of anabolic pyruvate synthesis by pyruvate: ferredoxinoxidoreductase from Hydrogenobacter thermophilus Takeshi Ikeda1,*, Masahiro Yamamoto1,, Hiroyuki Arai1, Daijiro Ohmori2, Masaharu Is
Trang 1of anabolic pyruvate synthesis by pyruvate: ferredoxin
oxidoreductase from Hydrogenobacter thermophilus
Takeshi Ikeda1,*, Masahiro Yamamoto1,, Hiroyuki Arai1, Daijiro Ohmori2, Masaharu Ishii1and Yasuo Igarashi1
1 Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan
2 Department of Chemistry, School of Medicine, Juntendo University, Chiba, Japan
Introduction
Pyruvate: ferredoxin oxidoreductase (POR; pyruvate
synthase, EC 1.2.7.1) catalyzes the thiamine
pyrophos-phate (TPP)-dependent oxidative decarboxylation of
pyruvate to form acetyl-CoA and CO2 POR contains
one or multiple iron-sulfur clusters in addition to TPP
[1]; the two electrons that arise during oxidation of pyruvate at the TPP site are sequentially transferred via the iron-sulfur cluster(s) to external electron accep-tors The physiological electron acceptor is a small iron-sulfur protein ferredoxin or FMN-containing
Keywords
Hydrogenobacter thermophilus; iron-sulfur
cluster; pyruvate: ferredoxin oxidoreductase;
reductive tricarboxylic acid cycle; thiamine
pyrophosphate
Correspondence
M Ishii, Department of Biotechnology,
Graduate School of Agricultural and Life
Sciences, The University of Tokyo, 1-1-1
Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Fax: +81 3 5841 5272
Tel: +81 3 5841 5143
E-mail: amishii@mail.ecc.u-tokyo.ac.jp
Present address
*Research Institute for Nanodevice and Bio
Systems, Hiroshima University, Japan
Institute of Biogeoscience, Japan Agency
for Marine-Earth Science and Technology
(JAMSTEC), Kanagawa, Japan
(Received 11 September 2009, revised 17
November 2009, accepted 19 November
2009)
doi:10.1111/j.1742-4658.2009.07506.x
Pyruvate: ferredoxin oxidoreductase (POR; EC 1.2.7.1) catalyzes the thia-mine pyrophosphate-dependent oxidative decarboxylation of pyruvate to form acetyl-CoA and CO2 The thermophilic, obligate chemolithoauto-trophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, assimilates CO2 via the reductive tricarboxylic acid cycle In this cycle, POR acts as pyruvate synthase catalyzing the reverse reaction (i.e reduc-tive carboxylation of acetyl-CoA) to form pyruvate The pyruvate synthesis reaction catalyzed by POR is an energetically unfavorable reaction and requires a strong reductant Moreover, the reducing equivalents must be supplied via its physiological electron mediator, a small iron-sulfur protein ferredoxin Therefore, the reaction is difficult to demonstrate in vitro and the reaction mechanism has been poorly understood In the present study,
we coupled the decarboxylation of 2-oxoglutarate catalyzed by 2-oxogluta-rate: ferredoxin oxidoreductase (EC 1.2.7.3), which generates sufficiently low-potential electrons to reduce ferredoxin, to drive the energy-demanding pyruvate synthesis by POR We demonstrate that H thermophilus POR catalyzes pyruvate synthesis from acetyl-CoA and CO2, confirming the operation of the reductive tricarboxylic acid cycle in this bacterium We also measured the electron paramagnetic resonance spectra of the POR intermediates in both the forward and reverse reactions, and demonstrate the intermediacy of a 2-(1-hydroxyethyl)- or 2-(1-hydroxyethylidene)-thia-mine pyrophosphate radical in both reactions The reaction mechanism of the reductive carboxylation of acetyl-CoA is also discussed
Abbreviations
DTNB, 5,5¢-dithiobis-(2-nitrobenzoic acid); EPR, electron paramagnetic resonance; HE-TPP, 2-(1-hydroxyethyl)- or 2-(1-hydroxyethylidene)-thiamine pyrophosphate; LDH, lactate dehydrogenase; OGOR, 2-oxoglutarate: ferredoxin oxidoreductase; OR, 2-oxoacid oxidoreductase; POR, pyruvate: ferredoxin oxidoreductase; TCA, tricarboxylic acid; TPP, thiamine pyrophosphate.
Trang 2flavodoxin By contrast to pyruvate dehydrogenase
multienzyme complex, which irreversibly catalyzes the
same reaction utilizing NAD+as an electron acceptor
in mitochondria and respiratory bacteria, POR can
also catalyze the reverse reaction (i.e the reductive
car-boxylation of acetyl-CoA) provided that a sufficiently
low-potential electron donor is available The reverse
reaction (pyruvate synthesis) is a central step for some
autotrophic bacteria because it serves to assimilate
CO2into cell carbon [1]
Hydrogenobacter thermophilus TK-6 is a
faculta-tive aerobic, thermophilic, obligate
chemolithoauto-trophic hydrogen-oxidizing bacterium [2] The optimum
growth temperature range for H thermophilus TK-6 is
70–75C Phylogenetic analyses of 16S ribosomal
RNA sequences have shown that the genus
Hydroge-nobacteris a member of the deepest branching order in
the domain Bacteria [3] H thermophilus assimilates
CO2 via the reductive tricarboxylic acid (TCA) cycle
[4], which is one of the microbial CO2 fixation
path-ways [5] The reductive TCA cycle is a reversal of the
oxidative TCA cycle, and is an endergonic anabolic
pathway that requires reducing equivalents to complete
the cycle [6,7] POR is one of the key enzymes of the
reductive TCA cycle, and catalyzes the anabolic
reduc-tive carboxylation of acetyl-CoA Two [4Fe-4S]
ferre-doxins, Fd1 and Fd2, from this bacterium are
considered to serve as low-potential electron donors
for this key reaction [8]
POR is distributed among archaea, bacteria and
anaerobic protozoa, and is a member of the 2-oxoacid
oxidoreductase (OR) family, which catalyzes the
oxida-tive decarboxylation of 2-oxoacids to their acyl- or
aryl-CoA derivatives [9] OR enzymes can be
homodi-meric [10,11], heterodihomodi-meric [12,13] or heterotetrahomodi-meric
[14], depending on the organism These three types of
OR are phylogenetically related and the
heterotetra-meric enzyme has been proposed to be the common
ancestor that underwent gene rearrangement and
fusion to generate homo- and heterodimeric ORs
[9,13,15] We recently found novel heteropentameric
ORs [POR and 2-oxoglutarate: ferredoxin
oxidoreduc-tase (OGOR; 2-oxoglutarate synthase, EC 1.2.7.3)] in
H thermophilus and its close relatives [16,17]; in these
organisms, the heteropentameric POR and OGOR
function as the key components of the reductive TCA
cycle and catalyze the anabolic reductive carboxylation
of acetyl-CoA and succinyl-CoA, respectively [17,18]
Four of the five subunits correspond to those of the
heterotetrameric ORs, suggesting that the
heteropenta-meric ORs might have evolved from an ancestral
het-erotetrameric enzyme by the acquisition of a unique
fifth polypeptide of unknown function Sequence
align-ments suggest that H thermophilus POR contains one TPP and three [4Fe-4S]2+⁄ 1+clusters per catalytic unit [17]; the enzyme contains all of the motifs required for cofactor binding that were identified in the crystal structure of the homodimeric Desulfovibrio africanus POR [19]
In most cases, the enzyme activity of POR is assayed
by monitoring the reduction of an artificial electron carrier, methyl viologen, during the oxidative decar-boxylation of pyruvate However, when the anabolic role of the novel heteropentameric POR in H thermo-philus is considered, the reverse reaction [i.e the reduc-tive carboxylation of acetyl-CoA (pyruvate synthesis)] needs to be assayed However, this reverse reaction has proven more difficult to study because a strong reduc-tant is required to drive the reaction Hence, the cata-lytic mechanism of the reductive carboxylation catalyzed by POR is poorly understood, whereas that
of the oxidative decarboxylation has been intensively investigated [20,21] In the present study, we developed
an assay system to demonstrate the reductive carboxyl-ation catalyzed by H thermophilus POR with reduced ferredoxin as an electron donor Specifically, we uti-lized another OR-family enzyme, OGOR from H ther-mophilus, to supply reduced ferredoxin for anabolic pyruvate synthesis mediated by POR OGOR also cat-alyzes the oxidative decarboxylation of 2-oxoglutarate using (oxidized) ferredoxin as an electron acceptor By coupling the OGOR decarboxylation, we demonstrate that H thermophilus POR catalyzes the anabolic reductive carboxylation of acetyl-CoA to form pyru-vate We also investigated the inter- and intramolecu-lar electron transfer during the reductive carboxylation
by electron paramagnetic resonance (EPR) spectros-copy to clarify its catalytic mechanism
Results
In vitro assay for pyruvate synthesis by POR: the coupled assay with OGOR and lactate
dehydrogenase (LDH; EC 1.1.1.27) Because the synthesis of pyruvate from acetyl-CoA and CO2 is an energetically unfavorable reaction with
a reduction potential of )540 mV [22], this reaction requires a strong reductant Pyruvate dehydrogenase multienzyme complex cannot catalyze the react-ion because the requisite electron donor, NADH (E0¢ =)320 mV), is much too weak an electron source to drive the transformation For the enzyme assay, the reducing power must be supplied in vitro by the physiological electron donor for POR, ferredoxin
A possible strategy is to couple the POR reaction to a
Trang 3ferredoxin-reducing enzyme For example, POR from
Moorella thermoacetica biosynthesizes pyruvate using
ferredoxin reduced by CO dehydrogenase [23];
Chloro-bium tepidum POR has been shown to catalyze
pyru-vate synthesis mediated by ferredoxin reduced by the
light-driven reactions of spinach chloroplasts or
Chloro-biumreaction centers [24,25] However, H thermophilus
does not possess these ferredoxin-reducing systems
In this bacterium, reducing equivalents are derived
from hydrogen oxidization catalyzed by multiple
hydrogenases [26], although the physiological electron
transfer pathway(s) from hydrogen to ferredoxin has
not yet been clarified Instead, we utilized OGOR from
this bacterium to reduce the low-potential ferredoxin
(Fig 1) The oxidative decarboxylation of
2-oxogluta-rate catalyzed by OGOR gene2-oxogluta-rates low-potential
electrons that reduce ferredoxins as follows:
2-oxoglut-arate + CoA fi succinyl-CoA + CO2+ H++ 2e),
E0¢ = )520 mV [27] The reactions catalyzed by POR
and OGOR were further coupled to the LDH reaction to
detect pyruvate formation spectrophotometrically
(Fig 1) Pyruvate generated by coupling the POR and
OGOR reactions was reductively converted to lactate
with NADH as an electron donor Thus, the rate of
pyru-vate formation was monitored as the decrease in A340as a
result of NADH oxidation The thermostable LDH from
a thermophilic bacterium Thermus caldophilus [28] was
used for the assay because the reaction was performed at
70C, which is the optimum temperature for H
thermo-philus Table 1 shows the overall reaction of this coupled
assay
Using the coupled assay with OGOR and LDH,
H thermophilusPOR was found to catalyze the
reduc-tive carboxylation of CoA Indeed,
acetyl-CoA-dependent NADH oxidation was observed with
either reduced Fd1 or Fd2 as an electron donor
(Fig 2) It was confirmed that pyruvate synthesis by
POR was rate-limiting in this coupled system A slight
decrease in A340 in the absence of acetyl-CoA was a
result of the spontaneous thermal degradation of
NADH [29] The reductive carboxylation depended on
the presence of POR, OGOR, LDH, ferredoxin,
2-oxo-glutarate and acetyl-CoA (data not shown), indicating that the coupled assay shown in Fig 1 proceeded as expected However, this reaction did not depend on the presence of NaHCO3 (CO2) and CoA (Fig 2B) Because CO2 was produced from 2-oxoglutarate by OGOR in this coupled assay, the addition of NaHCO3 was not necessarily required for the total reaction (Fig 1, dashed arrow) By contrast to CO2, CoA was
an essential substrate to initiate this coupled reaction; nevertheless, the reaction actually proceeded without the addition of CoA with a higher reaction rate than
Fig 1 Schematic representation of the coupled enzyme assay.
The reductive carboxylation catalyzed by POR was coupled with
the OGOR and LDH reactions Fdox, oxidized ferredoxin; Fdred,
reduced ferredoxin.
Table 1 Enzymatic reactions.
POR (reductive carboxylation)
Acetyl-CoA + CO2+ 2 · Fd red fi pyruvate + CoA + 2 · Fd ox
OGOR (oxidative decarboxylation)
2-Oxoglutarate + CoA + 2 · Fd ox
fi succinyl-CoA + CO 2 + 2 · Fd red
fi lactate + succinyl-CoA + NAD + a
Protons are omitted from the reactions for simplicity Fd ox , oxidized ferredoxin; Fdred, reduced ferredoxin.
A
B
Fig 2 Reductive carboxylation catalyzed by POR in the coupled enzyme assay The assay mixture contained 1 m M acetyl-CoA,
10 m M NaHCO3, 10 m M 2-oxoglutarate, 0.5 m M CoA, 0.2 m M NADH, 1 m M fructose 1,6-bisphosphate, 10 m M MgCl 2 , 1 m M di-thiothreitol, 0.5 m M TPP, 0.03 U of OGOR, 0.2 U of LDH and
10 l M Fd1 (A) or Fd2 (B) in 100 m M Hepes buffer (pH 8.0) Open circles, complete reaction; filled circles, acetyl-CoA omitted; open squares, NaHCO3omitted; filled squares, CoA omitted.
Trang 4that of the complete reaction (Fig 2B, filled square).
This was the result of a trace amount of CoA in the
assay mixture CoA quantification by the
5,5¢-dithiobis-(2-nitrobenzoic acid) (DTNB) assay showed
that 100 mm acetyl-CoA stock solution contained
2.4 mm CoA, corresponding to 24 lm CoA in the
stan-dard assay mixture (i.e without the addition of CoA
solution) The Km value for CoA of the OGOR
enzyme is reported to be 80 lm [30] Thus,
decarboxyl-ation of 2-oxoglutarate can proceed as a result of CoA
contamination of the acetyl-CoA solution When the
reaction commences, CoA is regenerated by the
reduc-tive carboxylation of acetyl-CoA (Fig 1, dashed
arrow) The reaction rate of this coupled assay was
significantly affected by the concentration of CoA
(Fig 2B) Although CoA was an essential substrate of
this assay, pyruvate synthesis by POR was inhibited in
the presence of excess CoA, which caused the reverse
reaction (oxidative decarboxylation of pyruvate) This
impasse prevented any further kinetic analysis of the
reaction
In this assay, the reductive carboxylation activity of
POR was determined to be 0.23 UÆmg)1 with 10 lm
Fd1, or 0.19 UÆmg)1 with 10 lm Fd2 These values
were comparable to those of the oxidative
decarboxyl-ation of pyruvate with Fd1 or Fd2 as an electron
acceptor (0.55 UÆmg)1 or 0.43 UÆmg)1, respectively;
data not shown), suggesting that H thermophilus POR
functions as an active pyruvate synthase
EPR measurements of POR during the oxidative
decarboxylation
The purified H thermophilus POR showed an EPR
sig-nal (g1,2,3= 1.973, 2.012 and 2.024) attributed to the
oxidized S = 1⁄ 2 [3Fe-4S]1+cluster [31] (Fig 3A) In
the dithionite-reduced state, the [3Fe-4S] signal
disap-peared and a new signal attributed to the reduced
S= 1⁄ 2 [4Fe-4S]1+ clusters was observed (Fig 3B)
This new signal is an overlap of a major signal with
g-values of 1.910, 1.922 and 2.040 and a minor signal
(approximately 4% of the major signal; determined by
spectral simulation) with g-values of 1.880, 2.003 and
2.020 (note that the minor feature around g = 2.00 in
Fig 3B was a result of this minor signal and not a
TPP radical intermediate described later) The relative
amount of the [3Fe-4S]1+ cluster in Fig 3A was
< 10% that of the [4Fe-4S]1+ clusters in Fig 3B
(determined by comparing the double integrals of the
EPR spectra), indicating that a substoichiometric
amount of the [3Fe-4S] cluster was derived from the
oxygen-sensitive [4Fe-4S] by partial oxidative damage,
as reported for other ORs [14,32–34] In the presence
of 20 mm pyruvate, POR showed a strong sharp signal centered at g = 2.0040 (Fig 3C), whereas 0.5 mm CoA did not affect the signal of POR (data not shown), indicating that the oxidative decarboxylation reaction begins with the binding of pyruvate, but not
A
B
C
D
E
F
Fig 3 EPR spectra of H thermophilus POR The purified POR was incubated with the components: (A) no substrate (as purified); (B) dithionite, (C) pyruvate; (D) pyruvate and CoA; (E) Fd1, OGOR, 2-oxoglutarate and CoA; (F) acetyl-CoA, Fd1, OGOR, 2-oxoglutarate and CoA Instrument settings were: temperature, 10 K; microwave power, 100 lW for (A), 250 lW for (B, D–F) or 1 lW for (C); micro-wave frequency, 9.024 GHz; modulation frequency, 100 kHz; mod-ulation amplitude, 0.2 mT The arrow indicates the signal of the TPP radical intermediate generated during the reductive carboxyla-tion of acetyl-CoA.
Trang 5CoA These results are consistent with a ping-pong
cat-alytic mechanism with pyruvate as the primary
sub-strate [35] (Note that the microwave power for
Fig 3C was 1 lW, 250-fold lower than that for the
others; the intensity of EPR signals is proportional to
the square root of the microwave power under
nonsat-urating conditions.) Although the [3Fe-4S]1+ signal in
Fig 3A disappeared at temperatures exceeding 30K,
the g = 2.0040 signal remained even at 70K (data
not shown), indicating that this signal was the result of
a TPP-radical intermediate Indeed, this radical is
pro-posed to be the common intermediate in pyruvate
decarboxylation catalyzed by all PORs [36], which is
generated by the binding of pyruvate to TPP and the
resultant decarboxylation Although the chemical
structure of this intermediate is still controversial [37–
39], it is often referred to as a 2-(1-hydroxyethyl)- or
2-(1-hydroxyethylidene)-TPP (HE-TPP) radical The
hyperfine structure of the radical was determined in
detail at 70K, at which the EPR signals of iron-sulfur
clusters are not detectable (Fig 4A) The hyperfine
splitting pattern is essentially the same as reported in
other studies [11,36–38,40,41] The HE-TPP radical is
generated by one-electron transfer; one of the two
elec-trons that are generated during decarboxylation of
pyruvate remains on the TPP intermediate, and the
other electron moves to the intramolecular iron-sulfur
cluster [42] Consistent with this mechanism, the
oxi-dized [3Fe-4S]1+ signal in Fig 3A disappeared in Fig 3C, indicating that the cluster was reduced by this electron However, no reduced [4Fe-4S] signal was detectable in Fig 3C This is probably because a reduced [4Fe-4S] cluster(s) can be reoxidized by a trace amount of oxygen [42] Because the redox potential
of [3Fe-4S] clusters is generally much higher than that of [4Fe-4S] clusters ()150 )100 mV versus )650 )250 mV) [43], the [3Fe-4S] cluster in the enzyme was not reoxidized Upon further addition of CoA, the signal of the HE-TPP radical markedly decreased, accompanied by concomitant formation of
a reduced [4Fe-4S] signal, which was similar to that of the dithionite-reduced POR (Fig 3D), indicating the second electron transfer from the radical to the [4Fe-4S] cluster(s) The presence of both pyruvate and CoA allows catalysis to proceed until all the oxygen is con-sumed [40], preventing reoxidation of the reduced [4Fe-4S] cluster(s) Because iron-sulfur clusters can receive only one electron at a time, multiple clusters should be reduced in this state These electrons are then readily released to external electron mediators Indeed, in the presence of Fd1, the rhombic
S= 1⁄ 2 [4Fe-4S]1+ signal of the reduced Fd1 [8] was clearly observed (data not shown)
EPR measurements of POR during the reductive carboxylation
Addition of 1 mm acetyl-CoA did not affect the EPR signal of POR (data not shown), indicating that elec-tron transfer from external elecelec-tron donors is a key step to initiate the carboxylation reaction To supply reducing equivalents to POR via ferredoxin, we utilized OGOR as described above In the presence of 2-oxoglutarate, CoA, OGOR and Fd1, the reduced [4Fe-4S] signal of POR was observed, overlapping with the rhombic [4Fe-4S]1+ signal of Fd1 (gz,y,x= 2.08, 1.94 and 1.92) [8] (Fig 3E) Upon further addition of acetyl-CoA, a signal with g = 2.0040 was observed (Fig 3F, arrow) (It was confirmed that this signal was not a result of the OGOR intermediate.) The hyperfine structure of this signal shows essentially the same hyperfine splitting pattern as that of the HE-TPP radi-cal intermediate observed during the decarboxylation
of pyruvate (Fig 4), indicating that the HE-TPP radi-cal was a common intermediate in both the oxidative and reductive reactions The HE-TPP radical was formed only after the reduction of the iron-sulfur clus-ters of the enzyme, suggesting that the electrons sup-plied via external ferredoxin molecules played an important role in forming the radical intermediate from TPP with bound acetyl-CoA
A
B
Fig 4 Hyperfine structures of the EPR signal of the TPP radical
intermediates (A) incubated with pyruvate and CoA (three scans);
(B) incubated with acetyl-CoA, Fd1, OGOR, 2-oxoglutarate and CoA
(five scans) Instrument settings were: temperature, 70 K;
micro-wave power, 1 lW for (A) or 100 lW for (B); modulation amplitude,
0.02 mT for (A) or 0.2 mT for (B); other settings were as described
in Fig 3 The higher power and wider modulation were used for (B)
to increase sensitivity because the amount of the radical
intermedi-ate was much less than for (A).
Trang 6In the present study, we demonstrate that H
thermo-philus POR catalyzes pyruvate synthesis from
acetyl-CoA and CO2, by the coupled assay with OGOR and
LDH (Fig 1) Although carboxylation activity is
gen-erally determined by monitoring the incorporation of
14CO2 to form [14C] pyruvate, OGOR catalyzes the
exchange reaction between CO2 and the carboxyl
group of 2-oxoglutarate [44], and therefore interferes
with the detection of [14C] pyruvate Instead, the rate
of pyruvate formation was determined by monitoring
the LDH-coupled oxidation of NADH to NAD+ The
coupled assay also demonstrated that Fd1 and Fd2
function as electron mediators for POR (and also for
OGOR) [45] in both the oxidative and reductive
reac-tions These results corroborate the operation of the
reductive TCA cycle in H thermophilus Specifically,
two irreversible reactions in the oxidative TCA cycle,
oxidative decarboxylation of pyruvate and
2-oxogluta-rate, are anabolically reversed by POR and OGOR,
respectively, as suggested by our early work [4], with
Fd1 and Fd2 acting as physiological electron donors
However, because this assay is a complex system
involving four proteins, kinetic analysis was not
possi-ble The substrates, CoA and CO2, were involved in
the two reactions catalyzed by POR and OGOR
(Fig 1, dashed arrows) In particular, CoA was a
sub-strate of OGOR as well as a product of POR, and
sig-nificantly affected the reaction rate of pyruvate
synthesis These problems were derived from the fact
that POR and OGOR are similar OR-family enzymes,
both reversibly catalyzing the CoA-dependent
oxidative decarboxylation of 2-oxoacids For further
analysis, an alternative enzyme that can generate
low-potential electrons to reduce ferredoxin is required
Thus far, two other enzymes that utilize ferredoxin as
an electron mediator have been purified from H
ther-mophilus: ferredoxin-NADP+ reductase (EC 1.18.1.2)
[46] and ferredoxin-dependent glutamate synthase
(EC 1.4.7.1) [47] However, the midpoint potentials of
the half reactions catalyzed by these enzymes are
higher than that mediated by OGOR, and are
there-fore unsuitable for the reduction of ferredoxin Thus,
the identification and characterization of enzymes that
transfer electrons to ferredoxins in vivo is of particular
importance for the improvement of this coupled assay
and also with respect to obtaining a deeper
under-standing of the metabolism of H thermophilus Indeed,
this would enable the kinetic analysis of the POR
reac-tions in both direcreac-tions In particular, the reaction rate
under physiological intracellular concentrations of
sub-strates needs to be determined to demonstrate that
H thermophilus POR functions toward pyruvate syn-thesis in vivo
To investigate the reaction mechanism of H thermo-philus POR, we measured the EPR spectra of the enzyme in the presence of various combinations of sub-strates Intra- and intermolecular electron transfer dur-ing the oxidative decarboxylation was essentially consistent with the catalytic cycle proposed by Menon and Ragsdale [36] We further measured the EPR spec-tra during the reductive carboxylation of acetyl-CoA, using OGOR to reduce ferredoxin as in the coupled assay In the presence of the reduced Fd1 and acetyl-CoA, the HE-TPP radical intermediate was formed (Figs 3F and 4B), indicating the intermediacy of the HE-TPP radical in both the oxidative and reductive reactions The results obtained also indicate that elec-tron transfer from external ferredoxin to the enzyme is
an indispensable step to form the radical in the reductive reaction From the data obtained in the present study, along with evidence available from the literature, we are able to propose the catalytic mechanism of the reductive carboxylation of acetyl-CoA (Fig 5) (1) The TPP carb-anion is generated by proton extraction from C2 carbon atom of the thiazolium ring by the tautomeric 4¢ imino group of the 4¢-aminopyrimidine ring [48], as is the case for all TPP-dependent enzymes; this process is also com-mon to the oxidative decarboxylation catalyzed by this enzyme (2) The nucleophilic TPP C2-carbanion attacks the carbonyl carbon of acetyl-CoA (as it attacks the car-bonyl carbon of pyruvate in the oxidative decarboxyl-ation) to form a transient tetrahedral intermediate (3) The tetrahedral intermediate undergoes CoA release and one-electron transfer to the adduct of TPP to form the HE-TPP radical intermediate It is not known whether CoA release and one-electron reduction occur
in a stepwise manner [possibly forming the acetyl-TPP
as an intermediate (3¢-a)] or simultaneously However,
we believe the latter process is more likely because dur-ing the oxidative decarboxylation the binddur-ing of CoA appears to be tightly coupled to electron transfer from the HE-TPP radical [49] (4) The generated HE-TPP radical is reduced to the HE-TPP C2a carbanion by a second electron transfer and then (5 and 6) the resultant carbanion attacks CO2, which might be tightly bound to the active site of the enzyme [37], to form pyruvate These latter steps (4, 5 and 6) correspond to the exchange reaction between CO2and the carboxyl group
of pyruvate catalyzed by this enzyme [50]
Further studies are being planed to confirm the above catalytic mechanism Moreover, the investigation
of the reductive reaction using the coupled system developed in this study is not only highly important itself, but also would provide further insights into the
Trang 7reverse, oxidative reaction and vice versa Thus, further
studies on the POR reactions in both directions would
lead to a deeper understanding of the overall reaction
mechanism of this enzyme
Materials and methods
Bacterial strains and growth conditions
Escherichia coli JM109 and BL21(DE3) were used as hosts
for derivatives of pUC19 and pET21c, respectively E coli
MV1184 was used as a host for the expression of T
caldo-philusLDH E coli strains were grown in tryptic soy broth
or LB medium at 37C When necessary, ampicillin
(100 lgÆmL)1) was added to the medium for plasmid
selection
Heterologous expression and purification of POR,
OGOR and ferredoxins
Because H thermophilus POR (UniProt accession numbers
Q9LBF7–Q9LBG1) is oxygen-sensitive, as is the case for
other ORs [51], the recombinant POR was expressed under
microaerobic conditions and purified under anaerobic
con-ditions as described previously [17] In preparation for EPR
spectroscopy, dithionite was removed from the purification buffers H thermophilus has two isozymes of OGOR, het-erodimeric Kor (UniProt accession numbers Q9AJL9 and Q9AJM0) and heteropentameric For (UniProt accession numbers Q93RA0–Q93RA4) [16,30] Because the former is much more active than the latter, we utilized the recombi-nant Kor in the present study Kor, Fd1 (UniProt accession number Q75VV9) and Fd2 (UniProt accession number Q4R2T6) were heterologously expressed and purified as described previously [8,52]
Heterologous expression and purification of LDH The plasmid, p8T4, carrying the gene encoding T caldophi-lus LDH (UniProt accession number P06150) [53] was a kind gift from Professor Hayao Taguchi (Tokyo University
of Science) LDH was heterologously expressed and purified
to apparent homogeneity (M Aoshima, A Nishiyama and
Y Igarashi, unpublished results) The enzyme activity of the recombinant LDH was assayed at 70C by monitoring the lactate-dependent NADH oxidation as the decrease in
A340 The standard assay mixture contained 1 mm lactate, 0.2 mm NADH and 1 mm fructose 1,6-bisphosphate (an allosteric effector of T caldophilus LDH) [28] in 100 mm Hepes buffer (pH 8.0 at 20C) The oxidation of NADH
N S H
H
N
H 3 C N
N
N S
H 3 C
O SCoA
TPP C2-carbanion
N S
OH
H3C
H3C
N S
HO SCoA
HE-TPP radical
e– CoASH
OH
H 3 C
N S
CO2
H 3 C
N S
HO COO–
H3C
O COO–
e–
HE-TPP C2 α-carbanion
4
5 6
1
N S
O
H 3 C
CoASH
3 ′-a 3 ′-b
e–
Fig 5 Proposed catalytic mechanism for the reductive carboxylation of acetyl-CoA catalyzed by POR The HE-TPP radical is illustrated on the basis of the model proposed by Barletta et al [57] with the unpaired electron on the C2a carbon, although its chemical structure is still controversial [37–39].
Trang 8was calculated using an extinction coefficient of
6200 m)1Æcm)1 One unit of enzyme activity was defined as
the oxidation of 1 lmolÆmin)1of NADH
POR enzyme assays
The oxidative decarboxylation activity of POR was assayed
at 70C by monitoring the ferredoxin-mediated reduction
of metronidazole [54] The standard assay mixture
con-tained 20 mm pyruvate, 0.5 mm CoA, 10 lm ferredoxin,
0.1 mm metronidazole, 10 mm MgCl2, 1 mm dithiothreitol
and 0.5 mm TPP in 100 mm Hepes buffer (pH 8.0 at
20C) The decrease in A320was measured under an argon
atmosphere The reduction of metronidazole was calculated
using an extinction coefficient of 9300 m)1Æcm)1 One unit
of enzyme activity was defined as the reduction of 2
lmolÆ-min)1 of metronidazole (corresponding to the
decarboxyl-ation of 1 lmolÆmin)1 of pyruvate on the assumption that
the bleaching of the chromophore is a one-electron process)
[55] The reductive carboxylation activity of POR was
determined at 70C by the coupled assay with OGOR and
LDH (see Results) The standard assay mixture contained
1 mm acetyl-CoA, 10 mm NaHCO3, 10 mm 2-oxoglutarate,
0.5 mm CoA, 0.2 mm NADH, 1 mm fructose
1,6-bisphos-phate, 10 mm MgCl2, 1 mm dithiothreitol, 0.5 mm TPP,
0.03 U of OGOR, 0.2 U of LDH and 10 lm ferredoxin
(Fd1 or Fd2) in 100 mm Hepes buffer (pH 8.0) The assay
mixture without NADH and acetyl-CoA was incubated at
70C under an argon atmosphere The reaction was started
by adding the NADH, acetyl-CoA and enzyme solutions to
the mixture, and the decrease in A340as a result of NADH
oxidation was measured One unit of enzyme activity was
defined as the reduction of 1 lmolÆmin)1 of NADH
(corresponding to the carboxylation of 1 lmolÆmin)1 of
acetyl-CoA)
Quantification of CoA
The concentration of CoA was quantified using DTNB,
which reacts with free thiol groups (e.g CoA-SH) to
pro-duce 2-nitro-5-thiobenzoate with an extinction coefficient of
13 600 m)1Æcm)1 at 412 nm [56] The assay mixture
contained 0.1 mm DTNB in 100 mm Tris–HCl buffer (pH
8.0) Measurement of A412was performed after the addition
of the sample solution
EPR measurements
The enzyme solution was incubated with a substrate(s) in
an EPR sample tube at 70C for 5–10 min under a gentle
argon flow that had passed through a deoxidizing column
(Gasclean GC-RP; Nikka Seiko, Tokyo, Japan) The
reac-tion was stopped by immersion of the tube in liquid
nitro-gen EPR spectra were measured on a JES-FA300
spectrometer (JEOL, Tokyo, Japan) using a cylindrical
cavity (TE101 mode) The measurement temperature was controlled with a JEOL ES-CT470 cryostat system and
a digital temperature indicator⁄ controller model 9650 (Scientific Instruments, West Palm Beach, FL, USA) The magnetic field was calibrated with a JEOL NMR field meter ES-FC5 The g-values were determined by spectral simulation using JEOL anisimu⁄ fa software, version 2.0.0
Acknowledgements
The authors thank Professor Hayao Taguchi (Tokyo University of Science) for the gift of the plasmid carry-ing the LDH gene from T caldophilus; Dr Miho Aoshima and Ms Ayako Nishiyama for the preparation
of the recombinant LDH; and Dr Ki-Seok Yoon (Iba-raki University) for helpful discussions This research was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science
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