The N-terminal 1–55 residues domain of pyruvate dehydrogenase from Escherichia coli assembles as a dimer in solution

2019 ◽  
Vol 32 (6) ◽  
pp. 271-276
Author(s):  
Yuanyuan Wang ◽  
Zemao Gong ◽  
Han Fang ◽  
Dongming Zhi ◽  
Hu Tao
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Critical Role ◽  
Size Exclusion ◽  
Titration Calorimetry ◽  
Stable Complex ◽  
Binding Assays ◽  
Exclusion Chromatography ◽  
Multiple Copies

Abstract The pyruvate dehydrogenase complex (PDHc) from Escherichia coli is a large protein complex consisting of multiple copies of the pyruvate dehydrogenase (E1ec), dihydrolipoamide acetyltransferase (E2ec) and dihydrolipoamide dehydrogenase (E3ec). The N-terminal domain (NTD, residues 1–55) of E1ec plays a critical role in the interaction between E1ec and E2ec and the whole PDHc activity. Using circular dichroism, size-exclusion chromatography and dynamic light scattering spectroscopy, we show that the NTD of E1ec presents dimeric assembly under physiological condition. Pull-down and isothermal titration calorimetry binding assays revealed that the E2ec peripheral subunit-binding domain (PSBD) forms a very stable complex with the NTD, indicating the isolated NTD functionally interacts with PSBD and the truncated E1ec (E1ec∆NTD) does not interact with PSBD. These findings are important to understand the mechanism of PDHc and other thiamine-based multi-component enzymes.

scholarly journals The plasticity of the pyruvate dehydrogenase complex confers a labile structure that is associated with its catalytic activity

2020 ◽  
Author(s):  
Jaehyoun Lee ◽  
Seunghee Oh ◽  
Saikat Bhattacharya ◽  
Ying Zhang ◽  
Laurence Florens ◽  
...  
Keyword(s):  
Ionic Strength ◽  
Pyruvate Dehydrogenase ◽  
Biochemical Analysis ◽  
Pyruvate Dehydrogenase Complex ◽  
Size Exclusion ◽  
Dependent Manner ◽  
Acetyl Coa ◽  
Cell Extracts ◽  
Exclusion Chromatography ◽  
Dehydrogenase Complex

ABSTRACTThe pyruvate dehydrogenase complex (PDC) is a multienzyme complex that plays a key role in energy metabolism by converting pyruvate to acetyl-CoA. An increase of nuclear PDC has been shown to be correlated with an increase of histone acetylation that requires acetyl-CoA. PDC has been reported to form a ~ 10 MDa macromolecular machine that is proficient in performing sequential catalytic reactions via its three components. In this study, we show that the PDC displays size versatility in an ionic strength-dependent manner using size exclusion chromatography of yeast cell extracts. Biochemical analysis in combination with mass spectrometry indicates that yeast PDC (yPDC) is a salt-labile complex that dissociates into sub-megadalton individual components even under physiological ionic strength. Interestingly, we find that each oligomeric component of yPDC displays a larger size than previously believed. In addition, we show that the mammalian PDC also displays this uncommon characteristic of salt-lability, although it has a somewhat different profile compared to yeast. We show that the activity of yPDC is reduced in higher ionic strength. Our results indicate that the structure of PDC may not always maintain its ~ 10 MDa organization, but is rather variable. We propose that the flexible nature of PDC may allow modulation of its activity.

scholarly journals The plasticity of the pyruvate dehydrogenase complex confers a labile structure that is associated with its catalytic activity

PLoS ONE ◽  
2020 ◽  
Vol 15 (12) ◽  
pp. e0243489
Author(s):  
Jaehyoun Lee ◽  
Seunghee Oh ◽  
Saikat Bhattacharya ◽  
Ying Zhang ◽  
Laurence Florens ◽  
...  
Keyword(s):  
Ionic Strength ◽  
Pyruvate Dehydrogenase ◽  
Biochemical Analysis ◽  
Pyruvate Dehydrogenase Complex ◽  
Size Exclusion ◽  
Dependent Manner ◽  
Acetyl Coa ◽  
Cell Extracts ◽  
Exclusion Chromatography ◽  
Dehydrogenase Complex

The pyruvate dehydrogenase complex (PDC) is a multienzyme complex that plays a key role in energy metabolism by converting pyruvate to acetyl-CoA. An increase of nuclear PDC has been shown to be correlated with an increase of histone acetylation that requires acetyl-CoA. PDC has been reported to form a ~ 10 MDa macromolecular machine that is proficient in performing sequential catalytic reactions via its three components. In this study, we show that the PDC displays size versatility in an ionic strength-dependent manner using size exclusion chromatography of yeast cell extracts. Biochemical analysis in combination with mass spectrometry indicates that yeast PDC (yPDC) is a salt-labile complex that dissociates into sub-megadalton individual components even under physiological ionic strength. Interestingly, we find that each oligomeric component of yPDC displays a larger size than previously believed. In addition, we show that the mammalian PDC also displays this uncommon characteristic of salt-lability, although it has a somewhat different profile compared to yeast. We show that the activity of yPDC is reduced in higher ionic strength. Our results indicate that the structure of PDC may not always maintain its ~ 10 MDa organization, but is rather variable. We propose that the flexible nature of PDC may allow modulation of its activity.

scholarly journals Role of protein–protein interactions in the regulation of pyruvate dehydrogenase kinase activity

2005 ◽  
Vol 387 (1) ◽  
pp. 147-153 ◽  
Author(s):  
Alina TUGANOVA ◽  
Kirill M. POPOV
Keyword(s):  
Protein Interactions ◽  
Pyruvate Dehydrogenase ◽  
Kinase Activity ◽  
Pyruvate Dehydrogenase Complex ◽  
Critical Role ◽  
Pyruvate Dehydrogenase Kinase ◽  
Mutual Orientation ◽  
Titration Calorimetry ◽  
Protein Protein Interactions ◽  
Glutathione S Transferase

The transacetylase component (E2) of PDC (pyruvate dehydrogenase complex) plays a critical role in the regulation of PDHK (pyruvate dehydrogenase kinase) activity. The present study was undertaken to investigate further the molecular mechanism by which E2 modulates the activity of PDHK. In agreement with the earlier results, it was found that the inner L2 (lipoyl-bearing domain 2) of E2 expressed with or without the C-terminal hinge region had little, if any, effect on the kinase activity, indicating a lack of direct allosteric effect of L2 on PDHK. In marked contrast, significant activation of PDHK was observed with the construct consisting of L2 and the E1BD (E1-binding domain) of E2 (L2-E1BD didomain) suggesting that co-localization and/or mutual orientation of PDHK and E1, facilitated by E2 binding, largely account for the activation of PDHK by the transacetylase component. Isothermal titration calorimetry and glutathione S-transferase pull-down assays established that binding of adenyl nucleotides to the PDHK molecule facilitated the release of L2 domain. In contrast, binding of the L2 domain caused a significant decrease in the affinity of PDHK for ATP. The cross-talk in binding of adenyl nucleotides and the L2 domain to PDHK may indicate the existence of a highly integrated mechanism whereby the exchange of lipoyl-bearing domains presented to PDHK by E2 is coupled with ADP/ATP exchange.

scholarly journals L-Alanine Prototrophic Suppressors Emerge from L-Alanine Auxotroph through Stress-Induced Mutagenesis in Escherichia coli

2021 ◽  
Vol 9 (3) ◽  
pp. 472
Author(s):  
Harutaka Mishima ◽  
Hirokazu Watanabe ◽  
Kei Uchigasaki ◽  
So Shimoda ◽  
Shota Seki ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Spontaneous Mutation ◽  
Point Mutations ◽  
Whole Genome Analysis ◽  
E Coli ◽  
Clone Pair ◽  
Induced Mutagenesis ◽  
Isogenic Mutants

In Escherichia coli, L-alanine is synthesized by three isozymes: YfbQ, YfdZ, and AvtA. When an E. coli L-alanine auxotrophic isogenic mutant lacking the three isozymes was grown on L-alanine-deficient minimal agar medium, L-alanine prototrophic mutants emerged considerably more frequently than by spontaneous mutation; the emergence frequency increased over time, and, in an L-alanine-supplemented minimal medium, correlated inversely with L-alanine concentration, indicating that the mutants were derived through stress-induced mutagenesis. Whole-genome analysis of 40 independent L-alanine prototrophic mutants identified 16 and 18 clones harboring point mutation(s) in pyruvate dehydrogenase complex and phosphotransacetylase-acetate kinase pathway, which respectively produce acetyl coenzyme A and acetate from pyruvate. When two point mutations identified in L-alanine prototrophic mutants, in pta (D656A) and aceE (G147D), were individually introduced into the original L-alanine auxotroph, the isogenic mutants exhibited almost identical growth recovery as the respective cognate mutants. Each original- and isogenic-clone pair carrying the pta or aceE mutation showed extremely low phosphotransacetylase or pyruvate dehydrogenase activity, respectively. Lastly, extracellularly-added pyruvate, which dose-dependently supported L-alanine auxotroph growth, relieved the L-alanine starvation stress, preventing the emergence of L-alanine prototrophic mutants. Thus, L-alanine starvation-provoked stress-induced mutagenesis in the L-alanine auxotroph could lead to intracellular pyruvate increase, which eventually induces L-alanine prototrophy.

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