 21 kDa, were newly synthesized upon exposure of cells to propylene or propylene oxide. The addition to glucose-grown cells of a variety of other aliphatic and chlorinated alkenes and epoxides, including ethylene, vinyl chloride (1-chloroethylene), cis- and trans-1,2-dichloroethylene, 1-chloropropylene, 1,3-dichloropropylene, 1-butylene, trans-2-butylene, isobutylene, ethylene oxide, epichlorohydrin (3-chloro-1,2-epoxypropane), 1,2-epoxybutane, cis- and trans-2,3-epoxybutane, and isobutylene oxide stimulated the synthesis of the five propylene-inducible polypeptides as well as increases in alkene monooxygenase and epoxidase activities. In contrast, acetylene, and a range of aliphatic and chlorinated alkanes, did not stimulate the synthesis of the propylene-inducible polypeptides or the increase in alkene monooxygenase and epoxidase activities. PMID:8572713

Understanding and Improving the Activity of Flavin Dependent Halogenases via Random and Targeted Mutagenesis

PubMed Central

Andorfer, Mary C.

2018-01-01

Flavin dependent halogenases (FDHs) catalyze the halogenation of organic substrates by coordinating reactions of reduced flavin, molecular oxygen, and chloride. Targeted and random mutagenesis of these enzymes has been used to both understand and alter their reactivity. These studies have led to insights into residues essential for catalysis and FDH variants with improved stability, expanded substrate scope, and altered site selectivity. Mutations throughout FDH structures have contributed to all of these advances. More recent studies have sought to rationalize the impact of these mutations on FDH function and to identify new FDHs to deepen our understanding of this enzyme class and to expand their utility for biocatalytic applications. PMID:29589959

Purification and properties of the hydroxylase component of methane monooxygenase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Patel, R.N.; Savas, J.C.

Methane monooxygenase from Methylobacterium sp. strain CRL-26 which catalyzes the oxygenation of hydrocarbons was resolved into two components, a hydroxylase and a flavoprotein. An anaerobic procedure was developed for the purification of the hydroxylase to homogeneity. The molecular weight of the hydroxylase as determined by gel filtration was 220,000, and that determined by sedimentation equilibrium analysis was about 225,000. The purified hydroxylase contained three nonidentical subunits with molecular weights of about 55,000, 40,000, and 20,000, in equal amounts as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that it is an ..cap alpha../sub 2/..beta gamma.. protein. Optical absorption spectra revealedmore » peaks near 408 and 280 nm, and fluorescence spectra revealed emission peaks at 490 and 630 nm. The purified hydroxylase contained 2.8 +/- 0.2 mol of iron and 0.5 +/- 0.1 mol of zinc per mol of protein but negligible amounts of acid-labile sulfide. The antisera prepared against the hydroxylase showed cross-reactivity with hydroxylase components in soluble extracts from other methanotrophs.« less

Distinct properties underlie flavin-based electron bifurcation in a novel electron transfer flavoprotein FixAB from Rhodopseudomonas palustris

DOE PAGES

Duan, H. Diessel; Lubner, Carolyn E.; Tokmina-Lukaszewska, Monika; ...

2018-02-09

A newly-recognized third fundamental mechanism of energy conservation in biology, electron bifurcation, uses free energy from exergonic redox reactions to drive endergonic redox reactions. Flavin-based electron bifurcation furnishes low potential electrons to demanding chemical reactions such as reduction of dinitrogen to ammonia. We employed the heterodimeric flavoenzyme FixAB from the diazotrophic bacterium Rhodopseudomonas palustris to elucidate unique properties that underpin flavin-based electron bifurcation.

Analysis of flavin oxidation and electron-transfer inhibition in Plasmodium falciparum dihydroorotate dehydrogenase.

PubMed

Malmquist, Nicholas A; Gujjar, Ramesh; Rathod, Pradipsinh K; Phillips, Margaret A

2008-02-26

Plasmodium falciparum dihydroorotate dehydrogenase (pfDHODH) is a flavin-dependent mitochondrial enzyme that provides the only route to pyrimidine biosynthesis in the parasite. Clinically significant inhibitors of human DHODH (e.g., A77 1726) bind to a pocket on the opposite face of the flavin cofactor from dihydroorotate (DHO). This pocket demonstrates considerable sequence variability, which has allowed species-specific inhibitors of the malarial enzyme to be identified. Ubiquinone (CoQ), the physiological oxidant in the reaction, has been postulated to bind this site despite a lack of structural evidence. To more clearly define the residues involved in CoQ binding and catalysis, we undertook site-directed mutagenesis of seven residues in the structurally defined A77 1726 binding site, which we term the species-selective inhibitor site. Mutation of several of these residues (H185, F188, and F227) to Ala substantially decreased the affinity of pfDHODH-specific inhibitors (40-240-fold). In contrast, only a modest increase in the Kmapp for CoQ was observed, although mutation of Y528 in particular caused a substantial reduction in kcat (40-100-fold decrease). Pre-steady-state kinetic analysis by single wavelength stopped-flow spectroscopy showed that the mutations had no effect on the rate of the DHO-dependent reductive half-reaction, but most reduced the rate of the CoQ-dependent flavin oxidation step (3-20-fold decrease), while not significantly altering the Kdox for CoQ. As with the mutants, inhibitors that bind this site block the CoQ-dependent oxidative half-reaction without affecting the DHO-dependent step. These results identify residues involved in inhibitor binding and electron transfer to CoQ. Importantly, the data provide compelling evidence that the binding sites for CoQ and species-selective site inhibitors do not overlap, and they suggest instead that inhibitors act either by blocking the electron path between flavin and CoQ or by stabilizing a

Fluorescence decay kinetics and imaging of NAD(P)H and flavins as metabolic indicators

NASA Astrophysics Data System (ADS)

Schneckenburger, Herbert; Koenig, Karsten

1992-07-01

The intrinsic fluorescence of various cell cultures in the blue and green spectral range has been attributed mainly to hydrated nicotinamide adenine dinucleotide (NADH) and flavin molecules. Their fluorescence decay curves were measured with subnanosecond resolution. The reduced coenzymes NADH and hydrated nicotinamide adenine dinucleotide phosphate NADPH, both showed a biexponential decay pattern in solution with similar time constants, but different relative intensities of the two components. They could thus be distinguished from one another as well as from their oxidized forms. The NADPH fluorescence of Saccharomyces cerevisiae was located within the cytoplasm and its organelles and was by about a factor of 4 higher for respiratory-deficient than for intact yeast strains. Intracellular flavin fluorescence showed a triexponential behavior--probably due to a superposition of protein-bound and free flavin molecules. The lifetime of the shortest component varied within the range of 0.20 to 0.50 ns between respiratory-deficient and intact yeast strains, and the relative intensity of this component was most pronounced for the intact strain DL1. Time- resolved fluorescence seems therefore to be an appropriate method of probe the function of the respiratory chain and--in the further step--to differentiate between various types of cells and tissues in medical diagnosis or environmental research.

SAR202 Genomes from the Dark Ocean Predict Pathways for the Oxidation of Recalcitrant Dissolved Organic Matter

PubMed Central

Landry, Zachary; Swan, Brandon K.; Herndl, Gerhard J.; Stepanauskas, Ramunas

2017-01-01

ABSTRACT Deep-ocean regions beyond the reach of sunlight contain an estimated 615 Pg of dissolved organic matter (DOM), much of which persists for thousands of years. It is thought that bacteria oxidize DOM until it is too dilute or refractory to support microbial activity. We analyzed five single-amplified genomes (SAGs) from the abundant SAR202 clade of dark-ocean bacterioplankton and found they encode multiple families of paralogous enzymes involved in carbon catabolism, including several families of oxidative enzymes that we hypothesize participate in the degradation of cyclic alkanes. The five partial genomes encoded 152 flavin mononucleotide/F420-dependent monooxygenases (FMNOs), many of which are predicted to be type II Baeyer-Villiger monooxygenases (BVMOs) that catalyze oxygen insertion into semilabile alicyclic alkanes. The large number of oxidative enzymes, as well as other families of enzymes that appear to play complementary roles in catabolic pathways, suggests that SAR202 might catalyze final steps in the biological oxidation of relatively recalcitrant organic compounds to refractory compounds that persist. PMID:28420738

Augmenter of liver regeneration: substrate specificity of a flavin-dependent oxidoreductase from the mitochondrial intermembrane space.

PubMed

Daithankar, Vidyadhar N; Farrell, Scott R; Thorpe, Colin

2009-06-09

Augmenter of liver regeneration (ALR) is both a growth factor and a sulfhydryl oxidase that binds FAD in an unusual helix-rich domain containing a redox-active CxxC disulfide proximal to the flavin ring. In addition to the cytokine form of ALR (sfALR) that circulates in serum, a longer form, lfALR, is believed to participate in oxidative trapping of reduced proteins entering the mitochondrial intermembrane space (IMS). This longer form has an 80-residue N-terminal extension containing an additional, distal, CxxC motif. This work presents the first enzymological characterization of human lfALR. The N-terminal region conveys no catalytic advantage toward the oxidation of the model substrate dithiothreitol (DTT). In addition, a C71A or C74A mutation of the distal disulfide does not increase the turnover number toward DTT. Unlike Erv1p, the yeast homologue of lfALR, static spectrophotometric experiments with the human oxidase provide no evidence of communication between distal and proximal disulfides. An N-terminal His-tagged version of human Mia40, a resident oxidoreductase of the IMS and a putative physiological reductant of lfALR, was subcloned and expressed in Escherichia coli BL21 DE3 cells. Mia40, as isolated, shows a visible spectrum characteristic of an Fe-S center and contains 0.56 +/- 0.02 atom of iron per subunit. Treatment of Mia40 with guanidine hydrochloride and triscarboxyethylphosphine hydrochloride during purification removed this chromophore. The resulting protein, with a reduced CxC motif, was a good substrate of lfALR. However, neither sfALR nor lfALR mutants lacking the distal disulfide could oxidize reduced Mia40 efficiently. Thus, catalysis involves a flow of reducing equivalents from the reduced CxC motif of Mia40 to distal and then proximal CxxC motifs of lfALR to the flavin ring and, finally, to cytochrome c or molecular oxygen.

Augmenter of Liver Regeneration: Substrate Specificity of a Flavin-dependent Oxidoreductase from the Mitochondrial Intermembrane Space†

PubMed Central

Daithankar, Vidyadhar N.; Farrell, Scott R.; Thorpe, Colin

2009-01-01

Augmenter of liver regeneration (ALR) is both a growth factor and a sulfhydryl oxidase that binds FAD in an unusual helix-rich domain containing a redox-active CxxC disulfide proximal to the flavin ring. In addition to the cytokine form of ALR (sfALR) that circulates in serum, a longer form, lfALR, is believed to participate in oxidative trapping of reduced proteins entering the mitochondrial intermembrane space (IMS). This longer form has an 80-residue N-terminal extension containing an additional, distal, CxxC motif. This work presents the first enzymological characterization of human lfALR. The N-terminal region conveys no catalytic advantage towards the oxidation of the model substrate dithiothreitol (DTT). In addition, C71A or C74A mutations of the distal disulfide do not increase the turnover number towards DTT. Unlike Erv1p, the yeast homolog of lfALR, static spectrophotometric experiments of the human oxidase provide no evidence for communication between distal and proximal disulfides. An N-terminal his-tagged version of human Mia40, a resident oxidoreductase of the IMS and a putative physiological reductant of lfALR, was subcloned and expressed in Escherichia coli BL21 DE3 cells. Mia40, as isolated, shows a visible spectrum characteristic of an Fe/S center and contains 0.56 ± 0.02 atoms of iron per subunit. Treatment of Mia40 with guanidine hydrochloride and triscarboxyethylphosphine hydrochloride during purification removed this chromophore. The resulting protein, with a reduced CxC motif, was a good substrate of lfALR. However, neither sfALR, nor lfALR mutants lacking the distal disulfide, could oxidize reduced Mia40 efficiently. Thus, catalysis involves a flow of reducing equivalents from the reduced CxC motif of Mia40, to distal- and then proximal CxxC motifs of lfALR, to the flavin ring, and, finally, to cytochrome c or molecular oxygen. PMID:19397338

Crystallographic, Spectroscopic, and Computational Analysis of a Flavin-C4a-Oxygen Adduct in Choline Oxidase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Orville, A.M.; Lountos, G. T.; Finnegan, S.

2009-02-03

Flavin C4a-OO(H) and C4a-OH adducts are critical intermediates proposed in many flavoenzyme reaction mechanisms, but they are rarely detected even by rapid transient kinetics methods. We observe a trapped flavin C4a-OH or C4a-OO(H) adduct by single-crystal spectroscopic methods and in the 1.86 {angstrom} resolution X-ray crystal structure of choline oxidase. The microspectrophotometry results show that the adduct forms rapidly in situ at 100 K upon exposure to X-rays. Density functional theory calculations establish the electronic structures for the flavin C4a-OH and C4a-OO(H) adducts and estimate the stabilization energy of several active site hydrogen bonds deduced from the crystal structure. Wemore » propose that the enzyme-bound FAD is reduced in the X-ray beam. The aerobic crystals then form either a C4a-OH or C4a-OO(H) adduct, but an insufficient proton inventory prevents their decay at cryogenic temperatures.« less

Crystallographic, Spectroscopic, and Computational Analysis of a Flavin C4a-Oxygen Adduct in Choline Oxidase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Orville, A.; Lountos, G; Finnegan, S

2009-01-01

Flavin C4a-OO(H) and C4a-OH adducts are critical intermediates proposed in many flavoenzyme reaction mechanisms, but they are rarely detected even by rapid transient kinetics methods. We observe a trapped flavin C4a-OH or C4a-OO(H) adduct by single-crystal spectroscopic methods and in the 1.86 {angstrom} resolution X-ray crystal structure of choline oxidase. The microspectrophotometry results show that the adduct forms rapidly in situ at 100 K upon exposure to X-rays. Density functional theory calculations establish the electronic structures for the flavin C4a-OH and C4a-OO(H) adducts and estimate the stabilization energy of several active site hydrogen bonds deduced from the crystal structure. Wemore » propose that the enzyme-bound FAD is reduced in the X-ray beam. The aerobic crystals then form either a C4a-OH or C4a-OO(H) adduct, but an insufficient proton inventory prevents their decay at cryogenic temperatures.« less

Efficiency of the energy transfer in the FMO complex using hierarchical equations on Graphics Processing Units

NASA Astrophysics Data System (ADS)

Kramer, Tobias; Kreisbeck, Christoph; Rodriguez, Mirta; Hein, Birgit

2011-03-01

We study the efficiency of the energy transfer in the Fenna-Matthews-Olson complex solving the non-Markovian hierarchical equations (HE) proposed by Ishizaki and Fleming in 2009, which include properly the reorganization process. We compare it to the Markovian approach and find that the Markovian dynamics overestimates the thermalization rate, yielding higher efficiencies than the HE. Using the high-performance of graphics processing units (GPU) we cover a large range of reorganization energies and temperatures and find that initial quantum beatings are important for the energy distribution, but of limited influence to the efficiency. Our efficient GPU implementation of the HE allows us to calculate nonlinear spectra of the FMO complex. References see www.quantumdynamics.de

Catalytic mechanism of phenylacetone monooxygenases for non-native linear substrates.

PubMed

Carvalho, Alexandra T P; Dourado, Daniel F A R; Skvortsov, Timofey; de Abreu, Miguel; Ferguson, Lyndsey J; Quinn, Derek J; Moody, Thomas S; Huang, Meilan

2017-10-11

Phenylacetone monooxygenase (PAMO) is the most stable and thermo-tolerant member of the Baeyer-Villiger monooxygenase family, and therefore it is an ideal candidate for the synthesis of industrially relevant compounds. However, its limited substrate scope has largely limited its industrial applications. In the present work, we provide, for the first time, the catalytic mechanism of PAMO for the native substrate phenylacetone as well as for a linear non-native substrate 2-octanone, using molecular dynamics simulations, quantum mechanics and quantum mechanics/molecular mechanics calculations. We provide a theoretical basis for the preference of the enzyme for the native aromatic substrate over non-native linear substrates. Our study provides fundamental atomic-level insights that can be employed in the rational engineering of PAMO for wide applications in industrial biocatalysis, in particular, in the biotransformation of long-chain aliphatic oils into potential biodiesels.

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. © 2013 International Union of Biochemistry and Molecular Biology, Inc.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Kyung-Jin, E-mail: kkj@postech.ac.kr; Kim, Sujin; Lee, Sujin

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 crystalmore » 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.« less

Effects of soluble flavin on heterogeneous electron transfer between surface-exposed bacterial cytochromes and iron oxides

NASA Astrophysics Data System (ADS)

Wang, Zheming; Shi, Zhi; Shi, Liang; White, Gaye F.; Richardson, David J.; Clarke, Thomas A.; Fredrickson, Jim K.; Zachara, John M.

2015-08-01

Dissimilatory iron-reducing bacteria can utilize insoluble Fe(Mn)-oxides as a terminal electron acceptor under anaerobic conditions. For Shewanella species specifically, evidence suggests that iron reduction is associated with the secretion of flavin mononucleotide (FMN) and riboflavin. However, the exact mechanism of flavin involvement is unclear; while some indicate that flavins mediate electron transfer (Marsili et al., 2008), others point to flavin serving as co-factors to outer membrane proteins (Okamoto et al., 2013). In this work, we used methyl viologen (MVrad +)-encapsulated, porin-cytochrome complex (MtrCAB) embedded liposomes (MELs) as a synthetic model of the Shewanella outer membrane to investigate the proposed mediating behavior of microbially produced flavins. The reduction kinetics of goethite, hematite and lepidocrocite (200 μM) by MELs ([MVrad +] ∼ 40 μM and MtrABC ⩽ 1 nM) were determined in the presence FMN at pH 7.0 in N2 atmosphere by monitoring the concentrations of MVrad + and FMN through their characteristic UV-visible absorption spectra. Experiments were performed where (i) FMN and Fe(III)-oxide were mixed and then reacted with the reduced MELs and (ii) FMN was reacted with the reduced MELs followed by addition of Fe(III)-oxide. The redox reactions proceeded in two steps: a fast step that was completed in a few seconds, and a slower one lasting over 400 s. For all three Fe(III)-oxides, the initial reaction rate in the presence of a low concentration of FMN (⩽1 μM) was at least a factor of five faster than those with MELs alone, and orders of magnitude faster than those by FMNH2, suggesting that FMN may serve as a co-factor that enhances electron transfer from outer-membrane c-cytochromes to Fe(III)-oxides. The rate and extent of the initial reaction followed the order of lepidocrocite > hematite > goethite, the same as their reduction potentials, implying thermodynamic control on reaction rate. For LEP, with the highest reduction

Redox Modulation of Flavin and Tyrosine Determines Photoinduced Proton-coupled Electron Transfer and Photoactivation of BLUF Photoreceptors

PubMed Central

Mathes, Tilo; van Stokkum, Ivo H. M.; Stierl, Manuela; Kennis, John T. M.

2012-01-01

Photoinduced electron transfer in biological systems, especially in proteins, is a highly intriguing matter. Its mechanistic details cannot be addressed by structural data obtained by crystallography alone because this provides only static information on a given redox system. In combination with transient spectroscopy and site-directed manipulation of the protein, however, a dynamic molecular picture of the ET process may be obtained. In BLUF (blue light sensors using FAD) photoreceptors, proton-coupled electron transfer between a tyrosine and the flavin cofactor is the key reaction to switch from a dark-adapted to a light-adapted state, which corresponds to the biological signaling state. Particularly puzzling is the fact that, although the various naturally occurring BLUF domains show little difference in the amino acid composition of the flavin binding pocket, the reaction rates of the forward reaction differ quite largely from a few ps up to several hundred ps. In this study, we modified the redox potential of the flavin/tyrosine redox pair by site-directed mutagenesis close to the flavin C2 carbonyl and fluorination of the tyrosine, respectively. We provide information on how changes in the redox potential of either reaction partner significantly influence photoinduced proton-coupled electron transfer. The altered redox potentials allowed us furthermore to experimentally describe an excited state charge transfer intermediately prior to electron transfer in the BLUF photocycle. Additionally, we show that the electron transfer rate directly correlates with the quantum yield of signaling state formation. PMID:22833672

Reduced flavin: NMR investigation of N5-H exchange mechanism, estimation of ionisation constants and assessment of properties as biological catalyst.

PubMed

Macheroux, Peter; Ghisla, Sandro; Sanner, Christoph; Rüterjans, Heinz; Müller, Franz

2005-11-25

The flavin in its FMN and FAD forms is a versatile cofactor that is involved in catalysis of most disparate types of biological reactions. These include redox reactions such as dehydrogenations, activation of dioxygen, electron transfer, bioluminescence, blue light reception, photobiochemistry (as in photolyases), redox signaling etc. Recently, hitherto unrecognized types of biological reactions have been uncovered that do not involve redox shuffles, and might involve the reduced form of the flavin as a catalyst. The present work addresses properties of reduced flavin relevant in this context. N(5)-H exchange reactions of the flavin reduced form and its pH dependence were studied using the 15N-NMR-signals of 15N-enriched, reduced flavin in the pH range from 5 to 12. The chemical shifts of the N(3) and N(5) resonances are not affected to a relevant extent in this pH range. This contrasts with the multiplicity of the N(5)-resonance, which strongly depends on pH. It is a doublet between pH 8.45 and 10.25 that coalesces into a singlet at lower and higher pH values. From the line width of the 15N(5) signal the pH-dependent rate of hydrogen exchange was deduced. The multiplicity of the 15N(5) signal and the proton exchange rates are little dependent on the buffer system used. The exchange rates allow an estimation of the pKa value of N(5)-H deprotonation in reduced flavin to be >or= 20. This value imposes specific constraints for mechanisms of flavoprotein catalysis based on this process. On the other hand the pK asymptotically equal to 4 for N(5)-H protonation (to form N(5)+-H2) would be consistent with a role of N(5)-H as a base.

Influence of Force Fields and Quantum Chemistry Approach on Spectral Densities of BChl a in Solution and in FMO Proteins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chandrasekaran, Suryanarayanan; Aghtar, Mortaza; Valleau, Stéphanie

2015-08-06

Studies on light-harvesting (LH) systems have attracted much attention after the finding of long-lived quantum coherences in the exciton dynamics of the Fenna–Matthews–Olson (FMO) complex. In this complex, excitation energy transfer occurs between the bacteriochlorophyll a (BChl a) pigments. Two quantum mechanics/molecular mechanics (QM/MM) studies, each with a different force-field and quantum chemistry approach, reported different excitation energy distributions for the FMO complex. To understand the reasons for these differences in the predicted excitation energies, we have carried out a comparative study between the simulations using the CHARMM and AMBER force field and the Zerner intermediate neglect of differential orbitalmore » (ZINDO)/S and time-dependent density functional theory (TDDFT) quantum chemistry methods. The calculations using the CHARMM force field together with ZINDO/S or TDDFT always show a wider spread in the energy distribution compared to those using the AMBER force field. High- or low-energy tails in these energy distributions result in larger values for the spectral density at low frequencies. A detailed study on individual BChl a molecules in solution shows that without the environment, the density of states is the same for both force field sets. Including the environmental point charges, however, the excitation energy distribution gets broader and, depending on the applied methods, also asymmetric. The excitation energy distribution predicted using TDDFT together with the AMBER force field shows a symmetric, Gaussian-like distribution.« less

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

DOE PAGES

Musumeci, Matias A.; Lozada, Mariana; Rial, Daniela V.; ...

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 putativemore » 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. As a result, this work allowed the identification of putative enzyme candidates with promising features from metagenomes, providing a suitable starting point for further developments.« less

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Musumeci, Matias A.; Lozada, Mariana; Rial, Daniela V.

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 putativemore » 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. As a result, this work allowed the identification of putative enzyme candidates with promising features from metagenomes, providing a suitable starting point for further developments.« less

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.

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

PubMed Central

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

2017-01-01

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. PMID:28397770

Shewanella secretes flavins that mediate extracellular electron transfer

PubMed Central

Marsili, Enrico; Baron, Daniel B.; Shikhare, Indraneel D.; Coursolle, Dan; Gralnick, Jeffrey A.; Bond, Daniel R.

2008-01-01

Bacteria able to transfer electrons to metals are key agents in biogeochemical metal cycling, subsurface bioremediation, and corrosion processes. More recently, these bacteria have gained attention as the transfer of electrons from the cell surface to conductive materials can be used in multiple applications. In this work, we adapted electrochemical techniques to probe intact biofilms of Shewanella oneidensis MR-1 and Shewanella sp. MR-4 grown by using a poised electrode as an electron acceptor. This approach detected redox-active molecules within biofilms, which were involved in electron transfer to the electrode. A combination of methods identified a mixture of riboflavin and riboflavin-5′-phosphate in supernatants from biofilm reactors, with riboflavin representing the dominant component during sustained incubations (>72 h). Removal of riboflavin from biofilms reduced the rate of electron transfer to electrodes by >70%, consistent with a role as a soluble redox shuttle carrying electrons from the cell surface to external acceptors. Differential pulse voltammetry and cyclic voltammetry revealed a layer of flavins adsorbed to electrodes, even after soluble components were removed, especially in older biofilms. Riboflavin adsorbed quickly to other surfaces of geochemical interest, such as Fe(III) and Mn(IV) oxy(hydr)oxides. This in situ demonstration of flavin production, and sequestration at surfaces, requires the paradigm of soluble redox shuttles in geochemistry to be adjusted to include binding and modification of surfaces. Moreover, the known ability of isoalloxazine rings to act as metal chelators, along with their electron shuttling capacity, suggests that extracellular respiration of minerals by Shewanella is more complex than originally conceived. PMID:18316736

Unique Features and Anti-microbial Targeting of Folate- and Flavin-Dependent Methyltransferases Required for Accurate Maintenance of Genetic Information.

PubMed

Myllykallio, Hannu; Sournia, Pierre; Heliou, Alice; Liebl, Ursula

2018-01-01

Comparative genome analyses have led to the discovery and characterization of novel flavin- and folate-dependent methyltransferases that mainly function in DNA precursor synthesis and post-transcriptional RNA modification by forming (ribo) thymidylate and its derivatives. Here we discuss the recent literature on the novel mechanistic features of these enzymes sometimes referred to as "uracil methyltransferases," albeit we prefer to refer to them as (ribo) thymidylate synthases. These enzyme families attest to the convergent evolution of nucleic acid methylation. Special focus is given to describing the unique characteristics of these flavin- and folate-dependent enzymes that have emerged as new models for studying the non-canonical roles of reduced flavin co-factors (FADH 2 ) in relaying carbon atoms between enzyme substrates. This ancient enzymatic methylation mechanism with a very wide phylogenetic distribution may be more commonly used for biological methylation reactions than previously anticipated. This notion is exemplified by the recent discovery of additional substrates for these enzymes. Moreover, similar reaction mechanisms can be reversed by demethylases, which remove methyl groups e.g., from human histones. Future work is now required to address whether the use of different methyl donors facilitates the regulation of distinct methylation reactions in the cell. It will also be of great interest to address whether the low activity flavin-dependent thymidylate synthases ThyX represent ancestral enzymes that were eventually replaced by the more active thymidylate synthases of the ThyA family to facilitate the maintenance of larger genomes in fast-growing microbes. Moreover, we discuss the recent efforts from several laboratories to identify selective anti-microbial compounds that target flavin-dependent thymidylate synthase ThyX. Altogether we underline how the discovery of the alternative flavoproteins required for methylation of DNA and/or RNA nucleotides

Expression, Purification, and Characterization of a Recombinant Flavin Reductase from the Luminescent Marine Bacterium "Photobacterium Leiognathi": A Set of Exercises for Students

ERIC Educational Resources Information Center

Crowley, Thomas E.

2010-01-01

In "Photobacterium," the flavin reductase encoded by "lux"G regenerates the reduced form of flavin mononucleotide (FMN). Reduced FMN is one of the substrates of the luciferase enzyme that catalyzes a light-emitting reaction. A set of experiments, that employs a "lux"G-expression plasmid construct (pGhis) and is suitable for an undergraduate…

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

[The release of flavin adenine dinucleotide upon local conformational transition in electron-transferring flavoprotein induced by trimethylamine dehydrogenase].

PubMed

Lomtev, A S; Bobrov, A G; Vekshin, N L

2004-01-01

The electron-transferring proteins, trimethylamine dehydrogenase (TMAD) and electron-transferring flavoprotein (ETF) from the bacterium Methylophilius methylotrophus, were studied in vitro by fluorescence spectroscopy. Flavin adenine dinucleotide (FAD) was found to be capable of a slow and spontaneous release from ETF, which is accompanied by an increase in flavin fluorescence. At a rather high ionic strength (0.1 M NaCl or 50 mM phosphate), the FAD release is sharply activated by TMAD preparations that induce a local conformational transition in ETF. The values of tryptophan fluorescence polarization and lifetime and the use of the Levshin-Perrin equation helped show that the size of protein particles remain unchanged upon the TMAD and ETF mixing; i.e., these proteins themselves do not form a stable complex with each other. The protein mixture did not release flavin from ETF in the presence of trimethylamine and formaldehyde. In this case, a stable complex between the proteins appeared to be formed under the action of formaldehyde. Upon a short-term incubation of ETF with ferricyanide, FAD was hydrolyzed to flavin mononucleotide (FMN) and AMP. This fact explains the previous detection of AMP in ETF preparations by some researches. A fluorescence method was proposed for distinguishing FAD from FMN in solution using ethylene glycol. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 3; see also http://www.maik.ru.

Importance of a serine proximal to the C(4a) and N(5) flavin atoms for hydride transfer in choline oxidase.

PubMed

Yuan, Hongling; Gadda, Giovanni

2011-02-08

Choline oxidase catalyzes the flavin-dependent, two-step oxidation of choline to glycine betaine with the formation of an aldehyde intermediate. In the first oxidation reaction, the alcohol substrate is initially activated to its alkoxide via proton abstraction. The substrate is oxidized via transfer of a hydride from the alkoxide α-carbon to the N(5) atom of the enzyme-bound flavin. In the wild-type enzyme, proton and hydride transfers are mechanistically and kinetically uncoupled. In this study, we have mutagenized an active site serine proximal to the C(4a) and N(5) atoms of the flavin and investigated the reactions of proton and hydride transfers by using substrate and solvent kinetic isotope effects. Replacement of Ser101 with threonine, alanine, cysteine, or valine resulted in biphasic traces in anaerobic reductions of the flavin with choline investigated in a stopped-flow spectrophotometer. Kinetic isotope effects established that the kinetic phases correspond to the proton and hydride transfer reactions catalyzed by the enzyme. Upon removal of Ser101, there is an at least 15-fold decrease in the rate constants for proton abstraction, irrespective of whether threonine, alanine, valine, or cysteine is present in the mutant enzyme. A logarithmic decrease spanning 4 orders of magnitude is seen in the rate constants for hydride transfer with increasing hydrophobicity of the side chain at position 101. This study shows that the hydrophilic character of a serine residue proximal to the C(4a) and N(5) flavin atoms is important for efficient hydride transfer.

Rhodococcus erythropolis DCL14 Contains a Novel Degradation Pathway for Limonene

PubMed Central

van der Werf, Mariët J.; Swarts, Henk J.; de Bont, Jan A. M.

1999-01-01

Strain DCL14, which is able to grow on limonene as a sole source of carbon and energy, was isolated from a freshwater sediment sample. This organism was identified as a strain of Rhodococcus erythropolis by chemotaxonomic and genetic studies. R. erythropolis DCL14 also assimilated the terpenes limonene-1,2-epoxide, limonene-1,2-diol, carveol, carvone, and (−)-menthol, while perillyl alcohol was not utilized as a carbon and energy source. Induction tests with cells grown on limonene revealed that the oxygen consumption rates with limonene-1,2-epoxide, limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and carveol were high. Limonene-induced cells of R. erythropolis DCL14 contained the following four novel enzymatic activities involved in the limonene degradation pathway of this microorganism: a flavin adenine dinucleotide- and NADH-dependent limonene 1,2-monooxygenase activity, a cofactor-independent limonene-1,2-epoxide hydrolase activity, a dichlorophenolindophenol-dependent limonene-1,2-diol dehydrogenase activity, and an NADPH-dependent 1-hydroxy-2-oxolimonene 1,2-monooxygenase activity. Product accumulation studies showed that (1S,2S,4R)-limonene-1,2-diol, (1S,4R)-1-hydroxy-2-oxolimonene, and (3R)-3-isopropenyl-6-oxoheptanoate were intermediates in the (4R)-limonene degradation pathway. The opposite enantiomers [(1R,2R,4S)-limonene-1,2-diol, (1R,4S)-1-hydroxy-2-oxolimonene, and (3S)-3-isopropenyl-6-oxoheptanoate] were found in the (4S)-limonene degradation pathway, while accumulation of (1R,2S,4S)-limonene-1,2-diol from (4S)-limonene was also observed. These results show that R. erythropolis DCL14 metabolizes both enantiomers of limonene via a novel degradation pathway that starts with epoxidation at the 1,2 double bond forming limonene-1,2-epoxide. This epoxide is subsequently converted to limonene-1,2-diol, 1-hydroxy-2-oxolimonene, and 7-hydroxy-4-isopropenyl-7-methyl-2-oxo-oxepanone. This lactone spontaneously rearranges to form 3-isopropenyl-6-oxoheptanoate. In

Mapping the Substrate Binding Site of Phenylacetone Monooxygenase from Thermobifida fusca by Mutational Analysis▿†

PubMed Central

Dudek, Hanna M.; de Gonzalo, Gonzalo; Torres Pazmiño, Daniel E.; Stępniak, Piotr; Wyrwicz, Lucjan S.; Rychlewski, Leszek; Fraaije, Marco W.

2011-01-01

Baeyer-Villiger monooxygenases catalyze oxidations that are of interest for biocatalytic applications. Among these enzymes, phenylacetone monooxygenase (PAMO) from Thermobifida fusca is the only protein showing remarkable stability. While related enzymes often present a broad substrate scope, PAMO accepts only a limited number of substrates. Due to the absence of a substrate in the elucidated crystal structure of PAMO, the substrate binding site of this protein has not yet been defined. In this study, a structural model of cyclopentanone monooxygenase, which acts on a broad range of compounds, has been prepared and compared with the structure of PAMO. This revealed 15 amino acid positions in the active site of PAMO that may account for its relatively narrow substrate specificity. We designed and analyzed 30 single and multiple mutants in order to verify the role of these positions. Extensive substrate screening revealed several mutants that displayed increased activity and altered regio- or enantioselectivity in Baeyer-Villiger reactions and sulfoxidations. Further substrate profiling resulted in the identification of mutants with improved catalytic properties toward synthetically attractive compounds. Moreover, the thermostability of the mutants was not compromised in comparison to that of the wild-type enzyme. Our data demonstrate that the positions identified within the active site of PAMO, namely, V54, I67, Q152, and A435, contribute to the substrate specificity of this enzyme. These findings will aid in more dedicated and effective redesign of PAMO and related monooxygenases toward an expanded substrate scope. PMID:21724896

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

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

Flavins secreted by bacterial cells of Shewanella catalyze cathodic oxygen reduction.

PubMed

Liu, Huan; Matsuda, Shoichi; Hashimoto, Kazuhito; Nakanishi, Shuji

2012-06-01

On Her Majesty's Secrete Service: Oxygen reduction is an important process for microbial fuel cells (MFCs) and microbiologically-influenced corrosion (MIC). We demonstrate that flavins secreted by anode-respiring Shewanella cells can catalyze cathodic oxygen reduction via adsorption on the cathode. The findings will provide new insight for developing methods to improve MFC performance and to prevent MIC. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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�

Kinetics and mechanisms of 1,5-dihydroflavin reduction of carbonyl compounds and flavin oxidation of alcohols. III. Oxidation of benzoin by flavin and reduction of benzil by 1,5-dihydroflavin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bruice, T.C.; Taulane, J.P.

1976-11-24

The oxidation of benzoin by lumiflavin-3-acetic acid (Fl/sub ox/) to provide benzil and 1,5-dihydrolumiflavin-3-acetic acid (FlH/sub 2/) is a readily reversible reaction. It has been established that the mechanism involves general base ionization of benzoin carbon acid (..cap alpha..-ketol) to yield endiolate anion, followed by partitioning of the endiolate anion back to benzoin through general acid proton donation and to benzil by reaction with Fl/sub ox/. The reaction of endiolate anion with Fl/sub ox/ is not subject to acid or base catalysis. Evidence that ionization of benzoin precedes its oxidation by Fl/sub ox/ stems from the observation that the ratemore » attributed to the latter process possesses a constant equal to that for racemization of (+)-benzoin and O/sub 2/ oxidation of benzoin and that this rate constant is characterized by a primary deuterium kinetic isotope effect (k/sup benzoin//k/sup ..cap alpha..-/sup 2/H-benzoin/) of 7.24 +- 1.5. Reduction of benzil to benzoin by FlH/sub 2/ is pH and buffer insensitive below the pK/sub a/ of FlH/sub 2/. These results are consistent with either general acid catalyzed attack of benzoin carbanion at the 4a-position of Fl/sub ox/, followed by a specific base catalyzed collapse of adduct to diketone and dihydroflavin (Scheme III), or to the uncatalyzed reaction of carbanion (endiolate anion) with flavin to provide a semidione-flavin radical pair which then goes on to diketone and dihydroflavin in a non-acid-base catalyzed reaction (Scheme V). These mechanisms are discussed in terms of the kinetics of reaction of other carbanion species with flavin.« less

Evidence for proton tunneling and a transient covalent flavin-substrate adduct in choline oxidase S101A.

PubMed

Uluisik, Rizvan; Romero, Elvira; Gadda, Giovanni

2017-11-01

The effect of temperature on the reaction of alcohol oxidation catalyzed by choline oxidase was investigated with the S101A variant of choline oxidase. Anaerobic enzyme reduction in a stopped-flow spectrophotometer was biphasic using either choline or 1,2-[ 2 H 4 ]-choline as a substrate. The limiting rate constants k lim1 and k lim2 at saturating substrate were well separated (k lim1 /k lim2 >9), and were >15-fold slower than for wild-type choline oxidase. Solvent deuterium kinetic isotope effects (KIEs) ~4 established that k lim1 probes the proton transfer from the substrate hydroxyl to a catalytic base. Primary substrate deuterium KIEs ≥7 demonstrated that k lim2 reports on hydride transfer from the choline alkoxide to the flavin. Between 15°C and 39°C the k lim1 and k lim2 values increased with increasing temperature, allowing for the analyses of H + and H - transfers using Eyring and Arrhenius formalisms. Temperature-independent KIE on the k lim1 value ( H2O k lim1 / D2O k lim1 ) suggests that proton transfer occurs within a highly reorganized tunneling-ready-state with a narrow distribution of donor-acceptor distances. Eyring analysis of the k lim2 value gave lines with the slope (choline) >slope (D-choline) , suggesting kinetic complexity. Spectral evidence for the transient occurrence of a covalent flavin-substrate adduct during the first phase of the anaerobic reaction of S101A CHO with choline is presented, supporting the notion that an important role of amino acid residues in the active site of flavin-dependent enzymes is to eliminate alternative reactions of the versatile enzyme-bound flavin for the reaction that needs to be catalyzed. Copyright © 2017 Elsevier B.V. All rights reserved.

Long-distance communication and signal amplification in systemic acquired resistance

PubMed Central

Shah, Jyoti; Zeier, Jürgen

2013-01-01

Systemic acquired resistance (SAR) is an inducible defense mechanism in plants that confers enhanced resistance against a variety of pathogens. SAR is activated in the uninfected systemic (distal) organs in response to a prior (primary) infection elsewhere in the plant. SAR is associated with the activation of salicylic acid (SA) signaling and the priming of defense responses for robust activation in response to subsequent infections. The activation of SAR requires communication by the primary infected tissues with the distal organs. The vasculature functions as a conduit for the translocation of factors that facilitate long-distance intra-plant communication. In recent years, several metabolites putatively involved in long-distance signaling have been identified. These include the methyl ester of SA (MeSA), the abietane diterpenoid dehydroabietinal (DA), the dicarboxylic acid azelaic acid (AzA), and a glycerol-3-phosphate (G3P)-dependent factor. Long-distance signaling by some of these metabolites also requires the lipid-transfer protein DIR1 (DEFECTIVE IN INDUCED RESISTANCE 1). The relative contribution of these factors in long-distance signaling is likely influenced by environmental conditions, for example light. In the systemic leaves, the AGD2-LIKE DEFENSE RESPONSE PROTEIN1 (ALD1)-dependent production of the lysine catabolite pipecolic acid (Pip), FLAVIN-DEPENDENT MONOOXYGENASE1 (FMO1) signaling, as well as SA synthesis and downstream signaling are required for the activation of SAR. This review summarizes the involvement and interaction between long-distance SAR signals and details the recently discovered role of Pip in defense amplification and priming that allows plants to acquire immunity at the systemic level. Recent advances in SA signaling and perception are also highlighted. PMID:23440336

Biotransformation of petroleum asphaltenes and high molecular weight polycyclic aromatic hydrocarbons by Neosartorya fischeri.

PubMed

Hernández-López, E Lorena; Perezgasga, Lucia; Huerta-Saquero, Alejandro; Mouriño-Pérez, Rosa; Vazquez-Duhalt, Rafael

2016-06-01

Neosartorya fischeri, an Aspergillaceae fungus, was evaluated in its capacity to transform high molecular weight polycyclic aromatics hydrocarbons (HMW-PAHs) and the recalcitrant fraction of petroleum, the asphaltenes. N. fischeri was able to grow in these compounds as sole carbon source. Coronene, benzo(g,h,i)perylene, and indeno(1,2,3-c,d)pyrene, together with the asphaltenes, were assayed for fungal biotransformation. The transformation of the asphaltenes and HMW-PAHs was confirmed by reverse-phase high-performance liquid chromatography (HPLC), nano-LC mass spectrometry, and IR spectrometry. The formation of hydroxy and ketones groups on the PAH molecules suggest a biotransformation mediated by monooxygenases such as cytochrome P450 system (CYP). A comparative microarray with the complete genome from N. fischeri showed three CYP monooxygenases and one flavin monooxygenase genes upregulated. These findings, together with the internalization of aromatic substrates into fungal cells and the microsomal transformation of HMW-PAHs, strongly support the role of CYPs in the oxidation of these recalcitrant compounds.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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. Here, we determined the 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 oxygenmore » activation. Our 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.« less

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

DOE PAGES

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

2016-12-22

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. Here, we determined the 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 oxygenmore » activation. Our 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.« less

Removal of arsenic from groundwater using low cost ferruginous manganese ore.

PubMed

Chakravarty, S; Dureja, V; Bhattacharyya, G; Maity, S; Bhattacharjee, S

2002-02-01

A low cost ferruginous manganese ore (FMO) has been studied for the removal of arsenic from groundwater. The major mineral phases present in the FMO are pyrolusite and goethite. The studied FMO can adsorb both AS(III) and As(V) without any pre-treatment, adsorption of As(III) being stronger than that of As(V). Both As(II) and As(V) are adsorbed by the FMO in the pH range of 2-8. Once adsorbed, arsenic does not get desorbed even on varying the pH in the range of 2-8. Presence of bivalent cations, namely, Ni2+, Co2+ Mg2+ enhances the adsorption capability of the FMO. The FMO has been successfully used for the removal of arsenic from six real groundwater samples containing arsenic in the range of 0.04-0.18 ppm. Arsenic removals are almost 100% in all the cases. The cost of the FMO is about 50-56 US$ per metric tonne.

Absorption and luminescence spectroscopy of mass-selected flavin adenine dinucleotide mono-anions

NASA Astrophysics Data System (ADS)

Giacomozzi, L.; Kjær, C.; Langeland Knudsen, J.; Andersen, L. H.; Brøndsted Nielsen, S.; Stockett, M. H.

2018-06-01

We report the absorption profile of isolated Flavin Adenine Dinucleotide (FAD) mono-anions recorded using photo-induced dissociation action spectroscopy. In this charge state, one of the phosphoric acid groups is deprotonated and the chromophore itself is in its neutral oxidized state. These measurements cover the first four optical transitions of FAD with excitation energies from 2.3 to 6.0 eV (210-550 nm). The S0 → S2 transition is strongly blue shifted relative to aqueous solution, supporting the view that this transition has a significant charge-transfer character. The remaining bands are close to their solution-phase positions. This confirms that the large discrepancy between quantum chemical calculations of vertical transition energies and solution-phase band maxima cannot be explained by solvent effects. We also report the luminescence spectrum of FAD mono-anions in vacuo. The gas-phase Stokes shift for S1 is 3000 cm-1, which is considerably larger than any previously reported for other molecular ions and consistent with a significant displacement of the ground and excited state potential energy surfaces. Consideration of the vibronic structure is thus essential for simulating the absorption and luminescence spectra of flavins.

Evidence from Studies with Acifluorfen for Participation of a Flavin-Cytochrome Complex in Blue Light Photoreception for Phototropism of Oat Coleoptiles 12

PubMed Central

Leong, Ta-Yan; Briggs, Winslow R.

1982-01-01

The diphenyl ether acifluorfen enhances the blue light-induced absorbance change in Triton X100-solubilized crude membrane preparations from etiolated oat (Avena sativa L. cv. Lodi) coleoptiles. Enhancement of the spectral change is correlated with a change in rate of dark reoxidation of a b-type cytochrome. Similar, although smaller, enhancement was obtained with oxyfluorfen, nitrofen, and bifenox. Light-minus-dark difference spectra in the presence and absence of acifluorfen, and the dithionite-reduced-minus oxidized difference spectrum indicate that acifluorfen is acting specifically at a blue light-sensitive cytochrome-flavin complex. Sodium azide, a flavin inhibitor, decreases the light-induced absorbance change significantly, but does not affect the dark reoxidation of the cytochrome. Hence, it is acting on the light reaction, suggesting that the photoreceptor itself is a flavin. Acifluorfen sensitizes phototropism in dark-grown oat seedlings such that the first positive response occurs with blue light fluences as little as one-third of those required to elicit the same response in seedlings grown in the absence of the herbicide. Both this increase in sensitivity to light and the enhancement of the light-induced cytochrome reduction vary with the applied acifluorfen concentration in a similar manner. The herbicide is without effect either on elongation or on the geotropic response of dark-grown oat seedlings, indicating that acifluorfen is acting specifically close to, or at the photoreceptor end of, the stimulus-response chain. It seems likely that the flavin-cytochrome complex serves to transduce the light signal into curvature in phototropism in oats, with the flavin moiety itself serving as the photoreceptor. PMID:16662593

Characterization of flavin-based fluorescent proteins: an emerging class of fluorescent reporters.

PubMed

Mukherjee, Arnab; Walker, Joshua; Weyant, Kevin B; Schroeder, Charles M

2013-01-01

Fluorescent reporter proteins based on flavin-binding photosensors were recently developed as a new class of genetically encoded probes characterized by small size and oxygen-independent maturation of fluorescence. Flavin-based fluorescent proteins (FbFPs) address two major limitations associated with existing fluorescent reporters derived from the green fluorescent protein (GFP)-namely, the overall large size and oxygen-dependent maturation of fluorescence of GFP. However, FbFPs are at a nascent stage of development and have been utilized in only a handful of biological studies. Importantly, a full understanding of the performance and properties of FbFPs as a practical set of biological probes is lacking. In this work, we extensively characterize three FbFPs isolated from Pseudomonas putida, Bacillus subtilis, and Arabidopsis thaliana, using in vitro studies to assess probe brightness, oligomeric state, maturation time, fraction of fluorescent holoprotein, pH tolerance, redox sensitivity, and thermal stability. Furthermore, we validate FbFPs as stable molecular tags using in vivo studies by constructing a series of FbFP-based transcriptional constructs to probe promoter activity in Escherichia coli. Overall, FbFPs show key advantages as broad-spectrum biological reporters including robust pH tolerance (4-11), thermal stability (up to 60°C), and rapid maturation of fluorescence (<3 min.). In addition, the FbFP derived from Arabidopsis thaliana (iLOV) emerged as a stable and nonperturbative reporter of promoter activity in Escherichia coli. Our results demonstrate that FbFP-based reporters have the potential to address key limitations associated with the use of GFP, such as pH-sensitive fluorescence and slow kinetics of fluorescence maturation (10-40 minutes for half maximal fluorescence recovery). From this view, FbFPs represent a useful new addition to the fluorescent reporter protein palette, and our results constitute an important framework to enable

Coupling Oxygen Consumption with Hydrocarbon Oxidation in Bacterial Multicomponent Monooxygenases.

PubMed

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

2015-09-15

active site; (ii) connects hydrophobic cavities in the hydroxylase leading from the exterior to the diiron active site, providing a pathway for O2 and methane, in the case of sMMO, to the reduced diiron center for O2 activation and substrate hydroxylation; (iii) closes the pore, as well as a channel in the case of four-component BMM enzymes, restricting proton access to the diiron center during formation of "Fe2O2" intermediates required for hydrocarbon oxidation; and (iv) inhibits undesired electron transfer to the Fe2O2 intermediates by blocking reductase binding during O2 activation. This mechanism is quite different from that adopted by cytochromes P450, a large class of heme-containing monooxygenases that catalyze reactions very similar to those catalyzed by the BMM enzymes. Understanding the timed enzyme control of substrate access has implications for designing artificial catalysts. To achieve multiple turnovers and tight coupling, synthetic models must also control substrate access, a major challenge considering that nature requires large, multimeric, dynamic protein complexes to accomplish this feat.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Isupov, Michail N.; Schröder, Ewald; Gibson, Robert P.

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.more » 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.« less

Evidence for Posttranslational Protein Flavinylation in the Syphilis Spirochete Treponema pallidum: Structural and Biochemical Insights from the Catalytic Core of a Periplasmic Flavin-Trafficking Protein.

PubMed

Deka, Ranjit K; Brautigam, Chad A; Liu, Wei Z; Tomchick, Diana R; Norgard, Michael V

2015-05-05

The syphilis spirochete Treponema pallidum is an important human pathogen but a highly enigmatic bacterium that cannot be cultivated in vitro. T. pallidum lacks many biosynthetic pathways and therefore has evolved the capability to exploit host-derived metabolites via its periplasmic lipoprotein repertoire. We recently reported a flavin-trafficking protein in T. pallidum (Ftp_Tp; TP0796) as the first bacterial metal-dependent flavin adenine dinucleotide (FAD) pyrophosphatase that hydrolyzes FAD into AMP and flavin mononucleotide (FMN) in the spirochete's periplasm. However, orthologs of Ftp_Tp from other bacteria appear to lack this hydrolytic activity; rather, they bind and flavinylate subunits of a cytoplasmic membrane redox system (Nqr/Rnf). To further explore this dichotomy, biochemical analyses, protein crystallography, and structure-based mutagenesis were used to show that a single amino acid change (N55Y) in Ftp_Tp converts it from an Mg(2+)-dependent FAD pyrophosphatase to an FAD-binding protein. We also demonstrated that Ftp_Tp has a second enzymatic activity (Mg(2+)-FMN transferase); it flavinylates protein(s) covalently with FMN on a threonine side chain of an appropriate sequence motif using FAD as the substrate. Moreover, mutation of a metal-binding residue (D284A) eliminates Ftp_Tp's dual activities, thereby underscoring the role of Mg(2+) in the enzyme-catalyzed reactions. The posttranslational flavinylation activity that can target a periplasmic lipoprotein (TP0171) has not previously been described. The observed activities reveal the catalytic flexibility of a treponemal protein to perform multiple functions. Together, these findings imply mechanisms by which a dynamic pool of flavin cofactor is maintained and how flavoproteins are generated by Ftp_Tp locally in the T. pallidum periplasm. Treponema pallidum, the syphilis spirochete, exploits its periplasmic lipoproteins for a number of essential physiologic processes. One of these, flavin

Investigations of blue light-induced reactive oxygen species from flavin mononucleotide on inactivation of E. coli.

PubMed

Liang, Ji-Yuan; Cheng, Chien-Wei; Yu, Chin-Hao; Chen, Liang-Yü

2015-02-01

The micronutrients in many cellular processes, riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide (FAD) are photo-sensitive to UV and visible light for generating reactive oxygen species (ROS). Produced from phosphorylation of riboflavin, FMN is more water-soluble and rapidly transformed into free riboflavin after ingestion. This study investigated the application of visible blue light with FMN to development of an effective antimicrobial treatment. The photosensitization of bacterial viability with FMN was investigated by light quality, intensity, time, and irradiation dosage. The blue light-induced photochemical reaction with FMN could inactivate Escherichiacoli by the generated ROS in damaging nucleic acids, which was validated. This novel photodynamic technique could be a safe practice for photo-induced inactivation of environmental microorganism to achieve hygienic requirements in food processing. Copyright © 2015 Elsevier B.V. All rights reserved.

Crystal structure of the bacterial luciferase/flavin complex provides insight into the function of the beta subunit.

PubMed

Campbell, Zachary T; Weichsel, Andrzej; Montfort, William R; Baldwin, Thomas O

2009-07-07

Bacterial luciferase from Vibrio harveyi is a heterodimer composed of a catalytic alpha subunit and a homologous but noncatalytic beta subunit. Despite decades of enzymological investigation, structural evidence defining the active center has been elusive. We report here the crystal structure of V. harveyi luciferase bound to flavin mononucleotide (FMN) at 2.3 A. The isoalloxazine ring is coordinated by an unusual cis-Ala-Ala peptide bond. The reactive sulfhydryl group of Cys106 projects toward position C-4a, the site of flavin oxygenation. This structure also provides the first data specifying the conformations of a mobile loop that is crystallographically disordered in both prior crystal structures [(1995) Biochemistry 34, 6581-6586; (1996) J. Biol. Chem. 271, 21956 21968]. This loop appears to be a boundary between solvent and the active center. Within this portion of the protein, a single contact was observed between Phe272 of the alpha subunit, not seen in the previous structures, and Tyr151 of the beta subunit. Substitutions at position 151 on the beta subunit caused reductions in activity and total quantum yield. Several of these mutants were found to have decreased affinity for reduced flavin mononucleotide (FMNH(2)). These findings partially address the long-standing question of how the beta subunit stabilizes the active conformation of the alpha subunit, thereby participating in the catalytic mechanism.

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...

Flavin Charge Transfer Transitions Assist DNA Photolyase Electron Transfer

NASA Astrophysics Data System (ADS)

Skourtis, Spiros S.; Prytkova, Tatiana; Beratan, David N.

2007-12-01

This contribution describes molecular dynamics, semi-empirical and ab-initio studies of the primary photo-induced electron transfer reaction in DNA photolyase. DNA photolyases are FADH--containing proteins that repair UV-damaged DNA by photo-induced electron transfer. A DNA photolyase recognizes and binds to cyclobutatne pyrimidine dimer lesions of DNA. The protein repairs a bound lesion by transferring an electron to the lesion from FADH-, upon photo-excitation of FADH- with 350-450 nm light. We compute the lowest singlet excited states of FADH- in DNA photolyase using INDO/S configuration interaction, time-dependent density-functional, and time-dependent Hartree-Fock methods. The calculations identify the lowest singlet excited state of FADH- that is populated after photo-excitation and that acts as the electron donor. For this donor state we compute conformationally-averaged tunneling matrix elements to empty electron-acceptor states of a thymine dimer bound to photolyase. The conformational averaging involves different FADH--thymine dimer confromations obtained from molecular dynamics simulations of the solvated protein with a thymine dimer docked in its active site. The tunneling matrix element computations use INDO/S-level Green's function, energy splitting, and Generalized Mulliken-Hush methods. These calculations indicate that photo-excitation of FADH- causes a π→π* charge-transfer transition that shifts electron density to the side of the flavin isoalloxazine ring that is adjacent to the docked thymine dimer. This shift in electron density enhances the FADH--to-dimer electronic coupling, thus inducing rapid electron transfer.

The NADH:flavin oxidoreductase Nox from Rhodococcus erythropolis MI2 is the key enzyme of 4,4'-dithiodibutyric acid degradation.

PubMed

Khairy, H; Wübbeler, J H; Steinbüchel, A

2016-12-01

The reduction of the disulphide bond is the initial catabolic step of the microbial degradation of the organic disulphide 4,4'-dithiodibutyric acid (DTDB). Previously, an NADH:flavin oxidoreductase from Rhodococcus erythropolis MI2 designated as Nox MI2 , which belongs to the old yellow enzyme (OYE) family, was identified. In the present study, it was proven that Nox MI2 has the ability to cleave the sulphur-sulphur bond in DTDB. In silico analysis revealed high sequence similarities to proteins of the flavin mononucleotide (FMN) reductase family identified in many strains of R. erythropolis. Therefore, nox was heterologously expressed in the pET23a(+) expression system using Escherichia coli strain BL21(DE3) pLysS, which effectively produces soluble active Nox MI2 . Nox MI2 showed a maximum specific activity (V max ) of 3·36 μmol min -1  mg -1 corresponding to a k cat of 2·5 s -1 and an apparent substrate K m of 0·6 mmol l -1 , when different DTDB concentrations were applied. No metal cofactors were required. Moreover, Nox MI2 had very low activity with other sulphur-containing compounds like 3,3'-dithiodipropionic acid (8·0%), 3,3'-thiodipropionic acid (7·6%) and 5,5'-dithiobis(2-nitrobenzoic acid) (8·0%). The UV/VIS spectrum of Nox MI2 revealed the presence of the cofactor FMN. Based on results obtained, Nox MI2 adds a new physiological substrate and mode of action to OYE members. It was unequivocally demonstrated in this study that an NADH:flavin oxidoreductase from Rhodococcus erythropolis MI2 (Nox MI2 ) is able to cleave the xenobiotic disulphide 4,4'-dithiodibutyric acid (DTDB) into two molecules of 4-mercaptobutyric acid (4MB) with concomitant consumption of NADH. Nox MI2 showed a high substrate specificity as well as high heat stability. This study provides the first detailed characterization of the initial cleavage of DTDB, which is considered as a promising polythioester precursor. © 2016 The Society for Applied Microbiology.

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 (LC 50 ) 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.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Carlin, DA; Bertolani, SJ; Siegel, JB

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.

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.

Toxicity of endophyte-infected ryegrass hay containing high ergovaline level in lactating ewes.

PubMed

Zbib, N; Repussard, C; Tardieu, D; Priymenko, N; Domange, C; Guerre, P

2015-08-01

The symbiotic association of var. (formerly named ) with perennial ryegrass () leads to the production of ergovaline (EV) and lolitrem B (LB) that are toxic for livestock. The objectives of this study were to determine the effects of feeding endophyte-infected ryegrass (SE+) hay on 16 lactating ewes (BW 80 ± 10 kg) in comparison with endophyte-free ryegrass (SE-) hay to investigate the putative mechanisms of action of EV and LB and to evaluate their persistence in milk and animal tissues. The mean EV and LB concentrations in SE+ hay were 851 and 884 μg/kg DM, respectively, whereas these alkaloids were below the limit of detection in SE- hay. No effect of SE+ was observed on animal health and skin temperature whereas prolactin decreased and significant differences between hays were observed from d 7 to 28 of the study ( < 0.03) but had no effect on milk production. Hematocrit and biochemical analyses of plasma revealed no significant difference between SE+ and SE-, whereas cortisol concentration differed significantly on d 28 ( = 0.001). Measurement of oxidative damage and antioxidant enzyme activities in plasma, liver, and kidneys revealed a slight increase in some enzyme activities involved in defense against oxidative damage in the SE+ fed ewes. Slight variations in the activities of hepatic and kidney flavin monooxygenase enzymes were observed, whereas in the kidney, glutathione -transferase activity decreased significantly ( = 0.002) in the SE+ fed ewes, whereas uridine diphosphate glucuronosyltransferase activity increased ( = 0.001). After 28 d of exposure of ewes to the SE+ hay, low EV and LB concentrations were measured in tissues. The highest concentration of EV was observed in the liver (0.68 μg/kg) whereas fat contained the highest concentration of LB (2.39 μg/kg). Both toxins were also identified at the trace level in milk.

Enzyme-Mediated Conversion of Flavin Adenine Dinucleotide (FAD) to 8-Formyl FAD in Formate Oxidase Results in a Modified Cofactor with Enhanced Catalytic Properties.

PubMed

Robbins, John M; Souffrant, Michael G; Hamelberg, Donald; Gadda, Giovanni; Bommarius, Andreas S

2017-07-25

Flavins, including flavin adenine dinucleotide (FAD), are fundamental catalytic cofactors that are responsible for the redox functionality of a diverse set of proteins. Alternatively, modified flavin analogues are rarely found in nature as their incorporation typically results in inactivation of flavoproteins, thus leading to the disruption of important cellular pathways. Here, we report that the fungal flavoenzyme formate oxidase (FOX) catalyzes the slow conversion of noncovalently bound FAD to 8-formyl FAD and that this conversion results in a nearly 10-fold increase in formate oxidase activity. Although the presence of an enzyme-bound 8-formyl FMN has been reported previously as a result of site-directed mutagenesis studies of lactate oxidase, FOX is the first reported case of 8-formyl FAD in a wild-type enzyme. Therefore, the formation of the 8-formyl FAD cofactor in formate oxidase was investigated using steady-state kinetics, site-directed mutagenesis, ultraviolet-visible, circular dichroism, and fluorescence spectroscopy, liquid chromatography with mass spectrometry, and computational analysis. Surprisingly, the results from these studies indicate not only that 8-formyl FAD forms spontaneously and results in the active form of FOX but also that its autocatalytic formation is dependent on a nearby arginine residue, R87. Thus, this work describes a new enzyme cofactor and provides insight into the little-understood mechanism of enzyme-mediated 8α-flavin modifications.

Signals of Systemic Immunity in Plants: Progress and Open Questions

PubMed Central

Ádám, Attila L.; Nagy, Zoltán Á.; Kátay, György; Mergenthaler, Emese; Viczián, Orsolya

2018-01-01

Systemic acquired resistance (SAR) is a defence mechanism that induces protection against a wide range of pathogens in distant, pathogen-free parts of plants after a primary inoculation. Multiple mobile compounds were identified as putative SAR signals or important factors for influencing movement of SAR signalling elements in Arabidopsis and tobacco. These include compounds with very different chemical structures like lipid transfer protein DIR1 (DEFECTIVE IN INDUCED RESISTANCE1), methyl salicylate (MeSA), dehydroabietinal (DA), azelaic acid (AzA), glycerol-3-phosphate dependent factor (G3P) and the lysine catabolite pipecolic acid (Pip). Genetic studies with different SAR-deficient mutants and silenced lines support the idea that some of these compounds (MeSA, DIR1 and G3P) are activated only when SAR is induced in darkness. In addition, although AzA doubled in phloem exudate of tobacco mosaic virus (TMV) infected tobacco leaves, external AzA treatment could not induce resistance neither to viral nor bacterial pathogens, independent of light conditions. Besides light intensity and timing of light exposition after primary inoculation, spectral distribution of light could also influence the SAR induction capacity. Recent data indicated that TMV and CMV (cucumber mosaic virus) infection in tobacco, like bacteria in Arabidopsis, caused massive accumulation of Pip. Treatment of tobacco leaves with Pip in the light, caused a drastic and significant local and systemic decrease in lesion size of TMV infection. Moreover, two very recent papers, added in proof, demonstrated the role of FMO1 (FLAVIN-DEPENDENT-MONOOXYGENASE1) in conversion of Pip to N-hydroxypipecolic acid (NHP). NHP systemically accumulates after microbial attack and acts as a potent inducer of plant immunity to bacterial and oomycete pathogens in Arabidopsis. These results argue for the pivotal role of Pip and NHP as an important signal compound of SAR response in different plants against different

Extracellular Electron Transport-Mediated Fe(III) Reduction by a Community of Alkaliphilic Bacteria That Use Flavins as Electron Shuttles

PubMed Central

Fuller, Samuel J.; McMillan, Duncan G. G.; Renz, Marc B.; Schmidt, Martin

2014-01-01

The biochemical and molecular mechanisms used by alkaliphilic bacterial communities to reduce metals in the environment are currently unknown. We demonstrate that an alkaliphilic (pH > 9) consortium dominated by Tissierella, Clostridium, and Alkaliphilus spp. is capable of using iron (Fe3+) as a final electron acceptor under anaerobic conditions. Iron reduction is associated with the production of a freely diffusible species that, upon rudimentary purification and subsequent spectroscopic, high-performance liquid chromatography, and electrochemical analysis, has been identified as a flavin species displaying properties indistinguishable from those of riboflavin. Due to the link between iron reduction and the onset of flavin production, it is likely that riboflavin has an import role in extracellular metal reduction by this alkaliphilic community. PMID:24141133

Interaction of Ammonia Monooxygenase from Nitrosomonas europaea with Alkanes, Alkenes, and Alkynes

PubMed Central

Hyman, Michael R.; Murton, Ian B.; Arp, Daniel J.

1988-01-01

Ammonia monooxygenase of Nitrosomonas europaea catalyzes the oxidation of alkanes (up to C8) to alcohols and alkenes (up to C5) to epoxides and alcohols in the presence of ammonium ions. Straight-chain, N-terminal alkynes (up to C10) all exhibited a time-dependent inhibition of ammonia oxidation without effects on hydrazine oxidation. PMID:16347810

Anti-mitochondrial flavoprotein autoantibodies of patients with myocarditis and dilated cardiomyopathy (anti-M7): interaction with flavin-carrying proteins, effect of vitamin B2 and epitope mapping

PubMed Central

Stähle, I; Brizzio, C; Barile, M; Brandsch, R

1999-01-01

Vitamin B2 and flavin cofactors are transported tightly bound to immunoglobulin in human serum. We reasoned that anti-mitochondrial flavoprotein autoantibodies (αFp-AB) present in the serum of patients with myocarditis and cardiomyopathy of unknown aetiology may form immunoglobulin aggregates with these serum proteins. However, immunodiffusion and Western blot assays demonstrated that the flavin-carrying proteins were not recognized by αFp-AB. Apparently the flavin moiety in the native protein conformation was inaccessible to αFp-AB. This conclusion was supported by the absence of an immunoreaction between the riboflavin-binding protein from egg white and αFP-AB. Intravenous application of vitamin B2 to rabbits immunized with 6-hydroxy-d-nicotine oxidase, a bacterial protein carrying covalently attached FAD, did not neutralize αFp-AB which had been raised in the serum of the animals. FAD-carrying peptides generated from 6-hydroxy-d-nicotine oxidase by trypsin and chymotrypsin treatment were not recognized by the αFp-AB, but those generated by endopeptidase Lys were. This demonstrates that the epitope recognized by αFp-AB comprises, besides the flavin moiety, protein secondary structure elements. PMID:10193410

CORRELATION OF FLAVIN-CONTAINING MONOOXYGENASE ACTIVITY, ALDICARB TOXICITY AND SALINITY IN THE EURYHALINE FISH, JAPANESE MEDAKA (ORYZIAS LATIPES). (R826109)

EPA Science Inventory

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Concl...

Down-regulation of flavin reductase and alcohol dehydrogenase-1 (ADH1) in metronidazole-resistant isolates of Trichomonas vaginalis

PubMed Central

Leitsch, David; Drinić, Mirjana; Kolarich, Daniel; Duchêne, Michael

2012-01-01

The microaerophilic parasite Trichomonas vaginalis is a causative agent of painful vaginitis or urethritis, termed trichomoniasis, and can also cause preterm delivery or stillbirth. Treatment of trichomoniasis is almost exclusively based on the nitroimidazole drugs metronidazole and tinidazole. Metronidazole resistance in T. vaginalis does occur and is often associated with treatment failure. In most cases, metronidazole-resistant isolates remain susceptible to tinidazole, but cross resistance between the two closely related drugs can be a problem. In this study we measured activities of thioredoxin reductase and flavin reductase in four metronidazole-susceptible and five metronidazole-resistant isolates. These enzyme activities had been previously found to be downregulated in T. vaginalis with high-level metronidazole resistance induced in the laboratory. Further, we aimed at identifying factors causing metronidazole resistance and compared the protein expression profiles of all nine isolates by application of two-dimensional gel electrophoresis (2DE). Thioredoxin reductase activity was nearly equal in all strains assayed but flavin reductase activity was clearly down-regulated, or even absent, in metronidazole-resistant strains. Since flavin reductase has been shown to reduce oxygen to hydrogen peroxide, its down-regulation could significantly contribute to the impairment of oxygen scavenging as reported by others for metronidazole-resistant strains. Analysis by 2DE revealed down-regulation of alcohol dehydrogenase 1 (ADH1) in strains with reduced sensitivity to metronidazole, an enzyme that could be involved in detoxification of intracellular acetaldehyde. PMID:22449940

Oxidoreductases on their way to industrial biotransformations.

PubMed

Martínez, Angel T; Ruiz-Dueñas, Francisco J; Camarero, Susana; Serrano, Ana; Linde, Dolores; Lund, Henrik; Vind, Jesper; Tovborg, Morten; Herold-Majumdar, Owik M; Hofrichter, Martin; Liers, Christiane; Ullrich, René; Scheibner, Katrin; Sannia, Giovanni; Piscitelli, Alessandra; Pezzella, Cinzia; Sener, Mehmet E; Kılıç, Sibel; van Berkel, Willem J H; Guallar, Victor; Lucas, Maria Fátima; Zuhse, Ralf; Ludwig, Roland; Hollmann, Frank; Fernández-Fueyo, Elena; Record, Eric; Faulds, Craig B; Tortajada, Marta; Winckelmann, Ib; Rasmussen, Jo-Anne; Gelo-Pujic, Mirjana; Gutiérrez, Ana; Del Río, José C; Rencoret, Jorge; Alcalde, Miguel

2017-11-01

Fungi produce heme-containing peroxidases and peroxygenases, flavin-containing oxidases and dehydrogenases, and different copper-containing oxidoreductases involved in the biodegradation of lignin and other recalcitrant compounds. Heme peroxidases comprise the classical ligninolytic peroxidases and the new dye-decolorizing peroxidases, while heme peroxygenases belong to a still largely unexplored superfamily of heme-thiolate proteins. Nevertheless, basidiomycete unspecific peroxygenases have the highest biotechnological interest due to their ability to catalyze a variety of regio- and stereo-selective monooxygenation reactions with H 2 O 2 as the source of oxygen and final electron acceptor. Flavo-oxidases are involved in both lignin and cellulose decay generating H 2 O 2 that activates peroxidases and generates hydroxyl radical. The group of copper oxidoreductases also includes other H 2 O 2 generating enzymes - copper-radical oxidases - together with classical laccases that are the oxidoreductases with the largest number of reported applications to date. However, the recently described lytic polysaccharide monooxygenases have attracted the highest attention among copper oxidoreductases, since they are capable of oxidatively breaking down crystalline cellulose, the disintegration of which is still a major bottleneck in lignocellulose biorefineries, along with lignin degradation. Interestingly, some flavin-containing dehydrogenases also play a key role in cellulose breakdown by directly/indirectly "fueling" electrons for polysaccharide monooxygenase activation. Many of the above oxidoreductases have been engineered, combining rational and computational design with directed evolution, to attain the selectivity, catalytic efficiency and stability properties required for their industrial utilization. Indeed, using ad hoc software and current computational capabilities, it is now possible to predict substrate access to the active site in biophysical simulations, and

Rationally engineered flavin-dependent oxidase reveals steric control of dioxygen reduction.

PubMed

Zafred, Domen; Steiner, Barbara; Teufelberger, Andrea R; Hromic, Altijana; Karplus, P Andrew; Schofield, Christopher J; Wallner, Silvia; Macheroux, Peter

2015-08-01

The ability of flavoenzymes to reduce dioxygen varies greatly, and is controlled by the protein environment, which may cause either a rapid reaction (oxidases) or a sluggish reaction (dehydrogenases). Previously, a 'gatekeeper' amino acid residue was identified that controls the reactivity to dioxygen in proteins from the vanillyl alcohol oxidase superfamily of flavoenzymes. We have identified an alternative gatekeeper residue that similarly controls dioxygen reactivity in the grass pollen allergen Phl p 4, a member of this superfamily that has glucose dehydrogenase activity and the highest redox potential measured in a flavoenzyme. A substitution at the alternative gatekeeper site (I153V) transformed the enzyme into an efficient oxidase by increasing dioxygen reactivity by a factor of 60,000. An inverse exchange (V169I) in the structurally related berberine bridge enzyme (BBE) decreased its dioxygen reactivity by a factor of 500. Structural and biochemical characterization of these and additional variants showed that our model enzymes possess a cavity that binds an anion and resembles the 'oxyanion hole' in the proximity of the flavin ring. We showed also that steric control of access to this site is the most important parameter affecting dioxygen reactivity in BBE-like enzymes. Analysis of flavin-dependent oxidases from other superfamilies revealed similar structural features, suggesting that dioxygen reactivity may be governed by a common mechanistic principle. Structural data are available in PDB database under the accession numbers 4PVE, 4PVH, 4PVJ, 4PVK, 4PWB, 4PWC and 4PZF. © 2015 FEBS.

The purification, crystallization and preliminary structural characterization of FAD-dependent monooxygenase PhzS, a phenazine-modifying enzyme from Pseudomonas aeruginosa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gohain, Neelakshi; Thomashow, Linda S.; USDA Agricultural Research Service, Root Disease and Biological Control Research Unit, Pullman, Washington 99164-6430

2006-10-01

PhzS, an FAD-dependent monooxygenase that catalyzes a reaction involved in the biosynthesis of the virulence factor pyocyanin in P. aeruginosa, was cloned, overexpressed and crystallized. Data collection from native and seleno-l-methionine-labelled crystals is reported. The blue chloroform-soluble bacterial metabolite pyocyanin (1-hydroxy-5-methyl-phenazine) contributes to the survival and virulence of Pseudomonas aeruginosa, an important Gram-negative opportunistic pathogen of humans and animals. Little is known about the two enzymes, designated PhzM and PhzS, that function in the synthesis of pyocyanin from phenazine-1-carboxylic acid. In this study, the FAD-dependent monooxygenase PhzS was purified and crystallized from lithium sulfate/ammonium sulfate/sodium citrate pH 5.5. Native crystalsmore » belong to space group C2, with unit-cell parameters a = 144.2, b = 96.2, c = 71.7 Å, α = γ = 90, β = 110.5°. They contain two monomers of PhzS in the asymmetric unit and diffract to a resolution of 2.4 Å. Seleno-l-methionine-labelled PhzS also crystallizes in space group C2, but the unit-cell parameters change to a = 70.6, b = 76.2, c = 80.2 Å, α = γ = 90, β = 110.5° and the diffraction limit is 2.7 Å.« less

Flavin redox bifurcation as a mechanism for controlling the direction of electron flow during extracellular electron transfer.

PubMed

Okamoto, Akihiro; Hashimoto, Kazuhito; Nealson, Kenneth H

2014-10-06

The iron-reducing bacterium Shewanella oneidensis MR-1 has a dual directional electronic conduit involving 40 heme redox centers in flavin-binding outer-membrane c-type cytochromes (OM c-Cyts). While the mechanism for electron export from the OM c-Cyts to an anode is well understood, how the redox centers in OM c-Cyts take electrons from a cathode has not been elucidated at the molecular level. Electrochemical analysis of live cells during switching from anodic to cathodic conditions showed that altering the direction of electron flow does not require gene expression or protein synthesis, but simply redox potential shift about 300 mV for a flavin cofactor interacting with the OM c-Cyts. That is, the redox bifurcation of the riboflavin cofactor in OM c-Cyts switches the direction of electron conduction in the biological conduit at the cell-electrode interface to drive bacterial metabolism as either anode or cathode catalysts. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Bacillus subtilis Fur represses one of two paralogous haem-degrading monooxygenases

PubMed Central

Gaballa, Ahmed

2011-01-01

Identification of genes regulated by the ferric uptake regulator (Fur) protein has provided insights into the diverse mechanisms of adaptation to iron limitation. In the soil bacterium Bacillus subtilis, Fur senses iron sufficiency and represses genes that enable iron uptake, including biosynthetic and transport genes for the siderophore bacillibactin and uptake systems for siderophores produced by other organisms. We here demonstrate that Fur regulates hmoA (formerly yetG), which encodes a haem monooxygenase. HmoA is the first characterized member of a divergent group of putative monooxygenases that cluster separately from the well-characterized IsdG family. B. subtilis also encodes an IsdG family protein designated HmoB (formerly YhgC). Unlike hmoA, hmoB is constitutively expressed and not under Fur control. HmoA and HmoB both bind haemin in vitro with approximately 1 : 1 stoichiometry and degrade haemin in the presence of an electron donor. Mutational and spectroscopic analyses indicate that HmoA and HmoB have distinct active site architectures and interact differently with haem. We further show that B. subtilis can use haem as an iron source, but that this ability is independent of HmoA and HmoB. PMID:21873409

Minimizing photodecomposition of flavin adenine dinucleotide fluorescence by the use of pulsed LEDs.

PubMed

Rösner, J; Liotta, A; Angamo, E A; Spies, C; Heinemann, U; Kovács, R

2016-11-01

Dynamic alterations in flavin adenine dinucleotide (FAD) fluorescence permit insight into energy metabolism-dependent changes of intramitochondrial redox potential. Monitoring FAD fluorescence in living tissue is impeded by photobleaching, restricting the length of microfluorimetric recordings. In addition, photodecomposition of these essential electron carriers negatively interferes with energy metabolism and viability of the biological specimen. Taking advantage of pulsed LED illumination, here we determined the optimal excitation settings giving the largest fluorescence yield with the lowest photobleaching and interference with metabolism in hippocampal brain slices. The effects of FAD bleaching on energy metabolism and viability were studied by monitoring tissue pO 2 , field potentials and changes in extracellular potassium concentration ([K + ] o ). Photobleaching with continuous illumination consisted of an initial exponential decrease followed by a nearly linear decay. The exponential decay was significantly decelerated with pulsed illumination. Pulse length of 5 ms was sufficient to reach a fluorescence output comparable to continuous illumination, whereas further increasing duration increased photobleaching. Similarly, photobleaching increased with shortening of the interpulse interval. Photobleaching was partially reversible indicating the existence of a transient nonfluorescent flavin derivative. Pulsed illumination decreased FAD photodecomposition, improved slice viability and reproducibility of stimulus-induced FAD, field potential, [K + ] o and pO 2 changes as compared to continuous illumination. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

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

PubMed

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

2014-06-01

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

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

2017-11-15

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 © 2016. Published by Elsevier Inc.

Monooxygenase, a Novel Beta-Cypermethrin Degrading Enzyme from Streptomyces sp

PubMed Central

Xiao, Ying; Deng, Yinyue; Chang, Changqing; Zhong, Guohua; Hu, Meiying; Zhang, Lian-Hui

2013-01-01

The widely used insecticide beta-cypermethrin has become a public concern because of its environmental contamination and toxic effects on mammals. In this study, a novel beta-cypermethrin degrading enzyme designated as CMO was purified to apparent homogeneity from a Streptomyces sp. isolate capable of utilizing beta-cypermethrin as a growth substrate. The native enzyme showed a monomeric structure with a molecular mass of 41 kDa and pI of 5.4. The enzyme exhibited the maximal activity at pH 7.5 and 30°C. It was fairly stable in the pH range from 6.5–8.5 and at temperatures below 10°C. The enzyme activity was significantly stimulated by Fe2+, but strongly inhibited by Ag+, Al3+, and Cu2+. The enzyme catalyzed the degradation of beta-cypermethrin to form five products via hydroxylation and diaryl cleavage. A novel beta-cypermethrin detoxification pathway was proposed based on analysis of these products. The purified enzyme was identified as a monooxygenase by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry analysis (MALDI-TOF-MS) and N-terminal protein sequencing. Given that all the characterized pyrethroid-degrading enzymes are the members of hydrolase family, CMO represents the first pyrethroid-degrading monooxygenase identified from environmental microorganisms. Taken together, our findings depict a novel pyrethroid degradation mechanism and indicate that the purified enzyme may be a promising candidate for detoxification of beta-cypermethrin and environmental protection. PMID:24098697

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

Morphology and morphometry of the meningo-orbital foramen as a result of plastic responses to the ambient temperature and its clinical relevance.

PubMed

Tomaszewska, Agnieszka; Zelaźniewicz, Agnieszka

2014-05-01

The meningo-orbital foramen (FMO) is an osteal opening, containing vessels providing an accessory blood supply to the orbit, situated close to the superior orbital fissure. Recent studies show FMO to be ubiquitous, with localization and occurrence varying, depending on a population, what may be due to environmental conditions (ie, temperature). It is often located near the operating area in surgeries in the orbital cavity, and its accurate localization allows avoiding unexpected bleeding during intervention. Because there is a lack of clarity in the literature concerning the morphology and the morphometry of the FMO, this study aimed to clarify the issue with clinical relevance. We studied dry adult human skulls (50 men and 33 women). The morphology and minimal distances between the FMO and standard anthropologic landmarks (nasion, frontomalare orbitale, supraorbital foramen, and zygomaticomaxillary suture) were measured, using MicroScribe G2L, a three-dimensional contact scanner. We compared the result with results of previous studies on populations from various climate zones. The FMO was present in 69.88% of the skulls (56.02% of orbits): in 60.34% of the skulls, the FMO was bilateral; and in 39.66%, unilateral. We observed 74 single, 10 double, and 2 triple foramina. The FMO was present mostly on the sphenoid and the frontal bone. There was no difference in minimal distances between the FMO and the anthropologic landmarks, depending on sex, except the distance to the nasion (shorter in women). The occurrence of the FMO in the population differed from that of other populations. The results show that it is possible that the morphology and the morphometry of the FMO depend on the climate zone or ambient temperature during growth, which should be considered before performing surgery in the orbital cavity.

Daily fluctuation of hepatic P450 monooxygenase activities in male rats is controlled by the suprachiasmatic nucleus but remains unaffected by adrenal hormones.

PubMed

Furukawa, T; Manabe, S; Watanabe, T; Sehata, S; Sharyo, S; Okada, T; Mori, Y

1999-09-01

Hepatic P450 monooxygenase activities, which strongly influence the efficacy and/or toxicity of drugs, are known to fluctuate daily. We also know that the P450 activities assessed by measurement of 7-alkoxycoumarin O-dealkylase (ACD) activities fluctuate daily, with apparently high values during the dark period in male rats. However, there is little knowledge about the factors that regulate daily fluctuation of P450 monooxygenase activities. In the present study using rats, we induced lesions in the suprachiasmatic nucleus (SCN) of the brain, the known site of the body's internal clock, and examined the effects on the daily fluctuation of the ACD activities to clarify the relationship between the SCN and the daily fluctuation of P450 monooxygenase activities. In addition, adrenalectomy was performed to re-evaluate the influence of adrenal hormones on the P450 activities. Our results indicated that daily fluctuations of the hepatic ACD activities were completely eliminated in the SCN-lesioned rats. However, the ACD activities in the adrenalectomized rats showed apparent daily fluctuations with high values during the dark period and low values during the light period. Therefore, this study demonstrated that the daily fluctuation of the hepatic P450 monooxygenase activities in male rats is controlled by the SCN but remains unaffected by the adrenal hormones.

The conserved baculovirus protein p33 (Ac92) is a flavin adenine dinucleotide-linked sulfhydryl oxidase

DOE Office of Scientific and Technical Information (OSTI.GOV)

Long, C.M.; Rohrmann, G.F.; Merrill, G.F., E-mail: merrillg@onid.orst.ed

2009-06-05

Open reading frame 92 of the Autographa californica baculovirus (Ac92) is one of about 30 core genes present in all sequenced baculovirus genomes. Computer analyses predicted that the Ac92 encoded protein (called p33) and several of its baculovirus orthologs were related to a family of flavin adenine dinucleotide (FAD)-linked sulfhydryl oxidases. Alignment of these proteins indicated that, although they were highly diverse, a number of amino acids in common with the Erv1p/Alrp family of sulfhydryl oxidases are present. Some of these conserved amino acids are predicted to stack against the isoalloxazine and adenine components of FAD, whereas others are involvedmore » in electron transfer. To investigate this relationship, Ac92 was expressed in bacteria as a His-tagged fusion protein, purified, and characterized both spectrophotometrically and for its enzymatic activity. The purified protein was found to have the color (yellow) and absorption spectrum consistent with it being a FAD-containing protein. Furthermore, it was demonstrated to have sulfhydryl oxidase activity using dithiothreitol and thioredoxin as substrates.« less

The conserved baculovirus protein p33 (Ac92) is a flavin adenine dinucleotide-linked sulfhydryl oxidase.

PubMed

Long, C M; Rohrmann, G F; Merrill, G F

2009-06-05

Open reading frame 92 of the Autographa californica baculovirus (Ac92) is one of about 30 core genes present in all sequenced baculovirus genomes. Computer analyses predicted that the Ac92 encoded protein (called p33) and several of its baculovirus orthologs were related to a family of flavin adenine dinucleotide (FAD)-linked sulfhydryl oxidases. Alignment of these proteins indicated that, although they were highly diverse, a number of amino acids in common with the Erv1p/Alrp family of sulfhydryl oxidases are present. Some of these conserved amino acids are predicted to stack against the isoalloxazine and adenine components of FAD, whereas others are involved in electron transfer. To investigate this relationship, Ac92 was expressed in bacteria as a His-tagged fusion protein, purified, and characterized both spectrophotometrically and for its enzymatic activity. The purified protein was found to have the color (yellow) and absorption spectrum consistent with it being a FAD-containing protein. Furthermore, it was demonstrated to have sulfhydryl oxidase activity using dithiothreitol and thioredoxin as substrates.

A conserved degron containing an amphipathic helix regulates the cholesterol-mediated turnover of human squalene monooxygenase, a rate-limiting enzyme in cholesterol synthesis.

PubMed

Chua, Ngee Kiat; Howe, Vicky; Jatana, Nidhi; Thukral, Lipi; Brown, Andrew J

2017-12-08

Cholesterol biosynthesis in the endoplasmic reticulum (ER) is tightly controlled by multiple mechanisms to regulate cellular cholesterol levels. Squalene monooxygenase (SM) is the second rate-limiting enzyme in cholesterol biosynthesis and is regulated both transcriptionally and post-translationally. SM undergoes cholesterol-dependent proteasomal degradation when cholesterol is in excess. The first 100 amino acids of SM (designated SM N100) are necessary for this degradative process and represent the shortest cholesterol-regulated degron identified to date. However, the fundamental intrinsic characteristics of this degron remain unknown. In this study, we performed a series of deletions, point mutations, and domain swaps to identify a 12-residue region (residues Gln-62-Leu-73), required for SM cholesterol-mediated turnover. Molecular dynamics and circular dichroism revealed an amphipathic helix within this 12-residue region. Moreover, 70% of the variation in cholesterol regulation was dependent on the hydrophobicity of this region. Of note, the earliest known Doa10 yeast degron, Deg1, also contains an amphipathic helix and exhibits 42% amino acid similarity with SM N100. Mutating SM residues Phe-35/Ser-37/Leu-65/Ile-69 into alanine, based on the key residues in Deg1, blunted SM cholesterol-mediated turnover. Taken together, our results support a model whereby the amphipathic helix in SM N100 attaches reversibly to the ER membrane depending on cholesterol levels; with excess, the helix is ejected and unravels, exposing a hydrophobic patch, which then serves as a degradation signal. Our findings shed new light on the regulation of a key cholesterol synthesis enzyme, highlighting the conservation of critical degron features from yeast to humans. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

P450 monooxygenases (P450ome) of the model white rot fungus Phanerochaete chrysosporium.

PubMed

Syed, Khajamohiddin; Yadav, Jagjit S

2012-11-01

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, 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. chrysosproium 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.

Cell-secreted flavins bound to membrane cytochromes dictate electron transfer reactions to surfaces with diverse charge and pH.

PubMed

Okamoto, Akihiro; Kalathil, Shafeer; Deng, Xiao; Hashimoto, Kazuhito; Nakamura, Ryuhei; Nealson, Kenneth H

2014-07-11

The variety of solid surfaces to and from which microbes can deliver electrons by extracellular electron transport (EET) processes via outer-membrane c-type cytochromes (OM c-Cyts) expands the importance of microbial respiration in natural environments and industrial applications. Here, we demonstrate that the bifurcated EET pathway of OM c-Cyts sustains the diversity of the EET surface in Shewanella oneidensis MR-1 via specific binding with cell-secreted flavin mononucleotide (FMN) and riboflavin (RF). Microbial current production and whole-cell differential pulse voltammetry revealed that RF and FMN enhance EET as bound cofactors in a similar manner. Conversely, FMN and RF were clearly differentiated in the EET enhancement by gene-deletion of OM c-Cyts and the dependency of the electrode potential and pH. These results indicate that RF and FMN have specific binding sites in OM c-Cyts and highlight the potential roles of these flavin-cytochrome complexes in controlling the rate of electron transfer to surfaces with diverse potential and pH.

Oxidation of Benzene to Phenol, Catechol, and 1,2,3-Trihydroxybenzene by Toluene 4-Monooxygenase of Pseudomonas mendocina KR1 and Toluene 3-Monooxygenase of Ralstonia pickettii PKO1

PubMed Central

Tao, Ying; Fishman, Ayelet; Bentley, William E.; Wood, Thomas K.

2004-01-01

Aromatic hydroxylations are important bacterial metabolic processes but are difficult to perform using traditional chemical synthesis, so to use a biological catalyst to convert the priority pollutant benzene into industrially relevant intermediates, benzene oxidation was investigated. It was discovered that toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1, toluene 3-monooxygenase (T3MO) of Ralstonia pickettii PKO1, and toluene ortho-monooxygenase (TOM) of Burkholderia cepacia G4 convert benzene to phenol, catechol, and 1,2,3-trihydroxybenzene by successive hydroxylations. At a concentration of 165 μM and under the control of a constitutive lac promoter, Escherichia coli TG1/pBS(Kan)T4MO expressing T4MO formed phenol from benzene at 19 ± 1.6 nmol/min/mg of protein, catechol from phenol at 13.6 ± 0.3 nmol/min/mg of protein, and 1,2,3-trihydroxybenzene from catechol at 2.5 ± 0.5nmol/min/mg of protein. The catechol and 1,2,3-trihydroxybenzene products were identified by both high-pressure liquid chromatography and mass spectrometry. When analogous plasmid constructs were used, E. coli TG1/pBS(Kan)T3MO expressing T3MO formed phenol, catechol, and 1,2,3-trihydroxybenzene at rates of 3 ± 1, 3.1 ± 0.3, and 0.26 ± 0.09 nmol/min/mg of protein, respectively, and E. coli TG1/pBS(Kan)TOM expressing TOM formed 1,2,3-trihydroxybenzene at a rate of 1.7 ± 0.3 nmol/min/mg of protein (phenol and catechol formation rates were 0.89 ± 0.07 and 1.5 ± 0.3 nmol/min/mg of protein, respectively). Hence, the rates of synthesis of catechol by both T3MO and T4MO and the 1,2,3-trihydroxybenzene formation rate by TOM were found to be comparable to the rates of oxidation of the natural substrate toluene for these enzymes (10.0 ± 0.8, 4.0 ± 0.6, and 2.4 ± 0.3 nmol/min/mg of protein for T4MO, T3MO, and TOM, respectively, at a toluene concentration of 165 μM). PMID:15240250

Fundamental Role of Methylenetetrahydrofolate Reductase 677 C → T Genotype and Flavin Compounds in Biochemical Phenotypes for Schizophrenia and Schizoaffective Psychosis

PubMed Central

Fryar-Williams, Stephanie

2016-01-01

The Mental Health Biomarker Project (2010–2016) explored variables for psychosis in schizophrenia and schizoaffective disorder. Blood samples from 67, highly characterized symptomatic cases and 67 gender and age matched control participants were analyzed for methyl tetrahydrofolate reductase (MTHFR) 677C → T gene variants and for vitamin B6, B12 and D, folate, unbound copper, zinc cofactors for enzymes in the methylation cycle, and related catecholamine pathways. Urine samples were analyzed for indole-catecholamines, their metabolites, and oxidative-stress marker, hydroxylpyrolline-2-one (HPL). Rating scales were Brief Psychiatric Rating Scale, Positive and Negative Syndrome Scale, Global Assessment of Function scale, Clinical Global Impression (CGI) score, and Social and Occupational Functioning Assessment Scale (SOFAS). Analysis used Spearman’s correlates, receiver operating characteristics and structural equation modeling (SEM). The correlative pattern of variables in the overall participant sample strongly implicated monoamine oxidase (MAO) enzyme inactivity so the significant role of MAO’s cofactor flavin adenine nucleotide and its precursor flavin adenine mononucleotide (FMN) within the biochemical pathways was investigated and confirmed as 71% on SEM of the total sample. Splitting the data sets for MTHFR 677C → T polymorphism variants coding for the MTHFR enzyme, discovered that biochemistry variables relating to the wild-type enzyme differed markedly in pattern from those coded by the homozygous variant and that the hereozygous-variant pattern resembled the wild-type-coded pattern. The MTHFR 677C → T-wild and -heterozygous gene variants have a pattern of depleted vitamin cofactors characteristic of flavin insufficiency with under-methylation and severe oxidative stress. The second homozygous MTHFR 677TT pattern related to elevated copper:zinc ratio and a vitamin pattern related to flavin sufficiency and risk of over-methylation. The

Fundamental Role of Methylenetetrahydrofolate Reductase 677 C → T Genotype and Flavin Compounds in Biochemical Phenotypes for Schizophrenia and Schizoaffective Psychosis.

PubMed

Fryar-Williams, Stephanie

2016-01-01

The Mental Health Biomarker Project (2010-2016) explored variables for psychosis in schizophrenia and schizoaffective disorder. Blood samples from 67, highly characterized symptomatic cases and 67 gender and age matched control participants were analyzed for methyl tetrahydrofolate reductase (MTHFR) 677C → T gene variants and for vitamin B6, B12 and D, folate, unbound copper, zinc cofactors for enzymes in the methylation cycle, and related catecholamine pathways. Urine samples were analyzed for indole-catecholamines, their metabolites, and oxidative-stress marker, hydroxylpyrolline-2-one (HPL). Rating scales were Brief Psychiatric Rating Scale, Positive and Negative Syndrome Scale, Global Assessment of Function scale, Clinical Global Impression (CGI) score, and Social and Occupational Functioning Assessment Scale (SOFAS). Analysis used Spearman's correlates, receiver operating characteristics and structural equation modeling (SEM). The correlative pattern of variables in the overall participant sample strongly implicated monoamine oxidase (MAO) enzyme inactivity so the significant role of MAO's cofactor flavin adenine nucleotide and its precursor flavin adenine mononucleotide (FMN) within the biochemical pathways was investigated and confirmed as 71% on SEM of the total sample. Splitting the data sets for MTHFR 677C → T polymorphism variants coding for the MTHFR enzyme, discovered that biochemistry variables relating to the wild-type enzyme differed markedly in pattern from those coded by the homozygous variant and that the hereozygous-variant pattern resembled the wild-type-coded pattern. The MTHFR 677C → T-wild and -heterozygous gene variants have a pattern of depleted vitamin cofactors characteristic of flavin insufficiency with under-methylation and severe oxidative stress. The second homozygous MTHFR 677TT pattern related to elevated copper:zinc ratio and a vitamin pattern related to flavin sufficiency and risk of over-methylation. The two

African Swine Fever Virus pB119L Protein Is a Flavin Adenine Dinucleotide-Linked Sulfhydryl Oxidase

PubMed Central

Rodríguez, Irene; Redrejo-Rodríguez, Modesto; Rodríguez, Javier M.; Alejo, Alí; Salas, José; Salas, María L.

2006-01-01

Protein pB119L of African swine fever virus belongs to the Erv1p/Alrp family of sulfhydryl oxidases and has been described as a late nonstructural protein required for correct virus assembly. To further our knowledge of the function of protein pB119L during the virus life cycle, we have investigated whether this protein possesses sulfhydryl oxidase activity, using a purified recombinant protein. We show that the purified protein contains bound flavin adenine dinucleotide and is capable of catalyzing the formation of disulfide bonds both in a protein substrate and in the small molecule dithiothreitol, the catalytic activity being comparable to that of the Erv1p protein. Furthermore, protein pB119L contains the cysteines of its active-site motif CXXC, predominantly in an oxidized state, and forms noncovalently bound dimers in infected cells. We also show in coimmunoprecipitation experiments that protein pB119L interacts with the viral protein pA151R, which contains a CXXC motif similar to that present in thioredoxins. Protein pA151R, in turn, was found to interact with the viral structural protein pE248R, which contains disulfide bridges and belongs to a class of myristoylated proteins related to vaccinia virus L1R, one of the substrates of the redox pathway encoded by this virus. These results suggest the existence in African swine fever virus of a system for the formation of disulfide bonds constituted at least by proteins pB119L and pA151R and identify protein pE248R as a possible final substrate of this pathway. PMID:16537584

African swine fever virus pB119L protein is a flavin adenine dinucleotide-linked sulfhydryl oxidase.

PubMed

Rodríguez, Irene; Redrejo-Rodríguez, Modesto; Rodríguez, Javier M; Alejo, Alí; Salas, José; Salas, María L

2006-04-01

Protein pB119L of African swine fever virus belongs to the Erv1p/Alrp family of sulfhydryl oxidases and has been described as a late nonstructural protein required for correct virus assembly. To further our knowledge of the function of protein pB119L during the virus life cycle, we have investigated whether this protein possesses sulfhydryl oxidase activity, using a purified recombinant protein. We show that the purified protein contains bound flavin adenine dinucleotide and is capable of catalyzing the formation of disulfide bonds both in a protein substrate and in the small molecule dithiothreitol, the catalytic activity being comparable to that of the Erv1p protein. Furthermore, protein pB119L contains the cysteines of its active-site motif CXXC, predominantly in an oxidized state, and forms noncovalently bound dimers in infected cells. We also show in coimmunoprecipitation experiments that protein pB119L interacts with the viral protein pA151R, which contains a CXXC motif similar to that present in thioredoxins. Protein pA151R, in turn, was found to interact with the viral structural protein pE248R, which contains disulfide bridges and belongs to a class of myristoylated proteins related to vaccinia virus L1R, one of the substrates of the redox pathway encoded by this virus. These results suggest the existence in African swine fever virus of a system for the formation of disulfide bonds constituted at least by proteins pB119L and pA151R and identify protein pE248R as a possible final substrate of this pathway.

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.

Evidence for Posttranslational Protein Flavinylation in the Syphilis Spirochete Treponema pallidum: Structural and Biochemical Insights from the Catalytic Core of a Periplasmic Flavin-Trafficking Protein

PubMed Central

Deka, Ranjit K.; Brautigam, Chad A.; Liu, Wei Z.

2015-01-01

ABSTRACT The syphilis spirochete Treponema pallidum is an important human pathogen but a highly enigmatic bacterium that cannot be cultivated in vitro. T. pallidum lacks many biosynthetic pathways and therefore has evolved the capability to exploit host-derived metabolites via its periplasmic lipoprotein repertoire. We recently reported a flavin-trafficking protein in T. pallidum (Ftp_Tp; TP0796) as the first bacterial metal-dependent flavin adenine dinucleotide (FAD) pyrophosphatase that hydrolyzes FAD into AMP and flavin mononucleotide (FMN) in the spirochete’s periplasm. However, orthologs of Ftp_Tp from other bacteria appear to lack this hydrolytic activity; rather, they bind and flavinylate subunits of a cytoplasmic membrane redox system (Nqr/Rnf). To further explore this dichotomy, biochemical analyses, protein crystallography, and structure-based mutagenesis were used to show that a single amino acid change (N55Y) in Ftp_Tp converts it from an Mg2+-dependent FAD pyrophosphatase to an FAD-binding protein. We also demonstrated that Ftp_Tp has a second enzymatic activity (Mg2+-FMN transferase); it flavinylates protein(s) covalently with FMN on a threonine side chain of an appropriate sequence motif using FAD as the substrate. Moreover, mutation of a metal-binding residue (D284A) eliminates Ftp_Tp’s dual activities, thereby underscoring the role of Mg2+ in the enzyme-catalyzed reactions. The posttranslational flavinylation activity that can target a periplasmic lipoprotein (TP0171) has not previously been described. The observed activities reveal the catalytic flexibility of a treponemal protein to perform multiple functions. Together, these findings imply mechanisms by which a dynamic pool of flavin cofactor is maintained and how flavoproteins are generated by Ftp_Tp locally in the T. pallidum periplasm. PMID:25944861

Evidence for posttranslational protein flavinylation in the syphilis spirochete Treponema pallidum: Structural and biochemical insights from the catalytic core of a periplasmic flavin-trafficking protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Deka, Ranjit K.; Brautigam, Chad A.; Liu, Wei Z.

The syphilis spirochete Treponema pallidum is an important human pathogen but a highly enigmatic bacterium that cannot be cultivated in vitro. T. pallidum lacks many biosynthetic pathways and therefore has evolved the capability to exploit host-derived metabolites via its periplasmic lipoprotein repertoire. We recently reported a flavin-trafficking protein in T. pallidum (Ftp_Tp; TP0796) as the first bacterial metal-dependent flavin adenine dinucleotide (FAD) pyrophosphatase that hydrolyzes FAD into AMP and flavin mononucleotide (FMN) in the spirochete’s periplasm. However, orthologs of Ftp_Tp from other bacteria appear to lack this hydrolytic activity; rather, they bind and flavinylate subunits of a cytoplasmic membrane redoxmore » system (Nqr/Rnf). To further explore this dichotomy, biochemical analyses, protein crystallography, and structure-based mutagenesis were used to show that a single amino acid change (N55Y) in Ftp_Tp converts it from an Mg²⁺-dependent FAD pyrophosphatase to an FAD-binding protein. We also demonstrated that Ftp_Tp has a second enzymatic activity (Mg²⁺-FMN transferase); it flavinylates protein(s) covalently with FMN on a threonine side chain of an appropriate sequence motif using FAD as the substrate. Moreover, mutation of a metal-binding residue (D284A) eliminates Ftp_Tp’s dual activities, thereby underscoring the role of Mg²⁺ in the enzyme-catalyzed reactions. The posttranslational flavinylation activity that can target a periplasmic lipoprotein (TP0171) has not previously been described. The observed activities reveal the catalytic flexibility of a treponemal protein to perform multiple functions. Together, these findings imply mechanisms by which a dynamic pool of flavin cofactor is maintained and how flavoproteins are generated by Ftp_Tp locally in the T. pallidum periplasm.« less

Evidence for posttranslational protein flavinylation in the syphilis spirochete Treponema pallidum: Structural and biochemical insights from the catalytic core of a periplasmic flavin-trafficking protein

DOE PAGES

Deka, Ranjit K.; Brautigam, Chad A.; Liu, Wei Z.; ...

2015-05-05

The syphilis spirochete Treponema pallidum is an important human pathogen but a highly enigmatic bacterium that cannot be cultivated in vitro. T. pallidum lacks many biosynthetic pathways and therefore has evolved the capability to exploit host-derived metabolites via its periplasmic lipoprotein repertoire. We recently reported a flavin-trafficking protein in T. pallidum (Ftp_Tp; TP0796) as the first bacterial metal-dependent flavin adenine dinucleotide (FAD) pyrophosphatase that hydrolyzes FAD into AMP and flavin mononucleotide (FMN) in the spirochete’s periplasm. However, orthologs of Ftp_Tp from other bacteria appear to lack this hydrolytic activity; rather, they bind and flavinylate subunits of a cytoplasmic membrane redoxmore » system (Nqr/Rnf). To further explore this dichotomy, biochemical analyses, protein crystallography, and structure-based mutagenesis were used to show that a single amino acid change (N55Y) in Ftp_Tp converts it from an Mg²⁺-dependent FAD pyrophosphatase to an FAD-binding protein. We also demonstrated that Ftp_Tp has a second enzymatic activity (Mg²⁺-FMN transferase); it flavinylates protein(s) covalently with FMN on a threonine side chain of an appropriate sequence motif using FAD as the substrate. Moreover, mutation of a metal-binding residue (D284A) eliminates Ftp_Tp’s dual activities, thereby underscoring the role of Mg²⁺ in the enzyme-catalyzed reactions. The posttranslational flavinylation activity that can target a periplasmic lipoprotein (TP0171) has not previously been described. The observed activities reveal the catalytic flexibility of a treponemal protein to perform multiple functions. Together, these findings imply mechanisms by which a dynamic pool of flavin cofactor is maintained and how flavoproteins are generated by Ftp_Tp locally in the T. pallidum periplasm.« less

Effects of soluble flavin on heterogeneous electron transfer between surface-exposed bacterial cytochromes and iron oxides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Zheming; Shi, Zhi; Shi, Liang

2015-08-25

Dissimilatory iron-reducing bacteria can utilize insoluble Fe(Mn)-oxides as a terminal electron acceptor under anaerobic conditions. For Shewanella species specifically, some evidence suggests that iron reduction is associated with the secretion of flavin mononucleotide (FMN) and riboflavin that are proposed to mediate electron transfer (Marsili et al., 2008). In this work, we used methyl viologen (MV•+)-encapsulated, porin-cytochrome complex (MtrCAB) embedded liposomes (MELs) as a synthetic model of the Shewanella outer membrane to investigate the proposed mediating behavior of secreted flavins. The reduction kinetics of goethite, hematite and lepidocrocite (200 µM) by MELs ([MV•+] ~ 42 µM and MtrABC ≤ 1 nM)more » were determined in the presence FMN at pH 7.0 in N2 atmosphere by monitoring the concentrations of MV•+ and FMN through their characteristic UV-visible absorption spectra. Experiments were performed where i) FMN and Fe(III)-oxide were mixed and then reacted with the reduced MELs and ii) FMN was reacted with the reduced MELs followed by addition of Fe(III)-oxide. The redox reactions proceeded in two steps: a fast step that was completed in a few seconds, and a slower one lasting over 400 seconds. For all three Fe(III)-oxides, the initial reaction rate in the presence of a low concentration of FMN (≤ 1 µM) was at least a factor of five faster than those with MELs alone, and orders of magnitude faster than those by FMNH2, suggesting that FMN may serve as a co-factor that enhances electron transfer from outer-membrane c-cytochromes to Fe(III)-oxides. The rate and extent of the initial reaction followed the order of lepidocrocite > hematite > goethite, the same as their reduction potentials, implying thermodynamic control on reaction rate. However, at higher FMN concentrations (> 1 µM), the reaction rates for both steps decreased and varied inversely with FMN concentration, indicating that FMN inhibited the MEL to Fe(III)-oxide electron

KDM1 Class Flavin-Dependent Protein Lysine Demethylases

PubMed Central

Burg, Jonathan M.; Link, Jennifer E.; Morgan, Brittany S.; Heller, Frederick J.; Hargrove, Amanda E.; McCafferty, Dewey G.

2015-01-01

Flavin-dependent, lysine-specific protein demethylases (KDM1s) are a subfamily of amine oxidases that catalyze the selective posttranslational oxidative demethylation of methyllysine side chains within protein and peptide substrates. KDM1s participate in the widespread epigenetic regulation of both normal and disease state transcriptional programs. Their activities are central to various cellular functions, such as hematopoietic and neuronal differentiation, cancer proliferation and metastasis, and viral lytic replication and establishment of latency. Interestingly, KDM1s function as catalytic subunits within complexes with coregulatory molecules that modulate enzymatic activity of the demethylases and coordinate their access to specific substrates at distinct sites within the cell and chromatin. Although several classes of KDM1 -selective small molecule inhibitors have been recently developed, these pan-active site inhibition strategies lack the ability to selectively discriminate between KDM1 activity in specific, and occasionally opposing, functional contexts within these complexes. Here we review the discovery of this class of demethylases, their structures, chemical mechanisms, and specificity. Additionally, we review inhibition of this class of enzymes as well as emerging interactions with coregulatory molecules that regulate demethylase activity in highly specific functional contexts of biological and potential therapeutic importance. PMID:25787087

Bacterial expression of human kynurenine 3-monooxygenase: Solubility, activity, purification☆

PubMed Central

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

2014-01-01

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

A Flavin Binding Cryptochrome Photoreceptor Responds to Both Blue and Red Light in Chlamydomonas reinhardtii[W

PubMed Central

Beel, Benedikt; Prager, Katja; Spexard, Meike; Sasso, Severin; Weiss, Daniel; Müller, Nico; Heinnickel, Mark; Dewez, David; Ikoma, Danielle; Grossman, Arthur R.; Kottke, Tilman; Mittag, Maria

2012-01-01

Cryptochromes are flavoproteins that act as sensory blue light receptors in insects, plants, fungi, and bacteria. We have investigated a cryptochrome from the green alga Chlamydomonas reinhardtii with sequence homology to animal cryptochromes and (6-4) photolyases. In response to blue and red light exposure, this animal-like cryptochrome (aCRY) alters the light-dependent expression of various genes encoding proteins involved in chlorophyll and carotenoid biosynthesis, light-harvesting complexes, nitrogen metabolism, cell cycle control, and the circadian clock. Additionally, exposure to yellow but not far-red light leads to comparable increases in the expression of specific genes; this expression is significantly reduced in an acry insertional mutant. These in vivo effects are congruent with in vitro data showing that blue, yellow, and red light, but not far-red light, are absorbed by the neutral radical state of flavin in aCRY. The aCRY neutral radical is formed following blue light absorption of the oxidized flavin. Red illumination leads to conversion to the fully reduced state. Our data suggest that aCRY is a functionally important blue and red light–activated flavoprotein. The broad spectral response implies that the neutral radical state functions as a dark form in aCRY and expands the paradigm of flavoproteins and cryptochromes as blue light sensors to include other light qualities. PMID:22773746

A Bacterial Multidomain NAD-Independent d-Lactate Dehydrogenase Utilizes Flavin Adenine Dinucleotide and Fe-S Clusters as Cofactors and Quinone as an Electron Acceptor for d-Lactate Oxidization

PubMed Central

Jiang, Tianyi; Guo, Xiaoting; Yan, Jinxin; Zhang, Yingxin; Wang, Yujiao; Zhang, Manman; Sheng, Binbin; Ma, Cuiqing; Xu, Ping

2017-01-01

ABSTRACT Bacterial membrane-associated NAD-independent d-lactate dehydrogenase (Fe-S d-iLDH) oxidizes d-lactate into pyruvate. A sequence analysis of the enzyme reveals that it contains an Fe-S oxidoreductase domain in addition to a flavin adenine dinucleotide (FAD)-containing dehydrogenase domain, which differs from other typical d-iLDHs. Fe-S d-iLDH from Pseudomonas putida KT2440 was purified as a His-tagged protein and characterized in detail. This monomeric enzyme exhibited activities with l-lactate and several d-2-hydroxyacids. Quinone was shown to be the preferred electron acceptor of the enzyme. The two domains of the enzyme were then heterologously expressed and purified separately. The Fe-S cluster-binding motifs predicted by sequence alignment were preliminarily verified by site-directed mutagenesis of the Fe-S oxidoreductase domain. The FAD-containing dehydrogenase domain retained 2-hydroxyacid-oxidizing activity, although it decreased compared to the full Fe-S d-iLDH. Compared to the intact enzyme, the FAD-containing dehydrogenase domain showed increased catalytic efficiency with cytochrome c as the electron acceptor, but it completely lost the ability to use coenzyme Q10. Additionally, the FAD-containing dehydrogenase domain was no longer associated with the cell membrane, and it could not support the utilization of d-lactate as a carbon source. Based on the results obtained, we conclude that the Fe-S oxidoreductase domain functions as an electron transfer component to facilitate the utilization of quinone as an electron acceptor by Fe-S d-iLDH, and it helps the enzyme associate with the cell membrane. These functions make the Fe-S oxidoreductase domain crucial for the in vivo d-lactate utilization function of Fe-S d-iLDH. IMPORTANCE Lactate metabolism plays versatile roles in most domains of life. Lactate utilization processes depend on certain enzymes to oxidize lactate to pyruvate. In recent years, novel bacterial lactate-oxidizing enzymes have been

Improved NADPH Regeneration for Fungal Cytochrome P450 Monooxygenase by Co-Expressing Bacterial Glucose Dehydrogenase in Resting-Cell Biotransformation of Recombinant Yeast.

PubMed

Jeon, Hyunwoo; Durairaj, Pradeepraj; Lee, Dowoo; Ahsan, Md Murshidul; Yun, Hyungdon

2016-12-28

Fungal cytochrome P450 (CYP) enzymes catalyze versatile monooxygenase reactions and play a major role in fungal adaptations owing to their essential roles in the production avoid metabolites critical for pathogenesis, detoxification of xenobiotics, and exploitation avoid substrates. Although fungal CYP-dependent biotransformation for the selective oxidation avoid organic compounds in yeast system is advantageous, it often suffers from a shortage avoid intracellular NADPH. In this study, we aimed to investigate the use of bacterial glucose dehydrogenase (GDH) for the intracellular electron regeneration of fungal CYP monooxygenase in a yeast reconstituted system. The benzoate hydroxylase FoCYP53A19 and its homologous redox partner FoCPR from Fusarium oxysporum were co-expressed with the BsGDH from Bacillus subtilis in Saccharomyces cerevisiae for heterologous expression and biotransformations. We attempted to optimize several bottlenecks concerning the efficiency of fungal CYP-mediated whole-cell-biotransformation to enhance the conversion. The catalytic performance of the intracellular NADPH regeneration system facilitated the hydroxylation of benzoic acid to 4-hydroxybenzoic acid with high conversion in the resting-cell reaction. The FoCYP53A19 +FoCPR+BsGDH reconstituted system produced 0.47 mM 4-hydroxybenzoic acid (94% conversion) in the resting-cell biotransformations performed in 50 mM phosphate buffer (pH 6.0) containing 0.5 mM benzoic acid and 0.25% glucose for 24 h at 30°C. The "coupled-enzyme" system can certainly improve the overall performance of NADPH-dependent whole-cell biotransformations in a yeast system.

Targeting kynurenine 3-monooxygenase (KMO): implications for therapy in Huntington's disease.

PubMed

Thevandavakkam, Mathuravani A; Schwarcz, Robert; Muchowski, Paul J; Giorgini, Flaviano

2010-12-01

Huntington's disease (HD) is an adult onset neurodegenerative disease caused by a polyglutamine expansion in the huntingtin protein. Recent work has shown that perturbation of kynurenine pathway (KP) metabolism is a hallmark of HD pathology, and that changes in brain levels of KP metabolites may play a causative role in this disease. The KP contains three neuroactive metabolites, the neurotoxins 3-hydroxykynurenine (3-HK) and quinolinic acid (QUIN), and the neuroprotectant kynurenic acid (KYNA). In model systems in vitro and in vivo, 3-HK and QUIN have been shown to cause neurodegeneration via a combination of excitotoxic mechanisms and oxidative stress. Recent studies with HD patient samples and in HD model systems have supported the idea that a shift away from the synthesis of KYNA and towards the formation of 3-HK and QUIN may trigger the neuropathological features observed in HD. The enzyme kynurenine 3-monooxygenase (KMO) is located at a critical branching point in the KP such that inhibition of this enzyme by either pharmacological or genetic means shifts the flux in the pathway towards the formation of KYNA. This intervention ameliorates disease-relevant phenotypes in HD models. Here we review the work implicating the KP in HD pathology and discuss the potential of KMO as a therapeutic target for this disorder. As several neurodegenerative diseases exhibit alterations in KP metabolism, this concept has broader implications for the treatment of brain diseases.

CLK-1/Coq7p is a DMQ mono-oxygenase and a new member of the di-iron carboxylate protein family.

PubMed

Rea, S

2001-12-14

Strains of Caenorhabditis elegans mutant for clk-1 exhibit a 20-40% increase in mean lifespan. clk-1 encodes a mitochondrial protein thought to be either an enzyme or regulatory molecule acting within the ubiquinone biosynthesis pathway. Here CLK-1 is shown to be related to the ubiquinol oxidase, alternative oxidase, and belong to the functionally diverse di-iron-carboxylate protein family which includes bacterioferritin and methane mono-oxygenase. Construction and analysis of a homology model indicates CLK-1 is a 2-polyprenyl-3-methyl-6-methoxy-1,4-benzoquinone mono-oxygenase as originally predicted. Analysis of known CLK-1/Coq7p mutations also supports this notion. These findings raise the possibility of developing CLK-1-specific inhibitors to test for lifespan extension in higher organisms.

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. Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.

Flavin binding to the deca-heme cytochrome MtrC: Insights from computational molecular simulation

DOE PAGES

Breuer, Marian; Rosso, Kevin  M.; Blumberger, Jochen

2015-12-15

Here, certain dissimilatory bacteria have the remarkable ability to use extracellular metal oxide minerals instead of oxygen as terminal electron sinks, using a process known as “extracellular respiration”. Specialized multiheme cytochromes located on the outer membrane of the microbe were shown to be crucial for electron transfer from the cell surface to the mineral. This process is facilitated by soluble, biogenic flavins secreted by the organism for the purpose of acting as an electron shuttle. However, their interactions with the outer-membrane cytochromes are not established on a molecular scale. Here, we study the interaction between the outer-membrane deca-heme cytochrome MtrCmore » from Shewanella oneidensis and flavin mononucleotide (FMN in fully oxidized quinone form) using computational docking. We find that interaction of FMN with MtrC is significantly weaker than with known FMN-binding proteins, but identify a mildly preferred interaction site close to heme 2 with a dissociation constant (K d) = 490 μM, in good agreement with recent experimental estimates, K d = 255 μM. The weak interaction with MtrC can be qualitatively explained by the smaller number of hydrogen bonds that the planar headgroup of FMN can form with this protein compared to FMN-binding proteins. Molecular dynamics simulation gives indications for a possible conformational switch upon cleavage of the disulphide bond of MtrC, but without concomitant increase in binding affinities according to this docking study. Overall, our results suggest that binding of FMN to MtrC is reversible and not highly specific, which may be consistent with a role as redox shuttle that facilitates extracellular respiration.« less

Fullerene-Assisted Photoinduced Charge Transfer of Single-Walled Carbon Nanotubes through a Flavin Helix.

PubMed

Mollahosseini, Mehdi; Karunaratne, Erandika; Gibson, George N; Gascón, Jose A; Papadimitrakopoulos, Fotios

2016-05-11

One of the greatest challenges with single-walled carbon nanotube (SWNT) photovoltaics and nanostructured devices is maintaining the nanotubes in their pristine state (i.e., devoid of aggregation and inhomogeneous doping) so that their unique spectroscopic and transport characteristics are preserved. To this effect, we report on the synthesis and self-assembly of a C60-functionalized flavin (FC60), composed of PCBM and isoalloxazine moieties attached on either ends of a linear, C-12 aliphatic spacer. Small amounts of FC60 (up to 3 molar %) were shown to coassembly with an organic soluble derivative of flavin (FC12) around SWNTs and impart effective dispersion and individualization. A key annealing step was necessary to perfect the isoalloxazine helix and expel the C60 moiety away from the nanotubes. Steady-state and transient absorption spectroscopy illustrate that 1% or higher incorporation of FC60 allows for an effective photoinduced charge transfer quenching of the encased SWNTs through the seamless helical encase. This is enabled via the direct π-π overlap between the graphene sidewalls, isoalloxazine helix, and the C60 cage that facilitates SWNT exciton dissociation and electron transfer to the PCBM moiety. Atomistic molecular simulations indicate that the stability of the complex originates from enhanced van der Waals interactions of the flexible spacer wrapped around the fullerene that brings the C60 in π-π overlap with the isoalloxazine helix. The remarkable spectral purity (in terms of narrow E(S)ii line widths) for the resulting ground-state complex signals a new class of highly organized supramolecular nanotube architecture with profound importance for advanced nanostructured devices.

Quenching of triplet-excited flavins by flavonoids. Structural assessment of antioxidative activity.

PubMed

Huvaere, Kevin; Olsen, Karsten; Skibsted, Leif H

2009-10-02

The mechanism of flavin-mediated photooxidation of flavonoids was investigated for aqueous solutions. Interaction of triplet-excited flavin mononucleotide with phenols, as determined by laser flash photolysis, occurred at nearly diffusion-controlled rates (k approximately 1.6 x 10(9) L mol(-1) s(-1) for phenol at pH 7, 293 K), but protection of the phenolic function by methylation inhibited reaction. Still, electron transfer was proposed as the dominating mechanism due to the lack of primary kinetic hydrogen/deuterium isotope effect and the low activation enthalpy (<20 kJ mol(-1)) for photooxidation. Activation entropy worked compensating in a series of phenolic derivatives, supporting a common oxidation mechanism. An ortho-hydroxymethoxy pattern was equally reactive (k approximately 2.3 x 10(9) L mol(-1) s(-1) for guaiacol at pH 7) as compounds with ortho-dihydroxy substitution (k approximately 2.4 x 10(9) L mol(-1) s(-1) for catechol at pH 7), which are generally referred to as good antioxidants. This refutes the common belief that stabilization of incipient phenoxyl radicals through intramolecular hydrogen bonding is the driving force behind the reducing activity of catechol-like compounds. Instead, such bonding improves ionization characteristics of the substrates, hence the differences in reactivity with (photo)oxidation of isolated phenols. Despite the similar reactivity, radicals from ortho-dihydroxy compounds are detected in high steady-state concentrations by electron paramagnetic resonance (EPR) spectroscopy, while those resulting from oxidation of ortho-hydroxymethoxy (or isolated phenolic) patterns were too reactive to be observed. The ability to deprotonate and form the corresponding radical anions at neutral pH was proposed as the decisive factor for stabilization and, consequently, for antioxidative action. Thus, substituting other ionizable functions for the ortho- or para-hydroxyl in phenolic compounds resulted in stable radical anion formation, as

Alkaloid cluster gene ccsA of the ergot fungus Claviceps purpurea encodes chanoclavine I synthase, a flavin adenine dinucleotide-containing oxidoreductase mediating the transformation of N-methyl-dimethylallyltryptophan to chanoclavine I.

PubMed

Lorenz, Nicole; Olsovská, Jana; Sulc, Miroslav; Tudzynski, Paul

2010-03-01

Ergot alkaloids are indole-derived secondary metabolites synthesized by the phytopathogenic ascomycete Claviceps purpurea. In wild-type strains, they are exclusively produced in the sclerotium, a hibernation structure; for biotechnological applications, submerse production strains have been generated by mutagenesis. It was shown previously that the enzymes specific for alkaloid biosynthesis are encoded by a gene cluster of 68.5 kb. This ergot alkaloid cluster consists of 14 genes coregulated and expressed under alkaloid-producing conditions. Although the role of some of the cluster genes in alkaloid biosynthesis could be confirmed by a targeted knockout approach, further functional analyses are needed, especially concerning the early pathway-specific steps up to the production of clavine alkaloids. Therefore, the gene ccsA, originally named easE and preliminarily annotated as coding for a flavin adenine dinucleotide-containing oxidoreductase, was deleted in the C. purpurea strain P1, which is able to synthesize ergot alkaloids in axenic culture. Five independent knockout mutants were analyzed with regard to alkaloid-producing capability. Thin-layer chromatography (TLC), ultrapressure liquid chromatography (UPLC), and mass spectrometry (MS) analyses revealed accumulation of N-methyl-dimethylallyltryptophan (Me-DMAT) and traces of dimethylallyltryptophan (DMAT), the first pathway-specific intermediate. Since other alkaloid intermediates could not be detected, we conclude that deletion of ccsA led to a block in alkaloid biosynthesis beyond Me-DMAT formation. Complementation with a ccsA/gfp fusion construct restored alkaloid biosynthesis. These data indicate that ccsA encodes the chanoclavine I synthase or a component thereof catalyzing the conversion of N-methyl-dimethylallyltryptophan to chanoclavine I.

Trichomonas vaginalis: metronidazole and other nitroimidazole drugs are reduced by the flavin enzyme thioredoxin reductase and disrupt the cellular redox system. Implications for nitroimidazole toxicity and resistance.

PubMed

Leitsch, David; Kolarich, Daniel; Binder, Marina; Stadlmann, Johannes; Altmann, Friedrich; Duchêne, Michael

2009-04-01

Infections with the microaerophilic parasite Trichomonas vaginalis are treated with the 5-nitroimidazole drug metronidazole, which is also in use against Entamoeba histolytica, Giardia intestinalis and microaerophilic/anaerobic bacteria. Here we report that in T. vaginalis the flavin enzyme thioredoxin reductase displays nitroreductase activity with nitroimidazoles, including metronidazole, and with the nitrofuran drug furazolidone. Reactive metabolites of metronidazole and other nitroimidazoles form covalent adducts with several proteins that are known or assumed to be associated with thioredoxin-mediated redox regulation, including thioredoxin reductase itself, ribonucleotide reductase, thioredoxin peroxidase and cytosolic malate dehydrogenase. Disulphide reducing activity of thioredoxin reductase was greatly diminished in extracts of metronidazole-treated cells and intracellular non-protein thiol levels were sharply decreased. We generated a highly metronidazole-resistant cell line that displayed only minimal thioredoxin reductase activity, not due to diminished expression of the enzyme but due to the lack of its FAD cofactor. Reduction of free flavins, readily observed in metronidazole-susceptible cells, was also absent in the resistant cells. On the other hand, iron-depleted T. vaginalis cells, expressing only minimal amounts of PFOR and hydrogenosomal malate dehydrogenase, remained fully susceptible to metronidazole. Thus, taken together, our data suggest a flavin-based mechanism of metronidazole activation and thereby challenge the current model of hydrogenosomal activation of nitroimidazole drugs.

An Inducible Propane Monooxygenase Is Responsible for N-Nitrosodimethylamine Degradation by Rhodococcus sp. Strain RHA1▿

PubMed Central

Sharp, Jonathan O.; Sales, Christopher M.; LeBlanc, Justin C.; Liu, Jie; Wood, Thomas K.; Eltis, Lindsay D.; Mohn, William W.; Alvarez-Cohen, Lisa

2007-01-01

Rhodococci are common soil heterotrophs that possess diverse functional enzymatic activities with economic and ecological significance. In this study, the correlation between gene expression and biological removal of the water contaminant N-nitrosodimethylamine (NDMA) is explored. NDMA is a hydrophilic, potent carcinogen that has gained recent notoriety due to its environmental persistence and emergence as a widespread micropollutant in the subsurface environment. In this study, we demonstrate that Rhodococcus sp. strain RHA1 can constitutively degrade NDMA and that activity toward this compound is enhanced by approximately 500-fold after growth on propane. Transcriptomic analysis of RHA1 and reverse transcriptase quantitative PCR assays demonstrate that growth on propane elicits the upregulation of gene clusters associated with (i) the oxidation of propane and (ii) the oxidation of substituted benzenes. Deletion mutagenesis of prmA, the gene encoding the large hydroxylase component of propane monooxygenase, abolished both growth on propane and removal of NDMA. These results demonstrate that propane monooxygenase is responsible for NDMA degradation by RHA1 and explain the enhanced cometabolic degradation of NDMA in the presence of propane. PMID:17873074

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chang, Chin -Yuan; Lohman, Jeremy; Cao, Hongnan

C-1027 is a chromoprotein enediyne antitumor antibiotic produced by Streptomyces globisporus. In the last step of biosynthesis of the (S)-3-chloro-5-hydroxy-beta-tyrosine moiety of the C-1027 enediyne chromophore, SgcE6 and SgcC compose a two-component monooxygenase that hydroxylates the C-5 position of (S)-3-chloro-beta-tyrosine. This two-component monooxygenase is remarkable for two reasons. (i) SgcE6 specifically reacts with FAD and NADH, and (ii) SgcC is active with only the peptidyl carrier protein (PCP)-tethered substrate. To address the molecular details of substrate specificity, we determined the crystal structures of SgcE6 and SgcC at 1.66 and 2.63 A resolution, respectively. SgcE6 shares a similar β-barrel fold withmore » the class I HpaC-like flavin reductases. A flexible loop near the active site of SgcE6 plays a role in FAD binding, likely by providing sufficient space to accommodate the AMP moiety of FAD, when compared to that of FMN-utilizing homologues. SgcC shows structural similarity to a few, other known FADH 2-dependent monooxygenases and sheds light on some biochemically but not structurally characterized homologues. In conclusion, the crystal structures reported here provide insights into substrate specificity, and comparison with homologues provides a catalytic mechanism of the two-component, FADH 2-dependent monooxygenase (SgcE6 and SgcC) that catalyzes the hydroxylation of a PCP-tethered substrate.« less

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

Competition between metals for binding to methanobactin enables expression of soluble methane monooxygenase in the presence of copper.

PubMed

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

2015-02-01

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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Production of the catalytic core of human peptidylglycine α-hydroxylating monooxygenase (hPHMcc) in Escherichia coli

PubMed Central

Handa, Sumit; Spradling, Tyler J.; Dempsey, Daniel R.; Merkler, David J.

2013-01-01

Most mammalian bioactive peptides possess a C-terminal amino acid amide moiety. The presence of the C-terminal amide is a significant impediment to the recombinant production of α-amidated peptides. α-Amidated peptides are produced in vivo by the enzymatic cleavage of a precursor with a C-terminal glycine residue. Peptidylglycine α-hydroxylating monooxygenase catalyzes the key step in the oxidation of the glycine-extended precursors to the α-amidated peptide. Herein, we detail the production of the catalytic core of human peptidylglycine α-hydroxylating monooxygenase (hPHMcc) in Escherichia coli possessing a N-terminal fusion to thioredoxin (Trx). Trx was fused to hPHMcc to enhance the yield of the resulting 52 kDa protein as a soluble and catalytically active enzyme. The Trx-hPHMcc-His6 fusion was purified to homogeneity and exhibited steady-state kinetic parameters that were similar to purified rat PHMcc. The bacterial production of recombinant hPHMcc will foster efforts to generate α-amidated peptides by the co-expression of hPHMcc and the α-amidated peptide precursors in E. coli or the in vitro amidation of recombinantly expressed α-amidated peptide precursors. PMID:22554821

Structures of almond hydroxynitrile lyase isoenzyme 5 provide a rationale for the lack of oxidoreductase activity in flavin dependent HNLs.

PubMed

Pavkov-Keller, Tea; Bakhuis, Janny; Steinkellner, Georg; Jolink, Fenneke; Keijmel, Esther; Birner-Gruenberger, Ruth; Gruber, Karl

2016-10-10

Hydroxynitrile lyases (HNLs) catalyze the asymmetric addition of HCN to aldehydes producing enantiomerically pure cyanohydrins. These enzymes can be heterologously expressed in large quantities making them interesting candidates for industrial applications. The HNLs from Rosaceae evolved from flavin dependent dehydrogenase/oxidase structures. Here we report the high resolution X-ray structure of the highly glycosylated Prunus amygdalus HNL isoenzyme5 (PaHNL5 V317A) expressed in Aspergillus niger and its complex with benzyl alcohol. A comparison with the structure of isoenzyme PaHNL1 indicates a higher accessibility to the active site and a larger cavity for PaHNL5. Additionally, the PaHNL5 complex structure with benzyl alcohol was compared with the structurally related aryl-alcohol oxidase (AAO). Even though both enzymes contain an FAD-cofactor and histidine residues at crucial positions in the active site, PaHNL5 lacks the oxidoreductase activity. The structures indicate that in PaHNLs benzyl alcohol is bound too far away from the FAD cofactor in order to be oxidized. Copyright © 2016 Elsevier B.V. All rights reserved.

Optimized formation of detergent micelles of beta-carotene and retinal production using recombinant human beta,beta-carotene 15,15'-monooxygenase.

PubMed

Kim, Nam-Hee; Kim, Yeong-Su; Kim, Hye-Jung; Oh, Deok-Kun

2008-01-01

The formation of beta-carotene detergent micelles and their conversion into retinal by recombinant human beta,beta-carotene 15,15'-monooxygenase was optimized under aqueous conditions. Toluene was the most hydrophobic among the organic solvents tested; thus, it was used to dissolve beta-carotene, which is a hydrophobic compound. Tween 80 was selected as the detergent because it supported the highest level of retinal production among all of the detergents tested. The maximum production of retinal was achieved in detergent micelles containing 200 mg/L of beta-carotene and 2.4% (w/v) Tween 80. Under these conditions, the recombinant enzyme produced 97 mg/L of retinal after 16 h with a conversion yield of 48.5% (w/w). The amount of retinal produced, which is the highest ever reported, is a result of the ability of our system to dissolve large amounts of beta-carotene.

New role of flavin as a general acid-base catalyst with no redox function in type 2 isopentenyl-diphosphate isomerase.

PubMed

Unno, Hideaki; Yamashita, Satoshi; Ikeda, Yosuke; Sekiguchi, Shin-Ya; Yoshida, Norie; Yoshimura, Tohru; Kusunoki, Masami; Nakayama, Toru; Nishino, Tokuzo; Hemmi, Hisashi

2009-04-03

Using FMN and a reducing agent such as NAD(P)H, type 2 isopentenyl-diphosphate isomerase catalyzes isomerization between isopentenyl diphosphate and dimethylallyl diphosphate, both of which are elemental units for the biosynthesis of highly diverse isoprenoid compounds. Although the flavin cofactor is expected to be integrally involved in catalysis, its exact role remains controversial. Here we report the crystal structures of the substrate-free and complex forms of type 2 isopentenyl-diphosphate isomerase from the thermoacidophilic archaeon Sulfolobus shibatae, not only in the oxidized state but also in the reduced state. Based on the active-site structures of the reduced FMN-substrate-enzyme ternary complexes, which are in the active state, and on the data from site-directed mutagenesis at highly conserved charged or polar amino acid residues around the active site, we demonstrate that only reduced FMN, not amino acid residues, can catalyze proton addition/elimination required for the isomerase reaction. This discovery is the first evidence for this long suspected, but previously unobserved, role of flavins just as a general acid-base catalyst without playing any redox roles, and thereby expands the known functions of these versatile coenzymes.

Nanoporous Mo2C functionalized 3D carbon architecture anode for boosting flavins mediated interfacial bioelectrocatalysis in microbial fuel cells

NASA Astrophysics Data System (ADS)

Zou, Long; Lu, Zhisong; Huang, Yunhong; Long, Zhong-er; Qiao, Yan

2017-08-01

An efficient microbial electrocatalysis in microbial fuel cells (MFCs) needs both high loading of microbes (biocatalysts) and robust interfacial electron transfer from microbes to electrode. Herein a nanoporous molybdenum carbide (Mo2C) functionalized carbon felt electrode with rich 3D hierarchical porous architecture is applied as MFC anode to achieve superior electrocatalytic performance. The nanoporous Mo2C functionalized anode exhibits strikingly improved microbial electrocatalysis in MFCs with 5-fold higher power density and long-term stability of electricity production. The great enhancement is attributed to the introduction of rough Mo2C nanostructural interface into macroporous carbon architecture for promoting microbial growth with great excretion of endogenous electron shuttles (flavins) and rich available nanopores for enlarging electrochemically active surface area. Importantly, the nanoporous Mo2C functionalized anode is revealed for the first time to have unique electrocatalytic activity towards redox reaction of flavins with more negative redox potential, indicating a more favourable thermodynamic driving force for anodic electron transfer. This work not only provides a promising electrode for high performance MFCs but also brings up a new insight into the effect of nanostructured materials on interfacial bioelectrocatalysis.

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

Genetic Analysis of Benzothiophene Biodesulfurization Pathway of Gordonia terrae Strain C-6

PubMed Central

Lian, Kehui; Zhang, Yue; Tian, Huimei; Ji, Kaihua; Li, Guoqiang

2013-01-01

Sulfur can be removed from benzothiophene (BT) by some bacteria without breaking carbon-carbon bonds. However, a clear mechanism for BT desulfurization and its genetic components have not been reported in literatures so far. In this study, we used comparative transcriptomics to study differential expression of genes in Gordonia terrae C-6 cultured with BT or sodium sulfate as the sole source of sulfur. We found that 135 genes were up-regulated with BT relative to sodium sulfate as the sole sulfur source. Many of these genes encode flavin-dependent monooxygenases, alkane sulfonate monooxygenases and desulfinase, which perform similar functions to those involved in the 4S pathway of dibenzothiophene (DBT) biodesulfurization. Three of the genes were found to be located in the same operon, designated bdsABC. Cell extracts of pET28a-bdsABC transfected E. coli Rosetta (DE3) converted BT to a phenolic compound, identified as o-hydroxystyrene. These results advance our understanding of enzymes involved in the BT biodesulfurization pathway. PMID:24367657

Development and molecular characterization of genic molecular markers for grain protein and calcium content in finger millet (Eleusine coracana (L.) Gaertn.).

PubMed

Nirgude, M; Babu, B Kalyana; Shambhavi, Y; Singh, U M; Upadhyaya, H D; Kumar, Anil

2014-03-01

Finger millet (Eleusine coracana (L.) Gaertn), holds immense agricultural and economic importance for its high nutraceuticals quality. Finger millets seeds are rich source of calcium and its proteins are good source of essential amino acids. In the present study, we developed 36 EST-SSR primers for the opaque2 modifiers and 20 anchored-SSR primers for calcium transporters and calmodulin for analysis of the genetic diversity of 103 finger millet genotypes for grain protein and calcium contents. Out of the 36 opaque2 modifiers primers, 15 were found polymorphic and were used for the diversity analysis. The highest PIC value was observed with the primer FMO2E33 (0.26), while the lowest was observed FMO2E27 (0.023) with an average value of 0.17. The gene diversity was highest for the primer FMO2E33 (0.33), however it was lowest for FMO2E27 (0.024) at average value of 0.29. The percentage polymorphism shown by opaque2 modifiers primers was 68.23%. The diversity analysis by calcium transporters and calmodulin based anchored SSR loci revealed that the highest PIC was observed with the primer FMCA8 (0.30) and the lowest was observed for FMCA5 (0.023) with an average value of 0.18. The highest gene diversity was observed for primer FMCA8 (0.37), while lowest for FMCA5 (0.024) at an average of 0.21. The opaque2 modifiers specific EST-SSRs could able to differentiate the finger millet genotypes into high, medium and low protein containing genotypes. However, calcium dependent candidate gene based EST-SSRs could broadly differentiate the genotypes based on the calcium content with a few exceptions. A significant negative correlation between calcium and protein content was observed. The present study resulted in identification of highly polymorphic primers (FMO2E30, FMO2E33, FMO2-18 and FMO2-14) based on the parameters such as percentage of polymorphism, PIC values, gene diversity and number of alleles.

Alkaloid Cluster Gene ccsA of the Ergot Fungus Claviceps purpurea Encodes Chanoclavine I Synthase, a Flavin Adenine Dinucleotide-Containing Oxidoreductase Mediating the Transformation of N-Methyl-Dimethylallyltryptophan to Chanoclavine I ▿

PubMed Central

Lorenz, Nicole; Olšovská, Jana; Šulc, Miroslav; Tudzynski, Paul

2010-01-01

Ergot alkaloids are indole-derived secondary metabolites synthesized by the phytopathogenic ascomycete Claviceps purpurea. In wild-type strains, they are exclusively produced in the sclerotium, a hibernation structure; for biotechnological applications, submerse production strains have been generated by mutagenesis. It was shown previously that the enzymes specific for alkaloid biosynthesis are encoded by a gene cluster of 68.5 kb. This ergot alkaloid cluster consists of 14 genes coregulated and expressed under alkaloid-producing conditions. Although the role of some of the cluster genes in alkaloid biosynthesis could be confirmed by a targeted knockout approach, further functional analyses are needed, especially concerning the early pathway-specific steps up to the production of clavine alkaloids. Therefore, the gene ccsA, originally named easE and preliminarily annotated as coding for a flavin adenine dinucleotide-containing oxidoreductase, was deleted in the C. purpurea strain P1, which is able to synthesize ergot alkaloids in axenic culture. Five independent knockout mutants were analyzed with regard to alkaloid-producing capability. Thin-layer chromatography (TLC), ultrapressure liquid chromatography (UPLC), and mass spectrometry (MS) analyses revealed accumulation of N-methyl-dimethylallyltryptophan (Me-DMAT) and traces of dimethylallyltryptophan (DMAT), the first pathway-specific intermediate. Since other alkaloid intermediates could not be detected, we conclude that deletion of ccsA led to a block in alkaloid biosynthesis beyond Me-DMAT formation. Complementation with a ccsA/gfp fusion construct restored alkaloid biosynthesis. These data indicate that ccsA encodes the chanoclavine I synthase or a component thereof catalyzing the conversion of N-methyl-dimethylallyltryptophan to chanoclavine I. PMID:20118373

Characterization of moclobemide N-oxidation in human liver microsomes.

PubMed

Hoskins, J; Shenfield, G; Murray, M; Gross, A

2001-07-01

1. Moclobemide underdergoes morpholine ring N-oxidation to form a major metabolite in plasma Rol2-5637. 2. The kinetics of moclobemide N-oxidation in human liver microsomes (HLM) (n = 6) have been investigated and the mixed-function oxidase enzymes catalysing this reaction have been identified using inhibition, enzyme correlation, altered pH and heat pretreatment experiments. 3. N-oxidation followed single enzyme Michealis-Menten kinetics (0.02-4.0 mm). Km app and Vmax ranged from 0.48 to 1.35 mM (mean +/- SD) 0.77 +/- 0.34 mM) and 0.22 to 2.15 nmol mg(-1) min(-1) (1.39 +/- 0.80 nmol mg(-1) respectively. 4. The N-oxidation of moclobemide strongly correlated with benzydamine N-oxidation a probe reaction for flavin-containing monoxygenase (FMO) activity (0.1 mM moclobemide, rs = 0.81, p < 0.005; 4 mM moclobemide, rs = 0.94, p = 0.0001). Correlations were observed between moclobemide N-oxidation and specific cytochromre P450 (CYP) activities at both moclobemide concentrations (0.1 mM moclobemide, CYP2C19 0.66, p < 0.05; 4 mM moclobemide, CYP2E1 rs = 0.56, p < 0.05). 5. The general P450 inhibitor, N-benzylimidazole, did not affect the rate of Rol2-5637 formation (0% inhibition versus control) (at 1.3 mM moclobemide. Furthermore, the rate of Ro12-5637 formation in HLM was unaffected by inhibitors Or substrates of specific P450s (< 10% inhibition versus control). 6. Heat pretreatment of HLM in the absence of NADPH (inactivating FMOs) resulted in 97% inhibition of Ro12-5637 formation. N-oxidation activity was greatest when incubated at pH 8.5. These results ilre consistent with the reaction being FMO medialtetd . 7. In conclusion, moclobemide N-oxidation activity has been observed in HLM in vitro and the reaction is predominantly catalysed by FMOs with a potentially small contribution from cytochrome P450 isoforms.

Isolation and Expression Analysis of CYP9A11 and Cytochrome P450 Reductase Gene in the Beet Armyworm (Lepidoptera: Noctuidae)

PubMed Central

Zhao, Chunqing; Feng, Xiaoyun; Tang, Tao; Qiu, Lihong

2015-01-01

Cytochrome P450 monooxygenases (CYPs), as an enzyme superfamily, is widely distributed in organisms and plays a vital function in the metabolism of exogenous and endogenous compounds by interacting with its obligatory redox partner, CYP reductase (CPR). A novel CYP gene (CYP9A11) and CPR gene from the agricultural pest insect Spodoptera exigua were cloned and characterized. The complete cDNA sequences of SeCYP9A11 and SeCPR are 1,931 and 3,919 bp in length, respectively, and contain open reading frames of 1,593 and 2,070 nucleotides, respectively. Analysis of the putative protein sequences indicated that SeCYP9A11 contains a heme-binding domain and the unique characteristic sequence (SRFALCE) of the CYP9 family, in addition to a signal peptide and transmembrane segment at the N-terminal. Alignment analysis revealed that SeCYP9A11 shares the highest sequence similarity with CYP9A13 from Mamestra brassicae, which is 66.54%. The putative protein sequence of SeCPR has all of the classical CPR features, such as an N-terminal membrane anchor; three conserved domain flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and nicotinamide adenine dinucleotide phosphate (NADPH) domain; and characteristic binding motifs. Phylogenetic analysis revealed that SeCPR shares the highest identity with HaCPR, which is 95.21%. The SeCYP9A11 and SeCPR genes were detected in the midgut, fat body, and cuticle tissues, and throughout all of the developmental stages of S. exigua. The mRNA levels of SeCYP9A11 and SeCPR decreased remarkably after exposure to plant secondary metabolites quercetin and tannin. The results regarding SeCYP9A11 and SeCPR genes in the current study provide foundation for the further study of S. exigua P450 system. PMID:26320261

Ligand complex structures of l-amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813 and its conformational change.

PubMed

Im, Dohyun; Matsui, Daisuke; Arakawa, Takatoshi; Isobe, Kimiyasu; Asano, Yasuhisa; Fushinobu, Shinya

2018-03-01

l-Amino acid oxidase/monooxygenase from Pseudomonas sp. AIU 813 (l-AAO/MOG) catalyzes both the oxidative deamination and oxidative decarboxylation of the α-group of l-Lys to produce a keto acid and amide, respectively. l-AAO/MOG exhibits limited specificity for l-amino acid substrates with a basic side chain. We previously determined its ligand-free crystal structure and identified a key residue for maintaining the dual activities. Here, we determined the structures of l-AAO/MOG complexed with l-Lys, l-ornithine, and l-Arg and revealed its substrate recognition. Asp238 is located at the ceiling of a long hydrophobic pocket and forms a strong interaction with the terminal, positively charged group of the substrates. A mutational analysis on the D238A mutant indicated that the interaction is critical for substrate binding but not for catalytic control between the oxidase/monooxygenase activities. The catalytic activities of the D238E mutant unexpectedly increased, while the D238F mutant exhibited altered substrate specificity to long hydrophobic substrates. In the ligand-free structure, there are two channels connecting the active site and solvent, and a short region located at the dimer interface is disordered. In the l-Lys complex structure, a loop region is displaced to plug the channels. Moreover, the disordered region in the ligand-free structure forms a short helix in the substrate complex structures and creates the second binding site for the substrate. It is assumed that the amino acid substrate enters the active site of l-AAO/MOG through this route. The atomic coordinates and structure factors (codes 5YB6, 5YB7, and 5YB8) have been deposited in the Protein Data Bank (http://wwpdb.org/). 1.4.3.2 (l-amino acid oxidase), 1.13.12.2 (lysine 2-monooxygenase).

Role of P450 monooxygenases in the degradation of the endocrine-disrupting chemical nonylphenol by the white rot fungus Phanerochaete chrysosporium.

PubMed

Subramanian, Venkataramanan; Yadav, Jagjit S

2009-09-01

The white rot fungus Phanerochaete chrysosporium extensively degraded the endocrine disruptor chemical nonylphenol (NP; 100% of 100 ppm) in both nutrient-limited cultures and nutrient-sufficient cultures. The P450 enzyme inhibitor piperonyl butoxide caused significant inhibition (approximately 75%) of the degradation activity in nutrient-rich malt extract (ME) cultures but no inhibition in defined low-nitrogen (LN) cultures, indicating an essential role of P450 monooxygenase(s) in NP degradation under nutrient-rich conditions. A genome-wide analysis using our custom-designed P450 microarray revealed significant induction of multiple P450 monooxygenase genes by NP: 18 genes were induced (2- to 195-fold) under nutrient-rich conditions, 17 genes were induced (2- to 6-fold) in LN cultures, and 3 were induced under both nutrient-rich and LN conditions. The P450 genes Pff 311b (corresponding to protein identification number [ID] 5852) and Pff 4a (protein ID 5001) showed extraordinarily high levels of induction (195- and 167-fold, respectively) in ME cultures. The P450 oxidoreductase (POR), glutathione S-transferase (gst), and cellulose metabolism genes were also induced in ME cultures. In contrast, certain metabolic genes, such as five of the peroxidase genes, showed partial downregulation by NP. This study provides the first evidence for the involvement of P450 enzymes in NP degradation by a white rot fungus and the first genome-wide identification of specific P450 genes responsive to an environmentally significant toxicant.

Role of P450 Monooxygenases in the Degradation of the Endocrine-Disrupting Chemical Nonylphenol by the White Rot Fungus Phanerochaete chrysosporium▿

PubMed Central

Subramanian, Venkataramanan; Yadav, Jagjit S.

2009-01-01

The white rot fungus Phanerochaete chrysosporium extensively degraded the endocrine disruptor chemical nonylphenol (NP; 100% of 100 ppm) in both nutrient-limited cultures and nutrient-sufficient cultures. The P450 enzyme inhibitor piperonyl butoxide caused significant inhibition (∼75%) of the degradation activity in nutrient-rich malt extract (ME) cultures but no inhibition in defined low-nitrogen (LN) cultures, indicating an essential role of P450 monooxygenase(s) in NP degradation under nutrient-rich conditions. A genome-wide analysis using our custom-designed P450 microarray revealed significant induction of multiple P450 monooxygenase genes by NP: 18 genes were induced (2- to 195-fold) under nutrient-rich conditions, 17 genes were induced (2- to 6-fold) in LN cultures, and 3 were induced under both nutrient-rich and LN conditions. The P450 genes Pff 311b (corresponding to protein identification number [ID] 5852) and Pff 4a (protein ID 5001) showed extraordinarily high levels of induction (195- and 167-fold, respectively) in ME cultures. The P450 oxidoreductase (POR), glutathione S-transferase (gst), and cellulose metabolism genes were also induced in ME cultures. In contrast, certain metabolic genes, such as five of the peroxidase genes, showed partial downregulation by NP. This study provides the first evidence for the involvement of P450 enzymes in NP degradation by a white rot fungus and the first genome-wide identification of specific P450 genes responsive to an environmentally significant toxicant. PMID:19542331

Impact of Mono-Fluorination on the Photophysics of the Flavin Chromophore.

PubMed

Reiffers, Anna; Torres Ziegenbein, Christian; Engelhardt, Alyn; Kühnemuth, Ralf; Gilch, Peter; Czekelius, Constantin

2018-03-31

Three mono-fluorinated derivatives of the flavin core system 10-methyl-isoalloxazine (MIA) were synthesized. Aqueous solutions of these compounds were characterized by steady-state and time-resolved spectroscopy. The positions for the fluorination (6, 7 and 8) were motivated by the nodal structure of the frontier orbitals of MIA. In comparison with MIA, the fluorination results in bathochromic (6F- and 7F-MIA) and hypsochromic (8F-MIA) shifts of the adiabatic excitation energy of the lowest allowed transition. Shifts of up to ~500 cm -1 were observed. These spectroscopic shifts go along with changes in fluorescence quantum yields and lifetimes. In addition, triplet yields are affected. For 7F-MIA, a 50% increase in the fluorescence quantum yield as well as a 50% decrease in triplet yield is observed rendering the compound interesting for fluorescence applications. The measured effects are discussed in terms of qualitative perturbation theory. © 2018 The American Society of Photobiology.

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.

Loss of Kynurenine 3-Mono-oxygenase Causes Proteinuria

PubMed Central

Deutsch, Konstantin; Bolanos-Palmieri, Patricia; Hanke, Nils; Schroder, Patricia; Staggs, Lynne; Bräsen, Jan H.; Roberts, Ian S.D.; Sheehan, Susan; Savage, Holly; Haller, Hermann

2016-01-01

Changes in metabolite levels of the kynurenine pathway have been observed in patients with CKD, suggesting involvement of this pathway in disease pathogenesis. Our recent genetic analysis in the mouse identified the kynurenine 3-mono-oxygenase (KMO) gene (Kmo) as a candidate gene associated with albuminuria. This study investigated this association in more detail. We compared KMO abundance in the glomeruli of mice and humans under normal and diabetic conditions, observing a decrease in glomerular KMO expression with diabetes. Knockdown of kmo expression in zebrafish and genetic deletion of Kmo in mice each led to a proteinuria phenotype. We observed pronounced podocyte foot process effacement on long stretches of the filtration barrier in the zebrafish knockdown model and mild podocyte foot process effacement in the mouse model, whereas all other structures within the kidney remained unremarkable. These data establish the candidacy of KMO as a causal factor for changes in the kidney leading to proteinuria and indicate a functional role for KMO and metabolites of the tryptophan pathway in podocytes. PMID:27020856

Loss of Kynurenine 3-Mono-oxygenase Causes Proteinuria.

PubMed

Korstanje, Ron; Deutsch, Konstantin; Bolanos-Palmieri, Patricia; Hanke, Nils; Schroder, Patricia; Staggs, Lynne; Bräsen, Jan H; Roberts, Ian S D; Sheehan, Susan; Savage, Holly; Haller, Hermann; Schiffer, Mario

2016-11-01

Changes in metabolite levels of the kynurenine pathway have been observed in patients with CKD, suggesting involvement of this pathway in disease pathogenesis. Our recent genetic analysis in the mouse identified the kynurenine 3-mono-oxygenase (KMO) gene (Kmo) as a candidate gene associated with albuminuria. This study investigated this association in more detail. We compared KMO abundance in the glomeruli of mice and humans under normal and diabetic conditions, observing a decrease in glomerular KMO expression with diabetes. Knockdown of kmo expression in zebrafish and genetic deletion of Kmo in mice each led to a proteinuria phenotype. We observed pronounced podocyte foot process effacement on long stretches of the filtration barrier in the zebrafish knockdown model and mild podocyte foot process effacement in the mouse model, whereas all other structures within the kidney remained unremarkable. These data establish the candidacy of KMO as a causal factor for changes in the kidney leading to proteinuria and indicate a functional role for KMO and metabolites of the tryptophan pathway in podocytes. Copyright © 2016 by the American Society of Nephrology.

Kynurenine 3-monooxygenase inhibition in blood ameliorates neurodegeneration

PubMed Central

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

2011-01-01

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

A copper-methionine interaction controls the pH-dependent activation of peptidylglycine monooxygenase.

PubMed

Bauman, Andrew T; Broers, Brenda A; Kline, Chelsey D; Blackburn, Ninian J

2011-12-20

The pH dependence of native peptidylglycine monooxygenase (PHM) and its M314H variant has been studied in detail. For wild-type (WT) PHM, the intensity of the Cu-S interaction visible in the Cu(I) extended X-ray absorption fine structure (EXAFS) data is inversely proportional to catalytic activity over the pH range of 3-8. A previous model based on more limited data was interpreted in terms of two protein conformations involving an inactive Met-on form and an active flexible Met-off form [Bauman, A. T., et al. (2006) Biochemistry 45, 11140-11150] that derived its catalytic activity from the ability to couple into vibrational modes critical for proton tunneling. The new studies comparing the WT and M314H variant have led to the evolution of this model, in which the Met-on form has been found to be derived from coordination of an additional Met residue, rather than a more rigid conformer of M314 as previously proposed. The catalytic activity of the mutant decreased by 96% because of effects on both k(cat) and K(M), but it displayed the same activity-pH profile with a maximum around pH 6. At pH 8, the reduced Cu(I) form gave spectra that could be simulated by replacement of the Cu(M) Cu-S(Met) interaction with a Cu-N/O interaction, but the data did not unambiguously assign the ligand to the imidazole side chain of H314. At pH 3.5, the EXAFS still showed the presence of a strong Cu-S interaction, establishing that the Met-on form observed at low pH in WT cannot be due to a strengthening of the Cu(M)-methionine interaction but must arise from a different Cu-S interaction. Therefore, lowering the pH causes a conformational change at one of the Cu centers that brings a new S donor residue into a favorable orientation for coordination to copper and generates an inactive form. Cys coordination is unlikely because all Cys residues in PHM are engaged in disulfide cross-links. Sequence comparison with the PHM homologues tyramine β-monooxygenase and dopamine β-monooxygenase

Effect of UGT2B10, UGT2B17, FMO3, and OCT2 Genetic Variation on Nicotine and Cotinine Pharmacokinetics and Smoking in African Americans

PubMed Central

Taghavi, Taraneh; St. Helen, Gideon; Benowitz, Neal L.; Tyndale, Rachel F.

2017-01-01

OBJECTIVES Nicotine metabolism rates differ greatly among individuals, even after controlling for variation in the major nicotine metabolizing enzyme, CYP2A6. In this study, the impact of genetic variation in alternative metabolic enzymes and transporters on nicotine and cotinine pharmacokinetics and smoking was investigated. METHODS We examined the impact of UGT2B10, UGT2B17, FMO3, NAT1, and OCT2 variation on pharmacokinetics and smoking (total nicotine equivalents and topography), before and after stratifying by CYP2A6 genotype in 60 African American smokers who received a simultaneous intravenous infusion of deuterium-labeled nicotine and cotinine. RESULTS Variants in UGT2B10 and UGT2B17 were associated with urinary glucuronidation ratios (glucuronide/free substrate). UGT2B10 rs116294140 was associated with significant alterations in cotinine and modest alterations in nicotine pharmacokinetics. These alterations, however, were not sufficient to change nicotine intake or topography. Neither UGT2B10 rs61750900, UGT2B17*2, FMO3 rs2266782, nor NAT1 rs13253389 altered nicotine or cotinine pharmacokinetics among all subjects (n=60); or among individuals with reduced CYP2A6 activity (n=23). The organic cation transporter OCT2 rs316019 significantly increased nicotine and cotinine Cmax (p=0.005, p=0.02, respectively) and decreased nicotine clearance (p=0.05). UGT2B10 rs116294140 had no significant impact on the plasma or urinary trans-3’-hydroxycotinine/cotinine ratio, commonly used as a biomarker of CYP2A6 activity. CONCLUSIONS We demonstrated that polymorphisms in genes other than CYP2A6 represent minor sources of variation in nicotine pharmacokinetics, insufficient to alter smoking in African Americans. The change in cotinine pharmacokinetics with UGT2B10 rs116294140 highlights the UGT2B10 gene as a source of variability in cotinine as a biomarker of tobacco exposure among African American smokers. PMID:28178031

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

PubMed

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

2016-02-01

Kynurenine monooxygenase (KMO) is an enzyme of the kynurenine (Kyn) pathway (KP), which is the major catabolic route of tryptophan. Kyn represents a branch point of the KP, being converted into the neurotoxin 3-hydroxykynurenine via KMO, neuroprotectant kynurenic acid, and anthranilic acid. As a result of this branch point, KMO is an attractive drug target for several neurodegenerative and/or neuroinflammatory diseases, especially Huntington's (HD), Alzheimer's (AD), and Parkinson's (PD) diseases. Although a neurological target, administration of KMO inhibitors in the periphery has demonstrated promising pharmacological results. In light of a recent crystal structure release and reports of preclinical candidates, here we provide a concise yet comprehensive update on the current state of research into the enzymology of KMO and related drug discovery efforts, highlighting areas where further work is required. Copyright © 2015 Elsevier Ltd. All rights reserved.

Hydrogen-bonding dynamics of free flavins in benzene and FAD in electron-transferring flavoprotein upon excitation.

PubMed

Sato, Kyousuke; Nishina, Yasuzou; Shiga, Kiyoshi; Tanaka, Fumio

2003-01-01

The dynamic natures of two hydrogen-bonding model systems, riboflavin tetrabutylate (RFTB)-trichloroacetic acid (TCA) and RFTB-phenol in benzene, and of electron-transferring flavoprotein (ETF) from pig kidney upon excitation of flavins was investigated by means of steady state and time-resolved fluorescence spectroscopy. In both model systems fluorescence intensities of RFTB decreased as TCA or phenol was added. The spectral characteristics of ETF under steady state excitation were quite similar to those of the RFTB-TCA system, but not to those of the RFTB-phenol system. The observed fluorescence decay curves of ETF fit well with the calculated decay curves with two lifetime components, as in the model systems. Averaged lifetime was 0.9 ns. The time-resolved fluorescence spectrum of ETF shifted toward longer wavelength with time after pulsed excitation, which was also observed in the RFTB-TCA system. In the RFTB-phenol system the emission spectrum did not shift at all with time. These results reveal that the dynamic nature of ETF can be ascribed to aliphatic hydrogen-bonding(s) of the isoalloxazine ring with surrounding amino acid(s). From the fluorescence characteristics of ETF in comparison with the model systems, human ETF and other flavoproteins, it was suggested that ETF from pig kidney does not contain Tyr-16 in the beta subunit, unlike human ETF.

New Role of Flavin as a General Acid-Base Catalyst with No Redox Function in Type 2 Isopentenyl-diphosphate Isomerase*S⃞

PubMed Central

Unno, Hideaki; Yamashita, Satoshi; Ikeda, Yosuke; Sekiguchi, Shin-ya; Yoshida, Norie; Yoshimura, Tohru; Kusunoki, Masami; Nakayama, Toru; Nishino, Tokuzo; Hemmi, Hisashi

2009-01-01

Using FMN and a reducing agent such as NAD(P)H, type 2 isopentenyl-diphosphate isomerase catalyzes isomerization between isopentenyl diphosphate and dimethylallyl diphosphate, both of which are elemental units for the biosynthesis of highly diverse isoprenoid compounds. Although the flavin cofactor is expected to be integrally involved in catalysis, its exact role remains controversial. Here we report the crystal structures of the substrate-free and complex forms of type 2 isopentenyl-diphosphate isomerase from the thermoacidophilic archaeon Sulfolobus shibatae, not only in the oxidized state but also in the reduced state. Based on the active-site structures of the reduced FMN-substrate-enzyme ternary complexes, which are in the active state, and on the data from site-directed mutagenesis at highly conserved charged or polar amino acid residues around the active site, we demonstrate that only reduced FMN, not amino acid residues, can catalyze proton addition/elimination required for the isomerase reaction. This discovery is the first evidence for this long suspected, but previously unobserved, role of flavins just as a general acid-base catalyst without playing any redox roles, and thereby expands the known functions of these versatile coenzymes. PMID:19158086

Desaturation reactions catalyzed by soluble methane monooxygenase.

PubMed

Jin, Y; Lipscomb, J D

2001-09-01

Soluble methane monooxygenase (MMO) is shown to be capable of catalyzing desaturation reactions in addition to the usual hydroxylation and epoxidation reactions. Dehydrogenated products are generated from MMO-catalyzed oxidation of certain substrates including ethylbenzene and cyclohexadienes. In the reaction of ethylbenzene, desaturation of ethyl C-H occurred along with the conventional hydroxvlations of ethyl and phenyl C-Hs. As a result, styrene is formed together with ethylphenols and phenylethanols. Similarly, when 1,3- and 1,4-cyclohexadienes were used as substrates, benzene was detected as a product in addition to the corresponding alcohols and epoxides. In all cases, reaction conditions were found to significantly affect the distribution among the different products. This new activity of MMO is postulated to be associated with the chemical properties of the substrates rather than fundamental changes in the nature of the oxygen and C-H activation chemistries. The formation of the desaturated products is rationalized by formation of a substrate cationic intermediate, possibly via a radical precursor. The cationic species is then proposed to partition between recombination (alcohol formation) and elimination (alkene production) pathways. This novel function of MMO indicates close mechanistic kinship between the hydroxylation and desaturation reactions catalyzed by the nonheme diiron clusters.

Activity-Based Protein Profiling of Ammonia Monooxygenase in Nitrosomonas europaea

DOE Office of Scientific and Technical Information (OSTI.GOV)

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

Nitrosomonas europaeais an aerobic nitrifying bacterium that oxidizes ammonia (NH 3) to nitrite (NO 2 ₋) 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 NH 4 +-dependent O 2uptake 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 usingmore » 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.« less

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

Relationships between laser powers and photoacoustic signal intensities of flavin adenine dinucleotide and beta-carotene dissolved in solutions

NASA Astrophysics Data System (ADS)

Imakubo, Keiichi

1994-10-01

Ar ion laser-induced photoacoustic spectroscopy has been performed on 0.01 mu M flavin adenine dinucleotide in H2O and 0.01 mu M beta-carotene in n-hexane where the optical absorption spectroscopy is not applicable. On the basis of the linear relationships between laser powers and photoacoustic signal intensities up to 500 mW, it may be concluded that laser power ranging from 10 to 50 mW is required for the successful observation of photoacoustic signals without any photochemical or photobiological effects.

Reduction of ferredoxin or oxygen by flavin-based electron bifurcation in Megasphaera elsdenii.

PubMed

Chowdhury, Nilanjan P; Kahnt, Jörg; Buckel, Wolfgang

2015-08-01

Over 50 years ago, it was reported that, in the anaerobic rumen bacterium Megasphaera elsdenii, the reduction of crotonyl-CoA to butyryl-CoA by NADH involved an electron transferring flavoprotein (Etf) as mediator [Baldwin RL, Milligan LP (1964) Biochim Biophys Acta 92, 421-432]. Purification and spectroscopic characterization revealed that this Etf contained 2 FAD, whereas, in the Etfs from aerobic and facultative bacteria, one FAD is replaced by AMP. Recently we detected a similar system in the related anaerobe Acidaminococcus fermentans that differed in the requirement of additional ferredoxin as electron acceptor. The whole process was established as flavin-based electron bifurcation in which the exergonic reduction of crotonyl-CoA by NADH mediated by Etf + butyryl-CoA dehydrogenase (Bcd) was coupled to the endergonic reduction of ferredoxin also by NADH. In the present study, we demonstrate that, under anaerobic conditions, Etf + Bcd from M. elsdenii bifurcate as efficiently as Etf + Bcd from A. fermentans. Under the aerobic conditions used in the study by Baldwin and Milligan and in the presence of catalytic amounts of crotonyl-CoA or butyryl-CoA, however, Etf + Bcd act as NADH oxidase producing superoxide and H2 O2 , whereas ferredoxin is not required. We hypothesize that, during bifurcation, oxygen replaces ferredoxin to yield superoxide. In addition, the formed butyryl-CoA is re-oxidized by a second oxygen molecule to crotonyl-CoA, resulting in a stoichiometry of 2 NADH consumed and 2 H2 O2 formed. As a result of the production of reactive oxygen species, electron bifurcation can be regarded as an Achilles' heel of anaerobes when exposed to air. © 2015 FEBS.

Influence of kynurenine 3-monooxygenase (KMO) gene polymorphism on cognitive function in schizophrenia.

PubMed

Wonodi, Ikwunga; McMahon, Robert P; Krishna, Nithin; Mitchell, Braxton D; Liu, Judy; Glassman, Matthew; Hong, L Elliot; Gold, James M

2014-12-01

Cognitive deficits compromise quality of life and productivity for individuals with schizophrenia and have no effective treatments. Preclinical data point to the kynurenine pathway of tryptophan metabolism as a potential target for pro-cognitive drug development. We have previously demonstrated association of a kynurenine 3-monooxygenase (KMO) gene variant with reduced KMO gene expression in postmortem schizophrenia cortex, and neurocognitive endophenotypic deficits in a clinical sample. KMO encodes kynurenine 3-monooxygenase (KMO), the rate-limiting microglial enzyme of cortical kynurenine metabolism. Aberration of the KMO gene might be the proximal cause of impaired cortical kynurenine metabolism observed in schizophrenia. However, the relationship between KMO variation and cognitive function in schizophrenia is unknown. This study examined the effects of the KMO rs2275163C>T C (risk) allele on cognitive function in schizophrenia. We examined the association of KMO polymorphisms with general neuropsychological performance and P50 gating in a sample of 150 schizophrenia and 95 healthy controls. Consistent with our original report, the KMO rs2275163C>T C (risk) allele was associated with deficits in general neuropsychological performance, and this effect was more marked in schizophrenia compared with controls. Additionally, the C (Arg452) allele of the missense rs1053230C>T variant (KMO Arg452Cys) showed a trend effect on cognitive function. Neither variant affected P50 gating. These data suggest that KMO variation influences a range of cognitive domains known to predict functional outcome. Extensive molecular characterization of this gene would elucidate its role in cognitive function with implications for vertical integration with basic discovery. Copyright © 2014 Elsevier B.V. All rights reserved.

Vibrational and electronic investigations, NLO, FMO analysis on a hetarylazoindole disperse dye by density functional theory

NASA Astrophysics Data System (ADS)

Çatıkkaş, Berna; Aktan, Ebru; Yalçın, Ergin

2016-08-01

This work deals with the optimized molecular structure, vibrational spectra, nonlinear optic (NLO) and frontier molecule orbital (FMO) properties of 1-Methyl-2-phenyl-3-(1,3,4-thiadiazol-2-yldiazenyl)-1H-indole (MPI) by quantum chemical calculations. The Fourier transform infrared (FT-MIR and FT-FIR) and Raman spectra of 1-Methyl-2-phenyl-3-(1,3,4-thiadiazol-2-yldiazenyl)-1H-indole (MPI) were recorded in the region (4000-400 cm-1 and 400-30 cm-1) and (3200-92 cm-1), respectively. The analysis and complete vibrational assignments of the fundamental modes of the MPI molecule were carried out by using the observed FT-IR and FT-Raman data and calculated Total Energy Distribution (TED) according to Scaled Quantum Mechanics procedure. The calculated geometrical parameters of the MPI molecule are in agreement with the obtained values from XRD studies. On the other hand, the difference between the scaled and observed wavenumber values of the most of the fundamentals are very small. 1H NMR and 13C NMR chemical shift values, and energy gap between LUMO-HOMO and molecular electrostatic potential (MEP) were investigated by using density functional theory (B3LYP) methods. UV/Visible spectra and λ maximum absorption values, the oscillator strengths in the chloroform, methanol and DMSO solvation in combination with different basis sets were calculated by using the time-dependent density functional theory (TD-DFT). Additionally, the predicted nonlinear optical (NLO) properties of the MPI are quite greater than that of urea at the B3LYP/6-31++G(d,p) level.

Biodegradation of trichloroethylene by Methylosinus trichosporium OB3b.

PubMed Central

Tsien, H C; Brusseau, G A; Hanson, R S; Waclett, L P

1989-01-01

The methanotroph Methylosinus trichosporium OB3b, a type II methanotroph, degraded trichloroethylene at rates exceeding 1.2 mmol/h per g (dry weight) following the appearance of soluble methane monooxygenase in continuous and batch cultures. Cells capable oxidizing trichloroethylene contained components of soluble methane monooxygenase as demonstrated by Western blot (immunoblot) analysis with antibodies prepared against the purified enzyme. Growth of cultures in a medium containing 0.25 microM or less copper sulfate caused derepression of the synthesis of soluble methane monooxygenase. In these cultures, the specific rates of methane and methanol oxidation did not change during growth, while trichloroethylene oxidation increased with the appearance of soluble methane monooxygenase. M. trichosporium OB3b cells that contained soluble methane monooxygenase also degraded vinyl chloride, 1,1-dichloroethylene, cis-1,2-dichloroethylene, and trans-1,2-dichloroethylene. Images PMID:2515801

Assessing and Modulating Kynurenine Pathway Dynamics in Huntington's Disease: Focus on Kynurenine 3-Monooxygenase.

PubMed

Sathyasaikumar, Korrapati V; Breda, Carlo; Schwarcz, Robert; Giorgini, Flaviano

2018-01-01

The link between disturbances in kynurenine pathway (KP) metabolism and Huntington's disease (HD) pathogenesis has been explored for a number of years. Several novel genetic and pharmacological tools have recently been developed to modulate key regulatory steps in the KP such as the reaction catalyzed by the enzyme kynurenine 3-monooxygenase (KMO). This insight has offered new options for exploring the mechanistic link between this metabolic pathway and HD, and provided novel opportunities for the development of candidate drug-like compounds. Here, we present an overview of the field, focusing on some novel approaches for interrogating the pathway experimentally.

Ultra-performance liquid chromatography tandem mass-spectrometry (uplc-ms/ms) for the rapid, simultaneous analysis of thiamin, riboflavin, flavin adenine dinucleotide, nicotinamide and pyridoxal in human milk

USDA-ARS?s Scientific Manuscript database

A novel, rapid and sensitive Ultra Performance Liquid-Chromatography tandem Mass-Spectrometry (UPLC-MS/MS) method for the simultaneous determination of several B-vitamins in human milk was developed. Resolution by retention time or multiple reaction monitoring (MRM) for thiamin, riboflavin, flavin a...

Coupled effects of methane monooxygenase and nitrogen source on growth and poly-β-hydroxybutyrate (PHB) production of Methylosinus trichosporium OB3b.

PubMed

Zhang, Tingting; Zhou, Jiti; Wang, Xiaowei; Zhang, Yu

2017-02-01

The coupled effects of nitrogen source and methane monooxygenase (MMO) on the growth and poly-β-hydroxybutyrate (PHB) accumulation capacity of methanotrophs were explored. The ammonia-supplied methanotrophs expressing soluble MMO (sMMO) grew at the highest rate, while N 2 -fixing bacteria expressing particulate MMO (pMMO) grew at the lowest rate. Further study showed that more hydroxylamine and nitrite was formed by ammonia-supplied bacteria containing pMMO, which might cause their slightly lower growth rate. The highest PHB content (51.0%) was obtained under nitrogen-limiting conditions with the inoculation of nitrate-supplied bacteria containing pMMO. Ammonia-supplied bacteria also accumulated a higher content of PHB (45.2%) with the expression of pMMO, while N 2 -fixing bacteria containing pMMO only showed low PHB production capacity (32.1%). The maximal PHB contents of bacteria expressing sMMO were low, with no significant change under different nitrogen source conditions. The low MMO activity, low cell growth rate and low PHB production capacity of methanotrophs continuously cultivated with N 2 with the expression of pMMO were greatly improved in the cyclic NO 3 - N 2 cultivation regime, indicating that long-term deficiency of nitrogen sources was detrimental to the activity of methanotrophs expressing pMMO. Copyright © 2016. Published by Elsevier B.V.

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.

Structure of a Thermobifida fusca lytic polysaccharide monooxygenase and mutagenesis of key residues

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kruer-Zerhusen, Nathan; Alahuhta, Markus; Lunin, Vladimir V.

Auxiliary activity (AA) enzymes are produced by numerous bacterial and fungal species to assist in the degradation of biomass. These enzymes are abundant but have yet to be fully characterized. Here, we report the X-ray structure of Thermobifida fusca AA10A (TfAA10A), investigate mutational characterization of key surface residues near its active site, and explore the importance of the various domains of Thermobifida fusca AA10B (TfAA10B). The structure of TfAA10A is similar to other bacterial LPMOs (lytic polysaccharide monooxygenases), including signs of photo-reduction and a distorted active site, with mixed features showing both type I and II copper coordination. The pointmore » mutation experiments of TfAA10A show that Trp82 and Asn83 are needed for binding, but only Trp82 affects activity. The TfAA10B domain truncation mutants reveal that CBM2 is crucial for the binding of substrate, but that the X1 module does not affect binding or activity. In TfAA10A, Trp82 and Asn83 are needed for binding, but only Trp82 affects activity. The TfAA10B domain truncation mutants reveal that CBM2 is crucial for substrate binding, but that the X1 module does not affect binding or activity. The structure of TfAA10A is similar to other bacterial lytic polysaccharide monooxygenases with mixed features showing both type I and II copper coordination. The role of LPMOs and the variability of abundance in genomes are not fully explored. LPMOs likely perform initial attacks into crystalline cellulose to allow larger processive cellulases to bind and attack, but the precise nature of their synergistic behavior remains to be definitively characterized.« less

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

Structure of a Thermobifida fusca lytic polysaccharide monooxygenase and mutagenesis of key residues

DOE PAGES

Kruer-Zerhusen, Nathan; Alahuhta, Markus; Lunin, Vladimir V.; ...

2017-11-30

Auxiliary activity (AA) enzymes are produced by numerous bacterial and fungal species to assist in the degradation of biomass. These enzymes are abundant but have yet to be fully characterized. Here, we report the X-ray structure of Thermobifida fusca AA10A (TfAA10A), investigate mutational characterization of key surface residues near its active site, and explore the importance of the various domains of Thermobifida fusca AA10B (TfAA10B). The structure of TfAA10A is similar to other bacterial LPMOs (lytic polysaccharide monooxygenases), including signs of photo-reduction and a distorted active site, with mixed features showing both type I and II copper coordination. The pointmore » mutation experiments of TfAA10A show that Trp82 and Asn83 are needed for binding, but only Trp82 affects activity. The TfAA10B domain truncation mutants reveal that CBM2 is crucial for the binding of substrate, but that the X1 module does not affect binding or activity. In TfAA10A, Trp82 and Asn83 are needed for binding, but only Trp82 affects activity. The TfAA10B domain truncation mutants reveal that CBM2 is crucial for substrate binding, but that the X1 module does not affect binding or activity. The structure of TfAA10A is similar to other bacterial lytic polysaccharide monooxygenases with mixed features showing both type I and II copper coordination. The role of LPMOs and the variability of abundance in genomes are not fully explored. LPMOs likely perform initial attacks into crystalline cellulose to allow larger processive cellulases to bind and attack, but the precise nature of their synergistic behavior remains to be definitively characterized.« less

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 k cat and k cat /K m comparable to or higher than kynurenine. However, kynurenines substituted in the 3-position are competitive inhibitors, with K I values lower than the K m for kynurenine. Bromination also enhances inhibition, and 3,5-dibromokynurenine is a potent competitive inhibitor with a K I value of 1.5μM. A pharmacophore model of KMO was developed, and predicted that 3,4-dichlorohippuric acid would be an inhibitor. The K I 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.

Feline morbillivirus, a previously undescribed paramyxovirus associated with tubulointerstitial nephritis in domestic cats

PubMed Central

Woo, Patrick C. Y.; Lau, Susanna K. P.; Wong, Beatrice H. L.; Fan, Rachel Y. Y.; Wong, Annette Y. P.; Zhang, Anna J. X.; Wu, Ying; Choi, Garnet K. Y.; Li, Kenneth S. M.; Hui, Janet; Wang, Ming; Zheng, Bo-Jian; Chan, K. H.; Yuen, Kwok-Yung

2012-01-01

We describe the discovery and isolation of a paramyxovirus, feline morbillivirus (FmoPV), from domestic cat (Felis catus). FmoPV RNA was detected in 56 (12.3%) of 457 stray cats (53 urine, four rectal swabs, and one blood sample) by RT-PCR. Complete genome sequencing of three FmoPV strains showed genome sizes of 16,050 bases, the largest among morbilliviruses, because of unusually long 5′ trailer sequences of 400 nt. FmoPV possesses identical gene contents (3′-N-P/V/C-M-F-H-L-5′) and is phylogenetically clustered with other morbilliviruses. IgG against FmoPV N protein was positive in 49 sera (76.7%) of 56 RT-PCR–positive cats, but 78 (19.4%) of 401 RT-PCR–negative cats (P < 0.0001) by Western blot. FmoPV was isolated from CRFK feline kidney cells, causing cytopathic effects with cell rounding, detachment, lysis, and syncytia formation. FmoPV could also replicate in subsequent passages in primate Vero E6 cells. Infected cell lines exhibited finely granular and diffuse cytoplasmic fluorescence on immunostaining for FmoPV N protein. Electron microscopy showed enveloped virus with typical “herringbone” appearance of helical N in paramyxoviruses. Histological examination of necropsy tissues in two FmoPV-positive cats revealed interstitial inflammatory infiltrate and tubular degeneration/necrosis in kidneys, with decreased cauxin expression in degenerated tubular epithelial cells, compatible with tubulointerstitial nephritis (TIN). Immunohistochemical staining revealed FmoPV N protein-positive renal tubular cells and mononuclear cells in lymph nodes. A case-control study showed the presence of TIN in seven of 12 cats with FmoPV infection, but only two of 15 cats without FmoPV infection (P < 0.05), suggesting an association between FmoPV and TIN. PMID:22431644

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

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

2015-11-01

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.

Equilibrium and ultrafast kinetic studies manipulating electron transfer: A short-lived flavin semiquinone is not sufficient for electron bifurcation

DOE PAGES

Hoben, John P.; Lubner, Carolyn E.; Ratzloff, Michael W.; ...

2017-06-14

Flavin-based electron transfer bifurcation is emerging as a fundamental and powerful mechanism for conservation and deployment of electrochemical energy in enzymatic systems. In this process, a pair of electrons is acquired at intermediate reduction potential (i.e. intermediate reducing power) and each electron is passed to a different acceptor, one with lower and the other with higher reducing power, leading to 'bifurcation'. It is believed that a strongly reducing semiquinone species is essential for this process, and it is expected that this species should be kinetically short-lived. We now demonstrate that presence of a short-lived anionic flavin semiquinone (ASQ) is notmore » sufficient to infer existence of bifurcating activity, although such a species may be necessary for the process. We have used transient absorption spectroscopy to compare the rates and mechanisms of decay of ASQ generated photochemically in bifurcating NADH-dependent ferredoxin-NADP + oxidoreductase and the non-bifurcating flavoproteins nitroreductase, NADH oxidase and flavodoxin. We found that different mechanisms dominate ASQ decay in the different protein environments, producing lifetimes ranging over two orders of magnitude. Capacity for electron transfer among redox cofactors vs. charge recombination with nearby donors can explain the range of ASQ lifetimes we observe. In conclusion, our results support a model wherein efficient electron propagation can explain the short lifetime of the ASQ of bifurcating NADH-dependent ferredoxin-NADP + oxidoreductase I, and can be an indication of capacity for electron bifurcation.« less

Equilibrium and ultrafast kinetic studies manipulating electron transfer: A short-lived flavin semiquinone is not sufficient for electron bifurcation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hoben, John P.; Lubner, Carolyn E.; Ratzloff, Michael W.

Flavin-based electron transfer bifurcation is emerging as a fundamental and powerful mechanism for conservation and deployment of electrochemical energy in enzymatic systems. In this process, a pair of electrons is acquired at intermediate reduction potential (i.e. intermediate reducing power) and each electron is passed to a different acceptor, one with lower and the other with higher reducing power, leading to 'bifurcation'. It is believed that a strongly reducing semiquinone species is essential for this process, and it is expected that this species should be kinetically short-lived. We now demonstrate that presence of a short-lived anionic flavin semiquinone (ASQ) is notmore » sufficient to infer existence of bifurcating activity, although such a species may be necessary for the process. We have used transient absorption spectroscopy to compare the rates and mechanisms of decay of ASQ generated photochemically in bifurcating NADH-dependent ferredoxin-NADP + oxidoreductase and the non-bifurcating flavoproteins nitroreductase, NADH oxidase and flavodoxin. We found that different mechanisms dominate ASQ decay in the different protein environments, producing lifetimes ranging over two orders of magnitude. Capacity for electron transfer among redox cofactors vs. charge recombination with nearby donors can explain the range of ASQ lifetimes we observe. In conclusion, our results support a model wherein efficient electron propagation can explain the short lifetime of the ASQ of bifurcating NADH-dependent ferredoxin-NADP + oxidoreductase I, and can be an indication of capacity for electron bifurcation.« less

Equilibrium and ultrafast kinetic studies manipulating electron transfer: A short-lived flavin semiquinone is not sufficient for electron bifurcation.

PubMed

Hoben, John P; Lubner, Carolyn E; Ratzloff, Michael W; Schut, Gerrit J; Nguyen, Diep M N; Hempel, Karl W; Adams, Michael W W; King, Paul W; Miller, Anne-Frances

2017-08-25

Flavin-based electron transfer bifurcation is emerging as a fundamental and powerful mechanism for conservation and deployment of electrochemical energy in enzymatic systems. In this process, a pair of electrons is acquired at intermediate reduction potential ( i.e. intermediate reducing power), and each electron is passed to a different acceptor, one with lower and the other with higher reducing power, leading to "bifurcation." It is believed that a strongly reducing semiquinone species is essential for this process, and it is expected that this species should be kinetically short-lived. We now demonstrate that the presence of a short-lived anionic flavin semiquinone (ASQ) is not sufficient to infer the existence of bifurcating activity, although such a species may be necessary for the process. We have used transient absorption spectroscopy to compare the rates and mechanisms of decay of ASQ generated photochemically in bifurcating NADH-dependent ferredoxin-NADP + oxidoreductase and the non-bifurcating flavoproteins nitroreductase, NADH oxidase, and flavodoxin. We found that different mechanisms dominate ASQ decay in the different protein environments, producing lifetimes ranging over 2 orders of magnitude. Capacity for electron transfer among redox cofactors versus charge recombination with nearby donors can explain the range of ASQ lifetimes that we observe. Our results support a model wherein efficient electron propagation can explain the short lifetime of the ASQ of bifurcating NADH-dependent ferredoxin-NADP + oxidoreductase I and can be an indication of capacity for electron bifurcation.

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

Active-site copper reduction promotes substrate binding of fungal lytic polysaccharide monooxygenase and reduces stability.

PubMed

Kracher, Daniel; Andlar, Martina; Furtmüller, Paul G; Ludwig, Roland

2018-02-02

Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-containing enzymes that oxidatively degrade insoluble plant polysaccharides and soluble oligosaccharides. Upon reductive activation, they cleave the substrate and promote biomass degradation by hydrolytic enzymes. In this study, we employed LPMO9C from Neurospora crassa , which is active toward cellulose and soluble β-glucans, to study the enzyme-substrate interaction and thermal stability. Binding studies showed that the reduction of the mononuclear active-site copper by ascorbic acid increased the affinity and the maximum binding capacity of LPMO for cellulose. The reduced redox state of the active-site copper and not the subsequent formation of the activated oxygen species increased the affinity toward cellulose. The lower affinity of oxidized LPMO could support its desorption after catalysis and allow hydrolases to access the cleavage site. It also suggests that the copper reduction is not necessarily performed in the substrate-bound state of LPMO. Differential scanning fluorimetry showed a stabilizing effect of the substrates cellulose and xyloglucan on the apparent transition midpoint temperature of the reduced, catalytically active enzyme. Oxidative auto-inactivation and destabilization were observed in the absence of a suitable substrate. Our data reveal the determinants of LPMO stability under turnover and non-turnover conditions and indicate that the reduction of the active-site copper initiates substrate binding. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Kynurenine 3-Monooxygenase: An Influential Mediator of Neuropathology.

PubMed

Parrott, Jennifer M; O'Connor, Jason C

2015-01-01

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

Kynurenine 3-Monooxygenase: An Influential Mediator of Neuropathology

PubMed Central

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

2015-01-01

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

Construction and application of a functional library of cytochrome P450 monooxygenases from the filamentous fungus Aspergillus oryzae.

PubMed

Nazir, K H M Nazmul Hussain; Ichinose, Hirofumi; Wariishi, Hiroyuki

2011-05-01

A functional library of cytochrome P450 monooxygenases from Aspergillus oryzae (AoCYPs) was constructed in which 121 isoforms were coexpressed with yeast NADPH-cytochrome P450 oxidoreductase in Saccharomyces cerevisiae. Using this functional library, novel catalytic functions of AoCYPs, such as catalytic potentials of CYP57B3 against genistein, were elucidated for the first time. Comprehensive functional screening promises rapid characterization of catalytic potentials and utility of AoCYPs.

Regulation of Camphor Metabolism: Induction and Repression of Relevant Monooxygenases in Pseudomonas putida NCIMB 10007.

PubMed

Willetts, Andrew; Masters, Pamela; Steadman, Carol

2018-05-07

For the first time, the differential rates of synthesis of all the key monooxygenases involved in the catabolism by Pseudomonas putida NCIMB 10007 of bicyclic ( rac )-camphor to ∆ 2,5 -3,4,4-trimethylpimelyl-CoA, the first aliphatic pathway intermediate, have been determined to help establish the relevant induction profile of each of the oxygen-dependent enzymes. The efficacy of both relevant substrates and pathway metabolites as inducers has been established. Further, inhibitors with characterised functionality have been used to indicate that the pertinent regulatory controls operate at the level of transcription of the corresponding genes.

Analytic second derivative of the energy for density-functional tight-binding combined with the fragment molecular orbital method

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakata, Hiroya, E-mail: hiroya.nakata.gt@kyocera.jp; Nishimoto, Yoshio; Fedorov, Dmitri G.

2016-07-28

The analytic second derivative of the energy is developed for the fragment molecular orbital (FMO) method combined with density-functional tight-binding (DFTB), enabling simulations of infrared and Raman spectra of large molecular systems. The accuracy of the method is established in comparison to full DFTB without fragmentation for a set of representative systems. The performance of the FMO-DFTB Hessian is discussed for molecular systems containing up to 10 041 atoms. The method is applied to the study of the binding of α-cyclodextrin to polyethylene glycol, and the calculated IR spectrum of an epoxy amine oligomer reproduces experiment reasonably well.

Chloroquine Binding Reveals Flavin Redox Switch Function of Quinone Reductase 2*

PubMed Central

Leung, Kevin K. K.; Shilton, Brian H.

2013-01-01

Quinone reductase 2 (NQO2) is an FAD-linked enzyme and the only known human target of two antimalarial drugs, primaquine (PQ) and chloroquine (CQ). The structural differences between oxidized and reduced NQO2 and the structural basis for inhibition by PQ and CQ were investigated by x-ray crystallography. Structures of oxidized NQO2 in complex with PQ and CQ were solved at 1.4 Å resolution. CQ binds preferentially to reduced NQO2, and upon reduction of NQO2-CQ crystals, the space group changed from P212121 to P21, with 1-Å decreases in all three unit cell dimensions. The change in crystal packing originated in the negative charge and 4–5º bend in the reduced isoalloxazine ring of FAD, which resulted in a new mode of CQ binding and closure of a flexible loop (Phe126–Leu136) over the active site. This first structure of a reduced quinone reductase shows that reduction of the FAD cofactor and binding of a specific inhibitor lead to global changes in NQO2 structure and is consistent with a functional role for NQO2 as a flavin redox switch. PMID:23471972

SAR202 Genomes from the Dark Ocean Predict Pathways for the Oxidation of Recalcitrant Dissolved Organic Matter.

PubMed

Landry, Zachary; Swan, Brandon K; Herndl, Gerhard J; Stepanauskas, Ramunas; Giovannoni, Stephen J

2017-04-18

Deep-ocean regions beyond the reach of sunlight contain an estimated 615 Pg of dissolved organic matter (DOM), much of which persists for thousands of years. It is thought that bacteria oxidize DOM until it is too dilute or refractory to support microbial activity. We analyzed five single-amplified genomes (SAGs) from the abundant SAR202 clade of dark-ocean bacterioplankton and found they encode multiple families of paralogous enzymes involved in carbon catabolism, including several families of oxidative enzymes that we hypothesize participate in the degradation of cyclic alkanes. The five partial genomes encoded 152 flavin mononucleotide/F420-dependent monooxygenases (FMNOs), many of which are predicted to be type II Baeyer-Villiger monooxygenases (BVMOs) that catalyze oxygen insertion into semilabile alicyclic alkanes. The large number of oxidative enzymes, as well as other families of enzymes that appear to play complementary roles in catabolic pathways, suggests that SAR202 might catalyze final steps in the biological oxidation of relatively recalcitrant organic compounds to refractory compounds that persist. IMPORTANCE Carbon in the ocean is massively sequestered in a complex mixture of biologically refractory molecules that accumulate as the chemical end member of biological oxidation and diagenetic change. However, few details are known about the biochemical machinery of carbon sequestration in the deep ocean. Reconstruction of the metabolism of a deep-ocean microbial clade, SAR202, led to postulation of new biochemical pathways that may be the penultimate stages of DOM oxidation to refractory forms that persist. These pathways are tied to a proliferation of oxidative enzymes. This research illuminates dark-ocean biochemistry that is broadly consequential for reconstructing the global carbon cycle. Copyright © 2017 Landry et al.

Hierarchically Porous N-Doped Carbon Nanotubes/Reduced Graphene Oxide Composite for Promoting Flavin-Based Interfacial Electron Transfer in Microbial Fuel Cells.

PubMed

Wu, Xiaoshuai; Qiao, Yan; Shi, Zhuanzhuan; Tang, Wei; Li, Chang Ming

2018-04-11

Interfacial electron transfer between an electroactive biofilm and an electrode is a crucial step for microbial fuel cells (MFCs) and other bio-electrochemical systems. Here, a hierarchically porous nitrogen-doped carbon nanotubes (CNTs)/reduced graphene oxide (rGO) composite with polyaniline as the nitrogen source has been developed for the MFC anode. This composite possesses a nitrogen atom-doped surface for improved flavin redox reaction and a three-dimensional hierarchically porous structure for rich bacterial biofilm growth. The maximum power density achieved with the N-CNTs/rGO anode in S. putrefaciens CN32 MFCs is 1137 mW m -2 , which is 8.9 times compared with that of the carbon cloth anode and also higher than those of N-CNTs (731.17 mW m -2 ), N-rGO (442.26 mW m -2 ), and the CNTs/rGO (779.9 mW m -2 ) composite without nitrogen doping. The greatly improved bio-electrocatalysis could be attributed to the enhanced adsorption of flavins on the N-doped surface and the high density of biofilm adhesion for fast interfacial electron transfer. This work reveals a synergistic effect from pore structure tailoring and surface chemistry designing to boost both the bio- and electrocatalysis in MFCs, which also provide insights for the bioelectrode design in other bio-electrochemical systems.

Demonstration that CobG, the monooxygenase associated with the ring contraction process of the aerobic cobalamin (vitamin B12) biosynthetic pathway, contains an Fe-S center and a mononuclear non-heme iron center.

PubMed

Schroeder, Susanne; Lawrence, Andrew D; Biedendieck, Rebekka; Rose, Ruth-Sarah; Deery, Evelyne; Graham, Ross M; McLean, Kirsty J; Munro, Andrew W; Rigby, Stephen E J; Warren, Martin J

2009-02-20

The ring contraction process that occurs during cobalamin (vitamin B(12)) biosynthesis is mediated via the action of two enzymes, CobG and CobJ. The first of these generates a tertiary alcohol at the C-20 position of precorrin-3A by functioning as a monooxygenase, a reaction that also forms a gamma lactone with the acetic acid side chain on ring A. The product, precorrin-3B, is then acted upon by CobJ, which methylates at the C-17 position and promotes ring contraction of the macrocycle by catalyzing a masked pinacol rearrangement. Here, we report the characterization of CobG enzymes from Pseudomonas denitrificans and Brucella melitensis. We show that both contain a [4Fe-4S] center as well as a mononuclear non-heme iron. Although both enzymes are active in vivo, the P. denitrificans enzyme was found to be inactive in vitro. Further analysis of this enzyme revealed that the mononuclear non-heme iron was not reducible, and it was concluded that it is rapidly inactivated once it is released from the bacterial cell. In contrast, the B. melitensis enzyme was found to be fully active in vitro and the mononuclear non-heme iron was reducible by dithionite. The reduced mononuclear non-heme was able to react with the oxygen analogue NO, but only in the presence of the substrate precorrin-3A. The cysteine residues responsible for binding the Fe-S center were identified by site-directed mutagenesis. A mechanism for CobG is presented.

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

Influence of kynurenine 3-monooxygenase (KMO) gene polymorphism on cognitive function in schizophrenia✰,✰✰

PubMed Central

Wonodi, Ikwunga; McMahon, Robert P.; Krishna, Nithin; Mitchell, Braxton D.; Liu, Judy; Glassman, Matthew; Hong, L. Elliot; Gold, James M.

2015-01-01

Background Cognitive deficits compromise quality of life and productivity for individuals with schizophrenia and have no effective treatments. Preclinical data point to the kynurenine pathway of tryptophan metabolism as a potential target for pro-cognitive drug development. We have previously demonstrated association of a kynurenine 3-monooxygenase (KMO) gene variant with reduced KMO gene expression in postmortem schizophrenia cortex, and neurocognitive endophenotypic deficits in a clinical sample. KMO encodes kynurenine 3-monooxygenase (KMO), the rate-limiting microglial enzyme of cortical kynurenine metabolism. Aberration of the KMO gene might be the proximal cause of impaired cortical kynurenine metabolism observed in schizophrenia. However, the relationship between KMO variation and cognitive function in schizophrenia is unknown. This study examined the effects of the KMO rs2275163C>T C (risk) allele on cognitive function in schizophrenia. Methods We examined the association of KMO polymorphisms with general neuropsychological performance and P50 gating in a sample of 150 schizophrenia and 95 healthy controls. Results Consistent with our original report, the KMO rs2275163C>T C (risk) allele was associated with deficits in general neuropsychological performance, and this effect was more marked in schizophrenia compared with controls. Additionally, the C (Arg452) allele of the missense rs1053230C>T variant (KMO Arg452Cys) showed a trend effect on cognitive function. Neither variant affected P50 gating. Conclusions These data suggest that KMO variation influences a range of cognitive domains known to predict functional outcome. Extensive molecular characterization of this gene would elucidate its role in cognitive function with implications for vertical integration with basic discovery. PMID:25464917

Discovery and characterization of a new family of lytic polysaccharide monooxygenases.

PubMed

Hemsworth, Glyn R; Henrissat, Bernard; Davies, Gideon J; Walton, Paul H

2014-02-01

Lytic polysaccharide monooxygenases (LPMOs) are a recently discovered class of enzymes capable of oxidizing recalcitrant polysaccharides. They are attracting considerable attention owing to their potential use in biomass conversion, notably in the production of biofuels. Previous studies have identified two discrete sequence-based families of these enzymes termed AA9 (formerly GH61) and AA10 (formerly CBM33). Here, we report the discovery of a third family of LPMOs. Using a chitin-degrading exemplar from Aspergillus oryzae, we show that the three-dimensional structure of the enzyme shares some features of the previous two classes of LPMOs, including a copper active center featuring the 'histidine brace' active site, but is distinct in terms of its active site details and its EPR spectroscopy. The newly characterized AA11 family expands the LPMO clan, potentially broadening both the range of potential substrates and the types of reactive copper-oxygen species formed at the active site of LPMOs.

Physical map location of the multicopy genes coding for ammonia monooxygenase and hydroxylamine oxidoreductase in the ammonia-oxidizing bacterium Nitrosomonas sp. strain ENI-11.

PubMed

Hirota, R; Yamagata, A; Kato, J; Kuroda, A; Ikeda, T; Takiguchi, N; Ohtake, H

2000-02-01

Pulsed-field gel electrophoresis of PmeI digests of the Nitrosomonas sp. strain ENI-11 chromosome produced four bands ranging from 1,200 to 480 kb in size. Southern hybridizations suggested that a 487-kb PmeI fragment contained two copies of the amoCAB genes, coding for ammonia monooxygenase (designated amoCAB(1) and amoCAB(2)), and three copies of the hao gene, coding for hydroxylamine oxidoreductase (hao(1), hao(2), and hao(3)). In this DNA fragment, amoCAB(1) and amoCAB(2) were about 390 kb apart, while hao(1), hao(2), and hao(3) were separated by at least about 100 kb from each other. Interestingly, hao(1) and hao(2) were located relatively close to amoCAB(1) and amoCAB(2), respectively. DNA sequence analysis revealed that hao(1) and hao(2) shared 160 identical nucleotides immediately upstream of each translation initiation codon. However, hao(3) showed only 30% nucleotide identity in the 160-bp corresponding region.

Physical Map Location of the Multicopy Genes Coding for Ammonia Monooxygenase and Hydroxylamine Oxidoreductase in the Ammonia-Oxidizing Bacterium Nitrosomonas sp. Strain ENI-11

PubMed Central

Hirota, Ryuichi; Yamagata, Akira; Kato, Junichi; Kuroda, Akio; Ikeda, Tsukasa; Takiguchi, Noboru; Ohtake, Hisao

2000-01-01

Pulsed-field gel electrophoresis of PmeI digests of the Nitrosomonas sp. strain ENI-11 chromosome produced four bands ranging from 1,200 to 480 kb in size. Southern hybridizations suggested that a 487-kb PmeI fragment contained two copies of the amoCAB genes, coding for ammonia monooxygenase (designated amoCAB1 and amoCAB2), and three copies of the hao gene, coding for hydroxylamine oxidoreductase (hao1, hao2, and hao3). In this DNA fragment, amoCAB1 and amoCAB2 were about 390 kb apart, while hao1, hao2, and hao3 were separated by at least about 100 kb from each other. Interestingly, hao1 and hao2 were located relatively close to amoCAB1 and amoCAB2, respectively. DNA sequence analysis revealed that hao1 and hao2 shared 160 identical nucleotides immediately upstream of each translation initiation codon. However, hao3 showed only 30% nucleotide identity in the 160-bp corresponding region. PMID:10633121

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

Perfect merohedral twinning combined with noncrystallographic symmetry potentially causes the failure of molecular replacement with low-homology search models for the flavin-dependent halogenase HalX from Xanthomonas campestris.

PubMed

Buss, Maren; Geerds, Christina; Patschkowski, Thomas; Niehaus, Karsten; Niemann, Hartmut H

2018-06-01

Flavin-dependent halogenases can be used as biocatalysts because they regioselectively halogenate their substrates under mild reaction conditions. New halogenases with novel substrate specificities will add to the toolbox of enzymes available to organic chemists. HalX, the product of the xcc-b100_4193 gene, is a putative flavin-dependent halogenase from Xanthomonas campestris. The enzyme was recombinantly expressed and crystallized in order to aid in identifying its hitherto unknown substrate. Native data collected to a resolution of 2.5 Å showed indications of merohedral twinning in a hexagonal lattice. Attempts to solve the phase problem by molecular replacement failed. Here, a detailed analysis of the suspected twinning is presented. It is most likely that the crystals are trigonal (point group 3) and exhibit perfect hemihedral twinning so that they appear to be hexagonal (point group 6). As there are several molecules in the asymmetric unit, noncrystallographic symmetry may complicate twinning analysis and structure determination.

Redox linked flavin sites in extracellular decaheme proteins involved in microbe-mineral electron transfer.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Edwards, Marcus J.; White, Gaye F.; Norman, Michael

2015-07-01

Extracellular microbe-mineral electron transfer is a major driving force for the oxidation of organic carbon in many subsurface environments. Extracellular multi-heme cytochromes of the Shewenella genus play a major role in this process but the mechanism of electron exchange at the interface between cytochrome and acceptor is widely debated. The 1.8 Å x-ray crystal structure of the decaheme MtrC revealed a highly conserved CX₈C disulfide that, when substituted for AX₈A, severely compromised the ability of S. oneidensis to grow under aerobic conditions. Reductive cleavage of the disulfide in the presence of flavin mononucleotide (FMN) resulted in the reversible formation ofmore » a stable flavocytochrome. Similar results were also observed with other decaheme cytochromes, OmcA, MtrF and UndA. The data suggest that these decaheme cytochromes can transition between highly reactive flavocytochromes or less reactive cytochromes, and that this transition is controlled by a redox active disulfide that responds to the presence of oxygen.« less

Determination of the human cytochrome P450 monooxygenase catalyzing the enantioselective oxidation of 2,2',3,5',6-pentachlorobiphenyl (PCB 95) and 2,2',3,4,4',5',6-heptachlorobiphenyl (PCB 183).

PubMed

Nagayoshi, Haruna; Kakimoto, Kensaku; Konishi, Yoshimasa; Kajimura, Keiji; Nakano, Takeshi

2017-10-17

2,2',3,5',6-Pentachlorobiphenyl (PCB 95) and 2,2',3,4,4',5',6-heptachlorobiphenyl (PCB 183) possess axial chirality and form the aS and aR enantiomers. The enantiomers of these congeners have been reported to accumulate in the human body enantioselectively via unknown mechanisms. In this study, we determined the cytochrome P450 (CYP) monooxygenase responsible for the enantioselective oxidization of PCB 95 and PCB 183, using a recombinant human CYP monooxygenase. We evaluated 13 CYP monooxygenases, namely CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2, CYP3A4, CYP3A5, CYP4F2, and aromatase (CYP19), and revealed that CYP2A6 preferably oxidizes aS-PCB 95 enantioselectively; however, it did not oxidize PCB 183. The enantiomer composition was elevated from 0.5 (racemate) to 0.54. In addition, following incubation with CYP2A6, the enantiomer fraction (EF) of PCB 95 demonstrated a time-dependent increase.

NAD(P)H:Flavin Mononucleotide Oxidoreductase Inactivation during 2,4,6-Trinitrotoluene Reduction

PubMed Central

Riefler, R. Guy; Smets, Barth F.

2002-01-01

Bacteria readily transform 2,4,6-trinitrotoluene (TNT), a contaminant frequently found at military bases and munitions production facilities, by reduction of the nitro group substituents. In this work, the kinetics of nitroreduction were investigated by using a model nitroreductase, NAD(P)H:flavin mononucleotide (FMN) oxidoreductase. Under mediation by NAD(P)H:FMN oxidoreductase, TNT rapidly reacted with NADH to form 2-hydroxylamino-4,6-dinitrotoluene and 4-hydroxylamino-2,6-dinitrotoluene, whereas 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene were not produced. Progressive loss of activity was observed during TNT reduction, indicating inactivation of the enzyme during transformation. It is likely that a nitrosodinitrotoluene intermediate reacted with the NAD(P)H:FMN oxidoreductase, leading to enzyme inactivation. A half-maximum constant with respect to NADH, KN, of 394 μM was measured, indicating possible NADH limitation under typical cellular conditions. A mathematical model that describes the inactivation process and NADH limitation provided a good fit to TNT reduction profiles. This work represents the first step in developing a comprehensive enzyme level understanding of nitroarene biotransformation. PMID:11916686

Biochemistry and structural studies of kynurenine 3-monooxygenase reveal allosteric inhibition by Ro 61-8048.

PubMed

Gao, Jingjing; Yao, Licheng; Xia, Tingting; Liao, Xuebin; Zhu, Deyu; Xiang, Ye

2018-04-01

The human kynurenine 3-monooxygenase (hKMO) is a potential therapeutic target for neurodegenerative and neurologic disorders. Inhibition of KMO by Ro 61-8048, a potent, selective, and the most widely used inhibitor of KMO, was shown effective in various models of neurodegenerative or neurologic disorders. However, the molecular basis of hKMO inhibition by Ro 61-8048 is not clearly understood. Here, we report biochemistry studies on hKMO and crystal structures of an hKMO homolog, pfKMO from Pseudomonas fluorescens, in complex with the substrate l-kynurenine and Ro 61-8048. We found that the C-terminal ∼110 aa are essential for the enzymatic activity of hKMO and the homologous C-terminal region of pfKMO folds into a distinct, all-α-helical domain, which associates with the N-terminal catalytic domain to form a unique tunnel in proximity to the substrate-binding pocket. The tunnel binds the Ro 61-8048 molecule, which fills most of the tunnel, and Ro 61-8048 is hydrogen bonded with several completely conserved residues, including an essential catalytic residue. Modification of Ro 61-8048 and biochemical studies of the modified Ro 61-8048 derivatives suggested that Ro 61-8048 inhibits the enzyme in an allosteric manner by affecting the conformation of the essential catalytic residue and by blocking entry of the substrate or product release. The unique binding sites distinguish Ro 61-8048 as a noncompetitive and highly selective inhibitor from other competitive inhibitors, which should facilitate further optimization of Ro 61-8048 and the development of new inhibitory drugs to hKMO.-Gao, J., Yao, L., Xia, T., Liao, X., Zhu, D., Xiang, Y. Biochemistry and structural studies of kynurenine 3-monooxygenase reveal allosteric inhibition by Ro 61-8048.

Secreted fungal sulfhydryl oxidases: sequence analysis and characterisation of a representative flavin-dependent enzyme from Aspergillus oryzae.

PubMed

Faccio, Greta; Kruus, Kristiina; Buchert, Johanna; Saloheimo, Markku

2010-08-20

Sulfhydryl oxidases are flavin-dependent enzymes that catalyse the formation of de novo disulfide bonds from free thiol groups, with the reduction of molecular oxygen to hydrogen peroxide. Sulfhydryl oxidases have been investigated in the food industry to remove the burnt flavour of ultraheat-treated milk and are currently studied as potential crosslinking enzymes, aiming at strengthening wheat dough and improving the overall bread quality. In the present study, potential sulfhydryl oxidases were identified in the publicly available fungal genome sequences and their sequence characteristics were studied. A representative sulfhydryl oxidase from Aspergillus oryzae, AoSOX1, was expressed in the fungus Trichoderma reesei. AoSOX1 was produced in relatively good yields and was purified and biochemically characterised. The enzyme catalysed the oxidation of thiol-containing compounds like glutathione, D/L-cysteine, beta-mercaptoethanol and DTT. The enzyme had a melting temperature of 57°C, a pH optimum of 7.5 and its enzymatic activity was completely inhibited in the presence of 1 mM ZnSO4. Eighteen potentially secreted sulfhydryl oxidases were detected in the publicly available fungal genomes analysed and a novel proline-tryptophan dipeptide in the characteristic motif CXXC, where X is any amino acid, was found. A representative protein, AoSOX1 from A. oryzae, was produced in T. reesei in an active form and had the characteristics of sulfhydryl oxidases. Further testing of the activity on thiol groups within larger peptides and on protein level will be needed to assess the application potential of this enzyme.

Analytic second derivative of the energy for density functional theory based on the three-body fragment molecular orbital method

NASA Astrophysics Data System (ADS)

Nakata, Hiroya; Fedorov, Dmitri G.; Zahariev, Federico; Schmidt, Michael W.; Kitaura, Kazuo; Gordon, Mark S.; Nakamura, Shinichiro

2015-03-01

Analytic second derivatives of the energy with respect to nuclear coordinates have been developed for spin restricted density functional theory (DFT) based on the fragment molecular orbital method (FMO). The derivations were carried out for the three-body expansion (FMO3), and the two-body expressions can be obtained by neglecting the three-body corrections. Also, the restricted Hartree-Fock (RHF) Hessian for FMO3 can be obtained by neglecting the density-functional related terms. In both the FMO-RHF and FMO-DFT Hessians, certain terms with small magnitudes are neglected for computational efficiency. The accuracy of the FMO-DFT Hessian in terms of the Gibbs free energy is evaluated for a set of polypeptides and water clusters and found to be within 1 kcal/mol of the corresponding full (non-fragmented) ab initio calculation. The FMO-DFT method is also applied to transition states in SN2 reactions and for the computation of the IR and Raman spectra of a small Trp-cage protein (PDB: 1L2Y). Some computational timing analysis is also presented.

From Cholesterogenesis to Steroidogenesis: Role of Riboflavin and Flavoenzymes in the Biosynthesis of Vitamin D12

PubMed Central

Pinto, John T.; Cooper, Arthur J. L.

2014-01-01

Flavin-dependent monooxygenases and oxidoreductases are located at critical branch points in the biosynthesis and metabolism of cholesterol and vitamin D. These flavoproteins function as obligatory intermediates that accept 2 electrons from NAD(P)H with subsequent 1-electron transfers to a variety of cytochrome P450 (CYP) heme proteins within the mitochondria matrix (type I) and the (microsomal) endoplasmic reticulum (type II). The mode of electron transfer in these systems differs slightly in the number and form of the flavin prosthetic moiety. In the type I mitochondrial system, FAD-adrenodoxin reductase interfaces with adrenodoxin before electron transfer to CYP heme proteins. In the microsomal type II system, a diflavin (FAD/FMN)-dependent cytochrome P450 oxidoreductase [NAD(P)H-cytochrome P450 reductase (CPR)] donates electrons to a multitude of heme oxygenases. Both flavoenzyme complexes exhibit a commonality of function with all CYP enzymes and are crucial for maintaining a balance of cholesterol and vitamin D metabolites. Deficits in riboflavin availability, imbalances in the intracellular ratio of FAD to FMN, and mutations that affect flavin binding domains and/or interactions with client proteins result in marked structural alterations within the skeletal and central nervous systems similar to those of disorders (inborn errors) in the biosynthetic pathways that lead to cholesterol, steroid hormones, and vitamin D and their metabolites. Studies of riboflavin deficiency during embryonic development demonstrate congenital malformations similar to those associated with genetic alterations of the flavoenzymes in these pathways. Overall, a deeper understanding of the role of riboflavin in these pathways may prove essential to targeted therapeutic designs aimed at cholesterol and vitamin D metabolism. PMID:24618756

Coenzyme Recognition and Gene Regulation by a Flavin Mononucleotide Riboswitch

DOE Office of Scientific and Technical Information (OSTI.GOV)

Serganov, A.; Huang, L; Patel, D

2009-01-01

The biosynthesis of several protein cofactors is subject to feedback regulation by riboswitches. Flavin mononucleotide (FMN)-specific riboswitches also known as RFN elements, direct expression of bacterial genes involved in the biosynthesis and transport of riboflavin (vitamin B2) and related compounds. Here we present the crystal structures of the Fusobacterium nucleatum riboswitch bound to FMN, riboflavin and antibiotic roseoflavin. The FMN riboswitch structure, centred on an FMN-bound six-stem junction, does not fold by collinear stacking of adjacent helices, typical for folding of large RNAs. Rather, it adopts a butterfly-like scaffold, stapled together by opposingly directed but nearly identically folded peripheral domains.more » FMN is positioned asymmetrically within the junctional site and is specifically bound to RNA through interactions with the isoalloxazine ring chromophore and direct and Mg{sup 2+}-mediated contacts with the phosphate moiety. Our structural data, complemented by binding and footprinting experiments, imply a largely pre-folded tertiary RNA architecture and FMN recognition mediated by conformational transitions within the junctional binding pocket. The inherent plasticity of the FMN-binding pocket and the availability of large openings make the riboswitch an attractive target for structure-based design of FMN-like antimicrobial compounds. Our studies also explain the effects of spontaneous and antibiotic-induced deregulatory mutations and provided molecular insights into FMN-based control of gene expression in normal and riboflavin-overproducing bacterial strains.« less

Discovery and characterization of a new family of lytic polysaccharide mono-oxygenases

PubMed Central

Hemsworth, Glyn R.; Henrissat, Bernard; Davies, Gideon J.; Walton, Paul H.

2014-01-01

Lytic polysaccharide mono-oxygenases (LPMOs) are a recently discovered class of enzymes capable of oxidizing recalcitrant polysaccharides. They currently attract much attention due to their potential use in biomass conversion, notably in the production of biofuels. Past work has identified two discrete sequence-based families of these enzymes termed AA9 (formerly GH61) and AA10 (formerly CBM33). Here we report the discovery of a third family of LPMOs. Using a chitin-degrading exemplar from Aspergillus oryzae, we show that the 3-D structure of the enzyme shares some features of the previous two classes of LPMOs, including a copper active centre featuring the histidine brace active site, but is distinct in terms of its active site details and its EPR spectroscopy. The new AA11 family expands the LPMO clan with the potential to broaden both the range of potential substrates and the types of reactive copper-oxygen species formed at the active site of LPMOs. PMID:24362702

Studies on the reaction mechanism of lactate oxidase. Formation of two covalent flavin-substrate adducts on reaction with glycollate.

PubMed

Massey, V; Ghisla, S; Kieschke, K

1980-04-10

L-Lactate oxidase from Mycobacterium smegmatis catalyzes the oxidative decarboxylation of glycollate, with formate, CO2, and H2O as the major products. In addition, some "uncoupling" of the normal reaction occurs, with glyoxylate and H2O adition, some "uncoupling" of the normal reaction occurs, with glyoxylate and H2O2 as products. Glyoxylate is also a substrate (presumably as its hydrate); in this case, the reaction products are oxalate and H2O2. Evidence is presented that the enzyme recognizes glycollate as a prochiral substrate, differentiating between the Re- and Si-faces of the alpha carbon atom. Two highly fluorescent species are formed concomitantly from the reaction with glycollate; they are proposed to be covalent alpha-glycollyl adducts to the reduced flavin position N(5). One of these adducts is labile and in rapid equilibrium with oxidized enzyme and glycollate, and with the complex of reduced enzyme and glyoxylate; this adduct is a catalytically competent intermediate. The other adduct is comparatively stable (t 1/2 for decay = 20 min at 25 degrees C) and does not react with O2. It is formed at a rate approximately 1% that of the catalytic adduct, but because of its lack of reaction with O2 and its stability, it gradually accumulates during catalytic turnover, resulting in catalytically incompetent enzyme. An isotope effect of approximately 4 is found in the reduction of oxidized enzyme flavin and in the formation of the labile fluorescent adduct, when alpha-2H2-glycollate or (R)-glycollate-2-d is used, but not with the (S)-glycollate-2-d enantiomer. It is concluded that the catalytic adduct is formed by hydrogen abstraction from the Re-face of glycollate.

Proposal for probing energy transfer pathway by single-molecule pump-dump experiment.

PubMed

Tao, Ming-Jie; Ai, Qing; Deng, Fu-Guo; Cheng, Yuan-Chung

2016-06-09

The structure of Fenna-Matthews-Olson (FMO) light-harvesting complex had long been recognized as containing seven bacteriochlorophyll (BChl) molecules. Recently, an additional BChl molecule was discovered in the crystal structure of the FMO complex, which may serve as a link between baseplate and the remaining seven molecules. Here, we investigate excitation energy transfer (EET) process by simulating single-molecule pump-dump experiment in the eight-molecules complex. We adopt the coherent modified Redfield theory and non-Markovian quantum jump method to simulate EET dynamics. This scheme provides a practical approach of detecting the realistic EET pathway in BChl complexes with currently available experimental technology. And it may assist optimizing design of artificial light-harvesting devices.

Proposal for probing energy transfer pathway by single-molecule pump-dump experiment

NASA Astrophysics Data System (ADS)

Tao, Ming-Jie; Ai, Qing; Deng, Fu-Guo; Cheng, Yuan-Chung

2016-06-01

The structure of Fenna-Matthews-Olson (FMO) light-harvesting complex had long been recognized as containing seven bacteriochlorophyll (BChl) molecules. Recently, an additional BChl molecule was discovered in the crystal structure of the FMO complex, which may serve as a link between baseplate and the remaining seven molecules. Here, we investigate excitation energy transfer (EET) process by simulating single-molecule pump-dump experiment in the eight-molecules complex. We adopt the coherent modified Redfield theory and non-Markovian quantum jump method to simulate EET dynamics. This scheme provides a practical approach of detecting the realistic EET pathway in BChl complexes with currently available experimental technology. And it may assist optimizing design of artificial light-harvesting devices.

Proposal for probing energy transfer pathway by single-molecule pump-dump experiment

PubMed Central

Tao, Ming-Jie; Ai, Qing; Deng, Fu-Guo; Cheng, Yuan-Chung

2016-01-01

The structure of Fenna-Matthews-Olson (FMO) light-harvesting complex had long been recognized as containing seven bacteriochlorophyll (BChl) molecules. Recently, an additional BChl molecule was discovered in the crystal structure of the FMO complex, which may serve as a link between baseplate and the remaining seven molecules. Here, we investigate excitation energy transfer (EET) process by simulating single-molecule pump-dump experiment in the eight-molecules complex. We adopt the coherent modified Redfield theory and non-Markovian quantum jump method to simulate EET dynamics. This scheme provides a practical approach of detecting the realistic EET pathway in BChl complexes with currently available experimental technology. And it may assist optimizing design of artificial light-harvesting devices. PMID:27277702

Molecular mechanism of lytic polysaccharide monooxygenases.

PubMed

Hedegård, Erik Donovan; Ryde, Ulf

2018-04-21

The lytic polysaccharide monooxygenases (LPMOs) are copper metalloenzymes that can enhance polysaccharide depolymerization through an oxidative mechanism and hence boost generation of biofuel from e.g. cellulose. By employing density functional theory in a combination of quantum mechanics and molecular mechanics (QM/MM), we report a complete description of the molecular mechanism of LPMOs. The QM/MM scheme allows us to describe all reaction steps with a detailed protein environment and we show that this is necessary. Several active species capable of abstracting a hydrogen from the substrate have been proposed previously and starting from recent crystallographic work on a substrate-LPMO complex, we investigate previously suggested paths as well as new ones. We describe the generation of the reactive intermediates, the abstraction of a hydrogen atom from the polysaccharide substrate, as well as the final recombination step in which OH is transferred back to the substrate. We show that a superoxo [CuO 2 ] + complex can be protonated by a nearby histidine residue (suggested by recent mutagenesis studies and crystallographic work) and, provided an electron source is available, leads to formation of an oxyl-complex after cleavage of the O-O bond and dissociation of water. The oxyl complex either reacts with the substrate or is further protonated to a hydroxyl complex. Both the oxyl and hydroxyl complexes are also readily generated from a reaction with H 2 O 2 , which was recently suggested to be the true co-substrate, rather than O 2 . The C-H abstraction by the oxyl and hydroxy complexes is overall favorable with activation barriers of 69 and 94 kJ mol -1 , compared to the much higher barrier (156 kJ mol -1 ) obtained for the copper-superoxo species. We obtain good structural agreement for intermediates for which structural data are available and the estimated reaction energies agree with experimental rate constants. Thus, our suggested mechanism is the most complete

Analytic second derivative of the energy for density functional theory based on the three-body fragment molecular orbital method

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nakata, Hiroya, E-mail: nakata.h.ab@m.titech.ac.jp; RIKEN, Research Cluster for Innovation, Nakamura Lab, 2-1 Hirosawa, Wako, Saitama 351-0198; Japan Society for the Promotion of Science, Kojimachi Business Center Building, 5-3-1 Kojimachi, Chiyoda-ku, Tokyo 102-0083

2015-03-28

Analytic second derivatives of the energy with respect to nuclear coordinates have been developed for spin restricted density functional theory (DFT) based on the fragment molecular orbital method (FMO). The derivations were carried out for the three-body expansion (FMO3), and the two-body expressions can be obtained by neglecting the three-body corrections. Also, the restricted Hartree-Fock (RHF) Hessian for FMO3 can be obtained by neglecting the density-functional related terms. In both the FMO-RHF and FMO-DFT Hessians, certain terms with small magnitudes are neglected for computational efficiency. The accuracy of the FMO-DFT Hessian in terms of the Gibbs free energy is evaluatedmore » for a set of polypeptides and water clusters and found to be within 1 kcal/mol of the corresponding full (non-fragmented) ab initio calculation. The FMO-DFT method is also applied to transition states in S{sub N}2 reactions and for the computation of the IR and Raman spectra of a small Trp-cage protein (PDB: 1L2Y). Some computational timing analysis is also presented.« less

Association study between kynurenine 3-monooxygenase gene and schizophrenia in the Japanese population.

PubMed

Aoyama, N; Takahashi, N; Saito, S; Maeno, N; Ishihara, R; Ji, X; Miura, H; Ikeda, M; Suzuki, T; Kitajima, T; Yamanouchi, Y; Kinoshita, Y; Yoshida, K; Iwata, N; Inada, T; Ozaki, N

2006-06-01

Several lines of evidence suggest that metabolic changes in the kynurenic acid (KYNA) pathway are related to the etiology of schizophrenia. The inhibitor of kynurenine 3-monooxygenase (KMO) is known to increase KYNA levels, and the KMO gene is located in the chromosome region associated with schizophrenia, 1q42-q44. Single-marker and haplotype analyses for 6-tag single nucleotide polymorphisms (SNPs) of KMO were performed (cases = 465, controls = 440). Significant association of rs2275163 with schizophrenia was observed by single-marker comparisons (P = 0.032) and haplotype analysis including this SNP (P = 0.0049). Significant association of rs2275163 and haplotype was not replicated using a second, independent set of samples (cases = 480, controls = 448) (P = 0.706 and P = 0.689, respectively). These results suggest that the KMO is unlikely to be related to the development of schizophrenia in Japanese.

Action of lytic polysaccharide monooxygenase on plant tissue is governed by cellular type.

PubMed

Chabbert, Brigitte; Habrant, Anouck; Herbaut, Mickaël; Foulon, Laurence; Aguié-Béghin, Véronique; Garajova, Sona; Grisel, Sacha; Bennati-Granier, Chloé; Gimbert-Herpoël, Isabelle; Jamme, Frédéric; Réfrégiers, Matthieu; Sandt, Christophe; Berrin, Jean-Guy; Paës, Gabriel

2017-12-19

Lignocellulosic biomass bioconversion is hampered by the structural and chemical complexity of the network created by cellulose, hemicellulose and lignin. Biological conversion of lignocellulose involves synergistic action of a large array of enzymes including the recently discovered lytic polysaccharide monooxygenases (LPMOs) that perform oxidative cleavage of cellulose. Using in situ imaging by synchrotron UV fluorescence, we have shown that the addition of AA9 LPMO (from Podospora anserina) to cellulases cocktail improves the progression of enzymes in delignified Miscanthus x giganteus as observed at tissular levels. In situ chemical monitoring of cell wall modifications performed by synchrotron infrared spectroscopy during enzymatic hydrolysis demonstrated that the boosting effect of the AA9 LPMO was dependent on the cellular type indicating contrasted recalcitrance levels in plant tissues. Our study provides a useful strategy for investigating enzyme dynamics and activity in plant cell wall to improve enzymatic cocktails aimed at expanding lignocelluloses biorefinery.

Overproduction and characterization of a lytic polysaccharide monooxygenase in Bacillus subtilis using an assay based on ascorbate consumption.

PubMed

Yu, Mi-Ji; Yoon, Sun-Hee; Kim, Young-Wan

2016-11-01

Lytic polysaccharide monooxygenases (LPMOs) are copper ion-containing enzymes that degrade crystalline polysaccharides, such as cellulose or chitin, through an oxidative mechanism. To the best of our knowledge, there are no assay methods for the direct characterization of LPMOs that degrade substrates without coupled enzymes. As such, in this study, a coupled enzyme-free assay method for LPMOs was developed, which is based on measuring the consumption of ascorbic acid used as an external electron donor for LPMOs. To establish this new assay method, a chitin-active LPMO from Bacillus atrophaeus (BatLPMO10) was cloned as a model enzyme. An expression system using B. subtilis as the host cell yielded a simple purification process without complicated periplasmic fractionation, as well as improved productivity by 3.7-fold higher than that of Escherichia coli BL21(DE3). At the optimum pH determined using a newly developed assay, BatLPMO10 showed the highest activity in terms of promoting chitin degradation by a chitinase. In addition, the assay method indicated that BatLPMO10 was inhibited by sodium ions, and BatLPMO10 and a chitinase mutually enhanced each other's activities upon degrading chitin as the substrate. In conclusion, this hydrolase-free ascorbate assay allows quantitative analysis of BatLPMO10 without a coupled enzyme. Copyright © 2016 Elsevier Inc. All rights reserved.

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

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

Reductive Dissolution of Goethite and Hematite by Reduced Flavins

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Zhi; Zachara, John M.; Wang, Zheming

2013-10-02

The abiotic reductive dissolution of goethite and hematite by the reduced forms of flavin mononucleotide (FMNH2) and riboflavin (RBFH2), electron transfer mediators (ETM) secreted by the dissimilatory iron-reducing bacterium Shewanella, was investigated under stringent anaerobic conditions. In contrast to the rapid redox reaction rate observed for ferrihydrite and lepidocrocite (Shi et al., 2012), the reductive dissolution of crystalline goethite and hematite was slower, with the extent of reaction limited by the thermodynamic driving force at circumneutral pH. Both the initial reaction rate and reaction extent increased with decreasing pH. On a unit surface area basis, goethite was less reactive thanmore » hematite between pH 4.0 and 7.0. AH2DS, the reduced form of the well-studied synthetic ETM anthraquinone-2,6-disulfonate (AQDS), yielded higher rates than FMNH2 under most reaction conditions, despite the fact that FMNH2 was a more effective reductant than AH2DS for ferryhydrite and lepidocrocite. Two additional model compounds, methyl viologen and benzyl viologen, were investigated under similar reaction conditions to explore the relationship between reaction rate and thermodynamic properties. Relevant kinetic data from the literature were also included in the analysis to span a broad range of half-cell potentials. Other conditions being equal, the surface area normalized initial reaction rate (ra) increased as the redox potential of the reductant became more negative. A non-linear, parabolic relationship was observed between log ra and the redox potential for eight reducants at pH 7.0, as predicted by Marcus theory for electron transfer. When pH and reductant concentration were fixed, log ra was positively correlated to the redox potential of four Fe(III) oxides over a wide pH range, following a non-linear parabolic relationship as well.« less

Electron-correlated fragment-molecular-orbital calculations for biomolecular and nano systems.

PubMed

Tanaka, Shigenori; Mochizuki, Yuji; Komeiji, Yuto; Okiyama, Yoshio; Fukuzawa, Kaori

2014-06-14

Recent developments in the fragment molecular orbital (FMO) method for theoretical formulation, implementation, and application to nano and biomolecular systems are reviewed. The FMO method has enabled ab initio quantum-mechanical calculations for large molecular systems such as protein-ligand complexes at a reasonable computational cost in a parallelized way. There have been a wealth of application outcomes from the FMO method in the fields of biochemistry, medicinal chemistry and nanotechnology, in which the electron correlation effects play vital roles. With the aid of the advances in high-performance computing, the FMO method promises larger, faster, and more accurate simulations of biomolecular and related systems, including the descriptions of dynamical behaviors in solvent environments. The current status and future prospects of the FMO scheme are addressed in these contexts.

A Novel F420-dependent Thioredoxin Reductase Gated by Low Potential FAD

PubMed Central

Susanti, Dwi; Loganathan, Usha; Mukhopadhyay, Biswarup

2016-01-01

A recent report suggested that the thioredoxin-dependent metabolic regulation, which is widespread in all domains of life, existed in methanogenic archaea about 3.5 billion years ago. We now show that the respective electron delivery enzyme (thioredoxin reductase, TrxR), although structurally similar to flavin-containing NADPH-dependent TrxRs (NTR), lacked an NADPH-binding site and was dependent on reduced coenzyme F420 (F420H2), a stronger reductant with a mid-point redox potential (E′0) of −360 mV; E′0 of NAD(P)H is −320 mV. Because F420 is a deazaflavin, this enzyme was named deazaflavin-dependent flavin-containing thioredoxin reductase (DFTR). It transferred electrons from F420H2 to thioredoxin via protein-bound flavin; Km values for thioredoxin and F420H2 were 6.3 and 28.6 μm, respectively. The E′0 of DFTR-bound flavin was approximately −389 mV, making electron transfer from NAD(P)H or F420H2 to flavin endergonic. However, under high partial pressures of hydrogen prevailing on early Earth and present day deep-sea volcanoes, the potential for the F420/F420H2 pair could be as low as −425 mV, making DFTR efficient. The presence of DFTR exclusively in ancient methanogens and mostly in the early Earth environment of deep-sea volcanoes and DFTR's characteristics suggest that the enzyme developed on early Earth and gave rise to NTR. A phylogenetic analysis revealed six more novel-type TrxR groups and suggested that the broader flavin-containing disulfide oxidoreductase family is more diverse than previously considered. The unprecedented structural similarities between an F420-dependent enzyme (DFTR) and an NADPH-dependent enzyme (NTR) brought new thoughts to investigations on F420 systems involved in microbial pathogenesis and antibiotic production. PMID:27590343

The Reaction Kinetics of 3-Hydroxybenzoate 6-Hydroxylase from Rhodococcus jostii RHA1 Provide an Understanding of the para-Hydroxylation Enzyme Catalytic Cycle*

PubMed Central

Sucharitakul, Jeerus; Tongsook, Chanakan; Pakotiprapha, Danaya; van Berkel, Willem J. H.; Chaiyen, Pimchai

2013-01-01

3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is an NADH-specific flavoprotein monooxygenase that catalyzes the para-hydroxylation of 3-hydroxybenzoate (3HB) to form 2,5-dihydroxybenzoate (2,5-DHB). Based on results from stopped-flow spectrophotometry, the reduced enzyme-3HB complex reacts with oxygen to form a C4a-peroxy flavin with a rate constant of 1.13 ± 0.01 × 106 m−1 s−1 (pH 8.0, 4 °C). This intermediate is subsequently protonated to form a C4a-hydroperoxyflavin with a rate constant of 96 ± 3 s−1. This step shows a solvent kinetic isotope effect of 1.7. Based on rapid-quench measurements, the hydroxylation occurs with a rate constant of 36 ± 2 s−1. 3HB6H does not exhibit substrate inhibition on the flavin oxidation step, a common characteristic found in most ortho-hydroxylation enzymes. The apparent kcat at saturating concentrations of 3HB, NADH, and oxygen is 6.49 ± 0.02 s−1. Pre-steady state and steady-state kinetic data were used to construct the catalytic cycle of the reaction. The data indicate that the steps of product release (11.7 s−1) and hydroxylation (36 ± 2 s−1) partially control the overall turnover. PMID:24129570

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

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.

Frequency, clinicopathological features and phylogenetic analysis of feline morbillivirus in cats in Istanbul, Turkey.

PubMed

Yilmaz, Huseyin; Tekelioglu, Bilge K; Gurel, Aydin; Bamac, Ozge E; Ozturk, Gulay Y; Cizmecigil, Utku Y; Altan, Eda; Aydin, Ozge; Yilmaz, Aysun; Berriatua, Eduardo; Helps, Chris R; Richt, Juergen A; Turan, Nuri

2017-12-01

Objectives The aim of the study was to investigate feline morbillivirus (FmoPV) frequency, phylogeny and associated pathology in cats in Istanbul, Turkey. Methods Samples from sick (n = 96) and dead ( n = 15) cats were analysed using reverse transcription PCR. Blood and urine analyses and histopathology were also performed. Results FmoPV RNA was detected in six cats (5.4%), including three sick (in the urine) and three dead cats (tissues). A significantly greater proportion of FmoPV RNA-positive cats had street access compared with non-infected cats. Blood samples from the morbillivirus-positive cats were negative for morbillivirus RNA. Tubular parenchymal cells, lymphoid and plasma cells in kidney and hepatocytes, lymphoid and plasma cells in liver from dead cats were also positive by immunohistochemistry for the viral N protein. Two FmoPV-positive cats were also positive for feline coronavirus RNA and one cat for feline immunodeficiency virus RNA and feline leukaemia virus proviral DNA. Phylogenetic analysis of the six FmoPV-positive cats showed that the strains were grouped into cluster D and had high similarity (98.5-100%) with strains from Japan and Germany. In the three FmoPV RNA-positive sick cats, respiratory, urinary and digestive system signs were observed as well as weight loss, fever and depression in some cats. Similar clinical signs were also seen in the morbillivirus RNA-negative sick cats. FmoPV RNA-positive cats had lower median red blood cell count, haemoglobin, albumin, albumin/globulin and urobilinogen and higher alanine transaminase, alkaline phosphatase and bilirubin compared with non-infected cats. Significant histopathology of FmoPV RNA-positive dead cats included tubulointerstitial nephritis characterised by severe granular and vacuolar degeneration of the epithelial cells of the cortical and medullary tubules as well as mononuclear cell infiltrates. Widespread lymphoid cell infiltrates were detected in the renal cortex and medullary

Analysis of Substrate Access to Active Sites in Bacterial Multicomponent Monooxygenase Hydroxylases: X-ray Crystal Structure of Xenon-Pressurized Phenol Hydroxylase from Pseudomonas sp. OX1†,‡

PubMed Central

McCormick, Michael S.; Lippard, Stephen J.

2011-01-01

In all structurally characterized bacterial multicomponent monooxygenase (BMM) hydroxylase proteins, a series of hydrophobic cavities in the α-subunit trace a conserved path from the protein exterior to the carboxylate-bridged diiron active site. The present study examines these cavities as a potential route for dioxygen transport to the active site by crystallographic characterization of a xenon-pressurized sample of the hydroxylase component of phenol hydroxylase from Pseudomonas sp. OX1. Computational analyses of the hydrophobic cavities in the hydroxylase α-subunits of phenol hydroxylase (PHH), toluene/o-xylene monooxygenase (ToMOH), and soluble methane monooxygenase (sMMOH) are also presented. The results, together with previous findings from crystallographic studies of xenon-pressurized sMMO hydroxylase, clearly identify the propensity for these cavities to bind hydrophobic gas molecules in the protein interior. This proposed functional role is supported by recent stopped flow kinetic studies of ToMOH variants (Song, et al., 2011). In addition to information about the Xe sites, the structure determination revealed significantly reduced regulatory protein binding to the hydroxylase in comparison to the previously reported structure of PHH, as well as the presence of a newly identified metal binding site in the α-subunit that adopts a linear coordination environment consistent with Cu(I), and a glycerol molecule bound to Fe1 in a fashion that is unique among hydrocarbon-diiron site adducts reported to date in BMM hydroxylase structures. Finally, a comparative analysis of the α-subunit structures of MMOH, ToMOH, and PHH details proposed routes for the other three BMM substrates, the hydrocarbon, electrons, and protons, comprising cavities, channels, hydrogen-bonding networks, and pores in the structures of their α-subunits. PMID:22136180

Remaining challenges in cellular flavin cofactor homeostasis and flavoprotein biogenesis

NASA Astrophysics Data System (ADS)

Giancaspero, Teresa Anna; Colella, Matilde; Brizio, Carmen; Difonzo, Graziana; Fiorino, Giuseppina Maria; Leone, Piero; Brandsch, Roderich; Bonomi, Francesco; Iametti, Stefania; Barile, Maria

2015-04-01

The primary role of the water-soluble vitamin B2 (riboflavin) in cell biology is connected with its conversion into FMN and FAD, the cofactors of a large number of dehydrogenases, oxidases and reductases involved in energetic metabolism, epigenetics, protein folding, as well as in a number of diverse regulatory processes. The problem of localisation of flavin cofactor synthesis events and in particular of the FAD synthase (EC 2.7.7.2) in HepG2 cells is addressed here by confocal microscopy in the frame of its relationships with kinetics of FAD synthesis and delivery to client apo-flavoproteins. FAD synthesis catalysed by recombinant isoform 2 of FADS occurs via an ordered bi-bi mechanism in which ATP binds prior to FMN, and pyrophosphate is released before FAD. Spectrophotometric continuous assays of the reconstitution rate of apo-D-aminoacid oxidase with its cofactor, allowed us to propose that besides its FAD synthesising activity, hFADS is able to operate as a FAD "chaperone". The physical interaction between FAD forming enzyme and its clients was further confirmed by dot blot and immunoprecipitation experiments carried out testing as a client either a nuclear or a mitochondrial enzyme that is lysine specific demethylase 1 (LSD1, EC 1.-.-.-) and dimethylglycine dehydrogenase (Me2GlyDH, EC 1.5.8.4), respectively which carry out similar reactions of oxidative demethylation, assisted by tetrahydrofolate used to form 5,10-methylene-tetrahydrofolate. A direct transfer of the cofactor from hFADS2 to apo-dimethyl glycine dehydrogenase was also demonstrated. Thus, FAD synthesis and delivery to these enzymes are crucial processes for bioenergetics and nutri-epigenetics of liver cells.

Enantiomeric behaviour of albendazole and fenbendazole sulfoxides in domestic animals: pharmacological implications.

PubMed

Capece, Bettencourt P S; Virkel, Guillermo L; Lanusse, Carlos E

2009-09-01

Albendazole and fenbendazole are methylcarbamate benzimidazole anthelmintics extensively used to control gastrointestinal parasites in domestic animals. These parent compounds are metabolised to albendazole sulfoxide and fenbendazole sulfoxide (oxfendazole), respectively. Both sulfoxide derivatives are anthelmintically active and are manufactured for use in animals. They metabolites have an asymmetric centre on their chemical structures and two enantiomeric forms of each sulfoxide have been identified in plasma, tissues of parasite location and within target helminths. Both the flavin-monooxygenase and cytochrome P450 systems are involved in the enantioselective biotransformation of these anthelmintic compounds in ruminant species. A relevant progress on the understanding of the relationship among enantioselective metabolism and systemic availability of each enantiomeric form has been achieved. This article reviews the current knowledge on the pharmacological implications of the enantiomeric behaviour of albendazole sulfoxide and oxfendazole in domestic animals.

Molecular insights into the enzymatic diversity of flavin-trafficking protein (Ftp; formerly ApbE) in flavoprotein biogenesis in the bacterial periplasm.

PubMed

Deka, Ranjit K; Brautigam, Chad A; Liu, Wei Z; Tomchick, Diana R; Norgard, Michael V

2016-02-01

We recently reported a flavin-trafficking protein (Ftp) in the syphilis spirochete Treponema pallidum (Ftp_Tp) as the first bacterial metal-dependent FAD pyrophosphatase that hydrolyzes FAD into AMP and FMN in the periplasm. Orthologs of Ftp_Tp in other bacteria (formerly ApbE) appear to lack this hydrolytic activity; rather, they flavinylate the redox subunit, NqrC, via their metal-dependent FMN transferase activity. However, nothing has been known about the nature or mechanism of metal-dependent Ftp catalysis in either Nqr- or Rnf-redox-containing bacteria. In the current study, we identified a bimetal center in the crystal structure of Escherichia coli Ftp (Ftp_Ec) and show via mutagenesis that a single amino acid substitution converts it from an FAD-binding protein to a Mg(2+)-dependent FAD pyrophosphatase (Ftp_Tp-like). Furthermore, in the presence of protein substrates, both types of Ftps are capable of flavinylating periplasmic redox-carrying proteins (e.g., RnfG_Ec) via the metal-dependent covalent attachment of FMN. A high-resolution structure of the Ftp-mediated flavinylated protein of Shewanella oneidensis NqrC identified an essential lysine in phosphoester-threonyl-FMN bond formation in the posttranslationally modified flavoproteins. Together, these discoveries broaden our understanding of the physiological capabilities of the bacterial periplasm, and they also clarify a possible mechanism by which flavoproteins are generated. © 2015 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

Elucidation of the mechanism of N-demethylation catalyzed by cytochrome P450 monooxygenase is facilitated by exploiting nitrogen-15 heavy isotope effects.

PubMed

Kwiecień, Renata A; Molinié, Roland; Paneth, Piotr; Silvestre, Virginie; Lebreton, Jacques; Robins, Richard J

2011-06-01

(15)N heavy isotope effects are especially useful when detail is sought pertaining to the reaction mechanism for the cleavage of a C-N bond. Their potential in assisting to describe the mechanism of N-demethylation of tertiary amines by the action of cytochrome P450 monooxygenase has been investigated. As a working model for the first step, oxidation of the N-methyl group to N-methoxyl, tropine and a cytochrome P450 monooxygenase reaction centre composed of a truncated heme with sulfhydryl as the axial ligand were used. It is apparent that this first step of the reaction proceeds via a hydrogen atom transfer mechanism. Transition states for this step are described for both the high spin ((4)TS(H)) and low spin ((2)TS(H)) pathways in both gas and solvation states. Hence, overall normal secondary (15)N KIE could be calculated for the reaction path modeled in the low spin state, and inverse for the reaction modeled in the high spin state. This partial reaction has been identified as the probable rate limiting step. The model for the second step, fission of the C-N bond, consisted of N-methoxylnortropine and two molecules of water. A transition state described for this step, TS(CN), gives a strongly inverse overall theoretical (15)N KIE. Copyright © 2011 Elsevier Inc. All rights reserved.

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.

The Divergent AmoC3 Subunit of Ammonia Monooxygenase Functions as Part of a Stress Response System in Nitrosomonas europaea

PubMed Central

Berube, Paul M.

2012-01-01

The ammonia monooxygenase of chemolithotrophic ammonia-oxidizing bacteria (AOB) catalyzes the first step in ammonia oxidation by converting ammonia to hydroxylamine. The monooxygenase of Nitrosomonas europaea is encoded by two nearly identical operon copies (amoCAB1,2). Several AOB, including N. europaea, also possess a divergent monocistronic copy of amoC (amoC3) of unknown function. Previous work suggested a possible functional role for amoC3 as part of the σE stress response regulon during the recovery of N. europaea from extended ammonia starvation, thus indicating its importance during the exit of cells from starvation. We here used global transcription analysis to show that expression of amoC3 is part of a general poststarvation cellular response system in N. europaea. We also found that amoC3 is required for an efficient response to some stress conditions, as deleting this gene impaired growth at elevated temperatures and recovery following starvation under high oxygen tensions. Deletion of the σ32 global stress response regulator demonstrated that the heat shock regulon plays a significant role in mediating the recovery of N. europaea from starvation. These findings provide the first described phenotype associated with the divergent AmoC3 subunit which appears to function as a stress-responsive subunit capable of maintaining ammonia oxidation activity under stress conditions. While this study was limited to starvation and heat shock, it is possible that the AmoC3 subunit may be responsive to other membrane stressors (e.g., solvent or osmotic shocks) that are prevalent in the environments of AOB. PMID:22544266

Purification of electron-transferring flavoprotein from Megasphaera elsdenii and binding of additional FAD with an unusual absorption spectrum.

PubMed

Sato, Kyosuke; Nishina, Yasuzo; Shiga, Kiyoshi

2003-11-01

Electron-transferring flavoprotein (ETF), its redox partner flavoproteins, i.e., D-lactate dehydrogenase and butyryl-CoA dehydrogenase, and another well-known flavoprotein, flavodoxin, were purified from the same starting cell paste of an anaerobic bacterium, Megasphaera elsdenii. The purified ETF contained one mol FAD/mol ETF as the sole non-protein component and bound almost one mol of additional FAD. This preparation is a better subject for investigations of M. elsdenii ETF than the previously isolated ETF, which contains varying amounts of FAD and varying percentages of modified flavins such as 6-OH-FAD and 8-OH-FAD. The additionally bound FAD shows an anomalous absorption spectrum with strong absorption around 400 nm. This spectral change is not due to a chemical modification of the flavin ring because the flavin released by KBr or guanidine hydrochloride is normal FAD. It is also not due to unknown small molecules because the same spectrum appears when ETF is reconstituted from its guanidine-denatured subunits and FAD. A similar anomalous spectrum was observed for AMP-free pig ETF under acidic conditions, suggesting a common flavin environment between pig and M. elsdenii ETFs.

Plant hemoglobins may be maintained in functional form by reduced flavins in the nuclei, and confer differential tolerance to nitro-oxidative stress.

PubMed

Sainz, Martha; Pérez-Rontomé, Carmen; Ramos, Javier; Mulet, Jose Miguel; James, Euan K; Bhattacharjee, Ujjal; Petrich, Jacob W; Becana, Manuel

2013-12-01

The heme of bacteria, plant and animal hemoglobins (Hbs) must be in the ferrous state to bind O(2) and other physiological ligands. Here we have characterized the full set of non-symbiotic (class 1 and 2) and 'truncated' (class 3) Hbs of Lotus japonicus. Class 1 Hbs are hexacoordinate, but class 2 and 3 Hbs are pentacoordinate. Three of the globins, Glb1-1, Glb2 and Glb3-1, are nodule-enhanced proteins. The O(2) affinity of Glb1-1 (50 pm) was the highest known for any Hb, and the protein may function as an O(2) scavenger. The five globins were reduced by free flavins, which transfer electrons from NAD(P)H to the heme iron under aerobic and anaerobic conditions. Class 1 Hbs were reduced at very fast rates by FAD, class 2 Hbs at slower rates by both FMN and FAD, and class 3 Hbs at intermediate rates by FMN. The members of the three globin classes were immunolocalized predominantly in the nuclei. Flavins were quantified in legume nodules and nuclei, and their concentrations were sufficient to maintain Hbs in their functional state. All Hbs, except Glb1-1, were expressed in a flavohemoglobin-deficient yeast mutant and found to confer tolerance to oxidative stress induced by methyl viologen, copper or low temperature, indicating an anti-oxidative role for the hemes. However, only Glb1-2 and Glb2 afforded protection against nitrosative stress induced by S-nitrosoglutathione. Because this compound is specifically involved in transnitrosylation reactions with thiol groups, our results suggest a contribution of the single cysteine residues of both proteins in the stress response. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

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

PubMed Central

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

1995-01-01

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

Inhibition of ammonia monooxygenase in Nitrosomonas europaea by carbon disulfide.

PubMed Central

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

1990-01-01

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

Sensor systems for bacterial reactors: A new flavin-phenol composite film for the in situ voltammetric measurement of pH.

PubMed

Casimero, Charnete; McConville, Aaron; Fearon, John-Joe; Lawrence, Clare L; Taylor, Charlotte M; Smith, Robert B; Davis, James

2018-10-16

Monitoring pH within microbial reactors has become an important requirement across a host of applications ranging from the production of functional foods (probiotics) to biofuel cell systems. An inexpensive and scalable composite sensor capable of monitoring the pH within the demanding environments posed by microbial reactors has been developed. A custom designed flavin derivative bearing an electropolymerisable phenol monomer was used to create a redox film sensitive to pH but free from the interferences that can impede conventional pH systems. The film was integrated within a composite carbon-fibre-polymer laminate and was shown to exhibit Nernstian behaviour (55 mV/pH) with minimal drift and robust enough to operate within batch reactors. Copyright © 2018 Elsevier B.V. All rights reserved.

Characterization of Two Mitochondrial Flavin Adenine Dinucleotide-Dependent Glycerol-3-Phosphate Dehydrogenases in Trypanosoma brucei

PubMed Central

Škodová, Ingrid; Verner, Zdeněk; Bringaud, Fréderic; Fabian, Peter

2013-01-01

Glycerol-3-phosphate dehydrogenases (G3PDHs) constitute a shuttle that serves for regeneration of NAD+ reduced during glycolysis. This NAD-dependent enzyme is employed in glycolysis and produces glycerol-3-phosphate from dihydroxyacetone phosphate, while its flavin adenine dinucleotide (FAD)-dependent homologue catalyzes a reverse reaction coupled to the respiratory chain. Trypanosoma brucei possesses two FAD-dependent G3PDHs. While one of them (mitochondrial G3PDH [mtG3PDH]) has been attributed to the mitochondrion and seems to be directly involved in G3PDH shuttle reactions, the function of the other enzyme (putative G3PDH [putG3PDH]) remains unknown. In this work, we used RNA interference and protein overexpression and tagging to shed light on the relative contributions of both FAD-G3PDHs to overall cellular metabolism. Our results indicate that mtG3PDH is essential for the bloodstream stage of T. brucei, while in the procyclic stage the enzyme is dispensable. Expressed putG3PDH-V5 was localized to the mitochondrion, and the data obtained by digitonin permeabilization, Western blot analysis, and immunofluorescence indicate that putG3PDH is located within the mitochondrion. PMID:24142106

Long distance electron-transfer mechanism in peptidylglycine alpha-hydroxylating monooxygenase: a perfect fitting for a water bridge.

PubMed

de la Lande, Aurélien; Martí, Sergio; Parisel, Olivier; Moliner, Vicent

2007-09-26

The active sites of copper enzymes have been the subject of many theoretical and experimental investigations from a number of years. Such studies have embraced topics devoted to the modeling of the first coordination sphere at the metallic cations up to the development of biomimetic, or bioinspired, catalytic systems. At least from the theoretical viewpoint, fewer efforts have been dedicated to elucidate how the two copper cations act concertedly in noncoupled dicopper enzymes such as peptidylglycine alpha-hydroxylating monooxygenase (PHM) and dopamine beta-monooxygenase (DbetaM). In these metalloenzymes, an electronic transfer is assumed between the two distant copper cations (11 A). Recent experimental results suggest that this transfer occurs through water molecules, a phenomenon which has been theoretically evidenced to be of high efficiency in the case of cytochrome b5 (Science, 2005, 310, 1311). In the present contribution dedicated to PHM, we overpass the common theoretical approaches dedicated to the electronic and geometrical structures of sites CuM or CuH restricted to their first coordination spheres and aim at directly comparing theoretical results to the experimentally measured activity of the PHM enzyme. To achieve this goal, molecular dynamics simulations were performed on wild-type and various mutants of PHM. More precisely, we provide an estimate of the electron-transfer efficiency between the CuM and CuH sites by means of such molecular dynamics simulations coupled to Marcus theory joined to the Beratan model to approximate the required coupling matrix elements. The theoretical results are compared to the kinetics measurements performed on wild and mutated PHM. The present work, the dynamic aspects of which are essential, accounts for the experimental results issued from mutagenesis. It supports the conclusion that an electronic transfer can occur between two copper(I) sites along a bridge involving a set of hydrogen and chemical bonds. Residue

Catalytic function of the mycobacterial binuclear iron monooxygenase in acetone metabolism.

PubMed

Furuya, Toshiki; Nakao, Tomomi; Kino, Kuniki

2015-10-01

Mycobacteria such as Mycobacterium smegmatis strain mc(2)155 and Mycobacterium goodii strain 12523 are able to grow on acetone and use it as a source of carbon and energy. We previously demonstrated by gene deletion analysis that the mimABCD gene cluster, which encodes a binuclear iron monooxygenase, plays an essential role in acetone metabolism in these mycobacteria. In the present study, we determined the catalytic function of MimABCD in acetone metabolism. Whole-cell assays were performed using Escherichia coli cells expressing the MimABCD complex. When the recombinant E. coli cells were incubated with acetone, a product was detected by gas chromatography (GC) analysis. Based on the retention time and the gas chromatography-mass spectrometry (GC-MS) spectrum, the reaction product was identified as acetol (hydroxyacetone). The recombinant E. coli cells produced 1.02 mM of acetol from acetone within 24 h. Furthermore, we demonstrated that MimABCD also was able to convert methylethylketone (2-butanone) to 1-hydroxy-2-butanone. Although it has long been known that microorganisms such as mycobacteria metabolize acetone via acetol, this study provides the first biochemical evidence for the existence of a microbial enzyme that catalyses the conversion of acetone to acetol. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

First molecular modeling report on novel arylpyrimidine kynurenine monooxygenase inhibitors through multi-QSAR analysis against Huntington's disease: A proposal to chemists!

PubMed

Amin, Sk Abdul; Adhikari, Nilanjan; Jha, Tarun; Gayen, Shovanlal

2016-12-01

Huntington's disease (HD) is caused by mutation of huntingtin protein (mHtt) leading to neuronal cell death. The mHtt induced toxicity can be rescued by inhibiting the kynurenine monooxygenase (KMO) enzyme. Therefore, KMO is a promising drug target to address the neurodegenerative disorders such as Huntington's diseases. Fiftysix arylpyrimidine KMO inhibitors are structurally explored through regression and classification based multi-QSAR modeling, pharmacophore mapping and molecular docking approaches. Moreover, ten new compounds are proposed and validated through the modeling that may be effective in accelerating Huntington's disease drug discovery efforts. Copyright © 2016 Elsevier Ltd. All rights reserved.

Development of a series of aryl pyrimidine kynurenine monooxygenase inhibitors as potential therapeutic agents for the treatment of Huntington's disease.

PubMed

Toledo-Sherman, Leticia M; Prime, Michael E; Mrzljak, Ladislav; Beconi, Maria G; Beresford, Alan; Brookfield, Frederick A; Brown, Christopher J; Cardaun, Isabell; Courtney, Stephen M; Dijkman, Ulrike; Hamelin-Flegg, Estelle; Johnson, Peter D; Kempf, Valerie; Lyons, Kathy; Matthews, Kimberly; Mitchell, William L; O'Connell, Catherine; Pena, Paula; Powell, Kendall; Rassoulpour, Arash; Reed, Laura; Reindl, Wolfgang; Selvaratnam, Suganathan; Friley, Weslyn Ward; Weddell, Derek A; Went, Naomi E; Wheelan, Patricia; Winkler, Christin; Winkler, Dirk; Wityak, John; Yarnold, Christopher J; Yates, Dawn; Munoz-Sanjuan, Ignacio; Dominguez, Celia

2015-02-12

We report on the development of a series of pyrimidine carboxylic acids that are potent and selective inhibitors of kynurenine monooxygenase and competitive for kynurenine. We describe the SAR for this novel series and report on their inhibition of KMO activity in biochemical and cellular assays and their selectivity against other kynurenine pathway enzymes. We describe the optimization process that led to the identification of a program lead compound with a suitable ADME/PK profile for therapeutic development. We demonstrate that systemic inhibition of KMO in vivo with this lead compound provides pharmacodynamic evidence for modulation of kynurenine pathway metabolites both in the periphery and in the central nervous system.

RNA interference of NADPH-cytochrome P450 reductase of the rice brown planthopper, Nilaparvata lugens, increases susceptibility to insecticides.

PubMed

Liu, Su; Liang, Qing-Mei; Zhou, Wen-Wu; Jiang, Yan-Dong; Zhu, Qing-Zi; Yu, Hang; Zhang, Chuan-Xi; Gurr, Geoff M; Zhu, Zeng-Rong

2015-01-01

NADPH-cytochrome P450 reductase (CPR) is essential for numerous biological reactions catalysed by microsomal cytochrome P450 monooxygenases (P450s). Knockdown of CPR in several insects leads to developmental defects and increased susceptibility to insecticides. However, information about the role of CPR in the brown planthopper, Nilaparvata lugens, is still unavailable. A full-length cDNA encoding CPR was cloned from N. lugens (NlCPR). The deduced amino acid sequence showed marked features of classical CPRs, such as an N-terminus membrane anchor, conserved domains for flavin mononucleotide, flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate binding, as well as an FAD-binding motif and catalytic residues. Phylogenetic analysis revealed that NlCPR was located in a branch along with bed bug and pea aphid hemipteran insects. NlCPR mRNA was detectable in all tissues and developmental stages of N. lugens, as determined by real-time quantitative PCR. NlCPR transcripts were most abundant in the abdomen in adults, and in first-instar nymphs. Injection of N. lugens with double-strand RNA (dsRNA) against NlCPR significantly reduced the transcription level of the mRNA, and silencing of NlCPR resulted in increased susceptibility in N. lugens to beta-cypermethrin and imidacloprid. The results provide first evidence that NlCPR contributes to the susceptibility to beta-cypermethrin and imidacloprid in N. lugens. © 2014 Society of Chemical Industry.

CYP79 P450 monooxygenases in gymnosperms: CYP79A118 is associated with the formation of taxiphyllin in Taxus baccata.

PubMed

Luck, Katrin; Jia, Qidong; Huber, Meret; Handrick, Vinzenz; Wong, Gane Ka-Shu; Nelson, David R; Chen, Feng; Gershenzon, Jonathan; Köllner, Tobias G

2017-09-01

Conifers contain P450 enzymes from the CYP79 family that are involved in cyanogenic glycoside biosynthesis. Cyanogenic glycosides are secondary plant compounds that are widespread in the plant kingdom. Their biosynthesis starts with the conversion of aromatic or aliphatic amino acids into their respective aldoximes, catalysed by N-hydroxylating cytochrome P450 monooxygenases (CYP) of the CYP79 family. While CYP79s are well known in angiosperms, their occurrence in gymnosperms and other plant divisions containing cyanogenic glycoside-producing plants has not been reported so far. We screened the transcriptomes of 72 conifer species to identify putative CYP79 genes in this plant division. From the seven resulting full-length genes, CYP79A118 from European yew (Taxus baccata) was chosen for further characterization. Recombinant CYP79A118 produced in yeast was able to convert L-tyrosine, L-tryptophan, and L-phenylalanine into p-hydroxyphenylacetaldoxime, indole-3-acetaldoxime, and phenylacetaldoxime, respectively. However, the kinetic parameters of the enzyme and transient expression of CYP79A118 in Nicotiana benthamiana indicate that L-tyrosine is the preferred substrate in vivo. Consistent with these findings, taxiphyllin, which is derived from L-tyrosine, was the only cyanogenic glycoside found in the different organs of T. baccata. Taxiphyllin showed highest accumulation in leaves and twigs, moderate accumulation in roots, and only trace accumulation in seeds and the aril. Quantitative real-time PCR revealed that CYP79A118 was expressed in plant organs rich in taxiphyllin. Our data show that CYP79s represent an ancient family of plant P450s that evolved prior to the separation of gymnosperms and angiosperms. CYP79A118 from T. baccata has typical CYP79 properties and its substrate specificity and spatial gene expression pattern suggest that the enzyme contributes to the formation of taxiphyllin in this plant species.

Clavine Alkaloids Gene Clusters of Penicillium and Related Fungi: Evolutionary Combination of Prenyltransferases, Monooxygenases and Dioxygenases

PubMed Central

Martín, Juan F.; Liras, Paloma

2017-01-01

The clavine alkaloids produced by the fungi of the Aspergillaceae and Arthrodermatacea families differ from the ergot alkaloids produced by Claviceps and Neotyphodium. The clavine alkaloids lack the extensive peptide chain modifications that occur in lysergic acid derived ergot alkaloids. Both clavine and ergot alkaloids arise from the condensation of tryptophan and dimethylallylpyrophosphate by the action of the dimethylallyltryptophan synthase. The first five steps of the biosynthetic pathway that convert tryptophan and dimethylallyl-pyrophosphate (DMA-PP) in chanoclavine-1-aldehyde are common to both clavine and ergot alkaloids. The biosynthesis of ergot alkaloids has been extensively studied and is not considered in this article. We focus this review on recent advances in the gene clusters for clavine alkaloids in the species of Penicillium, Aspergillus (Neosartorya), Arthroderma and Trychophyton and the enzymes encoded by them. The final products of the clavine alkaloids pathways derive from the tetracyclic ergoline ring, which is modified by late enzymes, including a reverse type prenyltransferase, P450 monooxygenases and acetyltransferases. In Aspergillus japonicus, a α-ketoglutarate and Fe2+-dependent dioxygenase is involved in the cyclization of a festuclavine-like unknown type intermediate into cycloclavine. Related dioxygenases occur in the biosynthetic gene clusters of ergot alkaloids in Claviceps purpurea and also in the clavine clusters in Penicillium species. The final products of the clavine alkaloid pathway in these fungi differ from each other depending on the late biosynthetic enzymes involved. An important difference between clavine and ergot alkaloid pathways is that clavine producers lack the enzyme CloA, a P450 monooxygenase, involved in one of the steps of the conversion of chanoclavine-1-aldehyde into lysergic acid. Bioinformatic analysis of the sequenced genomes of the Aspergillaceae and Arthrodermataceae fungi showed the presence of

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. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Imino-Oxy Acetic Acid Dealkylation as Evidence for an Inner-Sphere Alcohol Intermediate in the Reaction Catalyzed by Peptidylglycine α-Hydroxylating Monooxygenase (PHM)

PubMed Central

McIntyre, Neil R.; Lowe, Edward W.; Merkler, David J.

2009-01-01

Peptidylglycine α-hydroxylating monooxygenase (PHM, EC 1.14.17.3) catalyzes the stereospecific hydroxylation of a glycyl α-carbon in a reaction that requires O2 and ascorbate. Subsequent dealkylation of the α-hydroxyglycine by another enzyme, peptidylamidoglycolate lyase (PAL. EC 4.3.2.5), yields a bioactive amide and glyoxylate. PHM is a non-coupled, type II dicopper monooxygenase which activates O2 at only a single copper atom, CuM. In this study, the PHM mechanism was probed using a non-natural substrate, benzaldehyde imino-oxy acetic acid (BIAA). PHM catalyzes the O-oxidative dealkylation of BIAA to benzaldoxime and glyoxylate with no involvement of PAL. The minimal kinetic mechanism for BIAA was shown to be steady-state ordered using primary deuterium kinetic isotope effects. The D(V/K)APPARENT, BIAA decreased from 14.7 ± 1.0 as [O2] → 0 to 1.0 ± 0.2 as [O2] → ∞ suggesting the dissociation rate constant from the PHM·BIAA complex decreases as [O2] increases; thereby, reducing the steady-state concentration of [PHM]free. BIAA was further used to differentiate between potential oxidative Cu/O species using a QM/MM reaction coordinate simulation to determine which species could yield product O-dealkylation that matched our experimental data. The results of this study provided compelling evidence for the presence of a covalently linked CuII-alkoxide intermediate with a quartet spin state responsible BIAA oxidation. PMID:19569683

Pesticides: an important but underused model for the environmental health sciences.

PubMed Central

Hodgson, E; Levi, P E

1996-01-01

Pesticides are high-volume, widely used, environmental chemicals and there is continuous debate concerning their possible role in many chronic human health effects. Because of their known structures, known rates of application, and the presence of a large occupationally exposed population, they are not only important in their own right but are ideal models for the effects of environmental chemicals on the population in general. For reasons that are not always clear, this potential has not been realized. These exposed populations represent an underused asset in the study of the human health effects of environmental contaminants. Chronic effects thought to involve pesticides include carcinogenesis, neurotoxicity, and reproductive and development effects. In this paper we attempt to summarize this concern and, relying to a large extent on studies in our own laboratory, to indicate the importance and present status of studies of the mammalian metabolism of pesticides and indicate the need for further use of this model. Aspects considered include the role of pesticides as substrates for xenobiotic-metabolizing enzymes such as cytochrome P450 and the flavin-containing monooxygenase and their role as inducers or inhibitors of metabolic enzymes. The interaction of pesticides with complex multienzyme pathways, the role of biological characteristics, particularly gender, in pesticide metabolism, and the special role of pesticides at portals of entry and in target tissues are also considered. PMID:8722114

Enterobacter aerogenes ZDY01 Attenuates Choline-Induced Trimethylamine N-Oxide Levels by Remodeling Gut Microbiota in Mice.

PubMed

Qiu, Liang; Yang, Dong; Tao, Xueying; Yu, Jun; Xiong, Hua; Wei, Hua

2017-08-28

Trimethylamine N -oxide (TMAO), which is transformed from trimethylamine (TMA) through hepatic flavin-containing monooxygenases, can promote atherosclerosis. TMA is produced from dietary carnitine, phosphatidylcholine, and choline via the gut microbes. Previous works have shown that some small molecules, such as allicin, resveratrol, and 3,3-dimethyl-1-butanol, are used to reduce circulating TMAO levels. However, the use of bacteria as an effective therapy to reduce TMAO levels has not been reported. In the present study, 82 isolates were screened from healthy Chinese fecal samples on a basal salt medium supplemented with TMA as the sole carbon source. The isolates belonged to the family Enterobacteriaceae, particularly to genera Klebsiella, Escherichia, Cronobacter , and Enterobacter . Serum TMAO and cecal TMA levels were significantly decreased in choline-fed mice treated with Enterobacter aerogenes ZDY01 compared with those in choline-fed mice treated with phosphate-buffered saline. The proportions of Bacteroidales family S24-7 were significantly increased, whereas the proportions of Helicobacteraceae and Prevotellaceae were significantly decreased through the administration of E. aerogenes ZDY01. Results indicated that the use of probiotics to act directly on the TMA in the gut might be an alternative approach to reduce serum TMAO levels and to prevent the development of atherosclerosis and "fish odor syndrome" through the effect of TMA on the gut microbiota.

Kynurenine 3-monooxygenase polymorphisms: relevance for kynurenic acid synthesis in patients with schizophrenia and healthy controls.

PubMed

Holtze, Maria; Saetre, Peter; Engberg, Göran; Schwieler, Lilly; Werge, Thomas; Andreassen, Ole A; Hall, Håkan; Terenius, Lars; Agartz, Ingrid; Jönsson, Erik G; Schalling, Martin; Erhardt, Sophie

2012-01-01

Patients with schizophrenia show increased brain and cerebrospinal fluid (CSF) concentrations of the endogenous N-methyl-D-aspartate receptor antagonist kynurenic acid (KYNA). This compound is an end-metabolite of the kynurenine pathway, and its formation indirectly depends on the activity of kynurenine 3-monooxygenase (KMO), the enzyme converting kynurenine to 3-hydroxykynurenine. We analyzed the association between KMO gene polymorphisms and CSF concentrations of KYNA in patients with schizophrenia and healthy controls. Fifteen single nucleotide polymorphisms (SNPs) were selected covering KMO and were analyzed in UNPHASED. We included 17 patients with schizophrenia and 33 controls in our study. We found an association between a KMO SNP (rs1053230), encoding an amino acid change of potential importance for substrate interaction, and CSF concentrations of KYNA. Given the limited sample size, the results are tentative until replication. Our results suggest that the nonsynonymous KMO SNP rs1053230 influences CSF concentrations of KYNA.

Bacterial Energy Sensor Aer Modulates the Activity of the Chemotaxis Kinase CheA Based on the Redox State of the Flavin Cofactor.

PubMed

Samanta, Dipanjan; Widom, Joanne; Borbat, Peter P; Freed, Jack H; Crane, Brian R

2016-12-09

Flagellated bacteria modulate their swimming behavior in response to environmental cues through the CheA/CheY signaling pathway. In addition to responding to external chemicals, bacteria also monitor internal conditions that reflect the availability of oxygen, light, and reducing equivalents, in a process termed "energy taxis." In Escherichia coli, the transmembrane receptor Aer is the primary energy sensor for motility. Genetic and physiological data suggest that Aer monitors the electron transport chain through the redox state of its FAD cofactor. However, direct biochemical data correlating FAD redox chemistry with CheA kinase activity have been lacking. Here, we test this hypothesis via functional reconstitution of Aer into nanodiscs. As purified, Aer contains fully oxidized FAD, which can be chemically reduced to the anionic semiquinone (ASQ). Oxidized Aer activates CheA, whereas ASQ Aer reversibly inhibits CheA. Under these conditions, Aer cannot be further reduced to the hydroquinone, in contrast to the proposed Aer signaling model. Pulse ESR spectroscopy of the ASQ corroborates a potential mechanism for signaling in that the resulting distance between the two flavin-binding PAS (Per-Arnt-Sim) domains implies that they tightly sandwich the signal-transducing HAMP domain in the kinase-off state. Aer appears to follow oligomerization patterns observed for related chemoreceptors, as higher loading of Aer dimers into nanodiscs increases kinase activity. These results provide a new methodological platform to study Aer function along with new mechanistic details into its signal transduction process. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Intermittent hypoxia activates peptidylglycine α-amidating monooxygenase in rat brain stem via reactive oxygen species-mediated proteolytic processing

PubMed Central

Sharma, Suresh D.; Raghuraman, Gayatri; Lee, Myeong-Seon; Prabhakar, Nanduri R.; Kumar, Ganesh K.

2009-01-01

Intermittent hypoxia (IH) associated with sleep apneas leads to cardiorespiratory abnormalities that may involve altered neuropeptide signaling. The effects of IH on neuropeptide synthesis have not been investigated. Peptidylglycine α-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the α-amidation of neuropeptides, which confers biological activity to a large number of neuropeptides. PAM consists of O2-sensitive peptidylglycine α-hydroxylating monooxygenase (PHM) and peptidyl-α-hydroxyglycine α-amidating lyase (PAL) activities. Here, we examined whether IH alters neuropeptide synthesis by affecting PAM activity and, if so, by what mechanisms. Experiments were performed on the brain stem of adult male rats exposed to IH (5% O2 for 15 s followed by 21% O2 for 5 min; 8 h/day for up to 10 days) or continuous hypoxia (0.4 atm for 10 days). Analysis of brain stem extracts showed that IH, but not continuous hypoxia, increased PHM, but not PAL, activity of PAM and that the increase of PHM activity was associated with a concomitant elevation in the levels of α-amidated forms of substance P and neuropeptide Y. IH increased the relative abundance of 42- and 35-kDa forms of PHM (∼1.6- and 2.7-fold, respectively), suggesting enhanced proteolytic processing of PHM, which appears to be mediated by an IH-induced increase of endoprotease activity. Kinetic analysis showed that IH increases Vmax but has no effect on Km. IH increased generation of reactive oxygen species in the brain stem, and systemic administration of antioxidant prevented IH-evoked increases of PHM activity, proteolytic processing of PHM, endoprotease activity, and elevations in substance P and neuropeptide Y amide levels. Taken together, these results demonstrate that IH activates PHM in rat brain stem via reactive oxygen species-dependent posttranslational proteolytic processing and further suggest that PAM activation may contribute to IH-mediated peptidergic neurotransmission in rat brain stem

Intermittent hypoxia activates peptidylglycine alpha-amidating monooxygenase in rat brain stem via reactive oxygen species-mediated proteolytic processing.

PubMed

Sharma, Suresh D; Raghuraman, Gayatri; Lee, Myeong-Seon; Prabhakar, Nanduri R; Kumar, Ganesh K

2009-01-01

Intermittent hypoxia (IH) associated with sleep apneas leads to cardiorespiratory abnormalities that may involve altered neuropeptide signaling. The effects of IH on neuropeptide synthesis have not been investigated. Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the alpha-amidation of neuropeptides, which confers biological activity to a large number of neuropeptides. PAM consists of O(2)-sensitive peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) activities. Here, we examined whether IH alters neuropeptide synthesis by affecting PAM activity and, if so, by what mechanisms. Experiments were performed on the brain stem of adult male rats exposed to IH (5% O(2) for 15 s followed by 21% O(2) for 5 min; 8 h/day for up to 10 days) or continuous hypoxia (0.4 atm for 10 days). Analysis of brain stem extracts showed that IH, but not continuous hypoxia, increased PHM, but not PAL, activity of PAM and that the increase of PHM activity was associated with a concomitant elevation in the levels of alpha-amidated forms of substance P and neuropeptide Y. IH increased the relative abundance of 42- and 35-kDa forms of PHM ( approximately 1.6- and 2.7-fold, respectively), suggesting enhanced proteolytic processing of PHM, which appears to be mediated by an IH-induced increase of endoprotease activity. Kinetic analysis showed that IH increases V(max) but has no effect on K(m). IH increased generation of reactive oxygen species in the brain stem, and systemic administration of antioxidant prevented IH-evoked increases of PHM activity, proteolytic processing of PHM, endoprotease activity, and elevations in substance P and neuropeptide Y amide levels. Taken together, these results demonstrate that IH activates PHM in rat brain stem via reactive oxygen species-dependent posttranslational proteolytic processing and further suggest that PAM activation may contribute to IH-mediated peptidergic

HHM motif at the CuH-site of peptidylglycine monooxygenase is a pH-dependent conformational switch.

PubMed

Kline, Chelsey D; Mayfield, Mary; Blackburn, Ninian J

2013-04-16

Peptidylglycine monooxygenase is a copper-containing enzyme that catalyzes the amidation of neuropeptides hormones, the first step of which is the conversion of a glycine-extended pro-peptide to its α-hydroxyglcine intermediate. The enzyme contains two mononuclear Cu centers termed CuM (ligated to imidazole nitrogens of H242, H244 and the thioether S of M314) and CuH (ligated to imidazole nitrogens of H107, H108, and H172) with a Cu-Cu separation of 11 Å. During catalysis, the M site binds oxygen and substrate, and the H site donates the second electron required for hydroxylation. The WT enzyme shows maximum catalytic activity at pH 5.8 and undergoes loss of activity at lower pHs due to a protonation event with a pKA of 4.6. Low pH also causes a unique structural transition in which a new S ligand coordinates to copper with an identical pKA, manifest by a large increase in Cu-S intensity in the X- ray absorption spectroscopy. In previous work (Bauman, A. T., Broers, B. A., Kline, C. D., and Blackburn, N. J. (2011) Biochemistry 50, 10819-10828), we tentatively assigned the new Cu-S interaction to binding of M109 to the H-site (part of an HHM conserved motif common to all but one member of the family). Here we follow up on these findings via studies on the catalytic activity, pH-activity profiles, and spectroscopic (electron paramagnetic resonance, XAS, and Fourier transform infrared) properties of a number of H-site variants, including H107A, H108A, H172A, and M109I. Our results establish that M109 is indeed the coordinating ligand and confirm the prediction that the low pH structural transition with associated loss of activity is abrogated when the M109 thioether is absent. The histidine mutants show more complex behavior, but the almost complete lack of activity in all three variants coupled with only minor differences in their spectroscopic properties suggests that unique structural elements at H are critical for functionality. The data suggest a more general

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. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Methanobactin from Methylocystis sp. Strain SB2 Affects Gene Expression and Methane Monooxygenase Activity in Methylosinus trichosporium OB3b

PubMed Central

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

2015-01-01

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. PMID:25616801

[Effect of flavin adenine dinucleotide on ultraviolet B induced damage in cultured human corneal epithelial cells].

PubMed

Sakamoto, Asuka; Nakamura, Masatsugu

2012-01-01

This study evaluated the effects of flavin adenine dinucleotide (FAD) on ultraviolet B (UV-B)-induced damage in cultured human corneal epithelial (HCE-T) cells. The cultured HCE-T cells were treated with 0.003125-0.05% FAD before exposure to 80 mJ/cm2 UV-B. Cell viability was measured 24 h after UV-B irradiation using the MTS assay. Reactive oxygen species (ROS) were detected 30 min after UV-B irradiation using 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate acetyl ester. Apoptosis was evaluated 4 h after UV-B irradiation in the caspase-3/7 activity assay. UV-B irradiation reduced cell viability and stimulated ROS production and caspase-3/7 activity in HCE-T cells. Pretreatment of UV-B irradiated HCE-T cells with FAD significantly attenuated cell viability reduction and inhibited the stimulation of both ROS production and caspase-3/7 activity due to UV-B exposure compared with those with vehicle (0% FAD). These results clarified that FAD inhibits ROS-mediated apoptosis by UV-B irradiation in HCE-T cells and suggest that FAD may be effective as a radical scavenger in UV-B-induced corneal damage.

Development of LC/MS/MS, high-throughput enzymatic and cellular assays for the characterization of compounds that inhibit kynurenine monooxygenase (KMO).

PubMed

Winkler, Dirk; Beconi, Maria; Toledo-Sherman, Leticia M; Prime, Michael; Ebneth, Andreas; Dominguez, Celia; Muñoz-Sanjuan, Ignacio

2013-09-01

Kynurenine monooxygenase (KMO) catalyzes the conversion of kynurenine to 3-hydroxykynurenine. Modulation of KMO activity has been implicated in several neurodegenerative diseases, including Huntington disease. Our goal is to develop potent and selective small-molecule KMO inhibitors with suitable pharmacokinetic characteristics for in vivo proof-of-concept studies and subsequent clinical development. We developed a comprehensive panel of biochemical and cell-based assays that use liquid chromatography/tandem mass spectrometry to quantify unlabeled kynurenine and 3-hydroxykynurenine. We describe assays to measure KMO inhibition in cell and tissue extracts, as well as cellular assays including heterologous cell lines and primary rat microglia and human peripheral blood mononuclear cells.

Structural and Functional Analysis of a Lytic Polysaccharide Monooxygenase Important for Efficient Utilization of Chitin in Cellvibrio japonicus*

PubMed Central

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

2016-01-01

Cellvibrio japonicus is 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 the CjLPMO10A 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 that CjLPMO10A is needed by C. japonicus to obtain efficient growth on both purified chitin and crab shell particles. PMID:26858252

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. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

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.

New Dimensions in Microbial Ecology-Functional Genes in Studies to Unravel the Biodiversity and Role of Functional Microbial Groups in the Environment.

PubMed

Imhoff, Johannes F

2016-05-24

During the past decades, tremendous advances have been made in the possibilities to study the diversity of microbial communities in the environment. The development of methods to study these communities on the basis of 16S rRNA gene sequences analysis was a first step into the molecular analysis of environmental communities and the study of biodiversity in natural habitats. A new dimension in this field was reached with the introduction of functional genes of ecological importance and the establishment of genetic tools to study the diversity of functional microbial groups and their responses to environmental factors. Functional gene approaches are excellent tools to study the diversity of a particular function and to demonstrate changes in the composition of prokaryote communities contributing to this function. The phylogeny of many functional genes largely correlates with that of the 16S rRNA gene, and microbial species may be identified on the basis of functional gene sequences. Functional genes are perfectly suited to link culture-based microbiological work with environmental molecular genetic studies. In this review, the development of functional gene studies in environmental microbiology is highlighted with examples of genes relevant for important ecophysiological functions. Examples are presented for bacterial photosynthesis and two types of anoxygenic phototrophic bacteria, with genes of the Fenna-Matthews-Olson-protein (fmoA) as target for the green sulfur bacteria and of two reaction center proteins (pufLM) for the phototrophic purple bacteria, with genes of adenosine-5'phosphosulfate (APS) reductase (aprA), sulfate thioesterase (soxB) and dissimilatory sulfite reductase (dsrAB) for sulfur oxidizing and sulfate reducing bacteria, with genes of ammonia monooxygenase (amoA) for nitrifying/ammonia-oxidizing bacteria, with genes of particulate nitrate reductase and nitrite reductases (narH/G, nirS, nirK) for denitrifying bacteria and with genes of methane

Crystal structure of 4-hydroxybutyryl-CoA dehydratase: radical catalysis involving a [4Fe-4S] cluster and flavin.

PubMed

Martins, Berta M; Dobbek, Holger; Cinkaya, Irfan; Buckel, Wolfgang; Messerschmidt, Albrecht

2004-11-02

Dehydratases catalyze the breakage of a carbon-oxygen bond leading to unsaturated products via the elimination of water. The 1.6-A resolution crystal structure of 4-hydroxybutyryl-CoA dehydratase from the gamma-aminobutyrate-fermenting Clostridium aminobutyricum represents a new class of dehydratases with an unprecedented active site architecture. A [4Fe-4S](2+) cluster, coordinated by three cysteine and one histidine residues, is located 7 A from the Re-side of a flavin adenine dinucleotide (FAD) moiety. The structure provides insight into the function of these ubiquitous prosthetic groups in the chemically nonfacile, radical-mediated dehydration of 4-hydroxybutyryl-CoA. The substrate can be bound between the [4Fe-4S](2+) cluster and the FAD with both cofactors contributing to its radical activation and catalytic conversion. Our results raise interesting questions regarding the mechanism of acyl-CoA dehydrogenases, which are involved in fatty acid oxidation, and address the divergent evolution of the ancestral common gene.

Kynurenine 3-monooxygenase polymorphisms: relevance for kynurenic acid synthesis in patients with schizophrenia and healthy controls

PubMed Central

Holtze, Maria; Saetre, Peter; Engberg, Göran; Schwieler, Lilly; Werge, Thomas; Andreassen, Ole A.; Hall, Håkan; Terenius, Lars; Agartz, Ingrid; Jönsson, Erik G.; Schalling, Martin; Erhardt, Sophie

2012-01-01

Background Patients with schizophrenia show increased brain and cerebrospinal fluid (CSF) concentrations of the endogenous N-methyl-d-aspartate receptor antagonist kynurenic acid (KYNA). This compound is an end-metabolite of the kynurenine pathway, and its formation indirectly depends on the activity of kynurenine 3-monooxygenase (KMO), the enzyme converting kynurenine to 3-hydroxykynurenine. Methods We analyzed the association between KMO gene polymorphisms and CSF concentrations of KYNA in patients with schizophrenia and healthy controls. Fifteen single nucleotide polymorphisms (SNPs) were selected covering KMO and were analyzed in UNPHASED. Results We included 17 patients with schizophrenia and 33 controls in our study. We found an association between a KMO SNP (rs1053230), encoding an amino acid change of potential importance for substrate interaction, and CSF concentrations of KYNA. Limitations Given the limited sample size, the results are tentative until replication. Conclusion Our results suggest that the nonsynonymous KMO SNP rs1053230 influences CSF concentrations of KYNA. PMID:21693093

Simulations of Chemical Reactions with the Frozen Domain Formulation of the Fragment Molecular Orbital Method.

PubMed

Nakata, Hiroya; Fedorov, Dmitri G; Nagata, Takeshi; Kitaura, Kazuo; Nakamura, Shinichiro

2015-07-14

The fully analytic first and second derivatives of the energy in the frozen domain formulation of the fragment molecular orbital (FMO) were developed and applied to locate transition states and determine vibrational contributions to free energies. The development is focused on the frozen domain with dimers (FDD) model. The intrinsic reaction coordinate method was interfaced with FMO. Simulations of IR and Raman spectra were enabled using FMO/FDD by developing the calculation of intensities. The accuracy is evaluated for S(N)2 reactions in explicit solvent, and for the free binding energies of a protein-ligand complex of the Trp cage protein (PDB: 1L2Y ). FMO/FDD is applied to study the keto-enol tautomeric reaction of phosphoglycolohydroxamic acid and the triosephosphate isomerase (PDB: 7TIM ), and the role of amino acid residue fragments in the reaction is discussed.

[Free radical reactions and energy transformation in microsome membranes. Arrhenius equation for the monooxygenase reaction].

PubMed

Dmitriev, L F

2001-01-01

The mechanism of coupling of the oxidation and activation of membrane enzymes was considered. It is obvious that microsomal monooxygenase uses the energy of NADPH oxidation for the activation of the terminal agent--cytochrome P-450. However, till now the mechanism of the transformation of this energy has not been discussed. It is supposed that the coupling process includes transformation of oxidation energy to kinetic energy, the energy of lipid pulsations. The mechanism proposed by us and the mechanism of energy transformation according to Mitchell are two independent mechanisms, both being of fundamental importance for biochemistry and biophysics of membranes. One approach uses the dielectric properties of membrane, and the other is based on the ability of hydrocarbon chains of phospholipids for rotamerization. A new empirical Arrhenius equation for membrane processes is offered. It accounts for the ability of membrane to reserve the energy in kinetic form (internal temperature). In conditions when membrane proteins cease to be acceptors of energy, the transfer of energy, i.e., transformation of the energy of NADPH oxidation into heat or light, occurs.

Crystallization of a fungal lytic polysaccharide monooxygenase expressed from glycoengineered Pichia pastoris for X-ray and neutron diffraction

DOE Office of Scientific and Technical Information (OSTI.GOV)

O'Dell, William B.; Swartz, Paul D.; Weiss, Kevin L.

Lytic polysaccharide monooxygenases (LPMOs) are carbohydrate-disrupting enzymes secreted by bacteria and fungi that break glycosidic bondsviaan oxidative mechanism. Fungal LPMOs typically act on cellulose and can enhance the efficiency of cellulose-hydrolyzing enzymes that release soluble sugars for bioethanol production or other industrial uses. The enzyme PMO-2 fromNeurospora crassa(NcPMO-2) was heterologously expressed inPichia pastoristo facilitate crystallographic studies of the fungal LPMO mechanism. Diffraction resolution and crystal morphology were improved by expressingNcPMO-2 from a glycoengineered strain ofP. pastorisand by the use of crystal seeding methods, respectively. These improvements resulted in high-resolution (1.20 Å) X-ray diffraction data collection at 100 K and themore » production of a largeNcPMO-2 crystal suitable for room-temperature neutron diffraction data collection to 2.12 Å resolution.« less

Development of a Rapid Fluorescence-Based High-Throughput Screening Assay to Identify Novel Kynurenine 3-Monooxygenase Inhibitor Scaffolds.

PubMed

Jacobs, K R; Guillemin, G J; Lovejoy, D B

2018-02-01

Kynurenine 3-monooxygenase (KMO) is a well-validated therapeutic target for the treatment of neurodegenerative diseases, including Alzheimer's disease (AD) and Huntington's disease (HD). This work reports a facile fluorescence-based KMO assay optimized for high-throughput screening (HTS) that achieves a throughput approximately 20-fold higher than the fastest KMO assay currently reported. The screen was run with excellent performance (average Z' value of 0.80) from 110,000 compounds across 341 plates and exceeded all statistical parameters used to describe a robust HTS assay. A subset of molecules was selected for validation by ultra-high-performance liquid chromatography, resulting in the confirmation of a novel hit with an IC 50 comparable to that of the well-described KMO inhibitor Ro-61-8048. A medicinal chemistry program is currently underway to further develop our novel KMO inhibitor scaffolds.

Aerobic biodegradation of N-nitrosodimethylamine (NDMA) by axenic bacterial strains.

PubMed

Sharp, Jonathan O; Wood, Thomas K; Alvarez-Cohen, Lisa

2005-03-05

The water contaminant N-nitrosodimethylamine (NDMA) is a probable human carcinogen whose appearance in the environment is related to the release of rocket fuel and to chlorine-based disinfection of water and wastewater. Although this compound has been shown to be biodegradable, there is minimal information about the organisms capable of this degradation, and little is understood of the mechanisms or biochemistry involved. This study shows that bacteria expressing monooxygenase enzymes functionally similar to those demonstrated to degrade NDMA in eukaryotes have the capability to degrade NDMA. Specifically, induction of the soluble methane monooxygenase (sMMO) expressed by Methylosinus trichosporium OB3b, the propane monooxygenase (PMO) enzyme of Mycobacterium vaccae JOB-5, and the toluene 4-monooxygenases found in Ralstonia pickettii PKO1 and Pseudomonas mendocina KR1 resulted in NDMA degradation by these strains. In each of these cases, brief exposure to acetylene gas, a suicide substrate for certain monooxygenases, inhibited the degradation of NDMA. Further, Escherichia coli TG1/pBS(Kan) containing recombinant plasmids derived from the toluene monooxygenases found in strains PKO1 and KR1 mimicked the behavior of the parent strains. In contrast, M. trichosporium OB3b expressing the particulate form of MMO, Burkholderia cepacia G4 expressing the toluene 2-monooxygenase, and Pseudomonas putida mt-2 expressing the toluene sidechain monooxygenase were not capable of NDMA degradation. In addition, bacteria expressing aromatic dioxygenases were not capable of NDMA degradation. Finally, Rhodococcus sp. RR1 exhibited the ability to degrade NDMA by an unidentified, constitutively expressed enzyme that, unlike the confirmed monooxygenases, was not inhibited by acetylene exposure. 2005 Wiley Periodicals, Inc.

Prediction of probable mutations in influenza virus hemagglutinin protein based on large-scale ab initio fragment molecular orbital calculations.

PubMed

Yoshioka, Akio; Fukuzawa, Kaori; Mochizuki, Yuji; Yamashita, Katsumi; Nakano, Tatsuya; Okiyama, Yoshio; Nobusawa, Eri; Nakajima, Katsuhisa; Tanaka, Shigenori

2011-09-01

Ab initio electronic-state calculations for influenza virus hemagglutinin (HA) trimer complexed with Fab antibody were performed on the basis of the fragment molecular orbital (FMO) method at the second and third-order Møller-Plesset (MP2 and MP3) perturbation levels. For the protein complex containing 2351 residues and 36,160 atoms, the inter-fragment interaction energies (IFIEs) were evaluated to illustrate the effective interactions between all the pairs of amino acid residues. By analyzing the calculated data on the IFIEs, we first discussed the interactions and their fluctuations between multiple domains contained in the trimer complex. Next, by combining the IFIE data between the Fab antibody and each residue in the HA antigen with experimental data on the hemadsorption activity of HA mutants, we proposed a protocol to predict probable mutations in HA. The proposed protocol based on the FMO-MP2.5 calculation can explain the historical facts concerning the actual mutations after the emergence of A/Hong Kong/1/68 influenza virus with subtype H3N2, and thus provides a useful methodology to enumerate those residue sites likely to mutate in the future. Copyright © 2011 Elsevier Inc. All rights reserved.

[The drop in toxicity and the rise in the effectiveness of antineoplastic chemotherapy by correcting the activity of liver monooxygenases: from the experiment to the clinical practice].

PubMed

Bogush, T A; Bogush, E A; Durnov, L A; Syrkin, A B

2002-01-01

The paper reviews both the data available in the literature and the authors' own results of long-term experimental and clinical investigations of the involvement of hepatic monooxygenases (HMO) in the biological activity of antitumor drugs. It reports data of evaluation of HMO activity in pediatric and adult cancer patients, which has shown a decrease in HMO activity in one third of patients without clinical signs of hepatopathy and two thirds of those with toxic hepatic damages after prior chemotherapy. Decreased HMO activity has been found to be stimulated with the enzyme inductor zyxorin. Altered biochemical parameters, such as total bilirubin, ALT and AST, can be corrected with HNO, even if they show a 10-fold deviation from the normal physiological level. The efficacy of zyxorin was tested in patients with advanced cancer and concomitant toxic or viral hepatic disorders (grades II-IV by the WHO classification). Stimulation of inhibited HMO activity allows both decrease and prevention of the manifestations of hepatic toxicity due to anticancer chemotherapy providing a beneficial effect, the dose of cytostatics being not reduced. The authors concluded that the findings provide strong evidence for their assumption that the efficiency of antitumor chemotherapy can be enhanced in patients with concurrent hepatic abnormality by stimulating monooxygenases whose activity is diminished in the majority of these patients.

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

PubMed

Morgenstern, Ingo; Powlowski, Justin; Tsang, Adrian

2014-11-01

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. © The Author 2014. Published by Oxford University Press.

Rat oesophageal cytochrome P450 (CYP) monooxygenase system: comparison to the liver and relevance in N-nitrosodiethylamine carcinogenesis.

PubMed

Pinto, L F; Moraes, E; Albano, R M; Silva, M C; Godoy, W; Glisovic, T; Lang, M A

2001-11-01

N-nitrosodiethylamine (NDEA) is able to induce tumours in the rat oesophagus. It has been suggested that this could be due to tissue specific expression of NDEA activating cytochrome P450 enzymes. We investigated this by characterizing the oesophageal monooxygenase complex of male Wistar rats and comparing it with that of the liver. Total amount of cytochrome P450, NADPH P450 reductase, cytochrome b5 and cytochrome b5 reductase of the oesophageal mucosa was approximately 7% of what was found in the liver. In addition, major differences were found in the cytochrome P450 isoenzyme composition between these organs: CYP 2B1/2B2 and CYP3A were found only in the liver, whereas CYP1A1 was constitutively expressed only in the oesophagus. Of the two well-known nitrosamine metabolizing enzymes, CYP2A3 was found only in the oesophagus whereas CYP2E1 was exclusively expressed in the liver. Catalytic studies, western blotting and RT-PCR analyses confirmed the expression of CYP2A3 in the oesophagus. CYP2A enzymes are known to be good catalysts of NDEA metabolism. Oesophageal microsomes had a K(m) for NDEA metabolism, which was about one-third of that of hepatic microsomes, but they showed similar activities when compared per nmol of total P450. NDEA activity in the oesophagus was significantly increased by coumarin (CO), which also induced oesophageal CYP2A3. Immunoinhibition of the microsomal NDEA activity showed that up to 70% of this reaction is catalysed by CYP2A3 in the oesophagus, whereas no inhibition of the hepatic NDEA activity could be achieved by the anti-CYP2A5 antibody. NDEA, but not N-nitrosodimethylamine (NDMA) inhibited the oesophageal metabolism of CO. The results of the present investigation show major differences in the enzyme composition of the oesophageal and hepatic monooxygenase complexes, and are in accordance with the hypothesis that the NDEA organotropism could, to a large extent, be due to the tissue specific expression of the activating enzymes.

Molecular Dynamics Analysis Reveals Structural Insights into Mechanism of Nicotine N-Demethylation Catalyzed by Tobacco Cytochrome P450 Mono-Oxygenase

PubMed Central

Wang, Shan; Yang, Shuo; An, Baiyi; Wang, Shichen; Yin, Yuejia; Lu, Yang; Xu, Ying; Hao, Dongyun

2011-01-01

CYP82E4, a cytochrome P450 monooxygenase, has nicotine N-demethylase (NND) activity, which mediates the bioconversion of nicotine into nornicotine in senescing tobacco leaves. Nornicotine is a precursor of the carcinogen, tobacco-specific nitrosamine. CYP82E3 is an ortholog of CYP82E4 with 95% sequence identity, but it lacks NND activity. A recent site-directed mutagenesis study revealed that a single amino acid substitution, i.e., cysteine to tryptophan at the 330 position in the middle of protein, restores the NND activity of CYP82E3 entirely. However, the same amino acid change caused the loss of the NND activity of CYP82E4. To determine the mechanism of the functional turnover of the two molecules, four 3D structures, i.e., the two molecules and their corresponding cys–trp mutants were modeled. The resulting structures exhibited that the mutation site is far from the active site, which suggests that no direct interaction occurs between the two sites. Simulation studies in different biological scenarios revealed that the mutation introduces a conformation drift with the largest change at the F-G loop. The dynamics trajectories analysis using principal component analysis and covariance analysis suggests that the single amino acid change causes the opening and closing of the transfer channels of the substrates, products, and water by altering the motion of the F-G and B-C loops. The motion of helix I is also correlated with the motion of both the F-G loop and the B-C loop and; the single amino acid mutation resulted in the curvature of helix I. These results suggest that the single amino acid mutation outside the active site region may have indirectly mediated the flexibility of the F-G and B-C loops through helix I, causing a functional turnover of the P450 monooxygenase. PMID:21858078

Sterol homeostasis requires regulated degradation of squalene monooxygenase by the ubiquitin ligase Doa10/Teb4

PubMed Central

Foresti, Ombretta; Ruggiano, Annamaria; Hannibal-Bach, Hans K; Ejsing, Christer S; Carvalho, Pedro

2013-01-01

Sterol homeostasis is essential for the function of cellular membranes and requires feedback inhibition of HMGR, a rate-limiting enzyme of the mevalonate pathway. As HMGR acts at the beginning of the pathway, its regulation affects the synthesis of sterols and of other essential mevalonate-derived metabolites, such as ubiquinone or dolichol. Here, we describe a novel, evolutionarily conserved feedback system operating at a sterol-specific step of the mevalonate pathway. This involves the sterol-dependent degradation of squalene monooxygenase mediated by the yeast Doa10 or mammalian Teb4, a ubiquitin ligase implicated in a branch of the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway. Since the other branch of ERAD is required for HMGR regulation, our results reveal a fundamental role for ERAD in sterol homeostasis, with the two branches of this pathway acting together to control sterol biosynthesis at different levels and thereby allowing independent regulation of multiple products of the mevalonate pathway. DOI: http://dx.doi.org/10.7554/eLife.00953.001 PMID:23898401

Adaptive frozen orbital treatment for the fragment molecular orbital method combined with density-functional tight-binding

NASA Astrophysics Data System (ADS)

Nishimoto, Yoshio; Fedorov, Dmitri G.

2018-02-01

The exactly analytic gradient is derived and implemented for the fragment molecular orbital (FMO) method combined with density-functional tight-binding (DFTB) using adaptive frozen orbitals. The response contributions which arise from freezing detached molecular orbitals on the border between fragments are computed by solving Z-vector equations. The accuracy of the energy, its gradient, and optimized structures is verified on a set of representative inorganic materials and polypeptides. FMO-DFTB is applied to optimize the structure of a silicon nano-wire, and the results are compared to those of density functional theory and experiment. FMO accelerates the DFTB calculation of a boron nitride nano-ring with 7872 atoms by a factor of 406. Molecular dynamics simulations using FMO-DFTB applied to a 10.7 μm chain of boron nitride nano-rings, consisting of about 1.2 × 106 atoms, reveal the rippling and twisting of nano-rings at room temperature.

Plasma peptidylglycine alpha-amidating monooxygenase (PAM) and ceruloplasmin are affected by age and copper status in rats and mice

PubMed Central

Prohaska, Joseph R.; Broderius, Margaret

2009-01-01

In an attempt to identify a sensitive and improved marker of mammalian copper status during neonatal development experiments compared two plasma cuproenzymes, peptidylglycine α-amidating monooxygenase (PAM ), an enzyme involved in peptide posttranslational activation, to ceruloplasmin (Cp), a ferroxidase involved in iron mobilization. Dietary Cu deficiency (Cu−) was studied in dams and offspring at postnatal age 3 (P3), P12, and P28. Rodent Cp activity rose during lactation whereas PAM activity fell. Reduction in Cp activity was more severe than reduction in PAM activity in Cu− offspring and dams. Cp activity was greater in rats than mice whereas PAM activity was similar in adults but greater in mouse than rat pups. Both cuproenzymes changed during neonatal development and when dietary copper was limiting. With proper controls, each enzyme can be used to assess copper status. PMID:16448835

Comparative study on nutrient composition, phytochemical, and functional characteristics of raw, germinated, and fermented Moringa oleifera seed flour

PubMed Central

Ijarotimi, Oluwole S; Adeoti, Oluwole A; Ariyo, Oluwaseun

2013-01-01

Moringa oleifera seeds were processed as raw M. oleifera (RMO), germinated M. oleifera (GMO), and fermented M. oleifera (FMO), and were evaluated for proximate, minerals, amino acids, fatty acids, phytochemicals/antinutrients, and functional properties. Protein content of GMO (23.69 ± 0.11 g/100 g) was higher than FMO (21.15 ± 0.08 g/100 g) and that of RMO (18.86 ± 0.09 g/100 g) (P < 0.05), respectively. Energy value of FMO (465.32 ± 0.48 kcal) was higher than GMO (438.62 ± 0.12 kcal) and that of RMO (409.04 ± 1.61 kcal), respectively. Mineral contents in GMO were significantly higher in iron, sodium, potassium, magnesium, and copper, while FMO were higher in calcium, phosphorus, and magnesium, and both were significantly lower than those in RMO (P < 0.05). Total essential amino acids (TEAAs) in FMO (31.07 mg/g crude protein) were higher than in GMO (26.52 mg/g crude protein), and were higher than that in RMO (23.56 mg/g crude protein). Linoleic acid (58.79 ± 0.02–62.05 ± 0.01 g/100 g) and behenic acid (0.13 ± 0.00–0.20 ± 0.06 g/100 g) were the predominant and least fatty acids, respectively. Phytochemical/antinutrient compositions in FMO samples were significantly lower than GMO, and both were significantly lower when compared with RMO samples (P < 0.05). The bulk density (pack and loose), foaming capacity, swelling capacity, and water absorption capacity (WAC) of FMO were significantly higher than those of GMO, and there was no significant difference between GMO and RMO samples. The study established that fermentation processing methods increased the protein content, essential amino acid, and polyunsaturated fatty acid profiles, and reduced antinutrient compositions of M. oleifera seed than germination processing techniques; hence, fermentation techniques should be encouraged in processing moringa seeds in food processing. PMID:24804056

Interdomain electron transfer in cellobiose dehydrogenase is governed by surface electrostatics.

PubMed

Kadek, Alan; Kavan, Daniel; Marcoux, Julien; Stojko, Johann; Felice, Alfons K G; Cianférani, Sarah; Ludwig, Roland; Halada, Petr; Man, Petr

2017-02-01

Cellobiose dehydrogenase (CDH) is a fungal extracellular oxidoreductase which fuels lytic polysaccharide monooxygenase with electrons during cellulose degradation. Interdomain electron transfer between the flavin and cytochrome domain in CDH, preceding the electron flow to lytic polysaccharide monooxygenase, is known to be pH dependent, but the exact mechanism of this regulation has not been experimentally proven so far. To investigate the structural aspects underlying the domain interaction in CDH, hydrogen/deuterium exchange (HDX-MS) with improved proteolytic setup (combination of nepenthesin-1 with rhizopuspepsin), native mass spectrometry with ion mobility and electrostatics calculations were used. HDX-MS revealed pH-dependent changes in solvent accessibility and hydrogen bonding at the interdomain interface. Electrostatics calculations identified these differences to result from charge neutralization by protonation and together with ion mobility pointed at higher electrostatic repulsion between CDH domains at neutral pH. In addition, we uncovered extensive O-glycosylation in the linker region and identified the long-unknown exact cleavage point in papain-mediated domain separation. Transition of CDH between its inactive (open) and interdomain electron transfer-capable (closed) state is shown to be governed by changes in the protein surface electrostatics at the domain interface. Our study confirms that the interdomain electrostatic repulsion is the key factor modulating the functioning of CDH. The results presented in this paper provide experimental evidence for the role of charge repulsion in the interdomain electron transfer in cellobiose dehydrogenases, which is relevant for exploiting their biotechnological potential in biosensors and biofuel cells. Copyright © 2016 Elsevier B.V. All rights reserved.

From micelles to bicelles: Effect of the membrane on particulate methane monooxygenase activity.

PubMed

Ro, Soo Y; Ross, Matthew O; Deng, Yue Wen; Batelu, Sharon; Lawton, Thomas J; Hurley, Joseph D; Stemmler, Timothy L; Hoffman, Brian M; Rosenzweig, Amy C

2018-05-08

Particulate methane monooxygenase (pMMO) is a copper-dependent, integral membrane metalloenzyme that converts methane to methanol in methanotrophic bacteria. Studies of isolated pMMO have been hindered by loss of enzymatic activity upon its removal from the native membrane. To characterize pMMO in a membrane-like environment, we reconstituted pMMOs from Methylococcus ( Mcc. ) capsulatus (Bath) and Methylomicrobium ( Mm. ) alcaliphilum 20Z into bicelles. Reconstitution into bicelles recovers methane oxidation activity lost upon detergent solubilization and purification without substantial alterations to copper content or copper electronic structure as observed by electron paramagnetic resonance (EPR) spectroscopy.. These findings suggest that loss of pMMO activity upon isolation is due to removal from the membranes rather than caused by loss of the catalytic copper ions. A 2.7 Å resolution crystal structure of pMMO from Mm. alcaliphilum 20Z revealed a mononuclear copper center in the PmoB subunit and indicated that the transmembrane PmoC subunit may be conformationally flexible. Finally, results from extended X-ray absorption fine structure (EXAFS) analysis of pMMO from Mm. alcaliphilum 20Z were consistent with the observed monocopper center in the PmoB subunit. These results underscore the importance of studying membrane proteins in a membrane-like environment, and provide valuable insight into pMMO function. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Sequence-based screening for self-sufficient P450 monooxygenase from a metagenome library.

PubMed

Kim, B S; Kim, S Y; Park, J; Park, W; Hwang, K Y; Yoon, Y J; Oh, W K; Kim, B Y; Ahn, J S

2007-05-01

Cytochrome P450 monooxygenases (CYPs) are useful catalysts for oxidation reactions. Self-sufficient CYPs harbour a reductive domain covalently connected to a P450 domain and are known for their robust catalytic activity with great potential as biocatalysts. In an effort to expand genetic sources of self-sufficient CYPs, we devised a sequence-based screening system to identify them in a soil metagenome. We constructed a soil metagenome library and performed sequence-based screening for self-sufficient CYP genes. A new CYP gene, syk181, was identified from the metagenome library. Phylogenetic analysis revealed that SYK181 formed a distinct phylogenic line with 46% amino-acid-sequence identity to CYP102A1 which has been extensively studied as a fatty acid hydroxylase. The heterologously expressed SYK181 showed significant hydroxylase activity towards naphthalene and phenanthrene as well as towards fatty acids. Sequence-based screening of metagenome libraries is expected to be a useful approach for searching self-sufficient CYP genes. The translated product of syk181 shows self-sufficient hydroxylase activity towards fatty acids and aromatic compounds. SYK181 is the first self-sufficient CYP obtained directly from a metagenome library. The genetic and biochemical information on SYK181 are expected to be helpful for engineering self-sufficient CYPs with broader catalytic activities towards various substrates, which would be useful for bioconversion of natural products and biodegradation of organic chemicals.

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.

A magnetic bead-based ligand binding assay to facilitate human kynurenine 3-monooxygenase drug discovery.

PubMed

Wilson, Kris; Mole, Damian J; Homer, Natalie Z M; Iredale, John P; Auer, Manfred; Webster, Scott P

2015-02-01

Human kynurenine 3-monooxygenase (KMO) is emerging as an important drug target enzyme in a number of inflammatory and neurodegenerative disease states. Recombinant protein production of KMO, and therefore discovery of KMO ligands, is challenging due to a large membrane targeting domain at the C-terminus of the enzyme that causes stability, solubility, and purification difficulties. The purpose of our investigation was to develop a suitable screening method for targeting human KMO and other similarly challenging drug targets. Here, we report the development of a magnetic bead-based binding assay using mass spectrometry detection for human KMO protein. The assay incorporates isolation of FLAG-tagged KMO enzyme on protein A magnetic beads. The protein-bound beads are incubated with potential binding compounds before specific cleavage of the protein-compound complexes from the beads. Mass spectrometry analysis is used to identify the compounds that demonstrate specific binding affinity for the target protein. The technique was validated using known inhibitors of KMO. This assay is a robust alternative to traditional ligand-binding assays for challenging protein targets, and it overcomes specific difficulties associated with isolating human KMO. © 2014 Society for Laboratory Automation and Screening.

Isolation and characterization of styrene metabolism genes from styrene-assimilating soil bacteria Rhodococcus sp. ST-5 and ST-10.

PubMed

Toda, Hiroshi; Itoh, Nobuya

2012-01-01

Styrene metabolism genes were isolated from styrene-assimilating bacteria Rhodococcus sp. ST-5 and ST-10. Strain ST-5 had a gene cluster containing four open reading frames which encoded styrene degradation enzymes. The genes showed high similarity to styABCD of Pseudomonas sp. Y2. On the other hand, strain ST-10 had only two genes which encoded styrene monooxygenase and flavin oxidoreductase (styAB). Escherichia coli transformants possessing the sty genes of strains ST-5 and ST-10 produced (S)-styrene oxide from styrene, indicating that these genes function as styrene degradation enzymes. Metabolite analysis by resting-cell reaction with gas chromatography-mass spectrometry revealed that strain ST-5 converts styrene to phenylacetaldehyde via styrene oxide by styrene oxide isomerase (styC) reaction. On the other hand, strain ST-10 lacked this enzyme, and thus accumulated styrene oxide as an intermediate. HPLC analysis showed that styrene oxide was spontaneously isomerized to phenylacetaldehyde by chemical reaction. The produced phenylacetaldehyde was converted to phenylacetic acid (PAA) in strain ST-10 as well as in strain ST-5. Furthermore, phenylacetic acid was converted to phenylacetyl-CoA by the catalysis of phenylacetate-CoA ligase in strains ST-5 and ST-10. This study proposes possible styrene metabolism pathways in Rhodococcus sp. strains ST-5 and ST-10. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Expression profiles of phases 1 and 2 metabolizing enzymes in human skin and the reconstructed skin models Episkin and full thickness model from Episkin.

PubMed

Luu-The, Van; Duche, Daniel; Ferraris, Corinne; Meunier, Jean-Roch; Leclaire, Jacques; Labrie, Fernand

2009-09-01

Episkin and full thickness model from Episkin (FTM) are human skin models obtained from in vitro growth of keratinocytes into the five typical layers of the epidermis. FTM is a full thickness reconstructed skin model that also contains fibroblasts seeded in a collagen matrix. To assess whether enzymes involved in chemical detoxification are expressed in Episkin and FTM and how their levels compare with the human epidermis, dermis and total skin. Quantification of the mRNA expression levels of phases 1 and 2 metabolizing enzymes in cultured Episkin and FTM and human epidermis, dermis and total skin using Realtime PCR. The data show that the expression profiles of 61 phases 1 and 2 metabolizing enzymes in Episkin, FTM and epidermis are generally similar, with some exceptions. Cytochrome P450-dependent enzymes and flavin monooxygenases are expressed at low levels, while phase 2 metabolizing enzymes are expressed at much higher levels, especially, glutathione-S-transferase P1 (GSTP1) catechol-O-methyl transferase (COMT), steroid sulfotransferase (SULT2B1b), and N-acetyl transferase (NAT5). The present study also identifies the presence of many enzymes involved in cholesterol, arachidonic acid, leukotriene, prostaglandin, eicosatrienoic acids, and vitamin D3 metabolisms. The present data strongly suggest that Episkin and FTM represent reliable and valuable in vitro human skin models for studying the function of phases 1 and 2 metabolizing enzymes in xenobiotic metabolisms. They could be used to replace invasive methods or laboratory animals for skin experiments.

Functional Analysis of Genes for Biosynthesis of Pyocyanin and Phenazine-1-Carboxamide from Pseudomonas aeruginosa PAO1

PubMed Central

Mavrodi, Dmitri V.; Bonsall, Robert F.; Delaney, Shannon M.; Soule, Marilyn J.; Phillips, Greg; Thomashow, Linda S.

2001-01-01

Two seven-gene phenazine biosynthetic loci were cloned from Pseudomonas aeruginosa PAO1. The operons, designated phzA1B1C1D1E1F1G1 and phzA2B2C2D2E2F2G2, are homologous to previously studied phenazine biosynthetic operons from Pseudomonas fluorescens and Pseudomonas aureofaciens. Functional studies of phenazine-nonproducing strains of fluorescent pseudomonads indicated that each of the biosynthetic operons from P. aeruginosa is sufficient for production of a single compound, phenazine-1-carboxylic acid (PCA). Subsequent conversion of PCA to pyocyanin is mediated in P. aeruginosa by two novel phenazine-modifying genes, phzM and phzS, which encode putative phenazine-specific methyltransferase and flavin-containing monooxygenase, respectively. Expression of phzS alone in Escherichia coli or in enzymes, pyocyanin-nonproducing P. fluorescens resulted in conversion of PCA to 1-hydroxyphenazine. P. aeruginosa with insertionally inactivated phzM or phzS developed pyocyanin-deficient phenotypes. A third phenazine-modifying gene, phzH, which has a homologue in Pseudomonas chlororaphis, also was identified and was shown to control synthesis of phenazine-1-carboxamide from PCA in P. aeruginosa PAO1. Our results suggest that there is a complex pyocyanin biosynthetic pathway in P. aeruginosa consisting of two core loci responsible for synthesis of PCA and three additional genes encoding unique enzymes involved in the conversion of PCA to pyocyanin, 1-hydroxyphenazine, and phenazine-1-carboxamide. PMID:11591691

1H NMR study of the hetero-association of flavin-mononucleotide with mutagenic dyes: ethidium bromide and proflavine

NASA Astrophysics Data System (ADS)

Evstigneev, M. P.; Mukhina, Yu. V.; Davies, D. B.

The hetero-association of the vitamin B2 derivative, flavin-mononucleotide (FMN), with a mutagenic dye, ethidium bromide (EB) or proflavine (PF), has been studied by 1D and 2D 500 MHz 1H NMR spectroscopy. The variations of proton chemical shifts of both the vitamin and dye as a function of concentration and temperature were analysed in terms of the structural and thermodynamical properties of the FMN-EB and FMN-PF complexes in solution. The structures of the complexes were also investigated by observed intermolecular ROE contacts and molecular mechanics calculations. The results show that the 1 : 1 hetero-association complexes in solution are more stable than the self-association complexes, which is consistent with formation of an intermolecular hydrogen-bond in the hetero-complexes of FMN-EB and FMN-PF. Hence it is possible that the toxicity of aromatic molecules such as EB and PF may be reduced in vitro by the presence of FMN, partly because of the known antimutagenic action of FMN and partly because it has been shown in this work that there is an effective intermolecular association between the mutagens and the vitamin.

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.

Detection and identification of 1-methylethyl and methyl radicals generated by irradiating tea tree (Melaleuca alternifolia) oil with visible light (436 nm) in the presence of flavin mononucleotide and ferrous ion.

PubMed

Mori, H-M; Iwahashi, H

2013-08-01

Here, we determined the electron spin resonance (ESR) spectra of standard reaction mixtures (I) containing 25 μM flavin mononucleotide (FMN), 0.018% tea tree (Melaleuca alternifolia) oil, 1.9 M acetonitrile, 20 mM phosphate buffer (pH 7.4), 0.1 M α-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN), and 1.0 mM FeSO₄(NH₄)₂SO₄ irradiated with 436 nm visible light (7.8 J/cm²). Prominent ESR signals (αN = 1.58 mT and αHβ = 0.26 mT) were detected, suggesting that free radicals form in the standard reaction. In order to know whether singlet oxygen (¹O₂) is involved in the radical formation or not, ESR measurement was performed for the standard D₂O reaction mixture (I) which contained 25 μM FMN, 0.0036% tea tree oil, 1.9 M acetonitrile-d3, 20 mM phosphate buffer (pH 7.4), 0.1 M 4-POBN and 1.0 mM FeSO₄ in D₂O. The ESR peak height of the standard D₂O reaction increased to 169 ± 24% of the control. Thus, ¹O₂ seems to be involved in the formation of the radicals because D₂O increases the lifetime of singlet oxygen. High-performance liquid chromatography-ESR-mass spectrometry analyses detected 1-methylethyl and methyl radicals in the standard reaction. The radicals appear to form through the reaction of ferrous ion with α-terpinene endoperoxide (ascaridole), which generated from the reaction of α-terpinene with ¹O₂. The 1-methylethyl and methyl radicals may exert a pro-oxidant effect under these conditions.

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.

Kynurenine 3-monooxygenase mediates inhibition of Th17 differentiation via catabolism of endogenous aryl hydrocarbon receptor ligands.

PubMed

Stephens, Geoffrey L; Wang, Qun; Swerdlow, Bonnie; Bhat, Geetha; Kolbeck, Roland; Fung, Michael

2013-07-01

The aryl hydrocarbon receptor (AhR) is a key transcriptional regulator of Th17-cell differentiation. Although endogenous ligands have yet to be identified, evidence suggests that tryptophan metabolites can act as agonists for the AhR. Tryptophan metabolites are abundant in circulation, so we hypothesized that cell intrinsic factors might exist to regulate the exposure of Th17 cells to AhR-dependent activities. Here, we find that Th17 cells preferentially express kynurenine 3-monooxygenase (KMO), which is an enzyme involved in catabolism of the tryptophan metabolite kynurenine. KMO inhibition, either with a specific inhibitor or via siRNA-mediated silencing, markedly increased IL-17 production in vitro, whereas IFN-γ production by Th1 cells was unaffected. Inhibition of KMO significantly exacerbated disease in a Th17-driven model of autoimmune gastritis, suggesting that expression of KMO by Th17 cells serves to limit their continuous exposure to physiological levels of endogenous AhR ligands in vivo. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Osmotic Stress Induces Expression of Choline Monooxygenase in Sugar Beet and Amaranth1

PubMed Central

Russell, Brenda L.; Rathinasabapathi, Bala; Hanson, Andrew D.

1998-01-01

Choline monooxygenase (CMO) catalyzes the committing step in the synthesis of glycine betaine, an osmoprotectant accumulated by many plants in response to salinity and drought. To investigate how these stresses affect CMO expression, a spinach (Spinacia oleracea L., Chenopodiaceae) probe was used to isolate CMO cDNAs from sugar beet (Beta vulgaris L., Chenopodiaceae), a salt- and drought-tolerant crop. The deduced beet CMO amino acid sequence comprised a transit peptide and a 381-residue mature peptide that was 84% identical (97% similar) to that of spinach and that showed the same consensus motif for coordinating a Rieske-type [2Fe-2S] cluster. A mononuclear Fe-binding motif was also present. When water was withheld, leaf relative water content declined to 59% and the levels of CMO mRNA, protein, and enzyme activity rose 3- to 5-fold; rewatering reversed these changes. After gradual salinization (NaCl:CaCl2 = 5.7:1, mol/mol), CMO mRNA, protein, and enzyme levels in leaves increased 3- to 7-fold at 400 mm salt, and returned to uninduced levels when salt was removed. Beet roots also expressed CMO, most strongly when salinized. Salt-inducible CMO mRNA, protein, and enzyme activity were readily detected in leaves of Amaranthus caudatus L. (Amaranthaceae). These data show that CMO most probably has a mononuclear Fe center, is inducibly expressed in roots as well as in leaves of Chenopodiaceae, and is not unique to this family. PMID:9489025

A Lytic Polysaccharide Monooxygenase with Broad Xyloglucan Specificity from the Brown-Rot Fungus Gloeophyllum trabeum and Its Action on Cellulose-Xyloglucan Complexes

PubMed Central

Kojima, Yuka; Várnai, Anikó; Ishida, Takuya; Sunagawa, Naoki; Petrovic, Dejan M.; Igarashi, Kiyohiko; Jellison, Jody; Goodell, Barry; Alfredsen, Gry; Westereng, Bjørge

2016-01-01

ABSTRACT Fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-rot fungi, such as Gloeophyllum trabeum, tend to have few LPMOs, and information on these enzymes is scarce. The genome of G. trabeum encodes four auxiliary activity 9 (AA9) LPMOs (GtLPMO9s), whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants of GtLPMO9A seem to be produced, a single-domain variant, GtLPMO9A-1, and a longer variant, GtLPMO9A-2, which contains a C-terminal domain comprising approximately 55 residues without a predicted function. We have overexpressed the phylogenetically distinct GtLPMO9A-2 in Pichia pastoris and investigated its properties. Standard analyses using high-performance anion-exchange chromatography–pulsed amperometric detection (HPAEC-PAD) and mass spectrometry (MS) showed that GtLPMO9A-2 is active on cellulose, carboxymethyl cellulose, and xyloglucan. Importantly, compared to other known xyloglucan-active LPMOs, GtLPMO9A-2 has broad specificity, cleaving at any position along the β-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic viscosity measurements to compare the hemicellulolytic action of GtLPMO9A-2 to that of a well-characterized hemicellulolytic LPMO, NcLPMO9C from Neurospora crassa revealed that GtLPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurements also revealed minor activity on glucomannan that could not be detected by the analysis of soluble products by HPAEC-PAD and MS and that was lower than the activity of NcLPMO9C. Experiments with copolymeric substrates showed an inhibitory effect of hemicellulose coating on cellulolytic LPMO activity and did not reveal additional activities of GtLPMO9A-2. These results provide insight into the LPMO potential of G. trabeum and provide a novel sensitive method, a measurement of dynamic viscosity, for monitoring LPMO activity. IMPORTANCE Currently, there are

A Lytic Polysaccharide Monooxygenase with Broad Xyloglucan Specificity from the Brown-Rot Fungus Gloeophyllum trabeum and Its Action on Cellulose-Xyloglucan Complexes.

PubMed

Kojima, Yuka; Várnai, Anikó; Ishida, Takuya; Sunagawa, Naoki; Petrovic, Dejan M; Igarashi, Kiyohiko; Jellison, Jody; Goodell, Barry; Alfredsen, Gry; Westereng, Bjørge; Eijsink, Vincent G H; Yoshida, Makoto

2016-11-15

Fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (LPMOs) to degrade plant polysaccharides. Brown-rot fungi, such as Gloeophyllum trabeum, tend to have few LPMOs, and information on these enzymes is scarce. The genome of G. trabeum encodes four auxiliary activity 9 (AA9) LPMOs (GtLPMO9s), whose coding sequences were amplified from cDNA. Due to alternative splicing, two variants of GtLPMO9A seem to be produced, a single-domain variant, GtLPMO9A-1, and a longer variant, GtLPMO9A-2, which contains a C-terminal domain comprising approximately 55 residues without a predicted function. We have overexpressed the phylogenetically distinct GtLPMO9A-2 in Pichia pastoris and investigated its properties. Standard analyses using high-performance anion-exchange chromatography-pulsed amperometric detection (HPAEC-PAD) and mass spectrometry (MS) showed that GtLPMO9A-2 is active on cellulose, carboxymethyl cellulose, and xyloglucan. Importantly, compared to other known xyloglucan-active LPMOs, GtLPMO9A-2 has broad specificity, cleaving at any position along the β-glucan backbone of xyloglucan, regardless of substitutions. Using dynamic viscosity measurements to compare the hemicellulolytic action of GtLPMO9A-2 to that of a well-characterized hemicellulolytic LPMO, NcLPMO9C from Neurospora crassa revealed that GtLPMO9A-2 is more efficient in depolymerizing xyloglucan. These measurements also revealed minor activity on glucomannan that could not be detected by the analysis of soluble products by HPAEC-PAD and MS and that was lower than the activity of NcLPMO9C. Experiments with copolymeric substrates showed an inhibitory effect of hemicellulose coating on cellulolytic LPMO activity and did not reveal additional activities of GtLPMO9A-2. These results provide insight into the LPMO potential of G. trabeum and provide a novel sensitive method, a measurement of dynamic viscosity, for monitoring LPMO activity. Currently, there are only a few methods

Evaluating cytochrome p450 in lesser scaup (Aythya affinis) and tree swallow (Tachycineta bicolor) by monooxygenase activity and immunohistochemistry: Possible nonlethal assessment by skin immunohistochemistry

USGS Publications Warehouse

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

2006-01-01

Six-month-old lesser scaup (Aythya affinis) and nestling tree swallows (Tachycineta bicolor) were injected intraperitoneally with beta-naphthoflavone (BNF) in corn oil or in vehicle alone. Liver samples were taken and stored at -80 degrees C until microsome preparation and monooxygenase assay. Skin samples were placed in buffered formalin for subsequent immunohistochemical (IHC) analysis for cytochrome P4501A (CYP1A). Lesser scaup treated with BNF at 20 or 100 mg/kg body weight showed approximately 6- to 18-fold increases in four monooxygenases (benzyloxyresorufin-O-dealkylase, ethoxyresorufin-O-dealkylase, methoxyresorufin-O-dealkylase, and pentoxyresorufin-O-dealkylase). No IHC response was observed for CYP1A in the skin of vehicle-injected ducks, whereas in the skin from BNF-treated ducks, the positive IHC response was of similar magnitude for both dose levels of BNF. Tree swallows injected with BNF at 100 mg/kg, but not at. 20 mg/kg, showed significant increases (approximately fivefold) in hepatic microsomal O-dealkylase activities. Cytochrome P4501A was undetectable by IHC response in skin from corn oil-treated swallows, but positive IHC responses were observed in the skin of one of five swallows at 20 mg/kg and four of five swallows at 100 mg/kg. Although these data do not allow construction of significant dose-response curves, the IHC responses for CYP1A in skin support the possible use of this nonlethal approach for biomonitoring contaminant exposure of birds. In addition, the CYP1A signal observed at the bases of emerging feathers suggest that these might provide less invasive sampling sites for IHC analysis of CYP1A.

Plasmonic bio-sensing for the Fenna-Matthews-Olson complex

NASA Astrophysics Data System (ADS)

Chen, Guang-Yin; Lambert, Neill; Shih, Yen-An; Liu, Meng-Han; Chen, Yueh-Nan; Nori, Franco

2017-01-01

We study theoretically the bio-sensing capabilities of metal nanowire surface plasmons. As a specific example, we couple the nanowire to specific sites (bacteriochlorophyll) of the Fenna-Matthews-Olson (FMO) photosynthetic pigment protein complex. In this hybrid system, we find that when certain sites of the FMO complex are subject to either the suppression of inter-site transitions or are entirely disconnected from the complex, the resulting variations in the excitation transfer rates through the complex can be monitored through the corresponding changes in the scattering spectra of the incident nanowire surface plasmons. We also find that these changes can be further enhanced by changing the ratio of plasmon-site couplings. The change of the Fano lineshape in the scattering spectra further reveals that “site 5” in the FMO complex plays a distinct role from other sites. Our results provide a feasible way, using single photons, to detect mutation-induced, or bleaching-induced, local defects or modifications of the FMO complex, and allows access to both the local and global properties of the excitation transfer in such systems.

In vitro activation of ammonia monooxygenase from Nitrosomonas europaea by copper.

PubMed Central

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

1993-01-01

The effect of copper on the in vivo and in vitro activity of ammonia monooxygenase (AMO) from the nitrifying bacterium Nitrosomonas europaea was investigated. The addition of CuCl2 to cell extracts resulted in 5- to 15-fold stimulation of ammonia-dependent O2 consumption, ammonia-dependent nitrite production, and hydrazine-dependent ethane oxidation. AMO activity was further stimulated in vitro by the presence of stabilizing agents, including serum albumins, spermine, or MgCl2. In contrast, the addition of CuCl2 and stabilizing agents to whole-cell suspensions did not result in any stimulation of AMO activity. The use of the AMO-specific suicide substrate acetylene revealed two populations of AMO in cell extracts. The low, copper-independent (residual) AMO activity was completely inactivated by acetylene in the absence of exogenously added copper. In contrast, the copper-dependent (activable) AMO activity was protected against acetylene inactivation in the absence of copper. However, in the presence of copper both populations of AMO were inactivated by acetylene. [14C]acetylene labelling of the 27-kDa polypeptide of AMO revealed the same extent of label incorporation in both whole cells and optimally copper-stimulated cell extracts. In the absence of copper, the label incorporation in cell extracts was proportional to the level of residual AMO activity. Other metal ions tested, including Zn2+, Co2+, Ni2+, Fe2+, Fe3+, Ca2+, Mg2+, Mn2+, Cr3+, and Ag+, were ineffective at stimulating AMO activity or facilitating the incorporation of 14C label from [14C]acetylene into the 27-kDa polypeptide. On the basis of these results, we propose that loss of AMO activity upon lysis of N. europaea results from the loss of copper from AMO, generating a catalytically inactive, yet stable and activable, form of the enzyme. Images PMID:8458839

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.

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

Novel hydrocarbon monooxygenase genes in the metatranscriptome of a natural deep-sea hydrocarbon plume.

PubMed

Li, Meng; Jain, Sunit; Baker, Brett J; Taylor, Chris; Dick, Gregory J

2014-01-01

Particulate membrane-associated hydrocarbon monooxygenases (pHMOs) are critical components of the aerobic degradation pathway for low molecular weight hydrocarbons, including the potent greenhouse gas methane. Here, we analysed pHMO gene diversity in metagenomes and metatranscriptomes of hydrocarbon-rich hydrothermal plumes in the Guaymas Basin (GB) and nearby background waters in the deep Gulf of California. Seven distinct phylogenetic groups of pHMO were present and transcriptionally active in both plume and background waters, including several that are undetectable with currently available polymerase chain reaction (PCR) primers. The seven groups of pHMOs included those related to a putative ethane oxidizing Methylococcaceae-like group, a group of the SAR324 Deltaproteobacteria, three deep-sea clades (Deep sea-1/symbiont-like, Deep sea-2/PS-80 and Deep sea-3/OPU3) within gammaproteobacterial methanotrophs, one clade related to Group Z and one unknown group. Differential abundance of pHMO gene transcripts in plume and background suggests niche differentiation between groups. Corresponding 16S rRNA genes reflected similar phylogenetic and transcriptomic abundance trends. The novelty of transcriptionally active pHMOs we recovered from a hydrocarbon-rich hydrothermal plume suggests there are significant gaps in our knowledge of the diversity and function of these enzymes in the environment. © 2013 Society for Applied Microbiology and John Wiley & Sons Ltd.

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.

Development of a Surface Plasmon Resonance Assay for the Characterization of Small-Molecule Binding Kinetics and Mechanism of Binding to Kynurenine 3-Monooxygenase.

PubMed

Poda, Suresh B; Kobayashi, Masakazu; Nachane, Ruta; Menon, Veena; Gandhi, Adarsh S; Budac, David P; Li, Guiying; Campbell, Brian M; Tagmose, Lena

2015-10-01

Kynurenine 3-monooxygenase (KMO), a pivotal enzyme in the kynurenine pathway, was identified as a potential therapeutic target for treating neurodegenerative and psychiatric disorders. In this article, we describe a surface plasmon resonance (SPR) assay that delivers both kinetics and the mechanism of binding (MoB) data, enabling a detailed characterization of KMO inhibitors for the enzyme in real time. SPR assay development included optimization of the protein construct and the buffer conditions. The stability and inhibitor binding activity of the immobilized KMO were significantly improved when the experiments were performed at 10°C using a buffer containing 0.05% n-dodecyl-β-d-maltoside (DDM) as the detergent. The KD values of the known KMO inhibitors (UPF648 and RO61-8048) from the SPR assay were in good accordance with the biochemical LC/MS/MS assay. Also, the SPR assay was able to differentiate the binding kinetics (k(a) and k(d)) of the selected unknown KMO inhibitors. For example, the inhibitors that showed comparable IC50 values in the LC/MS/MS assay displayed differences in their residence time (τ = 1/k(d)) in the SPR assay. To better define the MoB of the inhibitors to KMO, an SPR-based competition assay was developed, which demonstrated that both UPF648 and RO61-8048 bound to the substrate-binding site. These results demonstrate the potential of the SPR assay for characterizing the affinity, the kinetics, and the MoB profiles of the KMO inhibitors.

Structural and molecular dynamics studies of a C1-oxidizing lytic polysaccharide monooxygenase from Heterobasidion irregulare reveal amino acids important for substrate recognition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Bing; Kognole, Abhishek A.; Wu, Miao

Lytic polysaccharide monooxygenases (LPMOs) are a group of recently discovered enzymes that play important roles in the decomposition of recalcitrant polysaccharides. Here, we report the biochemical, structural, and computational characterization of an LPMO from the white-rot fungus Heterobasidion irregulare (HiLPMO9B). This enzyme oxidizes cellulose at the C1 carbon of glycosidic linkages. The crystal structure of HiLPMO9B was determined at 2.1 A resolution using X-ray crystallography. Unlike the majority of the currently available C1-specific LPMO structures, the HiLPMO9B structure contains an extended L2 loop, connecting ..beta..-strands ..beta..2 and ..beta..3 of the ..beta..-sandwich structure. Molecular dynamics (MD) simulations suggest roles for bothmore » aromatic and acidic residues in the substrate binding of HiLPMO9B, with the main contribution from the residues located on the extended region of the L2 loop (Tyr20) and the LC loop (Asp205, Tyr207, and Glu210). Asp205 and Glu210 were found to be involved in the hydrogen bonding with the hydroxyl group of the C6 carbon of glucose moieties directly or via a water molecule. Two different binding orientations were observed over the course of the MD simulations. In each orientation, the active-site copper of this LPMO preferentially skewed toward the pyranose C1 of the glycosidic linkage over the targeted glycosidic bond. This study provides additional insight into cellulose binding by C1-specific LPMOs, giving a molecular-level picture of active site substrate interactions.« less

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

A Modified Shuttle Plasmid Facilitates Expression of a Flavin Mononucleotide-Based Fluorescent Protein in Treponema denticola ATCC 35405

PubMed Central

Godovikova, Valentina; Goetting-Minesky, M. Paula; Shin, Jae M.; Kapila, Yvonne L.; Rickard, Alexander H.

2015-01-01

Oral pathogens, including Treponema denticola, initiate the dysregulation of tissue homeostasis that characterizes periodontitis. However, progress of research on the roles of T. denticola in microbe-host interactions and signaling, microbial communities, microbial physiology, and molecular evolution has been hampered by limitations in genetic methodologies. This is typified by an extremely low transformation efficiency and inability to transform the most widely studied T. denticola strain with shuttle plasmids. Previous studies have suggested that robust restriction-modification (R-M) systems in T. denticola contributed to these problems. To facilitate further molecular genetic analysis of T. denticola behavior, we optimized existing protocols such that shuttle plasmid transformation efficiency was increased by >100-fold over prior reports. Here, we report routine transformation of T. denticola ATCC 35405 with shuttle plasmids, independently of both plasmid methylation status and activity of the type II restriction endonuclease encoded by TDE0911. To validate the utility of this methodological advance, we demonstrated expression and activity in T. denticola of a flavin mononucleotide-based fluorescent protein (FbFP) that is active under anoxic conditions. Addition of routine plasmid-based fluorescence labeling to the Treponema toolset will enable more-rigorous and -detailed studies of the behavior of this organism. PMID:26162875

Tirandamycin biosynthesis is mediated by co-dependent oxidative enzymes

NASA Astrophysics Data System (ADS)

Carlson, Jacob C.; Li, Shengying; Gunatilleke, Shamila S.; Anzai, Yojiro; Burr, Douglas A.; Podust, Larissa M.; Sherman, David H.

2011-08-01

Elucidation of natural product biosynthetic pathways provides important insights into the assembly of potent bioactive molecules, and expands access to unique enzymes able to selectively modify complex substrates. Here, we show full reconstitution, in vitro, of an unusual multi-step oxidative cascade for post-assembly-line tailoring of tirandamycin antibiotics. This pathway involves a remarkably versatile and iterative cytochrome P450 monooxygenase (TamI) and a flavin adenine dinucleotide-dependent oxidase (TamL), which act co-dependently through the repeated exchange of substrates. TamI hydroxylates tirandamycin C (TirC) to generate tirandamycin E (TirE), a previously unidentified tirandamycin intermediate. TirE is subsequently oxidized by TamL, giving rise to the ketone of tirandamycin D (TirD), after which a unique exchange back to TamI enables successive epoxidation and hydroxylation to afford, respectively, the final products tirandamycin A (TirA) and tirandamycin B (TirB). Ligand-free, substrate- and product-bound crystal structures of bicovalently flavinylated TamL oxidase reveal a likely mechanism for the C10 oxidation of TirE.

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.

Neutron and Atomic Resolution X-ray Structures of a Lytic Polysaccharide Monooxygenase Reveal Copper-Mediated Dioxygen Binding and Evidence for N-Terminal Deprotonation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bacik, John-Paul; Mekasha, Sophanit; Forsberg, Zarah

A 1.1 Å resolution, room-temperature X-ray structure and a 2.1 Å resolution neutron structure of a chitin-degrading lytic polysaccharide monooxygenase domain from the bacterium Jonesia denitrificans (JdLPMO10A) show a putative dioxygen species equatorially bound to the active site copper. We found that both structures show an elongated density for the dioxygen, most consistent with a Cu(II)-bound peroxide. The coordination environment is consistent with Cu(II). Furthermore, in the neutron and X-ray structures, difference maps reveal the N-terminal amino group, involved in copper coordination, is present as a mixed ND 2 and ND –, suggesting a role for the copper ion inmore » shifting the pK a of the amino terminus.« less

Neutron and Atomic Resolution X-ray Structures of a Lytic Polysaccharide Monooxygenase Reveal Copper-Mediated Dioxygen Binding and Evidence for N-Terminal Deprotonation

DOE PAGES

Bacik, John-Paul; Mekasha, Sophanit; Forsberg, Zarah; ...

2017-05-08

A 1.1 Å resolution, room-temperature X-ray structure and a 2.1 Å resolution neutron structure of a chitin-degrading lytic polysaccharide monooxygenase domain from the bacterium Jonesia denitrificans (JdLPMO10A) show a putative dioxygen species equatorially bound to the active site copper. We found that both structures show an elongated density for the dioxygen, most consistent with a Cu(II)-bound peroxide. The coordination environment is consistent with Cu(II). Furthermore, in the neutron and X-ray structures, difference maps reveal the N-terminal amino group, involved in copper coordination, is present as a mixed ND 2 and ND –, suggesting a role for the copper ion inmore » shifting the pK a of the amino terminus.« less

A Xylenol Orange-Based Screening Assay for the Substrate Specificity of Flavin-Dependent para-Phenol Oxidases.

PubMed

Ewing, Tom A; van Noord, Aster; Paul, Caroline E; van Berkel, Willem J H

2018-01-14

Vanillyl alcohol oxidase (VAO) and eugenol oxidase (EUGO) are flavin-dependent enzymes that catalyse the oxidation of para -substituted phenols. This makes them potentially interesting biocatalysts for the conversion of lignin-derived aromatic monomers to value-added compounds. To facilitate their biocatalytic exploitation, it is important to develop methods by which variants of the enzymes can be rapidly screened for increased activity towards substrates of interest. Here, we present the development of a screening assay for the substrate specificity of para -phenol oxidases based on the detection of hydrogen peroxide using the ferric-xylenol orange complex method. The assay was used to screen the activity of VAO and EUGO towards a set of twenty-four potential substrates. This led to the identification of 4-cyclopentylphenol as a new substrate of VAO and EUGO and 4-cyclohexylphenol as a new substrate of VAO. Screening of a small library of VAO and EUGO active-site variants for alterations in their substrate specificity led to the identification of a VAO variant (T457Q) with increased activity towards vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) and a EUGO variant (V436I) with increased activity towards chavicol (4-allylphenol) and 4-cyclopentylphenol. This assay provides a quick and efficient method to screen the substrate specificity of para -phenol oxidases, facilitating the enzyme engineering of known para- phenol oxidases and the evaluation of the substrate specificity of novel para -phenol oxidases.

Nonphotochemical Hole-Burning Studies of Energy Transfer Dynamics in Antenna Complexes of Photosynthetic Bacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matsuzaki, Satoshi

2001-01-01

This thesis contains the candidate's original work on excitonic structure and energy transfer dynamics of two bacterial antenna complexes as studied using spectral hole-burning spectroscopy. The general introduction is divided into two chapters (1 and 2). Chapter 1 provides background material on photosynthesis and bacterial antenna complexes with emphasis on the two bacterial antenna systems related to the thesis research. Chapter 2 reviews the underlying principles and mechanism of persistent nonphotochemical hole-burning (NPHB) spectroscopy. Relevant energy transfer theories are also discussed. Chapters 3 and 4 are papers by the candidate that have been published. Chapter 3 describes the application ofmore » NPHB spectroscopy to the Fenna-Matthews-Olson (FMO) complex from the green sulfur bacterium Prosthecochloris aestuarii; emphasis is on determination of the low energy vibrational structure that is important for understanding the energy transfer process associated within three lowest energy Q y-states of the complex. The results are compared with those obtained earlier on the FMO complex from Chlorobium tepidum. In Chapter 4, the energy transfer dynamics of the B800 molecules of intact LH2 and B800-deficient LH2 complexes of the purple bacterium Rhodopseudomonas acidophila are compared. New insights on the additional decay channel of the B800 ring of bacteriochlorophyll a (BChl a) molecules are provided. General conclusions are given in Chapter 5. A version of the hole spectrum simulation program written by the candidate for the FMO complex study (Chapter 3) is included as an appendix. The references for each chapter are given at the end of each chapter.« less

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

Learning from oligosaccharide soaks of crystals of an AA13 lytic polysaccharide monooxygenase: crystal packing, ligand binding and active-site disorder.

PubMed

Frandsen, Kristian E H; Poulsen, Jens Christian Navarro; Tovborg, Morten; Johansen, Katja S; Lo Leggio, Leila

2017-01-01

Lytic polysaccharide monooxygenases (LPMOs) are a class of copper-dependent enzymes discovered within the last ten years. They oxidatively cleave polysaccharides (chitin, lignocellulose, hemicellulose and starch-derived), presumably making recalcitrant substrates accessible to glycoside hydrolases. Recently, the first crystal structure of an LPMO-substrate complex was reported, giving insights into the interaction of LPMOs with β-linked substrates (Frandsen et al., 2016). The LPMOs acting on α-linked glycosidic bonds (family AA13) display binding surfaces that are quite different from those of LPMOs that act on β-linked glycosidic bonds (families AA9-AA11), as revealed from the first determined structure (Lo Leggio et al., 2015), and thus presumably the AA13s interact with their substrate in a distinct fashion. Here, several new structures of the same AA13 enzyme, Aspergillus oryzae AA13, are presented. Crystals obtained in the presence of high zinc-ion concentrations were used, as they can be obtained more reproducibly than those used to refine the deposited copper-containing structure. One structure with an ordered zinc-bound active site was solved at 1.65 Å resolution, and three structures from crystals soaked with maltooligosaccharides in solutions devoid of zinc ions were solved at resolutions of up to 1.10 Å. Despite similar unit-cell parameters, small rearrangements in the crystal packing occur when the crystals are depleted of zinc ions, resulting in a more occluded substrate-binding surface. In two of the three structures maltooligosaccharide ligands are bound, but not at the active site. Two of the structures presented show a His-ligand conformation that is incompatible with metal-ion binding. In one of these structures this conformation is the principal one (80% occupancy), giving a rare atomic resolution view of a substantially misfolded enzyme that is presumably rendered inactive.

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

Pharmacokinetics, Metabolism, and Excretion of the Antiviral Drug Arbidol in Humans

PubMed Central

Deng, Pan; Zhong, Dafang; Yu, Kate; Zhang, Yifan; Wang, Ting

2013-01-01

Arbidol is a broad-spectrum antiviral drug that is used clinically to treat influenza. In this study, the pharmacokinetics, metabolism, and excretion of arbidol were investigated in healthy male Chinese volunteers after a single oral administration of 200 mg of arbidol hydrochloride. A total of 33 arbidol metabolites were identified in human plasma, urine, and feces. The principal biotransformation pathways included sulfoxidation, dimethylamine N-demethylation, glucuronidation, and sulfate conjugation. The major drug-related component in the plasma was sulfinylarbidol (M6-1), followed by unmetabolized arbidol, N-demethylsulfinylarbidol (M5), and sulfonylarbidol (M8). The exposures of M5, M6-1, and M8, as determined by the metabolite-to-parent area under the plasma concentration-time curve from 0 to t (AUC0-t) ratio, were 0.9 ± 0.3, 11.5 ± 3.6, and 0.5 ± 0.2, respectively. In human urine, glucuronide and sulfate conjugates were detected as the major metabolites, accounting for 6.3% of the dose excreted within 0 to 96 h after drug administration. The fecal specimens mainly contained the unchanged arbidol, accounting for 32.4% of the dose. Microsomal incubation experiments demonstrated that the liver and intestines were the major organs that metabolize arbidol in humans. CYP3A4 was the major isoform involved in arbidol metabolism, whereas the other P450s and flavin-containing monooxygenases (FMOs) played minor roles. These results indicated possible drug interactions between arbidol and CYP3A4 inhibitors and inducers. Further investigations are needed to understand the importance of M6-1 in the efficacy and safety of arbidol, because of its high plasma exposure and long elimination half-life (25.0 h). PMID:23357765

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.

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

PubMed

Syed, Khajamohiddin; Porollo, Aleksey; Lam, Ying Wai; Grimmett, Paul E; Yadav, Jagjit S

2013-04-01

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 found to possess a broad oxidizing capability toward structurally diverse hydrocarbons belonging to mutagenic/carcinogenic fused-ring higher-molecular-weight polycyclic aromatic hydrocarbons (HMW-PAHs), endocrine-disrupting long-chain alkylphenols (APs), and crude oil aliphatic hydrocarbon n-alkanes. A homology-based three-dimensional (3D) model revealed the presence of an extraordinarily large active-site cavity in CYP63A2 compared to the mammalian PAH-oxidizing (CYP3A4, CYP1A2, and CYP1B1) and bacterial aliphatic-hydrocarbon-oxidizing (CYP101D and CYP102A1) P450s. This structural feature in conjunction with ligand docking simulations suggested potential versatility of the enzyme. Experimental characterization using recombinantly expressed CYP63A2 revealed its ability to oxidize HMW-PAHs of various ring sizes, including 4 rings (pyrene and fluoranthene), 5 rings [benzo(a)pyrene], and 6 rings [benzo(ghi)perylene], with the highest enzymatic activity being toward the 5-ring PAH followed by the 4-ring and 6-ring PAHs, in that order. Recombinant CYP63A2 activity yielded monohydroxylated PAH metabolites. The enzyme was found to also act as an alkane ω-hydroxylase that oxidized n-alkanes with various chain lengths (C9 to C12 and C15 to C19), as well as alkyl side chains (C3 to C9) in alkylphenols (APs). CYP63A2 showed preferential oxidation of long-chain APs and alkanes. To our knowledge, this is the first P450 identified from any of the biological kingdoms that possesses such broad substrate specificity toward structurally diverse xenobiotics (PAHs, APs, and alkanes), making it a potent enzyme biocatalyst candidate to handle mixed pollution (e.g., crude oil spills).

CYP63A2, a Catalytically Versatile Fungal P450 Monooxygenase Capable of Oxidizing Higher-Molecular-Weight Polycyclic Aromatic Hydrocarbons, Alkylphenols, and Alkanes

PubMed Central

Syed, Khajamohiddin; Porollo, Aleksey; Lam, Ying Wai; Grimmett, Paul E.

2013-01-01

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 found to possess a broad oxidizing capability toward structurally diverse hydrocarbons belonging to mutagenic/carcinogenic fused-ring higher-molecular-weight polycyclic aromatic hydrocarbons (HMW-PAHs), endocrine-disrupting long-chain alkylphenols (APs), and crude oil aliphatic hydrocarbon n-alkanes. A homology-based three-dimensional (3D) model revealed the presence of an extraordinarily large active-site cavity in CYP63A2 compared to the mammalian PAH-oxidizing (CYP3A4, CYP1A2, and CYP1B1) and bacterial aliphatic-hydrocarbon-oxidizing (CYP101D and CYP102A1) P450s. This structural feature in conjunction with ligand docking simulations suggested potential versatility of the enzyme. Experimental characterization using recombinantly expressed CYP63A2 revealed its ability to oxidize HMW-PAHs of various ring sizes, including 4 rings (pyrene and fluoranthene), 5 rings [benzo(a)pyrene], and 6 rings [benzo(ghi)perylene], with the highest enzymatic activity being toward the 5-ring PAH followed by the 4-ring and 6-ring PAHs, in that order. Recombinant CYP63A2 activity yielded monohydroxylated PAH metabolites. The enzyme was found to also act as an alkane ω-hydroxylase that oxidized n-alkanes with various chain lengths (C9 to C12 and C15 to C19), as well as alkyl side chains (C3 to C9) in alkylphenols (APs). CYP63A2 showed preferential oxidation of long-chain APs and alkanes. To our knowledge, this is the first P450 identified from any of the biological kingdoms that possesses such broad substrate specificity toward structurally diverse xenobiotics (PAHs, APs, and alkanes), making it a potent enzyme biocatalyst candidate to handle mixed pollution (e.g., crude oil spills). PMID:23416995

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%. Copyright © 2016 Elsevier Inc. All rights reserved.

Catalytic diversity and homotropic allostery of two Cytochrome P450 monooxygenase like proteins from Trichoderma brevicompactum.

PubMed

Hussain, Razak; Kumari, Indu; Sharma, Shikha; Ahmed, Mushtaq; Khan, Tabreiz Ahmad; Akhter, Yusuf

2017-12-01

Trichothecenes are the secondary metabolites produced by Trichoderma spp. Some of these molecules have been reported for their ability to stimulate plant growth by suppressing plant diseases and hence enabling Trichoderma spp. to be efficiently used as biocontrol agents in modern agriculture. Many of the proteins involved in the trichothecenes biosynthetic pathway in Trichoderma spp. are encoded by the genes present in the tri cluster. Tri4 protein catalyzes three consecutive oxygenation reaction steps during biosynthesis of isotrichodiol in the trichothecenes biosynthetic pathway, while tri11 protein catalyzes the C4 hydroxylation of 12, 13-epoxytrichothec-9-ene to produce trichodermol. In the present study, we have homology modelled the three-dimensional structures of tri4 and tri11 proteins. Furthermore, molecular dynamics simulations were carried out to elucidate the mechanism of their action. Both tri4 and tri11 encode for cytochrome P450 monooxygenase like proteins. These data also revealed effector-induced allosteric changes on substrate binding at an alternative binding site and showed potential homotropic negative cooperativity. These analyses also showed that their catalytic mechanism relies on protein-ligand and protein-heme interactions controlled by hydrophobic and hydrogen-bonding interactions which orient the complex in optimal conformation within the active sites.

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

Studies on the neuroprotective action of kynurenine mono-oxygenase inhibitors in post-ischemic brain damage.

PubMed

Moroni, Flavio; Carpenedo, Raffaella; Cozzi, Andrea; Meli, Elena; Chiarugi, Alberto; Pellegrini-Giampietro, Domenico E

2003-01-01

Kynurenine 3-mono-oxygenase (KMO) inhibitors facilitate kynurenic acid (KYNA) neosynthesis and reduce the formation of 3OH-kynurenine (3-HK) and quinolinic acid (QUIN). They also attenuate post-ischemic brain damage and decrease glutamate (Glu) content in brain extracellular spaces. To investigate KMO mechanism(s) of neuroprotection, we performed experiments in gerbils subjected to bilateral carotid occlusion and in organotypic rat hippocampal slice cultures exposed to oxygen and glucose deprivation (OGD). In gerbils, direct application of KYNA (100 nM, through reverse microdialysis in the hippocampus) completely prevented the increase in Glu output induced by transient (5 min) occlusion of the carotids. In rat hippocampal slices exposed for 30 min to OGD, KMO inhibitors (m-nitrobenzoyl)-alanine (mNBA, 30-100 microM) or 3,4-dimethoxy-[-N-4-(nitrophenyl)thiazol-2yl]-benzenesulfonamide (Ro 61-8048, 1-10 microM) reduced post-ischemic neuronal death and increased KYNA concentrations in the incubation medium. KYNA may antagonize glycineb or alpha7 nicotinic acetylcholine receptors but the concentrations in the incubation medium never reached values that could efficiently antagonize receptor function. On the contrary, 3-HK (1-10 microM) added to slices exposed to OGD in the presence of KMO inhibitors completely prevented the neuroprotective effects of the inhibitors. Our findings suggest that KMO inhibitors reduce OGD-induced pyramidal cell death by decreasing 3-HK (and possibly QUIN) synthesis.

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

Enterococcus faecium WEFA23 from infant lessens high-fat-diet-induced hyperlipidemia via cholesterol 7-alpha-hydroxylase gene by altering the composition of gut microbiota in rats.

PubMed

Huang, Fuqing; Zhang, Fen; Xu, Di; Zhang, Zhihong; Xu, Feng; Tao, Xueying; Qiu, Liang; Wei, Hua

2018-06-20

Enterococcus faecium WEFA23 is a potential probiotic strain from Chinese infants with the ability to decrease cholesterol levels. Aiming to explore the mechanism of E. faecium WEFA23 in lowering cholesterol in vivo, we examined the gene transcriptions related to cholesterol metabolism, the composition of bile acids in feces, the synthesis of trimethylamine N-oxide (TMAO) in liver, and the composition of the gut microbiota of rats. We found that E. faecium WEFA23 enhanced the synthesis of bile acids by promoting cholesterol excretion, upregulating the genes transcript level relevant to cholesterol decomposition and transportation, and downregulating the genes involved in cholesterol synthesis. In addition, E. faecium WEFA23 not only downregulated the transcript levels of farnesoid X receptor and fibroblast growth factor 15 as well as flavin-containing monooxygenase 3, but also decreased the TMAO production followed by increasing the CYP7A1 transcript level. Furthermore, when orally administered to rats for 35 d, E. faecium WEFA23 improved the gut microbiota diversity of rats fed a high-fat diet. Therein, the ratio of Bacteroidetes to Firmicutes and the abundance of Rikenellaceae increased, whereas the number of Veillonellaceae decreased. These results suggest that reduction of cholesterol level by E. faecium WEFA23 might be related to the changes in the gut microbiota. Our finding provides important information on lowering cholesterol by E. faecium and reveals that Enterococcus spp. might have the potential to decrease the TMAO level. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Effect of tree thinning and skidding trails on hydrological connectivity in two Japanese forest catchments

NASA Astrophysics Data System (ADS)

López-Vicente, Manuel; Sun, Xinchao; Onda, Yuichi; Kato, Hiroaki; Gomi, Takashi; Hiraoka, Marino

2017-09-01

Land use composition and patterns influence the hydrological response in mountainous and forest catchments. In plantation forest, management operations (FMO) modify the spatial and temporal dynamics of overland flow processes. However, we found a gap in the literature focussed on modelling hydrological connectivity (HC) in plantation forest under different FMO. In this study, we simulated HC in two steep paired forest subcatchments (K2 and K3, 33.2 ha), composed of Japanese cypress (Chamaecyparis obtusa Endl.) and Japanese cedar (Cryptomeria japonica D. Don) plantations (59% of the total area) against a tree thinning intensity of 50% at different time. Additionally, construction of new skidding trails and vegetation recovery was simulated on five thinning-based scenarios that covered a 40-month test period (July 2010 - October 2013). As a future scenario, six check-dams located in the main streams were proposed to reduce sediment and radionuclide delivery. An updated version of Borselli's index of runoff and sediment connectivity was run, using the D-infinity flow accumulation algorithm and exploiting three 0.5-m resolution digital elevation models. On the basis of the pre-FMO scenario, HC increased at catchment scale owing to tree thinning and the new skidding trails. This change was more noticeable within the area affected by the FMO, where HC increased by 11.4% and 10.5% in the cypress and cedar plantations in K2 respectively and by 8.8% in the cedar plantation in K3. At hillslope plot and stream scales, the evolution in the values of HC was less evident, except the increment (by 5.4%) observed in the streams at K2 after the FMO. Progressive vegetation recovery after the FMO triggered a slight reduction of connectivity in all compartments of both subcatchments. Forest roads and especially skidding trails presented the highest values of HC, appearing as the most efficient features connecting the different vegetation patches with the stream network. The spatial