(Received for publication, November 18, 1996)
From the Mikrobielle Genetik, Universität Tübingen, Waldhäuserstrasse 70/8, 72076 Tübingen, Germany
One of the steps involved in the biosynthesis of the lantibiotic epidermin is the oxidative decarboxylation reaction of peptides catalyzed by the flavoenzyme EpiD. EpiD catalyzes the formation of a (Z)-enethiol derivative from the C-terminal cysteine residue of the precursor peptide of epidermin and related peptides. The UV-visible spectra of the reaction products of EpiD are pH-dependent, indicating that the enethiol side chain is converted to an enethiolate anion. The pKa value of the enethiol group was determined to be 6.0 and is substantially lower than the pKa value of the thiol side chain of cysteine residues. The increased acid strength of the enethiol side chain compared with that of the thiol group is attributed to the resonance stabilization of the negative charge of the anion.
Several new posttranslational modification reactions, such as
dehydration of serine and threonine residues, thioether formation, and
formation of D-alanine residues from L-serine
residues, are involved in the biosynthesis of the ribosomally
synthesized lantibiotics (reviewed in Refs. 1 and 2). The lantibiotic
epidermin contains the ,
-unsaturated amino acid
didehydroaminobutyric acid,
S-[(Z)-2-aminovinyl]-D-cysteine, 3-methyl-lanthionine, and meso-lanthionine (3). The
formation of epidermin from the precursor peptide EpiA (4) includes the oxidative decarboxylation of the C-terminal cysteine residue of EpiA to
a (Z)-enethiol catalyzed by the FMN-containing enzyme EpiD
(5, 6). Two reducing equivalents from the C-terminal cysteine residue
are removed, a double bond is formed, and the coenzyme FMN is reduced
to FMNH2. The decarboxylation occurs spontaneously or is
also catalyzed by EpiD. The (Z)-enethiol derivative is the intermediate in the formation of the C-terminal
S-[(Z)-2-aminovinyl]-D-cysteine residue of epidermin (6). The unusual enethiol structure has been
confirmed by using mass spectrometry, tandem mass spectrometry, UV-visible spectroscopy, two-dimensional NMR spectroscopy, and conversion of the enethiol to a mixed disulfide with
5,5
-dithiobis(2-nitrobenzoic acid) (Ellman's reagent (7, 8)). EpiD
has a low substrate specificity, and most of the peptides with the
sequence [V/I/L/(M)/F/Y/W]-[A/S/V/T/C/(I/L)]-C at the C terminus
are substrates of EpiD, as elucidated by analysis of the reaction of
EpiD with single peptides and peptide libraries, respectively (7).
Here we report the further characterization of the reaction products of EpiD. We demonstrate that at physiological pH, the dominant form of the reaction product is the enethiolate anion and that the pKa value of the enethiol group is 6.0. We assume that the enethiolate anion is strongly nucleophilic and is the substrate for the Michael addition-like formation of S-[(Z)-2-aminovinyl]-D-cysteine. Furthermore, we discuss the possibility that enzymes exist in nature that catalyze the formation of enethiol groups from non-C-terminal cysteine residues.
SFNSYVC and SFNSYTC were synthesized as
described previously (7). For generation of the enethiol derivative of
SFNSYVC, approximately 25 mg of peptide were dissolved in 50 ml of 20 mM Tris/HCl (pH 8.0) containing 4 mM
DTT1 and 1 mg of MBP-EpiD (5). For generation
of the enethiol derivative of SFNSYTC, approximately 12 mg of peptide
and 500 µg of MBP-EpiD were used. After 25 min of incubation at
37 °C, 500 µl of trifluoroacetic acid was added. The reaction
mixture was then separated by reversed-phase chromatography using a
ProRPC HR10/10 column (Pharmacia Biotech Inc.). Peptides were eluted
with a linear gradient of 0-80% acetonitrile, 0.1% trifluoroacetic
acid in 150 ml at a flow rate of 2 ml/min. Peptides with a high
absorbance at 260 nm were collected and diluted with H2O,
0.1% trifluoroacetic acid. DTT was added to 3 mM, and the
samples were subjected to a second reversed-phase chromatography using
a µRPC C2/C18 SC 2.1/10 column (Pharmacia). Peptides were eluted with
a linear gradient of 0-50% acetonitrile, 0.1% trifluoroacetic acid
in 5.8 ml at a flow rate of 200 µl/min. The absorbance was measured
simultaneously at 214, 260, and 280 nm. The modified peptide was
identified by the high
A260/A214 ratio and
collected by the peak fractionation method using the
SMARTTM system (Pharmacia, Freiburg, FRG). Samples were
dried with a vacuum concentrator (yields: approximately 4.4 mg of
SFNSYV-NH-CH=CH-SH and 1 mg of SFNSYT-NH-CH=CH-SH, respectively)
and stored at 70 °C. The modified peptides were analyzed by
electrospray mass spectrometry as described recently (7).
UV-visible spectra were obtained with a Beckman DU 7500 spectrophotometer. Spectral data were recorded in the range of 200-600 nm at 21 °C at various pH values in quartz cuvettes (1-cm light path; the reference cuvette contained the used buffers and on occasion 4 mM DTT). For determination of the pKa, 0.6-ml aliquots of a stock solution of the oxidatively decarboxylated peptide SFNSYV-NH-CH=CH-SH were mixed with 0.15 ml of 50 mM sodium phosphate (pKa = 2.15, 7.20, and 12.38), 50 mM sodium citrate buffer (pKa = 3.13, 4.76, and 6.40) (final buffer concentration was 10 mM each for phosphate and citrate). The pH of the buffers was adjusted by adding HCl. pH values of the 10 mM phosphate/citrate buffers and of the buffered peptide solutions were determined using a Knick pH meter (761 Calimatic).
The reaction products of the flavoenzyme EpiD are oxidatively decarboxylated peptides with a C-terminal enethiol side chain (6-8). Recently, we demonstrated that the reaction products of EpiD have an absorption maximum at 260 nm when measured in 0.1% trifluoroacetic acid, 30% acetonitrile, 70% H2O (6, 7). In order to determine the acid strength of the -SH group of enethiol-containing peptides, we investigated whether the UV-visible spectra of SFNSYV-NH-CH=CH-SH and SFNSYT-NH-CH=CH-SH are pH-dependent. It is well established that the thiolate anion of cysteine can be detected by its absorbance at 240 nm (9, 10) and that the pKa value of the -SH group of cysteine-containing peptides is in the range of 8.5-9.5.
The UV-visible spectrum of the peptide SFNSYV-NH-CH=CH-SH purified
by reversed-phase chromatography was determined in the pH range
2.5-8.0. To ensure that spectral changes were not due to oxidation of
the peptide to the dimer, the UV spectra were recorded in the presence
of 4 mM DTT. Interestingly, the UV spectrum was
pH-dependent (Fig. 1) with an absorption
maximum of 259 nm under acidic conditions (pH 4.2) and of 283 nm at pH 7.0. Due to ionization of the tyrosine residue, the absorbance
increased significantly below 260 nm at pH 8.0; however, there was no
further shift of the absorption maximum to wavelengths greater than 283 nm (data not shown). Similar pH-dependent spectral changes
were also observed for the peptide SFNSYT-NH-CH=CH-SH (data not
shown but see Fig. 2).
The pH dependence of the UV spectrum of the oxidatively decarboxylated
peptide indicates the ionization of the enethiol form to the
enethiolate anion form (Fig. 1, A and B). From
the UV spectra, the apparent pKa value of the
enethiol group was determined to be pH 6.0 (Fig. 1B). The
molar extinction coefficient () of SFNSYV-NH-CH=CH-SH at 259 nm
and of SFNSYV-NH-CH=CH-S
at 283 nm was determined to
be at least
6,800 and
7,200 M
1 cm
1, respectively. However,
this value may even be higher due to error in weighing out the small
amounts of available peptide and due to the presence of sodium chloride
and water in the peptide sample. Assuming that the peptides SFNSYVC and
SFNSYV-NH-CH=CH-SH have the same
214 value,
259 of SFNSYV-NH-CH=CH-SH and
283 of
SFNSYV-NH-CH=CH-S
were calculated to be approximately
10,000 M
1 cm
1.
In order to confirm the pH dependence of the UV spectrum of enethiol-containing peptides, the reaction mixture containing the substrate peptide SFNSYTC (821 m/z), the reaction product, and MBP-EpiD was analyzed by reversed-phase chromatography under acidic conditions (0.1% trifluoroacetic acid, approximately pH 2.0) and under neutral conditions (25 mM TEAA, pH 7.0, Fig. 2). The reaction product (775 m/z) was identified by high resolution electrospray mass spectrometry as a monomer (data not shown). Degradation products (741 and 759 m/z) of the reaction product were also present. The A260/A280 ratio of the reaction product was 2.3 in 0.1% trifluoroacetic acid/acetonitrile/H2O and 0.6 in 25 mM TEAA/acetonitrile/H2O (compare Fig. 2, A2 and B2). Comparable results were obtained for the reaction products of SFNSYVC and SFNSYCC (data not shown).
The determined pKa value of 6.0 for the enethiol group of SFNSYV-NH-CH=CH-SH is significantly (3 pH units) lower than the reported pKa value for the thiol group of cysteine residues in small peptides. Peptides with a C-terminal cysteine residue in which the negative charge of the carboxyl group is removed (cysteine amides, a negative charge in the vicinity of the thiol group decreases the acidity of the thiol group (11)), as is also the case for the investigated oxidatively decarboxylated peptides SFNSYV-NH-CH=CH-SH and SFNSYT-NH-CH=CH-SH, are reported to have a pKa value in the range of pH 8.8 (12). The increased acid strength of the enethiol group compared with the acid strength of the thiol group is explained by a resonance effect in which the negative charge on the sulfur anion is delocalized in the enethiolate. Delocalization of the negative charge is the reason for stabilization of enolate ions so that the acidity of enols is increased compared with alcohols (13).
For several enzymes, such as the protein thiol-disulfide oxidoreductase DsbA from Escherichia coli, human glutathione transferase, and cysteine peptidases, it has been shown that the catalytic active cysteine thiol groups have a pKa value near 4.0 (14-16). This low pKa value is partially explained by stabilization of the thiolate anion by interaction with histidine or lysine side chains in the tertiary structure of the proteins. It is very unlikely that the peptides SFNSYV-NH-CH=CH-SH and SFNSYT-NH-CH=CH-SH have a defined three-dimensional structure and that this structure is responsible for stabilization of the enethiolate anion.
There is a strong correlation between the low pKa value of the thiol group of Cys30 of DsbA and the redox potential of DsbA (17). The two cysteine residues of the active site Cys30-Pro31-His32-Cys33 form a disulfide bridge, and disulfide-bonded DsbA is a potent oxidant that generates protein disulfide bonds (18, 19). Because of the correlation between the pKa value of the thiol group and the redox potential, we are interested in determining the redox potential of peptides containing both a cysteine residue and a C-terminal enethiol group (e.g. oxidatively decarboxylated precursor peptide EpiA), and we are interested in investigating the reaction between protein thiol-disulfide oxidoreductases and such substrates.
As known for thiols (20), we assume that enethiols are far more reactive as the enethiolate anions. Therefore, reasonable chemical intuition suggests that the intermediate in the Michael addition-like formation of the S-[(Z)-2-aminovinyl]-D-cysteine residue of epidermin is the enethiolate and not the enethiol form of posttranslationally modified precursor peptide EpiA. Furthermore, we postulate that in the case of lanthionine formation the thiolate anions of the involved cysteine residues are stabilized by interaction with positive charges within the precursor peptide or by interaction with the enzyme responsible for the thioether formation (EpiC and/or EpiB). Glutathione transferase catalyzes a similar reaction, the nucleophilic addition of glutathione to xenobiotic compounds containing an electrophilic center. It has been discussed that the pKa of the thiol group of glutathione is shifted from 9.0 in aqueous solution to 6.6 in the active site of glutathione transferase so that the predominant species in the active site is the thiolate anion of glutathione (21).
A fascinating question is whether enzymes exist in nature that catalyze
the formation of enethiols/enethiolates from cysteine residues that are
not located at the C terminus. Principally, such a reaction can be
involved in the formation of the thiazole backbone modification of the
gyrase inhibitor microcin B17, whose structure has been elucidated
recently (22, 23) (Fig. 3). Enzymes involved in microcin
B17 modification have not been characterized, and there is no
experimental evidence for the existence of the proposed
enethiol-forming activity in microcin B17 modification. By converting
the thiol side chain of cysteine residues in peptides and proteins to
enethiol/enethiolates the (pH dependence of the) activity and stability
of proteins may be influenced to a large extent. However, nothing is
known about the stability of these enethiol side chains. Peptides with
a C-terminal enethiolate group are unstable, but they are stabilized in
the presence of zinc ions (6-8). In addition to the flavoprotein EpiD,
another enzyme catalyzing an ,
-dehydrogenation reaction of
peptides has already been characterized; heme-containing
L-tryptophan 2
,3
-oxidase from Chromobacterium
violaceum is responsible for the synthesis of
,
-dehydrotryptophan derivatives (24, 25).
We thank Christoph Kempter for assisting in electrospray mass spectrometry and Karen A. Brune for critically reading the manuscript.