Structures of Thiamin-Dependent Enzymes

Structures of Thiamin-Dependent Enzymes 4. The Variety of Enzymatic Reactions Involving Thiamin 5. Oxidative Decarboxylation and 2-Acetylthiamin Diphosphate. 6. Thiamin Coenzymes in Nerve Action 753. .. Table 14-4 Some Pyruvoyl Enzymes... 

The crystal structures of thiamin-dependent enzymes (see next section) as well as modeling102 103 suggest that lactylthiamin pyrophosphate has the conformation shown in Eq. 14-21. If so, it would be formed by the addition of the ylid to the carbonyl of pyruvate in accord with stereoelectronic principles, and the carboxylate group would also be in the correct orientation for elimination to form the enamine in Eq. 14-21, step b.82 83a A transient 380- to 440-nm absorption band arising during the action of pyruvate decarboxylase has been attributed to the enamine. 

The reaction path of thiamine-dependent catalysis is essentially unchanged in the presence of an apoenzyme, except that the enzyme active site residues increase reaction rates and yields and influence the substrate and product specificity. The X-ray crystal structures of TDP-dependent enzymes have clarified this view and permitted an understanding of the roles of the individual amino acids of the active site in activating and controlling the thiazolium reactivity [36-40]. 

Acyloins (a-hydroxy ketones) are formed enzymatically by a mechanism similar to the classical benzoin condensation. The enzymes that can catalyze reactions of this type arc thiamine dependent. In this sense, the cofactor thiamine pyrophosphate may be regarded as a natural- equivalent of the cyanide catalyst needed for the umpolung step in benzoin condensations. Thus, a suitable carbonyl compound (a -synthon) reacts with thiamine pyrophosphate to form an enzyme-substrate complex that subsequently cleaves to the corresponding a-carbanion (d1-synthon). The latter adds to a carbonyl group resulting in an a-hydroxy ketone after elimination of thiamine pyrophosphate. Stereoselectivity of the addition step (i.e., addition to the Stand Re-face of the carbonyl group, respectively) is achieved by adjustment of a preferred active center conformation. A detailed discussion of the mechanisms involved in thiamine-dependent enzymes, as well as a comparison of the structural similarities, is found in references 1 -4. 

Figure 28. Modification of the nucleophile mechanism for the reaction of nitrosoarene compounds with thiamine-dependent enzymes. Structure A is the putative reaction intermediate at the enzyme active-site. B is active acetate when R=H.

Berthold CL, P Moussatche, NGJ Richards, Y Lindqvist (2005) Structural basis for activation of the thiamin diphosphate-dependent enzyme oxalyl-CoA decarboxylase by adenosine diphosphate. J Biol Chem 280 41645-41654. 

M. S. Hasson, A. Muscate, M. J. McLeish, L. S. Polovnikova, J. A. Gerlt, G. L. Kenyon, G. A. Petsko, D. Ringe, The crystal structure of benzoylformate decarboxylase at 1.6 A resolution, diversity of catalytic residues in thiamin diphosphate-dependent enzymes. Biochemistry 1998, 37, 9918-9930. 

By 1998, X-ray structures had been determined for four thiamin diphosphate-dependent enzymes (1) a bacterial pyruvate oxidase,119120 (2) yeast and bacterial pyruvate decarboxylases,121 122c (3) transketolase,110123124 and (4) benzoylformate decarboxylase.1243 Tire reactions catalyzed by these enzymes are all quite different, as are the sequences of the proteins. However, the thiamin diphosphate is bound in a similar way in all of them. 

An X-ray structure of a thiamine dependent transketolase enzyme was determined by Schneider et al. after isolation from Saccharomyces cerevisiae in the 1990s and is shown in Fig. 10 (Sundstrom et al. 1993 Nilsson et al. 1997). The thiamine cofactor is embedded in a narrow channel in the centre of the enzyme. From the complex surrounding of the heart of this enzyme it seems to be obvious that chemical reactions at the catalytically active site in this channel proceed inevitably with high selectivities. 

Y. Lindqvist, G. Schneider, U. Ermler, and M. Sundstrom. 1992. Three-dimensional structure of transketolase, a thiamine diphosphate dependent enzyme, at 2.5 A resolution EMBO J. 11 2373-2379. (PubMed)... 

Arjunan, P., et al. (1996). Crystal structure of the thiamin diphosphate-dependent enzyme pyruvate decarboxylase from the yeast saccharomyces cerevisiae at 2.3 A Resolution. J. Mol. Biol. 256, 590-600... 

An elegant work is reported concerning the oxidation of 2-alkyl and 2-benzylthia-zolium salts, in the presence of a base, with the scope of finding a structural relationship for the thiamine-bound intermediate which intervene in the oxidative decarboxylation of a-ketoacids catalysed by thiamin diphosphate-dependent enzymes. 2-Alkyl and 2-benzylthiazolium salts, which are not electroactive, can be transformed into electroactive species by treatment with the base (trimethylsilyl)amide. Subsequent anodic oxidation affords the corresponding symmetrical dimers, by an EC mechanism (Scheme 72). As expected, the stabilizing effect of the substituents R, R at the a-carbon on the radical cation follows the order H < Me < OMe. When R is aryl, electron-donating p-substituents again enhance the enamine oxidation. 

Konig, S., Schellenberger, A., Neee, H., Schneider, G. (1994), Specificity of coenzyme binding in thiamin diphosphate-dependent enzymes. Crystal structures of yeast transketolase in complex with analogs of thiamin diphosphate, J. Biol. Chem. 269, 10879-10882. 

Structure of the thiamine- and flavin-dependent enzyme pyruvate oxidase. Science 259, 965-967. 

Muller, Y.A., and Schulz, G.E., 1993. Structure of the thiamine- and flavin-dependent enzyme pyruvate oxidase. Science. 259 965-967. 

This arrangement is found in aldolase, rioflavin, niacin, thiamin and pyridoxal dependent enzymes. Here we shall limit ourselves to models of NAD (nicotinamide-adeninedinueleotide). In structure (2) we see the anion... 

Most known thiamin diphosphate-dependent reactions (Table 14-2) can be derived from the five halfreactions, a through e, shown in Fig. 14-3. Each halfreaction is an a cleavage which leads to a thiamin- bound enamine (center, Fig. 14-3) The decarboxylation of an a-oxo acid to an aldehyde is represented by step b followed by a in reverse. The most studied enzyme catalyzing a reaction of this type is yeast pyruvate decarboxylase, an enzyme essential to alcoholic fermentation (Fig. 10-3). There are two 250-kDa isoenzyme forms, one an a4 tetramer and one with an ( P)2 quaternary structure. The isolation of ohydroxyethylthiamin diphosphate from reaction mixtures of this enzyme with pyruvate52 provided important verification of the mechanisms of Eqs. 14-14,14-15. Other decarboxylases produce aldehydes in specialized metabolic pathways indolepyruvate decarboxylase126 in the biosynthesis of the plant hormone indoIe-3-acetate and ben-zoylformate decarboxylase in the mandelate pathway of bacterial metabolism (Chapter 25).1243/127... 

Benzoylformate decarboxylases purified from Acinetobacter calcoaceticus [81] and Pseudomonas putida [85] were shown to have similar properties. Like pyruvate decarboxylase this enzyme is thiamine pyrophosphate- and magnesium-dependent and has a tetrameric structure. The molecular weight of a subunit is 58,000 for A. calcoaceticus [81] and 57,000-57,500 for P. putida [86,87], as estimated from sodium dodecyl sulfate-polyamide gel electophoresis (SDS-PAGE). 

---