Propionate CoA-transferase from
Clostridium propionicum
Cloning of the gene and identi®cation of glutamate 324 at the active site
Thorsten Selmer, Angela Willanzheimer and Marc Hetzel
FB Biologie, Philipps-Universita
È
t, Marburg, Germany
Propionate CoA-transferase from Clostridium propionicum
has been puri®ed and the gene encoding the enzyme has been
cloned and sequenced. The enzyme was rapidly and irre-
versibly inactivated by sodium borohydride o r h ydroxyl-
amine in the presence of propionyl-CoA. The reduction of
the t hiol ester between a catalytic site glutamate and Co A
with borohydride and the cleavage by hydroxylamine were
used to introduce a site-speci®c label, which was followed
by MALDI-TOF-MS. This allowed the identi®cation of
glutamate 324 at the active site. Propionate CoA-transferase
and similar proteins deduced from the genomes of
Escherichia c oli, Staphylococcus aureus, Bacillus halodurans
and Aeropyrum pernix are proposed to form a novel subclass
of CoA-transferases. Secondary structure element predic-
tions were generated and compared to known crystal
structures in the d atabases. A h igh degree of structural
similarity w as obse rved between the arrange ment o f s ec-
ondary structure elements i n these proteins and glutaconate
CoA-transferase f rom Acid aminoc occus fermentans.
Keywords: Clostridium propionicum; alanine metabolism;
CoA-transferase; active site; thiol ester.
Clostridium propionicum has been isolated as an alanine
fermenting organism from the black mud of San Francisco
bay [ 1]. Th e fermentation products were acetate, ammonia,

ized as a homotetrameric enzyme (a
4
) with an apparent
molecular subunit m ass o f 6 7 kDa [6]. Although a prefer-
ence of (R)- lactate over (S)-lactate was observed, the
enzyme exhibited a rather broad substrate speci®city for
monocarboxylic acids including acrylate, propionate and
butyrate whereas dicarboxylic acids were not used.
The g eneral mechanism for the C oA-transferases has
been suggested to proceed via the successive formation o f
a m ixed anhydride between the C oA-donor carboxylic
acid an d an essential glutamate residue of the enzyme,
followed by t he formation of an e nzyme-CoA thiol ester
intermediate. The product is then f ormed by a n inverted
sequence of these steps with the acceptor carboxylate [7].
More recently, a number of CoA-transferases have been
discovered, which apparently do not follow this general
mechanism [8,9]. Formation of an enzyme-CoA thiol ester
can be detected either by site-speci®c cleavage of the
polypeptide chain [10], reduction of the t hiol ester w ith
sodium borohydride [11±13] or directly by mass spect-
rometry [14]. Alternatively the mixed anhydride interme-
diates can be detected by the formation of stable
derivatives of the catalytic glutamate and hydroxamic
acids [15] or indirectly by oxygen exchange experiments
[14,16].
To date, only the crystal structure of glutaconate CoA-
transferase (EC 2.8.3.12) from Acidaminococcus fermentans
has been solved. In contrast to the homotetrameric propi-
onate CoA-transferase of C. propionicum,theformer

chemicals were of the highest grade available and from
common commercial sources. C. propionicum (DSMZ
1682) was purchased from the German collection of
microorganisms and cell cultures (DSMZ, Braunsc hweig,
Germany).
Synthesis of acyl-CoA substrates
Acetyl-, butyryl- and propionyl-CoA were prepared from
the corresponding anhyd rides and CoA by the method of
Simon & Shemin [18]. All CoA-derivatives were puri®ed as
described previously [14].
Enzyme assay
The enzyme test for propionate CoA-transferase activity
was carried out at 25 °C as described previously [19].
Cultivation and storage of microorganisms
C. pr opionicum was cultivated in a complex medium
containing
D
,
L
-alanine as the s ole s ource of energy, as
described previously [6]. Freshly prepared anaerobic media
were inoculated with 5 to 20% stationary or late exponential
precultures and grown for 24 to 36 h at 37 °C. The cells
were harvested by centrifugation and stored at )80 °C.
Preparation of cell free extracts of
C. propionicum
Frozen cells (20 g) were suspended i n 100 mL of 25 m
M
potassium ph osphate, 1 m
M

. The solution was centrifuged for 45 m in at
100 000 g and app lied i n four aliquots to a Resource-Pheä
column (1 mL vol., Pharmacia) equilibrated with 1
M
(NH
4
)
2
SO
4
in buffer A. The column was washed with
5 m L of the starting buffer and the proteins were eluted in a
50-mL linear gradient from 1 to 0
M
(NH
4
)
2
SO
4
.The
pooled enzyme was dialysed overnight against 5 m
M
of each
boric, citric and phosphoric acids and 5 m
M
Tris adjusted to
pH 7.0 with KOH (buffer B) and then applied on a
Resource-Qä column (1 mL vol., Pharmacia) equilibrated
with buffer B. T he protein w as eluted by a linear gradient

acetate. The enzymes in volved are p yru-
vate:glutamate transaminase ( 1), g lutamate dehydrogenase (2)
(R)-lactate dehydrogenase (3), propionate C oA-transferase (4)
(R)lactoyl-CoA dehydratase (5), acrylyl-CoA reduc tase complex (6),
pyruvate:formate lyase (7), pho sphotransacetylase (8), acetate kinase
(9) and formate dehydrogenase (10). 2OG, 2-oxoglutarate. Ac-CoA,
acetyl-CoA. Ac-P
i
, acetylphosphate.
Ó FEBS 2002 Propionate CoA-transferase (Eur. J. Biochem. 269) 373
N-Terminal sequencing
Puri®ed e nzyme (20 lg) was subjected to SDS/PAGE and
blotted onto a poly(vinylidene di¯uoride) m embrane. A fter
staining of the membrane w ith Coomassie blue R 450, the
protein band was cut out and subjected t o N -terminal
sequencing by gas-phase Edman degradation.
Generation and puri®cation of internal peptides
The p uri®ed enzyme (20 0 lg) was freeze-dried, reductively
carboxymethylated and digested with trypsin as described
previously [14]. T he peptides were puri®ed by RP-HPLC
using a Supelco sil-LC318 column (4.6 ´ 250 mm, 5 lm,
300 A
Ê
) equilibrated with 0.1% ( v/v) tri¯uoroacetic acid and
eluted with a line ar gradient of 0±42% (v/v) acetonitrile
within 42 min. The elution of the peptides was monitored
simultaneously at 210 and 280 nm. Peptides exhibiting an
absorbance at 280 nm were re-applied to the same column
using an identical gradient in the presence of 0.1% (v/v)
hexa¯uoroacetone-ammonia, pH 6.0, and analysed by

2 m in at 25 °Cin50m
M
potassium phosphate, pH 7.0,
either in the presence o r absence of 100 l
M
propionyl-
CoA. Either hydroxylamine hydrochloride (pH 7.5,
200 m
M
®nal concentration) or sodium borohydride
(20 m
M
®nal concentration) were added and the reaction
was a llowed t o proceed for another 10 min at 25 °C.
Aliquots of t he samples were assayed for CoA-transferase
activity. The samples were reductively carboxymethylated
andthebufferwasexchangedbygel®ltrationtoyield
50 m
M
ammonium acetate, pH 8.0, 10% (v/v) acetonitrile.
Each sample was s plit into four equal volumes and
digested for 16 h at 37 °C i n the presence of either 2%
(w/w) chymotrypsin, endoproteinase AspN, endoprotein-
ase GluC ( V8 protease) or 2% ( w/w) trypsin. The samples
were acidi®ed with 0.01 vo l. of 2
M
tri¯uoroacetic acid and
analysed by MALDI-TOF-MS.
RESULTS
Puri®cation of propionate CoA-transferase

. In addition to the predominating polypeptide with an
apparent molecular mass of 60 kDa in SDS/PAGE, t wo
faint bands were observed around 40 and 20 kDa. These
additional bands were completely absent when the puri®ed
enzyme was incubated in t he presence of sodium acetate
followed by gel ®ltration on Sephadex G25, but accounted
for up to 30% of the total protein when the enzyme was
incubated with 100 l
M
propionyl-CoA, prior to sample
preparation. These data indicated that a small but signi-
®cant fraction of the puri®ed CoA-transferase w as trapped
as the enzyme-CoA thiol ester intermediate. It has been
Fig. 2. The p uri®cation of propionate CoA-tran sferase. ACoomassie
blue staine d 10% SDS/PAGE is shown. The arrows indicate the
position of two faint bands at 40 kDa and 20 kDa in lanes 2±4 which
are absent in lane 5. Cell free extract (lane 1, 50 lg), Q-Sepharose (lane
2, 10 lg), Resource-Phe (lane 3, 5 lg) , Resource-Q (lane 4, 5 lg). The
protein in lane 5 is ide ntical to lane 4 , but has been i ncub ated with
200 m
M
sodium acetate prior sample preparation.
374 T. Selmer et al. (Eur. J. Biochem. 269) Ó FEBS 2002
previously shown that glutamate thiol ester-containing
proteins, such as CoA-transferases and a2-macroglobulin,
are site-speci®cally cleaved at elevated temperature [22]; a
nucleophilic attack of the p eptidyl amide nitrogen of the
glutamyl residue to the thiol ester carbonyl, releases the thiol
forming a protein-bound 4-oxoproline residue. The peptide
bond between this residue and the preceding amino acid is

genomic DNA from C. propionicum in a k-ZAP-Express
vector. Two clones were isolated and excised from the phage
to yield the corresponding plasmid, which was subsequently
sequenced. The clones contained identical inserts of 2.7-kb
and e ncoded t he complete 524 amino-acid ORF corre-
sponding to the propionate CoA-transferase (Fig. 4).
According to the amino-acid sequence, an average molec-
ular mass of 56 553 Da and a n isoelectric point of 4.9 1 was
predicted for the encoded p rotein. B oth values w ere in
agreement with the observed molecular mass and the elution
of the enzyme during the ®nal puri®cation step.
In addition to the CoA-transferase gene, a second ORF
(lcdB), encoding 122 C-terminal amino acids of a protein
similar to the 2-hydroxyglutaryl-CoA dehydratase b subunit
of A. fermentans, was detected upstream of the transferase
gene. This gene probably encodes one subunit of the (R)-
lactoyl-CoA d ehydratase required in the reductive pathway
from alanine to propionate. Directly downstream of the
propionate CoA-transferase gene (pct), an AT-rich region
was found that resembles rho-independent termination
signals. The nucleotide sequence of the full insert has b een
deposited under t he accession number AJ276553 in the
EMBL nucleotide sequence database.
Sequence analysis
The amino-acid sequence of propionate CoA-transferase
was compared to o ther proteins in the database using the
BLAST
algorithm [24,25]. The protein was most similar t o a
putative acetoacetate:acetyl-CoA CoA-transferase from
B. ha lodu rans (B84137, 56% identity, 519 amino-acid

Resource-Q 1306 15 85 37 41
Table 2. N-terminal sequencing and PCR-primer deduction. The N-terminal amino acid sequences of the puri®ed, blotted protein and of two internal
peptides are shown. These sequ ences have been used to deduce a degenerated prime r pair for ampli®cation of a propionate CoA-transferase speci®c
probe f rom C. propionicum genomic DNA. M  AorC,N  A, G, C or T, R  AorG,H  A, C o r T, Y  CorT.
Region/peptide Amino-acid sequence Deduced PCR primer
N-Terminus MRKVPIITADEAAKLIK-D Sense: 5¢-ATGMGNAARGTNCCNATHATHACN
GCNGAYGCTGC-3¢
Peptide 1 YIAGHWATVPALGK Antisense: 5¢)CCNARNGCNGGNCANATNGCC-3¢
Peptide 2 GTYADESGNITFEKEVAPLEGTSV-QA Not used
Ó FEBS 2002 Propionate CoA-transferase (Eur. J. Biochem. 269) 375
Deinococcus radiodurans, B acillus s ubtilis, Streptomyces
coelicolor, Heliobacter pylori, Mycobacterium tuberculo-
sis, Haemophilus in¯uenzae and Clostridium acetobutylicum.
These latter e nzymes belong to the 3 -oxoadipate CoA-
transferase protein superfam ily and consist of t wo diff erent
subunits. The similarity of these latter sequences to propi-
onate CoA-tansferase was lower (23±28%) and restricted to
the larger subunit of these enzymes (232±255 amino-acids
overlap). However, when the C-terminal half of the amino-
acid sequence of propionate-CoA-transferase was used for
database search, t his part o f the sequ ence showed similarity
to the smaller subunits of the latter enzymes.
The catalytic glutamate residue of hetero-oligomeric
enzymes b elonging to the 3-oxoadipate CoA-transferase
superfamily is found in the small subun it and is located
within a so-called (S)ENG motif [11]. This characteristic
motif is not found in the sequence of propionate CoA-
transferase or i n any of the putative proteins from
B. halodurans, E. coli, A. pe rnix or S. aureus.Therefore,a
multiple sequence alignment o f the b subunits of 3-oxoadi-

M
) o r hydroxylamine (pH 7.5, 2 00 m
M
),
the en zyme w as rapidly and irreversibly inactivated. The
inactivation was strictly dependent on the presence of
propionyl-CoA.
The inactivated proteins and controls, which had been
incubated with the reagents but without propionyl-CoA,
were subjected to reductive carboxymethylation and desalt-
ed by gel ®ltration. Aliquots of t hese samples were digested
for 16 h at 37 °C with 2% (w/w) of either chymotrypsin,
endoprotease-AspN, en doprotease-GluC o r trypsin. The
peptides were analysed by MALDI-TOF-MS without
puri®cation. Altho ugh only around 30±50% of the predict-
ed peptides were detected in one particular digest, all four
samples together c overed the full amino-acid sequence
predicted by the gene.
The molecular masses of peptides carrying the proposed
catalytic glutamate 324 were found in all s amples except t he
endoprotease-GluC digest. The masses of these peptides
exclusively s howed differences for inactivate d samples and
controls. As summarized in T able 3, the derivatives showed
the predicted mass differences of )14 Da and +15 Da for
sodium borohydri de and hydroxylamine inactivated
enzyme, r espectively. In particular the observation of a
chymotryptic peptide comprising amino acids 322±338 was
very crucial, since this peptide contains the glutamate 324 as
the sole c arboxylate. Therefore, the tentative assignment of
glutamate 324 has been con®rmed by these experiments.

transferase from Bacillus halodurans and o ther proteins
with as yet u nknown function from Escherichia coli,
Aeropyrum pernix and Staphylococcus aureus. O ur data
strongly suggest that these genes encode CoA-transferases.
As shown in F ig. 4 , the proteins align well, and in
particular the glutamate residue 324 is c onserved a mong
all these proteins. It seems therefore reasonable to
conclude that these proteins form a novel subclass of
CoA-transferases. These en zymes lack t he characteristic
(S)ENG consensus motif of members of th e 3-oxoadipate
CoA-transferase superfamily and e xhibit either a homool-
igomeric or monomeric quarternary structure. It is
reasonable to s uggest that a gene fusion could h ave
occurred during the evolution of the former enzymes.
Such a natural g ene fusion has also been suggested for the
mammalian oxoadipate CoA-transferase [30]. In agree-
ment with this proposal, i t has been shown t hat the two
subunits of glutaconate CoA-transferase from A. fermen-
tans could b e f used with genetic tools to y ield an active
enzyme composed of a single polypeptide [31].
Fig. 4. Sequence alignment of propionate
CoA-transferase and similar p roteins derived
from genome projects. The p ropionate CoA-
transferase from Clostridium propionicum
(Cpro) is compared to proteins encoded in the
genomes o f Bacillus h alodurans (Bhal),
Eschrichia coli (Ecol) and Aeropyrum pernix
(Aper). The sequ ence s have b een align ed using
CLUSTALW
. Residues i dentical in at least three

the lack of this e lement (Fig. 5C) is not surprising when the
homooligomeric structure o f propionate CoA-transferase is
taken i nto a ccount. A n a dditional i nteresting difference
between both structures i s the lack of a subdomain formed
by two b sheets and one a helix connecting the bsheet 2 of
the b subunit, which carries the catalytic glutamate, with the
inner l ayer of a helices (Fig. 5B, triangles 3 and 4 and circle
3, respectively). This subdomain is thought to be involved in
the substrate binding of glutaconate CoA-transferase. It has
been suggested that two speci®c serine residues, Ser78 (in
subunit A) and Ser68 (in subunit B ), are involved i n the
formation of hydrogen bonds with the e-carboxylate of
glutaryl-CoA and t hat the latter s erine is located within this
subdomain. It is remarkable that both residues are appar-
ently replaced by stretches of rather hydrophobic residues in
propionate CoA-transferase and it is reasonable to conclude
that the additional subdomain in glutaconate CoA-trans-
Table 3. Mass spectrometry of peptides derived from controls and i nactivated p ropionate CoA -transferase. Propionate CoA-transferase w as i ncu-
bated in the absence (control) or presence of propionyl-CoA with either hydroxylamine (200 m
M
) or sodium borohydride (20 m
M
) as described in
Material and methods. The prote in was reductivel y carboxymethylated and subsequently digested with the proteases as indicated. The p eptides
were analysed by MALDI-TOF-MS as described elsewhere [14]. The molecular masses expected for the controls (E
324
 COOH), hydroxyl-
amine- (E
324
 CONHOH) or borohydride-treated (E

D
-
PSSM
provided by the Imperial Cancer Research Fold Recognition Server. These elements have
been aligned to the structure of glutaconate CoA-transferase. Elements homologous to the a subunit of glutaconate CoA-transferase are given in
black, those h omologous to the b subunit in white. T he a helices are shown by circles a nd the bsh eets by triangles. The position of t he catalytic
glutamates is marked by a dot.
378 T. Selmer et al. (Eur. J. Biochem. 269) Ó FEBS 2002
ferase represents an adaptation for t he binding o f a
dicarboxyl-CoA by the latter enzyme.
During the course of our research, an interesting obser-
vation was made. A ll attempts to exp ress the cloned gene
from C. propionicum in E. coli failed (A. Willanzheimer,
unpublished observations)
22
; the transformed E. coli cells
carrying an isopropyl thio-b-
D
-galactoside-inducible expres-
sion vector exhibited no g rowth defect until the p rotein was
induced by isopropyl thio-b-
D
-galactoside. Upon induction
of the protein, E. coli stopped growing. T hese observations
may point to a severe impairment of the cellular metabolism
of E. coli by the C. prop ionicum enzyme. Although the
reason for this impairment of growth for the h ost has not
been elucidated as yet, a likely explanation could be t he
formation of lactoyl-CoA and other short-chain fatty acid
CoA-thiolesters by the enzyme. Such reactions are predicted

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