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Nigel S. Scrutton - One of the best experts on this subject based on the ideXlab platform.
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deuterium isotope effects during carbon hydrogen bond cleavage by Trimethylamine dehydrogenase implications for mechanism and vibrationally assisted hydrogen tunneling in wild type and mutant enzymes
Journal of Biological Chemistry, 2001Co-Authors: Jaswir Basran, Michael J Sutcliffe, Nigel S. ScruttonAbstract:His-172 and Tyr-169 are components of a triad in the active site of Trimethylamine dehydrogenase (TMADH) comprising Asp-267, His-172, and Tyr-169. Stopped-flow kinetic studies with Trimethylamine as substrate have indicated that mutation of His-172 to Gln reduces the limiting rate constant for flavin reduction ∼10-fold (Basran, J., Sutcliffe, M. J., Hille, R., and Scrutton, N. S. (1999) Biochem. J. 341, 307–314). A kinetic isotope effect (KIE =k H/k D) accompanies flavin reduction by H172Q TMADH, the magnitude of which varies significantly with solution pH. With Trimethylamine, flavin reduction by H172Q TMADH is controlled by a single macroscopic ionization (pK a = 6.8 ± 0.1). This ionization is perturbed (pK a = 7.4 ± 0.1) in reactions with perdeuterated Trimethylamine and is responsible for the apparent variation in the KIE with solution pH. At pH 9.5, where the functional group controlling flavin reduction is fully ionized, the KIE is independent of temperature in the range 277–297 K, consistent with vibrationally assisted hydrogen tunneling during breakage of the substrate C–H bond. Y169F TMADH is ∼4-fold more compromised than H172Q TMADH for hydrogen transfer, which occurs non-classically. Studies with Y169F TMADH suggest partial thermal excitation of substrate prior to hydrogen tunneling by a vibrationally assisted mechanism. Our studies illustrate the varied effects of compromising mutations on tunneling regimes in enzyme molecules.
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Trimethylamine dehydrogenase and electron transferring flavoprotein
Sub-cellular biochemistry, 2000Co-Authors: Nigel S. Scrutton, Michael J SutcliffeAbstract:Many biological electron transfer reactionsoe.g. harnessing solar energy, metabolism, defence against toxic compounds and pathogensorely on the coexistence of protein-protein complexes in dissociation equilibrium with their constitutive reactants. The mechanism of electron transfer between weakly associating electron transfer partners has been the focus of intensive research activity in recent years. An in-depth understanding of these reactions requires knowledge of the role of protein dynamics in complex assembly, and the geometries of the complex that are compatible with interprotein electron transfer. Knowledge of the role of electrostatics in guiding complex assembly during a diffusional encounter is central to our understanding of electron transfer processes between redox partners. The role of electrostatics, and that of other interactions (e.g. hydrophobic), in maintaining complex structure is also of central importance in maximizing electronic coupling between neighbouring redox centers. In addition to the
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involvement of a flavin iminoquinone methide in the formation of 6 hydroxyflavin mononucleotide in Trimethylamine dehydrogenase a rationale for the existence of 8alpha methyl and c6 linked covalent flavoproteins
Biochemistry, 1997Co-Authors: Martin Mewies, Jaswir Basran, Leonard C Packman, Russ Hille, Nigel S. ScruttonAbstract:In Trimethylamine dehydrogenase, substrate is bound in the active site via cation−π bonding to three aromatic residues (Tyr-60, Trp-264, and Trp-355). Mutation of one of these residues (Trp-355 → Leu, mutant W355L) influences the chemistry of the flavin mononucleotide in the active site, enabling derivatization to 6-hydroxy-FMN. The W355L mutant is purified as a mixture of deflavo, natural 6-S-cysteinyl-FMN, and inactive 6-hydroxy-FMN forms, and the enzyme is severely compromised in its ability to oxidatively demethylate Trimethylamine. Analysis of samples of the native and recombinant wild-type Trimethylamine dehydrogenases also revealed the presence of 6-hydroxy-FMN, but at much reduced levels compared with that of the W355L enzyme. Unlike that for a C30A mutant of Trimethylamine dehydrogenase, addition of substrate to the W355L Trimethylamine dehydrogenase is not required for the production of 6-hydroxy-FMN. A mechanism is proposed for the 6-hydroxylation of FMN in Trimethylamine dehydrogenase that inv...
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assembly of redox centers in the Trimethylamine dehydrogenase of bacterium w3a1 properties of the wild type enzyme and a c30a mutant expressed from a cloned gene in escherichia coli
Journal of Biological Chemistry, 1994Co-Authors: Nigel S. Scrutton, Leonard C Packman, F S Mathews, R J Rohlfs, Russ HilleAbstract:Abstract In Trimethylamine dehydrogenase, the enzyme-bound FMN is covalently linked to Cys-30 by a 6-S-cysteinyl FMN bond. The role played by this bond in catalysis has been investigated using a recombinant wild-type Trimethylamine dehydrogenase and a Cys-30 to Ala-30 mutant, both expressed from a cloned gene (tmd) in the heterologous host Escherichia coli. The recombinant wild-type and C30A enzymes were found to be quantitatively associated with the 4Fe-4S center and ADP which are both present in the enzyme isolated from bacterium W3A1. In contrast to the enzyme isolated from bacterium W3A1, however, both recombinant proteins contained less than stoichiometric amounts of flavin and were refractory to reconstitution by FMN. The FMN in the recombinant wild-type enzyme was shown to be covalently linked to the protein, and the enzyme possessed catalytic properties similar to its counterpart isolated from bacterium W3A1. It is envisaged that flavinylation proceeds via a nucleophilic attack by the thiolate of Cys-30 at C-6 of the isoalloxazine ring of enzyme-bound FMN. The C30A mutant was found to bind FMN noncovalently and to also catalyze the demethylation of Trimethylamine. The major effect of removing the 6-S-cysteinyl FMN bond is to raise the apparent Km for Trimethylamine by 2 orders of magnitude and to diminish the apparent kcat for the reaction by only a factor of 2. Therefore, the 6-S-cysteinyl FMN bond is not essential for catalysis, but it is required for efficient functioning of the enzyme at micromolar concentrations of substrate.
Marjaliisa Riekkola - One of the best experts on this subject based on the ideXlab platform.
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modified zeolitic imidazolate framework 8 as solid phase microextraction arrow coating for sampling of amines in wastewater and food samples followed by gas chromatography mass spectrometry
Journal of Chromatography A, 2017Co-Authors: Hangzhen Lan, Tuukka Ronkko, Jevgeni Parshintsev, Kari Hartonen, Ning Gan, Motolani Sakeye, Jawad Sarfraz, Marjaliisa RiekkolaAbstract:Abstract In this study, a novel solid phase microextration (SPME) Arrow was prepared for the sampling of volatile low molecular weight alkylamines (Trimethylamine (TMA) and triethylamine (TEA)) in wastewater, salmon and mushroom samples before gas chromatographic separation with mass spectrometer as detector. Acidified zeolitic imidazolate framework-8 (A-ZIF-8) was utilized as adsorbent and poly(vinyl chloride) (PVC) as the adhesive. The custom SPME Arrow was fabricated via a physical adhesion: (1) ZIF-8 particles were suspended in a mixture of tetrahydrofuran (THF) and PVC to form a homogeneous suspension, (2) a non-coated stainless steel SPME Arrow was dipped in the ZIF-8/PVC suspension for several times to obtain a uniform and thick coating, (3) the pore size of ZIF-8 was modified by headspace exposure to hydrochloric acid in order to increase the extraction efficiency for amines. The effect of ZIF-8 concentration in PVC solution, dipping cycles and aging temperature on extraction efficiency was investigated. In addition, sampling parameters such as NaCl concentration, sample volume, extraction time, potassium hydroxide concentration, desorption temperature and desorption time were optimized. The Arrow-to-Arrow reproducibilities (RSDs) for five ZIF-8 coated Arrows were 15.6% and 13.3% for TMA and TEA, respectively. The extraction with A-ZIF-8/PVC Arrow was highly reproducible for at least 130 cycles without noticeable decrease of performance (RSD −1 for both TMA and TEA. The limit of quantitation (LOQ) was 1 ng mL −1 for both TMA and TEA. The method was successfully applied to the determination of TMA and TEA in wastewater, salmon and mushroom samples giving satisfactory selectivity towards the studied amines.
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solid phase microextraction arrow for the sampling of volatile amines in wastewater and atmosphere
Journal of Chromatography A, 2015Co-Authors: Aku Helin, Tuukka Ronkko, Jevgeni Parshintsev, Kari Hartonen, Beat Schilling, Thomas Laubli, Marjaliisa RiekkolaAbstract:A new method is introduced for the sampling of volatile low molecular weight alkylamines in ambient air and wastewater by utilizing a novel SPME Arrow system, which contains a larger volume of sorbent compared to a standard SPME fiber. Parameters affecting the extraction, such as coating material, need for preconcentration, sample volume, pH, stirring rate, salt addition, extraction time and temperature were carefully optimized. In addition, analysis conditions, including desorption temperature and time as well as gas chromatographic parameters, were optimized. Compared to conventional SPME fiber, the SPME Arrow had better robustness and sensitivity. Average intermediate reproducibility of the method expressed as relative standard deviation was 12% for dimethylamine and 14% for Trimethylamine, and their limit of quantification 10μg/L and 0.13μg/L respectively. Working range was from limits of quantification to 500μg/L for dimethylamine and to 130μg/L for Trimethylamine. Several alkylamines were qualitatively analyzed in real samples, while target compounds dimethyl- and Trimethylamines were quantified. The concentrations in influent and effluent wastewater samples were almost the same (∼80μg/L for dimethylamine, 120μg/L for Trimethylamine) meaning that amines pass the water purification process unchanged or they are produced at the same rate as they are removed. For the air samples, preconcentration with phosphoric acid coated denuder was required and the concentration of Trimethylamine was found to be around 1ng/m(3). The developed method was compared with optimized method based on conventional SPME and advantages and disadvantages of both approaches are discussed.
Jian Xiang - One of the best experts on this subject based on the ideXlab platform.
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shifts in methanogen community structure and function across a coastal marsh transect effects of exotic spartina alterniflora invasion
Scientific Reports, 2016Co-Authors: Junji Yuan, Weixin Ding, Hojeong Kang, Jian XiangAbstract:Invasion of Spartina alterniflora in coastal areas of China increased methane (CH4) emissions. To elucidate the underlying mechanisms, we measured CH4 production potential, methanogen community structure and biogeochemical factors along a coastal wetland transect comprised of five habitat regions: open water, bare tidal flat, invasive S. alterniflora marsh and native Suaeda salsa and Phragmites australis marshes. CH4 production potential in S. alterniflora marsh was 10 times higher than that in other regions, and it was significantly correlated with soil organic carbon, dissolved organic carbon and Trimethylamine concentrations, but was not correlated with acetate or formate concentrations. Although the diversity of methanogens was lowest in S. alterniflora marsh, invasion increased methanogen abundance by 3.48-fold, compared with native S. salsa and P. australis marshes due to increase of facultative Methanosarcinaceae rather than acetotrophic and hydrogenotrophic methanogens. Ordination analyses suggested that Trimethylamine was the primary factor regulating shift in methanogen community structure. Addition of Trimethylamine increased CH4 production rates by 1255-fold but only by 5.61- and 11.4-fold for acetate and H2/CO2, respectively. S. alterniflora invasion elevated concentration of non-competitive Trimethylamine, and shifted methanogen community from acetotrophic to facultative methanogens, which together facilitated increased CH4 production potential.
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methane production potential and methanogenic archaea community dynamics along the spartina alterniflora invasion chronosequence in a coastal salt marsh
Applied Microbiology and Biotechnology, 2014Co-Authors: Junji Yuan, Weixin Ding, Jian XiangAbstract:Invasion by the exotic species Spartina alterniflora, which has high net primary productivity and superior reproductive capacity compared with native plants, has led to rapid organic carbon accumulation and increased methane (CH4) emission in the coastal salt marsh of China. To elucidate the mechanisms underlying this effect, the methanogen community structure and CH4 production potential as well as soil organic carbon (SOC), dissolved organic carbon, dissolved organic acids, methylated amines, aboveground biomass, and litter mass were measured during the invasion chronosequence (0–16 years). The CH4 production potential in the S. alterniflora marsh (range, 2.94–3.95 μg kg−1 day−1) was significantly higher than that in the bare tidal mudflat. CH4 production potential correlated significantly with SOC, acetate, and Trimethylamine concentrations in the 0–20 cm soil layer. The abundance of methanogenic archaea also correlated significantly with SOC, and the dominant species clearly varied with S. alterniflora-driven SOC accumulation. The acetotrophic Methanosaetaceae family members comprised a substantial proportion of the methanogenic archaea in the bare tidal mudflat while Methanosarcinaceae family members utilized methylated amines as substrates in the S. alterniflora marsh. Ordination analysis indicated that Trimethylamine concentration was the primary factor inducing the shift in the methanogenic archaea composition, and regressive analysis indicated that the facultative family Methanosarcinaceae increased linearly with Trimethylamine concentration in the increasingly sulfate-rich salt marsh. Our results indicate that increased CH4 production during the S. alterniflora invasion chronosequence was due to increased levels of the non-competitive substrate Trimethylamine and a shift in the methanogenic archaea community.
Bogdan Zygmunt - One of the best experts on this subject based on the ideXlab platform.
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determination of volatile aliphatic amines in air by solid phase microextraction coupled with gas chromatography with flame ionization detection
Journal of Chromatography A, 2003Co-Authors: Jacek Namieśnik, Anna Jastrzebska, Bogdan ZygmuntAbstract:Practical aspects of the application of solid-phase microextraction (SPME) to the determination of volatile aliphatic amines in air are described. Analytes included methylamine (MA), ethylamine (EA), dimethylamine (DMA), diethylamine (DEA), Trimethylamine (TMA) and triethylamine (TEA). New SPME stationary phases were examined. The effects of relative humidity and temperature on analytes uptake were taken into account in analysis. Gas chromatography (GC) with flame ionization detector (FID) was used for the final analysis.
Emily P. Balskus - One of the best experts on this subject based on the ideXlab platform.
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Molecular Basis of C-N Bond Cleavage by the Glycyl Radical Enzyme Choline Trimethylamine-Lyase.
Cell chemical biology, 2016Co-Authors: Smaranda Bodea, Michael A. Funk, Emily P. Balskus, Catherine L. DrennanAbstract:Deamination of choline catalyzed by the glycyl radical enzyme choline Trimethylamine-lyase (CutC) has emerged as an important route for the production of Trimethylamine, a microbial metabolite associated with both human disease and biological methane production. Here, we have determined five high-resolution X-ray structures of wild-type CutC and mechanistically informative mutants in the presence of choline. Within an unexpectedly polar active site, CutC orients choline through hydrogen bonding with a putative general base, and through close interactions between phenolic and carboxylate oxygen atoms of the protein scaffold and the polarized methyl groups of the trimethylammonium moiety. These structural data, along with biochemical analysis of active site mutants, support a mechanism that involves direct elimination of Trimethylamine. This work broadens our understanding of radical-based enzyme catalysis and will aid in the rational design of inhibitors of bacterial Trimethylamine production.
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characterization of choline Trimethylamine lyase expands the chemistry of glycyl radical enzymes
ACS Chemical Biology, 2014Co-Authors: Smaranda Craciun, Jonathan A Marks, Emily P. BalskusAbstract:The recently identified glycyl radical enzyme (GRE) homologue choline Trimethylamine-lyase (CutC) participates in the anaerobic conversion of choline to Trimethylamine (TMA), a widely distributed microbial metabolic transformation that occurs in the human gut and is linked to disease. The proposed biochemical function of CutC, C–N bond cleavage, represents new reactivity for the GRE family. Here we describe the in vitro characterization of CutC and its activating protein CutD. We have observed CutD-mediated formation of a glycyl radical on CutC using EPR spectroscopy and have demonstrated that activated CutC processes choline to Trimethylamine and acetaldehyde. Surveys of potential alternate CutC substrates uncovered a strict specificity for choline. Homology modeling and mutagenesis experiments revealed essential CutC active site residues. Overall, this work establishes that CutC is a GRE of unique function and a molecular marker for anaerobic choline metabolism.
