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| thiamine(1+) monophosphate |
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| CHEBI:9533 |
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| A thiamine phosphate that is thiamin(1+) in which the hydroxy group is replaced by a phosphate group. |
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This entity has been manually annotated by the ChEBI Team.
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| ZINC000001532839 |
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Molfile
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SDF
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more structures >>
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Ideal conformer Mrv1927 07192111173D starting HoverWatcher_5 Time for openFile(TPS - Ideal conformer Mrv1927 07192111173D 40 41 0 0 0 0 999 V2000 6.6400 1.9250 1.4110 C 0 0 0 0 0 0 0 0 0 0 0 0 1.3700 -0.8600 -0.7330 N 0 3 0 0 0 4 0 0 0 0 0 0 1.3620 -2.0670 -0.2780 C 0 0 0 0 0 0 0 0 0 0 0 0 0.0150 -2.3020 0.7480 S 0 0 0 0 0 0 0 0 0 0 0 0 -0.4910 -0.6350 0.4800 C 0 0 0 0 0 0 0 0 0 0 0 0 0.3920 -0.0660 -0.3470 C 0 0 0 0 0 0 0 0 0 0 0 0 0.2780 1.3670 -0.8000 C 0 0 0 0 0 0 0 0 0 0 0 0 -1.7040 0.0300 1.0770 C 0 0 1 0 0 0 0 0 0 0 0 0 -2.9030 -0.1660 0.1470 C 0 0 1 0 0 0 0 0 0 0 0 0 -4.0530 0.4650 0.7120 O 0 0 0 0 0 0 0 0 0 0 0 0 4.6130 2.0900 0.0710 N 0 0 0 0 0 0 0 0 0 0 0 0 5.5260 1.3070 0.6060 C 0 0 0 0 0 0 0 0 0 0 0 0 5.4930 -0.0020 0.4580 N 0 0 0 0 0 0 0 0 0 0 0 0 4.5250 -0.5810 -0.2470 C 0 0 0 0 0 0 0 0 0 0 0 0 4.4940 -1.9570 -0.4010 N 0 0 0 0 0 0 0 0 0 0 0 0 3.5410 0.2210 -0.8280 C 0 0 0 0 0 0 0 0 0 0 0 0 3.6160 1.5880 -0.6450 C 0 0 0 0 0 0 0 0 0 0 0 0 2.4240 -0.3900 -1.6350 C 0 0 1 0 0 0 0 0 0 0 0 0 -5.4970 0.4570 0.0000 P 0 0 1 0 0 5 0 0 0 0 0 0 -5.9010 -0.9380 -0.2810 O 0 0 0 0 0 0 0 0 0 0 0 0 -6.5800 1.1430 0.9740 O 0 0 0 0 0 0 0 0 0 0 0 0 -5.4180 1.2820 -1.3800 O 0 0 0 0 0 0 0 0 0 0 0 0 6.3390 1.9920 2.4570 H 0 0 0 0 0 0 0 0 0 0 0 0 7.5340 1.3060 1.3300 H 0 0 0 0 0 0 0 0 0 0 0 0 6.8530 2.9230 1.0300 H 0 0 0 0 0 0 0 0 0 0 0 0 2.1000 -2.8200 -0.5120 H 0 0 0 0 0 0 0 0 0 0 0 0 -0.3920 1.4250 -1.6580 H 0 0 0 0 0 0 0 0 0 0 0 0 -0.1210 1.9740 0.0130 H 0 0 0 0 0 0 0 0 0 0 0 0 1.2630 1.7390 -1.0820 H 0 0 0 0 0 0 0 0 0 0 0 0 -1.9210 -0.4140 2.0480 H 0 0 0 0 0 0 0 0 0 0 0 0 -1.5100 1.0960 1.2000 H 0 0 0 0 0 0 0 0 0 0 0 0 -2.6850 0.2780 -0.8250 H 0 0 0 0 0 0 0 0 0 0 0 0 -3.0960 -1.2320 0.0230 H 0 0 0 0 0 0 0 0 0 0 0 0 5.1810 -2.5070 0.0070 H 0 0 0 0 0 0 0 0 0 0 0 0 3.7840 -2.3710 -0.9160 H 0 0 0 0 0 0 0 0 0 0 0 0 2.8720 2.2390 -1.0800 H 0 0 0 0 0 0 0 0 0 0 0 0 2.8100 -1.2300 -2.2120 H 0 0 0 0 0 0 0 0 0 0 0 0 2.0150 0.3590 -2.3140 H 0 0 0 0 0 0 0 0 0 0 0 0 -7.4740 1.1730 0.6090 H 0 0 0 0 0 0 0 0 0 0 0 0 -5.1590 2.2070 -1.2720 H 0 0 0 0 0 0 0 0 0 0 0 0 1 12 1 0 0 0 0 1 23 1 0 0 0 0 1 24 1 0 0 0 0 1 25 1 0 0 0 0 2 3 2 0 0 0 0 2 6 1 0 0 0 0 2 18 1 0 0 0 0 3 4 1 0 0 0 0 3 26 1 0 0 0 0 4 5 1 0 0 0 0 5 6 2 0 0 0 0 5 8 1 0 0 0 0 6 7 1 0 0 0 0 7 27 1 0 0 0 0 7 28 1 0 0 0 0 7 29 1 0 0 0 0 8 9 1 0 0 0 0 8 30 1 0 0 0 0 8 31 1 0 0 0 0 9 10 1 0 0 0 0 9 32 1 0 0 0 0 9 33 1 0 0 0 0 10 19 1 0 0 0 0 11 12 2 0 0 0 0 11 17 1 0 0 0 0 12 13 1 0 0 0 0 13 14 2 0 0 0 0 14 15 1 0 0 0 0 14 16 1 0 0 0 0 15 34 1 0 0 0 0 15 35 1 0 0 0 0 16 17 2 0 0 0 0 16 18 1 0 0 0 0 17 36 1 0 0 0 0 18 37 1 0 0 0 0 18 38 1 0 0 0 0 19 20 2 0 0 0 0 19 21 1 0 0 0 0 19 22 1 0 0 0 0 21 39 1 0 0 0 0 22 40 1 0 0 0 0 M CHG 1 2 1 M END): 17 ms reading 40 atoms ModelSet: haveSymmetry:false haveUnitcells:false haveFractionalCoord:false 1 model in this collection. 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| Thiamine monophosphate, also known as ThMP and TMP, is a phosphate ester of thiamine.
It is an intermediate from the hydrolysis of thiamine diphosphate to free thiamine by alkaline phosphatase. The conversion of ThMP to thiamine cannot be facilitated by acid hydrolysis. ThMP is also enzymatically synthesized by thiamine-phosphate pyrophosphorylase, which combines thiazole in its monophosphate form and pyrimidine as a pyrophosphate.
The physiological function of ThMP has not been identified. |
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Read full article at Wikipedia
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InChI=1S/C12H17N4O4PS/c1- 8-11(3-4-20-21(17,18)19)22-7-16(8)6-10-5-14-9(2)15-12(10)13/h5,7H,3-4,6H2,1-2H3,(H3-,13,14,15,17,18,19)/p+1 |
| HZSAJDVWZRBGIF-UHFFFAOYSA-O |
| CC1=C(CCOP(O)(O)=O)SC=[N+]1CC1=C(N)N=C(C)N=C1 |
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Mus musculus
(NCBI:txid10090)
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Source: BioModels - MODEL1507180067
See:
PubMed
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Synechococcus elongatus
(NCBI:txid32046)
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Found in
endometabolome
From MetaboLights
See:
MetaboLights Study
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Arabidopsis thaliana
(NCBI:txid3702)
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See:
PubMed
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Brassica napus
(NCBI:txid3708)
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Found in
leaf lamina
(BTO:0000719).
From MetaboLights
See:
MetaboLights Study
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Micromonas pusilla
(NCBI:txid38833)
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From MetaboLights
See:
MetaboLights Study
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Saccharomyces cerevisiae
(NCBI:txid4932)
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Source: yeast.sf.net
See:
PubMed
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Escherichia coli
(NCBI:txid562)
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See:
PubMed
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Trypanosoma brucei
(NCBI:txid5691)
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From MetaboLights
See:
MetaboLights Study
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Ruegeria pomeroyi
(NCBI:txid89184)
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Found in
endometabolome
From MetaboLights
See:
MetaboLights Study
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Homo sapiens
(NCBI:txid9606)
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See:
PubMed
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Bronsted base
A molecular entity capable of accepting a hydron from a donor (Bronsted acid).
(via organic amino compound )
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plant metabolite
Any eukaryotic metabolite produced during a metabolic reaction in plants, the kingdom that include flowering plants, conifers and other gymnosperms.
human metabolite
Any mammalian metabolite produced during a metabolic reaction in humans (Homo sapiens).
Escherichia coli metabolite
Any bacterial metabolite produced during a metabolic reaction in Escherichia coli.
Saccharomyces cerevisiae metabolite
Any fungal metabolite produced during a metabolic reaction in Baker's yeast (Saccharomyces cerevisiae ).
mouse metabolite
Any mammalian metabolite produced during a metabolic reaction in a mouse (Mus musculus).
Arabidopsis thaliana metabolite
Any plant metabolite that is produced by Arabidopsis thaliana.
mammalian metabolite
Any animal metabolite produced during a metabolic reaction in mammals.
water-soluble vitamin (role)
Any vitamin that dissolves in water and readily absorbed into tissues for immediate use. Unlike the fat-soluble vitamins, they are not stored in the body and need to be replenished regularly in the diet and will rarely accumulate to toxic levels since they are quickly excreted from the body via urine.
(via B vitamin )
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nutraceutical
A product in capsule, tablet or liquid form that provide essential nutrients, such as a vitamin, an essential mineral, a protein, an herb, or similar nutritional substance.
(via B vitamin )
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View more via ChEBI Ontology
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3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-4-methyl-5-[2-(phosphonooxy)ethyl]-1,3-thiazol-3-ium
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2-[3-[(4-amino-2-methyl-pyrimidin-5-yl)methyl]-4-methyl-thiazol-3-ium-5-yl]ethyl dihydrogen phosphate
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PDBeChem
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2-[3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-4-methyl-1,3-thiazol-3-ium-5-yl]ethyl dihydrogen phosphate
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ChEBI
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2-[3-[(4-azanyl-2-methyl-pyrimidin-5-yl)methyl]-4-methyl-1,3-thiazol-3-ium-5-yl]ethyl dihydrogen phosphate
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ChEBI
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3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-4-methyl-5-[2-(phosphonooxy)ethyl]thiazolium
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ChEBI
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thiamin monophosphate
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KEGG COMPOUND
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thiamin phosphate
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KEGG COMPOUND
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thiamine monophosphate
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KEGG COMPOUND
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thiamine phosphate
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KEGG COMPOUND
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TMP
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KEGG COMPOUND
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vitamin B1 monophosphate
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ChEBI
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vitamin B1 phosphate
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ChEBI
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10023-48-0
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CAS Registry Number
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DrugBank
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1063443
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Gmelin Registry Number
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Gmelin
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3916670
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Reaxys Registry Number
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Reaxys
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Huang Y, Gibson RA, Green TJ (2020)
Measuring thiamine status in dried blood spots.
Clinica chimica acta; international journal of clinical chemistry 509, 52-59 [PubMed:32505772]
[show Abstract]
BackgroundA reliable and robust method with minimum sample collection requirement for thiamine assay is needed in clinical and research settings.MethodsA simple and robust assay for three vitamers (thiamine, Th; thiamine monophosphate, TMP; and thiamine diphosphate, TDP) using a 6.35-mm dried blood spot (DBS) disc was developed, validated and applied.ResultsWe were able to quantify accurately thiamine status covering all major vitamers Th, TMP and TDP with acceptable recovery (90%-114%), limit of quantification (TDP: 3.0 nM, TMP and Th: 1.5 nM), linearity (TDP: LOQ 400 nM, TMP and Th: LOQ 50 nM, all R2 > 0.99), imprecision (coefficient variation < 4.3% for TDP, <10.0% for TMP and < 12.6% for Th) and stability at -20 °C for up to 42 days. By recruiting 20 healthy participants, we cross compared finger capillary DBS with venous whole blood and venous blood pre-spotted on filter papers. The results demonstrated minimum bias between methods. A preliminary dosing study showed the method had excellent sensitivity after a single dose of supplemental thiamine.ConclusionsWe have developed and clinically validated a simple, robust, accurate and sensitive assay for the analysis of thiamine status in DBS, suitable for large-scale population studies. | Jensen O, Matthaei J, Blome F, Schwab M, Tzvetkov MV, Brockmöller J (2020)
Variability and Heritability of Thiamine Pharmacokinetics With Focus on OCT1 Effects on Membrane Transport and Pharmacokinetics in Humans.
Clinical pharmacology and therapeutics 107, 628-638 [PubMed:31593619]
[show Abstract]
Thiamine is substrate of the hepatic uptake transporter organic cation transporter 1 (OCT1), and pathological lipid metabolism was associated with OCT1-dependent thiamine transport. However, it is unknown whether clinical pharmacokinetics of thiamine is modulated by OCT1 genotype. We analyzed thiamine transport in vitro, thiamine blood concentrations after high-dose and low-dose (nutritional) intake, and heritability of thiamine and thiamine-phosphate blood concentrations. The variant OCT1*2 had reduced and OCT1*3 to OCT1*6 had deficient thiamine uptake activity. However, pharmacokinetics of thiamine did not differ depending on OCT1 genotype. Further studies in primary human hepatocytes indicated that several cation transporters, including OCT1, OCT3, and THTR-2, contribute to hepatic uptake of thiamine. As much as 54% of the variation in thiamine and 75% in variation of thiamine monophosphate plasma concentrations was determined by heritable factors. Apparently, thiamine is not useful as a probe drug for OCT1 activity, but the high heritability, particularly of thiamine monophosphate, may stimulate further genomic research. | Sullivan AH, Dranow DM, Horanyi PS, Lorimer DD, Edwards TE, Abendroth J (2019)
Crystal structures of thiamine monophosphate kinase from Acinetobacter baumannii in complex with substrates and products.
Scientific reports 9, 4392 [PubMed:30867460]
[show Abstract]
Thiamine monophosphate kinase (ThiL) catalyzes the last step of thiamine pyrophosphate (TPP) synthesis, the ATP-dependent phosphorylation of thiamine monophosphate (TMP) to thiamine pyrophosphate. We solved the structure of ThiL from the human pathogen A. baumanii in complex with a pair of substrates TMP and a non-hydrolyzable adenosine triphosphate analog, and in complex with a pair of products TPP and adenosine diphosphate. High resolution of the data and anomalous diffraction allows for a detailed description of the binding mode of substrates and products, and their metal environment. The structures further support a previously proposed in-line attack reaction mechanism and show a distinct variability of metal content of the active site. | Schmidt A, Pratsch H, Schreiner MG, Mayer HK (2017)
Determination of the native forms of vitamin B1 in bovine milk using a fast and simplified UHPLC method.
Food chemistry 229, 452-457 [PubMed:28372200]
[show Abstract]
The aim of this study was to develop a high-throughput UHPLC method for the determination vitamin B1 active compounds; thiamin, thiamin monophosphate and thiamin diphosphate in bovine milk. In order to sustain the native vitamin B1 phosphorus esters, sample preparation is crucial. Various acids as well as commonly used enzymes and their enzyme mixtures were compared. Method accuracy was confirmed using certified reference material as well as comparison with the corresponding CEN method, and was found to be satisfactory. Studied milk samples showed significant amounts of thiamin monophosphate, which can make up to 53.9% of the total vitamin B1 content in commercial milk, and up to 78% in raw milk. Moreover, a tremendous variation of the total content of vitamin B1 was observed between single cows, which ranged from 0.24mg/L up to 0.54mg/L of total vitamin B1. | Rodionov DA, Leyn SA, Li X, Rodionova IA (2017)
A Novel Transcriptional Regulator Related to Thiamine Phosphate Synthase Controls Thiamine Metabolism Genes in Archaea.
Journal of bacteriology 199, e00743-16 [PubMed:27920295]
[show Abstract]
Thiamine (vitamin B1) is a precursor of thiamine pyrophosphate (TPP), an essential coenzyme in the central metabolism of all living organisms. Bacterial thiamine biosynthesis and salvage genes are controlled at the RNA level by TPP-responsive riboswitches. In Archaea, TPP riboswitches are restricted to the Thermoplasmatales order. Mechanisms of transcriptional control of thiamine genes in other archaeal lineages remain unknown. Using the comparative genomics approach, we identified a novel family of transcriptional regulators (named ThiR) controlling thiamine biosynthesis and transport genes in diverse lineages in the Crenarchaeota phylum as well as in the Halobacteria and Thermococci classes of the Euryarchaeota ThiR regulators are composed of an N-terminal DNA-binding domain and a C-terminal ligand-binding domain, which is similar to the archaeal thiamine phosphate synthase ThiN. By using comparative genomics, we predicted ThiR-binding DNA motifs and reconstructed ThiR regulons in 67 genomes representing all above-mentioned lineages. The predicted ThiR-binding motifs are characterized by palindromic symmetry with several distinct lineage-specific consensus sequences. In addition to thiamine biosynthesis genes, the reconstructed ThiR regulons include various transporters for thiamine and its precursors. Bioinformatics predictions were experimentally validated by in vitro DNA-binding assays with the recombinant ThiR protein from the hyperthermophilic archaeon Metallosphaera yellowstonensis MK1. Thiamine phosphate and, to some extent, TPP and hydroxyethylthiazole phosphate were required for the binding of ThiR to its DNA targets, suggesting that ThiR is derepressed by limitation of thiamine phosphates. The thiamine phosphate-binding residues previously identified in ThiN are highly conserved in ThiR regulators, suggesting a conserved mechanism for effector recognition.ImportanceThiamine pyrophosphate is a cofactor for many essential enzymes for glucose and energy metabolism. Thiamine or vitamin B1 biosynthesis and its transcriptional regulation in Archaea are poorly understood. We applied the comparative genomics approach to identify a novel family of regulators for the transcriptional control of thiamine metabolism genes in Archaea and reconstructed the respective regulons. The predicted ThiR regulons in archaeal genomes control the majority of thiamine biosynthesis genes. The reconstructed regulon content suggests that numerous uptake transporters for thiamine and/or its precursors are encoded in archaeal genomes. The ThiR regulon was experimentally validated by DNA-binding assays with Metallosphaera spp. These discoveries contribute to our understanding of metabolic and regulatory networks involved in vitamin homeostasis in diverse lineages of Archaea. | Hsieh WY, Liao JC, Wang HT, Hung TH, Tseng CC, Chung TY, Hsieh MH (2017)
The Arabidopsis thiamin-deficient mutant pale green1 lacks thiamin monophosphate phosphatase of the vitamin B1 biosynthesis pathway.
The Plant journal : for cell and molecular biology 91, 145-157 [PubMed:28346710]
[show Abstract]
Thiamin diphosphate (TPP, vitamin B1 ) is an essential coenzyme present in all organisms. Animals obtain TPP from their diets, but plants synthesize TPPde novo. We isolated and characterized an Arabidopsis pale green1 (pale1) mutant that contained higher concentrations of thiamin monophosphate (TMP) and less thiamin and TPP than the wild type. Supplementation with thiamin, but not the thiazole and pyrimidine precursors, rescued the mutant phenotype, indicating that the pale1 mutant is a thiamin-deficient mutant. Map-based cloning and whole-genome sequencing revealed that the pale1 mutant has a mutation in At5g32470 encoding a TMP phosphatase of the TPP biosynthesis pathway. We further confirmed that the mutation of At5g32470 is responsible for the mutant phenotypes by complementing the pale1 mutant with constructs overexpressing full-length At5g32470. Most plant TPP biosynthetic enzymes are located in the chloroplasts and cytosol, but At5g32470-GFP localized to the mitochondrion of the root, hypocotyl, mesophyll and guard cells of the 35S:At5g32470-GFP complemented plants. The subcellular localization of a functional TMP phosphatase suggests that the complete vitamin B1 biosynthesis pathway may involve the chloroplasts, mitochondria and cytosol in plants. Analysis of PALE1 promoter-uidA activity revealed that PALE1 is mainly expressed in vascular tissues of Arabidopsis seedlings. Quantitative RT-PCR analysis of TPP biosynthesis genes and genes encoding the TPP-dependent enzymes pyruvate dehydrogenase, α-ketoglutarate dehydrogenase and transketolase revealed that the transcript levels of these genes were upregulated in the pale1 mutant. These results suggest that endogenous levels of TPP may affect the expression of genes involved in TPP biosynthesis and TPP-dependent enzymes. | Mimura M, Zallot R, Niehaus TD, Hasnain G, Gidda SK, Nguyen TN, Anderson EM, Mullen RT, Brown G, Yakunin AF, de Crécy-Lagard V, Gregory JF, McCarty DR, Hanson AD (2016)
Arabidopsis TH2 Encodes the Orphan Enzyme Thiamin Monophosphate Phosphatase.
The Plant cell 28, 2683-2696 [PubMed:27677881]
[show Abstract]
To synthesize the cofactor thiamin diphosphate (ThDP), plants must first hydrolyze thiamin monophosphate (ThMP) to thiamin, but dedicated enzymes for this hydrolysis step were unknown and widely doubted to exist. The classical thiamin-requiring th2-1 mutation in Arabidopsis thaliana was shown to reduce ThDP levels by half and to increase ThMP levels 5-fold, implying that the THIAMIN REQUIRING2 (TH2) gene product could be a dedicated ThMP phosphatase. Genomic and transcriptomic data indicated that TH2 corresponds to At5g32470, encoding a HAD (haloacid dehalogenase) family phosphatase fused to a TenA (thiamin salvage) family protein. Like the th2-1 mutant, an insertional mutant of At5g32470 accumulated ThMP, and the thiamin requirement of the th2-1 mutant was complemented by wild-type At5g32470 Complementation tests in Escherichia coli and enzyme assays with recombinant proteins confirmed that At5g32470 and its maize (Zea mays) orthologs GRMZM2G148896 and GRMZM2G078283 are ThMP-selective phosphatases whose activity resides in the HAD domain and that the At5g32470 TenA domain has the expected thiamin salvage activity. In vitro and in vivo experiments showed that alternative translation start sites direct the At5g32470 protein to the cytosol and potentially also to mitochondria. Our findings establish that plants have a dedicated ThMP phosphatase and indicate that modest (50%) ThDP depletion can produce severe deficiency symptoms. | Hayashi M, Nosaka K (2015)
Characterization of Thiamin Phosphate Kinase in the Hyperthermophilic Archaeon Pyrobaculum calidifontis.
Journal of nutritional science and vitaminology 61, 369-374 [PubMed:26639844]
[show Abstract]
Thiamin pyrophosphate is an essential cofactor in all living systems. In its biosynthesis, the thiamin structure is initially formed as thiamin phosphate from a thiazole and a pyrimidine moiety, and then thiamin pyrophosphate is synthesized from thiamin phosphate. Many eubacterial cells directly synthesize thiamin pyrophosphate by the phosphorylation of thiamin phosphate by thiamin phosphate kinase (ThiL), whereas this final step occurs in two stages in eukaryotic cells and some eubacterial cells: hydrolysis of thiamin phosphate to free thiamin and its pyrophosphorylation by thiamin pyrophosphokinase. In addition, some eubacteria have thiamin kinase, a salvage enzyme that converts the incorporated thiamin from the environment to thiamin phosphate. This final step in thiamin biosynthesis has never been experimentally investigated in archaea, although the putative thiL genes are found in their genome database. In this study, we observed thiamin phosphate kinase activity in the soluble fraction of the hyperthermophilic archaeon Pyrobaculum calidifontis. On the other hand, neither thiamin pyrophosphokinase nor thiamin kinase activity was detected, suggesting that in this archaeon the phosphorylation of thiamin phosphate is only way to synthesize thiamin pyrophosphate and it cannot use exogenous thiamin for the salvage synthesis of thiamin pyrophosphate. We also investigated the kinetic properties of thiamin phosphate kinase activity using the recombinant ThiL protein from P. calidifontis. Furthermore, the results obtained by site-directed mutagenesis suggest that the Ser196 of ThiL protein plays a pivotal role in the catalytic process. | Hayashi M, Kobayashi K, Esaki H, Konno H, Akaji K, Tazuya K, Yamada K, Nakabayashi T, Nosaka K (2014)
Enzymatic and structural characterization of an archaeal thiamin phosphate synthase.
Biochimica et biophysica acta 1844, 803-809 [PubMed:24583237]
[show Abstract]
Studies on thiamin biosynthesis have so far been achieved in eubacteria, yeast and plants, in which the thiamin structure is formed as thiamin phosphate from a thiazole and a pyrimidine moiety. This condensation reaction is catalyzed by thiamin phosphate synthase, which is encoded by the thiE gene or its orthologs. On the other hand, most archaea do not seem to have the thiE gene, but instead their thiD gene, coding for a 2-methyl-4-amino-5-hydroxymethylpyrimidine (HMP) kinase/HMP phosphate kinase, possesses an additional C-terminal domain designated thiN. These two proteins, ThiE and ThiN, do not share sequence similarity. In this study, using recombinant protein from the hyperthermophile archaea Pyrobaculum calidifontis, we demonstrated that the ThiN protein is an analog of the ThiE protein, catalyzing the formation of thiamin phosphate with the release of inorganic pyrophosphate from HMP pyrophosphate and 4-methyl-5-β-hydroxyethylthiazole phosphate (HET-P). In addition, we found that the ThiN protein can liberate an inorganic pyrophosphate from HMP pyrophosphate in the absence of HET-P. A structure model of the enzyme-product complex of P. calidifontis ThiN domain was proposed on the basis of the known three-dimensional structure of the ortholog of Pyrococcus furiosus. The significance of Arg320 and His341 residues for thiN-coded thiamin phosphate synthase activity was confirmed by site-directed mutagenesis. This is the first report of the experimental analysis of an archaeal thiamin synthesis enzyme. | Kolos IK, Makarchikov AF (2014)
[Identification of thiamine monophosphate hydrolyzing enzymes in chicken liver].
Ukrainian biochemical journal 86, 39-49 [PubMed:25816604]
[show Abstract]
In animals, thiamine monophosphate (TMP) is an intermediate on the path of thiamine diphosphate, the coenzyme form of vitamin B1, degradation. The enzymes involved in TMP metabolism in animal tissues are not identified hitherto. The aim of this work was to study TMP hydrolysis in chicken liver. Two phosphatases have been found to contribute to TMP hydrolysis in liver homogenate. The first one, possessing a maximal activity at pH 6.0, is soluble, whereas the second one represents a membrane-bound enzyme with a pH optimum of 9.0. Membrane-bound TMPase activity was enhanced 1.7-fold by 5 mM Mg2+ ions and strongly inhibited by levamisole in uncompetitive manner with K1 of 53 μM, indicating the involvement of alkaline phosphatase. An apparent Km of alkaline phosphatase for TMP was calculated from the Hanes plot to be 0.6 mM. The soluble TMPase has an apparent Km of 0.7 mM; this enzyme is Mg2+ independent and insensitive to levamisole. As estimated by gel filtration on a Toyopearl HW-55 column, the soluble enzyme has a molecular mass of 17.8 kDa, TMPase activity being eluted simultaneously with peaks of flavinmononucleotide and p-nitrophenyl phosphatase activity. Thus, TMP appears to be a physiological substrate for a low-molecular weight acid phosphatase, also known as low-molecular-weight protein phosphotyrosine phosphatase. | Buchholz M, Drotleff AM, Ternes W (2012)
Thiamin (vitamin B₁) and thiamin phosphate esters in five cereal grains during maturation
Journal of Cereal Science. 56, 109-114 [Agricola:IND44724844]
[show Abstract]
Non-phosphorylated thiamin (T), thiamin monophosphate (TMP), and thiamin diphosphate (TDP) were extracted from whole grains of wheat (Triticum aestivum, cv. Dekan), triticale (Triticosecale, cv. SW Talentro), rye (Secale cereale, cv. Agronom), oats (Avena sativa, cv. Dominik), and barley (Hordeum vulgare, cv. Merlot) at different growth stages (from inflorescence emergence to full ripeness of the grains) and determined with high-performance liquid chromatography and fluorescence detection. No thiamin triphosphate or adenosine thiamin triphosphate was found in these cereals. Total thiamin levels (the sum of T and its phosphate esters) determined on a dry matter (DM) basis were found to be quite similar in all grain samples throughout the analysis period, ranging from 5.59 (wheat) to 13.00nmol/g DM (oats). In spikelets 3–5 days after flowering, the total thiamin content in all cereals was 40–75% T, 5–13% TMP, and 18–46% TDP. Generally, T increased and TDP decreased continuously with grain development, with maximum T (88–99%), and minimum TDP (2–10%) at ripeness. No TMP was detected in mature grains. The results indicate that variation in T, TMP, and TDP levels is probably due above all to the conversion of phosphorylated thiamin phosphate esters into less highly or non-phosphorylated forms. | Saab-Rincón G, Olvera L, Olvera M, Rudiño-Piñera E, Benites E, Soberón X, Morett E (2012)
Evolutionary walk between (β/α)(8) barrels: catalytic migration from triosephosphate isomerase to thiamin phosphate synthase.
Journal of molecular biology 416, 255-270 [PubMed:22226942]
[show Abstract]
The functionally versatile (β/α)(8) barrel scaffold was used to migrate triosephosphate isomerase (TPI) to thiamin phosphate synthase (TPS) activity, two enzymes that share the same fold but catalyze unrelated reactions through different mechanisms. The high sensitivity of the selection methodology was determinant to succeed in finding proteins with the desired activity. A combination of rational design and random mutagenesis was used to achieve the desired catalytic migration. One of the parallel directed evolution strategies followed resulted in TPI derivatives able to complement the thiamin phosphate auxotrophy phenotype of an Escherichia coli strain deleted of thiE, the gene that codes for TPS. Successive rounds of directed evolution resulted in better complementing TPI variants. Biochemical characterization of some of the evolved TPI clones demonstrated that the K(m) for the TPS substrates was similar to that of the native TPS; however and in agreement with the very slow complementation phenotype, the k(cat) was 4 orders of magnitude lower, indicating that substrate binding played a major role on selection. Interestingly, the crystal structure of the most proficient variant showed a slightly modified TPI active site occupied by a thiamin-phosphate-like molecule. Substitution of key residues in this region reduced TPS activity, strongly suggesting that this is also the catalytic site for the evolved TPS activity. The presence of the TPS reaction product at the active site explains the fast inactivation of the enzyme observed. In conclusion, by combining rational design, random mutagenesis and a very sensitive selection, it is possible to achieve enzymatic activity migration. | Stuetz W, Carrara VI, McGready R, Lee SJ, Biesalski HK, Nosten FH (2012)
Thiamine diphosphate in whole blood, thiamine and thiamine monophosphate in breast-milk in a refugee population.
PloS one 7, e36280 [PubMed:22768031]
[show Abstract]
BackgroundThe provision of high doses of thiamine may prevent thiamine deficiency in the post-partum period of displaced persons.Methodology/principal findingsThe study aimed to evaluate a supplementation regimen of thiamine mononitrate (100 mg daily) at the antenatal clinics in Maela refugee camp. Women were enrolled during antenatal care and followed after delivery. Samples were collected at 12 weeks post partum. Thiamine diphosphate (TDP) in whole blood and thiamine in breast-milk of 636 lactating women were measured. Thiamine in breast-milk consisted of thiamine monophosphate (TMP) in addition to thiamine, with a mean TMP to total thiamine ratio of 63%. Mean whole blood TDP (130 nmol/L) and total thiamine in breast-milk (755 nmol/L) were within the upper range reported for well-nourished women. The prevalence of women with low whole blood TDP (<65 nmol/L) was 5% and with deficient breast-milk total thiamine (<300 nmol/L) was 4%. Whole blood TDP predicted both breast-milk thiamine and TMP (R(2) = 0.36 and 0.10, p<0.001). A ratio of TMP to total thiamine ≥63% was associated with a 7.5 and 4-fold higher risk of low whole blood TDP and deficient total breast-milk thiamine, respectively. Routine provision of daily 100 mg of thiamine mononitrate post-partum compared to the previous weekly 10 mg of thiamine hydrochloride resulted in significantly higher total thiamine in breast-milk.Conclusions/significanceThiamine supplementation for lactating women in Maela refugee camp is effective and should be continued. TMP and its ratio to total thiamine in breast-milk, reported for the first time in this study, provided useful information on thiamine status and should be included in future studies of breast-milk thiamine. | Paul D, Chatterjee A, Begley TP, Ealick SE (2010)
Domain organization in Candida glabrata THI6, a bifunctional enzyme required for thiamin biosynthesis in eukaryotes.
Biochemistry 49, 9922-9934 [PubMed:20968298]
[show Abstract]
THI6 is a bifunctional enzyme found in the thiamin biosynthetic pathway in eukaryotes. The N-terminal domain of THI6 catalyzes the ligation of the thiamin thiazole and pyrimidine moieties to form thiamin phosphate, and the C-terminal domain catalyzes the phosphorylation of 4-methyl-5-hydroxyethylthiazole in a salvage pathway. In prokaryotes, thiamin phosphate synthase and 4-methyl-5-hydroxyethylthiazole kinase are separate gene products. Here we report the first crystal structure of a eukaryotic THI6 along with several complexes that characterize the active sites responsible for the two chemical reactions. THI6 from Candida glabrata is a homohexamer in which the six protomers form a cage-like structure. Each protomer is composed of two domains, which are structurally homologous to their monofunctional bacterial counterparts. Two loop regions not found in the bacterial enzymes provide interactions between the two domains. The structures of different protein-ligand complexes define the thiazole and ATP binding sites of the 4-methyl-5-hydroxyethylthiazole kinase domain and the thiazole phosphate and 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate binding sites of the thiamin phosphate synthase domain. Our structural studies reveal that the active sites of the two domains are 40 Å apart and are not connected by an obvious channel. Biochemical studies show 4-methyl-5-hydroxyethylthiazole phosphate is a substrate for THI6; however, adenosine diphospho-5β-ethyl-4-methylthiazole-2-carboxylic acid, the product of THI4, is not a substrate for THI6. This suggests that an unidentified enzyme is necessary to produce the substrate for THI6 from the THI4 product. | McCulloch KM, Kinsland C, Begley TP, Ealick SE (2008)
Structural studies of thiamin monophosphate kinase in complex with substrates and products.
Biochemistry 47, 3810-3821 [PubMed:18311927]
[show Abstract]
Thiamin monophosphate kinase (ThiL) catalyzes the ATP-dependent phosphorylation of thiamin monophosphate (TMP) to form thiamin pyrophosphate (TPP), the active form of vitamin B 1. ThiL is a member of a small ATP binding superfamily that also includes the purine biosynthetic enzymes, PurM and PurL, NiFe hydrogenase maturation protein, HypE, and selenophosphate synthase, SelD. The latter four enzymes are believed to utilize phosphorylated intermediates during catalysis. To understand the mechanism of ThiL and its relationship to the other superfamily members, we determined the structure of Aquifex aeolicus ThiL (AaThiL) with nonhydrolyzable AMP-PCP and TMP, and also with the products of the reaction, ADP and TPP. The results suggest that AaThiL utilizes a direct, inline transfer of the gamma-phosphate of ATP to TMP rather than a phosphorylated enzyme intermediate. The structure of ThiL is compared to those of PurM, PurL, and HypE, and the ATP binding site is compared to that of PurL, for which nucleotide complexes are available. | Soriano EV, Rajashankar KR, Hanes JW, Bale S, Begley TP, Ealick SE (2008)
Structural similarities between thiamin-binding protein and thiaminase-I suggest a common ancestor.
Biochemistry 47, 1346-1357 [PubMed:18177053]
[show Abstract]
ATP-binding cassette (ABC) transporters are responsible for the transport of a wide variety of water-soluble molecules and ions into prokaryotic cells. In Gram-negative bacteria, periplasmic-binding proteins deliver ions or molecules such as thiamin to the membrane-bound ABC transporter. The gene for the thiamin-binding protein tbpA has been identified in both Escherichia coli and Salmonella typhimurium. Here we report the crystal structure of TbpA from E. coli with bound thiamin monophosphate. The structure was determined at 2.25 A resolution using single-wavelength anomalous diffraction experiments, despite the presence of nonmerohedral twinning. The crystal structure shows that TbpA belongs to the group II periplasmic-binding protein family. Equilibrium binding measurements showed similar dissociation constants for thiamin, thiamin monophosphate, and thiamin pyrophosphate. Analysis of the binding site by molecular modeling demonstrated how TbpA binds all three forms of thiamin. A comparison of TbpA and thiaminase-I, a thiamin-degrading enzyme, revealed structural similarity between the two proteins, especially in domain 1, suggesting that the two proteins evolved from a common ancestor. | Edwards TE, Ferré-D'Amaré AR (2006)
Crystal structures of the thi-box riboswitch bound to thiamine pyrophosphate analogs reveal adaptive RNA-small molecule recognition.
Structure (London, England : 1993) 14, 1459-1468 [PubMed:16962976]
[show Abstract]
Riboswitches are noncoding mRNA elements that bind small-molecule metabolites with high affinity and specificity, and they regulate the expression of associated genes. The thi-box riboswitch can exhibit a 1000-fold higher affinity for thiamine pyrophosphate over closely related noncognate compounds such as thiamine monophosphate. To understand the chemical basis of thi-box pyrophosphate specificity, we have determined crystal structures of an E. coli thi-box bound to thiamine pyrophosphate, thiamine monophosphate, and the structural analogs benfotiamine and pyrithiamine. When bound to monophosphorylated compounds, the RNA elements that recognize the thiamine and phosphate moieties of the ligand move closer together. This allows the riboswitch to recognize the monophosphate in a manner similar to how it recognizes the beta-phosphate of thiamine pyrophosphate. In the pyrithiamine complex, the pyrophosphate binding site is largely unstructured. These results show how the riboswitch can bind to various metabolites, and why the thi-box preferentially binds thiamine pyrophosphate. | Ihara H, Matsumoto T, Shino Y, Hashizume N (2005)
Assay values for thiamine or thiamine phosphate esters in whole blood do not depend on the anticoagulant used.
Journal of clinical laboratory analysis 19, 205-208 [PubMed:16170809]
[show Abstract]
We compared the whole blood, plasma, and erythrocyte (red blood cell (RBC)) concentrations of thiamine and thiamine phosphate esters in the presence of heparin or EDTA as anticoagulants. Three blood specimens were collected from each of 24 healthy volunteers into evacuated collection tubes containing the following anticoagulants: heparin, Na2EDTA, or K2EDTA. The concentrations of nonphosphorylated free thiamine (T), thiamine monophosphate (TMP), thiamine diphosphate (TDP), and thiamine triphosphate (TTP) were determined by the NH2-column HPLC method. The anticoagulant used had no effect on the concentrations obtained in whole blood and plasma of thiamine or any of the above thiamine compounds (P>0.05). RBCs were isolated by centrifugation and washed with isotonic saline, and the cell counts of the washed cells were adjusted to their whole blood values. In the washed RBCs with any anticoagulant, the concentrations of T, TMP, and TDP expressed either as nmol/L of whole blood or a ratio to hemoglobin were significantly lower (P<0.05) than those in whole blood. | Mancinelli R, Ceccanti M, Guiducci MS, Sasso GF, Sebastiani G, Attilia ML, Allen JP (2003)
Simultaneous liquid chromatographic assessment of thiamine, thiamine monophosphate and thiamine diphosphate in human erythrocytes: a study on alcoholics.
Journal of chromatography. B, Analytical technologies in the biomedical and life sciences 789, 355-363 [PubMed:12742126]
[show Abstract]
An isocratic HPLC procedure for the assessment of thiamine (T), thiamine monophosphate (TMP) and thiamine diphosphate (TDP) in human erythrocytes is described. Several aspects of the procedure make it suitable for both clinical and research purposes: limits of detection and quantification of 1 and 2.5 nmol/l, respectively, recovery of 102% on average (range 93-112%), intra- and inter-day precisions within 5 and 9%, respectively, total elution time 15 min. This analytical methodology was applied to a case-control study on erythrocyte samples from 103 healthy subjects and 36 alcohol-dependent patients at risk of thiamine deficiency. Mean control values obtained were: T=89.6+/-22.7 nmol/l, TMP=4.4+/-6.6 nmol/l and TDP=222.23+/-56.3 nmol/l. T and TDP mean values of alcoholics were significantly lower than those of control cases: T=69.4+/-35.9 nmol/l (P<0.001) and TDP=127.4+/-62.5 nmol/l (P<10(-5)). The diagnostic role of TDP was evaluated and a significant role for thiamine was established in the study of alcohol related problems. | Zhao R, Gao F, Goldman ID (2002)
Reduced folate carrier transports thiamine monophosphate: an alternative route for thiamine delivery into mammalian cells.
American journal of physiology. Cell physiology 282, C1512-7 [PubMed:11997266]
[show Abstract]
Although the reduced folate carrier RFC1 and the thiamine transporters THTR-1 and THTR-2 share approximately 40% of their identity in protein sequence, RFC1 does not transport thiamine and THTR-1 and THTR-2 do not transport folates. In the present study, we demonstrate that transport of thiamine monophosphate (TMP), an important thiamine metabolite present in plasma and cerebrospinal fluid, is mediated by RFC1 in L1210 murine leukemia cells. Transport of TMP was augmented by a factor of five in cells (R16) that overexpress RFC1 and was markedly inhibited by methotrexate, an RFC1 substrate, but not by thiamine. At a near-physiological concentration (50 nM), TMP influx mediated by RFC1 in wild-type L1210 cells was approximately 50% of thiamine influx mediated by thiamine transporter(s). Within 1 min, the majority of TMP transported into R16 cells was hydrolyzed to thiamine with a component metabolized to thiamine pyrophosphate, the active enzyme cofactor. These data suggest that RFC1 may be one of the alternative transport routes available for TMP in some tissues when THTR-1 is mutated in the autosomal recessive disorder thiamine-responsive megaloblastic anemia. | Molina JA, Jiménez-Jiménez FJ, Hernánz A, Fernández-Vivancos E, Medina S, de Bustos F, Gómez-Escalonilla C, Sayed Y (2002)
Cerebrospinal fluid levels of thiamine in patients with Alzheimer's disease.
Journal of neural transmission (Vienna, Austria : 1996) 109, 1035-1044 [PubMed:12111441]
[show Abstract]
Thiamine is an essential cofactor for several important enzymes involved in brain oxidative metabolism, such as the alpha-ketoglutarate dehydrogenase complex (KGDHC), pyruvate-dehydrogenase complex (PDHC), and transketolase. Some investigators reported decreased thiamine-diphosphate levels and decreased activities of KGDHC, pyruvate-dehydrogenase complex and transketolase in the brain tissue of Alzheimer's disease (AD) patients. We measured cerebrospinal (CSF) levels of thiamine-diphosphate, thiamine-monophosphate, free thiamine, and total thiamine, using ion-pair reversed phase high performance liquid chromatography, in 33 patients with sporadic AD and 32 matched controls. The mean CSF levels of thiamine-derivatives did not differ significantly from those of controls, while the mean plasma levels of thiamine-diphosphate, free and total thiamine were significantly lower in the AD-patient group. CSF and plasma thiamine levels were not correlated with age, age at onset, duration of the disease, and scores of the MiniMental State Examination, with the exception of plasma thiamine-diphosphate with MiniMental State Examination (r = 0.41, p < 0.05) in the AD-patients group. CSF and plasma values did not predict dementia progression, assessed with the MiniMental State Examination scores. These results suggest that CSF thiamine levels are not related with the risk for and the progression of AD. | Peapus DH, Chiu HJ, Campobasso N, Reddick JJ, Begley TP, Ealick SE (2001)
Structural characterization of the enzyme-substrate, enzyme-intermediate, and enzyme-product complexes of thiamin phosphate synthase.
Biochemistry 40, 10103-10114 [PubMed:11513589]
[show Abstract]
Thiamin phosphate synthase catalyzes the formation of thiamin phosphate from 4-amino-5-(hydroxymethyl)-2-methylpyrimidine pyrophosphate and 5-(hydroxyethyl)-4-methylthiazole phosphate. Several lines of evidence suggest that the reaction proceeds via a dissociative mechanism. The previously determined crystal structure of thiamin phosphate synthase in complex with the reaction products, thiamin phosphate and magnesium pyrophosphate, provided a view of the active site and suggested a number of additional experiments. We report here seven new crystal structures primarily involving crystals of S130A thiamin phosphate synthase soaked in solutions containing substrates or products. We prepared S130A thiamin phosphate synthase with the intent of characterizing the enzyme-substrate complex. Surprisingly, in three thiamin phosphate synthase structures, the active site density cannot be modeled as either substrates or products. For these structures, the best fit to the electron density is provided by a model that consists of independent pyrimidine, pyrophosphate, and thiazole phosphate fragments, consistent with a carbenium ion intermediate. The resulting carbenium ion is likely to be further stabilized by proton transfer from the pyrimidine amino group to the pyrophosphate to give the pyrimidine iminemethide, which we believe is the species that is observed in the crystal structures. | Chiu HJ, Reddick JJ, Begley TP, Ealick SE (1999)
Crystal structure of thiamin phosphate synthase from Bacillus subtilis at 1.25 A resolution.
Biochemistry 38, 6460-6470 [PubMed:10350464]
[show Abstract]
The crystal structure of Bacillus subtilis thiamin phosphate synthase complexed with the reaction products thiamin phosphate and pyrophosphate has been determined by multiwavelength anomalous diffraction phasing techniques and refined to 1.25 A resolution. Thiamin phosphate synthase is an alpha/beta protein with a triosephosphate isomerase fold. The active site is in a pocket formed primarily by the loop regions, residues 59-67 (A loop, joining alpha3 and beta2), residues 109-114 (B loop, joining alpha5 and beta4), and residues 151-168 (C loop, joining alpha7 and beta6). The high-resolution structure of thiamin phosphate synthase complexed with its reaction products described here provides a detailed picture of the catalytically important interactions between the enzyme and the substrates. The structure and other mechanistic studies are consistent with a reaction mechanism involving the ionization of 4-amino-2-methyl-5-hydroxymethylpyrimidine pyrophosphate at the active site to give the pyrimidine carbocation. Trapping of the carbocation by the thiazole followed by product dissociation completes the reaction. The ionization step is catalyzed by orienting the C-O bond perpendicular to the plane of the pyrimidine, by hydrogen bonding between the C4' amino group and one of the terminal oxygen atoms of the pyrophosphate, and by extensive hydrogen bonding and electrostatic interactions between the pyrophosphate and the enzyme. | Komeda Y, Tanaka M, Nishimune T (1988)
A th-1 Mutant of Arabidopsis thaliana Is Defective for a Thiamin-Phosphate-Synthesizing Enzyme: Thiamin Phosphate Pyrophosphorylase.
Plant physiology 88, 248-250 [PubMed:16666289]
[show Abstract]
We have examined the activity of the thiamin phosphate pyrophosphorylase in Arabidopsis thaliana wild type and in a mutant (th-1) which requires exogenous thiamin for growth. Mutant and wild-type plants grown in 1 x 10(-7) molar thiamin were used for the examination of the production of thiamin and thiamin monophosphate (TMP) using 4-methyl-5-hydroxyethylthiazole phosphate and 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate as substrates. While the wild-type strain formed both thiamin and TMP, the th-1 mutant did not. When TMP was added to the extracts, the th-1 mutant, as well as wild type, produced thiamin. Accordingly, it was concluded that the th-1 mutant was defective in the activity of TMP pyrophosphorylase. Some of the characteristics of the enzyme from the wild-type plant were examined. The optimum temperature for the reaction is 45 degrees C, and the K(m) values for the substrates are 2.7 x 10(-6) molar for 4-methyl-5-hydroxyethylthiazole phosphate and 1.8 x 10(-6) molar for 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate. |
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