Thiamin, structure

Thiamin, structure of, 530, 1045 thiazolium ring in, 530 Thiamin diphosphate, p/Ca of, 1151 reaction with pyruvate, 1151-1153 structure of. 1151 ylide from. 1151 Thiazole, basicity of. 948 thio-, thioester name ending, 787 Thioacetal, synthesis of, 743 Thioanisole, electrostatic potential map of. 777... 

An extract from the soluble stromal proteins of purified and intact spinach-leaf chloroplasts was prepared by lysis of the cells in buffer, centrifugation of the suspension of broken cells, and concentration of the supernatant with removal of insoluble material. This extract contained all of the enzymes involved in the condensation of the cyclic moieties of thiamine, thiazole, and pyramine. Thus, the synthesis of thiamine in this extract following the addition of pyramine and putative precursors was a proof that the system had the possibility of building the thiazole. It was found that L-tyrosine was the donor of the C-2 carbon atom of thiazole, as in E. coli. Also, as in E. coli cells, addition of 1 -deoxy-D-f/irco-pen-tulose permitted synthesis of the thiamine structure. The relevant enzymes were localized by gel filtration in a fraction covering the 50- to 350-kDa molecular-mass range. This fraction was able to catalyze the formation of the thiazole moiety of thiamine from 0.1 -mM 1-deoxy-D-t/ireo-pentulose at the rate of 220 pmol per mg of protein per hour, in the presence of ATP and Mg2+. 

Determination of structural features. The ultraviolet spectrum has been of value in the determination of the structure of several vitamins. Thus the presence of an a-naphthoquinone system in vitamin K was first detected by this means. Also the 4-methylthiazole and the 2 5-dimethyl-6-aminopyridine system was first identified in vitamin Bj (thiamine), a- and /3-Ionones can be distinguished since the former contains two conjugated chromophores and the latter three conjugated chromophores. 

Thiamine, whose structure is shown in Figure 18.17, is known as vitamin Bj and is essential for the prevention of beriberi, a nervous system disease that has occurred in the Far East for centuries and has resulted in considerable sickness and death in these countries. (As recently as 1958, it was the fourth leading cause of death in the Philippine Islands.) It was shown in 1882 by the director-general of the medical department of the Japanese nayt that beriberi could be prevented by dietary modifications. Ten years later, Christiaan Eijkman, a Dutch medical scientist working in Java, began research that eventually showed that thiamine was the... 

Other common five-membered heterocyclic amines include imidazole and thiazole. Imidazole, a constituent of the amino acid histidine, has two nitrogens, only one of which is basic. Thiazole, the five-membered ring system on which the structure of thiamin (vitamin Bt) is based, also contains a basic nitrogen that is alkylated in thiamin to form a quaternary ammonium ion. 

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 1 shows the chemical structure ofvitamin B1 or thiamin (3-(4-amino-2-methyl-pyrimidin-5-ylmethyl)-5-(2-hydroxyethyl)-4-methylthiazolium) and its coenzyme form thiaminpyrophosphate (TPP). 

Vitamin B1. Figure 1 Structure of thiamin and its coenzyme form thiaminpyrophosphate (TPP). 

The thiazolecarboxylic acid structure (40) was also guessed in a similar way, from tracer experiments. The unknown compound was converted into the thiamine thiazole by heating at 100°C and pH 2. On paper electrophoresis, it migrated as an anion at pH 4. Tracer experiments indicated that it incorporated C-l and C-2 of L-tyrosine, and the sulfur of sulfate. The synthetic acid was prepared by carboxylation of the lithium derivative of the thiamine thiazole, and the derivatives shown in Scheme 19 were obtained by conventional methods. Again, the radioactivity of the unknown, labeled with 35S could not be separated from structure 40, added as carrier, and the molar radioactivity remained constant through several recrystallizations and the derivatizations of Scheme 17. 

The same is true of the thiazole acid 40. Although discovered as a growth factor, it is unable to sustain the growth of a thiazole-deficient mutant of E. coli in a liquid medium. It does not decarboxylate in water solution at pH 7. Phosphate 41 (Scheme 17) is also biologically inactive. In any case, if there is only one metabolic route to the thiazole of thiamine, the very structures of 39 and 40 show that they cannot both be intermediates. 

Nevertheless, the isolation of these metabolites was interesting in two respects. First, the structure of the thiazole glycol stimulated the research of functionalized carbohydrate chains as precursors of thiazole. Second, the thiazolecar-boxylic acid 40 can be secreted by derepressed cells in relatively high amounts, 0.24 nmol per mg of dried cells, which is nearly half the amount of synthesized thiamine. The presence of this free thiazolic derivative in the cells contrasts with... 

Calcium hyaluronate, 376-377 Calcium pectate, 353 Calcium welan, structure, 432-434 Candida utilis, thiamine synthesis,... 

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. 

The recognition of their structure permits the determination of vitamins by the tools of analytical chemistry, but while such methods are widely used in industrial production, the minute quantities in body fluids and tissues limit the purely chemical approach to a few members of this group present in relatively high concentration, e.g., vitamin C (K5). Microchemical methods are in use for the determination of thiamine, riboflavin, and some of the fat-soluble vitamins, based on the most sensitive colorimetric and, in particular, fluorometric techniques. Vitamin D, on the other hand, is determined by animal assay. 

Most coenzymes have aromatic heterocycles as major constituents. While enzymes possess purely protein structures, coenzymes incorporate non-amino acid moieties, most of them aromatic nitrogen het-erocycles. Coenzymes are essential for the redox biochemical transformations, e.g., nicotinamide adenine dinucleotide (NAD, 13) and flavin adenine dinucleotide (FAD, 14) (Scheme 5). Both are hydrogen transporters through their tautomeric forms that allow hydrogen uptake at the termini of the quinon-oid chain. Thiamine pyrophosphate (15) is a coenzyme that assists the decarboxylation of pyruvic acid, a very important biologic reaction (Scheme 6). 

Coenzymes complement the catalytic action of the amino-acid functional groups. They are bound to apoenzymes (apoproteins) either covalently or non-covalently. It is interesting to note that non-covalently-bound coenzymes are polyanions at neutral pH as exemplified by the structures of glutathione (GSH) [17] and thiamine pyrophosphate [18]. Shinkai and Kunitake (1976b, 1977a) demonstrated the efficient binding of glutathione and coenzyme A (a polyphosphate) to cationic micelles and cationic polysoaps. Thus, the combina- ... 

Polarography has been used to study the structure of thiamine phosphates190 and for the determination of phosphatase activity of milk.191... 

The crystal structure of MPT synthase and the simultaneously determined NMR structure of the MoaD-related ThiS protein involved in thiamine biosynthesis [37] unambiguously demonstrated the evolutionary relationship between a subset of enzymes involved in the biosynthesis of S-containing cofactors (e.g. Moco, thiamine and certain EeS-clusters) and the process of ubiquitin activation. MoaD displays significant structural homology to human ubiquitin (Figure 3.3B and C), resulting in a superposition with a root mean square (rms) deviation of 3.6 A for 68 equivalent Ca atoms out of 76 residues in ubiquitin. The key secondary structure... 

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]. 

TPP-dependent reactions in detail in Chapter 15. At this stage, we should merely examine the structure of thiamine, and correlate its properties with our knowledge of heterocycles. 

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