The Pyruvate Dehydrogenase Complex of E. coli K-12 Structure and Synthesis

Author(s):  
U. Henning ◽  
O. Vogel ◽  
W. Busch ◽  
J. E. Flatgaard
2008 ◽  
Vol 190 (11) ◽  
pp. 3851-3858 ◽  
Author(s):  
Youngnyun Kim ◽  
L. O. Ingram ◽  
K. T. Shanmugam

ABSTRACT Under anaerobic growth conditions, an active pyruvate dehydrogenase (PDH) is expected to create a redox imbalance in wild-type Escherichia coli due to increased production of NADH (>2 NADH molecules/glucose molecule) that could lead to growth inhibition. However, the additional NADH produced by PDH can be used for conversion of acetyl coenzyme A into reduced fermentation products, like alcohols, during metabolic engineering of the bacterium. E. coli mutants that produced ethanol as the main fermentation product were recently isolated as derivatives of an ldhA pflB double mutant. In all six mutants tested, the mutation was in the lpd gene encoding dihydrolipoamide dehydrogenase (LPD), a component of PDH. Three of the LPD mutants carried an H322Y mutation (lpd102), while the other mutants carried an E354K mutation (lpd101). Genetic and physiological analysis revealed that the mutation in either allele supported anaerobic growth and homoethanol fermentation in an ldhA pflB double mutant. Enzyme kinetic studies revealed that the LPD(E354K) enzyme was significantly less sensitive to NADH inhibition than the native LPD. This reduced NADH sensitivity of the mutated LPD was translated into lower sensitivity of the appropriate PDH complex to NADH inhibition. The mutated forms of the PDH had a 10-fold-higher Ki for NADH than the native PDH. The lower sensitivity of PDH to NADH inhibition apparently increased PDH activity in anaerobic E. coli cultures and created the new ethanologenic fermentation pathway in this bacterium. Analogous mutations in the LPD of other bacteria may also significantly influence the growth and physiology of the organisms in a similar fashion.


1993 ◽  
Vol 289 (1) ◽  
pp. 81-85 ◽  
Author(s):  
J Quinn ◽  
A G Diamond ◽  
A K Masters ◽  
D E Brookfield ◽  
N G Wallis ◽  
...  

The dihydrolipoamide acetyltransferase subunit (E2p) of mammalian pyruvate dehydrogenase complex has two highly conserved lipoyl domains each modified with a lipoyl cofactor bound in amide linkage to a specific lysine residue. A sub-gene encoding the inner lipoyl domain of human E2p has been over-expressed in Escherichia coli. Two forms of the domain have been purified, corresponding to lipoylated and non-lipoylated species. The apo-domain can be lipoylated in vitro with partially purified E. coli lipoate protein ligase, and the lipoylated domain can be reductively acetylated by human E1p (pyruvate dehydrogenase). Availability of the two forms will now allow detailed biochemical and structural studies of the human lipoyl domains.


2015 ◽  
Vol 84 (1) ◽  
pp. 320-328 ◽  
Author(s):  
Kirsten M. Schutte ◽  
Debra J. Fisher ◽  
Marie D. Burdick ◽  
Borna Mehrad ◽  
Amy J. Mathers ◽  
...  

Chemokines are best recognized for their role within the innate immune system as chemotactic cytokines, signaling and recruiting host immune cells to sites of infection. Certain chemokines, such as CXCL10, have been found to play an additional role in innate immunity, mediating CXCR3-independent killing of a diverse array of pathogenic microorganisms. While this is still not clearly understood, elucidating the mechanisms underlying chemokine-mediated antimicrobial activity may facilitate the development of novel therapeutic strategies effective against antibiotic-resistant Gram-negative pathogens. Here, we show that CXCL10 exerts antibacterial effects on clinical and laboratory strains ofEscherichia coliand report that disruption of pyruvate dehydrogenase complex (PDHc), which converts pyruvate to acetyl coenzyme A, enablesE. colito resist these antimicrobial effects. Through generation and screening of a transposon mutant library, we identified two mutants with increased resistance to CXCL10, both with unique disruptions of the gene encoding the E1 subunit of PDHc,aceE. Resistance to CXCL10 also occurred following deletion of eitheraceForlpdA, genes that encode the remaining two subunits of PDHc. Although PDHc resides within the bacterial cytosol, electron microscopy revealed localization of immunogold-labeled CXCL10 to the bacterial cell surface in both theE. coliparent andaceEdeletion mutant strains. Taken together, our findings suggest that while CXCL10 interacts with an as-yet-unidentified component on the cell surface, PDHc is an important mediator of killing by CXCL10. To our knowledge, this is the first description of PDHc as a key bacterial component involved in the antibacterial effect of a chemokine.


RSC Advances ◽  
2016 ◽  
Vol 6 (6) ◽  
pp. 4310-4320 ◽  
Author(s):  
Haifeng He ◽  
Jiangtao Feng ◽  
Junbo He ◽  
Qin Xia ◽  
Yanliang Ren ◽  
...  

Optimal binding mode of novel E. coli PDHc E1 inhibitor 9d.


1989 ◽  
Vol 573 (1 Alpha-Keto Ac) ◽  
pp. 400-401
Author(s):  
S. STEWART ◽  
J. BORVÁK ◽  
P. BOIVIN ◽  
T. TWOMEY ◽  
W. H. SCOUTEN

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