Action of the thiamine antagonist bacimethrin on thiamine biosynthesis

Bacimethrin is an analog of the 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) moiety of thiamine and inhibits the growth of Salmonella enterica serovar Typhimurium on a defined medium. Two classes of mutants that had increased bacimethrin resistance were isolated and characterized. Results showed that overexpression of the thi operon or specific lesions in thiD resulted in a bacimethrin-resistant phenotype. Phenotypic analyses of the thiD mutants suggested that they had a specific defect in one of the two kinase activities associated with this gene product and, further, that ThiD and not PdxK was primarily responsible for salvage of HMP from the medium.     

Identification of the two missing bacterial genes involved in thiamine salvage: thiamine pyrophosphokinase and thiamine kinase

The genes encoding thiamine kinase in Escherichia coli (ycfN) and thiamine pyrophosphokinase in Bacillus subtilis (yloS) have been identified. This study completes the identification of the thiamine salvage enzymes in bacteria.     

New function of the amino group of thiamine diphosphate in thiamine catalysis

In this work, we investigated the rate of formation of the central intermediate of the transketolase reaction with thiamine diphosphate (ThDP) or 4′-methylamino-ThDP as cofactors and its stability using stopped-flow spectroscopy and circular dichroism (CD) spectroscopy. The intermediates of the transketolase reaction were analyzed by NMR spectroscopy. The kinetic stability of the intermediate was shown to be dependent on the state of the amino group of the coenzyme. The rates of the intermediate formation were the same in the case of the native and methylated ThDP, but the rates of the protonation or oxidation of the complex in the ferricyanide reaction were significantly higher in the complex with methylated ThDP. A new negative band was detected in the CD spectrum of the complex transketolase—4′-methylamino-ThDP corresponding to the protonated dihydroxyethyl-4′-methylamino-ThDP released from the active sites of the enzyme. These data suggest that transketolase in the complex with the NH²-methylated ThDP exhibits dihydroxyethyl-4′-methylamino-ThDP-synthase activity. Thus, the 4′-amino group of the coenzyme provides kinetic stability of the central intermediate of the transketolase reaction, dihydroxyethyl-ThDP.     

Phosphorylation of some thiamine analogs by yeast thiamine pyrophosphokinase]

A rapid efficient method of separation of the thiamine pyrophosphokinase reaction products (ATP: thiamine pyrophosphotransferase) on the column packed with DEAE-Sephadex A-25 and their subsequent identification by direct spectrophotometry is suggested. Phosphorylation of some thiamine analogs substituted at the second position of the pyrimidine ring was studied. It was shown that in addition to thiamine, the enzyme transfers the pyrophosphate group to some of its derivatives. The vitamin analogs devoid of quaternary nitrogen in the thiazole cycle, do not form pyrophosphate ethers (thus being unable to act as substrates), whereas 2'-phenoxythiamine, 2'-methoxythiamine and especially 2'-phenylthiamine are phosphorylated at a greater rate than does the "true" substrate, thiamine, under similar conditions.     

Inhibition of thiamine transport in Saccharomyces cerevisiae by thiamine disulfides.

Both thiamine disulfide and O-benzoyl thiamine disulfide, which are thiolfrom derivatives of thiamine, strongly inhibited thiamine transport in Saccharomyces cerevisiae. The inhibition appeared to be due to a high affinity of the analogs for yeast cell membranes, in which thiamine transport component(s) may be integrated.     

Biosynthesis of thiamine triphosphate and identification of thiamine diphosphate-binding proteins in the rat liver hyaloplasm

The nature of the thiamine diphosphate binding proteins from rat liver hyaloplasm was studied. When [14C]thiamine was used as a marker, a [14C]thiamine diphosphate-containing electrophoretically homogeneous protein preparation was isolated from the liver soluble fraction and classified as transketolase. No other non-enzymatic proteins which bind thiamine diphosphate and can serve as substrates in the reaction of thiamine diphosphate synthesis in the hyaloplasm were found. It was shown that the phosphate group is transferred by rat liver thiamine diphosphate kinase to the free (but not to the protein-bound) thiamine diphosphate as it was believed earlier.     

Thiamine deficiency in rats affects thiamine metabolism possibly through the formation of oxidized thiamine pyrophosphate

BackgroundThiamine deficiency (TD) has a number of features in common with the neurodegenerative diseases development and close relationship between TD and oxidative stress (OS) has been repeatedly reported in the literature. The aim of this study is to understand how alimentary TD, accompanied by OS, affects the expression and level of two thiamine metabolism proteins in rat brain, namely, thiamine transporter 1 (THTR1) and thiamine pyrophosphokinase (TPK1), and what factors are responsible for the observed changes.MethodsThe effects of OS caused by TD on the THTR1and TPK1 expression in rat cortex, cerebellum and hippocampus were examined. The levels of active and oxidized forms of ThDP (enzymatically measured) in the blood and brain, ROS and SH-groups in the brain were also analyzed.ResultsTD increased the expression of THTR1 and protein level in all studied regions. In contrast, expression of TPK1 was depressed. TD-induced OS led to the accumulation of ThDP oxidized inactive form (ThDP_ox) in the blood and brain. In vitro reduction of ThDP_ox by dithiothreitol regenerates active ThDP suggesting that ThDP_ox is in disulfide form. A single high-dose thiamine administration to TD animals had no effect on THTR1 expression, partly raised TPK1 mRNA and protein levels, but is unable to normalize TPK1 enzyme activity. Brain and blood ThDP levels were increased in these conditions, but ThDP_ox was not decreased.General significanceIt is likely, that the accumulation of ThDP_ox in tissue could be seen as a potential marker of neurocellular dysfunction and thiamine metabolic state.     

Interaction of rat brain thiamine kinase with thiamine and its derivatives

Participation of the enzyme which provides the phosphorylation of thiamine to thiamindiphosphate (TDP) thiaminkinase (thiaminpyrophosphokinase, KF 2.7.6.2) of rat brain in the realization of thiamine action on the syntheses of acethylcholine (AC) was studied. The thiamine and its structure analogue, which differ the nature of the radicals in the 3-d and 5-e positions of the thiazollium cycle were used: 3-[(4-amino-2methylpyrimidinyl-5)methyl]-4-methylthiazolium chloride, 3-decyloxycarbonylmethyl-4-metyl-5-beta-hydrozyethylthiazolium chloride, 3-decyloxycarbonylmethyl-4-methylthiazolium chloride. All salts in the concentrations lower then Km render active influence on thiaminkinase. The analysis of data shows the presence of the regulation site on the enzyme distinguishing from the active enzyme centre and participating in the interaction with which the hydrophobic fragments of thiamine molecule participating. The comparative studies of thiamine and above mentioned derivatives influence on the inclusion of the labelled carbon with [2-(14)C] pyruvate in acethylcholine confirm an assumption about the key-role of the thiamine interaction with thiaminkinase (meaning its phosphorilation) regarding its action on the acethylcholine syntheses, and probably, on the function of the nervous cells as a whole.     

The role of bound thiamine pyrophosphate in the synthesis of thiamine triphosphate in rat liver.

Thiamine pyrophosphate-ATP phosphoryltransferase, the enzyme that catalyzes the synthesis of thiamine triphosphate, has been found in the supernatant fraction of rat liver. The substrate for the enzyme is endogenous, bound thiamine pyrophosphate, since the addition of exogenous thiamine pyrophosphate had no effect. Thus, when a rat liver supernatant was incubated with gamma-labelled [32P]ATP, thiamine [32P]triphosphate was formed whereas the incubation of thiamine [32P]pyrophosphate with ATP did not produce thiamine [32P]triphosphate. The endogenous thiamine pyrophosphate was found to be bound to a high molecular weight protein which comes out in the void volume of Sephadex G-75, and is not dialyzable. The activity that catalyzes the formation of thiamine triphosphate has an optimum pH between 6 and 6.5, a linear time course of thiamine triphosphate synthesis up to 30 min, and is not affected by Ca2+, cyclic GMP and sulfhydryl reagents.     

Isotopic labeling of thiamine

Thiamine of high specific radioactivity has been prepared by catalytic incorporation of tritium using ³H₂O and platinum. Purification of a crude commercially available product is described and the radiolabeling pattern is determined by NMR analysis of deuterated thiamine.