T. Werther et al.
Studies on oxalyl CoA decarboxylase of E. coli
both enzyme species, the C-terminal part of the subun-
its is not involved in crystal packing contacts. From
these results, it may be concluded that the prime effect
of ADP activation on the enzyme conformation is the
freezing of this part of the subunit to reduce its flexi-
bility and thus to shield the active site from the envi-
ronment. This is likely to enhance the rate of cofactor
activation (deprotonation of the C2 atom of ThDP
[21]) as well as the rate of decarboxylation [22]. A sim-
ilar activation mechanism is probably operative in
pyruvate decarboxylases from yeast species [21].
Experimental procedures
Unless otherwise stated, all chemicals and reagents were
purchased from Sigma-Aldrich Chemie GmbH (Steinheim,
Germany), VWR International GmbH (Darmstadt,
Germany) or AppliChem GmbH (Darmstadt, Germany),
and were of the highest available purity.
Protein expression and purification
The plasmid pMS470-115 ⁄ 6 ⁄ 5 was generously supplied by
Johannes Steinreiber (Dept. for Organic Chemistry, Univer-
sity of Graz, Austria). It carries the gene for oxalyl CoA
decarboxylase from E. coli under the control of a Tac pro-
moter, and was used to transform E. coli BL21 cells. The
cells were grown at 30 ꢀC in 2 · YT-ampicillin medium (1%
w ⁄ v yeast extract, 2% w ⁄ v tryptone, 1% w ⁄ v NaCl and
50 lgÆmL)1 ampicillin) in shaking flasks. When the solution
had reached an absorbance of 0.8 at 600 nm, expression of
EcODC was induced by adding 0.5 mm isopropyl thio-b-d-
galactopyranoside. After 10 h of growth at 30 ꢀC, corre-
sponding to an absorbance at 600 nm of 3.5–3.8, the cells
were harvested by centrifugation (2800 g, 20 min, 4 ꢀC).
Approximately 20 g of cells were suspended in 40 mL 0.1 m
Although the crystal structures of the holoenzyme
species from E. coli and O. formigenes are virtually
identical, the enzymes differ in their kinetic behaviour.
This difference is not obvious from the crystal struc-
ture of the ThDP binding sites formed by identical
amino acid residues in both species. However, in the
case of OfODC, a thiazolon cofactor analogue was
found at the active site even though ThDP was added
to the crystallization mixture. The reason for this strik-
ing difference is as yet unclear. The significantly higher
affinities of the substrate oxalyl CoA and the inhibitor
CoA for EcODC may be caused by two additional
hydrogen bond interactions (S265 and N404) in the
substrate binding site found for PADP in this enzyme
species. The corresponding side chains in OfODC
(A267 and M409) do not tend to form hydrogen bonds
with either the substrate or the inhibitor. Thus, these
structural differences could well be the reason for the
kinetic differences seen between the two enzyme
species. On the other hand, the differing kinetic con-
stants could be also partially due to the different
assays used, our novel continuous spectroscopic one
for EcODC and the discontinuous HPLC-based assay
for OfODC. The continuous assay appears to be the
more reliable and more direct approach, as whole
progress curves can be conveniently recorded.
sodium
phosphate,
pH
7.0,
containing
0.1 mm
ThDP ⁄ MgSO4, 5% v ⁄ v glycerol, 1 mm phenylmethanesulfo-
nyl fluoride, 1 mm dithiothreitol (DTT) and 1 mm EDTA,
and disrupted using
a French press (five passages at
1200 bar). The homogenate was clarified by centrifugation
(70 000 g, 30 min), and the supernatant was diluted to
40 mg proteinÆmL)1 using the same buffer. Nucleic acids
were eliminated by streptomycin sulfate precipitation (0.1%
w ⁄ v, 30 min agitation at 8 ꢀC, and 25 min centrifugation at
70 000 g). After two subsequent ammonium sulfate precipi-
tations (15 g ⁄ 100 mL each), the pellet was resuspended in
25 mm Tris ⁄ HCl, pH 7.5. The protein solution was dialysed
twice for 5 h against 25 mm Tris ⁄ HCl, pH 7.5, 1 mm DTT,
with or without 150 mm NaCl, and then further purified by
anion-exchange chromatography using Q-Sepharose (GE
Healthcare, Munich, Germany; column size, diameter
26 · length 100 mm). Elution was performed with a linear
gradient of 500 mL of 100–400 mm NaCl in 25 mm
Tris ⁄ HCl, pH 7.5. The EcODC-containing fractions, eluting
at 150–300 mm NaCl, were pooled and precipitated by
adding 32 g ammonium sulfate per 100 mL. After centrifu-
gation (40 000 g, 15 min), the pellet was resuspended in
50 mm MES ⁄ NaOH, pH 6.5, 0.2 m ammonium sulfate,
applied on Superdex 200 (GE Healthcare; column size,
diameter 26 · length 600 mm), and eluted at a flow rate of
0.5 mLÆmin)1 using the same buffer. Eluted fractions were
analysed by SDS–PAGE. EcODC-containing fractions with
> 95% homogeneity were pooled, flash-frozen in liquid
nitrogen, and stored at )80 ꢀC. The identity of the purified
enzyme was confirmed using a combination of tryptic diges-
tion and MALDI-TOF mass spectrometry.
The identical architecture of the ADP binding sites
of both species means that no structural explanation is
possible for the differing activating effects of ADP.
However, electron density for ADP was found in the
crystal structure of OfODC, even when no ligand was
added [3]. ADP was clearly detectable in the structure
of EcODC only if the ligand was present during
crystallization. The poor ADP activation of EcODC
presumably reflects the minor physiological relevance
of oxalate degradation for the energy metabolism of
E. coli. Thus, it is conceivable that non-oxalotrophic
bacteria only require enzymes for oxalate detoxifica-
tion under certain conditions [9]. Future studies of
other putative oxalyl CoA decarboxylases are required
to unravel this phenomenon, as well as the molecular
basis of ADP activation.
FEBS Journal 277 (2010) 2628–2640 Journal compilation ª 2010 FEBS. No claim to original US government works
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