Thiamin diphosphate in biological chemistry: new aspectsof thiamin metabolism, especially triphosphate derivatives acting other than as cofactors Lucien Bettendorff and Pierre Wins GIGA-
Trang 1Thiamin 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, Baˆt B36, Tour de
Pathologie 2, e´tage +1, Avenue de l’Hoˆpital,
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 Thithi-amin 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.
Trang 2plants, 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]).
Trang 3are 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 + Piby 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 aminoformylamino-pyrimidine
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 Kmvalues in the 10)6–10)5m range for THTR-1 [25] and in the 10)8–10)7m 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
Trang 4cause 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 [32P]ThTP could phosphorylate proteins in vitro Indeed, in postsynaptic membranes from Torpedo marmorata, [32P]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 (kcat 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,
kcat 0.5 min)1 at physiological pH) [46] However,
Trang 5after 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)6m) [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 Mg2+, Ca2+ or Mn2+ 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 NO3 ) 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 Mg2+ as activator, Ca2+ 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 conformamuta-tional 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
Trang 6triphosphatase 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 Mg2+ and ThTP determined
from NMR titration experiments Although the free
enzyme has an open cleft form, the ternary complex
[ThTPase–Mg2+–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
1H-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 + Pi(PPi), 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
Trang 7that 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
IV Evidence from studies on model systems J Am
Chem Soc 80, 3719–3726
3 Kale S, Ulas G, Song J, Brudvig GW, Furey W &
Jordan F (2008) Efficient coupling of catalysis and
dynamics in the E1 component of Escherichia coli
pyruvate dehydrogenase multienzyme complex Proc
Natl Acad Sci USA 105, 1158–1163
4 Kluger R & Tittmann K (2008) Thiamin diphosphate
catalysis: enzymic and nonenzymic covalent
intermedi-ates Chem Rev 108, 1797–1833
5 Kawasaki T (1992) Vitamin B1: thiamin In Modern
Chromatographic Analysis of Vitamins(De Leenheer
AP, Lambert WE & Nelis HJ, eds), pp 319–354
Marcel Dekker, New York
6 Makarchikov AF, Lakaye B, Gulyai IE, Czerniecki J,
Coumans B, Wins P, Grisar T & Bettendorff L (2003)
Thiamin triphosphate and thiamin triphosphatase
activ-ities: from bacteria to mammals Cell Mol Life Sci 60,
1477–1488
7 Bettendorff L, Wirtzfeld B, Makarchikov AF,
Maz-zucchelli G, Fre´de´rich M, Gigliobianco T, Gangolf M,
De Pauw E, Angenot L & Wins P (2007) Discovery of
a natural thiamin adenine nucleotide Nat Chem Biol 3,
211–212
8 Shi Q, Karuppagounder SS, Xu H, Pechman D, Chen
H & Gibson GE (2007) Responses of the mitochondrial
alpha-ketoglutarate dehydrogenase complex to thiamin
deficiency may contribute to regional selective vulnera-bility Neurochem Int 50, 921–931
9 Navarro D, Zwingmann C & Butterworth RF (2008) Region-selective alterations of glucose oxidation and amino acid synthesis in the thiamin-deficient rat brain:
a re-evaluation using1H⁄13
C nuclear magnetic reso-nance spectroscopy J Neurochem 106, 603–612
10 Karuppagounder SS, Xu H, Shi Q, Chen LH, Pedrini
S, Pechman D, Baker H, Beal MF, Gandy SE & Gib-son GE (2008) Thiamin deficiency induces oxidative stress and exacerbates the plaque pathology in Alzhei-mer’s mouse model Neurobiol Aging doi: 10.1016/j.neu-robiolaging.2007.12.013
11 Webb ME, Marquet A, Mendel RR, Rebeille F & Smith AG (2007) Elucidating biosynthetic pathways for vitamins and cofactors Nat Prod Rep 24, 988–1008
12 Begley TP, Chatterjee A, Hanes JW, Hazra A & Ealick
SE (2008) Cofactor biosynthesis – still yielding fascinat-ing new biological chemistry Curr Opin Chem Biol 12, 118–125
13 Murata K (1982) Actions of two types of thiaminase on thiamin and its analogues Ann NY Acad Sci 378, 146– 156
14 Jenkins AH, Schyns G, Potot S, Sun G & Begley TP (2007) A new thiamin salvage pathway Nat Chem Biol
3, 492–497
15 Onozuka M, Konno H, Kawasaki Y, Akaji K & Nosaka K (2008) Involvement of thiaminase II encoded
by the THI20 gene in thiamin salvage of Saccharomy-ces cerevisiae FEMS Yeast Res 8, 266–275
16 Montange RK & Batey RT (2008) Riboswitches: emerging themes in RNA structure and function Annu Rev Biophys 37, 117–133
17 Winkler W, Nahvi A & Breaker RR (2002) Thiamin derivatives bind messenger RNAs directly to regulate bacterial gene expression Nature 419, 952–956
18 Mironov AS, Gusarov I, Rafikov R, Lopez LE, Shata-lin K, Kreneva RA, Perumov DA & Nudler E (2002) Sensing small molecules by nascent RNA A mechanism
to control transcription in bacteria Cell 111, 747–756
19 Soriano EV, Rajashankar KR, Hanes JW, Bale S, Begley TP & Ealick SE (2008) Structural similarities between thiamin-binding protein and thiaminase-I sug-gest a common ancestor Biochemistry 47, 1346–1357
20 Enjo F, Nosaka K, Ogata M, Iwashima A & Nishimura
H (1997) Isolation and characterization of a thiamin transport gene, THI10, from Saccharomyces cerevisiae
J Biol Chem 272, 19165–19170
21 Singleton CK (1997) Identification and characterization
of the thiamin transporter gene of Saccharomyces cere-visiae Gene 199, 111–121
22 Vogl C, Klein CM, Batke AF, Schweingruber ME & Stolz J (2008) Characterization of Thi9, a novel thiamin (vitamin B1) transporter from Schizosaccharomy-ces pombe J Biol Chem 283, 7379–7389
Trang 823 Ganapathy V, Smith SB & Prasad PD (2004) SLC19:
the folate⁄ thiamin transporter family Pflu¨gers Arch
447, 641–646
24 Zhao R, Gao F & Goldman ID (2002) Reduced folate
carrier transports thiamin monophosphate: an
alterna-tive route for thiamin delivery into mammalian cells
Am J Physiol Cell Physiol 282, C1512–C1517
25 Dutta B, Huang W, Molero M, Kekuda R, Leibach
FH, Devoe LD, Ganapathy V & Prasad PD (1999)
Cloning of the human thiamin transporter, a member
of the folate transporter family J Biol Chem 274,
31925–31929
26 Eudy JD, Spiegelstein O, Barber RC, Wlodarczyk BJ,
Talbot J & Finnell RH (2000) Identification and
charac-terization of the human and mouse SLC19A3 gene: a
novel member of the reduced folate family of
micronutrient transporter genes Mol Genet Metab 71,
581–590
27 Said HM, Balamurugan K, Subramanian VS &
Mar-chant JS (2004) Expression and functional contribution
of hTHTR-2 in thiamin absorption in human intestine
Am J Physiol Gastrointest Liver Physiol 286, G491–G498
28 Fleming JC, Tartaglini E, Steinkamp MP, Schorderet
DF, Cohen N & Neufeld EJ (1999) The gene mutated
in thiamin-responsive anaemia with diabetes and
deaf-ness (TRMA) encodes a functional thiamin transporter
Nat Genet 22, 305–308
29 Nosaka K, Kaneko Y, Nishimura H & Iwashima A
(1993) Isolation and characterization of a thiamin
pyrophosphokinase gene, THI80, from
Saccharo-myces cerevisiae J Biol Chem 268, 17440–17447
30 Bettendorff L, Wins P & Lesourd M (1994) Subcellular
localization and compartmentation of thiamin
deriva-tives in rat brain Biochim Biophys Acta 1222, 1–6
31 Marobbio CM, Vozza A, Harding M, Bisaccia F,
Palmieri F & Walker JE (2002) Identification and
reconstitution of the yeast mitochondrial transporter for
thiamin pyrophosphate EMBO J 21, 5653–5661
32 Lindhurst MJ, Fiermonte G, Song S, Struys E, De
Leonardis F, Schwartzberg PL, Chen A, Castegna A,
Verhoeven N, Mathews CK et al (2006) Knockout of
Slc25a19 causes mitochondrial thiamin pyrophosphate
depletion, embryonic lethality, CNS malformations, and
anemia Proc Natl Acad Sci USA 103, 15927–15932
33 Fraccascia P, Sniekers M, Casteels M & Van Veldhoven
PP (2007) Presence of thiamin pyrophosphate in
mammalian peroxisomes BMC Biochem 8, 10
34 Sano S, Matsuda Y & Nakagawa H (1988) Thiamin
pyrophosphatase (nucleoside diphosphatase) in the
Golgi apparatus is distinct from microsomal nucleoside
diphosphatase J Biochem 103, 678–681
35 Sano S, Matsuda Y & Nakagawa H (1988) Type B
nucleoside-diphosphatase of rat brain Purification and
properties of an enzyme with high thiamin
pyrophos-phatase activity Eur J Biochem 171, 231–236
36 Kawasaki T, Egi Y & Sanemori H (1977) Specific thia-min monophosphate phosphohydrolase in Micrococ-cus denitrificans J Bacteriol 130, 542–544
37 Csillik B, Mihaly A, Krisztin-Peva B, Farkas I & Knyihar-Csillik E (2008) Mitigation of nociception via transganglionic degenerative atrophy: possible mecha-nism of vinpocetine-induced blockade of retrograde axoplasmic transport Ann Anat 190, 140–145
38 Bettendorff L (1994) Thiamin in excitable tissues: reflections on a non-cofactor role Metab Brain Dis 9, 183–209
39 Lakaye B, Wirtzfeld B, Wins P, Grisar T & Bettendorff
L (2004) Thiamin triphosphate, a new signal required for optimal growth of Escherichia coli during amino acid starvation J Biol Chem 279, 17142–17147
40 Bettendorff L, Kolb HA & Schoffeniels E (1993) Thia-min triphosphate activates an anion channel of large unit conductance in neuroblastoma cells J Membr Biol
136, 281–288
41 Sabirov RZ & Okada Y (2005) ATP release via anion channels Purinergic Signal 1, 311–328
42 Nghieˆm HO, Bettendorff L & Changeux JP (2000) Specific phosphorylation of Torpedo 43K rapsyn by endogenous kinase(s) with thiamin triphosphate as the phosphate donor FASEB J 14, 543–554
43 Bettendorff L (1994) The compartmentation of phos-phorylated thiamin derivatives in cultured neuroblas-toma cells Biochim Biophys Acta 1222, 7–14
44 Voskoboyev AI, Chernikevich IP & Luchko VS (1987) Studies on thiamin diphosphate kinase (EC 2.7.4.15) from brewer’s yeast: purification and some properties Biomed Biochim Acta 46, 3–13
45 Shikata H, Koyama S, Egi Y, Yamada K & Kawasaki
T (1989) Cytosolic adenylate kinase catalyzes the syn-thesis of thiamin triphosphate from thiamin diphos-phate Biochem Int 18, 933–941
46 Miyoshi K, Egi Y, Shioda T & Kawasaki T (1990) Evidence for in vivo synthesis of thiamin triphosphate
by cytosolic adenylate kinase in chicken skeletal muscle
J Biochem (Tokyo) 108, 267–270
47 Gigliobianco T, Lakaye B, Makarchikov AF, Wins P & Bettendorff L (2008) Adenylate kinase-independent thiamin triphosphate accumulation under severe energy stress in Escherichia coli BMC Microbiol 8, 16
48 Makarchikov AF, Wins P, Janssen E, Wieringa B, Grisar T & Bettendorff L (2002) Adenylate kinase 1 knockout mice have normal thiamin triphosphate levels Biochim Biophys Acta 1592, 117–121
49 Bettendorff L, Michel-Cahay C, Grandfils C, De Rycker C & Schoffeniels E (1987) Thiamin triphosphate and membrane-associated thiamin phosphatases in the electric organ of Electrophorus electricus J Neurochem
49, 495–502
50 Egi Y, Koyama S, Shikata H, Yamada K & Kawasaki
T (1986) Content of thiamin phosphate esters in
Trang 9mammalian tissues – an extremely high concentration
of thiamin triphosphate in pig skeletal muscle Biochem
Int 12, 385–390
51 Bettendorff L, Peeters M, Wins P & Schoffeniels E
(1993) Metabolism of thiamin triphosphate in rat brain:
correlation with chloride permeability J Neurochem 60,
423–434
52 Barchi RL & Braun PE (1972) A membrane-associated
thiamin triphosphatase from rat brain Properties of the
enzyme J Biol Chem 247, 7668–7673
53 Matsuda T, Tonomura H, Baba A & Iwata H (1991)
Membrane-associated thiamin triphosphatase in rat
skeletal muscle Int J Biochem 23, 1111–1114
54 Bettendorff L, Wins P & Schoffeniels E (1988) Thiamin
triphosphatase from Electrophorus electric organ is
anion-dependent and irreversibly inhibited by
4,4¢-diiso-thiocyanostilbene-2,2¢disulfonic acid Biochem Biophys
Res Commun 154, 942–947
55 Hashitani Y & Cooper JR (1972) The partial
purifica-tion of thiamin triphosphatase from rat brain J Biol
Chem 247, 2117–2119
56 Makarchikov AF & Chernikevich IP (1992) Purification
and characterization of thiamin triphosphatase from
bovine brain Biochim Biophys Acta 1117, 326–332
57 Lakaye B, Makarchikov AF, Antunes AF, Zorzi W,
Coumans B, De Pauw E, Wins P, Grisar T &
Bette-ndorff L (2002) Molecular characterization of a specific
thiamin triphosphatase widely expressed in mammalian
tissues J Biol Chem 277, 13771–13777
58 Iyer LM & Aravind L (2002) The catalytic domains of
thiamin triphosphatase and CyaB-like adenylyl cyclase
define a novel superfamily of domains that bind organic
phosphates BMC Genomics 3, 33
59 Szyniarowski P, Lakaye B, Czerniecki J, Makarchikov
AF, Wins P, Margineanu I, Coumans B, Grisar T &
Bettendorff L (2005) Pig tissues express a catalytically
inefficient 25-kDa thiamin triphosphatase: insight in the
catalytic mechanisms of this enzyme Biochim Biophys
Acta 1725, 93–102
60 Gong C, Smith P & Shuman S (2006)
Structure–func-tion analysis of Plasmodium RNA triphosphatase and
description of a triphosphate tunnel metalloenzyme superfamily that includes Cet1-like RNA triphosphata-ses and CYTH proteins RNA 12, 1468–1474
61 Lima CD, Wang LK & Shuman S (1999) Structure and mechanism of yeast RNA triphosphatase: an essential component of the mRNA capping apparatus Cell 99, 533–543
62 Keppetipola N, Jain R & Shuman S (2007) Novel triphosphate phosphohydrolase activity of Clostrid-ium thermocellumTTM, a member of the triphosphate tunnel metalloenzyme superfamily J Biol Chem 282, 11941–11949
63 Song J, Bettendorff L, Tonelli M & Markley JL (2008) Structural basis for the catalytic mechanism of mammalian 25-kDa thiamin triphosphatase J Biol Chem 283, 10939–10948
64 Lakaye B, Verlaet M, Dubail J, Czerniecki J, Bontems
S, Makarchikov AF, Wins P, Piette J, Grisar T & Bettendorff L (2004) Expression of 25 kDa thiamin triphosphatase in rodent tissues using quantitative PCR and characterization of its mRNA Int J Biochem Cell Biol 36, 2032–2041
65 Kovacevic Z, Fu D & Richardson DR (2008) The iron-regulated metastasis suppressor, Ndrg-1: identification
of novel molecular targets Biochim Biophys Acta 1783, 1981–1992
66 Suryo Rahmanto Y, Dunn L & Richardson D (2007) Identification of distinct changes in gene expression after modulation of melanoma tumor antigen p97 (mel-anotransferrin) in multiple models in vitro and in vivo Carcinogenesis 26, 2172–2183
67 Czerniecki J, Chanas G, Verlaet M, Bettendorff L, Makarchikov AF, Leprince P, Wins P, Grisar T & Lakaye B (2004) Neuronal localization of the 25-kDa specific thiamin triphosphatase in rodent brain Neuro-science 125, 833–840
68 Makarchikov AF, Brans A & Bettendorff L (2007) Thiamin diphosphate adenylyl transferase from E coli: functional characterization of the enzyme synthesizing adenosine thiamin triphosphate BMC Biochem 8, 17