Poster: Enzymology

Abs # 257: Why does dihydrolipoamide acetyltransferase need more than one lipoyl-domain?

Presenter:	Broz, Amanda K, akbbb0@mizzou.edu
Authors	Broz, Amanda K (A)   Mooney, Brian P (A)   Miernyk, Jan A (A) (B)  Randall, Doug D (A)
Affiliations:	(A): University of Missouri, Columbia (B): USDA-ARS Genetics Research Unit

The mitochondrial pyruvate dehydrogenase complex (mtPDC) is critical in regulating carbon flux into the Krebs cycle. Minimally, PDC consists of multiple copies of three enzymes: pyruvate dehydrogenase (E1), dihydrolipoamide acetyltransferase (E2), and dihydrolipoamide dehydrogenase (E3). The mtPDCs additionally include intrinsic kinase and phosphatase regulatory enzymes. The component proteins assemble non-covalently into a 8 MDa complex. The mtPDC core is a 60-mer of E2 subunits with a pentagonal dodecahedron structure. Flowering plant mitochondria all contain E2 with one lipoyl domain. However, some dicot plants exhibit an additional E2 with two lipoyl domains. We have identified three A. thaliana mtPDC E2 genes, a di-lipoyl form (E2II) plus two distinct mono-lipoyl forms (E2IA and E2IB). The deduced amino acid sequences encoded by the E2IA and E2IB proteins are closely related to the mono-lipoyl E2 protein from maize. Analysis of the three forms of A. thaliana mtPDC E2 by RT-PCR revealed expression varied within and between organs. Both E2IA and E2IB cDNAs have been cloned into bacterial expression vectors with and without affinity-fusion tags. These affinity tags will allow separation of the E2 proteins in vitro. Verification of core formation and activity of these recombinant E2s will be presented (e.g. E2IB studies show an enzymatically active icosahedral 60mer). We will also compare the effects of the various E2 cores on the binding and activity of the other components of mtPDC. To demonstrate that the A. thaliana E2 genes code for mitochondrial proteins, fusion constructs are being made between the full length E2 sequence in frame with a red fluorescent protein. These constructs will be expressed in tobacco BY-2 cells containing a mitochondrial GFP.