Pyruvate decarboxylase structure

A thiamin diphosphate binding fold revealed by comparison of the crystal structures of transketolase, pyruvate oxidase and pyruvate decarboxylase. Structure 1, 95-103. 

Hohmann S, Cederberg H. (1990). Autoregulation may control the expression of yeast pyruvate decarboxylase structural genes PDCl and PDC5. Eur J Biochem, 188, 615-621. 

D. Dohritzsch, S. Konig, G. Schneider, G. Lu, High resolution crystal structure of pyruvate decarboxylase from Zymomonas mohilis. Implications for substrate activation in pyruvate decarboxylases. J. Biol. Chem. 1998, 273, 20 196-20 204. 

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. 

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. 

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... 

Protein design by site-directed mutagenesis on pyruvate decarboxylase became possible after the 3D-structure of the enzyme from Saccharomyces uvarum had become available [35], Based on sequence comparison and secondary structure prediction, the 3D-structure of the yeast enzyme served as a model for PDCZ.m. [163], The point mutations which have been introduced into the two enzymes (Tables 4 and 5) concern catalytically important residues as well as significant side-chain interactions at the domain interface of the dimer. Besides, site-directed mutagenesis offered a powerful tool to improve the car-boligase reaction of PDCZ.m. with respect to the synthesis of (P)-PAC [163,164,170]. 

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... 

Despite the apparent simplicity of this highly exergonic reaction (AG° = -33.5 kJ/mol), its mechanism is one of the most complex known. The pyruvate dehydrogenase complex is a large multienzyme structure that contains three enzyme activities pyruvate dehydrogenase (Ej), also known as pyruvate decarboxylase, dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3). Each enzyme activity is present in multiple copies. Table 9.3 summarizes the number... 

Pyruvate decarboxylase, which contains TPP, catalyzes the formation of the HETPP. Using lipoic acid as a cofactor, dihydrolipoyl transacety-lase converts the hydroxyethyl group of HETPP to acetyl-CoA. Dihydrolipoyl dehydrogenase reoxidizes the reduced lipoic acid. (Refer to Figure 9.9 for the structure of lipoic acid.)... 

On the basis of the crystal structure of a Bacillus stearothermophilus pyruvate dehydrogenase subcomplex formed between the heterotetrameric El and the peripheral subunit binding domain of E2 with an evident stmctural dissymmetry of the two active sites, a direct active center communication via an acidic proton tunnel has been proposed (Frank et ak, 2004). According to this, one active site is in a closed state with an activated cofactor even before a substrate molecule is engaged, whereas the activation of the second active site is coupled to decarboxylation in the first site. Our own kinetic NMR studies on human PDH El (unpublished) support the model suggested, but similar studies on related thiamin enzymes, such as pyruvate decarboxylase, transketolase or pyruvate oxidase reveal that half-of-the-sites reactivity is a unique feature of ketoacid dehydrogenases. In line with this. X-ray crystallography studies on intermediates in transketolase catalysis indicated an active site occupancy close to unity in both active sites (Fiedler et al., 2002 and G. Schneider, personal communication). 

Figure 16.3. Crystal structure of active site residues of pyruvate decarboxylase from Zymomonas mobilis /ith the enzyme-bound cofactor ThDP in its typical V-conformation.

A FIGURE 3-21 Structure and function of pyruvate dehydrogenase, a large multimeric enzyme complex that converts pyruvate into acetyl CoA. (a) The complex consists of 24 copies of pyruvate decarboxylase (Ei), 24 copies of lipoamide transacetylase (E2), and 12 copies of dihydrolipoyl dehydrogenase (E3). The El and E3 subunits are bound to the outside of the core formed by the E2 subunits, (b) The reactions catalyzed by the complex include several enzyme-bound intermediates (not shown). The tight structural integration of the three enzymes increases the rate of the overall reaction and minimizes possible side reactions. 

Mesecar, A. D., Stoddard, B. L., Koshland Jr., D. E. (1997). Orbital steering in the catalytic power of enzymes small structural changes with large catalytic consequences, Science, 277, 202-206. Recent example Meyer, D., Neumann, R, Parthier, C., Friedemann, R., Nemeria, N., Jordan, F., Tittmann, K. (2010). Double Duty for a Conserved Glutamate in Pyruvate Decarboxylase Evidence of the Participation in Stereoelectronically Controlled Decarboxylation and in Protonation of the Nascent Carbanion/Enamine Intermediate. Biochemistry, 49, 8197-8212. 

Bran B, Sahm H. (1986). Cloning and expression of the structural gene for pyruvate decarboxylase of Zymomonas mobilis in Escherichia coli. Arch Microbiol, 144, 296-301. 

Hohmann S. (1991). Characterization of PDC6, a third structural gene for pyruvate decarboxylase in Saccharvmyces cerevisiae. J Bacterial, 173, 7963-7969. 

Schellenberger, A. Structure and mechanism of action of the active centre of yeast pyruvate decarboxylase. Angew. Chem. Int. Ed. Engl. 6, 1024-1035 (1967)... 

Before the molecular mechanism of the pyruvic decarboxylase reaction is discussed, it might be appropriate to summarize briefly modern concepts on the mechanism of action of coenzymes. Modern research in that field suggests that the coenzyme functions in biochemical reactions by virtue of its unique molecular properties, and that the apoenzyme acts mainly as a basic or acidic catalyst and provides a structural skeleton that brings substrate and coenzyme in close contact. 

Figure 4.1 Formation of ThDP ylide. (a) Proton transfer from the C2 atom to the N4 imino group forms the ThDP ylide, which is the first activation step of all ThDP-dependent enz5mes. (b) Structure of ThDP bound to pyruvate decarboxylase. Glu51 residue facilitates ylide formation.

Candy, J.M., and Duggleby, R.G., 1998. Structure and properties of pyruvate decarboxylase and site-directed mutagenesis of the Zymomonas mobilis enzyme. Biochimica et Biophysica Acta. 1385 323-338. 

Figure 5.4 Structural formulae of thiamin phosphate esters. At present, five natural thiamin phosphate derivatives have been described thiamin monophosphate (ThMP) thiamin diphopshate (ThDP) thiamin triphosphate (ThTP) adenosine thiamin diphosphate (AThDP) and adenosine thiamin triphosphate (AThTP). Catalytic intermediates, such as for instance a-hydroxyethyl thiamine diphosphate formed by the action of yeast pyruvate decarboxylase (EC 4.1.1.1), are not considered here.

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