Expression, purification, and structural analysis of the trimeric form of the catalytic domain of the Escherichia coli dihydrolipoamide succinyltransferase

The dihydrolipoamide succinyltransferase (E2o) component of the alpha-ketog lutarate dehydrogenase complex catalyzes the transfer of a succinyl group f rom the S-succinyldihydrolipoyl moiety to coenzyme A. E2o is normally a 24- mer, but is found as a trimer when E2o is expressed with a C-terminal [His] (6) tag. The crystal structure of the trimeric form of the catalytic domain (CD) of the Escherichia coli E2o has been solved to 3.0 Angstrom resolutio n using the Molecular Replacement method. The refined model contains an int act trimer in the asymmetric unit and has an R-factor of 0.257 (R-free = 0. 286) for 18,699 reflections between 10.0 and 3.0 Angstrom resolution. The c ore of tE2oCD (residues 187-396) superimposes onto that of the cubic E2oCD with an RMS difference of 0.4 Angstrom for all main-chain atoms. The C-term inal end of tE2oCD (residues 397-404) rotates by an average of 37 degrees c ompared to cubic E2oCD, disrupting the normal twofold interface. Despite th e alteration of quaternary structure, the active site of tE2oCD shows no si gnificant differences from that of the cubic E2oCD, although several side c hains in the active site are more ordered in the trimeric form of E2oCD. An alysis of the available sequence data suggests that the majority of E2 comp onents have active sites that resemble that of E. coli E2oCD, The remaining E2 components can be divided into three groups based on active-site sequen ce similarity. Analysis of the surface properties of both crystal forms of E. coli E2oCD suggests key residues that may be involved in the protein-pro tein contacts that occur between the catalytic and lipoyl domains of E2o.