Structure of the Pyruvate Dehydrogenase Multienzyme Complex E1 Component fromEscherichia coliat 1.85 Å Resolution^,

Abstract

The crystal structure of the recombinant thiamin diphosphate-dependent E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) has been determined at a resolution of 1.85 A. The E. coli PDHc E1 component E1p is a homodimeric enzyme and crystallizes with an intact dimer in an asymmetric unit. Each E1p subunit consists of three domains: N-terminal, middle, and C-terminal, with all having alpha/beta folds. The functional dimer contains two catalytic centers located at the interface between subunits. The ThDP cofactors are bound in the "V" conformation in clefts between the two subunits (binding involves the N-terminal and middle domains), and there is a common ThDP binding fold. The cofactors are completely buried, as only the C2 atoms are accessible from solution through the active site clefts. Significant structural differences are observed between individual domains of E1p relative to heterotetrameric multienzyme complex E1 components operating on branched chain substrates. These differences may be responsible for reported alternative E1p binding modes to E2 components within the respective complexes. This paper represents the first structural example of a functional pyruvate dehydrogenase E1p component from any species. It also provides the first representative example for the entire family of homodimeric (alpha2) E1 multienzyme complex components, and should serve as a model for this class of enzymes.

Communication between Thiamin Cofactors in the Escherichia coli Pyruvate Dehydrogenase Complex E1 Component Active Centers

“…The three crystal structures studied here are all very similar (in a global sense) to the previously reported crystal structure of the E1ec-ThDP complex (12). The main-chain folds are identical, and two subunits are present in an asymmetric unit.…”

“…There is no clear electron density for the thiazolium and 4-aminopyrimidine rings. The observed diphosphate group interaction, nevertheless, is similar to that observed in the E1ec-ThDP structure (12). The octahedrally coordinated Mg 2ϩ binding site contains three protein ligands, two oxygen atoms from the diphosphate and one water molecule.…”

“…For crystallization of the E571A-ThDP, the protein concentration was 25.6 mg/ml, whereas for the E235A-ThDP, it was 28 mg/ml with the ThDP concentrations at both 0.5 and 5 mM in the protein solution. In all cases, the crystals grew within 4 -6 weeks and were isomorphous to E1ec-ThDP crystals (12). The crystals typically have dimensions of 0.15 ϫ 0.20 ϫ 0.30 mm.…”

“…The E571A-ThDP data set was truncated at 2.1 Å resolution, whereas the E235A-ThDP data sets were truncated at 1.96 and 1.98 Å resolution for the complexes of E235A crystallized with 0.5 and 5 mM ThDP, respectively. Structure Determination and Refinement-Because the E1ec-ThDP crystals are isomorphous with the E571A-ThDP and E235A-ThDP crystals, the atomic coordinates of the E1ec-ThDP structure, refined to 1.85 Å resolution (12), were used as the starting model. The initial model included 1602 amino acids, with the cofactors ThDP and Mg 2ϩ along with neighboring active site residues omitted.…”

Efficient coupling of catalysis and dynamics in the E1 component of Escherichia coli pyruvate dehydrogenase multienzyme complex

“…E1ec is a thiamin diphosphate (ThDP)-dependent ␣ 2 homodimer with mass 198,948 Da and catalyzes the reactions shown in supporting information (SI) Scheme I. The crystal structure of E1ec complexed with ThDP revealed two disordered regions near the active site with no discernible electron density (2), spanning residues 401-413 (inner loop) and 541-557 (outer loop), which become ordered in the presence of C2␣-phosphonolactylThDP (PLThDP), a stable analogue of the first ThDP-bound predecarboxylation covalent intermediate C2␣-lactylThDP (LThDP) (3) (Fig. 1).…”