Role of acetyl-coenzyme A synthetase in leaves of Arabidopsis thaliana

Acetyl-coenzyme A synthetase (ACS) is a plastidic enzyme that forms acetyl-coenzyme A (acetyl-CoA) from acetate and coenzyme A using the energy from ATP. Traditionally it has been thought to be the major source for the production of acetyl-CoA destined for fatty acid formation. Recent work suggested that the accumulation of lipids in developing Arabidopsis seeds was more closely correlated with the expression of pyruvate dehydrogenase complex than with the expression of ACS, suggesting that most of the carbon for fatty acid formation in the plastids of seeds comes from pyruvate rather than from acetate. To explore the role of this enzyme, Arabidopsis plants with altered amounts of ACS were generated by overexpressing its cDNA in either the sense or the antisense configuration. The resulting plants had in vitro enzyme activities that ranged from about 5% to over 400% of wild-type levels. The rate of [1-14C]acetate conversion into fatty acids was closely related to the in vitro ACS activity, showing that the amount of enzyme clearly limited the capacity of leaves to convert exogenous acetate to fatty acids. There was, however, no relationship between the ACS level and the capacity of the plants to incorporate 14CO2 into 14C-labeled fatty acids. These data strongly support the idea that, although plants can convert acetate into fatty acids, relatively little carbon moves through this pathway under normal conditions.     

Sugars have potential to curtail oxygenase activity of Rubisco

Sugars play a critical role in regulating overall cellular metabolism and owing to their general compatibility with various cellular events plants invariably show enhanced levels of sugars for maintaining desired osmoticum under osmotic stress. Sugars (sucrose and trehalose) and sugar-alcohols (glycerol, mannitol, inositol, and sorbitol) with the exception of sorbitol lowered oxygenase activity of Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase, EC 4.1.1.39) without altering carboxylase activity under unstressed conditions. Most interestingly, these solutes including sorbitol fully curtailed NaCl-induced enhancement in oxygenase activity, even at concentrations as low as 50mM. However, none of these solutes could alleviate NaCl-suppressed carboxylase activity. In summary, our findings demonstrate that one of the most important roles of sugars and sugar-alcohols in plants exposed to salt stress is to curtail oxygenase activity of Rubisco.     

丙二醛对菠菜叶片中光合羧化酶和细胞保护酶活性的影响

经丙二醛(MDA)处理的菠菜(Spinacia oleracea L.)叶片无细胞提取物中的三种与光合作用碳素还原环有关的酶 RuBPC、PEPC、GAPDH 和三种能防御活性氧毒害的酶 SOD、CAT 和 POD 的活性皆有不同程度的降低。在较低浓度的 MDA 影响下,RuBPC/O 和 GAPDH的活性已受到明显的抑制。MDA 对纯 POD(辣根)和 CAT(牛肝)的活性同样具有抑制作用,其作用是不可逆的。CAT 与 MDA 的反应引起吸收峰向长波方向漂移。半胱氨酸对 HR-POD 活性的下降具有部分保护作用。结果认为体内积累 MDA 时,由于 MDA 对酶的损伤作用,可能导致细胞代谢的进一步紊乱。     

Anaerobic acetate oxidation to CO2 by Desulfotomaculum acetoxidans

Desulfotomaculum acetoxidans has been proposed to oxidize acetate to CO₂ via an oxidative acetyl-CoA/carbon monoxide dehydrogenase pathway rather than via the citric acid cycle. We report here the presence of the enzyme activities required for the operation of the novel pathway. In cell extracts the following activities were found (values in brackets=specific activities and apparent K _m; 1 U·mg^-1=1 mol·min^-1·mg protein^-1 at 37°C): Acetate kinase (6.3 U·mg^-1; 2 mM acetate; 2.4 mM ATP); phosphate acetyltransferase (60 U·mg^-1, 0.4 mM acetylphosphate; 0.1 mM CoA); carbon monoxide dehydrogenase (29 U·mg^-1; 13% carbon monoxide; 1.3 mM methyl viologen); 5,10-methylenetetrahydrofolate reductase (3 U·mg^-1, 0.06 mM CH₃–FH₄); methylenetetrahydrofolate dehydrogenase (3.6 U·mg^-1, 0.9 mM NAD, 0.1 mM CH₂=FH₄); methenyltetrahydrofolate cyclohydrolase (0.3 U·mg^-1); formyltetrahydrofolate synthetase (3 U·mg^-1, 1.4 mM FH₄, 0.4 mM ATP, 13 mM formate); and formate dehydrogenase (10 U·mg^-1, 0.4 mM formate, 0.5 mM NAD). The specific activities are sufficient to account for the in vivo acetate oxidation rate of 0.26 U·mg^-1.Non-standard abbreviations FH₄ Tetrahydrofolate - CHO-FH₄ N¹⁰-formyltetrahydrofolate - CHFH₄ N⁵,N¹⁰-methenyltetrahydrofolate - CH₂=FH₄ N⁵,N¹⁰-methylenetetrahydrofolate - CH₃–FH₄ N⁵-methyltetrahydrofolate - MOPS morpholinopropane sulfonic acid - DTT d,l-1,4-dithiothreitol - TRIS tris-(hydroxymethyl)-aminomethane - Ap₅A p¹,P⁵-di(adenosine-5)pentaphosphate - MV methyl viologen     

Characterization of acetyl-CoA: l-lysine N6-acetyltransferase,which catalyses the first step of carbon catabolism from lysine in Saccharomyces cerevisiae

The carbon catabolism of l-lysine starts in Saccharomyces cerevisiae with acetylation by an acetyl-CoA: l-lysine N⁶-acetyltransferase. The enzyme is strongly induced in cells grown on l-lysine as sole carbon source and has been purified about 530-fold. Its activity was specific for acetyl-CoA and, in addition to l-lysine, 5-hydroxylysine and thialysine act as acetyl acceptor. The following apparent Michaelis constants were determined: acetyl-CoA 0.8 mM, l-lysine 5.8 mM, dl-5-hydroxylysine 2.8 mM, l-thialysine 100 mM. The enzyme had a maximum activity at pH 8.5 and 37°C. Its molecular mass, estimated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, was 52 kDa. Since the native molecular mass, determined by gel filtration, was 48 kDa, the enzyme is a monomer.     

Spectroscopic and potentiometric characterization of cytochromes in two Sporomusa species and their expression during growth on selected substrates

The homoacetogenic bacteria Sporomusa ovata and Sporomusa sphaeroides were grown on betaine, betaine + formate, and acetoin in the absence of carbon dioxide, and the formation of membrane-bound cytochromes was determined. In S. sphaeroides, the growth substrate had little influence on the expression of cytochromes. In contrast, membranes from betaine-or acetoin-grown S. ovata cells had an 11-or 3-fold higher cytochrome b content than cells grown on betaine + formate. The cytochrome c content was reduced below the detection level after growth on the latter two substrates. The cytochromes in the membranes of S. sphaeroides and S. ovata were characterized by low-temperature difference spectroscopy, hemochrome difference spectroscopy, and redox potentiometry. Membranes of S. ovata were shown to contain two b-type cytochromes with E_m,7=-153±10 mV and E_m,7=-226±14 mV and two c-type cytochromes with E_m,7=-86±6 mV and E_m,7=-265±10 mV. In S. sphaeroides also two b-type cytochromes with E_m,7=-165±7 mV and E_m,7=-241±2 mV and two c-type cytochromes with E_m,7=-101±4 mV and E_{m, 8.5}=-338±9 mV could be distinguished. Cell extracts of S. sphaeroides were shown to contain all the enzymes of the acetyl-CoA (Wood) pathway. The degradation pathways of the substrates tested and the possible role of the cytochromes are discussed.Abbreviations E_m,7 midpoint potential at pH 7 and 25°C - H₄F tetrahydrofolate     

2,3,7,8-tetrachlorodibenzo-p-dioxin induced alterations of pyruvate carboxylase levels and lactate dehydrogenase isozyme shifts in C57BL/6J male mice

A dose-dependent reduction of hepatic pyruvate carboxylase levels and activity occurs in C57BL/6J male mice given 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) i.p. in a corn oil carrier. The dose range was from 1 to 75 μg/kg body weight and the analysis was done 8 days postinjection. At the maximum TCDD level investigated, we found a 10-fold reduction in pyruvate carboxylase activity. Furthermore, TCDD at a dose of 1 μg/kg body weight blocks corn oil induction of an increase in the amount of pyruvate carboxylase in liver protein extracts. At doses beyond those required to initiate a reduction in pyruvate carboxylase, lactate dehydrogenase isozyme patterns shift. This is accompanied by an increase in blood lactic acid levels. We propose that TCDD-mediated reduction in pyruvate carboxylase and lactate dehydrogenase isozyme shifts may represent a major component in TCDD toxicity.     

Acetyl-CoA decarbonylase/synthase complex from Archaeoglobus fulgidus

The acetyl-CoA decarbonylase/synthase (ACDS) multienzyme complex catalyzes the reversible cleavage and synthesis of acetyl-CoA in methanogens. This report of the enzyme complex in Archaeoglobus fulgidus demonstrates the existence of a functional ACDS complex in an organism that is not a methanogen. The A. fulgidus enzyme complex contained five subunits of 89, 72, 50, 49.5, and 18.5 kDa, and it catalyzed the overall synthesis of acetyl-CoA according to the following reaction: w CO₂ + 2 Fd_red(Fe²⁺) + 2 H⁺ + CH₃– H₄SPt + CoA ⇌ acetyl-CoA + H₄SPt + 2 Fd_ox(Fe³⁺) + H₂O where Fd is ferredoxin, and CH₃–H₄SPt and H₄SPt denote N ⁵-methyl-tetrahydrosarcinapterin and tetrahydrosarcinapterin, respectively. Received: 27 October 1997 / Accepted: 29 January 1998     

Pyruvate metabolism of the hyperthermophilic archaebacterium Pyrococcus furiosus

The hyperthermophilic anaerobe Pyrococcus furiosus was found to grow on pyruvate as energy and carbon source. Growth was dependent on yeast extract (0.1%). The organism grew with doublings times of about 1 h up to cell densities of 1–2×10⁸ cells/ml. During growth 0.6–0.8 mol acetate and 1.2–1.5 mol CO₂ and 0.8 mol H₂ were formed per mol of pyruvate consumed. The molar growth yield was 10–11 g cells(dry weight)/mol pyruvate. Cell suspensions catalyzed the conversion of 1 mol of pyruvate to 0.6–0.8 mol acetate, 1.2–1.5 mol CO₂, 1.2 mol H₂ and 0.03 mol acetoin. After fermentation of [3-¹⁴C]pyruvate the specific radioactivities of pyruvate, CO₂ and acetate were equal to 1:0.01:1. Cellfree extracts contained the following enzymatic activities: pyruvate: ferredoxin (methyl viologen) oxidoreductase (0.2 U mg^-1, T=60°C, with Clostridium pasteurianum ferredoxin as electron acceptor; 1.4 U mg^-1 at 90°C, with methyl viologen as electron acceptor); acetyl-CoA synthetase (ADP forming) [acetyl-CoA+ADP+Piacetate+ATP+CoA] (0.34 U mg^-1, T=90°C), and hydrogen: methyl viologen oxidoreductase (1.75 U mg^-1). Phosphate acetyl-transferase activity, acetate kinase activity, and carbon monoxide:methyl viologen oxidoreductase activity could not be detected. These findings indicate that the archaebacterium P. furiosus ferments pyruvate to acetate, CO₂ and H₂ involving only three enzymes, a pyruvate:ferredoxin oxidoreductase, a hydrogenase and an acetyl-CoA synthetase (ADP forming).Non-standard abbreviations DTE dithioerythritol - MV methyl viologen - MOPS morpholinopropane sulfonic acid - Tricine N-tris(hydroxymethyl)-methylglycine Part of the work was performed at the Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universität, Karlvon-Frisch-Strasse, W-3550 Marburg/Lahn, Federal Republic of Germany     

Evolutionary divergence in the two kinds of subunits of ribulose diphosphate carboxylase isolated from different species ofNicotiana

A ² analysis was made of previously reported values for the amino acid composition of the large and small subunits of ribulose diphosphate carboxylase (E.C. 4, 1, 1, 39) isolated from five species ofNicotiana. The distributions of these values were then compared with published values for hemoglobin chains and cytochromec's of diverse origins. It was concluded that evolutionary diversity in the large and small subunits of RuDP carboxylase occurs even within the limited taxonomic category of the genusNicotiana. The large subunit was as stable towards mutation during evolution as the chains and cytochromes, whereas the small subunit was much less stable. The hypothesis is discussed that the large subunit played a more significant role than the small subunit in the enzyme function and/or structural integrity of the oligomeric protein. It was also speculated that the addition of small subunits to the molecule, which may have been a recent evolutionary event, enables the fixation of many evolutionarily favorable mutations. Thus, survival of the enzymatic activity in changing environments is favored.