CRYSTAL-STRUCTURE OF THE TRUNCATED CUBIC CORE COMPONENT OF THE ESCHERICHIA-COLI 2-OXOGLUTARATE DEHYDROGENASE MULTIENZYME COMPLEX

The dihydrolipoamide succinyltransferase (E2o) component of the 2-oxog lutarate dehydrogenase multienzyme complex is composed of 24 subunits arranged with 432 point group symmetry. The catalytic domain (CD) of t he E2o component catalyzes the transfer of a succinyl group from the S -succinyldihydrolipoyl moiety to coenzyme A. The crystal structure of the Escherichia coli E2oCD has been solved to 3.0 Angstrom resolution using molecular replacement phases derived from the structure of the c atalytic domain from the Azotobacter vinelandii dihydrolipoamide acety ltransferase (E2pCD). The refined model of the E. coli E2oCD consists of residues 172 to 404 and has an R-factor of 0.205 (R-free = 0.249) f or 9696 reflections between 20.0 and 3.0 Angstrom resolution. Although both E2oCD and E2pCD form 24mers, subtle changes in the orientations of two helices in E2oCD increase the stability of the E2oCD 24mer in c omparison to the less stable A. vinelandii E2pCD 24mer. Like E2pCD and chloramphenicol acetyltransferase (CAT), the active site of E2oCD is located in the middle of a channel formed at the interface between two S-fold related subunits. Two of the active-site residues (His375 and Thr323) have a similar orientation to their counterparts in E2pCD and CAT. A third catalytic residue (Asp379) assumes a conformation similar to the corresponding residue in E2pCD (Asn614), but different from it s counterpart in CAT (Asp199). Binding of the substrates to E2oCD is p roposed to induce a change in the conformation of Asp379, allowing thi s residue to form a salt bridge with Arg184 that is analogous to that formed between Asp199 and Arg18 in CAT. Computer models of the active site of E2o complexed with dihydrolipoamide and with coenzyme A led to the identification of the probable succinyl-binding pocket. The resid ues which form this pocket (Ser330, Ser333, and His348) are probably r esponsible for E2o's substrate specificity. (C) 1998 Academic Press.