Pyrimidine Moiety Of Thiamine Research Articles

Induction of the Sugar-Phosphate Stress Response Allows Saccharomyces cerevisiae 2-Methyl-4-Amino-5-Hydroxymethylpyrimidine Phosphate Synthase To Function in Salmonella enterica.

Thiamine pyrophosphate is a required cofactor for all forms of life. The pyrimidine moiety of thiamine, 2-methyl-4-amino-5-hydroxymethylpyrimidine phosphate (HMP-P), is synthesized by different mechanisms in bacteria and plants compared to fungi. In this study, Salmonella enterica was used as a host to probe requirements for activity of the yeast HMP-P synthase, Thi5p. Thi5p synthesizes HMP-P from histidine and pyridoxal-5-phosphate and was reported to use a backbone histidine as the substrate, which would mean that it was a single-turnover enzyme. Heterologous expression of Thi5p did not complement an S. enterica HMP-P auxotroph during growth with glucose as the sole carbon source. Genetic analyses described here showed that Thi5p was activated in S. enterica by alleles of sgrR that induced the sugar-phosphate stress response. Deletion of ptsG (encodes enzyme IICB [EIICB] of the phosphotransferase system [PTS]) also allowed function of Thi5p and required sgrR but not sgrS. This result suggested that the role of sgrS in activation of Thi5p was to decrease PtsG activity. In total, the data herein supported the hypothesis that one mechanism to activate Thi5p in S. enterica grown on minimal medium containing glucose (minimal glucose medium) required decreased PtsG activity and an unidentified gene regulated by SgrR. This work describes a metabolic link between the sugar-phosphate stress response and the yeast thiamine biosynthetic enzyme Thi5p when heterologously expressed in Salmonella enterica during growth on minimal glucose medium. Suppressor analysis (i) identified a mutant class of the regulator SgrR that activate sugar-phosphate stress response constitutively and (ii) determined that Thi5p is conditionally active in S. enterica. These results emphasized the power of genetic systems in model organisms to uncover enzyme function and underlying metabolic network structure.

Aminoimidazole Carboxamide Ribotide Exerts Opposing Effects on Thiamine Synthesis in Salmonella enterica.

In Salmonella enterica, the thiamine biosynthetic intermediate 5-aminoimidazole ribotide (AIR) can be synthesized de novo independently of the early purine biosynthetic reactions. This secondary route to AIR synthesis is dependent on (i) 5-amino-4-imidazolecarboxamide ribotide (AICAR) accumulation, (ii) a functional phosphoribosylaminoimidazole-succinocarboxamide (SAICAR) synthetase (PurC; EC 6.3.2.6), and (iii) methionine and lysine in the growth medium. Studies presented here show that AICAR is a direct precursor to AIR in vivo. PurC-dependent conversion of AICAR to AIR was recreated in vitro. Physiological studies showed that exogenous nutrients (e.g., methionine and lysine) antagonize the inhibitory effects of AICAR on the ThiC reaction and decreased the cellular thiamine requirement. Finally, genetic results identified multiple loci that impacted the effect of AICAR on thiamine synthesis and implicated cellular aspartate levels in AICAR-dependent AIR synthesis. Together, the data here clarify the mechanism that allows conditional growth of a strain lacking the first five biosynthetic enzymes, and they provide additional insights into the complexity of the metabolic network and its plasticity. In bacteria, the pyrimidine moiety of thiamine is derived from aminoimidazole ribotide (AIR), an intermediate in purine biosynthesis. A previous study described conditions under which AIR synthesis is independent of purine biosynthesis. This work is an extension of that previous study and describes a new synthetic pathway to thiamine that depends on a novel thiamine precursor and a secondary activity of the biosynthetic enzyme PurC. These findings provide mechanistic details of redundancy in the synthesis of a metabolite that is essential for nucleotide and coenzyme biosynthesis. Metabolic modifications that allow the new pathway to function or enhance it are also described.

THIAMINE – INDUCED FORMATION OF THE MONOPYRROLE MOIETY OF PRODIGIOSIN

Thiamine stimulates the production of a red pigment , which is chromatographically and spectrophotometrically identical to prodigiosin , by growing cultures of serratia marcescens mutant 9-3-3 . this mutant is blocked in the formation of 2- methyl -3- amyl pyrorol( MAP),the monopyrrole moiety of prodigiosin , but accumulates 4-methoxy-2, 2-bipyrrole -5- carboxaldehyde (MBC) and can couple this compound with( MAP) to form prodigiosin . Addition of thiamine caused production of( MAP) , and as little as 0.02 mg of thiamine / ml in peptone- glycerol medium stimulated production of measurable amounts of prodigiosin. Phosphate saltes and another type of peptone decreased the thiamine- induced formation of prodigiosin ,yeast extract and glycerol enhanced formation of this substance. Thiamine also enhanced production of prodigiosin by wiled – type Strain Nima of S. marcescens . The pyrimidine moiety of thiamine was also 10% as effective as the vitamin ; the thiazol moiety only 4% , and the two moieties together , 25% . Thiamine did not stimulate production of prodigiosin biosynthesis as strain 9-3-3 . This is not surprising since strain 9-3-3 originated as a result of two mutational events one event may involve thiamine directly , and the other may involve the biosynthesis of( MAP).

THIAMINE – INDUCED FORMATION OF THE MONOPYRROLE MOIETY OF PRODIGIOSIN

Thiamine stimulates the production of a red pigment , which is chromatographically and spectrophotometrically identical to prodigiosin , by growing cultures of serratia marcescens mutant 9-3-3 . this mutant is blocked in the formation of 2- methyl -3- amyl pyrorol( MAP),the monopyrrole moiety of prodigiosin , but accumulates 4-methoxy-2, 2-bipyrrole -5- carboxaldehyde (MBC) and can couple this compound with( MAP) to form prodigiosin . Addition of thiamine caused production of( MAP) , and as little as 0.02 mg of thiamine / ml in peptone- glycerol medium stimulated production of measurable amounts of prodigiosin. Phosphate saltes and another type of peptone decreased the thiamine- induced formation of prodigiosin ,yeast extract and glycerol enhanced formation of this substance. Thiamine also enhanced production of prodigiosin by wiled – type Strain Nima of S. marcescens . The pyrimidine moiety of thiamine was also 10% as effective as the vitamin ; the thiazol moiety only 4% , and the two moieties together , 25% . Thiamine did not stimulate production of prodigiosin biosynthesis as strain 9-3-3 . This is not surprising since strain 9-3-3 originated as a result of two mutational events one event may involve thiamine directly , and the other may involve the biosynthesis of( MAP).

Raman and surface-enhanced Raman study of thiamine at different pH values

Raman spectroscopy was employed for evidencing the protonated and unprotonated thiamine molecular species in aqueous solutions at different acid and basic pH. The recorded Raman spectra were assigned on the basis of density functional theory (DFT) calculations. Also, at different pH values of the gold sol, the adsorption of the two molecular species to colloidal metal particles was monitored by means of surface-enhanced Raman scattering (SERS). At pH values of 3, 4 and 5, the SERS spectra revealed the coexistence of unprotonated and protonated thiamine molecules adsorbed on the gold surface. The adsorption was concluded taking place through the N3′ and N1′ atoms from the pyrimidine moiety of thiamine. The orientation to the gold surface of the protonated and unprotonated thiamine molecular species was discussed.

Biosynthesis of the pyrimidine moiety of thiamine independent of the PurF enzyme (Phosphoribosylpyrophosphate amidotransferase) in Salmonella typhimurium: incorporation of stable isotope-labeled glycine and formate.

Genetic analyses have suggested that the pyrimidine moiety of thiamine can be synthesized independently of the first enzyme of de novo purine synthesis, phosphoribosylpyrophosphate amidotransferase (PurF), in Salmonella typhimurium. To obtain biochemical evidence for and to further define this proposed synthesis, stable isotope labeling experiments were performed with two compounds, [2-13C]glycine and [13C]formate. These compounds are normally incorporated into thiamine pyrophosphate (TPP) via steps in the purine pathway subsequent to PurF. Gas chromatography-mass spectrometry analyses indicated that both of these compounds were incorporated into the pyrimidine moiety of TPP in a purF mutant. This result clearly demonstrated that the pyrimidine moiety of thiamine was being synthesized in the absence of the PurF enzyme and strongly suggested that this synthesis utilized subsequent enzymes of the purine pathway. These results were consistent with an alternative route to TPP that bypassed only the first enzyme in the purine pathway. Experiments quantitating cellular thiamine monophosphate (TMP) and TPP levels suggested that the alternative route to TPP did not function at the same capacity as the characterized pathway and determined that levels of TMP and TPP in the wild-type strain were significantly altered by the presence of purines in the medium.

Transition and Group IIB Metal Complexes With “Active Aldehyde” Derivatives of Thiamine

The Zn2+, Cd2+, Hg2+, Co2+ and Ni2+ ions produce zwitterionic type complexes with the ligands (L), 2-(α-hydroxy-benzyl)thiamine=HBT and 2-(α-hydroxy-cyclohexyl-methyl)thiamine = HCMT, of the type MLCl3. The ligands are in the S conformation, the metals are bound to N1, of the pyrimidine moiety of thiamine and the complexes have a trigonally distorted tetrahedral structure, as the crystal structure of the complex Zn(HCMT)Cl3 (orthorombic, a=14.4 b=14.1 c=17.4 β=105.6O V=3392A3 R=13.8%), the one and two dimensional 1H nmr spectra of the Zn2+, Cd2+ and Hg2+ complexes and the electronic spectra of the Co2+ and Ni2+ complexes show. A brief review of the previous techniques (structure of the Hg(HBT)Cl3 complex, IR-Raman spectra, 13C nmr in solution and solid state etc) used to characterize these complexes, is also given here and the proper conclusions drawn.

Investigation on Incorporation of Glycine into The Pyrimidine Moiety of Thiamin in Eucaryote, Saccharomyces cerevisiae

Investigation on Incorporation of Glycine into The Pyrimidine Moiety of Thiamin in Eucaryote, Saccharomyces cerevisiae

Incorporation of histidine into the pyrimidine moiety of thiamin in Saccharomyces cerevisiae

To investigate the incorporation of histidine into the pyrimidine moiety of thiamin in eukaryotes, Saccharomyces cerevisiae was grown in a synthetic medium in the presence of 15N- or 14C-labeled histidine. Two 15N-atoms of DL-[1,3-15N2]histidine were incorporated into the N-3 and amino-N atom at C-4 of pyrimidine. Furthermore, incorporation of the 15N-amino group of aspartate, the origin of the N-1 of histidine, into the N-3 of pyrimidine shows that N-3 and the amino-N atom at C-4 of pyrimidine are derived from N-1 and N-3 of histidine, respectively. In contrast, the 15N atom of DL-[amino-15N]histidine was not incorporated into the molecule, whereas L-[2-14C]histidine was incorporated directly into the pyrimidine. We conclude that N-1, C-2, and N-3 of histidine are the origins of the N-3, C-4, and amino-N at C-4 of the pyrimidine in thiamin synthesized by S. cerevisiae.

Transport of thiamine and 4-methyl-5-hydroxyethylthiazole by Salmonella typhimurium

The transport of thiamine and 4-methyl-5-hydroxyethylthiazole (MHET), its thiazole moiety, was studied using whole cells of Salmonella typhimurium. It was found that the bacteria possessed an active transport system for thiamine that had K m 0.21 μM and V max 33 nmol·min −1·(mg dry wt. cells) −1. Transport of thiamine was glucose dependent, whereas MHET uptake was dependent on both glucose and 2-methyl-4-amino-5-hydroxymethylpyrimidine (MAHMP), the pyrimidine moiety of thiamine. Uptake of both thiamine and MHET was severely curtailed by cyanide, azide, N-ethylmaleimide and carbonyl cyanide m-chlorophenylhydrazone. Oxythiamine inhibited thiamine, but not MHET, uptake and thiamine slightly inhibited MHET uptake. 2-Methyl-4-amino-5-methoxymethylpyrimidine and 4-amino-5-hydroxymethylpyrimidine were unable to replace MAHMP as stimulators of MHET uptake, but 2-methyl-4-amino-5-aminomethylpyrimidine was marginally effective in this regard. Similar results were obtained with attempts to replace MAHMP as a growth requirement for a purD mutant of Salmonella typhimurium. MHET uptake showed saturation kinetics only in the presence of MAHMP, and is not otherwise actively transported.

Precursor of carbon atom five and hydroxymethyl carbon atom of the pyrimidine moiety of thiamin in Escherichia coli.

The pyrimidine moiety of thiamin can be synthesized in bacteria from 5-aminoimidazole ribotide (AIR), an intermediate in purine biosynthesis. To transform the imidazole ring of AIR to the pyrimidine, the bond between C-4 and C-5 of the imidazole ring should be ruptured and a two-carbon unit should be inserted. We investigated a precursor of the two-carbon unit which should be inserted to form the pyrimidine. To examine the possibility of the two-carbon compound as a precursor which is formed from a three-carbon intermediate in the glycolytic pathway and its three-carbon derivative by decarboxylation in cells, we studied the incorporation of [2-14C]glycerol, [2-14C]pyruvate, [U-14C]alanine and [3-14C]serine into the pyrimidine by Escherichia coli. Glycerol, pyruvate and alanine were not incorporated into the pyrimidine. Radioactive carbon of [3-14C]serine was incorporated into C-2 of the pyrimidine via one-carbon units pool. Then, we investigated the possibility of a ribose moiety of AIR being a precursor of the two-carbon unit. As ribose has low permeability into E. coli cells, the ribose moiety of AIR was labeled with [14C]glucose which was added to the growth medium. The results show that two radioactive carbons of [U-14C]glucose were incorporated into the pyrimidine and radioactive carbon of [6-14C]glucose was incorporated into hydroxymethyl carbon attached to C-5 of the pyrimidine. The dilution rates of the specific radioactivity of the pyrimidine from [U-14C]glucose and [6-14C]glucose well coincide with those of the ribose moiety of the nucleotide (AMP). Radioactive carbon of [1-14C]glucose was not incorporated into the pyrimidine and nucleotide. It is concluded that the two-carbon fragment derived from C-4' and C-5' of the ribose moiety of AIR can be incorporated into the pyrimidine moiety of thiamin as the precursor of C-5 and hydroxymethyl carbon.

An X-ray evidence for the ring-stacking interaction between indole ring and the pyrimidine moiety of thiamin: Crystal structure of thiamin indole-3-propionate

The crystal structure of thiamin indole-3-propionate was determined by X-ray diffraction as a model for the possible thiamin coenzyme-tryptophan residue interaction at the binding site of thiamin pyrophosphate dependent enzymes. There is an intermolecular stacking interaction of the indole ring with the pyrimidine ring, but not with the positively charged thiazolium ring, of thiamin retaining the characteristic F-conformation. Although this association is due to dipole-dipole interaction between both aromatic rings, charge-transfer interaction cannot be ruled out in solution state because the absorption spectrum shows the characteristic charge-transfer band.

The biosynthesis of thiamine. Syntheses of [1,1,1,5-2H4]-1-deoxy-D-threo-2-pentulose and incorporation of this sugar in biosynthesis of thiazole by Escherichia coli cells

Non-growing, washed cells of Escherichia coli, depressed for the synthesis of thiamine, were incubated in the presence of [1,1,1,5-2H4]-1-deoxy-D-threo-2-pentulose (9) in a medium containing the pyrimidine moiety of thiamine, L-tyrosine, and glucose. The thiamine thus biosynthesized was extracted and cleaved to give 5-(2-hydroxyethyl)-4-methylthiazole (HET) which was examined as the trifluoroacetate derivative by electron-impact mass spectrometry. The distribution of the label in the fragments indicated that the pentulose (9) was a precursor of the C5-chain of HET without C–C bond cleavage. Several routes to 1-deoxypentuloses are described. Condensation of 2,4-O-benzylidene-D-[4-2H1]threose (23) with trideuteriomethylmagnesium iodide gave the protected 1-deoxypentitols (24) and (25). Brominolysis of the mixed dibutylstannylidenes then afforded [1,1,1,5-2H4]-3,5-O-benzylidene-1-deoxy-D-threo-2-pentulose (26), which was converted into the free sugar (9) by acidic hydrolysis. 1-Deoxy-D-erythro-2-pentulose was prepared in similar manner. Condensation of 2-([2H3]-methyl)-1,3-dithian with 2,3-O-isopropylidene-D-glyceraldehyde, followed by a C-3 epimerization step also led, after deprotection, to a mixture of [1,1,1-2H3]-1-deoxy-D-erythro- and [1,1,1-2H3]-1-deoxy-D-threo-2-pentulose, (5) and (6).

Investigation on Localization of Radioactive Carbon in the Pyrimidine Moiety of Thiamin from Saccharomyces cerevisiae Grown in the Presence of [<14>^C] Formate

Investigation on Localization of Radioactive Carbon in the Pyrimidine Moiety of Thiamin from Saccharomyces cerevisiae Grown in the Presence of [<14>^C] Formate

Incorporation of glycine in pyrimidine and thiazole moieties of thiamine in Escherichia coli

[U− 14C]Glycine was incorporated into hydroxymethylpyrimidine and thiamine by a thiamine regulatory mutant of Escherichia coli with 5.1 and 2.5 × dilutions, respectively. The incorporation of 14C radioactivity into the pyrimidine moiety of thiamine was effectively decreased by the addition of hydroxymethylpyrimidine to the medium in the presence of adenine, whereas the incorporation into the thiazole moiety was decreased by thiazole in the presence of phenylalanine.

Studies on Vitamin B<SUB>1</SUB> and Related Compounds. CIX. A Novel Cleavage of Thiamin and Its Homologues by the Reaction with Aromatic Aldehydes

A novel cleavage reaction of thiamin to give 4-amino-2, 5-dimethylpyrimidine (III) and 2-benzoyl-5-(2-hydroxyethyl)-4-methylthiazole (IV) has been discovered. The reaction involves the treatment of thiamin chloride hydrochloride (I) with two mole equivalents of triethylamine and an excess of benzaldehyde in methanol. The finding was further extended to the reactions of thiamin and its homologs with a variety of aromatic aldehydes to give several new 2-acylthiazoles. The pyrimidine moiety of thiamin was indispensable for these reactions. On the other hand, the reaction of thiamin with cinnamaldehyde gave 2, 9a-dimethyl-9-(2-hydroxyethyl)-7-(3-methoxy-3-phenylpropionyl)-5, 9, 9a, -10-tetrahydro-7H-pyrimido [4, 5-d] thiazolo [3, 4-a] pyrimidine (XV).

Thiamine-induced formation of the monopyrrole moiety of prodigiosin.

Thiamine stimulates the production of a red pigment, which is chromatographically and spectrophotometrically identical to prodigiosin, by growing cultures of Serratia marcescens mutant 9-3-3. This mutant is blocked in the formation of 2-methyl-3-amylpyrrole (MAP), the monopyrrole moiety of prodigiosin, but accumulates 4-methoxy-2,2,'-bipyrrole-5-carboxaldehyde (MBC) and can couple this compound with MAP to form prodigiosin. Addition of thiamine caused production of MAP, and as little as 0.02 mg of thiamine per ml in a peptone-glycerol medium stimulated production of measurable amounts of prodigiosin. Phosphate salts and another type of peptone decreased the thiamine-induced formation of prodigiosin; yeast extract and glycerol enhanced the formation of this substance. Thiamine also enhanced production of prodigiosin by wild-type strain Nima of S. marcescens. The thiamine antagonists, oxythiamine and pyrithiamine, inhibited thiamine-induced production of MAP and of prodigiosin by the mutant strain 9-3-3, formation of prodigiosin by the wild-type strain Nima, and production of MAP by another mutant, strain WF. The pyrimidine moiety of thiamine was only 10% as effective as the vitamin; the thiazole moiety, only 4%; and the two moieties together, 25%. Various other vitamins tested did not stimulate formation of prodigiosin by strain 9-3-3. Thiamine did not stimulate production of prodigiosin by a single-step mutant that showed the same phenotypic block in prodigiosin biosynthesis as strain 9-3-3. This is not surprising since strain 9-3-3 originated as a result of two mutational events. One event may involve thiamine directly, and the other may involve the biosynthesis of MAP. Thiamine is probably involved in the regulation of the biosynthesis of MAP, because the vitamin or inhibitory antagonists must be added during the early phases of growth in order to be effective.

Acetate as a methyl group precursor in the pyrimidine moiety of thiamine

Acetate as a methyl group precursor in the pyrimidine moiety of thiamine

GROWTH STIMULATING EFFECT OF 4-METHYL-5(2-HYDROXYETHYL) THIAZOLE ON THIAMINE-DEFICIENT RATS.

It is well known that 4-methyl-5 (2-hydroxyethyl) thiazole (MHET) can replace thiamine as a nutrient for some bacteria (1). Abderhalden (2,3) without success attempted to demonstrate that MHET could replace thiamine in the diet of the rat. Since then, MHET and the pyrimidine moiety of thiamine (2-methyl-4-amino-5-hydroxymethyl pyrimidine) have been isolated from urine of rats after givin large doses of thiamine (4,5), suggesting that the rat is capable of cleaving thiamine to give these two products. Also Van Eys (6) has identified MHET as a component of glycerol phosphate dehydrogenase, which indicates a strong possibility that MHET is biologically active. It, therefore, seemed desirable to investigate once again the possibility that MHET is biologically active. As workers in the past have apparently achieved essentially negative results (2,3,6,8) our experimental approach involved 3 special considerations.1. From chemical considerations it seemed possible that MHET, if not carefully prepared, may not be...

The biosynthesis of thiamine: V. Studies concerning precursors of the pyrimidine moiety

The fact that neither radioactive uracil nor known precursors of this pyrimidine (orotic acid and aspartic acid) are incorporated in any significant quantities into the pyrimidine moiety of thiamine by Escherichia coli indicates that this specific pyrimidine compound is made by a biosynthetic pathway that is different from that by which other pyrimidines are made. Other radioactive compounds that are not incorporated into this pyrimidine to any significant extent are methionine-C 14H 3 and serine-3-C 14. Formate-C 14 is incorporated relatively efficiently and acetate-2-C 14 and glycine-2-C 14 are also incorporated, but to a lesser extent by several fold than is formate.