scholarly journals Utilization of l-threonine by a species of Arthrobacter. A novel catabolic role for ‘aminoacetone synthase’

1969 ◽  
Vol 112 (5) ◽  
pp. 657-671 ◽  
Author(s):  
D. McGilvray ◽  
J G Morris
Keyword(s):  
Culture Medium ◽  
Tricarboxylic Acid Cycle ◽  
Glyoxylate Cycle ◽  
Sole Source ◽  
Short Term ◽  
Acetyl Coa ◽  
Threonine Dehydrogenase ◽  
Coa Ligase ◽  
Serine Pathway ◽  
Aldolase Activity

1. A species of Arthrobacter (designated Arthrobacter 9759) was isolated from soil by its ability to grow aerobically on l-threonine as sole source of carbon atoms, nitrogen atoms and energy; the organism also grew well on other sources of carbon atoms including glycine, but no growth was obtainable on aminoacetone or dl-1-aminopropan-2-ol. 2. During growth on threonine, 14C from l-[U−14C]threonine was rapidly incorporated into glycine and citrate, and thereafter into serine, alanine, aspartate and glutamate. 3. With extracts of threonine-grown cells supplied with l-[U−14C]threonine, evidence was obtained of the NAD and CoA-dependent catabolism of l-threonine to produce acetyl-CoA plus glycine. Short-term incorporation studies in which [2−14C]acetate and [2−14C]glycine were supplied (a) to cultures growing on threonine, and (b) to extracts of threonine-grown cells, showed that the acetyl-CoA was metabolized via the tricarboxylic acid cycle and glyoxylate cycle whereas the glycine was converted into pyruvate via the folate-dependent ‘serine pathway’. 4. The threonine-grown organism contained ‘biosynthetic’ threonine dehydratase and a potent NAD-linked l-threonine dehydrogenase but possessed no l-threonine aldolase activity. 5. Evidence was obtained that the acetyl-CoA and glycine produced from l-threonine had their immediate origin in the α-amino-β-oxobutyrate formed by the threonine dehydrogenase; the CoA-dependent cleavage of this compound was catalysed by an α-amino-β-oxobutyrate CoA-ligase, which was identified with ‘aminoacetone synthase’. A continuous spectrophotometric assay of this enzyme was developed, and it was found to be inducibly synthesized only during growth on threonine and not during growth on acetate plus glycine. 6. By using a reconstituted mixture of separately purified l-threonine dehydrogenase and α-amino-β-oxobutyrate CoA-ligase (i.e. ‘aminoacetone synthase’), l-[U−14C]threonine was broken down to [14C]glycine plus [14C]acetyl-CoA (trapped as [14C]citrate). 7. There was no evidence of aminoacetone metabolism by Arthrobacter 9759 even though a small amount of this amino ketone appeared in the culture medium during growth on threonine.

THE TRICARBOXYLIC ACID AND GLYOXYLATE CYCLES IN XANTHOMONAS PHASEOLI (XP8)

1959 ◽  
Vol 5 (1) ◽  
pp. 1-8 ◽  
Author(s):  
N. B. Madsen ◽  
R. M. Hochster
Keyword(s):  
Tricarboxylic Acid Cycle ◽  
Glyoxylate Cycle ◽  
Sole Source ◽  
Tricarboxylic Acid ◽  
Acid Cycle ◽  
Main Pathway ◽  
The Individual ◽  
Acetate Medium ◽  
Terminal Oxidation ◽  
The Glyoxylate Cycle

Cell-free extracts of Xanthomonas phaseoli contain the individual enzymes of the tricarboxylic acid cycle, and it is suggested that this is the main pathway for the terminal oxidation of carbohydrate in this organism. X. phaseoli can grow on a medium containing acetate as the sole source of carbon. Cell-free extracts of such acetate-grown organisms contain the enzymes of the glyoxylate cycle, and it is concluded that the operation of this cycle permits the initial stages of synthesis of complex cell material from acetate at a rate sufficiently high to account for the observed rate of growth on the acetate medium. The two enzymes required to modify a tricarboxylic acid cycle into a glyoxylate cycle are present in very small amounts (malate synthetase) or absent entirely (isocitritase) in extracts of glucose-grown X. phaseoli.

scholarly journals Metabolic homoeostasis of l-threonine in the normally-fed rat. Importance of liver threonine dehydrogenase activity

1983 ◽  
Vol 214 (3) ◽  
pp. 687-694 ◽  
Author(s):  
M I Bird ◽  
P B Nunn
Keyword(s):  
Dehydrogenase Activity ◽  
High Protein Diet ◽  
Fed State ◽  
Threonine Dehydratase ◽  
Threonine Dehydrogenase ◽  
Threonine Aldolase ◽  
Coa Ligase ◽  
Metabolic States ◽  
Aldolase Activity

Threonine dehydratase, threonine aldolase and threonine dehydrogenase activities were assayed in livers of rats that had been normally-fed, starved for 72 h, fed a high-protein diet or normally-fed and injected with glucagon or cortisone. A modified continuous spectrophotometric assay for threonine aldolase overcame interference resulting from threonine dehydratase activity and revealed that threonine aldolase activity was very low in rat liver, irrespective of the metabolic state of the animal. The concentration of free threonine was determined in livers of animals subjected to the same treatments as described above. Using Michaelis-Menten kinetics to estimate enzyme activities in vivo at intracellular threonine concentrations it was calculated that in the normally-fed state, 87% of the threonine degraded was catabolized by threonine dehydrogenase. In other metabolic states (except in glucagon-treated animals) threonine dehydratase was the major enzyme catalysing threonine catabolism. It was concluded that threonine dehydrogenase activity plays a hitherto unrecognized role in the metabolic homoeostasis of threonine in the normally-fed rat and that this enzyme activity, in association with 2-amino-3-oxobutyrate CoA-ligase, accounts for the known rate of glycine formation from threonine in the rat.

PARTICIPATION OF THE GLYOXYLATE CYCLE IN THE METABOLISM OF ETHANOL BY CASTOR BEAN ENDOSPERM TISSUES

1966 ◽  
Vol 44 (4) ◽  
pp. 423-432 ◽  
Author(s):  
Carol A. Peterson ◽  
E. A. Cossins
Keyword(s):  
Castor Bean ◽  
Tricarboxylic Acid Cycle ◽  
Glyoxylate Cycle ◽  
Experimental Period ◽  
Tricarboxylic Acid ◽  
Ethanol Metabolism ◽  
Short Term ◽  
Tissue Slices ◽  
Acidic Amino Acids ◽  
The Glyoxylate Cycle

The kinetics and pathway of ethanol metabolism in endosperm tissues of the germinating castor bean-have been studied by incubating tissue slices with micromolar quantities of ethanol-1-14C and ethanol-2-14C. In short term experiments, ethanol-14C was incorporated into the organic acids and acidic amino acids. When the experimental period was increased up to 1 hour, large amounts of ethanol-2-14C were incorporated into the sugars, and ethanol-1-14C was extensively incorporated into the respiratory carbon dioxide. Incorporation of ethanol-14C was stimulated by incubation of the tissues with glyoxylate. Ethanol metabolism was markedly inhibited by iodoacetate and malonate. These inhibitors also changed the distribution of14C in the products isolated. Isotopic competition studies indicated that ethanol was incorporated into the acids of the glyoxylate and the tricarboxylic acid cycles at rates substantially lower than acetate.The results are interpreted as being consistent with a metabolism of ethanol mainly via the glyoxylate cycle with some cycling of ethanol carbon through the tricarboxylic acid cycle.

scholarly journals Evidence that ACN1 (acetate non-utilizing 1) prevents carbon leakage from peroxisomes during lipid mobilization in Arabidopsis seedlings

2011 ◽  
Vol 437 (3) ◽  
pp. 505-513 ◽  
Author(s):  
Elizabeth Allen ◽  
Annick Moing ◽  
Jonathan A. D. Wattis ◽  
Tony Larson ◽  
Mickaël Maucourt ◽  
...  
Keyword(s):  
Carbon Sink ◽  
Glyoxylate Cycle ◽  
Eicosenoic Acid ◽  
Primary Metabolism ◽  
Acetyl Coa ◽  
Primary Metabolite ◽  
Lipid Mobilization ◽  
Transcript Levels ◽  
Chain Acyl ◽  
Acetate Accumulation

ACN1 (acetate non-utilizing 1) is a short-chain acyl-CoA synthetase which recycles free acetate to acetyl-CoA in peroxisomes of Arabidopsis. Pulse-chase [2-13C]acetate feeding of the mutant acn1–2 revealed that acetate accumulation and assimilation were no different to that of wild-type, Col-7. However, the lack of acn1–2 led to a decrease of nearly 50% in 13C-labelling of glutamine, a major carbon sink in seedlings, and large decreases in primary metabolite levels. In contrast, acetyl-CoA levels were higher in acn1–2 compared with Col-7. The disappearance of eicosenoic acid was slightly delayed in acn1–2 indicating only a small effect on the rate of lipid breakdown. A comparison of transcript levels in acn1–2 and Col-7 showed that induced genes included a number of metabolic genes and also a large number of signalling-related genes. Genes repressed in the mutant were represented primarily by embryogenesis-related genes. Transcript levels of glyoxylate cycle genes also were lower in acn1–2 than in Col-7. We conclude that deficiency in peroxisomal acetate assimilation comprises only a small proportion of total acetate use, but this affects both primary metabolism and gene expression. We discuss the possibility that ACN1 safeguards against the loss of carbon as acetate from peroxisomes during lipid mobilization.

Proliferation and agglutinability of primary and transformed human epithelial cells in culture

1976 ◽  
Vol 21 (3) ◽  
pp. 553-561
Author(s):  
M.A. Ricard ◽  
R.J. Hay
Keyword(s):  
Culture Medium ◽  
Monolayer Culture ◽  
Primary Cultures ◽  
Short Term ◽  
Chloromethyl Ketone ◽  
Normal Populations ◽  
Short Term Exposure ◽  
Cell Strains ◽  
Human Placentae

Primary epithelial populations (HAM) were obtained by dissociation of the amniotic membrane stripped from human placentae. Agglutinability of cells from such normal populations and of cells from the transformed epithelial line WISH was then compared using concavanalin A as mediator. Extensive similar studies have previously been reported with cell strains isolated from other species. Freshly dissociated HAM cells from primary cultures agglutinated much less readily than did cells from WISH populations. Furthermore, the former exhibited a drastic decline in agglutinability as a function of time in suspension culture after trypsinization. Short-term exposure (60 h) of HAM cells in monolayer culture to 5-bromodeoxyuridine (BrdU) elicited heightened agglutinability detectable through 22 days in vitro. Addition of the protease inhibitors n-tosyl-L-lysyl-chloromethyl ketone (TLCK) or p-tosyl-L-arginine-methyl ester (TAME) to the culture medium inhibited proliferation of the WISH line by 40–50% while effecting only a 10–15% inhibition of HAM cells. These results also confirm data with other cell species indicating that high proteolytic activity at the surface of transformed cells may be related to the rapid proliferation rate.

13C isotopomer model for estimation of anaplerotic substrate oxidation via acetyl-CoA

1996 ◽  
Vol 271 (4) ◽  
pp. E788-E799 ◽  
Author(s):  
F. M. Jeffrey ◽  
C. J. Storey ◽  
A. D. Sherry ◽  
C. R. Malloy
Keyword(s):  
Biochemical Analysis ◽  
Tricarboxylic Acid Cycle ◽  
Tricarboxylic Acid ◽  
Acetyl Coa ◽  
Acid Cycle ◽  
Propionate Metabolism ◽  
13C Nuclear Magnetic Resonance ◽  
Isotopomer Analysis ◽  
Tricarboxylic Acid Cycle Intermediates ◽  
Anaplerotic Pathway

A previous model using 13C nuclear magnetic resonance isotopomer analysis provided for direct measurement of the oxidation of 13C-enriched substrates in the tricarboxylic acid cycle and/or their entry via anaplerotic pathways. This model did not allow for recycling of labeled metabolites from tricarboxylic acid cycle intermediates into the acetyl-CoA pool. An extension of this model is now presented that incorporates carbon flow from oxaloacetate or malate to acetyl-CoA. This model was examined using propionate metabolism in the heart, in which previous observations indicated that all of the propionate consumed was oxidized to CO2 and water. Application of the new isotopomer model shows that 2 mM [3-13C]propionate entered the tricarboxylic acid cycle as succinyl-CoA (an anaplerotic pathway) at a rate equal to 52% of tricarboxylic acid cycle turnover and that all of this carbon entered the acetyl-CoA pool and was oxidized. This was verified using standard biochemical analysis; from the rate (mumol.min-1.g dry wt-1) of propionate uptake (4.0 +/- 0.7), the estimated oxygen consumption (24.8 +/- 5) matched that experimentally determined (24.4 +/- 3).

scholarly journals Novel Pathway for the Degradation of 2-Chloro-4-Nitrobenzoic Acid by Acinetobacter sp. Strain RKJ12

2011 ◽  
Vol 77 (18) ◽  
pp. 6606-6613 ◽  
Author(s):  
Dhan Prakash ◽  
Ravi Kumar ◽  
R. K. Jain ◽  
B. N. Tiwary
Keyword(s):  
Salicylic Acid ◽  
Tricarboxylic Acid Cycle ◽  
Sole Source ◽  
Degradation Pathway ◽  
Ring Cleavage ◽  
Muconic Acid ◽  
Content Type ◽  
Nitrobenzoic Acid ◽  
Catabolic Genes ◽  
Catechol 1,2 Dioxygenase

ABSTRACTThe organismAcinetobactersp. RKJ12 is capable of utilizing 2-chloro-4-nitrobenzoic acid (2C4NBA) as a sole source of carbon, nitrogen, and energy. In the degradation of 2C4NBA by strain RKJ12, various metabolites were isolated and identified by a combination of chromatographic, spectroscopic, and enzymatic activities, revealing a novel assimilation pathway involving both oxidative and reductive catabolic mechanisms. The metabolism of 2C4NBA was initiated by oxidativeorthodehalogenation, leading to the formation of 2-hydroxy-4-nitrobenzoic acid (2H4NBA), which subsequently was metabolized into 2,4-dihydroxybenzoic acid (2,4-DHBA) by a mono-oxygenase with the concomitant release of chloride and nitrite ions. Stoichiometric analysis indicated the consumption of 1 mol O2per conversion of 2C4NBA to 2,4-DHBA, ruling out the possibility of two oxidative reactions. Experiments with labeled H218O and18O2indicated the involvement of mono-oxygenase-catalyzed initial hydrolytic dechlorination and oxidative denitration mechanisms. The further degradation of 2,4-DHBA then proceeds via reductive dehydroxylation involving the formation of salicylic acid. In the lower pathway, the organism transformed salicylic acid into catechol, which was mineralized by theorthoring cleavage catechol-1,2-dioxygenase tocis, cis-muconic acid, ultimately forming tricarboxylic acid cycle intermediates. Furthermore, the studies carried out on a 2C4NBA−derivative and a 2C4NBA+transconjugant demonstrated that the catabolic genes for the 2C4NBA degradation pathway possibly reside on the ∼55-kb transmissible plasmid present in RKJ12.

Isolation and characterization of the ethynylestradiol-biodegrading microorganism Fusarium proliferatum strain HNS-1

2002 ◽  
Vol 45 (12) ◽  
pp. 175-179 ◽  
Author(s):  
J.H. Shi ◽  
Y. Suzuki ◽  
B.-D. Lee ◽  
S. Nakai ◽  
M. Hosomi
Keyword(s):  
Culture Medium ◽  
Polar Compounds ◽  
Sole Source ◽  
Order Rate Constant ◽  
Fusarium Proliferatum ◽  
Rrna Gene ◽  
28S Rrna ◽  
Isolation And Characterization ◽  
Phenolic Group

We cultivated hundreds of sediment, soil, and manure samples taken from rivers and farms in a medium containing ethynylestradiol (EE2) as the sole source of carbon, so that microorganisms in the samples would acclimatize to the presence of EE2. Finally, we isolated an EE2-degrading microorganism, designated as strain HNS-1, from a cowshed sample. Based on its partial nucleotide sequence (563 bp) of the 28S rRNA gene, strain HNS-1 was identified as Fusarium proliferatum. Over 15 days, F. proliferatum strain HNS-1 removed 97% of EE2 at an initial concentration of 25 mg.L−1, with a first-order rate constant of 0.6 d−1. Unknown products of EE2 degradation, which may be more polar compounds that have a phenolic group, remained in the culture medium.

scholarly journals Association of the malate dehydrogenase-citrate synthase metabolon is modulated by intermediates of the Krebs tricarboxylic acid cycle

2021 ◽  
Author(s):  
Joy Omini ◽  
Izabela Wojciechowska ◽  
Aleksandra Skirycz ◽  
Hideaki Moriyama ◽  
Toshihiro Obata
Keyword(s):  
Malate Dehydrogenase ◽  
Metabolic Flux ◽  
Citrate Synthase ◽  
Tricarboxylic Acid Cycle ◽  
Feedback Regulation ◽  
Dynamic Structure ◽  
Tca Cycle ◽  
Tricarboxylic Acid ◽  
Enzyme Complex ◽  
Acetyl Coa

Mitochondrial malate dehydrogenase (MDH)-citrate synthase (CS) multi-enzyme complex is a part of the Krebs tricarboxylic acid (TCA) cycle 'metabolon' which is enzyme machinery catalyzing sequential reactions without diffusion of reaction intermediates into a bulk matrix. This complex is assumed to be a dynamic structure involved in the regulation of the cycle by enhancing metabolic flux. Microscale Thermophoresis analysis of the porcine heart MDH-CS complex revealed that substrates of the MDH and CS reactions, NAD+ and acetyl-CoA, enhance complex association while products of the reactions, NADH and citrate, weaken the affinity of the complex. Oxaloacetate enhanced the interaction only when it was presented together with acetyl-CoA. Structural modeling using published CS structures suggested that the binding of these substrates can stabilize the closed format of CS which favors the MDH-CS association. Two other TCA cycle intermediates, ATP, and low pH also enhanced the association of the complex. These results suggest that dynamic formation of the MDH-CS multi-enzyme complex is modulated by metabolic factors responding to respiratory metabolism, and it may function in the feedback regulation of the cycle and adjacent metabolic pathways.

scholarly journals The Apparent Malate Synthase Activity of Rhodobacter sphaeroides Is Due to Two Paralogous Enzymes, (3S)-Malyl-Coenzyme A (CoA)/β-Methylmalyl-CoA Lyase and (3S)- Malyl-CoA Thioesterase

2010 ◽  
Vol 192 (5) ◽  
pp. 1249-1258 ◽  
Author(s):  
Tobias J. Erb ◽  
Lena Frerichs-Revermann ◽  
Georg Fuchs ◽  
Birgit E. Alber
Keyword(s):  
Rhodobacter Sphaeroides ◽  
Coenzyme A ◽  
Glyoxylate Cycle ◽  
Condensation Reaction ◽  
Malate Synthase ◽  
Acetyl Coa ◽  
Acetyl Coenzyme A ◽  
Claisen Condensation ◽  
Enzyme Functions ◽  
The Glyoxylate Cycle

ABSTRACT Assimilation of acetyl coenzyme A (acetyl-CoA) is an essential process in many bacteria that proceeds via the glyoxylate cycle or the ethylmalonyl-CoA pathway. In both assimilation strategies, one of the final products is malate that is formed by the condensation of acetyl-CoA with glyoxylate. In the glyoxylate cycle this reaction is catalyzed by malate synthase, whereas in the ethylmalonyl-CoA pathway the reaction is separated into two proteins: malyl-CoA lyase, a well-known enzyme catalyzing the Claisen condensation of acetyl-CoA with glyoxylate and yielding malyl-CoA, and an unidentified malyl-CoA thioesterase that hydrolyzes malyl-CoA into malate and CoA. In this study the roles of Mcl1 and Mcl2, two malyl-CoA lyase homologs in Rhodobacter sphaeroides, were investigated by gene inactivation and biochemical studies. Mcl1 is a true (3S)-malyl-CoA lyase operating in the ethylmalonyl-CoA pathway. Notably, Mcl1 is a promiscuous enzyme and catalyzes not only the condensation of acetyl-CoA and glyoxylate but also the cleavage of β-methylmalyl-CoA into glyoxylate and propionyl-CoA during acetyl-CoA assimilation. In contrast, Mcl2 was shown to be the sought (3S)-malyl-CoA thioesterase in the ethylmalonyl-CoA pathway, which specifically hydrolyzes (3S)-malyl-CoA but does not use β-methylmalyl-CoA or catalyze a lyase or condensation reaction. The identification of Mcl2 as thioesterase extends the enzyme functions of malyl-CoA lyase homologs that have been known only as “Claisen condensation” enzymes so far. Mcl1 and Mcl2 are both related to malate synthase, an enzyme which catalyzes both a Claisen condensation and thioester hydrolysis reaction.

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