Levels of coenzyme F420, coenzyme M, hydrogenase, and methylcoenzyme M methylreductase in acetate-grown Methanosarcina

Methanosarcina barkeri strain 227 maintained on an acetate medium for 2 years was found to possess hydrogenase, methylcoenzyme M methylreductase, coenzyme F420, and coenzyme M. The levels of these constituents in acetate-grown cells were similar to those found in cells of the same strain grown on methanol or hydrogen and carbon dioxide.     

Levels of coenzyme F420, coenzyme M, hydrogenase, and methylcoenzyme M methylreductase in acetate-grown Methanosarcina.

Methanosarcina barkeri strain 227 maintained on an acetate medium for 2 years was found to possess hydrogenase, methylcoenzyme M methylreductase, coenzyme F420, and coenzyme M. The levels of these constituents in acetate-grown cells were similar to those found in cells of the same strain grown on methanol or hydrogen and carbon dioxide.     

PQQ, the elusive coenzyme

The recently discovered redox coenzyme, PQQ (methoxatin), is widely distributed. Quantitation of protein-bound PQQ has been difficult, but unique redox cycling reactions, which reflect its striking biological properties, reveal trace amounts. PQQ is a potential target for drugs.     

Synthesis of acetyl coenzyme A from carbon monoxide, methyltetrahydrofolate, and coenzyme A by enzymes from Clostridium thermoaceticum

Two purified fractions from Clostridium thermoaceticum are shown to catalyze the following reaction: CO + CH3THF + CoA ATP leads to CH3COCoA + THF. The methyltetrahydrofolate (CH3THF) gives rise to the methyl group of the acetyl-coenzyme A (CoA) and the carbon monoxide (CO) and CoA to its carboxyl thio ester group. The role of ATP is unknown. One of the protein fractions (F2) is a methyltransferase, whereas the other fraction (F3) contains CO dehydrogenase and a methyl acceptor which is postulated to be a corrinoid enzyme. The methyltransferase catalyzes the transfer of the methyl group to the methyl acceptor, and the CO is converted to a formyl derivative by the CO dehydrogenase. By a mechanism that is as yet unknown, the formyl derivative in combination with CoA and the methyl of the methyl acceptor are converted to acetyl-CoA. It is also shown that fraction F3 catalyzes the reversible exchange of 14C from [1-14C]acetyl-CoA into 14CO and that ATP is required, but not the methyltransferase. It is proposed that these reactions are part of the mechanism which enables certain autotrophic bacteria to grow on CO. It is postulated that CH3THF is synthesized from CO and tetrahydrofolate which then, as described above, is converted to acetyl-CoA. The acetyl-CoA then serves as a precursor in other anabolic reactions. A similar autotropic pathway may occur in bacteria which grow on carbon dioxide and hydrogen.     

Levels of acetyl coenzyme A, reduced and oxidized coenzyme A, and coenzyme A in disulfide linkage to protein in dormant and germinated spores and growing and sporulating cells of Bacillus megaterium.

Dormant spores of Bacillus megaterium were found to contain approximately 850 pmol of coenzyme A (CoA) per milligram of dry weight. Of this total, less than 1.5% was acetyl-CoA, 25% was CoA-disulfide, 43% was in disulfide linkage to protein, and the remainder was the free thiol. Dormand spores of Bacillus cereus and Clostridium bifermentans contained 700 and 600 pmol of CoA per milligram of dry weight, respectively; in both species approximately 45% of the CoA 45% of the CoA was in disulfide linkage to protein. During germination of spores of all three species, greater than 75% of the CoA-protein disulfides were cleaved. In B. megaterium, cleavage of these disulfides during spore germination did not require exogenous metabolites and occurred at about the same time as the initiation of germination. Much of the CoA was converted to acetyl-CoA at this time. Dormant spores also contained reduced nicotinamide adenine dinucleotide-dependent CoA-disulfide reductase at levels higher than those in other stages of growth. The level of total CoA in the growing cells was two- to three-fold higher than in spores. This level remained constant throughout growth and sporulation, but less than 2% of the total cellular CoA was in disulfide linkage to protein until late in sporulation. The CoA-protein disulfides accumulated exclusively within the developing spore at about the time when dipicolinic acid was accumulated.     

Acetyl coenzyme A synthetase catalyzed reactions of coenzyme A with alpha, beta-unsaturated carboxylic acids

alpha, beta-Unsaturated coenzyme A (CoA) thioesters including acrylyl CoA, methacrylyl CoA, and propiolyl CoA were synthesized by catalysis with acetyl CoA synthetase (EC 6.2.1.1.). After isolation from the enzymatic reactions, the products were found to be the result of 1,4 addition of CoASH to the double bond and addition of water to the triple bond of the initial acyl CoA adducts. Structural determinations of these products by 1H NMR, 13C NMR, and the chemical reactions leading to their formation are described.     

L-methionine decarboxylase from Dryopteris filix-mas: purification, characterization, substrate specificity, abortive transamination of the coenzyme, and stereochemical courses of substrate decarboxylation and coenzyme transamination

L-Methionine decarboxylase from the male fern Dryopteris filix-mas has been purified 256-fold from acetone powder extracts to very near homogeneity. The enzyme is membrane-associated and requires detergent for solubilization during the initial extraction. The enzyme is a homodimer of subunit Mr 57,000 and shows a pH optimum at approximately 5.0 with 20 mM (2S)-methionine as substrate. The specific activity, kcat, for methionine is approximately 50 mol s(-1) (mol of active site)(-1) at pH 4.5 and below. A wide range of straight- and branched-chain (2S)-alkylamino acids are substrates for the enzyme. The values for the rate of decarboxylation, Vmax, and for the apparent Michaelis constant, Km, however, vary with structure and with the chirality at C-3. The pH dependence of V and V/K has been examined for three substrates: (2S)-methionine, valine, and leucine. Pyridoxal 5'-phosphate (PLP) is required for activity, and in the absence of excess PLP, the activity of the enzyme in incubations reduced with respect to time. The addition of PLP fully restores the activity, indicating that an abortive decarboxylation-transamination accompanies the normal decarboxylation reaction. The occurrence of the abortive reaction was confirmed by showing that [35S]methionine is converted to labeled 3-(methylthio)propionaldehyde while [4'-3H]PLP is converted to labeled pyridoxamine 5'-phosphate (PMP). The decarboxylation of (2S)-methionine gave 3-(methylthio)-1-aminopropane. Preparation of the N-camphanamide derivative of the amine allowed the C-1 methylene protons to be distinguished by 1H NMR spectroscopy. Synthetic samples of the camphanamide were prepared in which each of the C-1 methylene protons was replaced by deuterium. When (2S)-methionine and the C-2 deuteriated isotopomer were incubated with the enzyme in deuterium oxide and protium oxide, respectively, and the products were converted to their camphanamide derivatives and analyzed by 1H NMR spectroscopy, it was evident that decarboxylation occurred with retention of configuration at C-2. When the decarboxylation of six other substrates was studied, examination of the N-camphanamide derivatives of the amines indicated that decarboxylation occurred stereospecifically and, by analogy, with retention of configuration at C-2. When tritiated pyridoxal phosphate was incubated with the enzyme, tritiated pyridoxamine phosphate was formed. Analysis of the chirality of the methylene group at C-4' indicated that, during abortive transamination, protonation occurred from the 4'-si face of the coenzyme, the same stereochemical result as that obtained for several bona fide transaminase enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of coenzyme Q10 intake on endogenous coenzyme Q content, mitochondrial electron transport chain, antioxidative defenses, and life span of mice

The main purpose of this study was to determine whether intake of coenzyme Q10, which can potentially act as both an antioxidant and a prooxidant, has an impact on indicators of oxidative stress and the aging process. Mice were fed diets providing daily supplements of 0, 93, or 371 mg CoQ10 /kg body weight, starting at 3.5 months of age. Effects on mitochondrial superoxide generation, activities of oxidoreductases, protein oxidative damage, glutathione redox state, and life span of male mice were determined. Amounts of CoQ9 and CoQ10, measured after 3.5 or 17.5 months of intake, in homogenates and mitochondria of liver, heart, kidney, skeletal muscle, and brain increased with the dosage and duration of CoQ10 intake in all the tissues except brain. Activities of mitochondrial electron transport chain oxidoreductases, rates of mitochondrial O2-* generation, state 3 respiration, carbonyl content, glutathione redox state of tissues, and activities of superoxide dismutase, catalase, and glutathione peroxidase, determined at 19 or 25 months of age, were unaffected by CoQ10 administration. Life span studies, conducted on 50 mice in each group, showed that CoQ10 administration had no effect on mortality. Altogether, the results indicated that contrary to the historical view, supplemental intake of CoQ10 elevates the endogenous content of both CoQ9 and CoQ10, but has no discernable effect on the main antioxidant defenses or prooxidant generation in most tissues, and has no impact on the life span of mice.     

Spectroscopic analysis of the interaction of rat liver short-chain, medium-chain, and long-chain acyl coenzyme A dehydrogenases with acyl coenzyme A substrates

We systematically studied the visual spectral changes of short-chain, medium-chain, and long-chain acyl coenzyme A (acyl-CoA) dehydrogenases, purified from rat liver mitochondria, that occur upon reaction with acyl-CoA in the absence of an electron acceptor (half-reaction). Acyl-CoA esters having various chain lengths were tested, and changes in the steady-state spectral parameters were correlated with the turnover number in the complete reaction, which represented the ability of an enzyme/substrate combination to produce an enoyl-CoA. The long-wavelength absorbance, centered around 580 nm, was observed only in the enzyme/substrate combinations in which enoyl-CoA product was produced at a significant rate in the complete reaction. There was a good correlation between the magnitudes of the long-wavelength absorbance and the turnover numbers. In contrast, the bleaching of the flavin chromophore at 450 nm was observed not only in the titration with preferred substrates but also in that with unfavorable substrates, which were shorter than favorable substrates. In the interaction with the shorter than favorable substrates, however, enoyl-CoA was not produced, nor did long-wavelength absorbance occur. When short-chain and medium-chain acyl-CoA dehydrogenases were reacted with longer than favorable substrate from which no enoyl-CoA was produced, neither the appearance of the long-wavelength absorbance nor bleaching of flavin chromophore was observed. These data suggest that the catalytic base, which abstracts alpha-proton, and flavin adenine dinucleotide are internally located, and the region containing these two sites may physically be in the form of crevice or pocket.     

The steady state concentrations of coenzyme A-SH and coenzyme A thioester, citrate, and isocitrate during tricarboxylate cycle oxidations in rabbit heart mitochondria.

The steady state mitochondrial content of coenzyme A-SH (CoA), acetyl-CoA, succinyl-CoA, and long chain acyl-CoA has been determined during the oxidation of palmitoylcarnitine by rabbit heart mitochondria. Variation of the substrate concentration during ADP-stimulated (state 3) respiration varies the mitochondrial content of long chain acyl-CoA and the rate of O2 uptake, and permits the conclusion that the Km of beta oxidation for intramitochondrial long chain acyl-CoA is approximately 1 nmol/mg of mitochondrial protein. At near saturating concentrations of palmitoylcarnitine, plus L-malate, the addition of ADP causes a decrease in acetyl-CoA, an increase in CoA and succinyl-CoA, and no clear change in long chain acyl-CoA content. These changes reverse upon the depletion of ADP (state 3 leads to 4 transition). Similar changes in CoA, acetyl-CoA, and succinyl-CoA are seen during state 4 leads to 3 leads to 4 transitions with pyruvate plus L-malate and octanoate plus L-malate as substrates. These results suggest a limitation of flux by citrate synthase during the controlled oxidation of these three substrates. The ratio acetyl-CoA/succinyl-CoA was determined not only during state 3 and state 4 oxidation of palmitoylcarnitine plus L-malate and pyruvate plus L-malate, but also during intermediate respiratory states (state 3 1/2) generated by adding glucose and varying amounts of hexokinase. These intermediate states are characterized by a high succinyl-CoA content, relative to either state 3 or state 4, and a suboptimal flux through citrate synthase, estimated either by pyruvate disappearance or by O2 uptake.     

Function of methanofuran,tetrahydromethanopterin, and coenzyme F420 in Archaeoglobus fulgidus

Archaeoglobus fulgidus is an extremely thermophilic archaebacterium that can grow at the expense of lactate oxidation with sulfate to CO₂ and H₂S. The organism contains coenzyme F₄₂₀, tetrahydromethanopterin, and methanofuran which are coenzymes previously thought to be unique for methanogenic bacteria. We report here that the bacterium contains methylenetetrahydromethanopterin: F₄₂₀ oxidoreductase (20 U/mg), methenyltetrahydromethanopterin cyclohydrolase (0.9 U/mg), formyltetrahydromethanopterin: methanofuran formyltransferase (4.4 U/mg), and formylmethanofuran: benzyl viologen oxidoreductase (35 mU/mg). Besides these enzymes carbon monoxide: methyl viologen oxidoreductase (5 U/mg), pyruvate: methyl viologen oxidoreductase (0.7 U/mg), and membranebound lactate: dimethylnaphthoquinone oxidoreductase (0.1 U/mg) were found. 2-Oxoglutarate dehydrogenase, which is a key enzyme of the citric acid cycle, was not detectable. From the enzyme outfit it is concluded that in A. fulgidus lactate is oxidized to CO₂ via a modified acetyl-CoA/carbon monoxide dehydrogenase pathway involving C₁-intermediates otherwise only used by methanogenic bacteria.Non-standard abbreviations APS adenosine 5-phosphosulfate - BV benzyl viologen - DCPIP 2,6-dichlorophenolindophenol - DMN 2,3-dimethyl-1,4-naphthoquinone - DTT DL-1,4-dithiothreitol - H₄F tetrahydrofolate - H₄MPT tetrahydromethanopterin - CH₂ H₄MPT, methylene-H₄MPT - CH H₄MPT, methenyl-H₄MPT - Mes morpholinoethane sulfonic acid - MFR methanofuran - Mops morpholinopropane sulfonic acid - MV methyl viologen - Tricine N-tris(hydroxymethyl)-methylglycine - U mol product formed per min