Saccharomyces cerevisiae, thiamine

Vitamin Bi is an essential co-factor for several enzymes of carbohydrate metabolism such as transketolase, pyruvate dehydrogenase (PDH), pyruvate decarboxylase and a-ketoglutarate dehydrogenase. To become the active co-factor thiamin pyrophosphate (TPP), thiamin has to be salvaged by thiamin pyrophosphokinase or synthesized de novo. In Escherichia coli and Saccharomyces cerevisiae thiamin biosynthesis proceeds via two branches that have to be combined. In the pyrimidine branch, 4-amino-5-hydroxymethy-2-methylpyrimidine (PIMP) is phosphorylated to 4-amino-2-methyl-5-hydroxymethyl pyrimidine diphosphate (PIMP-PP) by the enzyme HMP/HMP-P kinase (ThiD) however, the step can also be catalyzed by pyridoxine kinase (PdxK), an enzyme also responsible for the activation of vitamin B6 (see below). The second precursor of thiamin biosynthesis, 5-(2-hydroxyethyl)-4-methylthiazole (THZ), is activated by THZ kinase (ThiM) to 4-methyl-5-(2-phosphoethyl)-thiazole (THZ-P), and then the thia-zole and pyrimidine moieties, HMP-PP and THZ-P, are combined to form thiamin phosphate (ThiP) by thiamin phosphate synthase (ThiE). The final step, pyrophosphorylation, yields TPP and is carried out by thiamin pyrophosphorylase (TPK). 

Transketolase from common yeast (Saccharomyces cerevisiae) is commercially available, but it is possible to work with a partially purified enzyme, isolated with little expense from spinach leaves.54 Transketolase catalyzes the transfer of a hydroxyacetyl group, reversibly from a ketose phosphate, or irreversibly from hydroxypyruvate to an acceptor aldose, phosphorylated or not.55 It requires thiamine pyrophosphate as a coenzyme, but only in catalytic amounts. In all the cases listed in Table V, the new chiral center, C-3 of the ketose, has the l-glycero configuration. 

ADH CD 3D HEThDP Mes nh PAC PDC PDCS.c. PDCS.u. PDCZ.w. So.5 ThDP v/S v max wt alcohol dehydrogenase circular dichroism three-dimensional 2-(hydroxyethyl)thiamine diphosphate 4-morpholineethanesulfonsaure Hill-coefficient phenylacetyl carbinol pyruvate decarboxylase PDC from Saccharomyces cerevisiae PDC from Saccharomyces uvarum PDC from Zymomonas mobilis substrate concentration necessary for half-maximal velocity thiamine diphosphate velocity vs substrate concentration maximal velocity wild-type... 

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. 

Hohmann S and Meacock PA (1998) Thiamin metabolism and thiamin diphosphate-dependent enzymes in the yeast Saccharomyces cerevisiae. genetic regulation. Bio-chimica et Biophysica Acta 1385, 201-19. 

Pyruvate is initially decarboxylated into ethanal by pyruvate decarboxylase. This enzyme needs magnesium and thiamine pyrophosphate as cofactors (Hohmann 1996). Thereafter, alcohol dehydrogenase reduces ethanal to ethanol, recycling the NADH to NAD+. There are three isoenzymes of alcohol dehydrogenase in Saccharomyces cerevisiae, but isoenzyme I is chiefly responsible for converting ethanal into ethanol (Gancedo 1988). Alcohol dehydrogenase uses zinc as cofactor (Ciriacy 1996). 

Biomimetic Synthesis of Solerone. We applied pyruvate decarboxylase [EC 4.1.1.1] (PDC) as key enzyme for the biomimetic synthesis elucidating the formation of solerone 1 figure 1). The thiamine diphosphate depending enzyme from Saccharomyces cerevisiae is responsible for the decarboxylation of pyruvate in the course of alcoholic fermentation. After loss of carbon dioxide from 2-oxoacids the resulting aldehyde is released. Alternatively, the cofactor-bound decarboxylation product can react with a further aldehyde. By the latter acyloin condensation a new carbon-carbon bond will be formed, thus opening a biosynthetic way to a-hydroxy carbonyl compounds 11J2). 

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... 

Deprotonation Rate of the C2-H of Thiamin Diphosphate in Transketolase from Saccharomyces cerevisiae... 

Fiedler, E., Thoeell, S., Sandalova, T., Golbik, R., Konig, S., Schneider, G. (2002), Snapshot of a key intermediate in enzymatic thiamin catalysis crystal stmeture of the a-carbanion of dihydroxyethyl)-thiamin diphosphate in the active site of transketolase from Saccharomyces cerevisiae, Proc. Nat. Acad. Sci. USA 99, 591-595. 

Figure 14 Thiamin pyrophosphate biosynthesis in Saccharomyces cerevisiae. The nudix hydrolase required for the removal of AMP from 38 has not yet been identified.

Saccharomyces cerevisiae (mutant resistant to 2-amino-4-methyl-5- -hy-droxyethylthiazole, an antimetabolite of 4-methyl-5-/l-hydroxyethylthia-zole, deficient in activity of both EC 2.5.1.3 and EC 2.7.1.50 [2] bifunctional enzyme with hydroxyethylthiazole kinase and thiamine-phosphate pyrophosphorylase activity [2]) [1, 2]... 

Kawasaki, Y. Copurification of hydroxyethylthiazole kinase and thiamine-phosphate pyrophosphorylase of Saccharomyces cerevisiae characterization of hydroxyethylthiazole kinase as a bifunctional enzyme in the thiamine biosynthetic pathway. J. Bacteriol., 175, 5153-5158 (1993)... 

Many of the micro-organisms proposed, even some that have been extensively used, show a certain lack of specificity their requirement for thiamine as a growth factor can also be met by the pyrimidine and thiazole moieties of the molecule, either singly or in combination. Such is the case for the yeast Saccharomyces cerevisiae and the mould Phycomyces blakesleeanus. 

Kneen MM, Stan R, Yep A, lyier RP, Saehuan C, McLeish MJ. Characterization of a thiamin diphosphate-dependent phenylpyruvate decarboxylase from Saccharomyces cerevisiae. FEBSJ. 2011 278 1842-1853. 

Leonian and Lilly (201) examined 10 strains of Saccharomyces cerevisiae for the effects on 72 hr. growth (25°) of omission of various vitamins from the media none of the strains showed decreased growth on omission of pyridoxin. Marchant (245) had strains which were much more sensitive to pyridoxin, and presumably were less effecient synthesizers of it than the strains examined by Leonian and Lilly. Two yeasts, Sacch. hanseniaspora valbyensis and yeast 2375, showed a marked stimulation w ith pyridoxin which could, for these two yeasts, replace a bios Vll solution. A strain of Sacch. cerevisiae has been trained to grow on a completely synthetic medium without added vitamins the yeast then synthesized thiamin, riboflavin. 

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