Thiamine diphosphate adenylyl transferase from E. coli: functional characterization of the enzyme synthesizing adenosine thiamine triphosphate

BACKGROUND: We have recently identified a new thiamine derivative, adenosine thiamine triphosphate (AThTP), in E. coli. In intact bacteria, this nucleotide is synthesized only in the absence of a metabolizable carbon source and quickly disappears as soon as the cells receive a carbon source such as glucose. Thus, we hypothesized that AThTP may be a signal produced in response to carbon starvation. RESULTS: Here we show that, in bacterial extracts, the biosynthesis of AThTP is carried out from thiamine diphosphate (ThDP) and ADP or ATP by a soluble high molecular mass nucleotidyl transferase. We partially purified this enzyme and characterized some of its functional properties. The enzyme activity had an absolute requirement for divalent metal ions, such as Mn2+ or Mg2+, as well as for a heat-stable soluble activator present in bacterial extracts. The enzyme has a pH optimum of 6.5-7.0 and a high Km for ThDP (5 mM), suggesting that, in vivo, the rate of AThTP synthesis is proportional to the free ThDP concentration. When ADP was used as the variable substrate at a fixed ThDP concentration, a sigmoid curve was obtained, with a Hill coefficient of 2.1 and an S0.5 value of 0.08 mM. The specificity of the AThTP synthesizing enzyme with respect to nucleotide substrate is restricted to ATP/ADP, and only ThDP can serve as the second substrate of the reaction. We tentatively named this enzyme ThDP adenylyl transferase (EC 2.7.7.65). CONCLUSION: This is the first demonstration of an enzyme activity transferring a nucleotidyl group on thiamine diphosphate to produce AThTP. The existence of a mechanism for the enzymatic synthesis of this compound is in agreement with the hypothesis of a non-cofactor role for thiamine derivatives in living cells

Adenylate kinase-independent thiamine triphosphate accumulation under severe energy stress in Escherichia coli

BACKGROUND: Thiamine triphosphate (ThTP) exists in most organisms and might play a role in cellular stress responses. In E. coli, ThTP is accumulated in response to amino acid starvation but the mechanism of its synthesis is still a matter of controversy. It has been suggested that ThTP is synthesized by an ATP-dependent specific thiamine diphosphate kinase. However, it is also known that vertebrate adenylate kinase 1 catalyzes ThTP synthesis at a very low rate and it has been postulated that this enzyme is responsible for ThTP synthesis in vivo. RESULTS: Here we show that bacterial, as vertebrate adenylate kinases are able to catalyze ThTP synthesis, but at a rate more than 106-fold lower than ATP synthesis. This activity is too low to explain the high rate of ThTP accumulation observed in E. coli during amino acid starvation. Moreover, bacteria from the heat-sensitive CV2 strain accumulate high amounts of ThTP (>50% of total thiamine) at 37 degrees C despite complete inactivation of adenylate kinase and a subsequent drop in cellular ATP. CONCLUSION: These results clearly demonstrate that adenylate kinase is not responsible for ThTP synthesis in vivo. Furthermore, they show that E. coli accumulate large amounts of ThTP under severe energy stress when ATP levels are very low, an observation not in favor of an ATP-dependent mechanisms for ThTP synthesis

Biosynthesis of thiamine

Thiamine (vitamin B1) is essential compound for all living things performing, in the form of thiamine diphosphate (ThDP), catalytic functions in the reactions of central and secondary metabolic pathways. There is no thiamine synthesis in animal cells, and therefore it must be continuously supplied with food. Most eubacteria, archaea, fungi, and plants are capable of synthesizing thiamine de novo or salvaging the products of its degradation. Biosynthesis of pyrimidine (as 4-amino-5-hydroxymethyl-2-methylpyrimidine diphosphate, HMP-PP) and thiazole (as 2-carboxy-4-methyl-5-β-hydroxyethylthiazole phosphate, HET-P) rings of the vitamin B1 molecule proceeds separately with their condensation into thiamine monophosphate (ThMP). In bacteria and archaea, ThMP is converted to ThDP by thiamine phosphate kinase (ThiL) while in eukaryotic cells it undergoes hydrolysis to thiamine, which is then phosphorylated to ThDP by thiamine pyrophosphokinase. Bacteria synthesize HET-P from 2-iminoacetate, 1-deoxy-D-xylulose-5-phosphate and ThiS-thiocarboxylate using at least 7 proteins (Dxs, ThiS, ThiF, ThiO, NifS, ThiG, and TenI in B. subtilis), while only two proteins, ThiC and ThiD, are involved in the formation of HMP-PP (from 5-aminoimidazol ribotide (AIR)). In fungi, HET-P is formed from NAD and glycine, the source of sulfur being the Cys residue of the active site of the THI4 protein, a suicidal enzyme that carries out only one catalytic cycle. Another suicidal enzyme, THI5, is involved in the synthesis of HMP-PP in fungal cells. This enzyme incorporate a nitrogen atom of the Hys residue of its active site into the pyridine ring of pyridylxal-5-phosphate when forming HMP-P, which is then phosphorylated by the THI20 protein to HMP-PP. In plants, like in fungi, the formation of HET-P proceeds under the action of the THI1 (THI4) protein, while HMP-PP is synthesized via the bacterial pathway from AIR with the participation of THIC and TH1 proteins. Archaea synthesize the thiazole moiety of the thiamine molecule by the eukaryotic THI4 mechanism, and the pyrimidine, by the bacterial/plant pathway. Depending on species thiamine biosynthesis is regulated by ThDP riboswitches or by transcription factors.Тиамин (витамин В1) необходим для жизнедеятельности всех известных организмов, выполняя в форме тиаминдифосфата (ТДФ) каталитические функции в реакциях центрального и вторичного метаболизма. В клетках животных тиамин не образуется и поэтому должен прстоянно поступать с пищей. Большинство эубактерий, архей, грибов и растений способны осуществлять биосинтез тиамина de novo либо использовать продукты его деградации. Биосинтез пиримидинового (в виде 4-амино-5-гидроксиметил-2-метилпиримидин дифосфата, HMP-PP) и тиазолового (в виде 2-карбокси-4-метил-5-β-гидроксиэтилтиазол фосфата, HET-P) колец молекулы витамина В1 протекает раздельно с их последующей конденсацией в тиаминмонофосфат (ТМФ). У бактерий и архей ТМФ превращается в ТДФ под действием тиаминфосфат-киназы (ThiL), а в клетках эукариот подвергается гидролизу до тиамина, который фосфорилируется до ТДФ тиаминпирофосфокиназой. Бактерии синтезируют HET-P из 2-иминоацетата, 1-дезокси-D-ксилулозо-5-фосфата и ThiS-тиокарбоксилата при помощи по крайней мере 7 белков (Dxs, ThiS, ThiF, ThiO, NifS, ThiG и TenI – у B. subtilis), тогда как в образование HMP-PP (из 5-аминоимидазолриботида (AIR)) вовлечены только два белка – ThiC и ThiD. У грибов HET-P образуется из NAD и глицина, при этом источником серы служит остаток Cys активного центра белка THI4 – суицидного фермента, осуществляющего лишь один каталитический цикл. В синтезе HMP-PP в клетках грибов задействован еще один суицидный фермент – THI5, включающий атом азота остатка Hys своего активного центра в пиридиновое кольцо пиридлксаль-5-фосфата в реакции образования HMP-P, который затем фосфорилируется белком THI20 до HMP-PP. В растениях образование HET-P протекает, как и у грибов, под действием белка THI1(THI4), тогда как HMP-PP синтезируется по бактериальному пути из AIR с участием белков THIС и TH1. Археи синтезируют тиазоловый гетероцикл молекулы тиамина по эукариотному THI4-механизму, а пиримидиновый – по бактериальному/растительному пути. Регуляция биосинтеза тиамина у разных видов организмов осуществляется благодаря наличию ТДФ-рибосвитчей и под контролем транскрипционных факторов

Transport of vitamin B1 in animals, plants and microorganisms

Vitamin B1, in the form of coenzyme thiamine diphosphate (ThDP), is indispensible for the life of almost all types of organisms. Plants, yeast, and many bacteria synthesize vitamin B1 de novo, while animal cells lack this ability and therefore must absorb thiamine constantly through specialized transport systems. Carrier proteins are expressed not only by cells of thiamine auxotrophic organisms, but also by organisms capable of its biosynthesis. During the biological evolution, there has been a significant divergence in the mechanisms of vitamin B1 transport. Prokaryotes carry out its uptake through ATP-dependent ABC-type transporters or using energy uncoupled facilitated diffusion mechanism through the PnuT transporter. Yeast and animal cells uptake thiamine by the mechanism of secondary active transport by proteins from the NCS1 and SLC19 families, respectively. ThDP synthesized in the cytosol of eukaryotic cells is imported into the mitochondrial matrix through transporters belonging to the MCF family.Витамин В1 в форме кофермента тиаминдифосфата (ТДФ) необходим для жизнедеятельности практически всех видов организмов. Растения, дрожжи и многие бактерии синтезируют витамин В1 de novo, тогда как клетки животных лишены такой способности и поэтому постоянно должны поглощать тиамин с помощью специализированных транспортных систем. Белки-переносчики экспрессируются не только клетками ауксотрофных по тиамину организмов, но тех, которые способны осуществлять его биосинтез. В ходе биологической эволюции произошла значительная дивергенция механизмов транспорта витамин В1. Прокариоты осуществляют его активный транспорт с помощью АТФ-зависимых транспортеров ABC-типа или используя энергонезависимый механизм облегченной диффузии через транспортер PnuT. В клетки дрожжей и животных тиамин переносится по механизму вторичного активного транспорта белками-транспортерами из семейств NCS1 и SLC19 соответственно. Синтезируемый в цитозоле клеток эукариот ТДФ импортируется в матрикс митохондриий транспортерами, принадлежащими семейству MCF

Concentration of paramagnetic centres at low-temperature thermal destruction of asphaltenes of heavy petroleum distillates

© Kazan Federal University (KFU).Changes of paramagnetic centers (PC) concentration in dispersed petroleum systems were studied in the process of low-temperature thermolysis. The kinetic model of PC concentration dynamics based on the processes of unpaired electrons formation during singlet-triplet transitions, weak chemical bonds dissociation and recombination of free radicals is proposed. PACS: 75.20.-g, 96.20.Dt, 02.30.H

Adenosine thiamine triphosphate and adenosine thiamine triphosphate hydrolase activity in animal tissues

Adenosine thiamine triphosphate (AThTP), a vitamin B1 containing nucleotide with unknown biochemi­cal role, was found previously to be present in various biological objects including bacteria, yeast, some human, rat and mouse tissues, as well as plant roots. In this study we quantify AThTP in mouse, rat, bovine and chicks. We also show that in animal tissues the hydrolysis of AThTP is catalyzed by a membrane-bound enzyme seemingly of microsomal origin as established for rat liver, which exhibits an alkaline pH optimum of 8.0-8.5 and requires no Mg2+ ions for activity. In liver homogenates, AThTP hydrolase obeys Michaelis-Menten kinetics with apparent Km values of 84.4 ± 9.4 and 54.6 ± 13.1 µМ as estimated from the Hanes plots for rat and chicken enzymes, respectively. The hydrolysis of AThTP has been found to occur in all samples examined from rat, chicken and bovine tissues, with liver and kidney being­ the most abundant in enzyme activity. In rat liver, the activity of AThTP hydrolase depends on the age of animals

Dysautonomia, A Heuristic Approach to a Revised Model for Etiology of Disease

Dysautonomia refers to a disease where the autonomic nervous system is dysfunctional. This may be a central control mechanism, as in genetically determined familial dysautonomia (Riley-Day Syndrome), or peripherally in the distribution of the sympathetic and parasympathetic systems. There are multiple reports of a number of different diseases associated with dysautonomia. The etiology of this association has never been explained. There are also multiple publications on dysautonomia associated with specific non-caloric nutritional deficiencies. Beriberi is the prototype of autonomic dysfunction. It is the best known nutritional deficiency disease caused by an imbalance between ingested calories and the vitamins required for their oxidation, particularly thiamin. Long thought to be abolished in modern medical thinking, there are occasional isolated reports of the full-blown disease in developed Western cultures

Identification of Inhibitors against Mycobacterium tuberculosis Thiamin Phosphate Synthase, an Important Target for the Development of Anti-TB Drugs

Tuberculosis (TB) continues to pose a serious challenge to human health afflicting a large number of people throughout the world. In spite of the availability of drugs for the treatment of TB, the non-compliance to 6–9 months long chemotherapeutic regimens often results in the emergence of multidrug resistant strains of Mycobacterium tuberculosis adding to the precariousness of the situation. This has necessitated the development of more effective drugs. Thiamin biosynthesis, an important metabolic pathway of M.tuberculosis, is shown to be essential for the intracellular growth of this pathogen and hence, it is believed that inhibition of this pathway would severely affect the growth of M.tuberculosis. In this study, a comparative homology model of M.tuberculosis thiamin phosphate synthase (MtTPS) was generated and employed for virtual screening of NCI diversity set II to select potential inhibitors. The best 39 compounds based on the docking results were evaluated for their potential to inhibit the MtTPS activity. Seven compounds inhibited MtTPS activity with IC50 values ranging from 20 – 100 µg/ml and two of these exhibited weak inhibition of M.tuberculosis growth with MIC99 values being 125 µg/ml and 162.5 µg/ml while one compound was identified as a very potent inhibitor of M.tuberculosis growth with an MIC99 value of 6 µg/ml. This study establishes MtTPS as a novel drug target against M.tuberculosis leading to the identification of new lead molecules for the development of antitubercular drugs. Further optimization of these lead compounds could result in more potent therapeutic molecules against Tuberculosis