A mutation affecting lipoamide dehydrogenase, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase activities in Saccharomyces cerevisiae

1986 ◽  
Vol 204 (1) ◽  
pp. 103-107 ◽  
Author(s):  
J. Richard Dickinson ◽  
Douglas J. Roy ◽  
Ian W. Dawes
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Pyruvate Dehydrogenase ◽  
Oxoglutarate Dehydrogenase ◽  
Lipoamide Dehydrogenase

scholarly journals Low immunogenicity of the common lipoamide dehydrogenase subunit (E3) of mammalian pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes

1985 ◽  
Vol 226 (2) ◽  
pp. 509-517 ◽  
Author(s):  
O L De Marcucci ◽  
A Hunter ◽  
J G Lindsay
Keyword(s):  
Rat Liver ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Sodium Dodecyl ◽  
Normal Component ◽  
Proteolytic Fragment ◽  
Multienzyme Complexes ◽  
The Common ◽  
Oxoglutarate Dehydrogenase ◽  
Lipoamide Dehydrogenase

The production of high-titre monospecific polyclonal antibodies against the purified pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes from ox heart is described. The specificity of these antisera and their precise reactivities with the individual components of the complexes were examined by immunoblotting techniques. All the subunits of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes were strongly antigenic, with the exception of the common lipoamide dehydrogenase component (E3). The titre of antibodies raised against E3 was, in both cases, less than 2% of that of the other subunits. Specific immunoprecipitation of the dissociated N-[3H]ethylmaleimide-labelled enzymes also revealed that E3 alone was absent from the final immune complexes. Strong cross-reactivity with the enzyme present in rat liver (BRL) and ox kidney (NBL-1) cell lines was observed when the antibody against ox heart pyruvate dehydrogenase was utilized to challenge crude subcellular extracts. The immunoblotting patterns again lacked the lipoamide dehydrogenase band, also revealing differences in the apparent Mr of the lipoate acetyltransferase subunit (E2) from ox kidney and rat liver. The additional 50 000-Mr polypeptide, previously found to be associated with the pyruvate dehydrogenase complex, was apparently not a proteolytic fragment of E2 or E3, since it could be detected as a normal component in boiled sodium dodecyl sulphate extracts of whole cells. The low immunogenicity of the lipoamide dehydrogenase polypeptide may be attributed to a high degree of conservation of its primary sequence and hence tertiary structure during evolution.

Modification of thiamine pyrophosphate dependent enzyme activity by oxythiamine in Saccharomyces cerevisiae cells

2005 ◽  
Vol 51 (10) ◽  
pp. 833-839 ◽  
Author(s):  
Adam Tylicki ◽  
Jan Czerniecki ◽  
Pawel Dobrzyn ◽  
Agnieszka Matanowska ◽  
Anna Olechno ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Pyruvate Dehydrogenase ◽  
Specific Activity ◽  
Pyruvate Decarboxylase ◽  
Yeast Cells ◽  
Thiamine Pyrophosphate ◽  
Decarboxylase Activity ◽  
Mitochondrial Enzymes ◽  
Oxoglutarate Dehydrogenase ◽  
Cytosolic Enzymes

Oxythiamine is an antivitamin derivative of thiamine that after phosphorylation to oxythiamine pyro phos phate can bind to the active centres of thiamine-dependent enzymes. In the present study, the effect of oxythiamine on the viability of Saccharomyces cerevisiae and the activity of thiamine pyrophosphate dependent enzymes in yeast cells has been investigated. We observed a decrease in pyruvate decarboxylase specific activity on both a control and an oxythiamine medium after the first 6 h of culture. The cytosolic enzymes transketolase and pyruvate decarboxylase decreased their specific activity in the presence of oxythiamine but only during the beginning of the cultivation. However, after 12 h of cultivation, oxythiamine-treated cells showed higher specific activity of cytosolic enzymes. More over, it was established by SDS–PAGE that the high specific activity of pyruvate decarboxylase was followed by an increase in the amount of the enzyme protein. In contrast, the mitochondrial enzymes, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase complexes, were inhibited by oxythiamine during the entire experiment. Our results suggest that the observed strong decrease in growth rate and viability of yeast on medium with oxythiamine may be due to stronger in hibition of mitochondrial pyruvate dehydrogenase than of cytosolic enzymes.Key words: pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, transketolase, pyruvate decarboxylase, activity, oxythiamine, inhibition.

Pyruvate dehydrogenase subcomplex with lipoamide dehydrogenase deficiency in a patient with lactic acidosis and branched chain ketoaciduria

1984 ◽  
Vol 140 (1) ◽  
pp. 59-64 ◽  
Author(s):  
Matuda Sadayuki ◽  
Kitano Akito ◽  
Sakaguchi Yusuke ◽  
Yoshino Makoto ◽  
Saheki Takeyori
Keyword(s):  
Lactic Acidosis ◽  
Pyruvate Dehydrogenase ◽  
Dehydrogenase Deficiency ◽  
Lipoamide Dehydrogenase ◽  
Branched Chain

scholarly journals Regulation of the PDA1 gene encoding the E1alpha subunit of the pyruvate dehydrogenase complex from Saccharomyces cerevisiae

1993 ◽  
Vol 218 (2) ◽  
pp. 405-411 ◽  
Author(s):  
Thibaut J. WENZEL ◽  
Marijke A. H. LUTTIK ◽  
Johan A. BERG ◽  
H. Yde STEENSMA
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Gene Encoding ◽  
Dehydrogenase Complex

Engineering of the pyruvate dehydrogenase bypass in Saccharomyces cerevisiae for high-level production of isoprenoids

2007 ◽  
Vol 9 (2) ◽  
pp. 160-168 ◽  
Author(s):  
Yoichiro Shiba ◽  
Eric M. Paradise ◽  
James Kirby ◽  
Dae-Kyun Ro ◽  
Jay D. Keasling
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Pyruvate Dehydrogenase ◽  
High Level ◽  
Level Production

scholarly journals Expressing a cytosolic pyruvate dehydrogenase complex to increase free fatty acid production in Saccharomyces cerevisiae

2020 ◽  
Vol 19 (1) ◽  
Author(s):  
Yiming Zhang ◽  
Mo Su ◽  
Ning Qin ◽  
Jens Nielsen ◽  
Zihe Liu
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Fatty Acids ◽  
Fatty Acid ◽  
Free Fatty Acid ◽  
Pyruvate Dehydrogenase ◽  
Unsaturated Fatty Acids ◽  
Pyruvate Dehydrogenase Complex ◽  
Cell Factory ◽  
Acetyl Coa ◽  
Fatty Acid Production

Abstract Background Saccharomyces cerevisiae is being exploited as a cell factory to produce fatty acids and their derivatives as biofuels. Previous studies found that both precursor supply and fatty acid metabolism deregulation are essential for enhanced fatty acid synthesis. A bacterial pyruvate dehydrogenase (PDH) complex expressed in the yeast cytosol was reported to enable production of cytosolic acetyl-CoA with lower energy cost and no toxic intermediate. Results Overexpression of the PDH complex significantly increased cell growth, ethanol consumption and reduced glycerol accumulation. Furthermore, to optimize the redox imbalance in production of fatty acids from glucose, two endogenous NAD+-dependent glycerol-3-phosphate dehydrogenases were deleted, and a heterologous NADP+-dependent glyceraldehyde-3-phosphate dehydrogenase was introduced. The best fatty acid producing strain PDH7 with engineering of precursor and co-factor metabolism could produce 840.5 mg/L free fatty acids (FFAs) in shake flask, which was 83.2% higher than the control strain YJZ08. Profile analysis of free fatty acid suggested the cytosolic PDH complex mainly resulted in the increases of unsaturated fatty acids (C16:1 and C18:1). Conclusions We demonstrated that cytosolic PDH pathway enabled more efficient acetyl-CoA provision with the lower ATP cost, and improved FFA production. Together with engineering of the redox factor rebalance, the cytosolic PDH pathway could achieve high level of FFA production at similar levels of other best acetyl-CoA producing pathways.

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