Unité de Bioénergétique et
Ingénierie des Protéines, IBSM, CNRS, 13402 Marseille Cedex
20, France
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TEXT |
Hydrogenase, which catalyzes the
reversible oxidation of molecular hydrogen, is involved in many
relevant anaerobic processes where hydrogen is oxidized or evolved
(1). This enzyme plays a central role in energy-generating
mechanisms of sulfate-reducing bacteria that exhibit a strictly
anaerobic respiration of sulfate (3, 28, 30). Among the
[NiFe] hydrogenases, the Desulfovibrio gigas enzyme is the
most studied of its kind. Extensive biochemical and biophysical
analyses (4, 7, 16, 38) have shown that it is a
heterodimeric, periplasmic protein exhibiting a molecular mass of 89 kDa (28- and 60-kDa subunits) and contains four redox centers: one
nickel center, one [3Fe-4S] cluster, and two [4Fe-4S] clusters. The
three-dimensional structure of the D. gigas hydrogenase determined at 2.85 Å resolution (43) revealed that the
small subunit coordinated the three iron-sulfur clusters and that the active site, composed of an NiFe cluster (12, 44), was
buried in the large subunit. Although much is known, progress in
understanding the molecular mechanism of the [NiFe] hydrogenase is
facilitated by a genetic system that allows the production of a
correctly matured and active Desulfovibrio enzyme. As yet,
such a system does not exist for D. gigas, and because
Desulfovibrio fructosovorans is genetically accessible, it
is a candidate for the expression of Desulfovibrio
recombinant hydrogenases. Such a system would be especially useful
because functional heterologous hydrogenases have not been produced in
Escherichia coli (46). The aim of the present
study was to explore the possibility of expressing the D. gigas [NiFe] hydrogenase operon in D. fructosovorans.
In this paper we report the description of the D. gigas
[NiFe] hydrogenase operon and the characterization of the recombinant
heterologous enzyme.
Sequence analysis of the D. gigas [NiFe] hydrogenase
operon.
The D. gigas genomic library was probed with a
PCR-amplified fragment which contained the D. gigas [NiFe]
hydrogenase structural genes (45). The hydrogenase operon
was sequenced by EUROGENTEC, Seraing, Belgium. The D. gigas
hyn operon contains only four genes: hynA encodes the
small subunit (reference 45 and this work), hynB encodes the large subunit (reference
45 and this work), and hynC and
hynD encode newly identified maturation proteins. No other
maturation genes were identified downstream from hynD. The
calculated molecular weights of the deduced amino acid sequences were
33,869 for the pre-small subunit, 28,322 for the mature small subunit,
61,397 for the pre-large subunit, and 59,620 for the mature large
subunit. Comparison between the deduced amino acid sequences of the
hydrogenases from D. fructosovorans (33) and D. gigas revealed that the small subunits shared 64%
identity and 81% homology and that the large subunits shared 64%
identity and 79% homology. Only the new regions of interest of the
D. gigas hydrogenase operon are presented in Fig.
1. The 400-bp region upstream from
hynA is likely to contain the 
70-type
promoter region since expression of the enzyme was obtained (see
below). The product of the hynC gene shares 64% identity with HynC from D. fructosovorans (35). It also
shares strong homologies with HyaD from E. coli hydrogenase
1 (24), HybD from E. coli hydrogenase 2 (27), HycI from E. coli hydrogenase 3 (32), HupD from Rhodobacter capsulatus
(9), Rhizobium leguminosarum (18), and
Bradyrhizobium japonicum (42), and HoxM from
Alcaligenes eutrophus (19) and Azotobacter
vinelandii (26). Proteins homologous to HynC have been
found to be responsible for the C-terminal cleavage of the [NiFe]
hydrogenase large subunits (5, 21, 32). The second gene
identified was named hynD, and the product of this gene
shares strong homologies with HypC/HupC from A. vinelandii (8), R. capsulatus (9), A. eutrophus (10), E. coli (20), B. japonicum (29), R. leguminosarum
(31), and Azotobacter chroococcum
(40). It is interesting to note that members of the
hyp/hup gene family, which have been shown to play a major role in the maturation process of the hydrogenase (5, 11, 27) by interacting with the large subunit (13), are
not usually present within the hydrogenase operons but rather are in
contiguous regions. Indeed, hynD is not present in the
D. fructosovorans operon in spite of the close relationship
which exists between D. gigas and D. fructosovorans. Nevertheless, the absence of large numbers of
genes encoding maturation proteins seems to be a characteristic of the
Desulfovibrio [NiFe] hydrogenase operons.
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FIG. 1.
Nucleotide and derived amino acid sequences of the DNA
region upstream and downstream from the hynA and
hynB structural genes of the D. gigas [NiFe]
hydrogenase. The ribosome binding sites (RBS) are overlined. The broken
upward arrows indicate the translation start sites, and the broken
downward arrows indicate the translation stop sites. The important
restriction sites used in pBCG4 construction are designated. The
sequences of the oligonucleotides used for PCR amplification are
indicated in boldface.
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Properties of the recombinant D. gigas [NiFe]
hydrogenase.
The 3.9-kb BstEII-XbaI fragment
(Fig. 1) which contains the hynABCD operon from D. gigas was cloned into pBMC6 (36). The resulting plasmid
was called pBCG4 and was introduced into D. fructosovorans
MR400 (hyn::npt [Knr]
hynABC) (34) by electrotransformation
(36). The [NiFe] hydrogenase activity measured in D. fructosovorans MR400 (pBCG4) was 0.15 ± 0.05 U/mg. The
soluble extract from D. fructosovorans MR400 (pBCG4) was
loaded onto a sodium dodecylsulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) gel and analyzed by an immunoblotting experiment with
antibodies directed against D. gigas [NiFe] hydrogenase (Fig. 2A, lane 2). On the
chemoluminogram, the large subunit (60 kDa) appeared as a strong band.
Its migration pattern was similar to that of the matured large subunit
of the native enzyme (Fig. 2A, lane 1), which suggests the presence of
a processed form. The lower-molecular-weight bands are attributed to a
nonspecific cross-reaction since they appeared after a long time
exposure, which was necessary to detect traces of the small subunit. On the other hand, the small subunit (28 kDa) was barely detectable, and
the faint band above it presented a 33-kDa molecular mass which might
correspond to the unprocessed form of that subunit. In order to check
whether exportation was responsible for the lack of the small subunit
in the soluble fraction, the membrane fraction was tested by Western
immunoblotting analysis. No accumulation of the precursor form of the
D. gigas small subunit was observed (data not shown). The
weak detection of the small subunit might therefore be the result of
its degradation, which can be due to a maturation failure. Indeed,
instability of the small subunit has been reported in the case of large
subunit maturation defects in A. eutrophus (11,
39). As a control, the [NiFe] hydrogenase activity was measured
in extracts from D. fructosovorans MR400 complemented with
its own homologous operon. In that case, an activity of 1.0 ± 0.4 U/mg was measured and no unprocessed forms were identified by Western
immunoblotting experiments (data not shown). It is therefore possible
to conclude that the small subunit of the heterologous recombinant
hydrogenase appears to be mainly unprocessed and that the presence of
the genes on a multicopy plasmid is not responsible for this partial
maturation. However, further characterization studies were carried out
to determine the origin of the hydrogenase activity measured in
D. fructosovorans MR400(pBCG4). The soluble protein fraction
was enriched up to a specific activity of 1.8 U/mg. This partially
purified enzyme exhibited catalytic properties similar to those of the
D. gigas native enzyme (14, 16). When the
hydrogenase activity was tested in a native PAGE gel the one activity
band that was present (Fig. 2B, lane 2) exhibited an
Rf of 0.40, which is identical to that of the
native enzyme (Fig. 2B, lane 1). Furthermore, in the partially purified
fraction, the molecular masses of the subunits of the heterologous
recombinant enzyme were found to be identical to those of the native
enzyme and no trace of the precursor forms of each subunit was detected
(Fig. 2B, lane 6).
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FIG. 2.
Analysis of the native and recombinant D. gigas [NiFe] hydrogenases. (A) SDS-PAGE and chemoluminescent
Western immunoblotting analysis of soluble extracts from the
recombinant D. gigas [NiFe] hydrogenase. Lane 1, purified
native hydrogenase (250 ng); lane 2, extract from D. fructosovorans MR400 (pBCG4) (60 µg); lane 3, extract from
D. fructosovorans MR400 (pBCFG14) (60 µg). (B)
Pairwise comparisons of pure wild-type D. gigas [NiFe]
hydrogenase (lanes 1, 3, and 5) and partially purified recombinant
D. gigas [NiFe] hydrogenase (lanes 2, 4, and 6). Lanes 1 (140 ng) and 2 (11 µg), hydrogenase activity staining (native PAGE
gel); lanes 3 (150 ng) and 4 (10 µg), enzyme detection by Western
blotting with D. gigas [NiFe] hydrogenase polyclonal
antibodies; lanes 5 and 6, Western blotting (240 ng and 11 µg,
respectively, on an SDS-PAGE gel).
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EPR spectroscopy of recombinant D. gigas [NiFe]
hydrogenases.
In order to check the correct arrangement of the
metal centers, the partially purified D. gigas hydrogenase
was studied by electron paramagnetic resonance (EPR) spectroscopy. The
D. fructosovorans homologous recombinant enzyme was also
analyzed as a control. As isolated in air, the enzymes showed, at low
temperature (15 K), nearly isotropic EPR signals centered at
g = 2.02, which arise from their oxidized
[3Fe-4S]1+ center (Fig. 3, signals a and
b). The D. gigas recombinant
hydrogenase [3Fe-4S]1+ signal exhibits a distinct line
shape which is identical to that previously observed for the native
enzyme (Fig. 3, signal b) and which is quite different from the signals
observed for the D. fructosovorans hydrogenase (Fig. 3,
signal a) (2, 17). Ni(III) EPR signals were also observed
for the oxidized recombinant hydrogenases as a mixture of the
well-known Ni-A (g = 2.31, 2.23, and 2.01) and Ni-B
(g = 2.33, 2.16, and 2.01) signals (Fig. 3, signals c and d) (7).
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FIG. 3.
EPR spectra given by recombinant hydrogenases from
D. fructosovorans (signals a, c, and e) and D. gigas (signals b, d, and f). Signals a to d are from oxidized
enzymes and signals e and f are from H2-reduced enzymes.
The experimental conditions used (for the signals indicated in
parentheses) were as follows: Temperature 15 K (a and b), 100 K (c and
d), and 6.5 K (e and f); microwave frequency, 9.420 GHz; microwave
power, 0.04 mW (a and b) and 10 mW (c to f); modulation amplitude, 0.1 mT (a and b) and 1 mT (c to f).
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Upon reduction of the recombinant hydrogenases with hydrogen gas, these
signals disappeared and were replaced by the Ni-C signal at
g = 2.19, 2.14, and 2.01. At very low temperature
(<10°K), the broad and fast-relaxing signal given by the reduced
[4Fe-4S]1+ clusters was detected, and the magnetic
coupling between the proximal [4Fe-4S]1+ center and the
Ni-C species led to the complex split Ni-C signal (15)
characterized by major features at g = 2.21 and 2.10 (Fig. 3, signals e and f). Thus, all the EPR signals given by the
D. gigas recombinant hydrogenase were found to be identical
to those observed with the native enzyme (2, 7), which
indicates that the four metal centers are present in the same
environment. Moreover, as the split Ni-C signal is very sensitive to
minor variations in the relative arrangement of the interacting
centers, it can be inferred that no structural changes occurred in the cloned enzyme. This suggests that the active fraction of the
heterologous recombinant [NiFe] hydrogenase was correctly matured. To
our knowledge, this is the first report of heterologous expression of a
hydrogenase, and at the present time 64% identity and 80% homology
constitute the threshold above which a heterologous recombinant
hydrogenase can be expressed in an active form.
Construction of the chimeric [NiFe] hydrogenase operon.
The
[NiFe] hydrogenase activity measured in D. fructosovorans
MR400 (pBCG4) (0.15 ± 0.05 U/mg) was about six times lower than when D. fructosovorans MR400 was complemented with its own
homologous operon (1.0 ± 0.4 U/mg). This strong decrease is
assumed to be due to a poor maturation efficiency of the heterologous
enzyme, even though a low expression level might also have an
additional effect. These two possibilities were tested by inserting
D. gigas structural genes in the D. fructosovorans operon. The resulting plasmid, which contained a
chimeric operon made up of the D. fructosovorans hydrogenase
promoter (35), the D. gigas hydrogenase
structural genes hynA and hynB (reference
45 and this work), and the D. fructosovorans
hynC gene (35), was called pBCFG14. Surprisingly, similar levels of activity were observed (0.16 ± 0.05 U/mg). This indicates that the presence of the D. fructosovorans
hydrogenase promoter did not increase the level of activity. The
immunoblotting experiments performed on the soluble extracts from
D. fructosovorans MR400 (pBCFG14) (Fig. 2B, lane 3) showed
the same pattern as in the case of D. fructosovorans
(pBCG4). The biochemical and EPR analyses conducted on the partially
purified hydrogenase activity revealed that the heterologous
hydrogenase expressed in D. fructosovorans MR400 (pBCFG14)
was absolutely identical to the native enzyme, as previously observed
in the case of D. fructosovorans MR400 (pBCG4). Considering
that the absence of hynC led to the expression of an
inactive enzyme (35), it is possible to assume that no other
C-terminal protease is at work in D. fructosovorans and therefore that the HynC C-terminal protease from D. fructosovorans was able to cleave the HynB large subunit from
D. gigas. This is quite surprising since we know from
studies performed on A. eutrophus (5, 39) and
E. coli (21, 22, 25, 32, 37) that these types of
proteases are so specific that they can distinguish between the
different [NiFe] hydrogenases present in these microorganisms. One
possible explanation for this discrepancy might lie in the amino acid
sequence around the protease cleavage site. The C-terminal regions from
the D. gigas and D. fructosovorans hydrogenases
are highly conserved while those from the three E. coli
hydrogenases (6, 24, 27) and the two A. eutrophus
hydrogenases (19, 41) are significantly different from each
other. In keeping with the results of previous site-directed
mutagenesis experiments (23), it is then possible to assume
that the amino acid sequence at the cleavage site makes a specific
recognition sequence for the C-terminal protease. In the same way, the
fact that the absence of hynD in the chimeric operon did not
decrease the level of activity of the recombinant hydrogenase suggests
that this function was likely to be supplied in trans by a
D. fructosovorans homologous gene. The low yield of D. gigas hydrogenase maturation in D. fructosovorans is an
intriguing observation which suggests that the processing might be
kinetically limited. All the components necessary for the correct
maturation of the heterologous hydrogenase are present but some steps
might be too slow, compared to structural gene expression, to allow the
structural subunits as a whole to be processed. Our experiments suggest
that neither the homologous HynC nor the heterologous HynD protein is
responsible for this rate-limiting processing since the presence of
either protein did not influence the level of activity.
Nucleotide sequence accession numbers.
The nucleotide
sequences described in this study were submitted to the EMBL and
GenBank data libraries and were assigned accession numbers AJ223628 and
AJ223629.
We acknowledge J. D. Wall for critically reading the
manuscript and for valuable suggestions. We are indebted to A. Bélaich for her assistance in genetic constructions. We thank
Long-Fei Wu for very helpful discussions. We are also grateful to
J. Bonicel for determination of the N-terminal amino acid
sequences of the subunits of the recombinant hydrogenase and to P. Sauve for the estimation of the molecular mass of the protein by
analytical ultracentrifugation.
This work was supported by grant BI02-CT94-2041 from the European
Commission Biotechnology Program.
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