From the Institut für Mikrobiologie,
Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Strasse
3, D-6099 Halle, Germany and the Forschungsstelle
"Enzymologie der Proteinfaltung" der Max-Planck-Gesellschaft,
Weinbergweg 22, D-06120 Halle, Germany
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ABSTRACT |
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Highly active D-proline
reductase was obtained from Clostridium sticklandii
by a modified purification scheme. The cytoplasmic enzyme had a
molecular mass of about 870 kDa and was composed of three subunits with
molecular masses of 23, 26, and 45 kDa. The 23-kDa subunit contained a
carbonyl group at its N terminus, which could either be labeled with
fluorescein thiosemicarbazide or removed by
o-phenylenediamine; thus, N-terminal sequencing became
feasible for this subunit. L-[14C]proline was
covalently bound to the 23-kDa subunit if proline racemase and
NaBH4 were added. Selenocysteine was detected in the 26-kDa
subunit, which correlated with an observed selenium content of 10.6 g-atoms in D-proline reductase. No other non-proteinaceous cofactor was identified in the enzyme. A 4.8-kilobase pair (kb) EcoRI fragment was isolated and sequenced containing the
two genes prdA and prdB. prdA coding for a
68-kDa protein was most likely translated as a proprotein that was
posttranslationally cleaved at a threonine-cysteine site to give the
45-kDa subunit and most probably a pyruvoyl-containing 23-kDa subunit.
The gene prdB encoded the 26-kDa subunit and contained an
in frame UGA codon for selenocysteine insertion.
prdA and prdB were transcribed together on a
transcript of 4.5 kb; prdB was additionally transcribed as
indicated by a 0.8-kb mRNA species.
D-Proline reductase (EC 1.4.1.6) is involved in amino
acid metabolism of several clostridia and catalyzes the reductive ring
cleavage of D-proline to 5-aminovalerate
(1-3).1 In a typical
Stickland reaction, this reduction is coupled to the oxidation of other
amino acids, but an utilization of electron donors like formate is also
possible (3, 5, 6). D-Proline reductase catalyzes the
cleavage of a carbon-nitrogen bond. A similar reaction is catalyzed by
glycine reductase, which cleaves glycine to acetyl phosphate and
ammonia (7, 8). Different reaction mechanisms were postulated for the
catalytic cascades of these enzymes (2, 3, 7, 9). D-Proline
is first activated by an enzyme-bound pyruvoyl group. Then, a
nucleophilic attack at the Clostridium sticklandii is the best characterized Stickland
type organism containing both D-proline reductase and
glycine reductase (3, 5, 17). In order to learn more about the regulation of proline and glycine reduction on a molecular level, we
started to purify D-proline reductase from C. sticklandii. So far, D-proline reductase has been
described as a membrane-bound homodecamer of 300 kDa containing a
pyruvoyl group in each subunit of 30 kDa (1-3). Evidence was presented
that the pyruvoyl moiety is located in a 4.6-kDa peptide that might be
connected to the enzyme by an ester bond between a serine and glutamate
residue (18, 19). Dithiols acted as electron donor for the purified D-proline reductase, whereas NADH is more effective than
dithiols in crude preparations (1). It was suggested that the electrons might be transferred by two proteins from NADH to D-proline
reductase, presumably involving an FAD-containing NADH dehydrogenase
and a 250-kDa iron-containing protein (3, 20). The specific activity of
D-proline reductase was 3 times higher in crude extracts if C. sticklandii was cultivated in the presence of selenite;
however, no selenium was detected in the purified enzyme preparation
(1).
During our studies, we developed a modified purification procedure for
D-proline reductase and characterized the enzyme as a
cytoplasmic protein with three subunits and a molecular mass of about
870,000 Da containing selenium in form of selenocysteine and a carbonyl
moiety, most probably a pyruvoyl group. These data were supported by
sequence analysis of the subunits of D-proline reductase
and their corresponding genes and gave a more complex and detailed view
of D-proline reductase than described so far (1, 3, 10,
20). The subunits of D-proline reductase were identified to
exhibit sequence similarities to the protein B of glycine reductase
(11) emphasizing a similar function of these proteins to split a
carbon-nitrogen bond.
Materials--
All chemicals were obtained from commercial
sources unless otherwise specified.
Growth of C. sticklandii--
C. sticklandii strain
HF, DSM 517T obtained from DSMZ (Braunschweig, Germany),
was grown in complex medium containing 50 mM D/L-proline (21) or in minimal medium at
30 °C as described previously (22); 1 µM selenite was
always added. D-Proline reductase was purified from cells
harvested in the late logarithmic growth phase. Escherichia
coli XL2 blue was obtained from Stratagene (Heidelberg, Germany)
and used for cloning purposes. It was grown in LB medium (23) or on
agar plates containing 1.5% (w/v) agar. Ampicillin was added (125 µg/ml) if needed;
isopropyl-1-thio- Enzyme Assay--
D-Proline reductase activity was
measured by quantifying the product 5-aminovalerate with
o-phthalaldehyde as described (24). The reaction mixture of
0.5 ml contained 100 mM potassium phosphate buffer, pH 8.0, 10 mM MgCl2, 20 mM DTT or NADH, 10 mM proline, and enzyme. The reaction was incubated at
30 °C and terminated after 30 min by addition of 0.3 ml of 5%
HClO4. After centrifugation to remove precipitated
material, 50 µl of the supernatant was added to 1.95 ml of the
fluorescence solution and the fluorescence was determined at 455 nm
using an excitation at 340 nm. The fluorescence solution was prepared
by adding 1 ml of an o-phthalaldehyde solution (80 mg/ml
ethanol) and 0.2 ml of 2-mercaptoethanol to 100 ml of 0.4 M
HBO3 buffer (pH 9.7, adjusted with KOH). One unit of
D-proline reductase activity converted 1 µmol of
D-proline to 5-aminovalerate in 1 min under standard
reaction conditions.
Purification of D-Proline Reductase--
About
30 g of cells were suspended in 50 ml of 50 mM
Tris-HCl buffer, pH 8.6, containing 1 mM EDTA and 1 mM DTT (TED buffer) and incubated with lysozyme (1 mg/ml),
DNase I (0.2 mg/ml), and PMSF (200 µM) for 30-45 min at
37 °C. This suspension was passed twice at 140 megapascals through a
French press cell. Unbroken cells and cell debris were removed by
centrifugation at 18,000 × g for 15 min. Subsequently,
the membrane fraction was removed by ultracentrifugation at
120,000 × g for 1 h. The resulting supernatant was termed crude extract. It was fractionated with solid ammonium sulfate between 25% and 60% saturation. Precipitates were collected by centrifugation at 20,000 × g for 20 min. The pellet
obtained at 60% saturation was dissolved in 50 ml of TED buffer
containing 0.7 M ammonium sulfate and applied to a
phenyl-Sepharose (Pharmacia, Freiburg, Germany) column (3.6 × 5.1 cm) equilibrated with TED buffer containing 0.7 M ammonium
sulfate. After washing the column with the same buffer, the enzyme was
eluted by a linear gradient of ammonium sulfate and then ethylene
glycol (0.7-0 M and 0-5% (v/v), respectively) in 1,150 ml of buffer. The active fractions were combined, concentrated to 30 ml, and dialyzed against TED buffer (3 × 2 liters). The solution
was applied to an aminohexane affinity column (Pharmacia) (5.6 × 2.4 cm) equilibrated with TED buffer containing 0.1 M KCl.
Activity was eluted with a linear KCl gradient (0.1-0.7 M)
in 300 ml. Active fractions were collected, concentrated, and applied
to a Superose-6 HR10/30 column (Pharmacia) equilibrated with TED buffer
containing 0.4 M KCl and eluted with the same buffer. The
active fractions contained the homogeneous enzyme and were used for
further characterization.
Molecular Mass Determination--
The native molecular
mass of D-proline reductase was estimated by gel filtration
using a fast protein liquid chromatography Superose-6 column
equilibrated with TED buffer containing 400 mM KCl and by
polyacrylamide gel electrophoresis. Calibration standards were blue
dextran (2,000,000), thyroglobulin (686,000), ferritin (440,000),
catalase (232,000), aldolase (158,000), and bovine serum albumin
(69,000). The molecular masses of the subunits of D-proline
reductase were determined by SDS-PAGE (25) and MALDI mass spectrometry
(Bruker-Franzen Analytik, Bremen, Germany).
Selenium and Metal Determination--
Selenium and metal content
of D-proline reductase was analyzed by ICPMS using an HP
4500 model (Hewlett Packard, Waldbronn, Germany).
Identification of Carbonyl Groups in Proline
Reductase--
Fluorescein thiosemicarbazide was used to detect
carbonyl groups (26). About 10 µg of purified protein in 10 µl were
incubated with 10 µl of potassium acetate buffer (300 mM,
pH 4.6) and 1 µl of fluorescein thiosemicarbazide (0.1% in
Me2SO) for 2 h at room temperature in the dark.
Protein was precipitated by chloroform/methanol (27) and separated by
SDS-PAGE. Carbonyl groups were identified by UV light on a
transilluminator or on a phosphorimager (Molecular Dynamics,
Krefeld, Germany).
Removal of N-terminal Reductive Coupling of D-Proline to
D-Proline Reductase--
14C-Labeled
L-proline (>8.33 GBq/mmol; Amersham, Braunschweig,
Germany) was used to identify the substrate-binding subunit of D-proline reductase. Purified D-proline
reductase (100 µg) and proline racemase present in crude extracts
(0.5 mg) were incubated under standard assay conditions (600 µl) with
[14C]proline. After 2 min of incubation at 30 °C, 150 µl of 0.2 M KBH4 was added to reduce the
adduct formed between enzyme and proline resulting in its covalent attachment.
Electrophoresis--
SDS-polyacrylamide gel electrophoresis was
performed as described (25). For molecular mass determination,
calibration kits from Sigma Chemie (Deisenhofen, Germany) were used as
standards. Proteins were stained with Serva Blue (Serva, Heidelberg, Germany).
Protein Analytical Methods--
Protein was determined by the
method of Bradford (29) with bovine serum albumin as a standard.
N-terminal amino acid sequence determinations were done by automated
Edman degradation in a protein sequencer (model 476A, Applied
Biosystems, Weiterstadt, Germany). To get internal amino acid sequence
information of proline reductase subunits, two approaches were used to
isolate internal peptides: (a) the subunits were separated
by SDS-PAGE and digested in the gel overnight at 37 °C with trypsin
in 120 mM NH4HCO3 buffer, pH 8.5;
(b) purified D-proline reductase (1 nmol) was
dissolved in 50 µl of 6 M guanidine hydrochloride
containing 250 mM Tris-HCl buffer, pH 8.5, reduced with 2.5 µl of 2-mercaptoethanol for 2 h in the dark at room temperature,
and alkylated with 2 µl of 4-vinylpyridine by incubation for 2 h
in the dark at room temperature. The pyridylethylated protein was
desalted and separated by RP chromatography. The 45-kDa subunit eluted
in a single peak; the 23- and 26-kDa subunits eluted close together.
The subunits were digested with endoproteinase LysC at 37 °C
overnight. After digestion the masses of the peptides were determined
by MALDI mass spectrometry on a reflectron-type time-of-flight mass
spectrometer (Bruker Franzen). A saturated solution of
Identification of Selenocysteine by Pyridylethylation--
To
derivatize the selenocysteine moiety of D-proline
reductase, protein was incubated in a volume of 50 µl containing 6 M guanidinium chloride, 0.25 M sodium phosphate
buffer, pH 7.0, 1 mM EDTA, 0.3 mg of
tricarboxyethylphosphine, and 2 µl of 4-vinylpyridine for 2 h at
37 °C in the dark under an N2 atmosphere, resulting in a
labeling of the selenocysteine residue and all cysteine residues. The
selenocysteine-containing peptide was desalted and subjected to Edman
degradation. A synthetic peptide containing selenocysteine (obtained
from S. Pegoraro, MPI für Biochemie, Munich, Germany) was used to
optimize the conditions for labeling and identification.
Standard Molecular Biology Techniques--
All standard
procedures were performed as described (23). Enzymes were used
according to the recommendations of the manufacturer. DNA sequence
determination was performed by the dideoxy chain-termination method
(30) using the Pharmacia ALF system (Pharmacia). Hybridizations were
carried out as described by the manufacturer using the DIG system from
Boehringer (Mannheim, Germany).
Cloning of the Genes Encoding D-Proline
Reductase--
The amino acid sequences obtained for
D-proline reductase were used to design degenerated primer
on the basis of the codon usage for C. sticklandii (31).
Primer 1 was 5'-AAAGARCATGCNAATGA-3', primer 2 was
5'-ATCRAAYTCYTCATCATTCATAACYTCAAT-3'. PCR amplification was carried out
using Taq DNA polymerase, an annealing temperature of
50 °C, and 30 cycles. A specific PCR fragment was obtained, cloned
into the pGEM-T vector (Promega, Mannheim, Germany), and sequenced. To
produce a genomic library of C. sticklandii, total DNA was
isolated (32), partially digested with EcoRI or
SauIIIA, and separated in a sucrose density gradient from
5% to 40% (w/v) sucrose for 24 h at 200,000 × g. 5-kb fragments of EcoRI-digested DNA and 3-kb
fragments of SauIIIA-digested DNA were isolated, dialyzed
against H2O, and cloned into the vector pBluescript SKII (Stratagene). After transformation of E. coli XL2 blue,
nearly 2400 EcoRI and 4000 SauIIIA clones were
obtained, which were stored as plasmid mixtures at Isolation of RNA from C. sticklandii and Northern Blot
Analysis--
C. sticklandii was grown in 30 ml of minimal
medium to midlogarithmic phase, harvested by centrifugation at
20,000 × g for 5 min, and RNA was isolated using the
RNeasy spin columns from Qiagen (Hilden, Germany). 10 µg of RNA was
separated in a denaturing agarose gel (1.5%) containing 37% formamide
and blotted onto a nylon membrane. Hybridization was done in 2× SSC
buffer containing 50% (v/v) formamide at 42 °C. The DIG system was
used for detection (Boehringer).
Localization of D-Proline Reductase--
After
disruption of cells from C. sticklandii by a French press
cell and ultracentrifugation, nearly all (99%) of the
D-proline reductase activity was recovered in the
supernatant, not in the membrane pellet. In the presence of high salt
(1 M KCl) or detergents (1.5% Triton X-100 or 1% sodium
deoxycholate), 0.2-1% of the total activity was present in the
pellet. Similar results were obtained using different methods for cell
disintegration (lysozyme, sonication) or preparation of protoplasts
before separation of cytoplasmic and membrane fractions (data not
shown). Substrate combinations or growth phase using complex or
synthetic media had no influence on the localization of
D-proline reductase activity being in all cases present in
the cytoplasmic fraction (data not shown).
Purification of D-Proline Reductase--
Compared with
previous results (1), an improved purification scheme was
established for D-proline reductase (Table
I). Ultracentrifugation, chromatography
on phenyl-Sepharose, and affinity chromatography with
aminohexane-Sepharose were highly effective for purification.
Subsequently, gel filtration on Superose-6 resulted in a homogeneous
enzyme preparation as judged by polyacrylamide gel electrophoresis
(Fig. 1). However, nearly 90% of the
activity was lost during the final gel filtration step. Other methods
tried did not remove impurities or give higher yields.
Purified D-proline reductase was highly specific for
D-proline; L-proline was not reduced to
5-aminovalerate. Due to an active proline racemase present in C. sticklandii (33), crude extracts exhibited D-proline
reductase activity even with L-proline as substrate. DTT
was used as artificial electron donor throughout purification.
NADH-dependent activity was nearly 3 times higher in crude
extracts but vanished after ammonium sulfate precipitation. The
proteins that were involved in electron transfer from NADH to
D-proline reductase were also present in the cytoplasm
(data not shown).
Molecular Mass and Subunit Structure of D-Proline
Reductase--
The molecular mass of D-proline reductase
was calculated using gel filtration on Superose-6 to be about 870 kDa
(data not shown). Native gel electrophoresis of the purified enzyme
showed a single band, which barely invaded into the gel also indicating a high molecular mass (data not shown). To completely denature D-proline reductase, as indicated by SDS-polyacrylamide gel
electrophoresis, the enzyme had to be incubated for 60 min at 94 °C.
Three subunits of D-proline reductase were found under
these denaturing conditions. Their molecular masses determined by
SDS-polyacrylamide gel electrophoresis (Fig. 1) and mass spectrometry
were 23, 26, and 45 kDa. Thus, a decameric structure can be anticipated
for the protomers of D-proline reductase, assuming that all
three subunits are present in the same stoichiometry. Attempts failed
to separate these subunits with detergents other than SDS (data not
shown). The subunits of D-proline reductase were
N-terminally sequenced (Fig. 2). However, the 23-kDa subunit was blocked but could successfully be deblocked by
treatment with o-phenylenediamine. To obtain further
sequence information, internal peptides were analyzed after digestion
with trypsin and separation by RP chromatography. Some isolated
peptides were N-terminally sequenced (Fig. 2), and nearly all peptides were analyzed by mass spectrometry and identified after the DNA sequence was available (data not shown).
Cofactor Content of D-Proline
Reductase--
Absorption spectra of purified D-proline
reductase showed only a peak at 280 nm but no characteristics that
might indicate bound cofactors like flavins, pyridoxal phosphate or
Fe/S centers. The addition from 1 µM up to 1 mM of Fe2+, FAD, FMN, NAD, and pyridoxal
phosphate did not stimulate enzyme activity. Mg2+ (10 mM) was essential for the reaction, but cations like
K+ (34) had no effect on enzyme activity. ICPMS was
employed to analyze the metal content of D-proline
reductase. However, no metal but 10.6 g-atom selenium per 870 kDa was
identified by ICPMS.
Detection of Selenocysteine and Cysteine--
To identify
selenocysteine as possible selenium compound, D-proline
reductase was pyridylethylated with 4-vinylpyridine and the
selenocysteine-containing peptide from Glu140 to
Lys212 of PrdB was isolated. Using normal reducing
conditions with 2-mercaptoethanol, no PTH amino acids were detected at
position Sec152 and Cys155, although cysteine
residues from other peptides were labeled under these conditions and
identified during amino acid sequencing. Therefore,
tricarboxyethylphosphine was used as reducing agent because it does not
react with 4-vinylpyridine and the selenol group can be directly
pyridylethylated after its formation. The peptide was desalted and
again alkylated, and Edman degradation was repeated (Fig. 2). Now, the
pyridylethylated selenocysteine eluted exactly at the position that was
revealed for selenocysteine using a synthetic peptide as standard. The
retention time was close to proline as standard during amino acid
sequencing (35). The cysteine adjacent to selenocysteine became also
labeled after the drastic reduction with tricarboxyethylphosphine. This
indicates that a mixed selenide-sulfide might be formed between
Sec152 and Cys155 preventing pyridylethylation
under mild reducing conditions (2-mercaptoethanol) even when the enzyme
was denatured.
Presence of Carbonyl Groups in D-Proline
Reductase--
Carbonyl groups were detected in the 23- and 26-kDa
subunit using labeling with fluorescein thiosemicarbazide (Fig.
3A). The 23-kDa subunit became
susceptible for N-terminal sequencing after treatment with
o-phenylenediamine that removes specifically Labeling of D-Proline Reductase with
[14C]Proline--
In all reaction mechanisms proposed so
far for D-proline reductase, the pyruvoyl group is supposed
to bind the substrate proline (1-3). By addition of NaBH4,
an adduct should be formed that is covalently bound to the enzyme.
Because only L-[14C]proline is commercially
available, crude extract of C. sticklandii containing
proline racemase (33) had to be added to allow labeling experiments.
The 23-kDa subunit became labeled to a high extent under such
conditions, indicating that it reacted with proline (Fig.
3B). Controls using a high molar excess of unlabeled proline (500/1) or no NaBH4 showed no labeling of the 23-kDa
subunit (Fig. 3B). If no NaBH4 was added, the
26-kDa subunit became slightly labeled, indicating that proline or a
reaction derivative might also be bound to this subunit during the
reaction cycle (Fig. 3B).
Cloning of the Genes Encoding D-Proline
Reductase--
The protein sequences obtained from the three subunits
were used to isolate the genes encoding D-proline
reductase. A homologous DNA fragment of 1.2 kb was generated by PCR
using DNA of C. sticklandii as a template and was identified
by sequencing to encode part of the prd-operon. This
fragment was used as a probe to isolate a 4.8-kb EcoRI clone
from the genomic library of C. sticklandii (Fig.
4). Southern blot hybridization revealed
that the genes encoding D-proline reductase were present as
single copy in the genome of C. sticklandii (data not
shown).
Identification and Characterization of the prd Operon--
Five
open reading frames were identified on the 4.8-kb DNA fragment (Figs. 2
and 4). The open reading frame prdA encoded a protein of 630 amino acids. The amino acid sequences determined for fragments of the
45-kDa subunit of D-proline reductase corresponded exactly
to the deduced sequence from Val1 to Thr425
(Fig. 2); the amino acids sequences obtained from the deblocked 23-kDa
subunit corresponded to the deduced amino acid sequence from
Ile427 to Lys630 of prdA (Fig. 2).
The C-terminal peptide of the 45-kDa subunit stopped with
Thr425. After removal of the N-terminal carbonyl group, the
N terminus of the 23-kDa subunit started with Ile427 (Fig.
2). Cys426 was encoded by prdA but not detected
in one of the peptides; therefore, this amino acid should be the
precursor of the
The open reading frame prdB encoded a protein of 242 amino
acids (25.6 kDa) corresponding to N-terminal and internal protein sequences determined for the 26-kDa subunit of D-proline
reductase (Fig. 2). An in-frame TGA codon was present that should code
for selenocysteine as was identified by amino acid determination.
The open reading frame orfX' upstream of prdA
encoded 237 amino acids of a truncated C-terminal part of a protein
that did not correspond to amino acid sequences obtained for subunits
of D-proline reductase. The sequence of orfX'
stopped at the EcoRI site at the 5' end of the clone. No
function of this protein can be derived from comparisons with sequences
deposited in data banks. Two putative open reading frames
orfY and orfZ' were identified downstream of
prdB (Figs. 2 and 4), which exhibited high similarities to
N-terminal and C-terminal parts of the prdA gene,
respectively (Fig. 5). They were
interrupted by a short stretch of nucleotides, which was localized at
the Cys426 site, the potential cleavage site of PrdA as
became evident from sequence alignments (Fig. 5).
orfX' was separated from prdA by 117 bp; the
region between prdA and prdB was 287 bp. The
start codon of prdA and prdB is preceded by a
sequence resembling the Shine-Dalgarno motif for ribosome binding (Fig.
2) (36). The region directly upstream of prdB contained a
putative promotor sequence TTAATG-(14 bp)-TAATAT, which was located 32 bp upstream of the start codon. A few bp upstream of this promotor, a
sequence motif resembling the structure of a typical E. coli
To analyze the transcription of these genes, probes were deduced
from prdA and prdB and used in Northern blot
experiments for hybridization against total RNA from C. sticklandii (Fig. 6). Both probes
hybridized with a 4.5-kb mRNA, indicating that prdA and
prdB formed a transcription unit. Additionally, the
prdB probe hybridized with a 0.8-kb mRNA that should be
an extra transcript of this 26-kDa selenoprotein.
In this study, D-proline reductase was found to be
located exclusively in the cytoplasm of C. sticklandii. In
contrast to former studies (1), no indications were now obtained that
the DTT-dependent D-proline reductase might be
a membrane-bound enzyme. At least it was readily soluble if associated
with the cytoplasmic membrane. This might apply also for the proteins
involved in electron transfer from NADH to D-proline
reductase. In our studies, these proteins were present in the soluble
cytoplasmic fraction after separation by ultracentrifugation, as was
also shown by another group (20). An association with the cytoplasmic
membrane might be indicated by a report that proline reduction was
coupled to the extrusion of H+ ions in
Clostridium sporogenes (40).
The D-proline reductase from C. sticklandii was purified by a modified purification scheme to
homogeneity. The enzyme exhibited a molecular mass of about 870 kDa and
consisted of three different subunits with molecular masses of 23, 26, and 45 kDa. This is in sharp contrast to earlier studies (1), where
D-proline reductase was characterized as homodecamer with a
molecular mass of 300 kDa consisting of one pyruvoyl-containing subunit
of 30 kDa (1, 2). The now obtained 23-kDa subunit also seems to contain
a pyruvoyl group, thus, it might be identical to the reported 30-kDa subunit. However, the amino acid composition reported for the 30-kDa
subunit (1) is strikingly different from the amino acid composition of
the 23-kDa (or the 26-kDa) subunit characterized in this study. The
three subunits present in our preparation were essential components of
active D-proline reductase, as indicated by the fact that
the specific activity for purified D-proline reductase was
much higher in our preparation (16 µmol min So far, we have no explanation for these inconsistent data, especially
for the fact that the enzyme prepared during earlier studies was still
active containing just one subunit. The strikingly lower specific
activity of the former preparations (1, 20) might indicate that the
other two subunits should have been present in the previous preparation
too, perhaps in a much lower non-detected amount; thus, both were not
identified as subunits of D-proline reductase. The
decameric structure previously proposed corresponds to our
calculations. However, the reported native molecular mass deviates
dramatically for both preparations, being about 300 kDa in former
studies (1, 20), but about 870 kDa in this study.
Genes encoding D-proline reductase have not been cloned
before, nor have they been identified during genome sequencing. Thus, for the first time the sequence of a D-proline reductase is
reported. The amino acid sequence data obtained by Edman degradation
and mass spectrometry were compared with the DNA sequence, revealing a
highly refined sequence of D-proline reductase. Subunits of D-proline reductase exhibited only similarities to the
subunits of the substrate-binding protein B of glycine reductase, which is encoded by grdEB (11) (Figs. 5 and
7). PrdB exhibited similarities (23%
identity) to GrdB (11), especially the region around the selenocysteine
was conserved; however, grdB encodes an extension of about
200 amino acids at its N terminus that was not present in
prdB. PrdA was similar to GrdE (16% identity), for both
proteins are posttranslationally cleaved to give a 23- and a 22-kDa
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-carbon of proline was presumed to be
carried out by an electron-donating dithiol (like
DTT)2 and the ring is cleaved
to give 5-aminovalerate after hydrolytic release from the pyruvoyl
adduct. Glycine reductase is composed of the proteins A, B, and C;
protein B binds glycine as Schiff base via an enzyme-bound carbonyl
group (11). A selenol anion attacks the
-carbon, and subsequently
the carbon-nitrogen bond is cleaved to give ammonium and probably a
protein B-bound carboxymethylselenoether that is transferred to protein
A. By action of protein C, the carbon unit is transformed to a protein
C-bound acetyl-enzyme intermediate leaving an oxidized protein A (12).
Subsequently, the acetyl group is released as acetyl phosphate from
protein C and its energy can be conserved by acetate kinase (13). To start a new reaction cycle, protein A is reduced by the thioredoxin system that obtains its electrons from NADPH (14). The genes encoding
protein A, protein B, protein C, thioredoxin, and thioredoxin reductase
are organized in an operon-like structure (11, 15). For
D-proline reductase, the existence of an analogous high
energy acyl-enzyme intermediate has been excluded, indicating that
proline reduction is not coupled to energy conservation by substrate
level phosphorylation (10). Despite this energetic disadvantage,
proline is preferentially utilized from a synthetic growth medium
containing both proline and glycine (16).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside (40 µg/ml) and
5-bromo-4-chloro-3-indolyl
-D-galactopyranoside (48 µg/ml) were added if necessary.
-Keto Acids--
-Keto acids can be
selectively removed from the N terminus of proteins by
o-phenylenediamine (28). One volume of D-proline reductase (10 µg) was incubated with two volumes of sodium acetate buffer (750 mM, pH 4.5) containing 120 mM
o-phenylenediamine for 17 h at 37 °C. For Edman
degradation, the protein was precipitated with chloroform/methanol (27)
and purified by SDS-PAGE.
-cyano-4-hydroxycinnamonic acid was used as matrix. Peptide
separation was done by RP chromatography using an ET 125/2 nucleosil
500-5C3 PPN column (Macherey & Nagel, Düren, Germany) and a high
pressure liquid chromatography system (Shimadzu, Duisburg, Germany).
Protein was precipitated by chloroform/methanol (27): to one volume of
protein solution, four volumes of methanol, three volumes of
H2O, and one volume of chloroform were added. After
centrifugation (10,000 × g, 1 min), the supernatant
was decanted and methanol (three volumes) was added. After another centrifugation, the precipitated protein was dried.
20 °C.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Purification of D-proline reductase from C. sticklandii
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Fig. 1.
Purification of D-proline
reductase from C. sticklandii. An
SDS-polyacrylamide gel (12%) electrophoresis was performed with the
different preparations obtained during purification of
D-proline reductase. Lane 1, marker proteins,
the molecular masses are shown in kDa; lane 2, crude
extracts (30 µg of protein); lane 3, ammonium sulfate
precipitation (30 µg of protein); lane 4, phenyl-Sepharose
(20 µg of protein); lane 5 and 6,
aminohexane-Sepharose (5 µg of protein); lane 7,
Superose-6 (5 µg of protein). The proteins were stained with Serva
Blue.
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Fig. 2.
Nucleotide and amino acid sequence of
D-proline reductase operon. Start and stop codons and
names of the open reading frames are in bold. Promotors,
putative ribosome binding sites, the proposed termination loop, and
protein sequences determined by Edmann degradation are underlined. Special sites are indicated:
SD, putative ribosome binding site; pyr,
precursor of the -keto acid that was removed by
o-phenylenediamine treatment; Sec, codon encoding
selenocysteine.
-keto acids
from the N terminus of proteins (25). Subsequently, the amino acid
sequence IGPASKENSRH ... (Fig. 2) was determined that corresponded
exactly to an internal region of the prdA gene product. Cys426 was the amino acid residue upstream of this sequence
as identified by DNA sequencing (Fig. 2). Thus, the terminal carbonyl
group in the 23-kDa subunit most likely derived from this cysteine
residue, which could be transformed to an N-terminal pyruvoyl group.
The 23-kDa subunit might therefore correspond to the
pyruvoyl-containing 30-kDa subunit previously described for
D-proline reductase (1). However, in former studies only
one subunit of D-proline reductase was reported (1) and the
pyruvoyl moiety was assigned to a small peptide of 4.6 kDa that could
be separated from the subunit after treatment with mild alkali (18,
19). We did not obtain such a peptide using similar conditions. The
carbonyl function associated with the 26-kDa subunit (Fig.
3A) could so far not be assigned to a specific amino
acid.
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Fig. 3.
Labeling of D-proline reductase
with fluorescein thiosemicarbazide (A) and
[14C]proline (B). A,
purified D-proline reductase was separated in a 12%
SDS-polyacrylamide gel and labeled with fluorescein thiosemicarbazide.
Lane 1, marker proteins, molecular masses are indicated in
kDa; lane 2, D-proline reductase (0.5 µg)
stained with Serva Blue; lane 3, D-proline
reductase (0.5 µg) labeled with fluorescein thiosemicarbazide.
B, D-proline reductase was labeled in the
presence of proline racemase with [14C]proline and
separated in a 12% SDS-polyacrylamide gel. The gel lane was cut into
pieces, and the radioactivity was determined in a scintillation
counter. Black columns represent an experiment
with NaBH4 added to the reaction mixture; open
columns represent an experiment without NaBH4
(control). Beside the diagram, a sample of D-proline
reductase separated in an SDS-polyacrylamide (12%) gel is shown as
reference. The molecular masses of the subunits are indicated.
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Fig. 4.
Structure of the sequenced
D-proline reductase gene region. The locations of the
cleavage site in prdA and of the selenocysteine in
prdB are indicated. The genes, the putative open reading
frames, and a possible organization of the transcripts are shown.
-keto acid blocking the 23-kDa subunit. The DNA
sequence upstream of Cys426 did not contain a typical
ribosome binding site or a start codon in a suitable distance. Thus,
prdA should encode a proprotein (
subunit) that is
posttranslationally cleaved to form the 45-kDa subunit and the
N-terminally blocked carbonyl-containing 23-kDa subunit of
D-proline reductase.
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Fig. 5.
Alignment of amino acid sequences of the
proproteins of D-proline reductase and glycine
reductase. The designations correspond to: PrdA, proprotein of
D-proline reductase from C. sticklandii; GrdE,
proprotein of the substrate-binding protein B of glycine reductase from
Eubacterium acidaminophilum (11); OrfY/OrfZ', open reading
frames localized downstream of the D-proline reductase
genes. Identical amino acid residues are printed in bold.
Between OrfY and OrfZ', the non-coding region is indicated by .
OrfZ' started with Val3. The N-terminal extension of 180 amino acids in PrdA is not shown.
54 promotor was identified (Fig. 2). No promoter-like
structure was identified upstream of prdA. The sequence
located downstream from prdB contained two inverted repeats
with the potential to form a hairpin structure. This structure is
similar to the Rho-independent transcription termination signal in
E. coli (37). orfY started downstream of this
secondary structure (62 bp downstream of prdB) and was separated from
orfZ' by a short nucleotide stretch. Both sequences were
preceded by a putative ribosome binding site, but no promotor-like
structure was identified. A mRNA structure similar to the secondary
mRNA structure necessary for selenocysteine incorporation in
E. coli (38, 39) was not identified in the prdB
gene, similar to the situation in grdA and grdB
(11) of glycine reductase.
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Fig. 6.
Northern blot analysis of the
D-proline reductase gene region. Total RNA (20 µg)
was isolated from C. sticklandii, separated, blotted onto a
nylon membrane, and hybridized. A PCR-amplified 0.5-kb DNA fragment
from an internal region of prdA (lane 2) or a
PCR-amplified 0.3-kb DNA fragment from an internal region of
prdB (lane 3) were used as probes. A RNA marker
is shown in lane 1, and the length of the marker fragments
are indicated in kb.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 mg
protein
1) than reported before varying between 0.08 to
0.26 µmol min
1 mg protein
1 using the same
assay with DTT as electron donor (1, 20). A more direct evidence was
obtained by the organization of the genes prdA and
prdB, forming an operon structure. The close relation of the
45- and 23-kDa subunits is also given by the identity of the amino acid
sequences of the analyzed peptides to the deduced sequence of
prdA indicating their translation as proprotein PrdA. A
threefold stimulation of the specific activity of D-proline reductase was observed in extracts if C. sticklandii was
grown in the presence of selenite (1). However, no labeling of
D-proline reductase by 75Se selenite was
detected in the former final preparation (1). We demonstrated that the
26-kDa subunit (PrdB) actually contained selenocysteine by identifying
this particular amino acid as pyridylethylated derivative. An in-frame
TGA codon in prdB (41) and the selenium content of the
holoenzyme as determined by ICPMS were two additional proofs for
D-proline reductase being a selenoprotein.
-keto acid (pyruvoyl)-containing subunit, respectively (Fig. 5). The N-terminal part (about 180 amino acids) of the prdA gene
product is not present in grdE; thus, both proproteins
differ substantially in mass (68 versus 48 kDa).
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Fig. 7.
Alignment of the selenocysteine-containing
subunit of D-proline reductase (PrdB) and the 47-kDa
subunit of protein B of glycine reductase. The designations
correspond to: PrdB, 26-kDa subunit of D-proline reductase
from C. sticklandii; GrdB, selenocysteine-containing subunit
of protein B of glycine reductase from E. acidaminophilum
(11). The selenoprotein A-like motif -UXXC- is underlined,
and identical residues are printed in bold. The N-terminal
extension in GrdB of about 200 amino acids is not shown.
prdA and prdB are transcribed together on a 4.5-kb mRNA that stops most likely with the stem loop structure downstream of prdB. In this case orfX' is probably cotranscribed with the structural genes of D-proline reductase. A possible function of orfX' is not obvious from the truncated sequence. The extra transcript of 0.8 kb for prdB might be necessary to achieve a sufficient level of the 26-kDa subunit due to the peculiarities of selenocysteine incorporation (41). The open reading frames orfY and orfZ' exhibited high similarities to both proteins of the prdA gene (Fig. 5). So far, no function can be assigned to these putative proteins.
The PrdA protein was translated as a proprotein that is cleaved to the
23- and 45-kDa subunits of D-proline reductase. The -keto acid blocking the new N terminus of the 23-kDa subunit should
be a pyruvoyl group because a cysteine residue was detected as the
precursor of this group. In previous studies (18), a labeled pyruvoyl
group was identified in D-proline reductase if C. sticklandii was grown in the presence of
[14C]serine, indicating that either serine or cysteine is
the precursor of this group. It is well established (42-45) that all
known pyruvoyl-containing enzymes are translated as a proenzyme (
subunit) that undergoes a cleavage reaction to generate two subunits
with the pyruvoyl group at the N terminus of the former C-terminal part
of the proprotein. In all these enzymes, a serine residue has been
identified as the precursor of the pyruvoyl group. In mutants of
phosphatidylserine decarboxylase of E. coli and human and
plant S-adenosylmethionione decarboxylase proenzymes, this serine
residue was exchanged by site-directed mutagenesis to threonine or
cysteine. In these mutants, processing of the proprotein occurred
in vivo and in vitro with 10-200 times decreased
processing rates (45, 46). In case of D-proline reductase,
the
subunit is encoded by prdA and the precursor of the
pyruvoyl group is a cysteine residue according to the derived sequences
as observed for GrdE of glycine reductase (11). The cleavage has to
occur between Thr425 and Cys426 via a thioester
(42, 43) to generate the observed
-keto acid group. Most likely, the
substrate proline reacted with this group, as indicated by the labeling
of the 23-kDa subunit with [14C]proline after reduction
with borohydride. D-Proline reductase and protein B of
glycine reductase (11) are so far the only known examples of
pyruvoyl-containing enzymes where the
subunit is split at the
N-terminal site of a cysteine residue and, thus, cysteine is the
precursor of the pyruvoyl group. Therefore, both enzymes should be
assigned to a new subclass of pyruvoyl-containing enzymes.
Based on structural and sequence data presented in this paper, we
postulate a modified reaction mechanism for the reduction of proline
involving both selenocysteine and cysteine moieties present in PrdB
(Fig. 8, A-E). First, an
adduct between the nitrogen of proline and the pyruvoyl group of the
23-kDa subunit is formed (B) (2, 3, 10). The formation of a
Schiff base might also be possible (2, 3), but according to Ref. 10 it
is not shown. The selenol anion of selenocysteine in PrdB attacks
nucleophilically the -carbon, resulting in cleavage of the N-C bond
of the proline ring (C) similar to protein B of glycine
reductase (11). This intermediate would be transformed to the oxidized
26-kDa protein containing a mixed selenide/sulfide group and to the
5-aminovalerate adduct at the 23-kDa protein (D). The final
product, 5-aminovalerate, is formed by hydrolysis (E).
Subsequently, the selenide-sulfide group of PrdB is reduced by,
e.g., artificial electron donors like DTT. The natural
electron donating system for D-proline reductase is quite
unknown (3, 20), except that NADH, not NADPH, is effective in crude
extracts. Thus, in contrast to glycine reductase (7, 11), electrons
will not be transferred via the strictly NADPH-dependent
thioredoxin system of C. sticklandii (4). Further studies
must prove if the protein A-like motif
(-U-X-X-C-) is actually a redox-active site in
case of D-proline reductase. However, the resistance of
Sec152 and Cys155 against alkylation after
reduction with 2-mercaptoethanol but not after reduction using
tricarboxyethylphosphine indicates a redox-active function of this
group. The presence of an NAD-dependent dihydrolipoamide
dehydrogenase (16) besides an NADPH-dependent thioredoxin
reductase (4) in C. sticklandii would be in line with the
observed indication that an FAD-containing protein might be involved in
proline reduction (3). In D-proline reductase, no
energy-rich intermediate or phosphorylated product has been identified,
in contrast to glycine reductase (10, 12, 13). Hence, one main
difference between proline and glycine reduction is the capability to
conserve energy by substrate level phosphorylation in case of glycine
reduction. However, for C. sporogenes, it has been shown
that proline reduction is coupled to the formation of a pH gradient
across the cytoplasmic membrane and, thus, proline reduction might be
used for energy conservation by a chemiosmotic mechanism (40).
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ACKNOWLEDGEMENTS |
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We thank Peter Planitz, Hewlett Packard GmbH, Waldbronn, Germany, for the ICPMS analysis and Melanie Friedenberger for technical assistance. We also thank S. Pegoraro from the Max-Planck-Institut für Biochemie, Martinsried, for providing us with a synthetic peptide containing selenocysteine.
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FOOTNOTES |
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* This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AJ130879.
§ To whom correspondence should be addressed. Tel.: 49-3455526350; Fax: 49-345-5527010; E-mail: j.andreesen{at}mikrobiologie.uni-halle.de.
1 Kenklies, J., Ziehn, R., Fritsche, K., Pich, A., and Andreesen, J. R., Microbiology 145, 819-826.
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ABBREVIATIONS |
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The abbreviations used are: DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; MALDI, matrix-assisted laser desorption ionization; kb, kilobase pair(s); bp, base pair(s); ICPMS, inductively coupled plasma mass spectrometry; PTH, phenylthiohydantoin; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; RP, reversed phase.
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