Thiamin phosphate

Thiamine can be considered to be the product of the quatemization of 4-methyl-5-(2-hydroxymethyl)thiazole (5) by an active derivative of 4-amino-5-(hydroxymethyl)-2-methyl pyrimidine (4) (Scheme 2). In living cells, pyramine can be activated by conversion into the diphosphate 7, via monophosphate 6, and the substrate of the enzyme responsible for the quatemization is not the thiamine thiazole, but its phosphate 8. The product of the condensation, thiamine phosphate (9), is finally converted into diphosphate 2—the biochemically active derivative—by hydrolysis to free thiamine, followed by diphosphorylation, or more directly, in some cases. Enzymes are known for all of the steps depicted in Scheme 2, and adenosine triphosphate (ATP) is, as usual, the phosphate donor. 

Thiamine Phosphates.—Thiamine thiazolone and thiothiazolone have been phos-phorylated in two steps to the corresponding diphosphates (8 X = O or S).31... 

The hypE proteins are 302-376 residues long and appear to consist of three domains. Domain 1 shows sequence identity to a domain from phosphoribosyl-aminoimida-zole synthetase which is involved in the fifth step in de novo purine biosynthesis and to a domain in thiamine phosphate kinase which is involved in the synthesis of the cofactor thiamine diphosphate (TDP). TDP is required by enzymes which cleave the bond adjacent to carbonyl groups, e.g. phosphoketolase, transketolase or pyruvate decarboxylase. Domain 2 also shows identity to a domain found in thiamine phosphate kinase. Domain 3 appears to be unique to the HypF proteins. 

S ATP + 2-methyl-4-amino-5-pho.sphomethylpyrimidine <1, 2, 3> (<1> specific for ATP [1] <3> enzyme has both 2-methyl-4-amino-5-hydroxy-methylpyrimidine phosphate kinase and thiamin-phosphate diphosphatase activities, involved in thiamine biosynthesis [3]) (Reversibility <1, 2, 3> [1,2, 3]) [1,2,3]... 

ATP thiamin-phosphate phosphotransferase kinase, thiamin monophosphate (phosphorylating) thiamin monophosphatase... 

S ATP -I- thiamine phosphate <1, 2> (<2> enzyme plays an important role in the thiamine diphosphate biosynthetic pathway, thiamine diphosphate is the regulatory molecule for thiamine synthesis and predicts the existence of a sensor/regulatory protein [2]) (Reversibility <1, 2> [1, 2])... 

The Thil gene, which encodes aother sulfurtransferase protein, is also needed.3743 The enzymology of the insertion of this sulfur into the thiazole is uncertain but may resemble that involved in synthesis of biotin, lipoic acid, and molybdopterin.374 Linkage of the two parts of the thiamin molecule (step d, Fig. 25-21) is catalyzed by thiamin phosphate synthase, evidently via an SN2 type reaction.377-37713... 

Figure 25-21 Proposed pathways for biosynthesis of thiamin phosphate and pyridoxol phosphate.

Commercial thiamine dietary supplements are prepared by synthesis Pyrimidine + thiazole nuclei synthesized separately and then condensed also build on pyrimidine with acelaiiudiiie. Precursors in the biosynthesis of thiamine include thiazole and pyrimidine pyrophosphate, with thiamine phosphate as an intermediate. In plants, production sites arc found in grain and cereal germ. 

Total thiamine Milk Enzymatic hydrolysis of protein with trypsin and thiamine phosphates to thiamine with claradiastase oxidation of thiamine to thiochrome using ferricyanide (derivatization stopped with sodium sulphite) thiochrome extracted with 1-butanol Analytical Nucleosil Phenyl (150 mm, 5 fi Macherey-Nagel). Isocratic methanol + acetonitrile + isobutanol + water (80 +10+10+5 v/v/v/v). 0.7 ml/min. Fluorescence 375/430 nm (ex/em). External standardization. 76 Recoveries 95% thiamine as thiochrome from milk. 

Total thiamine Baby food cereals cookies Acid hydrolysis with 0.1 M hydrochloric acid at 100°C for 30 min enzymatic hydrolysis of thiamine phosphates to thiamine with takadiastase at 47°C for 3 h weak ion-exchange (methyl-carboxylatein, add form) solid-phase extraction/ cleanup Analytical Lichrospher 100RP-18 (125 X 4 mm, 5 /u.m Merck ). Isocratic methanol + 10 mM phosphate buffer, pH 2.8 containing 5 mM sodium hexane-sulphonate + tri-ethylamine (15 + 84.9 + 0.1, v/v/v). 1.0 ml/min. UV absorbance 254 nm. External standardization. 79 Linear range = LoD to 7 /zg/ml thiamine, r = 0.9995. Reproducibility CV < 3% using food samples (n = 10). Recoveries 92.1-96.0% thiamine using baby food (n = 3). Samples spiked before hydrolysis. 

AutoDock4 and Dock6 used with a homology model of thiamin phosphate synthetase NCI diversity set II docked in homology model. 39 compounds tested, 25 showed activity, and seven have >20% inhibition at 100 pg/mL. One compound also had an MIC99 of 6 pg/mL Khare et al. (89)... 

Khare G, Kar R, Tyagi AK (2011) Identification of inhibitors against Mycobacterium tuberculosis thiamin phosphate synthase, an important target for the development of anti-TB drugs. PLoS One 6 e22441... 

Dietary thiamin phosphates are hydrolyzed by intestinal phosphatases, and the resultant free thiamin is absorbed by active transport in the duodenum and proximal jejunum, with little absorption in the rest of the small intestine. 

HerouxM and Butterworth RF (1995) Regional alterations of thiamine phosphate esters and of thiamine diphosphate-dependent enzymes in relation to function in experimental Wernicke s encephalopathy. Neurochemistry Research 20,87-93. 

Laforenza U, Patrini C, Rindi G (1988) Distribution of thiamine, thiamine phosphates, and thiamine metabolizing enzymes in neuronal and gUal cell enriched fractions of rat brain. J Neurochem 51(3) 730-735... 

Figure 4 Biosynthesis of thiamine (vitamin ). 37, aminoimidazole ribotide 38, 2-methyl-4-amino-5-hydroxymethyl-pyrimidine phosphate 39, pyridoxal 5 -phosphate 40, histidine 41, 2-methyl-4-amino-5-hydroxymethyl-pyrimidine pyrophosphate 42, 4-methyl-5-p-hydroxyethylthiazole phosphate 43,1 -deoxy-D-xylulose 5-phosphate 44, 5-ADP-D-ribulose 45, thiamine phosphate 46, thiamine pyrophosphate.

The thiamin phosphate synthase-catalyzed formation of thiamin phosphate from 4-amino-5-(hydroxymethyl)-2-methylpyrimidine pyrophosphate and 4-methyl-5-( 1 -hydroxyethyl)thiazole phosphate has been studied. A mechanism was proposed, and the substituent effects of the pyrimidine ring upon the TS discussed <2001B10095>. 

---