Refined Crystal Structure of the Catalytic Domain of Dihydrolipoyl Transacetylase (E2p) from Azotobacter vinelandii at 2·6 Å Resolution

Dihydrolipoyl transacetylase (E2p) is both structurally and functionally the central enzyme of the pyruvate dehydrogenase multienzyme complex. The crystal structure of the catalytic domain, i.e. residues 382 to 637, of Azotobacter vinelandii E2p (E2pCD) was solved by multiple isomorphous replacement and refined by energy minimization procedures. The final model contains 2182 protein atoms and 37 ordered water molecules. The R-factor is 18·7% for 10,344 reflections between 10·0 and 2·6 Å resolution. The root-mean-square shift deviation from the ideal values is 0·017 Å for bond lengths and 3·3° for bond angles. The N-terminal residues 382 to 394 are disordered and not visible in the electron density map, otherwise all residues have well-defined density. The catalytic domain forms an oligomer of 24 subunits, having octahedral 432 symmetry. In the E2pCD crystals, the 24 subunits are related by the crystallographic symmetry. The cubic arrangement of subunits gives rise to a large hollow cube with edges of 120 Å. The faces of the cube have pores of diameter of 30 Å. The true building block of the cube is the E2p trimer, eight of which occupy the corners of the cube. Two levels of intermolecular contacts can be distinguished: (1) the extensive interactions between 3-fold related subunits leading to a tightly associated trimer; and (2) the interactions along the 2-fold axis leading to the assembly of the trimers into the cubic 24-mer. Each subunit has a topology similar to chloramphenicol acetyltransferase (CAT) and comprises a central β-sheet surrounded by five α-helices. The comparison of the two proteins indicates a large rotation of the N-terminal residues 395 to 426 of E2pCD, which reshapes the substrate binding site and extends the interaction between threefold related subunits. The catalytic centre consists of a 30 Å long channel extending from the "inner" side of the trimmer to the "outer" side, where inner and outer refer to the position in the 24-meric cubic core of the pyruvate dehydrogenase complex and correspond with CoA and lipoamide binding sites, respectively. The active site is formed by the residues with the lowest mobility as indicated by the atomic B -factors. Five proline residues surround the active site. The side-chain of His610, which, by analogy with CAT, is most likely involved in catalysis, is stabilized in its unusual conformation by the salt-bridge between Asp609 and Arg611, and by contacts with the side-chains of Val435, Tyr608, Leu425′ and Ile571′, the latter two residues being located on a threefold related subunit. At the N terminus of the protein, residues 395 to 402 form an extended arm. Since they are part of the linker connecting the catalytic to the E1/E3 binding domain, their conformation is suggestive that the linker might consist of segments of rather inflexible extended polypeptide chain connected to each other by more flexible "hinge" residues