Thiamine mechanism

The pathways for thiamine biosynthesis have been elucidated only partiy. Thiamine pyrophosphate is made universally from the precursors 4-amino-5-hydroxymethyl-2-methylpytimidinepyrophosphate [841-01-0] (47) and 4-methyl-5-(2-hydroxyethyl)thiazolephosphate [3269-79-2] (48), but there appear to be different pathways ia the eadier steps. In bacteria, the early steps of the pyrimidine biosynthesis are same as those of purine nucleotide biosynthesis, 5-Aminoimidazole ribotide [41535-66-4] (AIR) (49) appears to be the sole and last common iatermediate ultimately the elements are suppHed by glycine, formate, and ribose. AIR is rearranged in a complex manner to the pyrimidine by an as-yet undetermined mechanism. In yeasts, the pathway to the pyrimidine is less well understood and maybe different (74—83) (Fig. 9). 

A number of the genes involved in the biosynthesis of thiamine in E. coli (89—92), i hium meliloti (93), B. suhtilis (94), and Schi saccharomycespomhe (95,96) have been mapped, cloned, sequenced, and associated with biosynthetic functions. Thiamine biosynthesis is tightly controlled by feedback and repression mechanisms limiting overproduction (97,98). A cost-effective bioprocess for production of thiamine will require significant additional progress. 

The pyruvate dehydrogenase complex (PDC) is a noncovalent assembly of three different enzymes operating in concert to catalyze successive steps in the conversion of pyruvate to acetyl-CoA. The active sites of ail three enzymes are not far removed from one another, and the product of the first enzyme is passed directly to the second enzyme and so on, without diffusion of substrates and products through the solution. The overall reaction (see A Deeper Look Reaction Mechanism of the Pyruvate Dehydrogenase Complex ) involves a total of five coenzymes thiamine pyrophosphate, coenzyme A, lipoic acid, NAD+, and FAD. 

The mechanism of the pyruvate dehydrogenase reaction is a tour de force of mechanistic chemistry, involving as it does a total of three enzymes (a) and five different coenzymes—thiamine pyrophosphate, lipoic acid, coenzyme A, FAD, and NAD (b). 

Based on the action of thiamine pyrophosphate in catalysis of the pyruvate dehydrogenase reaction, suggest a suitable chemical mechanism for the pyruvate decarboxylase reaction in yeast ... 

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. 

Fermenting baker s yeast also catalyzes the 1,4-addition of a formal trifluoroethanol-d1-synthon to a,/i-unsaturated aldehydes, to give optically active l,l,l-trifluoro-2-hydroxy-5-alka-nones52. Presumably, the mechanism involves oxidation of the alcohol to the corresponding aldehyde followed by an umpolung step with thiamine pyrophosphate and Michael addition to the a,/i-unsaturated aldehyde. For example, l,l,l-trifluoro-2-hydroxy-5-hexanone (yield 26%, ee 93%) is thus obtained from trifluoroethanol and l-bnten-3-one. 

Some kinds of fish and Crustacea contain thiaminases. These enzymes cleave thiamin and thus inactivate the vitamin. Some plant phenols, e.g., chlorogenic acid, may possess antithiaminic properties, too, though their mechanism of action is so far not well understood. 

The suggestion that eNOS-derived NO is implicated in neuronal cell death mechanisms in thiamine deficiency contrasts with current views in cerebral ischemia in which increased eNOS-derived NO is thought to play a neuro-protective role by virtue of its vasodilatory potential. 

Pannunzio, P., Hazell, A. S., Pannunzio, M., Rama Rao, K. V. and Butterworth, R. F. Thiamine deficiency results in metabolic acidosis and energy failure in cerebellar granule cells an in vitro model for the study of cell death mechanisms in Wernicke s encephalopathy. /. Neurosci. Res. 62 286-292, 2000. 

However, cyanide ion is not suitable for inducing a benzoin-type condensation between two aliphatic aldehydes, since the basic character of this ion induces an aldol condensation between them. In Nature, nevertheless, condensations of this type take place easily. As Breslow proposed in 1958 [8], such condensations are catalysed by thiamine pyrophosphate 6 (or cocarboxylase), the active part of which is the conjugate base of the "thiazolium cation present in it. According to Breslow [8a], the mechanism is, in fact, identical to that described for the cyanide ion (see Scheme 5.7) that is to say, the conjugate base of thiamine (TPP ) reacts with an "aldehyde equivalent -such as an a-ketoacid 2- to generate the corresponding "active aldehyde" 8 with umpoled reactivity, which then reacts with the electrophile to give finally, after elimination of "thiamine anion", a 1,2-D system (9). 

The finding that thiamine, and even simple thiazolium ring derivatives, can perform many reactions in the absence of the host apoenzyme has allowed detailed analyses of its chemistry [33, 34]. In 1958 Breslow first proposed a mechanism for thiamine catalysis to this day, this mechanism remains as the generally accepted model [35]. NMR deuterium exchange experiments were enlisted to show that the thiazolium C2-proton of thiamine was exchangeable, suggesting that a carbanion zwitterion could be formed at that center. This nucleophilic carbanion was proposed to interact with sites in the substrates. The thiazolium thus acts as an electron sink to stabilize a carbonyl carbanion generated by deprotonation of an aldehydic carbon or decarboxylation of an a-keto acid. The nucleophilic carbonyl equivalent could then react with other electro-... 

Breslow and co-workers elucidated the currently accepted mechanism of the benzoin reaction in 1958 using thiamin 8. The mechanism is closely related to Lapworth s mechanism for cyanide anion catalyzed benzoin reaction (Scheme 2) [28, 29], The carbene, formed in situ by deprotonation of the corresponding thiazolium salt, undergoes nucleophilic addition to the aldehyde. A subsequent proton transfer generates a nucleophilic acyl anion equivalent known as the Breslow intermediate IX. Subsequent attack of the acyl anion equivalent into another molecule of aldehyde generates a new carbon - carbon bond XI. A proton transfer forms tetrahedral intermediate XII, allowing for collapse to produce the a-hydroxy ketone accompanied by liberation of the active catalyst. As with the cyanide catalyzed benzoin reaction, the thiazolylidene catalyzed benzoin reaction is reversible [30]. 

Its injection was decreased to the greatest extent (by almost 24%). Thiamine resulted in an increase in the total volume of distribution of 1 of about 47.4%, the expansion of the central volume being about 13.3% greater than that of the peripheral volume. The mechanism of the changes in the volume of distribution of I is not apparent. 

During the first 3 h after Intravenous injection of 1 that followed administration of thiamine, urinary excretion of the oxime was about 12.7% below that during the corresponding period of the control experiment during the remainder of the run, it was 62.2% above that during the same period of the control experiment. Inasmuch as intravenous injection of 900 mg of sodium jg-aminohippurate with I decreased by only 6.3% the urinary excretion of I during the first 3 h after its administration, the tubular transport mechanisms for 1 and for jg-amino-hippurate probably are different. 

Benzoylformate decarboxylase (BFD EC 4.1.1.7) belongs to the class of thiamine diphosphate (ThDP)-dependent enzymes. ThDP is the cofactor for a large number of enzymes, including pyruvate decarboxylase (PDC), benzaldehyde lyase (BAL), cyclohexane-1,2-dione hydrolase (CDH), acetohydroxyacid synthase (AHAS), and (lR,6] )-2-succinyl-6-hydroxy-2,4-cyclohexadiene-l-carboxylate synthase (SHCHC), which all catalyze the cleavage and formation of C-C bonds [1]. The underlying catalytic mechanism is summarized elsewhere [2] (see also Chapter 2.2.3). 

M. Muller, G. A. Sprenger, Thiamine-dependent enzymes as catalysts of C-C-bonding reactions The role of orphan enzymes, in Thiamine Catalytic Mechanisms and Role in Normal and Disease States, Marcel Dekker, New York, 2004, pp. 77-91. 

Reddy et al. (1983) concluded that NO inactivation of iron-sulfur proteins was the probable mechanism of botulinal inhibition in nitrite-tteated foods. In support of this conclusion, Carpenter et al. (1987) observed decreased activity of clostridial pyruvate-ferredoxin oxidoteductase and lower cytochrome c reducing ability by ferredoxin in extracts of cells treated with nitrite. NO tteatment also inhibits yeast pyruvate decarboxylase (a non-iron-sulfur protein) and py-ruvate-ferredoxin oxidoteductase from C. perfringens (McMindes and Siedler, 1988). They suggested that thiamine-dependent decarboxylation of pyruvate may be an additional site for antimicrobial effects of NO. 

The first examples of mechanism must be divided into two principal classes the chemistry of enzymes that require coenzymes, and that of enzymes without cofactors. The first class includes the enzymes of amino-acid metabolism that use pyridoxal phosphate, the oxidation-reduction enzymes that require nicotinamide adenine dinucleotides for activity, and enzymes that require thiamin or biotin. The second class includes the serine esterases and peptidases, some enzymes of sugar metabolism, enzymes that function by way of enamines as intermediates, and ribonuclease. An understanding of the mechanisms for all of these was well underway, although not completed, before 1963. 

The next coenzyme for which a mechanism was established was thiamin pyrophosphate [3]. Ronald Breslow used nmr spectroscopy to show that the hydrogen atom at C-2 of a thiazolium salt rapidly exchanges with deuterium in even slightly alkaline solutions (6), so that the coenzyme offers an anionic centre for catalysis (Breslow, 1957). With this established, Breslow could confidently offer the pathway shown in Scheme 2 for the action of the... 

Thiamin itself (in the absence of enzyme) had previously been shown to catalyse the formation of acetoin from acetaldehyde, albeit in very poor yield (Ukai et al., 1943 Mizuhara et al., 1951 Mizuhara and Handler, 1954). The reaction parallels the formation of benzoin from benzaldehyde, catalysed by cyanide ion. The mechanism of the latter reaction had been suggested in 1903 by Arthur Lapworth, who had shown how an aldehyde, R—CHO, could be converted into the equivalent of the anion R—C=0- (Lapworth, 1903). It is this idea that Breslow carried over to thiamin pyrophosphate and used to... 

Fig. 9. A schematic drawing of a possible mechanism for the reaction catalyzed by the pyruvate dehydrogenase complex. The three enzymes Elf E2, and E3 are located so that lipoic acid covalently linked to E2 can rotate between the active sites containing thiamine pyrophosphate (TPP) and pyruvate (Pyr) on Elt CoA on E2, and FAD on E3. Acetyl-CoA and GTP are allosteric effectors of E, and NAD+ is an inhibitor of the overall reaction.

Lewin, L.M. Brown, G.M. The Biosynthesis of thiamine. III. Mechanism of enzymatic formation of the pyrophosphate ester of 2-methyl-4-ammo-5-hy-droxymethylpyrimidine. J. Biol. Chem., 236, 2768-2771 (1961)... 

Amprolium (Fig. 5.7) is a vitamin IT analogue. It is a competitive antagonist of the thiamine transport mechanism. Amprolium has been used as a coccidi-ostat mainly in chickens, laying hens, turkeys, and ruminants. It is available as a soluble powder for addition to drinking water (60-240 mg/L) or as a premix, usually in combination with ethopabate and/or sulfaquinoxaline, for mixing with the feed (125-500 mg/kg feed). A withdrawal period of 3 days is required for chickens. 

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