SEQUENTIAL H-1 AND N-15 NUCLEAR-MAGNETIC-RESONANCE ASSIGNMENTS AND SECONDARY STRUCTURE OF THE N-TERMINAL LIPOYL DOMAIN OF THE DIHYDROLIPOYLTRANSACETYLASE COMPONENT OF THE PYRUVATE-DEHYDROGENASE COMPLEX FROM AZOTOBACTER-VINELANDII

The N-terminal lipoyl domain (79 residues) of the transacetylase compo nent of the pyruvate dehydrogenase complex from Azotobacter vinelandii has been sub-cloned and produced in Escherichia coli. Over-expression exceeds the capacity of E. coli cells to lipoylate all expressed lipo yl domain, but addition of lipoic acid to the growth medium results in expression of fully lipolylated domain. A two-dimensional homo- and h eteronuclear NMR study of the lipoyl domain has resulted in sequential H-1 and N-15 resonance assignments of the unlipoylated form of the pr otein. Small differences in chemical shift values for protons of resid ues in the vicinity of the lipoyl-lysine residue are observed for the lipoylated form of the domain, suggesting that the conformation of the lipoyl domain is not altered significantly by the coupled cofactor. F rom nuclear Overhauser effects, backbone coupling constants and slowly exchanging amide protons, two antiparallel beta-sheets, each containi ng four strands, were identified. The lipoyl-lysine residue is exposed to the solvent and located in a type-I turn between two strands. The N- and C-terminal residues of the folded chain are close together in t he other sheet. Preliminary data on the relative three-dimensional ori entation of the two beta-sheets are presented. Comparison with the sol ution structure of the lipoyl domain of the Bacillus stearothermophilu s pyruvate dehydrogenase complex shows resemblance to a large extent, despite the sequence identity of 31%.