Under physiological conditions, R-lactyl-CoA is only effectively dehydrated, because the very small equilibrium concentration of the unsaturated compound 2c is irreversibly trapped by th
Trang 1(R)-2-hydroxyacyl-CoA and 2-enoyl-CoA
Anutthaman Parthasarathy1, Wolfgang Buckel1and David M Smith2
1 Laboratory for Microbiology, Philipps-Universita¨t, Marburg, Germany
2 Centre for Computational Solutions in the Life Sciences, Rudjer Boskovic Institute, Zagreb, Croatia
Introduction
Dehydratases catalyze a,b-eliminations of water from
hydroxy compounds, and form a large class of
enzymes; over 100 different types are listed in the
enzyme nomenclature database (EC 4.2.1.–) In most
cases, the hydroxyl group is located in the b-position
of an adjacent carboxylate, CoA-thioester or ketone,
and the a-proton to be removed is thus activated In
a-amino acid fermentation pathways, however,
dehy-dratases are found, whose substrates are
a-hydroxya-cyl-CoA derivatives [1,2] In such cases, the
b-hydrogen to be removed during a,b-elimination of
water has an approximate pKa of 40 The requisite
activation of this proton is achieved by transient
addition of one high-energy electron to the thioester
carbonyl, forming a ketyl radical anion (3, Scheme 1)
This allows the elimination of the a-hydroxyl group
[3,4] (3 fi 4) and lowers the pKa of the b-hydrogen
in the resulting enoxy radical intermediate (4) by at least 26 units [5] Recycling of the initiatory electron from the second ketyl intermediate thus produced (5) yields enoyl-CoA (2) and completes the catalytic cycle
Recently, the mechanism shown in Scheme 1 has received strong support with the reported observation
of the allylic ketyl radical intermediate (5) in the enyzmatic dehydration of (R)-2-hydroxyisocaproyl-CoA [1a, R = CH(CH3)2; Fig 1].[4] However, no such observations have yet been possible for the analogous dehydrations of (R)-2-hydroxyglutaryl-CoA (1b, R = CH2CO2H) or (R)-lactyl-CoA (1c, R = H) Whereas the equilibrium constants (K) of b-hydrox-yacyl-CoA, c-hydroxyacyl-CoA or d-hydroxyacyl-CoA typically lie around unity [6], the situation is much less clear for the a-hydroxyacyl-CoA derivatives For
Keywords
ab initio calculations; enzymes; kinetics;
solvent effects; substituent effects
Correspondence
D M Smith, Centre for Computational
Solutions in the Life Sciences, Rudjer
Boskovic Institute, Bijenicka 54, 10000
Zagreb, Croatia
Fax: +385 1 456 1182
Tel: +385 1 456 1182
E-mail: David.Smith@irb.hr
(Received 20 December 2009, revised
25 January 2010, accepted 28 January
2010)
doi:10.1111/j.1742-4658.2010.07597.x
A combined experimental and computational approach has been applied to investigate the equilibria between several a-hydroxyacyl-CoA compounds and their 2-enoyl-CoA derivatives In contrast to those of their b, c and d counterparts, the equilibria for the a-compounds are relatively poorly char-acterized, but qualitatively they appear to be unusually sensitive to substit-uents Using a variety of techniques, we have succeeded in measuring the equilibrium constants for the reactions beginning from 2-hydroxyglutaryl-CoA and lactyl-2-hydroxyglutaryl-CoA A complementary computational evaluation of the equilibrium constants shows quantitative agreement with the measured values By examining the computational results, we arrive at an explanation
of the substituent sensitivity and provide a prediction for the, as yet unmeasured, equilibrium involving 2-hydroxyisocaproyl-CoA
Abbreviations
Nbs2, 5,5¢-dithiobis(2-nitrobenzoate); TFA, trifluoroacetic acid; THF, trifluoroacetic acid.
Trang 2example, it has recently been determined that the
enzy-matic dehydration of 1a (also known as
2-hydroxy-4-methylpentanoyl-CoA), derived from the amino acid
(S)-leucine, to isocaprenoyl-CoA
(4-methyl-2-pente-noyl-CoA, probably the E-isomer, 2a), occurs
irrevers-ibly, within the limits of detection [7] In contrast, the
equilibrium of the dehydration of (R)-lactyl-CoA to
acryloyl-CoA (1c fi 2c) strongly favors the hydroxy
compound Under physiological conditions,
(R)-lactyl-CoA is only effectively dehydrated, because the very
small equilibrium concentration of the unsaturated
compound (2c) is irreversibly trapped by the
consecu-tive reductase, resulting in propionyl-CoA [8,9] With
(R)-2-hydroxyglutaryl-CoA (1b) and
(E)-glutaconyl-CoA (2b) as substrates, the equilibrium appears to lie
more in the middle [10], although the value of Kb, like
those of Kaand Kc, is presently unknown
In order to place the known qualitative results on a
more solid footing, we adopted a combined
experimen-tal and computational approach to characterize the
equilibria shown in Fig 1 Specifically, using a bidirec-tional kinetic analysis, we present experimental deter-minations of Kb and Kc The obtained values are supported and rationalized by high-level ab initio molecular orbital calculations, which are also used to obtain a value for the elusive Ka
Results and Discussion For the determination of the experimental value of Kb,
we performed activity measurements in both the for-ward and reverse directions, requiring the preparation
of both 1b and 2b Although an enzymatic method was potentially applicable for the production of 1b, the incubations in that case also yielded the thermo-dynamically more favorable (R)-4-hydroxyglutaryl-CoA, which is not dehydrated [11] To avoid this complication, a chemical method for the preparation
of 1b was used; namely, (R)-2-hydroxyglutaryl-CoA (1b) was prepared by the direct reaction of CoASH and (R)-butyrolactone-5-carboxylchloride in aqueous NaHCO3 [11] After the thiol had been consumed, the reaction was acidified to pH 0.5 and incubated at
25C for 3 h until the equilibrium between lactone-CoA and 2-hydroxyglutaryl-lactone-CoA (1 : 4) was estab-lished Prior to use, the mixture was neutralized, whereby equilibration was terminated It was shown that the remaining lactone-CoA did not interfere with the dehydration In the case of glutaconyl-CoA (2b) preparation, there were no complicating factors, and the target compound was prepared by incubation of glutaconate with acetyl-CoA, catalyzed by glutaconate-CoA transferase from Acidaminococcus fermentans produced in Escherichia coli [12]
For our source of 2-hydroxyglutaryl-CoA dehydra-tase, we employed the strictly anaerobic bacterium Clostridium symbiosum, which is involved in the fermentation of glutamate to ammonia, CO2, acetate, butyrate, and H2 The heterodimeric enzyme
O SCoA
SCoA
R H H
OH
H H
H
H2O
2-hydroxyisocaproyl-CoA (1a)
2-hydroxyglutaryl-CoA (1b)
lactyl-CoA (1c)
E-isocaprenoyl-CoA (2a) + H2 O
E-glutaconyl-CoA (2b) + H2 O
acryloyl-CoA (2c) + H2 O
Kconc. = [2] [H2O]
[1] [2]
[1]
K = K
R=CH(CH3)2
R=CH2CO2H
R=H
K a >> 1
K b ~ 1
K c << 1
K conc.
55.5
=
Fig 1 Dehydrations of the
a-hydroxyacyl-CoA derivatives discussed in this work As
the biochemical experiments were
performed in dilute aqueous conditions, we
employ K, as opposed to K conc , for
convenience.
Scheme 1 Electron recycling mechanism for the conversion of
a-hydroxyacyl-CoA derivatives to the corresponding enoyl-CoA
compounds.
Trang 3(48 + 43 kDa) contains, per mole (91 kDa), 1 mol of
FMNH2 and 8 mol of iron + 8 mol of sulfur,
proba-bly as two [4Fe–4S] clusters It has to be activated by
incubation with a reducing agent, ATP, and Mg2+,
mediated by a homodimeric protein (2· 27 kDa) with
one [4Fe–4S]+ cluster between the two subunits
Thereby, one electron is transferred from the activator
protein to the dehydratase, driven by hydrolysis of two
molecules of ATP Prior to use in our activity
mea-surements, 2-hydroxyglutaryl-CoA dehydratase,
puri-fied from C symbiosum, was activated under strict
anaerobic conditions with ATP, MgCl2 and dithionite,
mediated by catalytic amounts of the activator from
A fermentansproduced in E coli [13]
The dehydration (1b fi 2b) and hydration
(2b fi 1b) reactions were followed
spectrophotometri-cally at 290 nm (e290 nm= 2.1 mm)1Æcm)1) Although
the absorbance maximum of enoyl-CoA (2b) lies at
260 nm (De260 nm= 6.0 mm)1Æcm)1), the longer
wave-length was chosen to avoid interference with the high
absorbance of the adenine moiety of CoA
(e260 nm= 16 mm)1Æcm)1) [7]
The activity measurements between 0 and 1 mm
(R)-2-hydroxyglutaryl-CoA (1b) and 0 and 5 mm
glutaco-nyl-CoA (2b) gave smooth Michaelis–Menten curves,
from which Km and kcat values could be calculated
by simulation In experiments starting with
(R)-2-hydroxyglutaryl-CoA (1b), we obtained a Km of
0.052 ± 0.003 mm and a kcat of 83 ± 8 s)1, resulting
in a specificity constant [kcat⁄ Km (1b)] of
1600 ± 300 s)1Æmm)1 Beginning the reaction with,
instead, (E)-glutaconyl-CoA (2b) resulted in a Km of
0.25 ± 0.02 mm and a kcatof 7.0 ± 0.7 s)1, associated
with a specificity constant [kcat⁄ Km (2b)] of
28 ± 6 s)1Æmm)1 Kbwas subsequently calculated from
the Briggs–Haldane equation:
Kb¼ ½kcat=Kmð1bÞ=½kcat=Kmð2bÞ ¼ 1600=28 ¼ 57 1:5
For the measurement of Kc, the equilibrium constant
for (R)-lactyl-CoA (1c) and acryloyl-CoA (2c), we
purified lactyl-CoA dehydratase from the strict
anaer-obe Clostridium propionicum to apparent homogeneity
Like 2-hydroxyglutaryl-CoA dehydratase, lactyl-CoA
dehydratase is a heterodimer (a, 48 kDa; b, 41 kDa)
containing two [4Fe–4S] clusters and substoichiometric
amounts of FMN and riboflavin [9,14] The purified
enzyme was treated with 3-pentynoyl-CoA to abolish a
slight acryloyl-CoA reductase activity [9,15] Activation
of the dehydratase occurred under conditions similar
to that used for the C symbiosum dehydratase in the
presence of Mg-ATP, dithionite, and the activator
from A fermentans [13] The CoA-thioesters were pre-pared from acrylate, (R)-lactate and 3-pentynoic acid [16] by the carbonyl-diimidazole method [17], and analyzed enzymatically and by MALDI-TOF MS [18] The kinetic parameters for the hydration of acryloyl-CoA (2c) to (R)-lactyl-CoA (1c) were measured as Km= 0.150 ± 0.004 mm and Vmax=
85 ± 6 UÆmg)1, yielding kcat= 126 ± 10 s)1 and [kcat⁄ Km (2c)] = 0.84 ± 0.05· 106s)1Æm)1 Because the equilibrium concentration of acryloyl-CoA (2c) is very low, it was more difficult to determine the kinetics
of (R)-lactyl-CoA dehydration (1c fi 2c) Reasonable estimates are given by: Km= 0.32 ± 0.02 mm and
Vmax= 3.0 ± 0.4 UÆmg)1, with kcat= 4.5 ± 0.6 s)1 and [kcat⁄ Km(1c)] = 1.41 ± 0.1· 104s)1Æm)1) Substi-tution of these data into the Briggs–Haldane equation yields:
Kc¼ ½kcat=Kmð1cÞ=½kcat=Kmð2cÞ ¼ 0:017 0:007 This low value of Kc corroborates the high redox potential of the acryloyl-CoA⁄ propionyl-CoA pair (E0¢ = + 69 mV) as compared with those of the higher homologs of 2-enoyl-CoA⁄ acyl-CoA (E0¢ =)10 mV) [19]
The relative magnitudes of Kb and Kc confirm an unexpectedly large ( 20 kJÆmol)1) substituent effect
on the dehydration equilibrium, arising from the pres-ence of a carboxymethyl group on the b-carbon in 1b
in place of a hydrogen atom in 1c This fact, combined with the similarly large effect that was apparent upon further substitution by the isopropyl group in 1a, led
us to perform ab initio molecular orbital calculations
in order to seek an explanation
To enable high-accuracy calculations, we elected to replace the adenylphosphopantetheine chain of CoA
by the S-CH3 group, resulting in the model systems shown in Fig 2 We expected this substitution to have only a minor effect on the individual equilibrium con-stants and, because it is adopted uniformly, there should be virtually no effect on the relative equilibrium constants Although our final goal was to compute the free energies of the reactions shown in Fig 1 under aqueous conditions, we elected to present the gas-phase results as well The rationale behind this is that the gas-phase calculations encompass the fundamental electronic effects governing the differences in the equi-librium constants By decomposing the final aqueous energy differences into a gas-phase component and a component related to solvation, we are thus able to comment on the extent to which each of these aspects influences the final result
Trang 4Table 1 shows the standard (referenced to 1 atm of
pressure) free energy change for each reaction in the
gas phase [1(g) fi 2(g)+ H2O(g); DG
ðgÞ] (Although 1 and 2 strictly represent the CoA thioester, for
simplic-ity, we keep the same notation for the methyl
thioest-ers used in the calculations.)
In the gas phase, the dehydration of 1c was found
to be mildly (2.9 kJÆmol)1) endergonic The
introduc-tion of a carboxymethyl substituent at the b-carbon
was found to preferentially stabilize the 2-enoyl
spe-cies (2b), such that dehydration of 2a was exergonic
by 1.6 kJÆmol)1 The small associated substituent
effect (4.5 kJÆmol)1) can be rationalized by the mild
(net) capability of the alkyl substituent to donate
electrons to the electron-deficient b-carbon in 2c
Indeed, acrylamide, with no substituent at the
b-posi-tion, has been shown to act as a toxic electrophilic
agent [20], an effect that should be more pronounced
in acryloyl-CoA (2c) The more electron-donating
iso-propyl substituent results in a larger preferential
sta-bilization for the enoyl species (2a), such that the
dehydration of 1a is exergonic by 9.0 kJÆmol)1 Both
of these relatively small, inherent (gas-phase)
substitu-ent effects are more in line with qualitative
expecta-tions than the values apparent from the measured
solution-phase equilibrium constants It thus appears that the explanation of the unusually large substituent effects is not related to fundamental electronic factors
at the molecular level
The standard free energy changes in aqueous solu-tion [1(aq) fi 2(aq)+ H2O(aq); DG
ðaqÞ, referenced to
1 molÆL)1] could be expected to preferentially favor the products, simply because of the sizeable solvation free energy of water [experimentally, DG
s (H2O) = )26.5 kJÆmol)1 [21]] This preference for the bimo-lecular products is reduced by the reference state correction (from 1 atm to 1 molÆL)1) of RT ln(~RT)
of 7.9 kJÆmol)1 (at 298 K) for each species to )18.6 kJÆmol)1 (~R= 0.082053 K)1) In the case of (R)-lactyl-CoA, the inherent product preference in solution is partially compensated for by the relatively large (absolute) value of DGs(1c) as compared with
DGs(2c) (Table 2) The result of these competing effects is that DGðaqÞ(C) is only 2.8 kJÆmol)1 less than the corresponding gas-phase value Despite the poten-tial uncertainties involved, the final calculated value for DG
ðaqÞ(C) (0.1 kJÆmol)1) is in very good agreement with that derived from the measured equilibrium con-stant of 0.017 (0.1 kJÆmol)1)
The absolute magnitude of the free energies of solvation of 1b and 2b are much larger than those of 1c and 2c, because of the presence of the hydrophilic
+
+
+
+
1a
1b
1c
2a
2b
2c
K calc. a = 1610
K expt. a > 1000
K calc. b = 8.42
K expt. b = 57
K calc. c = 0.02
K expt. c = 0.017
Fig 2 Comparison of the calculated
and experimental equilibrium constants
determined in this work.
Table 1 Experimentally determined and calculated values for the
equilibria represented by Fig 1.
Equilibrium
Calculated [G3(MP2)] Experimental
DG
ðgÞ
a,b DG ðaqÞ a,c K d DG
ðaqÞ a,c K d
a
kJÆmol)1 at 298 K. b 1 atm reference. c 1 molÆL)1 reference.
d Dimensionless, K = Kconc.⁄ 55.5 (see Fig 1).
Table 2 Calculated free energies of solvation (DG
s ) for the species shown in Fig 1.
DG
a The calculated free energy of solvation (in kJÆmol)1) for x(g) (1 molÆL-1) fi x (aq) (1 molÆL)1) The final DG
ðaqÞ values in Table 1 are corrected by RT ln(~ R T) for each species See text.
Trang 5carboxylic acid groups Even though the hydroxyacyl
species (1b) species is again solvated more strongly
than the enoyl one (2b), the difference between them,
and hence the associated effect on DGðaqÞ(B), is much
smaller than for reaction C The favorable
contribu-tion from DG
s (H2O) is therefore counteracted to a
much smaller extent, resulting in the value for
DG
ðaqÞ(B) being 13.6 kJÆmol)1 more negative than
DG
ðgÞ (B) The corresponding calculated value of
Kb= 8.42 deviates somewhat from the measured
value of Kb= 57 In terms of energy, however, the
discrepancy of only 4.7 kJÆmol)1 is certainly within
acceptable limits for a solution-phase property
The more hydrophobic nature of 1a and 2a is
reflected in their less favorable solvation free energies
(Table 2) In this case, however, it is the enoyl species
(2a) that is better solvated than the hydroxyacyl one
(1a) This serves to slightly reinforce the favorable
effect of DGs (H2O) rather than to counteract it, as
occurred for equilibria B and C The final result is that
equilibrium A is predicted to lie very far to the right,
with associated values of DG
ðaqÞ(A) =)28.3 kJÆmol)1
and Ka= 1610 Given the good agreement between
theory and experiment obtained for equilibria B and
C, we are confident that these values provide a
reason-ably accurate description of the thermodynamics of
2-hydroxyisocaproyl-CoA dehydration They are
cer-tainly consistent with the fact that the equilibrium
concentration of 1a was not detectable in experiments
concerning its conversion into 2a [7] The precise
experimental determination of Ka, however, still
remains a challenge for the future
Conclusion
In summary, and in agreement with previous
qualita-tive observations, the measurements presented here
confirm an unusually large effect of the substituent at
the b-carbon on the equilibrium constants of the
dehy-dration reactions shown Fig 1 Molecular orbital
cal-culations show that the inherent substituent effects, as
reflected in the gas-phase data, are less drastic The
condensed-phase calculations reproduce the measured
values and reveal that the large effects are primarily
due to a complex interplay of competing effects
con-nected to the solvation process The calculations
pre-dict that the, as yet, unmeasured equilibrium involving
(R)-2-hydroxyisocaproyl-CoA very strongly favors the
product Further work is required to determine to
what extent this is connected with the successful
obser-vation of the proposed penultimate intermediate (5) in
the dehydration mechanism (Scheme 1) for reaction A
and not for reactions B or C
Experimental procedures Chemical synthesis of CoA thioesters
Acrylyl-CoA was synthesized under a gentle stream of N2
by reacting a three-fold molar excess of acrylyl chloride in dry acetonitrile with free CoASH dissolved in 0.5 mL of aqueous 0.5 m NaHCO3 The solution was stirred at room temperature until no yellow color of free thiol was obtained with 5,5¢-dithiobis(2-nitrobenzoate) (Nbs2) [22,23,24] The
pH was adjusted with 5 m HCl to 2 and the solution was stored at )20 C In view of the instability of the com-pound, it was prepared and purified the day before use 2-Hydroxyglutaryl-CoA was synthesized from commer-cial (R)-2-oxo-tetrafuran carboxylic acid that was converted
to the corresponding acid chloride by reacting with an excess of oxalyl chloride at 60C for 3 h The excess oxalyl chloride was removed by evaporation under reduced pres-sure Then, a three-fold excess of the acid chloride was dissolved in dry acetonitrile and reacted with CoASH in 0.5 m NaHCO3at room temperature, and the pH was low-ered to 6 to obtain the CoA Finally, the lactone-CoA was equilibrated with (R)-2-hydroxyglutaryl-lactone-CoA at
pH 1 and 25C for 3 h The equilibration was stopped by raising the pH to 8.0 The resulting (R)-2-hydroxyglutaryl-CoA contained about 3% lactone as analyzed by MALDI-TOF MS
3-Pentynoyl-CoA was synthesized by the procedure used for making 3-pentynoyl pantetheine [15,9] CoASH (40 lmol) was suspended in 5 mL of dry acetone (CaSO4) Another flask contained 60 lmol of dicyclohexylcarbodii-mide in 5 mL of dry tetrahydrofuran (THF) To the THF solution was added 0.1 g of 3-pentynoic acid The THF and acetone solutions were mixed quickly and stirred overnight
in a sealed flask at 4C The solution was filtered on a sin-tered glass filter to remove dicyclohexylurea The solvent was removed with a rotary evaporator to yield oil, which was treated with diethyl ether and dried under vacuum
Butyryl-CoA, propionyl-CoA and acetyl-CoA were synthesized from their anhydrides [25] To CoA (25 lmol)
in 1 mL of 0.5 m KHCO3, 35 lmol of the respective anhy-dride in 0.5 mL of acetonitrile was added After dilution to
5 mL with water, the mixture was reacted at room tempera-ture until the Nsb2test was negative, and then acidified to
pH 2 (R)-Lactyl-CoA was synthesized from (R)-lactate and CoASH using 1,1¢-carbonyldiimidazole [17]
Enzymatic synthesis of glutaconyl-CoA with glutaconate CoA-transferase
The incubation contained, in 5 mL of 50 mm potassium phosphate (pH 7.0), 20 lmol of acetyl-CoA, 300 lmol of glutaconic acid, and 5 U of recombinant transferase After
1 h at 37C, the mixture was acidified to pH 2 and filtered through a 1 kDa membrane (Amicon; Amersham
Trang 6Biosciences, Freiburg, Germany; now part of GE
Health-care, Munich, Germany) to remove precipitated protein
Purification of CoA thioesters by reverse-phase
chromatography
All CoA thioesters were purified by reverse-phase
chroma-tography through Sep-Pak C18 columns (Waters, Milford,
MA, USA) The reaction mixtures at pH 2 were freed from
solvents under reduced pressure and from precipitated
proteins by ultrafiltration They were then loaded onto
C18 columns washed with methanol and equilibrated with
0.1% (v⁄ v) trifluoroacetic acid (TFA) After washing with
three volumes of the same solution, elution was performed
with 0.1% TFA in 50% acetonitrile (v⁄ v) The eluted CoA
esters were freed from acetonitrile on a Speed-Vac
concen-trator (Bachofer, Reutlingen, Germany) and vacuum-dried
on a lyophilizer (Alpha1-4; Christ Instruments, San Diego,
CA, USA) The lyophilized powders were stored at)80 C
until further use
MALDI-TOF MS
The CoA thioester samples were purified as described
above, and the lyophilized samples were dissolved in
10–40 lL of water Acetyl-CoA or free CoA was used as
internal standard The matrix was
a-cyano-4-hydroxycin-namic acid (Sigma) dissolved in 70% acetonitrile⁄ 0.1%
TFA One microliter of each sample was mixed with 1 lL
of a-cyano-4-hydroxycinnamic acid or
a-cyano-3-hydroxy-cinnamic acid as matrix, and spotted onto a gold plate in a
dilution series Measurements were performed with a
355 nm laser in positive reflector mode with a delayed
extraction and a positive polarity on the Proteomics
Ana-lyzer 4800 mass spectrometer (Applied Biosystems,
Fra-mingham, MA, USA) at the MPI for Terrestrial
Microbiology, Marburg, Germany The acceleration voltage
was 20 000 V, the grid voltage was 58%, and the delay time
was 50 ns The ratio of reflector voltage was 1.00–1.12 An
average of 0.5% of acceleration was laid on the guidewire
The mass range measured was 700–1000 Da For each
spec-trum, more than 1000 shots were accumulated
Enzymatic assays
All spectrophotometric assays were performed on
Ultro-spec 1100 pro Ultro-spectrophotometers from Amersham
Bio-sciences, installed under aerobic or anaerobic conditions as
needed, or a Uvikon 943 double-beam spectrophotometer
from Kontron Instruments (Zurich, Switzerland) Quartz
cuvettes were used for measurements below 320 nm, and
disposable plastic cuvettes for measurements above 320 nm
2-Hydroxyglutaryl-CoA dehydratase activity was
mea-sured under strict anaerobic conditions (d = 1 cm, total
volume 0.5 mL at 25C) with 50 mm Tris ⁄ HCl (pH 8.0),
5 mm MgCl2, 5 mm dithiothreitol, 0.4 mm ATP, and 0.1 mm dithionite, as well as the activator from A fermen-tans and dehydratase from C symbiosum After incubation for 5 min, the reaction was started by addition of (R)-2-hy-droxyglutaryl-CoA The formation of (E)-glutaconyl-CoA was measured at 290 nm (e290 nm= 2.2 mm)1Æcm)1) [7] In the reverse direction, (E)-glutaconyl-CoA was used as sub-strate The kinetic constants were determined with 2.0 lg of dehydratase (specific activity of 54 lmol min)1Æmg)1 pro-tein) and 0.6 lg of activator, using either 0.02–1.0 mm (R)-2-hydroxyglutaryl-CoA or 0.1–5.0 mm glutaconyl-CoA Under these conditions, the minimum substrate⁄ enzyme ratio was 450 : 1 The data were fitted to the Michaelis– Menten equation using the excel program In the routine assays during purification of the dehydratase, (R)-2-hy-droxyglutaryl-CoA was replaced by (R)-2-hydroxyglutarate, acetyl-CoA, and glutaconate CoA-transferase
Prior to the assay of lactyl-CoA dehydratase from C pro-pionicum, the crude enzyme fractions or the purified enzyme were incubated for 30 min under anaerobic conditions with
5 mm 3-pentynoyl-CoA, which is a reported inactivator of acrylyl-CoA reductase [9], whose activity interferes with the assay under the applied reducing conditions [26] The pro-tein fraction was freed from the inhibitor by passing it over
a 1 mL PD-10 Spintrap G-25 column (GE Healthcare) equilibrated with anaerobic buffer, and concentrating via a Centricon 30 kDa filter (Millipore Corporation, Billerica,
MA, USA) The assay was then performed exactly as that for 2-hydroxyglutaryl-CoA dehydratase, except that acrylyl-CoA or lactyl-acrylyl-CoA was used as substrate The recombinant activator from A fermentans could be used instead of the activator from C propionicum, which is very unstable and has never been purified completely [9] The kinetic constants were determined with 2.0 lg of dehydratase (specific activity
of 85 lmolÆmin)1Æmg)1 protein) and 0.6 lg of activator, using either 0.2–10 mm lactyl-CoA or 0.01–2.0 mm acrylyl-CoA, and evaluated as above Under these conditions, the minimum substrate⁄ enzyme ratio was 370 : 1 Acrylyl-CoA reductase activity was measured with propionyl-CoA and ferricenium hexafluorophosphate as electron acceptor [26] The concentrations of CoASH, acetyl-CoA and glutaco-nyl-CoA (or any other CoA-ester substrate of glutaconate-CoA transferase) were determined in a single assay using Nbs2, oxaloacatate, citrate synthase, and transferase [23,24,27] Similarly, lactyl-CoA and acrylyl-CoA were determined in the same assay, with glutaconate-CoA trans-ferase being replaced by propionate CoA-transtrans-ferase [28]
Enzyme purification
Prior to use, columns, Centricon filters, centrifuge tubes, pipette tips and other plastic materials were stored in a glovebox (Coy Labs, Ann Arbor, MI, USA) for at least
Trang 724 h All operations during the purifications were
per-formed in this box Buffers and solutions were degassed
under reduced pressure, purged with nitrogen, and
prere-duced with 2 mm dithiothreitol Protein was determined by
the Bradford method [29]
C symbiosumHB25 was grown on glutamate, yeast
extract, thioglycollate, and biotin [30], whereas C
propioni-cumDSM 1682 required alanine, yeast extract, and cysteine
[26] Production and purification of the recombinant
activa-tor from A fermentans has been described elsewhere [31]
Purification of 2-hydroxyglutaryl-CoA
dehydratase from C symbiosum [32]
Frozen cells were suspended in 50 mL of buffer A (50 mm
Mops, pH 7.2) under anaerobic conditions and disrupted
by ultrasonication in the anaerobic chamber The cell-free
extract was clarified by centrifugation at 100 000 g for 1 h
at 4C on an Optima L-90K Ultracentrifuge (Beckman
Coulter, Brea, CA, USA), and applied to a
DEAE–Sepha-rose column equilibrated with buffer A The column was
washed with buffer A, and elution was performed by
run-ning a linear gradient of 0–0.7 m NaCl The active fractions
eluted around 0.35 m NaCl The pooled fractions were
combined, and desalted by filtration through a Centricon
membrane (30 kDa cut-off); solid ammonium sulfate was
then added to 1 m final concentration This solution was
loaded onto a phenyl–Sepharose column pre-equilibrated
with buffer A containing 1 m ammonium sulfate After
washing, the proteins were eluted with a gradient of 1–
0.3 m ammonium sulfate The active fractions starting from
0.5 m ammonium sulfate were combined, desalted, and
loaded onto a Q-Sepharose column pre-equilibrated with
buffer A After washing of the column, elution was
per-formed with a gradient of 0–0.5 m NaCl The most active
and pure fractions, which eluted around 0.3 m NaCl, were
combined, desalted, concentrated, and stored at )80 C
until further use; the yield was 34% (based on the activity
of the cell-free extract)
Purification of lactyl-CoA dehydratase from
C propionicum
The following buffers were used: A, 25 mm Tris⁄ HCl,
1 mm MgCl2, 1 mm EDTA, and 2 mm dithiothreitol; B,
1.5 m NaCl in A; C, 1.5 m ammonium sulfate in A; and D,
150 mm NaCl in A Pre-equilibration of the columns
allowed purification in about one working day Frozen cells
(12 g) were suspended in 20 mL of buffer A and opened by
ultrasonication The cell-free extract was clarified by
ultra-centrifugation for 45 min at 100 000 g and applied onto a
Source 15Q column (1.6· 15 cm) equilibrated with
fer A After washing of the column with 25 mL of
buf-fer A, the proteins were eluted in a linear gradient of
0–0.33 m NaCl with 100 mL of buffer B Two brownish peaks were obtained The first eluting peak contained the activator (5 mm ATP⁄ MgCl2was added, and the fractions were stored on ice), and the second peak was found to be lactyl-CoA dehydratase The relevant fractions were com-bined, desalted, and stored on ice After addition of solid ammonium sulfate to a final concentration of 1.5 m, the solution was sterile-filtered and applied to a Source 15Phe column (1.0· 10 cm) equilibrated with buffer C The col-umn was washed with 20 mL of buffer C, and the proteins were eluted in a gradient of 1.5–0 m ammonium sulfate The brownish fractions were pooled, and the sample was concentrated to about 400 lL with a 100 kDa cut-off Centricon membrane The concentrated sample (200 lL) was applied to a Superdex 200 column (HR 1.0⁄ 30) equili-brated with buffer D, and 0.5 mL fractions were collected The pure dehydratase (yield 32%) and the still impure acti-vator were not frozen The dehydratase preparation lost its activity at a rate of 10–15% per day when stored in the glovebox on ice water Owing to the brownish color of the dehydratase, a ‘blind’ purification could be performed in order to save time and specific activity The purity of the enzymes was checked by SDS⁄ PAGE [33]
Computational methods
Calculations were carried out with gaussian 03 [34] The geometry of each of the model systems shown in Fig 2 was optimized with the B3-LYP⁄ 6-31G(d) level of theory Frequency calculations were performed to derive appropriate thermochemical corrections Improved relative energies, in the gas phase, were obtained using the G3(MP2) model chemistry [35] The free energies of solvation [21] were obtained using a polarizable continuum model, with Uni-ted Atom Topological Model cavities at the B3-LYP⁄ 6-311G(d,p) level of theory [36] The combination of the G3(MP2) gas-phase free energies [DG
ðgÞ, 1 atm reference] with the free energies of solvation [DG
s,
1 molÆL)1(g) fi 1 molÆL)1(aq)] [21] and a reference state correction of RT ln(~RT) [1 atm(g) fi 1 molÆL)1(g)] yields the relative free energies in solution [DGðaqÞ], corresponding
to a standard state of 1 molÆL)1 It is these values that were used to determine Kconc, which were corrected to K according to Fig 1 Gaussian archive entries of the gas-phase and solution calculations of the 1a, 2a, 1b, 2b, 1c, 2c and H2O can be found in Table S1
Acknowledgements
We gratefully acknowledge support (of D M Smith)
by the Croatian Ministry of Science (project 098-0982933-2937) and the EC (FP6 contract 043749) Work in Marburg was supported by the Max-Planck Society, Deutsche Forschungsgemeinschaft and the
Trang 8Fonds der chemischen Industrie We thank T Selmer,
Fachhochschule Aachen, Germany, for advice on the
purification of lactyl-CoA dehydratase
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Supporting information The following supplementary material is available: Table S1 Gaussian archive entries of the gas-phase and solution calculations of the species 1a, 2a, 1b, 2b, 1c, 2c, and H2O
This supplementary material can be found in the online version of this article
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