scholarly journals Crystal structure of the catalytic subunit of bovine pyruvate dehydrogenase phosphatase

2020 ◽  
Vol 76 (7) ◽  
pp. 292-301
Author(s):  
Youzhong Guo ◽  
Weihua Qiu ◽  
Thomas E. Roche ◽  
Marvin L. Hackert
Keyword(s):  
Crystal Structure ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Ion Pairs ◽  
Catalytic Subunit ◽  
Regulatory Subunit ◽  
Amino Acid Residues ◽  
Cell Parameters ◽  
Twofold Symmetry Axis ◽  
Pyruvate Dehydrogenase Phosphatase

Mammalian pyruvate dehydrogenase (PDH) activity is tightly regulated by phosphorylation and dephosphorylation, which is catalyzed by PDH kinase isomers and PDH phosphatase isomers, respectively. PDH phosphatase isomer 1 (PDP1) is a heterodimer consisting of a catalytic subunit (PDP1c) and a regulatory subunit (PDP1r). Here, the crystal structure of bovine PDP1c determined at 2.1 Å resolution is reported. The crystals belonged to space group P3221, with unit-cell parameters a = b = 75.3, c = 173.2 Å. The structure was solved by molecular-replacement methods and refined to a final R factor of 21.9% (R free = 24.7%). The final model consists of 402 of a possible 467 amino-acid residues of the PDP1c monomer, two Mn2+ ions in the active site, an additional Mn2+ ion coordinated by His410 and His414, two MnSO4 ion pairs at special positions near the crystallographic twofold symmetry axis and 226 water molecules. Several new features of the PDP1c structure are revealed. The requirements are described and plausible bases are deduced for the interaction of PDP1c with PDP1r and other components of the pyruvate dehydrogenase complex.

Regulation of pyruvate dehydrogenase activity through phosphorylation at multiple sites

2001 ◽  
Vol 358 (1) ◽  
pp. 69-77 ◽  
Author(s):  
Elena KOLOBOVA ◽  
Alina TUGANOVA ◽  
Igor BOULATNIKOV ◽  
Kirill M. POPOV
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Phosphorylation Site ◽  
Enzymic Activity ◽  
Mammalian Tissues ◽  
Activity State ◽  
The Individual ◽  
Pyruvate Dehydrogenase Phosphatase ◽  
Thiamin Pyrophosphate ◽  
Dehydrogenase Complex

The enzymic activity of the mammalian pyruvate dehydrogenase complex is regulated by the phosphorylation of three serine residues (sites 1, 2 and 3) located on the E1 component of the complex. Here we report that the four isoenzymes of protein kinase responsible for the phosphorylation and inactivation of pyruvate dehydrogenase (PDK1, PDK2, PDK3 and PDK4) differ in their abilities to phosphorylate the enzyme. PDK1 can phosphorylate all three sites, whereas PDK2, PDK3 and PDK4 each phosphorylate only site 1 and site 2. Although PDK2 phosphorylates site 1 and 2, it incorporates less phosphate in site 2 than PDK3 or PDK4. As a result, the amount of phosphate incorporated by each isoenzyme decreases in the order PDK1>PDK3PDK4>PDK2. Significantly, binding of the coenzyme thiamin pyrophosphate to pyruvate dehydrogenase alters the rates and stoichiometries of phosphorylation of the individual sites. First, the rate of phosphorylation of site 1 by all isoenzymes of kinase is decreased. Secondly, thiamin pyrophosphate markedly decreases the amount of phosphate that PDK1 incorporates in sites 2 and 3 and that PDK2 incorporates in site 2. In contrast, the coenzyme does not significantly affect the total amount of phosphate incorporated in site 2 by PDK3 and PDK4, but instead decreases the rate of phosphorylation of this site. Furthermore, pyruvate dehydrogenase complex phosphorylated by the individual isoenzymes of kinase is reactivated at different rates by pyruvate dehydrogenase phosphatase. Both isoenzymes of phosphatase (PDP1 and PDP2) readily reactivate the complex phosphorylated by PDK2. When pyruvate dehydrogenase is phosphorylated by other isoenzymes, the rates of reactivation decrease in the order PDK4PDK3> PDK1. Taken together, results reported here strongly suggest that the major determinants of the activity state of pyruvate dehydrogenase in mammalian tissues include the phosphorylation site specificity of isoenzymes of kinase in addition to the absolute amounts of kinase and phosphatase protein expressed in mitochondria.

scholarly journals Pyruvate Dehydrogenase Phosphatase Deficiency: Identification of the First Mutation in Two Brothers and Restoration of Activity by Protein Complementation

2005 ◽  
Vol 90 (7) ◽  
pp. 4101-4107 ◽  
Author(s):  
Mary C. Maj ◽  
Neviana MacKay ◽  
Valeriy Levandovskiy ◽  
Jane Addis ◽  
E. Regula Baumgartner ◽  
...  
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Genetic Basis ◽  
Pyruvate Dehydrogenase Complex ◽  
Protein A ◽  
Wild Type ◽  
Cell Extracts ◽  
Steady State Levels ◽  
Pyruvate Dehydrogenase Phosphatase ◽  
Protein Complementation ◽  
Elevated Lactate

Abstract Context: Pyruvate dehydrogenase phosphatase (PDP) deficiency has been previously reported as an enzymopathy, but the genetic basis for such a defect has never been established. Objective: The aim of this study was to identify the cause of the defect in two patients who presented with PDP deficiency. Patients: We studied two brothers of consanguineous parents who presented with neonatal hypotonia, elevated lactate, and less than 25% native pyruvate dehydrogenase complex (PDHc) activity in skin fibroblasts compared with controls. The activity of the complex could be restored to normal values by preincubation of the cells with dichloroacetate or by treating cell extracts with calcium. Results: These two individuals were found to be homozygous for a 3-bp deletion in the coding sequence of the PDP isoform 1 (PDP1), which removes the amino acid residue leucine from position 213 of the protein. A recombinant version of this protein was synthesized and found to have a very reduced (<5%) ability to activate purified PDHc. Reduced steady-state levels of PDP1 in the patient’s fibroblasts coupled with the low catalytic activity of the mutant PDP1 resulted in native PDHc activity being reduced, but this could be corrected by the addition of recombinant PDP1 (wild type). Conclusion: We have identified mutations in PDP1 in two brothers with PDP deficiency and have proven that the mutation is disease-causing. This is the first demonstration of human disease due to a mutation in PDP1.

One-Step Purification of the Recombinant Catalytic Subunit of Pyruvate Dehydrogenase Phosphatase

2000 ◽  
Vol 20 (1) ◽  
pp. 128-131 ◽  
Author(s):  
Wahn Soo Choi ◽  
Jiangong Yan ◽  
Diane B. McCarthy ◽  
Seung Hee Park ◽  
Lester J. Reed
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Catalytic Subunit ◽  
One Step ◽  
Pyruvate Dehydrogenase Phosphatase
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