Oxidative decarboxylation of tris-(p-carboxyltetrathiaaryl)methyl radical EPR probes by peroxidases and related hemeproteins: Intermediate formation and characterization of the corresponding cations

2010 ◽  
Vol 502 (1) ◽  
pp. 74-80 ◽  
Author(s):  
Christophe Decroos ◽  
Yun Li ◽  
Asma Soltani ◽  
Yves Frapart ◽  
Daniel Mansuy ◽  
...  
Keyword(s):  
Intermediate Formation ◽  
Oxidative Decarboxylation ◽  
Methyl Radical

scholarly journals Characterization of an archaeal malic enzyme from the hyperthermophilic archaeonThermococcus kodakaraensisKOD1

Archaea ◽  
2005 ◽  
Vol 1 (5) ◽  
pp. 293-301 ◽  
Author(s):  
Wakao Fukuda ◽  
Yulia Sari Ismail ◽  
Toshiaki Fukui ◽  
Haruyuki Atomi ◽  
Tadayuki Imanaka
Keyword(s):  
Enzyme Activity ◽  
Malic Enzyme ◽  
Hyperthermophilic Archaea ◽  
Oxidative Decarboxylation ◽  
Limited Information ◽  
Gene Encoding ◽  
Malic Enzyme Activity ◽  
Reductive Carboxylation ◽  
Reaction Direction

Although the interconversion between C4 and C3 compounds has an important role in overall metabolism, limited information is available on the properties and regulation of enzymes acting on these metabolites in hyperthermophilic archaea. Malic enzyme is one of the enzymes involved in this interconversion, catalyzing the oxidative decarboxylation of malate to pyruvate as well as the reductive carboxylation coupled with NAD(P)H. This study focused on the enzymatic properties and expression profile of an uncharacterized homolog of malic enzyme identified in the genome of a heterotrophic, hyperthermophilic archaeonT hermococcus kodakaraensisKOD1 (Tk-Mae). The amino acid sequence ofTk-Mae was 52–58% identical to those of malic enzymes from bacteria, whereas the similarities to the eukaryotic homologs were lower. Several catalytically important regions and residues were conserved in the primary structure ofTk-Mae. The recombinant protein, which formed a homodimer, exhibited thermostable malic enzyme activity with strict divalent cation dependency. The enzyme preferred NADP+rather than NAD+, but did not catalyze the decarboxylation of oxaloacetate, unlike the usual NADP-dependent malic enzymes. The apparent Michaelis constant (Km) ofTk-Mae for malate (16.9 mM) was much larger than those of known enzymes, leading to no strong preference for the reaction direction. Transcription of the gene encodingTk-Mae and intracellular malic enzyme activity inT. kodakaraensiswere constitutively weak, regardless of the growth substrates. Possible roles ofTk-Mae are discussed based on these results and the metabolic pathways ofT. kodakaraensisdeduced from the genome sequence.

scholarly journals Capture of micrococcin biosynthetic intermediates reveals C-terminal processing as an obligatory step for in vivo maturation

2016 ◽  
Vol 113 (44) ◽  
pp. 12450-12455 ◽  
Author(s):  
Kathryn D. Bewley ◽  
Philip R. Bennallack ◽  
Mark A. Burlingame ◽  
Richard A. Robison ◽  
Joel S. Griffitts ◽  
...  
Keyword(s):  
Phase Ii ◽  
Oxidative Decarboxylation ◽  
Affinity Tag ◽  
Bioactive Natural Products ◽  
Precursor Peptide ◽  
Processing Step ◽  
Substrate Peptide ◽  
Processing Steps

Thiopeptides, including micrococcins, are a growing family of bioactive natural products that are ribosomally synthesized and heavily modified. Here we use a refactored, modular in vivo system containing the micrococcin P1 (MP1) biosynthetic genes (TclIJKLMNPS) from Macrococcus caseolyticus str 115 in a genetically tractable Bacillus subtilis strain to parse the processing steps of this pathway. By fusing the micrococcin precursor peptide to an affinity tag and coupling it with catalytically defective enzymes, biosynthetic intermediates were easily captured for analysis. We found that two major phases of molecular maturation are separated by a key C-terminal processing step. Phase-I conversion of six Cys residues to thiazoles (TclIJN) is followed by C-terminal oxidative decarboxylation (TclP). This TclP-mediated oxidative decarboxylation is a required step for the peptide to progress to phase II. In phase II, Ser/Thr dehydration (TclKL) and peptide macrocycle formation (TclM) occurs. A C-terminal reductase, TclS, can optionally act on the substrate peptide, yielding MP1, and is shown to act late in the pathway. This comprehensive characterization of the MP1 pathway prepares the way for future engineering efforts.

scholarly journals Dynamics of cetyltrimethylammonium bromide mediated reaction of phenylsulfinylacetic acid with Cr(VI): Treatment of pseudo-phase models

2015 ◽  
Vol 80 (8) ◽  
pp. 1019-1034 ◽  
Author(s):  
Perumal Subramaniam ◽  
Thamil Selvi
Keyword(s):  
Cetyltrimethylammonium Bromide ◽  
Initial Rate ◽  
Intermediate Formation ◽  
Oxidative Decarboxylation ◽  
Micellar Phase ◽  
Cooperative Model ◽  
Rate Acceleration ◽  
Rate Retardation ◽  
Phase Models ◽  
Rate Profile

micellar effect % Hammett correlation % Piszkiewicz cooperative model KR nema The influence of cetyltrimethylammonium bromide, CTon the oxidative decarboxylation of phenylsulfinylacetic acid, PSAA and several meta- and para-substituted PSAAs by Cr(VI) was investigated in 95 % H2O-5 % CH3CN medium. The rate profile displayed a peculiar trend with an initial rate increase at low [CTAB] followed by sharp rate inhibition at higher [CTAB]. The initial rate acceleration can be explained by strong binding of SO42- on the positively charged micellar surface. The specific partitioning of PSAA in micellar phase by hydrophobic interaction and the oxidizing species, HCrO3+ in aqueous phase by electrostatic repulsion accounted the rate retardation at higher [CTAB]. The Hammett plot with different substituted PSAAs showed an excellent correlation affording negative ? value which supports the proposed mechanism involving a sulfonium cation intermediate formation. The obtained ? value in CTmedium is found to be slightly lower than that in aqueous medium. The quantitative analysis of rate data for the inhibition shown by CTwas performed using Menger-Portnoy and Piszkiewicz pseudo-phase models. The binding constant for PSAA with micelle was evaluated from Piszkiewicz cooperative model.

scholarly journals Purification and Characterization of (Per)Chlorate Reductase from the Chlorate-Respiring Strain GR-1

1999 ◽  
Vol 181 (21) ◽  
pp. 6706-6711 ◽  
Author(s):  
Servé W. M. Kengen ◽  
Geoffrey B. Rikken ◽  
Wilfred R. Hagen ◽  
Cees G. van Ginkel ◽  
Alfons J. M. Stams
Keyword(s):  
Electron Paramagnetic Resonance Spectroscopy ◽  
Bacterial Species ◽  
Intermediate Formation ◽  
Resonance Spectroscopy ◽  
Purification And Characterization ◽  
Paramagnetic Resonance ◽  
Terminal Electron Acceptor ◽  
Key Enzyme ◽  
Chlorate Reductase

ABSTRACT Strain GR-1 is one of several recently isolated bacterial species that are able to respire by using chlorate or perchlorate as the terminal electron acceptor. The organism performs a complete reduction of chlorate or perchlorate to chloride and oxygen, with the intermediate formation of chlorite. This study describes the purification and characterization of the key enzyme of the reductive pathway, the chlorate and perchlorate reductase. A single enzyme was found to catalyze both the chlorate- and perchlorate-reducing activity. The oxygen-sensitive enzyme was located in the periplasm and had an apparent molecular mass of 420 kDa, with subunits of 95 and 40 kDa in an α3β3 composition. Metal analysis showed the presence of 11 mol of iron, 1 mol of molybdenum, and 1 mol of selenium per mol of heterodimer. In accordance, quantitative electron paramagnetic resonance spectroscopy showed the presence of one [3Fe-4S] cluster and two [4Fe-4S] clusters. Furthermore, two different signals were ascribed to Mo(V). The Km values for perchlorate and chlorate were 27 and <5 μM, respectively. Besides perchlorate and chlorate, nitrate, iodate, and bromate were also reduced at considerable rates. The resemblance of the enzyme to nitrate reductases, formate dehydrogenases, and selenate reductase is discussed.

Characterization of 3-isopropylmalate dehydrogenase from extremely halophilic archaeon Haloarcula japonica

2021 ◽  
Author(s):  
Shintaro Nagaoka ◽  
Noriko Sugiyama ◽  
Rie Yatsunami ◽  
Satoshi Nakamura
Keyword(s):  
Analytical Ultracentrifugation ◽  
Maximum Activity ◽  
Oxidative Decarboxylation ◽  
Halophilic Archaeon ◽  
Leucine Biosynthesis ◽  
First Report ◽  
Haloarcula Japonica ◽  
Extremely Halophilic Archaeon ◽  
Isopropylmalate Dehydrogenase

Abstract 3-Isopropylmalate dehydrogenase (IPMDH) catalyzes oxidative decarboxylation of (2R, 3S)-3-isopropylmalate to 2-oxoisocaproate in leucine biosynthesis. In this study, recombinant IPMDH (HjIPMDH) from an extremely halophilic archaeon, Haloarcula japonica TR-1, was characterized. Activity of HjIPMDH increased as KCl concentration increased, and the maximum activity was observed at 3.0 M KCl. Analytical ultracentrifugation revealed that HjIPMDH formed a homotetramer at high KCl concentrations, and it dissociated to a monomer at low KCl concentrations. Additionally, HjIPMDH was thermally stabilized by higher KCl concentrations. This is the first report on haloarchaeal IPMDH.

A DFT STUDY ON THE REACTION MECHANISM OF SrO + CH4

2008 ◽  
Vol 07 (02) ◽  
pp. 189-203 ◽  
Author(s):  
SONG QIN ◽  
HUA-QING YANG ◽  
SONG QIN ◽  
JIAN XU ◽  
DIAN-YONG TANG ◽  
...  
Keyword(s):  
Reaction Mechanism ◽  
Molecular Complex ◽  
Reaction Pathway ◽  
Intermediate Formation ◽  
Methanol Conversion ◽  
The Other ◽  
Main Reaction ◽  
Methyl Radical ◽  
Formation Pathway ◽  
Bond Forming

The reaction mechanism of SrO + CH 4 has been investigated on a singlet spin state potential energy surface at the B3LYP/SDD, AUG-cc-pVQZ//B3LYP/SDD, 6-311+G(2df, 2p) levels of theory. Initially, the reactants give two molecule–molecule complexes: (i) a collinear C – H approach to O -end of the Sr – O bond forming SrOCH 4 molecular complex with C 3v symmetry, and (ii) a side-on to side-on approach of C – H bond to Sr – O forming OSrCH 4 molecular complex with C 1 symmetry. From SrOCH 4, the SrOH + CH 3 radicals are formed by the direct abstraction of a hydrogen atom from CH 4 moiety. From OSrCH 4, the gas-phase methane to methanol conversion by SrO is suggested to involve the SrO insertion into a C – H bond of CH 4 to produce the hydroxy and methoxy intermediates, HOSrCH 3 and HSrOCH 3, and the reaction pathway via the hydroxy intermediate ( HOSrCH 3) is energetically more favorable than the other one via the methoxy intermediate ( HSrOCH 3). These channels to form ( SrOH + CH 3) and ( Sr + CH 3 OH ) are expected to compete with each other, and the formation of methyl radical via the direct Sr – C cleavage from HOSrCH 3 is energetically more preferable. On the other hand, the intermediates HSrOCH 3 and HOSrCH 3 are predicted to be the energetically preferred configuration in the reaction of Sr + CH 3 OH , which is precisely the reverse reaction of methane hydroxylation. For the reaction of MO ( M = Mg , Ca , Sr ) with CH 4, the two main reaction pathways, methyl formation via a-TS1 and hydroxyl intermediate formation via b-TS1, are expected to compete with each other, and the preference of methyl formation pathway via a-TS1 would follow the sequence: MgO > CaO > SrO .

scholarly journals 4-Methyl-2-oxopentanoate oxidation by rat skeletal-muscle mitochondria

1979 ◽  
Vol 182 (2) ◽  
pp. 353-360 ◽  
Author(s):  
V W M Van Hinsbergh ◽  
J H Veerkamp ◽  
J F C Glatz
Keyword(s):  
Skeletal Muscle ◽  
Intermediate Formation ◽  
Inhibitory Effect ◽  
Oxidative Decarboxylation ◽  
Rat Skeletal Muscle ◽  
Mitochondrial Inner Membrane ◽  
Skeletal Muscle Mitochondria ◽  
Intact Muscle ◽  
Km Value ◽  
Branched Chain

1. Oxidative decarboxylation of 4-methyl-2-oxopentanoate (2-oxoisocaproate) by mitochondria of rat skeletal muscle showed biphasic kinetics. Two apparent Km values of 9.1 micronM and 0.78 mM were established. In broken mitochondria the rate of oxidation was lower and only the higher apparent Km value was found. 2. Isovalerylcarnitine inhibited 4-methyl-2-oxopentanoate oxidation in the presence and absence of carnitine, but isovaleryl-CoA had no inhibitory effect. 3. Addition of ADP enhanced 4-methyl-2-oxopentanolate oxidation. Malate, succinate and 2-oxoglutarate additionally increased the rate of oxidation, but in the absence of ADP succinate and 2-oxoglutarate inhibited. 4. Addition of rotenone and simultaneous addition of carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP) and valinomycin markedly decreased 4-methyl-2-oxopentanoate oxidation. 5. These observations indicate that the branched-chain 2-oxo acid dehydrogenase complex is situated on the inner side of the mitochondrial inner membrane. 6. In mitochondria and homogenates CO2 was only produced by oxidative decarbosylation of 4-methyl-2-oxopentanoate. In intact muscle oxidation of this oxo acid proceeds more to completeness. 7. The physiological significance of intermediate formation during oxidation of branched-chain amino acids is discussed.

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