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From the
Received for publication, October 5, 2001, and in revised form, December 3, 2001
The formation of
S-hydroxymethylglutathione from formaldehyde and
glutathione is a central reaction in the consumption of the cytotoxin
formaldehyde in some methylotrophic bacteria as well as in many other
organisms. We describe here the discovery of an enzyme from
Paracoccus denitrificans that accelerates this spontaneous
condensation reaction. The rates of
S-hydroxymethylglutathione formation and cleavage were
determined under equilibrium conditions via two-dimensional proton
exchange NMR spectroscopy. The pseudo first order rate constants
k1* were estimated from the temperature dependence of the reaction and the signal to noise ratio of the uncatalyzed reaction. At 303 K and pH 6.0 k1*
was found to be 0.02 s Formaldehyde is a highly toxic compound due to nonspecific
reactivity with proteins and nucleic acids (1). It is liberated as a
result of demethylation reactions in mammals (2) or from environmental
sources (3), and it is a central intermediate upon growth of
methylotrophic bacteria on one-carbon substrates like methanol or
methane (4). The most widespread enzymatic system for the conversion of
formaldehyde is the glutathione
(GSH)1-linked oxidation
pathway, which has been found in bacteria, mammals, and plants. In
autotrophic methylotrophic bacteria like Paracoccus denitrificans and Rhodobacter sphaeroides as well as
methylotrophic yeasts, it is involved in the complete oxidation of
methanol to carbon dioxide (5-8). In higher organisms, as well as
non-methylotrophic bacteria, such as Escherichia coli,
glutathione-linked formaldehyde oxidation serves to detoxify the
one-carbon unit (9, 10).
The glutathione-dependent formaldehyde
conversion to formate starts with the adduct formation, formaldehyde
reacts with the SH group of glutathione producing
S-hydroxymethylglutathione (Reaction 1) (11). This reaction
is considered to proceed in vivo uncatalyzed by a specific
enzyme (6, 7, 10, 11). The product of this reaction,
S-hydroxymethylglutathione, is oxidized by
glutathione-dependent formaldehyde dehydrogenase (GS-FDH)
(Reaction 2), which belongs to the class III alcohol dehydrogenases and
has been characterized from various organisms (6, 7, 9, 12). The enzyme
has been shown to be induced upon formaldehyde stress in different microorganisms (10, 13). In the subsequent enzymatic reaction, S-formylglutathione hydrolase (FGH) regenerates glutathione
and forms formate (Reaction 3) (14), which can be further oxidized to
carbon dioxide.
To determine S-hydroxymethylglutathione formation from
formaldehyde and glutathione in P. denitrificans, we used
proton exchange NMR spectroscopy (15). The method is based on the
finding that the protons at the C NMR Measurements--
Rates of
S-hydroxymethylglutathione formation from formaldehyde and
glutathione were determined under equilibrium conditions via EXSY (16).
NMR spectra were acquired at a 1H frequency of 600.13 MHz
on a DRX600 spectrometer (Bruker) and processed with the program
XWINNMR (Bruker). The assays were performed in NMR tubes ( Bacterial Growth and Enzyme
Purification--
P. denitrificans (DSM413), E. coli DH5 In most organisms, the conversion of exogenous or
endogenous formaldehyde proceeds by addition to glutathione prior to
oxidation by GS-FDH. To address the question of whether an enzyme
exists which catalyzes the formation of
S-hydroxymethylglutathione from formaldehyde and
glutathione, we analyzed cell extracts of P. denitrificans
grown under methylotrophic conditions. The rates of
formaldehyde-glutathione condensation were determined by
one-dimensional and two-dimensional proton exchange NMR spectroscopy.
Recording of the standard spectra was performed at pH 6.0, 303 K
(30 °C) and under aerobic conditions, since P. denitrificans is an aerobic mesophilic bacterium. To increase the
accuracy of the analysis, a product/educt ratio of 1:1 was aspired and
achieved by using a ratio of glutathione to formaldehyde of 2:1 (10.8 mM glutathione, 5 mM formaldehyde). This ratio
was used throughout this study.
In Fig. 2, the aliphatic
regions of a one-dimensional proton NMR spectrum and a two-dimensional
1H homonuclear EXSY NMR spectrum of glutathione and
S-hydroxymethylglutathione at equilibrium, in the absence
(A) and in the presence (B) of cell extract from
methanol grown P. denitrificans, are shown. From the
one-dimensional spectra, the relative populations
pGSH = 0.52 and pGSCH2OH = 0.48 were obtained by integration of the signals 1 and 1' (Fig. 2,
A and B). Integration of the signals 2 and 2'
yields the same values for both species. From the two-dimensional spectrum in the presence of cell extract (Fig. 2B) the peak
volumes of the protons 1 and 2 were obtained and
k1* = 0.24 s
ACCELERATED PUBLICATION
A Glutathione-dependent Formaldehyde-activating
Enzyme (Gfa) from Paracoccus denitrificans Detected and
Purified via Two-dimensional Proton Exchange NMR
Spectroscopy*,
§,§¶
,**,
Max-Planck-Institut für terrestrische
Mikrobiologie, Karl-von-Frisch-Strasse, 35043 Marburg, Germany, the
¶ Institut für Organische Chemie der Universität
Frankfurt, Marie-Curie-Strasse 11, 60439 Frankfurt a.M., Germany, and
the Max-Planck-Institut für biophysikalische Chemie, Am Fassberg
11, 37077 Göttingen, Germany
ABSTRACT
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1 for the spontaneous reaction. A
10-fold increase of the rate constant was observed upon addition of
cell extract from P. denitrificans grown in the presence of
methanol corresponding to a specific activity of 35 units
mg1. Extracts of cells grown in the presence of succinate
revealed a lower specific activity of 11 units mg1. The
enzyme catalyzing the conversion of formaldehyde and glutathione was
purified and named glutathione-dependent
formaldehyde-activating enzyme (Gfa). The gene gfa is
located directly upstream of the gene for
glutathione-dependent formaldehyde dehydrogenase, which catalyzes the subsequent oxidation of
S-hydroxymethylglutathione. Putative proteins with sequence
identity to Gfa from P. denitrificans are present also in
Rhodobacter sphaeroides, Sinorhizobium meliloti, and
Mesorhizobium loti.
INTRODUCTION
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In this study, we investigated whether the
condensation of formaldehyde and glutathione (Reaction 1) proceeds
indeed only non-enzymatically in vivo. We have chosen
P. denitrificans as a model organism, since it is a
facultative methylotroph and converts high amounts of formaldehyde
during energy metabolism upon growth on methanol by glutathione-linked
enzymes. Glutathione-dependent formaldehyde dehydrogenase
and S-formylglutathione hydrolase have been shown to be
essential for growth of the autotrophic bacterium in the presence of
methanol (6, 14).
atom of the thiol
group of the cysteine part in glutathione and
S-hydroxymethylglutathione exhibit different chemical shifts
and that the saturation transfer kinetics of these protons can be
followed by proton exchange NMR spectroscopy (EXSY) (Fig.
1). We used the two-dimensional EXSY
approach to detect the activity of an previously unknown enzyme and
used it for purification of the enzyme from cell extracts. To our
knowledge this is the first time that EXSY has been successfully
applied to find a previously unknown enzyme.
View larger version (11K):
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Fig. 1.
Formation of
S-hydroxymethylglutathione (GSCH2OH) from
formaldehyde and GSH
( 
-L-glutamyl-L-cysteinyl-glycine).
Two diasteriotopic protons at C have different chemical
shifts and were used for the analysis of EXSY (see Fig. 2).
EXPERIMENTAL PROCEDURES
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5 mm)
with 0.6 ml of reaction mixture. Standard assays contained 10.8 mM GSH and 5 mM formaldehyde in 120 mM potassium phosphate buffer pH 6.0 (H2O/D2O = 9:1) if not otherwise noted. Exchange rates v1 = k1*
[GSH] = v2 = k2*
[GSCH2OH] (see Fig. 1) were calculated from the
concentrations of GSH and GSCH2OH in equilibrium which were
obtained by integration of one-dimensional spectra yielding the
[GSH]/[GSCH2OH] ratio (see Fig. 2). From the ratios,
the relative populations pGSH = [GSH]/([GSH] + [GSCH2OH]) and pGSCH2OH = [GSCH2OH]/([GSH] + [GSCH2OH]) were
calculated, whereby [GSH] + [GSCH2OH] equals the GSH
concentration added. GSH was considered to be fully protonated, since
measurements were performed between pH 5.5 and 6.5 and the
pKa of GSH is 9.12. The second order rate constants
k1 and k2 were defined from k1* and k2* and the
equilibrium concentration of formaldehyde: v1 = k1[GSH][HCHO] = v2 = k2[GSCH2OH][H2O].
[H2O] was considered to be constant, since measurements
were performed in aqueous solution. The exchange rates
v1 and v2 were calculated
from the concentration of GSH, HCHO, and GSCH2OH, and the
rate constants k1 and k2,
which are related to the relative populations
pGSH and pGSCH2OH, and the peak volumes Vij and the mixing time
m by the expression Vij = (exp (
Rm))ij. For definition of
Vij and R, see Ref. 16. The enzyme
activities were calculated from the exchange rates
v1 of the GSCH2OH formation and
converted from the unit mM s1 to µmol
min1 (=1 unit).
, and Methylobacterium extorquens AM1 were
cultivated as described previously (4, 10). For enzyme
purification from methanol-grown P. denitrificans, 20 g
of wet cells were resuspended in 120 mM potassium phosphate buffer and broken by a French press. Purification of Gfa was performed by four chromatographic steps at 4 °C. The soluble fraction of the
cell extract was loaded onto a DEAE-Sephacel (Sigma) column equilibrated with 50 mM potassium phosphate, pH 7.0. Protein was eluted with the following gradient steps of NaCl in this
buffer: 60 ml of 0 mM, 60 ml of 150 mM, 60 ml
of 200 mM, 60 ml of 250 mM, and 60 ml of 500 mM. Gfa was eluted at 150 mM NaCl. Active fractions were diluted 1:2 in 10 mM potassium phosphate
buffer, pH 7.0, and loaded onto a hydroxyapatite column (Bio-Rad)
equilibrated in the same buffer. Protein was eluted with a stepwise
increasing potassium phosphate gradient (10-250 mM in 275 ml). Gfa was recovered in the flow-through of the column, which was
subjected to chromatography on Q-Sepharose (Amersham Biosciences,
Inc.) in 120 mM potassium phosphate buffer. Protein
was eluted with an increasing gradient of NaCl (0-300 mM
in 450 ml). Gfa was eluted at 60 mM NaCl. Active fractions
were pooled and diluted 1:2 in 50 mM potassium phosphate buffer, pH 7.0, and loaded onto a Mono Q column (Amersham Biosciences, Inc.). Protein was eluted with an increasing gradient of NaCl in this
buffer (0-500 mM in 100 ml). Gfa was eluted at 400 mM NaCl. GS-FDH was measured photometrically and purified
as described previously (6).
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1 and
k2* = 0.21 s1 calculated (16).
From the data, an exchange rate v1 was obtained corresponding to 41 units for the rate in the presence of cell extract
of methanol grown P. denitrificans, which has 35 units mg1 cell extract protein (Table
I). Without cell extract (Fig.
2A) a spontaneous rate of only 5 units was determined. No
increase in the spontaneous rate was observed if supernatant of
denatured and centrifuged cell extract from P. denitrificans
was applied, indicating that the observed activity is the result of
enzyme catalysis. Addition of purified GS-FDH from P. denitrificans, which oxidizes
S-hydroxymethylglutathione (Reaction 2), did not result in
higher S-hydroxymethylglutathione formation from
formaldehyde and glutathione (Table I). This shows that the observed
acceleration is catalyzed by a separate enzyme distinct from GS-FDH.
Analysis of cell extract of P. denitrificans grown in the
presence of succinate revealed that enzymatic formaldehyde conversion
is still clearly detectable with an activity of 11 units
mg1 amounting to one-third of the activity in comparison
to cells grown in the presence of the one-carbon substrate. Activity of GS-FDH, which was measured as a control enzyme, was not detectable upon
growth in the presence of succinate and shows a more pronounced effect
of induction (Table I; Ref. 6).
View larger version (22K):
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Fig. 2.
Aliphatic region of the one-dimensional and
two-dimensional EXSY NMR spectra of the
C protons of glutathione and
S-hydroxmethylglutathione at equilibrium without
(A) and with (B) cell extract from
P. denitrificans at 303 K and pH 6.0. Two-dimensional 1H homonuclear EXSY NMR spectra (400 ms
mixing time) are shown in the center and one-dimensional proton NMR
spectra above and lateral. The signals H(
)1 and
H()2 of the one-dimensional spectra are from protons of
glutathione and the signals H()1' and
H()2' from protons of
S-hydroxymethylglutathione (Fig. 1). Diagonal peaks of the
glutathione and S-hydroxymethylglutathione protons in
chemical exchange are labeled with 11 and 1'1' or 22 and 2'2'.
Off-diagonal cross-peaks arising from chemical exchange are labeled
with 1'1 and 11' or 2'2 and 22'. These exchange cross-peaks are clearly
visible in the presence of cell extract of P. denitrificans
(1.04 mg), whereas in the absence of cell extract they are hardly
detectable because of the signal to noise ratio (B). The NMR
spectra were acquired at a 1H frequency of 600.13 MHz on a
DRX600 spectrometer (Bruker) and processed as described under
"Experimental Procedures." The NMR tube ( 5 mm) contained 0.6 ml
of a reaction mixture made up of 10.8 mM glutathione, 5 mM formaldehyde, 60 µl of D2O, 1.04 mg of
cell extract protein of methanol grown P. denitrificans
(B) in 120 mM potassium phosphate buffer, pH
6.0.
Effect of cell extracts of different organisms on the rate of
S-hydroxymethylglutathione formation in 120 mM potassium
phosphate buffer, pH 6.0, and 303 K (30 °C)
5 mm).
The 0.6-ml reaction mixture contained 10.8 mM glutathione,
5 mM formaldehyde, 60 µl of D2O, and 1.04 mg of
cell extract protein if not otherwise noted. Where indicated, denatured
cell extract protein was applied, which was boiled for 5 min at
95 °C and centrifuged. A unit of enzyme activity was defined as the
formation of 1 µmol of S-hydroxymethylglutathione from
formaldehyde and glutathione per min minus the spontaneous reaction
rate without enzyme added. The activity of GS-FDH is given as a control
and was measured photometrically with NAD as electron acceptor to
exclude an effect of the dehydrogenase on the exchange rates (6). For
definition of k1* and calculation of the
activities, see "Experimental Procedures." LB, Luria-Bertani
medium; ND = not detectable.
The influence of temperature and pH upon the rate of
S-hydroxymethylglutathione formation from formaldehyde and
glutathione was analyzed. The rate of the spontaneous reaction
versus the accelerated rate in the presence of cell extract
of methanol-grown P. denitrificans was determined between
293 and 333 K (20-60 °C). In both cases, the rate of
S-hydroxymethylglutathione formation increased about
3-fold when the temperature was raised from 293 K to 303 K (20 and
30 °C). The increase of the spontaneous rate was linear up to 333 K
(60 °C), whereas determination of the enzyme-promoted rates, by
addition of cell extract, above 323 K (50 °C) was not possible due
to protein denaturation. Dependence of the pH on the rate of
S-hydroxymethylglutathione formation was determined between
pH 5.5 and 6.5. The spontaneous rate increased with higher pH; the rate
k1* without cell extract was only 0.03 s1 at pH 5.5 and 0.45 s1 at pH 6.5. In the
presence of cell extract from P. denitrificans the rate was
always higher. At pH values higher than 6.5 the determination was
rather difficult due to instability of
S-hydroxymethylglutathione in vitro (17).
The enzyme that catalyzes the formation of
S-hydroxymethylglutathione,
glutathione-dependent formaldehyde-activating enzyme, Gfa,
was purified from P. denitrificans as described under
"Experimental Procedures." The enzyme activity was detected via NMR
measurements. After four chromatographic steps, preparations contained
only one polypeptide with an apparent molecular mass of 21 kDa, as revealed by SDS-PAGE and exhibited a specific activity of 350 units
mg1. Purification was about 24-fold with a yield of 6%.
UV/visible spectroscopy did not reveal the presence of a chromophoric
prosthetic group. The N-terminal amino acid sequence of the 21-kDa
polypeptide was determined (MVDTSGVKIHPAVDNG; terminal methionine
cleaved off to 90%) and matched exactly that predicted for the
orf2 gene product (6, 14). We now assign this gene as
gfa. Gfa from P. denitrificans shows high
sequence identity to putative proteins known from the complete genome
sequences of the -proteobacteria R. sphaeroides
(72%),2
Sinorhizobium meliloti (75%) (19), and
Mesorhizobium loti (61%) (20). Putative proteins
with sequence identities of about 63% could also be identified in the
currently unfinished genome sequences of the -proteobacteria
Thiobacillus ferrooxidans and Shewanella putrefaciens.3
Interestingly, gfa from P. denitrificans is located directly upstream from flhA
coding for GS-FDH (or GD-FALDH) (6) (Fig. 3). In R. sphaeroides
(7)2 and T. ferrooxidans,3 the same
arrangement of genes for the putative glutathione-dependent proteins could be found, whereas in M. loti the arrangement
of the two genes is inverted (20). In S. meliloti, the genes
for a putative Gfa and a putative GS-FDH are located about 13 kb apart on the pSymB megaplasmid (Fig. 3). This genome region also includes a
putative methanol dehydrogenase structural gene (19). In S. putrefaciens, the gene for a protein with sequence identity to Gfa
is located directly downstream of a putative iron containing alcohol
dehydrogenase.3 No more additional putative proteins with
sequence identity to Gfa from P. denitrificans could be
identified. Therefore Gfa is not conserved in all organisms that have
been shown to contain GS-FDH, i.e. E. coli
(10).
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In this study, we detected and purified a novel glutathione-dependent formaldehyde-activating enzyme Gfa from the facultative methylotrophic bacterium P. denitrificans. The condensation of formaldehyde and glutathione to S-hydroxymethylglutathione is the first step in the widespread glutathione-linked conversion of formaldehyde and was thought to occur without enzymatic catalysis in vivo.
Gfa is not the first example of a protein that catalyzes the condensation of formaldehyde and a cofactor to form an adduct in the process of energy metabolism. It was recently shown that the methylotrophic proteobacterium M. extorquens AM1 possesses a tetrahydromethanopterin-linked formaldehyde-activating enzyme, Fae, which catalyzes the condensation of formaldehyde and tetrahydromethanopterin producing methylene tetrahydromethanopterin (22). Fae is present in all heterotrophic methylotrophic proteobacteria we tested that contain tetrahydromethanopterin-dependent enzymes.4 Both formaldehyde-converting enzymes, Gfa and Fae, are composed of one type of subunit of about 20 kDa and lack a chromophoric prosthetic group. In addition, both enzymes are encoded next to genes for enzymes involved in further oxidation of the cofactor-bound one-carbon unit to carbon dioxide (6, 22). The primary sequences of Gfa and Fae do not reveal any sequence identity to each other and have obviously evolved independently, which is not too surprising, since the cofactors are very different, and binding of formaldehyde occurs either to the sulfur atom of glutathione or the N5,N10 nitrogen atoms of tetrahydromethanopterin.
Tetrahydromethanopterin-dependent enzymes are restricted to methylotrophic proteobacteria and methanogenic archaea, whereas the glutathione-linked formaldehyde dehydrogenase is widespread in procarya and eucarya (6, 7, 9, 12). Nevertheless, the presence of Gfa appears to be limited. It might be that Gfa is present only in organisms that produce and consume large amounts of intracellular formaldehyde, whereas the spontaneous formation of S-hydroxymethylglutathione would be sufficient for detoxification of exogenous formaldehyde, which may occur in the environment. In this respect it is interesting to discuss the bacteria that contain a Gfa homolog. Methanol consumption of the nitrogen-fixing bacteria S. meliloti and M. loti appears likely, since they contain open reading frames for putative proteins with high sequence identity to Gfa as well as putative proteins for S-hydroxymethylglutathione oxidation and methanol dehydrogenase structural genes (19, 20). A functional active Gfa homolog could also be expected in R. sphaeroides where the role of glutathione-linked formaldehyde dehydrogenase has been shown under both photosynthetic and aerobic respiratory conditions (8). S. putrefaciens is able to grow anaerobically in the presence of formate and proposed to form free formaldehyde intracellulary (21). A Thiobacillus species, Thiobacillus thioparus, also forms formaldehyde upon growth on methyl mercaptan (18). The same might be true for T. ferrooxidans, which possesses putative proteins for Gfa and glutathione-linked formaldehyde dehydrogenase.
We cannot rule out that another glutathione-linked formaldehyde-activating enzyme might have evolved that is shared by other organisms. We observed a slight increase in S-hydroxymethylglutathione formation in cell extracts of E. coli, which was, however, not induced by formaldehyde stress like GS-FDH so that the presence of glutathione-linked formaldehyde activation could not be demonstrated.
At present it is not clear whether Gfa serves solely as an enzyme or
can also serve as a formaldehyde scavenger to prevent unspecific
binding of the toxin. In this respect, it is interesting to note that
in P. denitrificans, Gfa activity could also be detected in
cells grown in the absence of methanol, whereas activity of GS-FDH is
not detectable under these growth conditions. Therefore it is likely
that the corresponding genes are under the control of different promotors.
| |
ACKNOWLEDGEMENT |
|---|
We thank Jochen Junker for revealing discussions.
| |
FOOTNOTES |
|---|
* This work was supported by the Max-Planck-Gesellschaft, the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie. All NMR measurements were conducted at the European Large Scale Facility for Biomolecular NMR (ERBCT95-0034) at the University of Frankfurt.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 on-line version of this article (available at
http://www.jbc.org) contains Supplemental Figs. 2 and 3.
§ These authors contributed equally to this work.
Supported by a Kekulé stipend of the Fonds der
Chemischen Industrie.
** Supported by the Peter and Traudl Engelhorn Stiftung.
To whom correspondence should be addressed: INRA/CNRS, BP27
Chemin de Borde Rouge, 31326 Castanet-Tolosan, France. Tel.:
33-5-61-28-54-58; Fax: 33-5-61-28-50-61; E-mail:
vorholt@toulouse.inra.fr.
Published, JBC Papers in Press, December 10, 2001, DOI 10.1074/jbc.C100579200
2 Sequence data was obtained from the Oak Ridge National Laboratory webpage at genome.ornl.gov/microbial/rsph/.
3 Preliminary sequence data was obtained from The Institute for Genomic Research website at www.tigr.org.
4 M. Goenrich and J. A. Vorholt, unpublished results.
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ABBREVIATIONS |
|---|
The abbreviations used are: GSH, glutathione; Gfa, glutathione-dependent formaldehyde-activating enzyme; GS-FDH (or GD-FALDH), glutathione-dependent formaldehyde dehydrogenase; FGH, S-formylglutathione hydrolase; Fae, tetrahydromethanopterin-dependent formaldehyde-activating enzyme; EXSY, proton exchange NMR spectroscopy.
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DISCUSSION
REFERENCES
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Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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