The backbone and tryptophan side-chain dynamics of both the reduced and oxidized forms of uniformly 15N-labeled Escherichia coli thioredoxin have been characterized using inverse-detected two-dimensional 1H-15N NMR spectroscopy. Longitudinal (T1) and transverse (T2) 15N relaxation time constants and steady-state (1H)-15N NOEs were measured for more than 90% of the protonated backbone nitrogen atoms and for the protonated indole nitrogen atoms of the two tryptophan residues. These data were analyzed by using a model free dynamics formalism to determine the generalized order parameter (S2), the effective correlation time for internal motions (tau e), and 15N exchange broadening contributions (Rex) for each residue, as well as the overall molecular rotational correlation time (tau m). The reduced and oxidized forms exhibit almost identical dynamic behavior on the picosecond to nanosecond time scale. The W31 side chain is significantly more mobile than the W28 side chain, consistent with the positions of W31 on the protein surface and W28 buried in the hydrophobic core. Backbone regions which are significantly more mobile than the average include the N-terminus, which is constrained in the crystal structure of oxidized thioredoxin by specific contacts with a Cu2+ ion, the C-terminus, residues 20-22, which constitute a linker region between the first alpha-helix and the second beta-strand, and residues 73-75 and 93-94, which are located adjacent to the active site. In contrast, on the microsecond to millisecond time scale, reduced thioredoxin exhibits considerable dynamic mobility in the residue 73-75 region, while oxidized thioredoxin exhibits no significant mobility in this region. The possible functional implications of the dynamics results are discussed.

Thiamin-dependent enzymes play key roles in sugar metabolism, typically catalyzing the decarboxylation of ␣-keto acids and the transfer of an aldehyde or an acyl group (1-5). Examples include the E1 components in pyruvate dehydrogenase complexes (PDHc), 2 pyruvate decarboxylase, transketolase, etc. Crystallographic studies (6 -12) have elucidated many of the structural, stereochemical, and biochemical details in the mechanism of action of these enzymes and in the catalytic role of the cofactor ThDP (thiamin diphosphate, vitamin B1 diphosphate, Fig. 1, top left). Despite the enormous contributions made by these and other studies to our understanding of how such enzymes function, important details still remain obscure. There are, for example, no detailed structural data on the first ThDP-bound intermediate in the presence of any enzyme (for example, ␣-lactylthiamin diphosphate (␣-LThDP) in PDHc E1 and pyruvate decarboxylase), which is postulated to form in the currently accepted mechanism of thiamin catalysis (Fig. 1, top, third object from the right). In an effort to obtain structural information pertaining to this key intermediate, we have determined the crystal structure of PDHc E1 from Escherichia coli in complex with ␣-phosphonolactylthiamin diphosphate (PLThDP).PLThDP is the product of the reaction between ThDP and methylacetylphosphonate, with the latter being an analogue of the true substrate pyruvate and a potent inhibitor of PDHc. The complex formed with PLThDP instead of ThDP therefore mimics the structure of the enzyme-bound, reactive tetrahedral intermediate ␣-LThDP (13) in the decarboxylation step of the PDHc E1 reaction. It differs from the complex formed with the true substrate only in the replacement of the carboxylate group by a methyl phosphonate (PO 3 Me) group. However, unlike the C2␣-CO 2 bond normally cleaved in the reaction with pyruvate, the C2␣-PO 3 Me bond remains intact. The reaction is therefore trapped in a pre-CO 2 release-like state, and the structure represents a covalently bound, pre-decarboxylation reaction intermediate analogue.There have been three covalently bound reaction intermediate structures reported for ThDP-dependent enzymes (11,12,14), but they all represented the planar enamine intermediate (Fig. 1, top right object) that exists only after decarboxylation. The E1-PLThDP structure is thus the first structural example of a covalently bound, pre-decarboxylation reaction intermediate analogue in any ThDP-dependent enzyme.* This work was supported by a grant from the Veterans Affairs Merit Review Program and National Institutes of Health Grant GM-61791 (to W. F.) and by National Institutes of Health Grant GM-62330 (to F. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The atomic coordinates and structure factors (codes 2G25 and 2G28)