MINIREVIEW
Reaction mechanisms of thiamin diphosphate enzymes:
redox reactions
Kai Tittmann
Albrecht-von-Haller-Institut fu
¨
r Pflanzenwissenschaften und Go
¨
ttinger Zentrum fu
¨
r Molekulare Biowissenschaften, Georg-August-Universita
¨
t
Go
¨
ttingen, Germany
Introduction
The oxidative decarboxylation of 2-keto acids, such
as pyruvate, branched-chain keto acids and ketoglu-
tarate, is a key reaction of intermediary metabolism
in virtually all organisms and is catalyzed by thiamin
diphosphate (ThDP)-dependent enzymes [1]. In view
of the central metabolic role of pyruvate, the various
biochemical reactions involving pyruvate are the
most intensely studied and are well understood.
Thus, they serve as prototypical reactions for the
enzymic oxidative conversion of 2-keto acids. Hence,
the present review mainly focuses on the reaction
mechanisms of ThDP enzymes that directly oxidize
pyruvate. Special emphasis is devoted to the nature
and reactivity of transient intermediates, the coupling

Caspari-Haus, Justus-von-Liebig-Weg 11,
D-37077 Go
¨
ttingen, Germany
Fax: +49 551 39 5749
Tel: +49 551 39 14430
E-mail:
(Received 7 November 2008, revised 3
February 2009, accepted 13 February 2009)
doi:10.1111/j.1742-4658.2009.06966.x
Amongst a wide variety of different biochemical reactions in cellular car-
bon metabolism, thiamin diphosphate-dependent enzymes catalyze the oxi-
dative decarboxylation of 2-keto acids. This type of reaction typically
involves redox coupled acyl transfer to CoA or phosphate and is mediated
by additional cofactors, such as flavins, iron-sulfur clusters or lipoamide
swinging arms, which transmit the reducing equivalents that arise during
keto acid oxidation to a final electron acceptor. EPR spectroscopic and
kinetic studies have implicated the intermediacy of radical cofactor
intermediates in pyruvate:ferredoxin oxidoreductase and an acetyl phos-
phate-producing pyruvate oxidase, whereas the occurrence of transient
on-pathway radicals in other enzymes is less clear. The structures of pyru-
vate:ferredoxin oxidoreductase and pyruvate oxidase with different enzymic
reaction intermediates along the pathway including a radical intermediate
were determined by cryo-crystallography and used to infer electron tunnel-
ing pathways and the potential roles of CoA and phosphate for an intimate
coupling of electron and acyl group transfer. Viable mechanisms of reduc-
tive acetylation in pyruvate dehydrogenase multi-enzyme complex, and of
electron transfer in the peripheral membrane enzyme pyruvate oxidase
from Escherichia coli, are also discussed.
Abbreviations

) and is composed of multiple copies of
three enzyme components: a ThDP-dependent pyru-
vate dehydrogenase (termed the E1 component), a
dihydrolipoamide transacetylase (E2 component),
which carries lipoyl groups covalently attached to
lysine residues [N
6
-(lipoyl)lysine, lipoamide], and lipoa-
mide dehydrogenase (E3 component) with a nonco-
valently yet tightly bound FAD cofactor [3]. In
mammals, PDHc contains an additional E3 binding
protein and specific kinases and phosphatases, which
control the activity of the complex by reversible phos-
phorylation ⁄ dephosphorylation of serine side chains in
E1 [4]. Initially, E1 catalyzes the irreversible decarbox-
ylation of pyruvate and the subsequent reductive acet-
ylation of an N
6
-(lipoyl)lysine in E2. E2 itself catalyzes
acyl group transfer from the reduced S-acety-
ldihydrolipoyl-lysine to CoA. Finally, E3 regenerates
the oxidized form of lipoamide and transfers the two
reducing equivalents to NAD
+
.
Pyruvate:ferredoxin oxidoreductase
In anaerobic organisms, acetyl-CoA is synthesized by
the enzyme pyruvate:ferredoxin oxidoreductase
(PFOR), which may contain one or multiple
[Fe

formation [7]. The reverse synthase reaction (pyruvate
formation) is central to CO
2
fixation in acetogenic and
green photosynthetic bacteria [8].
Acetyl phosphate-producing pyruvate oxidases
In Lactobacillae such as Lactobacillus plantarum or
Lactobacillus delbrueckii, which are unable to synthe-
size hemes and thus lack a respiratory chain for oxi-
dative phosphorylation, ATP is mainly generated by
fermentation of carbohydrates with lactic acid as a
final product. Under aerobic growth conditions, some
Lactobacillae convert carbohydrates to the high-
energy metabolite acetyl phosphate, which in turn is
used for ATP synthesis. A key reaction of this path-
way is the oxidative decarboxylation of pyruvate by
the enzyme pyruvate oxidase (POX) that requires
ThDP, Mg
2+
and FAD as cofactors [9,10]. After
binding and decarboxylation of pyruvate, the reduc-
ing equivalents are transferred to the neighboring
FAD cofactor. The flavin is then reoxidized by the
final electron acceptor dioxygen to yield hydrogen
peroxide (Eqn 3).
POX : pyruvate þ phosphate + oxygen + H
þ
! acetyl phosphate + CO
2
+H

H
2
ð4Þ
K. Tittmann Redox reactions of thiamin diphosphate enzymes
FEBS Journal 276 (2009) 2454–2468 ª 2009 The Author Journal compilation ª 2009 FEBS 2455
Reaction intermediates in the ThDP
catalyzed oxidation of pyruvate
The oxidative decarboxylation of pyruvate in PDHc,
PFOR and POX involves a series of covalent ThDP
intermediates and analogous elementary reactions
(Fig. 1) [13]. In pioneering studies on models, Breslow
[14] identified C2 of the ThDP thiazolium as the
reactive center that, in its carbanionic form, attacks
the substrate carbonyl yielding, in the case of pyru-
vate, the tetrahedral pre-decarboxylation intermediate
2-lactyl-ThDP. Decarboxylation of the latter gives the
resonant a-carbanion ⁄ enamine forms of 2-(1-hydroxy-
ethyl)-ThDP (HEThDP). The enamine is sometimes
(and more accurately) referred to as 2-(1-hydroxye-
thylidene)-ThDP and formally represents the C2a-
deprotonated conjugate base of HEThDP in a
resonance stabilized form. Essentially, all steps
encompassing binding and decarboxylation of pyru-
vate are common to PDHc, PFOR and POX. Reac-
tion sequences diverge at the HEThDP
carbanion ⁄ enamine intermediate, which is highly
reducing and may undergo one-electron or two-elec-
tron oxidation by proximal redox cofactors. The
[Fe
4

state.
The occurrence of 2-lactyl-ThDP, HEThDP and
AcThDP as reaction intermediates in ThDP enzymes
has been confirmed by 1H NMR spectroscopy after
acid quench isolation [17]. EPR spectroscopy was
employed to detect radical ThDP intermediates [18].
Thermodynamic aspects of pyruvate
oxidation
In PDHc, PFOR and POX, the thermodynamically
favorable oxidation of pyruvate is coupled to forma-
tion of the ‘energy-rich’ metabolites acetyl-CoA and
acetyl-phosphate, which serve either as chemically acti-
vated building blocks in anabolic pathways, or for
ATP synthesis because the group transfer potential of
the acetyl-CoA thioester (DG°
¢
= )35.7 kJÆmol
)1
) and
the acetyl phosphate acid anhydride (DG°
¢
= )44.8
kJÆmol
)1
) exceeds that of ATP (DG°
¢
= 31.8 kJÆmol
)1
)
[19].

+
⁄ NAD
+
,2H
+
]=
)0.32 V), ubiquinone (E°
¢
[dihydroquinone ⁄ quinone,
2H
+
] = +0.10 V) or oxygen (E°
¢
= +0.29 V for
the O
2
⁄ H
2
O
2
couple). The redox potentials of
additional cofactors directing electron transfer from
the ThDP enamine onto final electron acceptors may
be modulated to some degree by the protein environ-
ment but are suspected to lie in between. Redox
potentials of [Fe
4
S
4
] clusters in PFOR and FAD in

14
C]pyruvate, whereas
no label could be detected after addition of
[1-
14
C]pyruvate, clearly suggesting the radical to be
formed after decarboxylation [18]. Second, the hyper-
fine splitting of the radical EPR signal was shown to
be dependent on the chemical nature of the substrate
methyl substituent (i.e. the number of nuclear spins at
C3 of pyruvate) [5]. When the EPR spectra were
recorded at temperatures below 20 K, spin coupling
between the ThDP-derived radical and the reduced
[Fe
4
S
4
]
1+
cluster was observable, indicating that the
two paramagnetic centers are located at a distance of
approximately 1 nm or less [18].
Subsequently, kinetic and spectroscopic studies on
PFORs from different organisms including Desulfovib-
rio africanus and Clostridium thermoaceticum suggested
a common reaction mechanism with an obligate tran-
sient ThDP-based radical, the lifetime of which criti-
cally depends on the presence of CoA [22]. Unlike the
archetypical PFOR from H. halobium, these PFORs
contain three [Fe

= )390 mV). No mag-
netic interaction between this cluster and the ThDP
radical was detectable, and it was concluded that the
reduced cluster is distant from the thiamin binding site
[23]. In the presence of pyruvate and CoA, all three
clusters become reduced. This exciting discovery on
different PFORs pinpointed a crucial role of CoA for
facilitating transfer of the second electron from the
ThDP radical to the iron-sulfur clusters.
Structural studies on PFOR and its ThDP radical
intermediate
In 1999, Fontecilla-Camps et al. solved the X-ray crys-
tallographic structure of the homodimeric PFOR from
D. africanus in the resting state at 2.3 A
˚
resolution
[24]. The ThDP cofactor is deeply buried within the
protein, and its reactive center, the thiazolium nucleus
of ThDP, is located approximately 10 A
˚
from the most
proximal [Fe
4
S
4
] cluster (referred to as cluster A, prox-
imal) (Fig. 2). Clusters A, B (medial) and C (distal) of
each subunit are separated by approximately 10–12 A
˚
,

ThDP with the substrate C2 was reported to be excep-
tionally long (1.86 A
˚
) prompting the authors to
suggest a r ⁄ n-type AcThDP radical in which the
unpaired spin is mostly confined to the acetyl moiety
and, to a lesser degree, to C2 of the cofactor [26].
Fig. 2. Stereo drawing of PFOR structure
(Protein Data Bank code: 1kek) in transpar-
ent surface representation. The ThDP radical
and the three [Fe
4
S
4
] clusters are shown as
sticks. Edge-to-edge distances between all
cofactors are indicated.
Redox reactions of thiamin diphosphate enzymes K. Tittmann
2458 FEBS Journal 276 (2009) 2454–2468 ª 2009 The Author Journal compilation ª 2009 FEBS
After fragmentation of the r ⁄ n-type cation radical and
formation of an acetyl radical, radical recombination
with a CoA thiyl radical was proposed to occur. By
contrast to p-type radicals with extensive delocalization
of the unpaired spin over aromatic systems, the pro-
posed r ⁄ n-type AcThDP radical must be regarded,
especially in view of the tenuously bonded acetyl moi-
ety, as an unstable high-energy intermediate and, thus,
its long lifetime, as demonstrated experimentally both
in the crystalline phase and in solution, is seemingly
counterintuitive.

r ⁄ n-type AcThDP radical proposed on the basis of
pure structural data [26]. Although which protonation
state pertains to the radical intermediate cannot be
clarified unambiguously, the observed
1
H- and
13
C-hyperfine splittings of the C2ß protons and C2
and C2a carbons would best correspond to an interme-
diate state between C2a O-protonated (HEThDP radi-
cal) and O-deprotonated (AcThDP radical) forms. The
close proximity of the cofactor’s exocyclic 4¢-amino
group demonstrated in the X-ray structure favors a
hydrogen-bonding interaction between C2a-O and N4¢
(Fig. 3B).
As noted above, addition of pyruvate to PFOR gen-
erates a stable ThDP radical and one reduced [Fe
4
S
4
]
cluster, which was more recently demonstrated to be
the medial cluster [28]. Rapid depletion of the thiamin
radical and reduction of all clusters is only achieved
after addition of CoA. What is the special role of CoA
for propagation of the second electron and the associ-
ated 10
5
-fold rate enhancement of electron transfer? In
pursuit of this question, Ragsdale proposed several

] cluster in
PFOR from C. thermoaceticum, even in the absence of
pyruvate [22]. However, such behavior has not been
reported for all PFORs and there was no EPR spectro-
scopic evidence for the putative CoA sulfur-based thiyl
radical. An additional intricacy of this mechanism is
the necessity of a structural rearrangement of CoA in
the course of catalysis: initially, the reactive thiol
group of CoA must be positioned proximal to an iron-
sulfur cluster and distant to ThDP but, after oxida-
tion–reduction, it would have to swing closer to ThDP.
Although a simple bond rotation could account for
such conformational transition, diffusion of the CoA
radical out of the active site and abortive side
reactions of the highly reactive thiyl radical could
successfully compete with radical recombination.
Furthermore, direct access to the clusters is sterically
occluded by different loops, so the structural confine-
ments of the active site channel render the proposed
double duty of CoA (cluster reduction and radical
recombination) unlikely, unless binding of CoA would
enforce large structural rearrangements of the protein.
Third, it was proposed that the rate enhancement of
electron transfer by CoA could result from a chemical
and kinetic coupling of oxidation–reduction and acyl
group transfer [7]. This mechanism would generate a
covalent adduct between the AcThDP-type radical and
CoA to form an anion radical, the reducing power of
which can be anticipated to be much higher than of a
charge-neutral AcThDP radical, thus increasing the

flexible, a ‘swinging arm’ that permits active site cou-
pling between E1, E2 and E3 components by rotation
of the lipoyl moiety itself and by additional movement
of the whole protein domain (‘swinging domain’) that
carries the lipoyl-lysine, thus providing a ‘super arm’
that is capable to span the gaps between the active
centers on the different components [2,30].
Oxidation–reduction chemistry of lipoic acid in
models and implications for reductive acetylation
in pyruvate dehydrogenase
Lipoic acid exists in an oxidized disulfide form with a
slightly strained five-membered dithiolane ring (LipS
2
)
and in the two-electron reduced acyclic dithiol form
(dihydrolipoic acid, Lip(SH)
2
). The standard redox
potential of the Lip(SH
2
) ⁄ LipS
2
couple has been
determined by polarographic analysis to be approxi-
mately )0.32 V (pH 7) and is thus more positive than
the two subsequent one-electron oxidation potentials
of thiazolium enamine models, making oxidation of
the enamine by LipS
2
thermodynamically favorable

observed that, in chemical models, reductive acetyla-
tion of lipoic acid by thiazolium enamine occurs
extremely slowly and requires the addition of a
mercury trapping reagent [33]. Subsequently, the
same laboratory used S-methylated lipoic acid
[LipS(SCH
3
)
+
] as a viable chemical model for the
S-protonated form of LipS
2
[34]. MS analysis revealed
the existence of a tetrahedral adduct with an S-C
Redox reactions of thiamin diphosphate enzymes K. Tittmann
2460 FEBS Journal 276 (2009) 2454–2468 ª 2009 The Author Journal compilation ª 2009 FEBS
linkage formed between lipoic acid and the thiazolium
C2a [34]. Very remarkably, LipS(SCH
3
)
+
easily oxi-
dizes thiazolium enamine models with second-order
rate constants that, in view of the effective molarity
of the lipoyl-lysine in the multi-enzyme complex, can
account for the observed turnover number of PDHc.
This intriguing observation suggests that reductive
acetylation in PDHc requires an acid ⁄ base catalyst to
protonate the dithiolane part of lipoamide. Two dif-
ferent mechanisms were envisioned to explain the cat-

formation of the 6-S isomer [35]. By invoking the prin-
ciple of microscopic reversibility, 8-(S)-acetyl-
dihydrolipoylamide is the chemically (and kinetically)
competent isomer for the physiologically relevant for-
ward reaction of E2 (i.e. the formation of acetyl-CoA)
and this must be formed in the preceding reductive
acetylation at E1.
A further compelling question concerns a possible
coupling of oxidation–reduction and acyl group trans-
fer. In principle, the two elementary reactions of reduc-
tive acetylation could occur simultaneously in a
tightly-coupled mechanism or, alternatively, in a step-
wise manner. Both mechanisms would involve the
tetrahedral adduct between reduced lipoamide and
AcThDP; however, AcThDP would be a compulsory
on-pathway intermediate only in the stepwise mecha-
nism. Frey et al. could isolate AcThDP in the steady
state of the overall reaction of PDHc by acid quench
trapping [36]. This finding is consistent with a stepwise
mechanism of oxidation–reduction and acyl group
transfer; however, it cannot disprove a coupled mecha-
nism because AcThDP could be generated from the
tetrahedral thiamin-lipoamide adduct in an equilibrium
side reaction.
Further support for a stepwise mechanism comes
from the observation that E1-bound AcThDP (formed
by enzymic conversion of 3-flouropyruvate) is a chemi-
cally competent acyl group donor to externally added
dihydrolipoamide [37]. In search of putative free radi-
cal intermediates that could be transiently formed in

[41,42]. A probable (and partially modeled) structural
snapshot of catalysis showing E2-bound lipoamide
prior to reaction with the planar HEThDP enamine
intermediate (atomic coordinates of HEThDP enamine
taken from POX) at the active center of E1 from
E. coli is illustrated in Fig. 4. The lipoamide molecule
was modeled into the substrate channel of E1 such
that (a) formation of 8-(S)-acetyl-dihydrolipoylamide
is more likely than of the 6-S isomer and (b)
protonation of the lipoamide dithiolane by His407
K. Tittmann Redox reactions of thiamin diphosphate enzymes
FEBS Journal 276 (2009) 2454–2468 ª 2009 The Author Journal compilation ª 2009 FEBS 2461
may occur (on the basis of structural considerations
and previously available functional data [41]). The
resultant model invokes active center residue His640 to
be important for deprotonation of the a-OH of the
HEThDP enamine.
Reaction mechanism of acetyl
phosphate-producing pyruvate oxidases
Chemical considerations of acetyl phosphate
formation by pyruvate oxidases
In phosphate-dependent pyruvate oxidases, such as
that from L. plantarum (LpPOX), thermodynamically
favorable oxidation of pyruvate is coupled to forma-
tion of the ‘energy-rich’ metabolite acetyl phosphate
that carries an acid anhydride linkage [10]. Owing to
its high group transfer potential, acetyl phosphate
may undergo favorable phosphotransfer to ADP to
give ATP, a process that is catalyzed by the enzyme
acetate kinase [19]. Besides ThDP and a divalent cat-

Besides overcoming the large barrier for expelling
acetyl phosphate from the tetrahedral phospho
adduct, the enzyme must also suppress hydrolytic
cleavage of the presumed AcThDP intermediate to
avoid decoupling of oxidation and acid anhydride
bond formation.
Molecular architecture of phosphate-dependent
pyruvate oxidases and implications for electron
transfer
The X-ray crystal structure of the homotetrameric
LpPOX was solved by Muller and Schulz [44] in the
early 1990s and serves as the structural prototype for
acetyl phosphate-producing POXs. As in all ThDP-
dependent enzymes that have been structurally charac-
terized to date, the active site is located at the interface
of two corresponding subunits constituting the cata-
lytic dimer (Fig. 5). The thiazolium of ThDP and the
redox active isoalloxazine of FAD are bound at
approximately 7 A
˚
edge-to-edge distance, with the
dimethylbenzene part of FAD pointing directly
towards the thiazolium. The flavin isoalloxazine is
markedly bent over the N5–N10 axis ($ 10–15°),
which is a structural feature that increases the driving
force of oxidation–reduction because the distorted con-
formation resembles the reduced state of the flavin,
thus increasing its oxidizing power. The widely-
accepted theoretical framework for biological electron
transfer (Dutton’s ruler) predicts that pure electron

(Phe479 and Phe121) contributed by different mono-
mers as way stations for electron transfer in a com-
bined through-space ⁄ through-bond mechanism [44]. In
support of this proposal, an arginine side chain sitting
atop Phe479 could partially offset the transiently
formed negative charge at the phenyl ring. These dif-
ferent possible modes notwithstanding, theoretical
treatment of oxidation–reduction between the HET-
hDP enamine and FAD might not be as straightfor-
ward as in other systems because electron transfer in
POX is definitely coupled to proton transfer (i.e.
deprotonation of C2a-OH of HEThDP and proton-
ation of FAD at N5 and N1). The tight and rigid
binding of both cofactors excludes a direct carbanion
mechanism with covalent linkage between C2a of the
HEThDP enamine and C4a of FAD, as suggested for
FAD-catalyzed oxidation of other organic substrates
(e.g. amino acids).
Besides the different hydrophobic active center resi-
dues considered above as being involved in electron
transfer, there are a few polar side chains (Glu, Gln)
in close vicinity to the ThDP cofactor, which are likely
to play important roles for catalysis and binding of
phosphate. This initially premature functional assign-
ment has been corroborated by kinetic and structural
analysis of different LpPOX variants (G. Wille and
K. Tittmann, unpublished results).
Kinetic and spectroscopic analysis of
oxidation–reduction in pyruvate oxidase
As considered above, the structural confinements of

pyruvate concentrations. The inability to observe radi-
cal intermediates cannot, however, rule out a two-step
sequential electron transfer mechanism because a
kinetic stabilization of radical intermediates requires
the transfer of the second electron (k
red
2
) to proceed at
a comparable rate or slower than that which occurs
for the first electron (k
red
1
). No transient radicals will
be kinetically stabilized when k
red
2
» k
red
1
. Initial
Fig. 5. Stereo drawing of the active site of
pyruvate oxidase from L. plantarum (Protein
Data Bank code: 1pox) showing the cofac-
tors ThDP and FAD and selected proximal
amino acid residues. The amino acid resi-
dues contributed by the corresponding
subunits are colored individually (green or
pink). The two Phe residues suggested to
be involved in electron transfer are
indicated.

$ 3s
)1
)isa
true electron transfer reaction [46]. In summary, the
kinetic experiments pinpointed a crucial role of phos-
phate for facilitating transfer of the second electron
from the HEThDP radical to the FAD radical,
whereas no such role is evident for transfer of the first
reducing equivalent from the HEThDP enamine to
FAD in the oxidized state.
A coupling mechanism of oxidation–reduction
and acyl transfer?
What could be the catalytic role of phosphate as a
mediator for electron transfer between HEThDP and
FAD? By theory, an enhanced rate of electron transfer
could result from a larger driving force of the reaction
(i.e. a change of the redox potentials of the donor
and ⁄ or acceptor pair), a shorter tunneling pathway
between donor and acceptor, a decreased reorganiza-
tion energy, an increased packing density in the inter-
reactant space, or as a result of a change in chemistry
of oxidation–reduction. At first, a scenario could be
envisioned in which phosphate bridges the pathway
(i.e. it binds in the active center crevice right inbetween
of HEThDP and FAD). Cryo-crystallographic studies
on LpPOX clearly argue against this mechanism. In
the X-ray structure of LpPOX with the HEThDP
enamine resembling the situation prior to electron
transfer, phosphate is not bound in the presumed
through-space tunneling pathway but rather in close

negative charge of which would certainly make it a
low-potential intermediate and thus increase the free
driving force of the reaction [46,47]. In an alternative
mechanism (Fig. 6B), phosphate could add to the
HEThDP cation radical, followed by homolytic frag-
mentation to the ThDP ylide and an O-protonated
acetyl phosphate radical [13,46]. The latter would then
transfer the second electron to FAD yielding O-pro-
tonated acetyl phosphate and two-electron reduced
FAD. The reaction is then completed by deprotona-
tion of acetyl phosphate.
Reaction mechanism of pyruvate
oxidase from Escherichia coli
Pyruvate oxidase from E. coli (EcPOX) catalyzes a
similar intramolecular redox reaction as LpPOX, also
involving two-electron oxidation of the HEThDP
enamine by a neighboring FAD cofactor. Unlike
LpPOX, EcPOX does not produce the energy-rich
product acetyl phosphate, but rather acetate [11].
Analysis of the reductive half-reaction by transient
kinetics revealed that no radical intermediates are
kinetically stabilized under all sets of conditions tested
[46]. This result, however, is not unexpected because
there is no chemical need for a kinetically stabilized
HEThDP radical as in PFOR or LpPOX, where nucle-
ophiles (CoA, phosphate) add to the ThDP-based radi-
cal in a coupling mechanism of acyl transfer and
oxidation–reduction.
Redox reactions of thiamin diphosphate enzymes K. Tittmann
2464 FEBS Journal 276 (2009) 2454–2468 ª 2009 The Author Journal compilation ª 2009 FEBS

Fig. 7. Superposition of the active sites of
EcPOX in the resting state (green; Protein
Data Bank code: 3ey9) and after proteolytic
activation (yellow; Protein Data Bank code:
3eya) in stereo view. The position of residue
Phe465 suspected to be involved in oxida-
tion–reduction is highlighted.
K. Tittmann Redox reactions of thiamin diphosphate enzymes
FEBS Journal 276 (2009) 2454–2468 ª 2009 The Author Journal compilation ª 2009 FEBS 2465
Conclusions
ThDP-dependent enzymes catalyze an amazing variety
of different chemical reactions, among which the
oxidative decarboxylation of 2-keto acids is a funda-
mental process of intermediary metabolism in all
organisms. ThDP enzyme-catalyzed oxidation of keto
acids is coupled to the formation of energy-rich meta-
bolites such as acyl-CoA conjugates or acyl phosphate
and the concomitant generation of reducing equiva-
lents. The underlying chemistry is demanding, as
demonstrated by the poor yields and rates observed in
models. ThDP enzymes have evolved a sui generis
versatility in combining the elegant radical chemistry
of ThDP in tandem with iron-sulfur clusters, flavins,
lipoic acid and CoA; delicately balanced acid ⁄ base
catalysis; and perfectly organized redox chains to
successfully meet this challenge.
Acknowledgements
Insightful discussions with Perry Frey, Sandro Ghisla,
Wolfgang Buckel and Steve Ragsdale are gratefully
acknowledged. This review is dedicated to Professor

synthetic bacterium. Proc Natl Acad Sci USA 55, 928–
934.
9 Hager LP & Lipmann F (1961) Coupling between phos-
phorylation and flavin adenine dinucleotide reduction
with the pyruvate oxidase of L. delbrueckii enzyme.
Proc Natl Acad Sci USA 47, 1768–1772.
10 Sedewitz B, Schleifer KH & Go
¨
tz F (1984) Purification
and biochemical-characterization of pyruvate oxidase
from Lactobacillus plantarum. J Bacteriol 160, 273–278.
11 Williams FR & Hager LP (1961) A crystalline flavin
pyruvate oxidase. J Biol Chem 236, PC36–PC37.
12 Koland JG, Miller MJ & Gennis RB (1984) Reconstitu-
tion of the membrane-bound, ubiquinone-dependent
pyruvate oxidase respiratory chain of Escherichia coli
with the cytochrome d terminal oxidase. Biochemistry
23, 445–453.
13 Kluger R & Tittmann K (2008) Thiamin diphosphate
catalysis: enzymic and nonenzymic covalent intermedi-
ates. Chem Rev 108, 1797–1833.
14 Breslow R (1957) The mechanism of thiamine action.
II. Rapid deuterium exchange in thiazolium salts. JAm
Chem Soc 79, 1762–1763.
15 Howie JK, Houts JJ & Sawyer DT (1977) Oxidation-
reduction chemistry of DL-alpha-lipoic acid, propane-
dithiol, and trimethylene disulfide in aprotic and in
aqueous-media. J Am Chem Soc 99, 6323–6326.
16 Gruys KJ, Halkides CJ & Frey PA (1987) Synthesis and
properties of 2-acetylthiamin pyrophosphate – an enzy-

phate radical. Biochemistry 36, 8484–8494.
23 Pieulle L, Guigliarelli B, Asso M, Dole F, Bernadac A
& Hatchikian EC (1995) Isolation and characterization
of the pyruvate-ferredoxin oxidoreductase from the sul-
fate-reducing bacterium Desulfovibrio africanus. Biochim
Et Biophys Acta-Protein Struct Mol Enzymol 1250, 49–
59.
24 Chabriere E, Charon MH, Volbeda A, Pieulle L,
Hatchikian EC & Fontecilla-Camps JC (1999) Crystal
structures of the key anaerobic enzyme pyruvate:ferre-
doxin oxidoreductase, free and in complex with pyru-
vate. Nat Struct Biol 6, 182–190.
25 Page CC, Moser CC, Chen XX & Dutton PL (1999)
Natural engineering principles of electron tunnelling in
biological oxidation–reduction. Nature 402, 47–52.
26 Chabriere E, Vernede X, Guigliarelli B, Charon MH,
Hatchikian EC & Fontecilla-Camps JC (2001) Crystal
structure of the free radical intermediate of pyru-
vate:ferredoxin oxidoreductase. Science 294, 2559–2563.
27 Mansoorabadi SO, Seravalli J, Furdui C, Krymov V,
Gerfen GJ, Begley TP, Melnick J, Ragsdale SW &
Reed GH (2006) EPR spectroscopic and computational
characterization of the hydroxyethylidene-thiamine
pyrophosphate radical intermediate of pyruvate:
ferredoxin oxidoreductase. Biochemistry 45, 7122–7131.
28 Astashkin AV, Seravalli J, Mansoorabadi SO, Reed
GH & Ragsdale SW (2006) Pulsed electron para-
magnetic resonance experiments identify the
paramagnetic intermediates in the pyruvate ferredoxin
oxidoreductase catalytic cycle. J Am Chem Soc 128,

sis of acetyl-Tpp and isolation of acetyl-Tpp as a
transient species in pyruvate-dehydrogenase catalyzed-
reactions. Biochemistry 28, 9071–9080.
37 Flournoy DS & Frey PA (1986) Pyruvate-dehydrogenase
and 3-fluoropyruvate – chemical competence of 2-acetyl-
thiamin pyrophosphate as an acetyl group donor to
dihydrolipoamide. Biochemistry 25, 6036–6043.
38 Frank RAW, Kay CWM, Hirsi J & Luisi BF (2008)
Off-pathway, oxygen-dependent thiamine radical in the
Krebs cycle. J Am Chem Soc 130, 1662–1668.
39 Aevarsson A, Seger K, Turley S, Sokatch JR & Hol
WG (1999) Crystal structure of 2-oxoisovalerate and
dehydrogenase and the architecture of 2-oxo acid dehy-
drogenase multienzyme complexes. Nat Struct Biol 6,
785–792.
40 Arjunan P, Nemeria N, Brunskill A, Chandrasekhar K,
Sax M, Yan Y, Jordan F, Guest JR & Furey W (2002)
Structure of the pyruvate dehydrogenase multienzyme
complex E1 component from Escherichia coli at
1.85 angstrom resolution. Biochemistry 41, 5213–5221.
41 Nemeria N, Arjunan P, Brunskill A, Sheibani F, Wei
W, Yan Y, Zhang S, Jordan F & Furey W (2002) Histi-
dine 407, a phantom residue in the E1 subunit of the
Escherichia coli pyruvate dehydrogenase complex, acti-
vates reductive acetylation of lipoamide on the E2 sub-
unit. An explanation for conservation of active sites
between the E1 subunit and transketolase. Biochemistry
41, 15459–15467.
42 Arjunan P, Sax M, Brunskill A, Chandrasekhar K,
Nemeria N, Zhang S, Jordan F & Furey W (2006) A

47 Wille G, Meyer D, Steinmetz A, Hinze E, Golbik R &
Tittmann K (2006) The catalytic cycle of a thiamin
diphosphate enzyme examined by cryocrystallography.
Nat Chem Biol 2, 324–328.
48 Cunningham CC & Hager LP (1971) Crystalline pyru-
vate oxidase from Escherichia coli. II. Activation by
phospholipids. J Biol Chem 246, 1575–1582.
49 Bertagnolli BL & Hager LP (1991) Activation of
Escherichia coli pyruvate oxidase enhances the oxidation
of hydroxyethylthiamin pyrophosphate. J Biol Chem
266, 10168–10173.
50 Neumann P, Weidner A, Pech A, Stubbs MT & Titt-
mann K (2008) Structural basis for membrane binding
and catalytic activation of the peripheral membrane
enzyme pyruvate oxidase from Escherichia coli. Proc
Natl Acad Sci USA 105 , 17390–17395.
51 Pang SS, Duggleby RG & Guddat LW (2002) Crystal
structure of yeast acetohydroxyacid synthase: a target
for herbicidal inhibitors. J Mol Biol 317, 249–262.
52 Kaplun A, Binshtein E, Vyazmensky M, Steinmetz A,
Barak Z, Chipman DM, Tittmann K & Shaanan B
(2008) Glyoxylate carboligase lacks the canonical active
site glutamate of thiamine-dependent enzymes. Nat
Chem Biol 4, 113–118.
Redox reactions of thiamin diphosphate enzymes K. Tittmann
2468 FEBS Journal 276 (2009) 2454–2468 ª 2009 The Author Journal compilation ª 2009 FEBS