Introduction
In all living cells, thiamine is essential in energy metabolism. Thiamine diphosphate (ThDP) is an essential cofactor for the metabolism of pyruvate, the end product of the glycolytic pathway; for the metabolism of 
-ketoglutarate, a central intermediate in the tricarboxylic acid pathway; and for the conversion of fructose-6-phosphate and xylulose-5-phosphate, intermediates in carbohydrate metabolism. ThDP also unlocks the chemistry of other processes in plant and microbial metabolism, including the production of acetolactate for amino acid biosynthesis and the decarboxylation of oxalyl coenzyme A in oxalate metabolism. Decades ago, bioorganic chemists unmasked essential aspects of the chemistry of ThDP and postulated rational chemical mechanisms for its biological functions1. In this issue, Tittmann and co-workers uncover the structures of three ThDP intermediates at the active site of pyruvate oxidase and show the ways these are correlated with the theory of ThDP action2.
Chemical synthesis of the compounds 2-(-hydroxyethyl)-ThDP, 2-(,
-dihydroxyethyl)-ThDP and 2-acetyl-ThDP (AcThDP) opened the door to testing the prevailing theories about the action of ThDP. Two lines of evidence proved that the compounds were chemically competent as intermediates. First, the authentic compounds were shown to be identical to intermediates produced by ThDP-dependent enzymes3, 4, 5, 6, 7. Further, these compounds were transformed by the enzymes into the expected products. The next advance came when Tittmann and co-workers performed transient kinetic analyses to observe the kinetic behavior of the presumed intermediates. Their rapid mix-quench NMR experiments demonstrated the transient production of several of the synthetic compounds as reaction intermediates. These included 2-(-hydroxyethyl)-ThDP, AcThDP and a species that seemed to be 2-lactyl-ThDP (LThDP), which had not previously been synthesized8.
The ThDP- and flavin-dependent pyruvate oxidase (POX) catalyzes the conversion of pyruvate, O2 and inorganic phosphate (Pi) into acetyl phosphate, CO2 and H2O2 by a mechanism that is not fully understood but involves several ThDP intermediates. The current study by Wille and associates reports the X-ray crystal structures of POX with three of the postulated ThDP intermediates bound at the active site1. Wille and colleagues used clever biochemical strategies and cryocrystallography to obtain these structures. S-LThDP is the dominant form at the steady state owing to rate limitation by decarboxylation, so that crystals that are soaked with substrate and frozen contain this intermediate. Reduction of the flavin by an external reducing agent blocks electron transfer within POX, so that 2-(-hydroxyethylidene)-ThDP accumulates and is observed in crystals frozen after addition of pyruvate. Exclusion of phosphate blocks acetyl transfer and allows AcThDP to accumulate and be observed in the frozen crystals. One of the intermediates, LThDP, has not been observed before at an enzymatic site, and details of its structure are revealed here for the first time, including its absolute stereochemical configuration. Moreover, the conformation of the carboxyl moiety turns out to be stereoelectronically ideal for the next step in the mechanism, the decarboxylation of this intermediate.
The structures of AcThDP and 2-(-hydroxyethylidene)-ThDP at the active site are also described, as well as that of a phosphonate analog of LThDP. LThDP has an optical center at C2 of the lactyl moiety. Synthetic LThDP would be racemic, but active sites generally are asymmetric and likely to accommodate only one stereoisomer. This is borne out by the structure of the POX-LThDP complex2. The structure shows S-LThDP at the active site, with the carboxylate plane positioned perpendicular to the plane of the thiazolium ring (Fig. 1).
Figure 1: S-LThDP.
The structure of POX with LThDP bound at the active site reveals the configuration at the optical center at C2 of the lactyl group in the S configuration, as it exists in the enzymatic site. P, phosphate. Boldface atoms define the stereocenter.
Full size image (26 KB)Other ThDP-dependent enzymes that process pyruvate also generate LThDP as the predecarboxylation species. It will be interesting to see whether they also generate the S stereoisomer of this new intermediate. LThDP is presumed to be an intermediate in the reactions of pyruvate with other ThDP-dependent enzymes, including pyruvate decarboxylase, pyruvate dehydrogenase, acetolactate synthase, pyruvate:ferridoxin oxidoreductase, the flavin-independent pyruvate oxidase and other pyruvate oxidoreductases. If these enzymes are all part of the same evolutionary family, one might expect S-LThDP to be a common intermediate within the family. However, the only way to determine whether this is true is by application of the biochemical and crystallographic methods used in the study of Wille and associates2.
