Abstract
The crystal structure of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) has been determined with phosphonolactylthiamin diphosphate (PLThDP) in its active site. PLThDP serves as a structural and electrostatic analogue of the natural intermediate alpha-lactylthiamin diphosphate (LThDP), in which the carboxylate from the natural substrate pyruvate is replaced by a phosphonate group. This represents the first example of an experimentally determined, three-dimensional structure of a thiamin diphosphate (ThDP)-dependent enzyme containing a covalently bound, pre-decarboxylation reaction intermediate analogue and should serve as a model for the corresponding intermediates in other ThDP-dependent decarboxylases. Regarding the PDHc-specific reaction, the presence of PLThDP induces large scale conformational changes in the enzyme. In conjunction with the E1-PLThDP and E1-ThDP structures, analysis of a H407A E1-PLThDP variant structure shows that an interaction between His-407 and PLThDP is essential for stabilization of two loop regions in the active site that are otherwise disordered in the absence of intermediate analogue. This ordering completes formation of the active site and creates a new ordered surface likely involved in interactions with the lipoyl domains of E2s within the PDHc complex. The tetrahedral intermediate analogue is tightly held in the active site through direct hydrogen bonds to residues His-407, Tyr-599, and His-640 and reveals a new, enzyme-induced, strain-related feature that appears to aid in the decarboxylation process. This feature is almost certainly present in all ThDP-dependent decarboxylases; thus its inclusion in our understanding of general thiamin catalysis is important.
Highlights
The crystal structure of the E1 component from the Escherichia coli pyruvate dehydrogenase multienzyme complex (PDHc) has been determined with phosphonolactylthiamin diphosphate (PLThDP) in its active site
The C2␣-C bond in ␣-LThDP is expected to be oriented relative to the thiazolium ring nearly the same as the C2␣-P bond seen in ␣-PLThDP, and this orientation had been predicted for both compounds [29]
Given the structural and electronic similarities, active site residues adjacent to C2␣, OH(2␣), CH3(2␣), and some of the phosphonyl and carboxylate oxygens are expected to have very nearly identical locations in both the catalytically active and the analogue intermediates, and the short N4Ј-O2␣ distance and outof-plane deformation of the C2–C2␣ bond observed in the E1-PLThDP complex should occur in the E1-␣-LThDP complex as well
Summary
Crystallization and Data Collection—The PDHc E1 from E. coli was purified and assayed according to published procedures [18]. Structure Determination and Refinement—Since the previous E1-ThDP crystals are isomorphous with both E1-PLThDP and H407A E1-PLThDP crystals (cell constants differing by less than 0.8 Å), atomic coordinates from the former structure were used as the starting model for both refinements. Examination of electron density difference maps clearly showed strong density for the intermediate analogue, PLThDP, and produced strong and interpretable electron density for two of the three previously unobserved regions, 401– 413 and 541–557. After including the PLThDP and the two missing regions, the model was refined with simulated annealing, and subsequent cycles consisted of positional and B factor refinement. The refinement procedure included periodic examinations of simulated annealing omit and difference maps, as well as the introduction of water molecules. For all data to 2.1 Å resolution, the R
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