Structure, Expression, and Protein Engineering of the Pyruvate Dehydrogenase Complex of Escherichia coli

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease characterized by the presence of antimitochondrial antibodies in the serum. It is possible that the PBC-specific immunoreactive trypsin-sensitive antigens on the inner mitochondrial membrane, termed M2, are important in the pathogenesis of this autoimmune disease. We have previously shown that a major M2"a" antigen is the E2 component of the pyruvate dehydrogenase multienzyme complex located within mitochondria. Analysis of the primary structure of the E2 components of all three 2-oxo acid dehydrogenase complexes reveals a high degree of homology with a similar highly segmented structure including lipoyl domains, E3-binding domains, C-terminal catalytic domains, and interdomain linker sequences. Immunoblotting of PBC patients' sera against purified E2 protein from 2-oxoglutarate dehydrogenase complex and branched-chain 2-oxo acid dehydrogenase complex reveals that these polypeptides are also autoantigens in this disease. Sera from 29 of 40 (72.5%) PBC patients gave a positive response against bovine 2-oxoglutarate dehydrogenase complex E2 and from 25 of 40 (62.5%) PBC patients gave a positive response against bovine branched-chain 2-oxo acid dehydrogenase complex E2. All 40 PBC patients (100%) have autoantibodies directed against at least one of the E2 components of the family of 2-oxo acid dehydrogenase complexes. Identification of these M2 mitochondrial autoantigens and detailed knowledge of their structure will allow important questions concerning this autoimmune disease to be addressed.

Chloramphenicol acetyltransferase [acetyl-CoA:chloramphenicol O3-acetyltransferase; EC 2.3.1.28] is the enzyme responsible for high-level bacterial resistance to the antibiotic chloramphenicol. It catalyzes the transfer of an acetyl group from acetyl CoA to the primary hydroxyl of chloramphenicol. The x-ray crystallographic structure of the type III variant enzyme from Escherichia coli has been determined and refined at 1.75-A resolution. The enzyme is a trimer of identical subunits with a distinctive protein fold. Structure of the trimer is stabilized by a beta-pleated sheet that extends from one subunit to the next. The active site is located at the subunit interface, and the binding sites for both chloramphenicol and CoA have been characterized. Substrate binding is unusual in that the two substrates approach the active site via clefts on opposite molecular "sides." A histidine residue previously implicated in catalysis is appropriately positioned to act as a general base catalyst in the reaction.