The Pyruvate Dehydrogenase Complex and homologous multienzyme complexes form enormous macromolecular assemblies (Mw 4-10 million Da). The complexes have important functions in central metabolism and are of great medical relevance as sites of mutations causing defects in humans and as potential drug targets in human parasites. The main enzyme components, each present in multiple copies, are called E1, E2 and E3. Structures of E2 and E3 from different sources have been determined. This includes the recently solved structures of the dodecahedral 60 subunit cores of pyruvate dehydrogenase multienzyme complexes from Enterococcus feacalis and Bacillus stearothermophilus (Izard et al., manuscript in preparation). No structural information is available for the E1 component which is the most important protein in terms of regulation of the complexes and from medical perspective. This information would also make it possible to build a more detailed model of a whole complex especially with additional information from structures of complexes of different components. We are currently focusing on E1 from different sources. We have obtained crystals of: A) Pseudomonas putida E1b, a heterotetramer (alpha2beta2) with a molecular weight of 160 kDa. B) Bacilllus stearothermophilus E1p (alpha2beta2) in complex with a fragment of E2p (binding domain and lipoyl domain) with a molecular weight of 170 kDa. C) Human E1b (alpha2beta2) with a molecular weight of 160 kDa. Native data has been collected for all three proteins, with and without cofactor TTP and a substrate analogue and resolution ranging from 2.8 to 2.4A (native data of A was collected at CHESS F1 to 2.6A resolution). Preliminary phases are available for A. MIR and MAD data (from a seleno-methionine derivate) analysis for this protein is in progress.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR001646-17
Application #
6220494
Study Section
Project Start
1999-08-15
Project End
2000-08-14
Budget Start
1998-10-01
Budget End
1999-09-30
Support Year
17
Fiscal Year
1999
Total Cost
Indirect Cost
Name
Cornell University
Department
Type
DUNS #
City
Ithaca
State
NY
Country
United States
Zip Code
14850
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