Condensation of ?-ketobutyrate and acetyl-CoA in baker's yeast

1965 ◽  
Vol 50 (2) ◽  
pp. 164-170 ◽  
Author(s):  
J. L. Canovas ◽  
M. Ruiz-Amil ◽  
M. Losada
1969 ◽  
Vol 112 (5) ◽  
pp. 755-762 ◽  
Author(s):  
J. J. Cazzulo ◽  
A. O. M. Stoppani

1. Pyruvate carboxylase from baker's yeast is inhibited by ADP, AMP and adenosine at pH8·0 in the presence of magnesium chloride concentrations equal to or higher than the ATP concentration. The adenine moiety is essential for the inhibitory effect. 2. In the absence of acetyl-CoA (an allosteric activator) ADP, AMP and adenosine are competitive inhibitors with respect to ATP. In the presence of acetyl-CoA, besides the effect with respect to ATP, AMP competes with acetyl-CoA, whereas ADP and adenosine are non-competitive inhibitors with respect to the activator. 3. Pyruvate carboxylase is inhibited by NADH. The inhibition is competitive with respect to acetyl-CoA and specific with respect to NADH, since NAD+, NADP+ and NADPH do not affect the enzyme activity. In the absence of acetyl-CoA, NAD+, NADH, NADP+ and NADPH do not inhibit pyruvate carboxylase. 4. Pyruvate carboxylase is inhibited by ADP, AMP and NADH at pH6·5, in the presence of 12mm-Mg2+, 0·75mm-Mn2+ and 0·5mm-ATP, medium conditions similar to those existing inside the yeast cell. The ADP and NADH effects are consistent with a regulation of enzyme activity by the intracellular [ATP]/[ADP] ratio and secondarily by NADH concentration. These mechanisms would supplement the already known control of yeast pyruvate carboxylase by acetyl-CoA and l-aspartate. Inhibition by AMP is less marked and its physiological role is perhaps limited.


1972 ◽  
Vol 126 (1) ◽  
pp. 27-34 ◽  
Author(s):  
B. Middleton ◽  
P. K. Tubbs

1. A purification of 3-hydroxy-3-methylglutaryl-CoA synthase from baker's yeast is described. This yields a preparation of average specific activity 2.1 units (μmol/min)/mg in which contamination by acetoacetyl-CoA thiolase is less than 0.2%. 2. The molecular weights of 3-hydroxy-3-methylglutaryl-CoA synthase and acetoacetyl-CoA thiolase from baker's yeast were determined by gel filtration on Sephadex G-200. The values obtained were 130000 and 190000 respectively. 3. 3-Hydroxy-3-methylglutaryl-CoA synthase is susceptible to irreversible inhibition by a wide variety of alkylating and acylating agents. The time-course of inhibition of the enzyme by some of these, including the active-site-directed inhibitor bromoacetyl-CoA, was studied in the presence and absence of substrates, products and product analogues. Acetyl-CoA, even when present at concentrations as low as 5μm, gives almost complete protection. Other acyl-CoA derivatives give some protection, but only at concentrations 10–30-fold higher. 4. These results are discussed with reference to an ordered reaction pathway in which acetyl-CoA reacts to give a covalent acetyl-enzyme intermediate.


mBio ◽  
2014 ◽  
Vol 5 (5) ◽  
Author(s):  
Barbara U. Kozak ◽  
Harmen M. van Rossum ◽  
Marijke A. H. Luttik ◽  
Michiel Akeroyd ◽  
Kirsten R. Benjamin ◽  
...  

ABSTRACTThe energetic (ATP) cost of biochemical pathways critically determines the maximum yield of metabolites of vital or commercial relevance. Cytosolic acetyl coenzyme A (acetyl-CoA) is a key precursor for biosynthesis in eukaryotes and for many industrially relevant product pathways that have been introduced intoSaccharomyces cerevisiae, such as isoprenoids or lipids. In this yeast, synthesis of cytosolic acetyl-CoA via acetyl-CoA synthetase (ACS) involves hydrolysis of ATP to AMP and pyrophosphate. Here, we demonstrate that expression and assembly in the yeast cytosol of an ATP-independent pyruvate dehydrogenase complex (PDH) fromEnterococcus faecaliscan fully replace the ACS-dependent pathway for cytosolic acetyl-CoA synthesis.In vivoactivity ofE. faecalisPDH required simultaneous expression ofE. faecalisgenes encoding its E1α, E1β, E2, and E3 subunits, as well as genes involved in lipoylation of E2, and addition of lipoate to growth media. A strain lacking ACS that expressed theseE. faecalisgenes grew at near-wild-type rates on glucose synthetic medium supplemented with lipoate, under aerobic and anaerobic conditions. A physiological comparison of the engineered strain and an isogenic Acs+reference strain showed small differences in biomass yields and metabolic fluxes. Cellular fractionation and gel filtration studies revealed that theE. faecalisPDH subunits were assembled in the yeast cytosol, with a subunit ratio and enzyme activity similar to values reported for PDH purified fromE. faecalis. This study indicates that cytosolic expression and assembly of PDH in eukaryotic industrial microorganisms is a promising option for minimizing the energy costs of precursor supply in acetyl-CoA-dependent product pathways.IMPORTANCEGenetically engineered microorganisms are intensively investigated and applied for production of biofuels and chemicals from renewable sugars. To make such processes economically and environmentally sustainable, the energy (ATP) costs for product formation from sugar must be minimized. Here, we focus on an important ATP-requiring process in baker’s yeast (Saccharomyces cerevisiae): synthesis of cytosolic acetyl coenzyme A, a key precursor for many industrially important products, ranging from biofuels to fragrances. We demonstrate that pyruvate dehydrogenase from the bacteriumEnterococcus faecalis, a huge enzyme complex with a size similar to that of a ribosome, can be functionally expressed and assembled in the cytosol of baker’s yeast. Moreover, we show that this ATP-independent mechanism for cytosolic acetyl-CoA synthesis can entirely replace the ATP-costly native yeast pathway. This work provides metabolic engineers with a new option to optimize the performance of baker’s yeast as a “cell factory” for sustainable production of fuels and chemicals.


2014 ◽  
Vol 13 (12) ◽  
pp. 3153-3160 ◽  
Author(s):  
Zakaria Al-Qodah ◽  
Mohammad Al-Shannag ◽  
Kholoud Alananbeh ◽  
Nahla Bouqellah ◽  
Eman Assirey ◽  
...  

Author(s):  
Ida N Jamal ◽  
Reiny A Tumbol ◽  
Remy E.P Mangindaan

Motile Aeromonas Septicaemia disease (MAS) attacking tilapia has increased in recent years as a consequence of intensive aquaculture activities, which led to losses in aquaculture industry. The agent causing MAS disease is Aeromonas hydrophila. The disease can be controlled with the β-glucan. As immunostimulants, β-glucans can also increase resistance in farmed tilapia. Studies on the use of β-glucan extracted from baker's yeast Saccharomyces cerevisiae was intended to evaluate the non-specific immune system of tilapia that were challenged with Aeromonas hydrophila. The method used was an experimental method with a completely randomized design consisting of four treatments with three replicats. The dose of β-glucan used as treatments were 0 mg.kg-1 fish (Control), 5 mg.kg-1 fish (B), 10 mg.kg-1 fish (C) and 20 mg.kg-1 fish (D), each treatment as injected three times at intervals of 3 days, the injection volume of 0.5 ml/fish for nine days and resistance surveillance for seven days. The results showed that the difference in the amount of β-glucan and the frequency of the injected real influence on total leukocytes, phagocytic activity and resistance. Total leukocytes, phagocytic activity and resistance to treatment was best achieved by the administration of C a dose of  10 mg.kg-1 of the fish© Penyakit Motil Aeromonas Septicaemia (MAS) yang menyerang ikan nila mengalami peningkatan selama beberapa tahun terakhir sebagai konsekuensi dari kegiatan akuakultur intensif, yang menyebabkan kerugian dalam industri budidaya. Agen utama penyebab penyakit MAS adalah Aeromonas hydrophila. Untuk mengendalikan penyakit tersebut dapat dilakukan dengan pemberian β-glukan. Sebagai imunostimulan, β-glukan juga dapat  meningkatkan resistensi pada ikan nila yang dibudidayakan. Pengkajian mengenai pemanfaatan β-glukan yang diekstrak dari ragi roti Saccharomyces cerevisiae dimaksudkan untuk menguji sistem imun non spesifik ikan nila yang diuji tantang dengan bakteri Aeromonas hydrophila. Metode yang digunakan yaitu metode eksperimen dengan rancangan acak lengkap yang terdiri dari empat perlakuan dan tiga ulangan. Dosis β-glukan  yang digunakan sebagai perlakuan sebesar 0 mg.kg-1 ikan (Kontrol), 5 mg.kg-1 ikan (B), 10 mg.kg-1 ikan (C) dan 20 mg.kg-1 ikan (D), masing-masing perlakuan diinjeksi sebanyak 3 kali dengan interval waktu 3 hari selama 9 hari, volume injeksi 0,5 mL/ekor ikan dan pengamatan resistensi selama tujuh hari. Hasil penelitian menunjukkan perbedaan jumlah β-glukan dan frekuensi pemberian yang diinjeksikan memberikan pengaruh nyata terhadap total leukosit, aktivitas fagositosis dan resistensi. Total leukosit, aktivitas fagositosis dan resistensi terbaik dicapai pada perlakuan C dengan dosis 10 mg.kg-1 ikan©


Author(s):  
Yeong-Hoon Han ◽  
Hyun Joong Kim ◽  
Tae-Rim Choi ◽  
Hun-Suk Song ◽  
Sun Mi Lee ◽  
...  

1978 ◽  
Vol 253 (7) ◽  
pp. 2392-2399 ◽  
Author(s):  
J.N. Siedow ◽  
S. Power ◽  
F.F. de la Rosa ◽  
G. Palmer

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