Active-site changes in the pyruvate dehydrogenase multienzyme complex E1 apoenzyme component from Escherichia coli observed at 2.32 Å resolution

The first enzymatic component, E1 (EC 1.2.4.1), of the pyruvate dehydrogenase multienzyme complex (PDHc) utilizes thiamine diphosphate (ThDP) and Mg²⁺ as cofactors. The structure of a branched-chain-specific E1 apoenzyme from the heterotetrameric α₂β₂ E1 family was recently reported and showed that disorder-to-order transformations in two active-site loops take place upon cofactor binding. To ascertain what effect the absence of cofactor may have in the homodimeric α₂ Escherichia coli PDHc E1, the corresponding apoenzyme has been prepared and its three-dimensional structure determined and analyzed at 2.32 Å by crystallographic methods. This represents the first reported apoenzyme structure for any E1 component from the homodimeric α₂ family. Electron-density features occurring in the region where the cofactor pyrimidine ring would normally be expected to bind are of size, shape and location compatible with water molecules that form a hydrogen-bonded linkage between residues Glu571 and Val192, which normally make conserved interactions with the ThDP cofactor. A histidine side chain that normally forms hydrogen bonds to ThDP is disordered in its absence and partially occupies two sites. Unlike in the reported heterotetrameric branched-chain apo-­E1, no disorder/order loop transformations are evident in apo-PDHc E1 relative to the holo-E1 enzyme (PDHc E1–ThDP–Mg²⁺). Differences in the extent of hydrogen-bonding networks found in the apo-E1 enzyme, the holo-E1 enzyme and in an inhibitor complex with bound thiamine 2-thiazolone diphosphate (ThTDP), PDHc E1–ThTDP–Mg²⁺, are described.