Báo cáo khoa học: Thiamin diphosphate in biological chemistry: new aspects of thiamin metabolism, especially triphosphate derivatives acting other than as cofactors pdf

MINIREVIEW Thiamin diphosphate in biological chemistry: new aspects of thiamin metabolism, especially triphosphate derivatives acting other than as cofactors Lucien Bettendorff and Pierre Wins GIGA-Neurosciences, University of Lie ` ge, Belgium Thiamin is best known for the cofactor role of its diphosphorylated derivative thiamin diphosphate (ThDP; Fig. 1) in many enzymes and multienzyme complexes [1]. The mechanism by which the thiamin moiety of ThDP exerts its coenzyme function by proton substitution on position 2 of the thiazolium ring was elucidated by Ronald Breslow in 1958 [2] and nowadays, a lot of work is devoted to understanding the interplay between ThDP and ThDP-dependent enzymes in catalysis [3,4]. However, thiamin phosphate derivatives other than ThDP exist in most organisms. In microorganisms and Keywords adenosine thiamin triphosphate; adenylate kinase; alarmone; Escherichia coli; regulation; riboswitch; thiamin transport; thiamin triphosphatase; thiamin triphosphate; triphosphate tunnel metalloenzymes Correspondence L. Bettendorff, GIGA-Neurosciences, University of Lie ` ge, Ba ˆ t. B36, Tour de Pathologie 2, e ´ tage +1, Avenue de l’Ho ˆ pital, 1, B-4000 Lie ` ge 1 (Sart Tilman), Belgium Fax: + 32 4 366 59 53 Tel: +32 4 366 59 67 E-mail: L.Bettendorff@ulg.ac.be (Received 9 October 2008, revised 26 February 2009, accepted 12 March 2009) doi:10.1111/j.1742-4658.2009.07019.x Prokaryotes, yeasts and plants synthesize thiamin (vitamin B1) via complex pathways. Animal cells capture the vitamin through specific high-affinity transporters essential for internal thiamin homeostasis. Inside the cells, thi- amin is phosphorylated to higher phosphate derivatives. Thiamin diphos- phate (ThDP) is the best-known thiamin compound because of its role as an enzymatic cofactor. However, in addition to ThDP, at least three other thiamin phosphates occur naturally in most cells: thiamin monophosphate, thiamin triphosphate (ThTP) and the recently discovered adenosine thiamin triphosphate. It has been suggested that ThTP has a specific neurophysio- logical role, but recent data favor a much more basic metabolic function. During amino acid starvation, Escherichia coli accumulate ThTP, possibly acting as a signal involved in the adaptation of the bacteria to changing nutritional conditions. In animal cells, ThTP can phosphorylate some pro- teins, but the physiological significance of this mechanism remains unknown. Adenosine thiamin triphosphate, recently discovered in E. coli, accumulates during carbon starvation and might act as an alarmone. Among the proteins involved in thiamin metabolism, thiamin transporters, thiamin pyrophosphokinase and a soluble 25-kDa thiamin triphosphatase have been characterized at the molecular level, in contrast to thiamin mono- and diphosphatases whose specificities remain to be proven. A solu- ble enzyme catalyzing the synthesis of adenosine thiamin triphosphate from ThDP and ADP or ATP has been partially characterized in E. coli , but the mechanism of ThTP synthesis remains elusive. The data reviewed here illustrate the complexity of thiamin biochemistry, which is not restricted to the cofactor role of ThDP. Abbreviations ABC, ATB-binding cassette; AK, adenylate kinase; AThTP, adenosine thiamin triphosphate; TenA, thiaminase II; ThDP, thiamin diphosphate; ThDPase, thiamin diphosphatase; ThMP, thiamin monophosphate; ThMPase, thiamin monophosphatase; ThTP, thiamin triphosphate; ThTPase, thiamin triphosphatase; TPK, thiamin pyrophosphokinase; TTM, triphosphate tunnel metalloenzyme. FEBS Journal 276 (2009) 2917–2925 ª 2009 The Authors Journal compilation ª 2009 FEBS 2917 plants, thiamin monophosphate (ThMP; Fig. 1) is formed during the final step of thiamin biosynthesis, but ThMP also exists in animal cells unable to carry out de novo synthesis of thiamin. In animal tissues, ThMP is a product of the enzymatic hydrolysis of ThDP and has no known physiological function. Thia- min triphosphate (ThTP; Fig. 1) was first suggested to exist in eukaryotic cells in the 1950s, but progress in the field was hampered by the lack of analytical tools available to measure the tiny amounts of ThTP occur- ring in most cells. This difficulty was overcome by the advent of HPLC techniques in the late 1970s and early 1980s [5]. Most of these techniques rely on the precol- umn derivatization of thiamin to highly fluorescent thiochrome derivatives, thereby increasing sensitivity and selectivity. Subsequent studies showed that ThTP is present in most cells studied to date, from bacteria to mammals [6]. Finally, the complexity of thiamin metabolism was further highlighted by the discovery of a hitherto unsuspected derivative: adenosine thiamin triphosphate (AThTP; Fig. 1) [7]. It is generally assumed that the pathologies linked to thiamin deficiency, mainly beriberi and Wernicke– Korsakoff syndrome, are the consequence of decreased ThDP levels, resulting in reduced activity of key enzymes for oxidative metabolism such as 2-oxoglutarate dehydrogenase [8,9]. Although oxida- tive metabolism is obviously very important for neu- ronal survival, general impairment of oxidative decarboxylation reactions does not explain the selec- tive vulnerability of certain brain areas to thiamin deficiency, for example the periventricular thalamic regions and mammillary bodies in Wernicke–Korsak- off syndrome. Likewise, it is difficult to link the decreased activity of ThDP-dependent enzymes to, for example, the observation that thiamin deficiency exac- erbates plaque pathology in a mouse model of Alzhei- mer’s disease [10]. Therefore, it is important to better understand the role of all thiamin derivatives and to characterize the enzymes involved in the metabolism of thiamin phosphate derivatives. Here, we focus on some recent developments in the field, such as thiamin biosynthesis, transport, thiamin triphosphate metabolism and the discovery of the new adenosine thiamin nucleotide. Thiamin biosynthesis and salvage De novo thiamin biosynthesis may occur in bacteria, some protozoa, plants and fungi [11,12]. The pathways Fig. 1. Scheme depicting the enzymatic interconversions of thiamin derivatives in mammalian cells. Free ThDP represents the high turnover ThDP pool, precursor of ThMP, ThTP and AThTP. This ‘rapid’ pool plays a pivotal role in the metabolism of phosphorylated thiamin deriva- tives in eukaryotic cells. The bound ThDP represents the low turnover cofactor ThDP pool, with ThDP mostly bound to apoenzymes. 1, Thiamin pyrophosphokinase; 2, thiamin diphosphatase; 3, thiamin monophosphatase; 4, ThTP synthase (unknown mechanism); 5, membrane-associated and soluble thiamin triphosphatases; 6, thiamin diphosphate-adenylyl transferase; 7, adenosine thiamin triphosphate hydrolase (postulated). Among all these enzymes, only thiamin pyrophosphokinase (1) and 25-kDa soluble thiamin triphosphatase (5) have been characterized at the molecular level. All the other conversions occur in intact cells or in cellular extracts but the enzymes have not yet been characterized and the genes identified (adapted and updated from Bettendorff [43]). Thiamin metabolism and thiamin phosphates L. Bettendorff and P. Wins 2918 FEBS Journal 276 (2009) 2917–2925 ª 2009 The Authors Journal compilation ª 2009 FEBS are complex, in particular because of the thiazole moi- ety, a heterocycle rarely encountered in natural prod- ucts. In all cases, the thiazole and pyrimidine moieties are synthesized separately and then assembled to form ThMP by thiamin-phosphate synthase (EC 2.5.1.3). The exact biosynthetic pathways may differ among organ- isms. In Escherichia coli and other Enterobacteriaceae, ThMP may be phosphorylated to the cofactor ThDP by a thiamin-phosphate kinase (ThMP + ATP « ThDP + ADP; EC 2.7.4.16). In most bacteria and in eukaryotes, ThMP is hydrolyzed to thiamin + P i by a thiamin monophosphatase (ThMPase). Thiamin may then be pyrophosphorylated to ThDP by thiamin diphosphokinase (thiamin + ATP « ThDP + AMP; EC 2.7.6.2). Thiamin can be degraded by thiaminases [13]. Thiaminase I (EC 2.5.1.2), a pyrimidine transferase able to use various acceptors, is found in shellfish, the viscera of some freshwater fish, fern species (Pter- idium aquilinum) and some microorganisms (Bacillus thiaminolyticus). The physiological significance of this enzyme is not known, but it is responsible for animal (grazing ruminants or horses in pastures) as well as human poisoning (reliance on shellfish or fish as main food). By contrast, thiaminase II (TenA; EC 3.5.99.2) is a hydrolase that cleaves thiamin in its thiazole and pyrimidine moieties. It is found in some microorganisms (Bacillus subtilis, for example) and its significance has recently been elucidated [14]. Indeed, TenA is involved in a salvage pathway recycling thia- min-degradation products, such as formylamino- pyrimidine formed in the soil, to aminopyrimidine and hydroxypyrimidine, a building block for the bio- synthesis of ThMP. Hydrolysis of aminopyrimidine to hydroxypyrimidine by TenA is 100 times faster than the hydrolysis of thiamin, and thiamin phosphate esters (representing nearly all the intracellular thia- min) are not hydrolyzed by TenA. This recycling does not seem to be limited to bacteria, but could also take place in yeast [15]. Thus, the thiaminase activity of TenA probably has no physiological rele- vance [14]. Thiamin biosynthesis is regulated by so-called ribos- witches [16], consisting of ThDP-sensing noncoding mRNA elements present in some mRNAs coding for enzymes involved in the thiamin biosynthetic pathways [17,18]. When plenty of ThDP is present, binding to the riboswitch induces a conformational change in the mRNA, sequestering the ribosome-binding site and preventing protein synthesis. This specific feedback mechanism is an alternative to the allosteric modula- tion of rate-limiting enzymes of metabolic pathways by metabolic end products. Thiamin transport into prokaryotic and eukaryotic cells In bacteria, thiamin uptake occurs through ATP-bind- ing cassette (ABC)-transporters, initiated with the binding of thiamin or one of its phosphate derivatives to the periplasmic protein component of the trans- porter [19]. Interestingly, thiaminase I from B. thia- minolyticus shares structural similarities with the periplasmic thiamin-binding protein of the ABC thiamin transporter from E. coli, suggesting that both proteins share a common ancestor [19]. In eukaryotes, a plasma membrane thiamin trans- porter was first cloned in yeast [20,21]. A second transporter has been discovered recently in Schizosac- charomyces pombe [22]. In animals, thiamin transport- ers regulate thiamin homeostasis within the whole organism with thiamin entry occurring in the small intestine and excretion in the kidneys. Three proteins, all belonging to the SLC19A solute carrier family, have been implicated in thiamin transport [23]. SLC19A1, a reduced folate transporter, does not carry thiamin, but is able to transport ThMP and ThDP [24]. Because ThMP is present in plasma and cerebro- spinal fluid, SLC19A1 might play a role in brain thia- min homeostasis [24] as well as in the absorption of ThDP from the intestine. SLC19A2 (thiamin trans- porter 1, THTR-1) [25] and SLC19A3 (THTR-2) [26] are specific plasma membrane thiamin ⁄ H + antiporters. Both transporters are quite ubiquitously expressed in mammalian tissues, with K m values in the 10 )6 –10 )5 m range for THTR-1 [25] and in the 10 )8 –10 )7 m range for THTR-2 [27]. In humans, mutations in SLC19A2 are responsible for thiamin-responsive megaloblastic anemia, characterized by diabetes, deafness and anemia [28]. Thiamin diphosphate biosynthesis and transport into mitochondria and peroxisomes Thiamin diphosphate is formed in the cytosol by an ATP : thiamin pyrophosphotransferase (thiamin diphosphokinase or thiamin pyrophosphokinase, TPK; EC 2.7.6.2). TPK is a homodimer of 46–56 kDa. Its sequence was first obtained from Saccharomyces cerevi- siae [29]. In the cytosol, a small part of the ThDP is free and has a rapid turnover, whereas another part binds to cytoplasmic transketolase with high affinity [30]. However, most of the ThDP synthesized is trans- ported into mitochondria by a carrier (SLC25A19) that has recently been characterized in yeast [31] and animals [32]. In humans, mutations in SLC25A19 L. Bettendorff and P. Wins Thiamin metabolism and thiamin phosphates FEBS Journal 276 (2009) 2917–2925 ª 2009 The Authors Journal compilation ª 2009 FEBS 2919 cause Amish lethal microcephaly, a disease generally fatal by the age of 6 months. Slc25a19 ) ⁄ ) mice also have central nervous system developmental defects, such as an open neural tube and do not survive embry- onic day 11. Cells cultured from Slc25a19 ) ⁄ ) mice are virtually devoid of intramitochondrial ThDP, resulting in impairment of oxidative metabolism. Some ThDP is also found in peroxisomes. Because these organelles do not contain thiamin pyrophospho- kinase activity, ThDP may be imported either by a specific transport mechanism or it may bind first to 2-hydroxyacyl-CoA lyase and then be imported with the enzyme as recently suggested [33]. Hydrolysis of thiamin diphosphate and thiamin monophosphate Hydrolysis of ThDP to ThMP may occur in most organisms and tissues, but to date no specific thiamin diphosphatase (ThDPase) has been characterized. Many phosphatases are able to hydrolyze ThDP as well as other thiamin phosphate derivatives, but gener- ally less efficiently than nucleoside diphosphates, as is the case of a liver microsomal nucleoside diphospha- tase [34]. ThDPase activity is often used as a specific marker of the Golgi apparatus. Indeed, a membrane- associated nucleoside diphosphatase with a slight preference for ThDP as substrate compared with nucleoside diphosphates has been purified from rat brain [35]. This enzyme has many properties in com- mon with the ThDPase in the Golgi and is different from the above-mentioned nucleoside diphosphatase [34]. Other enzymes, especially from liver can hydro- lyze both ThDP and nucleoside diphosphates but their physiological function is probably to hydrolyze the latter compounds. ThMP can be rapidly hydrolyzed to thiamin in cul- tured cells [24] but, except for one report in bacteria [36], no specific ThMPase has yet been characterized. ThMPase activity is used as a marker for spinal chord small diameter dorsal root ganglions involved in noci- ception [37]. ThMP is used as substrate in those stud- ies because, in contrast to other phosphatase substrates, it is not as easily hydrolyzed by lysosomal acid phosphatase present in these preparations. Thiamin triphosphate and its potential roles As mentioned earlier, the existence of ThTP was first suggested in the 1950s and it was thought to have a cofactor-independent neurophysiological role [38]. However, recent results show that ThTP is present in most tissues and in most organisms studied to date [6], suggesting that it might have a much more basic role in cellular metabolism. In E. coli, ThTP is synthesized in response to amino acid starvation and seems required for optimal growth under these conditions [39]. We suggested that ThTP may be a signal pro- duced in response to changes in the nutritional envi- ronment of the bacteria. In multicellular organisms, the role of ThTP remains enigmatic. It was shown to activate maxi-anion chan- nels in inside-out patches of neuroblastoma cells [40]. These channels are thought to play a role in swelling- induced ATP release [41]. The activation of maxi-anion channels may be dependent on phosphorylation by ThTP. Indeed, ThTP, like ATP, contains two phosphoanhydride bonds with a high phosphate energy transfer potential. Therefore, we tested whether [ 32 P]ThTP could phosphorylate proteins in vitro. Indeed, in postsynaptic membranes from Torpedo marmorata,[ 32 P]ThTP could phosphorylate rapsyn, a protein required for the clustering of acetylcholine receptors at the neuromuscular junction [42]. Other, as yet unidentified, proteins were also phosphorylated in rodent brain. It is important to determine whether pro- tein phosphorylation by ThTP could be part of a new physiological signaling pathway. Enzymatic synthesis of thiamin triphosphate The biosynthesis of ThTP was observed in vivo in organisms such as bacteria [39], in cultured neuroblas- toma cells [43] as well as in rat brain [30]. However, the mechanism of ThTP synthesis remains an enigma. The earliest reports proposed that an ATP : ThDP phosphotransferase (ThDP kinase) catalyzes the reac- tion ThDP + ATP « ThTP + ADP. Such an enzyme system was purified [44] as a high molecular mass multisubunit complex, but the rate of reaction was very slow (k cat  1 min )1 ). Actually, it is not cer- tain if the product of the reaction was authentic ThTP and, with our present knowledge, it appears more likely that the compound formed was AThTP which is indeed formed under these conditions (see below). In the late 1980s and early 1990s, Kawasaki and co-workers [45,46] showed that vertebrate adenylate kinase isoform 1 (AK1; EC 2.7.4.3), which is predomi- nant in skeletal muscle cytoplasm, is able to synthesize ThTP according to the reaction ThDP + ADP « ThTP + AMP. They suggested that the in vivo syn- thesis of ThTP occurs through this reaction, although the rate of reaction was very low (for chicken AK1, k cat  0.5 min )1 at physiological pH) [46]. However, Thiamin metabolism and thiamin phosphates L. Bettendorff and P. Wins 2920 FEBS Journal 276 (2009) 2917–2925 ª 2009 The Authors Journal compilation ª 2009 FEBS after heat inactivation of AK in E. coli (E. coli has only one AK isoform), ThTP levels are increased rather than decreased [47] and transgenic mice lacking AK1 have normal ThTP levels [48]. Our results suggest that ThTP synthesis according to the above reaction is a general property of AKs [47], but that it is not of general physiological significance. Possible exceptions to this rule are Electrophorus electricus electric organ [49], as well as pig [50] and chicken [46] skeletal mus- cles. Those tissues have a very high ThTP content, a situation that may result from the combined effects of a high cytosolic AK1 activity and a lack of a specific soluble ThTPase activity (see below). This raises the possibility that, in animals, ThTP might actually be formed not in the cytosol, but in a different cellular compartment. In fact, except for the very low ThTP- synthesizing activity of AK1, we never observed a rapid net synthesis of ThTP in soluble prepara- tions from any biological source (B. Wirtzfeld, A. F. Makarchikov and L. Bettendorff, unpublished results). Therefore, it is possible that ThDP is not formed in the cytosol but in a different subcellular compartment. Subcellular fractionation of rat brain showed that the highest ThTP levels were found in the mitochon- drial and synaptosomal fractions, the latter being also rich in mitochondria [30]. Furthermore, when a rat brain homogenate was incubated with ThDP, ThTP was formed inside closed compartments [51]. The nature of these compartments and the mechanism are now under investigation in our laboratory. Hydrolysis of thiamin triphosphate In most mammalian cells, the steady-state concentra- tions of ThTP remain low (10 )7 –10 )6 m) [6], probably because of the presence of one or several ThTP-hydro- lyzing enzymes with sufficient specificity and catalytic efficiency. ThTP hydrolysis has been relatively well studied, because the reaction can be more readily dem- onstrated in cell extracts than ThTP synthesis. Early studies have shown the presence of a soluble and a membrane-associated enzyme able to hydrolyze ThTP in rat tissues. The latter enzyme was found to be associated with particulate fractions (nuclear, synaptosomal and micro- somal), was activated by Mg 2+ ,Ca 2+ or Mn 2+ and had a pH optimum around 6.5 [52]. Membrane-associ- ated ThTPases were also described in electric organs [49] and skeletal muscle [53], but to date all attempts to purify these enzymes have failed. Although they appear to be distinct from membrane-associated ATPases, their specificity for ThTP is not established and their catalytic efficiency could not be quantified. Membrane-associated ThTPases from electric organs and skeletal muscle are strongly activated by anions [53,54], in particular by the chaotropic I ) and NO 3 ) . This is different from membrane-associated ThTPases from other tissues such as the brain, that are inhibited by these anions [53]. The cytosolic ThTPase (EC 3.6.1.28) is a soluble protein requiring Mg 2+ as activator, Ca 2+ being inhibitory, and having an alkaline pH optimum ( 9.0) [55]. The enzyme is expressed in most mamma- lian tissues and was first purified from bovine brain [56]. It is a low molecular mass protein ( 25 kDa) with high catalytic efficiency and nearly absolute speci- ficity for ThTP. Molecular characterization of the human 25-kDa ThTPase [57] revealed that the sequence does not closely resemble that of any other protein identified in mammalian genomes. However, bioinformatic analyses suggest that the mammalian 25-kDa ThTPases and the CyaB adenylyl cyclase from Aeromonas hydrophila define a superfamily of domains that can be traced back to the last universal common ancestor [58]. This domain, called the CYTH (CyaB, thiamin triphosphatase) domain, includes enzymes that require divalent metal ions for activity and would play various roles at the interface of organic polyphosphate and nucleotide metabolism. Surprisingly, there is no evidence that a member of this protein superfamily exists in birds, the only known major class where it would be absent. The pig ThTPase, though retaining the CYTH signature, is practically devoid of ThTPase activity probably as a result of a Glu85 fi Lys muta- tion leading to conformational changes [59]. Indepen- dent of this analysis by Iyer & Aravind [58], Shuman and co-workers [60] defined a family of metal-depen- dent phosphohydrolases which they called ‘triphos- phate tunnel metalloenzymes’ (TTM) because their active site is usually located within a topologically closed hydrophilic b-barrel [60]. The proteins of this family have common features with CYTH domains, such as the EXEXK signature, which is a divalent cat- ion-binding motif. The founding member of the TTM superfamily was the yeast RNA triphosphatase Cet1 [61], but homologous sequences have been found in archaeal and bacterial species. One of these proteins from Clostridium thermocellum was recently found to hydrolyze inorganic triphosphate with a much higher catalytic efficiency than ATP [62]. There was no adenylyl cyclase activity. It is not known whether any of these enzymes from the TTM family would be able to bind or to hydrolyze ThTP. It appears that the ‘CYTH–TTM’ superfamily includes enzymes with vari- ous catalytic properties (adenylyl cyclase or inorganic L. Bettendorff and P. Wins Thiamin metabolism and thiamin phosphates FEBS Journal 276 (2009) 2917–2925 ª 2009 The Authors Journal compilation ª 2009 FEBS 2921 triphosphatase in some bacteria, RNA triphosphatase in yeast, ThTPase in some animal species) but with important common features: (a) the activity always requires divalent metal cations, and (b) there is speci- ficity for substrates containing a triphosphate group. The structure of recombinant mouse 25-kDa ThT- Pase has been determined recently [63] and the residues responsible for binding Mg 2+ and ThTP determined from NMR titration experiments. Although the free enzyme has an open cleft form, the ternary complex [ThTPase–Mg 2+ –ThTP] tends to adopt a closed tun- nel-fold, suggesting that mammalian 25-kDa ThTPases may be considered true members of the TTM super- family of proteins. Another important question is to know why ThTP should be hydrolyzed at all? If ThTP is indeed a signaling molecule, hydrolysis by ThTPases might terminate its action in the same way as phosphodi- esterases terminate the action of cAMP. This may be true in E. coli, where the appearance of ThTP is gener- ally transient and followed by rapid hydrolysis [7,39]. However, in mammalian cells, ThTP seems to be continuously formed and hydrolyzed [30,43]. An important question therefore concerns the regu- lation of soluble ThTPase activity, which would ulti- mately control cytoplasmic ThTP concentrations. ThTPase contains several consensus sequences for phosphorylation by protein kinase C and casein kinase 2 [57,64]. However, it was shown recently that the iron-regulated metastasis suppressor Ndgr-1 upregu- lates 25-kDa ThTPase expression in several cancer cell models [65]. ThTPase expression was inversely corre- lated to melanoma tumor antigen p97 (MTf), an iron- binding protein expressed at high levels in melanoma cells [66]. The significance of these results is unclear, but they may suggest that ThTPase expression is linked to the degree of differentiation of cells. Indeed, in the adult rodent brain, ThTPase is mainly found in the highly differentiated pyramidal and Purkinje neurons [67]. Adenosine thiamin triphosphate, a new thiamin compound As mentioned above, incubation of E. coli in amino acid-deficient medium in the presence of a suitable car- bon source led to the accumulation of ThTP. How- ever, in the absence of a carbon source, no ThTP was observed, but an additional peak was detected. This raised the possibility of the existence of a new thiamin compound. Purification and analysis by MS and 1 H-NMR showed that this compound is adenosine thiamin triphosphate or thiaminylated ATP [7], a com- pound not previously described. In E. coli, AThTP is synthesized according to the reaction ThDP + ADP (ATP) « AThTP + P i (PP i ), probably by a thi- amin diphosphate adenylyl transferase [68]. Note that both ATP and ADP may be substrates, but not other nucleotides. This enzyme is probably a high molecular mass complex requiring a low molecular mass activa- tor. When E. coli accumulate AThTP, addition of glucose leads, within minutes, to its complete dis- appearance. This experiment strongly suggests the existence of at least one AThTP-hydrolyzing enzyme that remains to be characterized. In E. coli, AThTP might act as an alarmone, signaling carbon starvation and ⁄ or a low energy charge. AThTP is also found at low levels in eukaryotic organisms [7], though we have no clue as to its role there. Conclusion The textbook view on the biochemical role of thiamin is that the vitamin, after entering the cells, is pyro- phosphorylated to ThDP, a cofactor for several enzymes. Decreased enzyme activities caused by decreased ThDP levels would be responsible for the symptoms observed during thiamin deficiency. For two decades, this view has nearly been raised to a dogma, with practically complete ignorance of other thiamin derivatives and the enzymes of thiamin metabolism (Fig. 1). In E. coli, a significant part of ThDP (up to 60%) can be rapidly converted to either ThTP or AThTP according to the metabolic state of the bacteria [7,39,47]. ThTP and AThTP could act as signals involved in the adaptation of the bacteria to stress con- ditions. Both compounds can be rapidly formed (within minutes) or hydrolyzed, suggesting the existence of a complete set of enzymes able to rapidly respond to changing environmental conditions. In eukaryotes, ThTP and AThTP seem to be less subject to rapid changes. Nevertheless, ThTP has a higher turnover than the bulk of ThDP [30,43]. In rat brain, it is con- stantly formed and hydrolyzed, but until now no spe- cific conditions under which it accumulates or disappears could be determined. AThTP is also formed in eukaryotes but its potential role in these organisms is completely unknown. It was previously suggested that ThTP might have a specific neurochemical role [38], but recent evidence is in favor of a much more basic role in cellular metabolism, possibly in the fine- tuning or integration of some metabolic processes. In addition to clarifying the precise roles of ThTP and AThTP, the enzymes leading to their synthesis and hydrolysis need to be characterized. It is surprising Thiamin metabolism and thiamin phosphates L. Bettendorff and P. Wins 2922 FEBS Journal 276 (2009) 2917–2925 ª 2009 The Authors Journal compilation ª 2009 FEBS that 50 years after the discovery of ThTP, the mecha- nism of its synthesis remains unclear. Membrane-asso- ciated ThTPases have been reported to exist in practically all organisms, including bacteria, but so far none has been characterized from a molecular point of view. Last, but not least, ThDPases and ThMPases, although probably playing a role in the maintenance of steady-state ThDP concentrations, have not been characterized at the molecular level. The intriguing complexity of thiamin metabolism raises numerous questions, many of which remain unanswered. In any event, the new developments described in this short review strongly suggest that the simplistic view that the cofactor ThDP is the only biologically active form of thiamin is no longer tenable. Acknowledgements LB is Research Director at the F.R.S FNRS. This work was supported by grants from the F.R.S FNRS. References 1 Frank RA, Leeper FJ & Luisi BF (2007) Structure, mechanism and catalytic duality of thiamin-dependent enzymes. Cell Mol Life Sci 64, 892–905. 2 Breslow R (1958) On the mechanism of thiamin action. 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MINIREVIEW Thiamin diphosphate in biological chemistry: new aspects of thiamin metabolism, especially triphosphate derivatives acting other than as cofactors Lucien Bettendorff and Pierre Wins GIGA-Neurosciences,. adenylate kinase; AThTP, adenosine thiamin triphosphate; TenA, thiaminase II; ThDP, thiamin diphosphate; ThDPase, thiamin diphosphatase; ThMP, thiamin monophosphate; ThMPase, thiamin monophosphatase;. hydrolyzed to thiamin + P i by a thiamin monophosphatase (ThMPase). Thiamin may then be pyrophosphorylated to ThDP by thiamin diphosphokinase (thiamin + ATP « ThDP + AMP; EC 2.7.6.2). Thiamin can be