Báo cáo khoa học: Analysis of the transcarbamoylation-dehydration reaction catalyzed by the hydrogenase maturation proteins HypF and HypE pot

Analysis of the transcarbamoylation-dehydration reaction catalyzed
by the hydrogenase maturation proteins HypF and HypE
Melanie Blokesch, Athanasios Paschos,* Anette Bauer, Stefanie Reissmann, Nikola Drapal and August Bo¨ck
Department Biologie I, Mikrobiologie, Ludwig-Maximilians-Universita
¨
tMu
¨
nchen, Mu
¨
nchen, Germany
The h ydrogenase maturation proteins HypF and HypE
catalyze the synthesis of the CN ligands of the active site iron
of the NiFe-hydrogenases using carbamoylphosphate as a
substrate. HypE protein from Escherichia coli was purified
from a transformant overexpressing the hypE gene from a
plasmid. Purified HypE in gel filtration experiments behaves
predominantly as a monomer. It does not contain statisti-
cally significant amounts o f metals or o f cofactors absorbing
in the UV and visible light range. The protein displays low
intrinsic ATPase activity with ADP and phosphate as the
products, the apparent K
m
being 25 l
M
and the k
cat
1.7 · 10
)3
s
)1
. Removal of the C-terminal cysteine r esidue

of HypE which accepts the carbamoyl mo iety from HypF
affected the K
m
(47 l
M
) but not significantly the k
cat
(2.1 · 10
)3
s
)1
). During the carbamoyltransfer reaction,
HypE and HypF enter a complex which is rather tight at
stoichiometric ratios of the two proteins. A mutant HypE
variant was generated by amino acid replacements in the
nucleoside triphosphate binding region, which showed no
intrinsic ATPase activity. The variant was a ctive as an
acceptor in the transcarbamoylation reaction but did not
dehydrate the thiocarboxamide to the thiocyanate. The
results obtained with the HypE variants and also with
mutant HypF forms are integrated to explain the co mplex
reaction pattern of protein HypF.
Keywords: NiFe hydrogenase; maturation; CN ligand syn-
thesis; hypE mutations; carbamoyl transfer.
Escherichia coli possesses four hydrogenases (Hyd1–Hyd4)
which are all members of the N iFe c lass [1,2]. In these
enzymes, the bimetallic active centre is hooked to the
protein via four cysteine thiolates whereby two of them act
as ligands bridging the iron and the nickel (for a review, see
[3]). The most intriguing feature, however, is that the iron

carries three diatomic, nonprotein ligands which in the
classical case consist of two cyanides and one carbon
monoxide [4,5]. The NiFe metal centre i s positioned in the
interior of the large subunit close to its interface with the
small subunit.
In addition to the operons coding for the structural
proteins of the four hydrogenases there are six genes,
designated hyp, whose products have a f unction in the
maturation of the enzymes. Most o f them a ct pleiotropically
in the synthesis of all four hydrogenases, in particular in the
synthesis and insertion of the metal centre [6,7]. Two of the
products of the hyp genes, namely HypA and HypB, are
involved in nickel insertion [8,9]. From the other four hyp
gene products, HypF and HypE have a function in the
synthesis of the cyanide ligands. HypF functions as a
carbamoyl transferase using carbamoylphosphate as a
substrate and transferring the carboxamido moiety in an
ATP-dependent reaction to the thiolate of the C-terminal
cysteine of the HypE protein yielding a protein-S-carbox-
amide [10–12]. Subsequent dehydration of the carboxamide
residue via ATP dependent activation of the oxygen and
dephosphorylation leads to HypE-thiocyanate. Chemical
model reactions demonstrated that the cyano group can be
nucleophilically transferred to an iron complex [12]. The
origin of the carbonyl group of the Fe ligands in the E. coli
hydrogenases is still unresolved. It is also open a s to w hether
the ligandation of iron takes place at the hydrogenase large
subunit apoprotein, which had accepted the iron before, or
whether i t o ccurs at some scaffold protein from which the
fully substituted m etal is then transferred to the target

protein. Our present information supports the latter model.
Arguments are that in cells deprived of carbamoylphos-
phate a complex accumulates which consists of the matur-
ation proteins HypC and HypD [13]. This complex is
resolved in a time-dependent manner upon supply of a
carbamoyl source delivered from citrulline. Moreover, the
complex occurs in two electrophoretically different forms, a
faster migr ating one from cells lack ing carbamoylphosphate
and a slower one when carbamoylphosphate is present but
the apoprotein of the large subunit is a bsent. On the basis of
these results it was speculated that the site of Fe ligandation
is at the HypC–HypD complex from where it is transferred
to the large subunit apoprotein. HypC was supposed to be
involved in this transfer as it also undergoes complex
formation with the precursor of the large subunit [14,15].
Protein HypE thus possesses a key role in the process. It
accepts the carbamoyl residue, dehydrates it to the cyano
moiety and appears to transfer it to the iron. In this
communication we describe the p rocedure for the purifica-
tion of the HypE protein, its chemical properties and kinetic
Correspondence to A. Bo
¨
ck, Department Biologie I, M ikrobiologie,
Ludwig-Maximilians-Universita
¨
tMu
¨
nchen, Maria-Ward-Strasse 1a,
D-80638 Mu
¨

nchen, Germany. Fax: + 49 89 218063857,
Tel.: + 49 89 21806116, E-mail:
*Present a ddress: Department of Biology, McMaster University,
1280 Main St. West, Hamilton, Ontario, L8S 4K1, Canada.
(Received 11 May 2004, revised 29 June 2004, accepted 7 July 2004)
Eur. J. Biochem. 271, 3428–3436 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04280.x
characterization relative to the substrate ATP. The analysis
of mutant variants of HypE demonstrates that the carb-
amoylation of th e C-terminal thiol and the d ehydration of
the S-carboxamide to the thiocyanate are independent
reactions and that HypE p er se is a ble to catalyze the
dehydration reaction. In addition, it is shown that during
the carbamoyltransfer reaction HypE forms a complex w ith
the HypF protein. The results reported for HypE and also
for selected mutant variants of HypF are integrated into a
model explaining the complex reaction pattern of the two
proteins.
Experimental procedures
E. coli
strains, plasmids and growth conditions
E. coli strain MC4100 [16] was u sed a s wild-type a nd DH 5a
[17] as host in transformations. DHP-E is a derivative of
MC4100, which carries an in-frame deletion in the hypE
gene [6]. The cultures were g rown at 37 °C in Luria broth
[18] or in the buffered rich medium (TGYEP) described by
Begg et al. [19]. Aerobic growth was achieved in rigorously
shaken Erlenmeyer flasks, anaerobic g rowth in standing
screw c ap flasks filled to the top. For the maintenance of t he
plasmids, ampicillin was added at a concentration of
100 lgÆmL

)1
.
Plasmid pHypE was constructed by removing a 3006 bp
large HpaI-StyI fragment, which contains most of the fhlA
gene from plasmid pSA3 [20], and by religation after
treatment w ith Klenow e nzyme. It had a size of 5156 bp and
carried the hypE gene. pHypE was t hen employed to
construct a variant lacking the codon for the C-terminal
cysteine. It was achieved by inverse PCR [21] e mploying the
overlapping primers cys336del-forward (5¢-C CGCGG AT
ATGATAATAAAATTCTAAATCTCCTATAG-3¢)and
cys336del-reverse (5¢-TCATATCCGCGG AAGCGGTT
CGGCGTGTGGTAAATC-3¢) which harbour a SacII
restriction site (bases in bold face letters) at their 5¢-ends.
After the PCR reaction, DpnI was added to the mixture to
digest the matrix plasmid pHypE. The PCR fragment was
purified via p assage over a QIAquick Sp in Column (Qiagen
GmbH, Hilden, Germany) and used directly to transfrom
strain DH5a following the method of Ansaldi et al.[21].
The selection of accurate clones was accomplished by mini-
preparation followed by Sac II digestion a nd its a uthenticity
was confirmed by sequencing.
For o verproduction of the wild-type HypE protein, a
plasmid was constructed by excision of a 1.6 kb KpnI-MluI
fragment f rom plasmid pSA3, treatment with Klenow
enzyme to remove the protruding ends and by cloning into
the SmaI r estricted vector pT7-7. I n this w ay the GTG start
codon of the hypE gene was out of frame relative to an A TG
of the vector. The plasmid was designated pTE-C2. I t
represents a derivative of pT7-7 co ntaining a (ÔhypD-hypE-

fhlAÕ) gene fragment from the E. coli chromosome.
N-Terminal amino acid sequencing of the purified protein
revealed the sequence MNNIQLAHG, which is in accord-
ance with the observation made by Lutz et al.[22]and
Jacobi et al. [6] that the translation of the hypE gene initiates
at the GTG cod on 42 bases upstream of the previously
assumed ATG codon. The GTG codon overlaps with the
TGA termination codon of hypD [6]. Hence the hypE gene
codes for a protein with 336 amino acids and a molecular
mass of 35.1 kDa. The C-terminal a mino acid is therefore
Cys336 rather than Cys322 as previously specified [12].
For overexpression of the hypED gene it was excised from
plasmid pHypED by restriction with MluI, trea tment with
Klenow enzyme and SpeI digestion and cloned into plasmid
pTE-C2 via replacement of a HindIII-fragment that was
treated with the Klenow enzyme and su bsequently cleaved
with SpeI. The plasmid was designated pTEC-ED.
Plasmid pTE-C2 was also used to construct hypE gene
variants coding for products with an amino acid exchange
in the nucleotide binding site of HypE. The variant
containing the D83N replacement was obtained by inverse
PCR employing primers t hat carried the desired mutation
[23]. The plasmids harbouring the wild-type and mutant
hypF genes have been described before [11].
For all constructions, the Expand High Fidelity PCR
System from Roche Diagnostics GmbH (Mannheim,
Germany) was employed. Amplified fragments generated
by use of overlapping primers w ere purified by passage o ver
a QIAquick Spin Column (Qiagen GmbH, Hilden, Ger-
many) and used directly to transform strain DH5a.The

authenticity of all constructs was verified by DNA sequen-
cing using an ABI PRISM
TM
310 sequencer (PE Applied
Biosystems, Weiterstadt, Germany).
Purification of HypE and HypF proteins
Overproduction of HypE and of i ts derivatives took place in
E. coli BL21(DE3) [24] transformed w ith the respective
plasmid. The following proced ure was developed for
purification of HypE from the wild-type and essentially
the same could be adopted for the mutant variants. The
transformants w ere grown aerobically in LB-medium in 2-L
Erlenmeyer flasks a t 37 °C until the culture reached an A
600
of 1. The expression was initiated by the addition of 0.5 m
M
isopropyl thio-b-
D
-galactoside follo wed by a furthe r 3-h
incubation period. The cells were collected by centrifugation
at 3000 g
1,2
, washed in a buffer containing 50 m
M
Tris/HCl,
1,2
pH 7.4, centrifuged again and taken up in 1 : 10 of the
volume of 50 m
M
Tris/HCl,

3
pH 7.4, 10 m
M
magnesium
acetate, 50 m
M
sodium chloride, 0.1 m
M
dithiothreitol and
0.5 lgÆmL
)1
each of leupeptin and p epstatin. A fter addition
of 20 lgÆmL
)1
each of phenylmethylsulfonyl fluoride and
DNAse I, the cells were broken by a passage through a
French Press cell at 118 Mpa.
The crude extract was clarified by centrifugation
(30 000 g for 30 min) and the supernatant was loaded on
a 35 mL DEAE-Sepharose Fast Flow Column (Pharmacia,
Freiburg, Germany), which had been equilibrated with
50 m
M
Tris/HCl,
4
pH 7.4, 10 m
M
magnesium acetate,
50 m
M

sodium chloride, and 0.1 m
M
dithiothreitol. Elution
was performed with a linear gradient of sodium chloride
reaching from 50 to 350 m
M
at a flow rate of 60 mLÆh
)1
.
The separation was followed via SDS/PAGE of each
fraction. HypE-containing fractions were sampled, brought
to an ammonium sulfate concentration of 30% saturation
andslowlystirredat0°C for 0.5 h. The precipitate
developed was collected by centrifugation at 15000 g for
30 min.
The precipitate was dissolved in a minimum of a buffer
containing 50 m
M
Tris/HCl
5
pH 7.4, 10 m
M
magnesium
Ó FEBS 2004 Hydrogenase maturation protein HypE (Eur. J. Biochem. 271) 3429
acetate, 100 m
M
sodium chloride, 0.1 m
M
dithiothreitol,
dialyzed against the same buffer a nd subjected to gel

filtration o ver a HiL oad
TM
16/60 Superdex
TM
75 pg column
(1.6 · 60 cm) (Pharmacia, Freiburg, Germany) at a flow
rate of 60 mLÆh
)1
. F ractions containing apparently homo-
genous HypE were sampled, dialyzed against the same
buffer containing 50% glycerol (v/v) and stored at )20 °C.
The purification of wild-type HypF protein has been
described [11]. The purified HypF mutant proteins investi-
gated were obtained employing an identical protocol.
Electrophoretic separations
Separation of proteins under denaturing conditions was
conducted by SDS/PAGE employing gels made up of 10%
or 12.5% polyacrylamide [25] and following the sample
denaturation condition indicated. Electrophoresis took
place at room temperature at a voltage of 150 V. For the
immunological detection of HypE protein, the separated
proteins were transferred onto a nitrocellulose membrane
(BioTrace NT; P all Corp., Dreieich, Germany)
6
,stainedwith
amidoblack and the m embrane was subjected t o a standard
immunoblotting procedure [14]. Polyclonal antibodies
directed against HypE protein or H ypF protein were used
in dilutions of 1 : 500 and 1 : 1000, respectively. D etection
of the a ntibody–antigen complex on the membrane

occurred by decoration with horseradish peroxidase cou-
pled to Staphylococcus aureus protein A (dilution 1 : 3000)
and by detection with the Lumi-Light Western Blotting
Substrate (Roche Diagn ostics GmbH, Mannheim, G er-
many) via exposure to WICORexÒ B+, Medical X-ray
screen films.
Separation of proteins under nondenaturing conditions
was achieved with the procedure described previously [14].
Transcarbamoylation/dehydration assays
The assay was performed by m ixing the HypE and HypF
proteins at the i ndicated concentrations in buffer containing
50 m
M
Tris/HCl,
7
pH 7.5, 100 m
M
KCl, 5 m
M
magnes-
ium acetate, 0.1 m
M
each of ATP and
14
C-labelled car-
bamoylphosphate in the presence or absence 0.1 m
M
dithiothreitol. The reaction was performed at 25 °Cfor
10–30 min. The radioactivity transferred to H ypE was
either determined by filtrating the samples through nitro-

cellulose filters and subsequent scintillation counting of the
label r etained by the filters as described previously [12] or by
separating the m ixture in polyacrylamide gels. To detect the
14
C-labelled forms of HypE, non denaturing PAGE [14] and
a Ômild -denaturingÕ SDS/PAGE was employed. In the latter
case, the proteins of the reaction mixture were mixed with
sample buffer containing dithiothreitol at a final concen-
tration of 100 m
M
,heatedfor10minto56°Cand
subjected to SDS/PAGE afterwards. All separations were
conducted in the cold room at a voltage of 110 V o r less.
After the separation, the p roteins were transferred onto
nitrocellulose membranes that were dried and exposed to
Tritium Storage Phosphor Screen Cassettes (Amersham
Biosciences, Freiburg, Germany). The radioactivity of the
screens w as scanned with a Storm 840 PhosphoImager
(Amersham Biosciences, F reiburg; Germany) and data were
analyzed using
IMAGEQUANT
5.2.
Determination of the ATPase activity
The hydrolysis of ATP by HypE was followed in assays
containing 50 m
M
Tris/HCl
8
pH 7.4, 100 m
M

KCl, 5 m
M
MgCl
2
,0.1m
M
dithiothreitol, 50 lgÆmL
)1
of bovine serum
albumin and HypE protein at the indicated concentration.
The reaction took place at 25 °C in a final volume of
100 lL. Ten microliter samples were taken, mixed with
500 lL (5%) charcoal suspended in 50 m
M
KH
2
PO
4
for
30 s and clarified by centrifugation. The radioactivity of
100 lL samples of the supernatant was determined in a
Liquid Scintillation Analyzer Tri-Carb 2100TR (Canberra
Packard GmbH, Dreieich, Germany).
Alternatively, ATP hydrolysis was followed in reaction
mixtures of identical composition except containing
[
32
P]ATP[aP]. After incubation, 1-lL samples were spotted
onto polyethyleneimine plates (Merck, D armstadt, Ger-
many) which were developed with 0.5

M
KH
2
PO
4
,pH3.4.
After drying, the plates were exposed to Storage Phosphor
Screen Cassettes (Molecular Dynamics) and the radioactiv-
ity of the screens was quantified in a Storm 840 Phospho-
Imager (Amersham Biosciences, Freiburg, Germany).
Enzymes and special chemicals
Enzymes for restriction and modification of DNA were
purchased from one of the following companies: MBI
Fermentas (St. L eon-Rot, Germany), N ew England Biolabs
(Frankfurt, Germany), Stratagene (Heidelberg, Germany)
Roche Molecular Biochemicals (Penzberg, Germany) and
Eurogentec (Ko
¨
ln, Germany). Oligonucleotides were syn-
thesized by MWG (Ebersberg, Germany) o r Interactiva
(Ulm, Germany). Carbamoylphosphate was obtained either
from Sigma (Deisenhofen, Germany) or ICN Biomedical
Inc. (Eschwege, Germany). It was provided in the form of
the dilithium salt and had a purity between 90 and 95%.
14
C-labelled carbamoylphosphate was purchased from
American Radiolabelled Chemicals Inc. (St. Louis, MO,
USA) at a specific radioactivity of 7 mCi Æmmol
)1
.Itwas

dissolved in water and distributed into small aliquots that
were stored at )80 °C.
Results
Sequence characteristics of the HypE protein
The in silico analysis of the amino acid sequence of the
HypE protein revealed similarities with those of the ThiL
protein (thiamin monophosphate kinase), the SelD protein
(monoselenophosphate synthetase) and the PurM protein
(aminoimidazole ribonucleotide synthetase) [26]. The simi-
larity embraces several positions and sequence stretches that
are assumed to be involved in the binding of ATP. The
determination of the crystal structure of the PurM protein
from E. coli in complex with its substrate then showed that
these residues indeed are involved in liganding ATP,
forming a novel nucleoside triphosphate binding site [26].
All members of the HypE family, in addition, possess the
strongly conserved C-terminal tetrapeptide PR[I/V]C [12].
The sequences of HypE and of SelD could be modelled
into the coordinates of the Pu rM 3D structure, which
suggested that the three proteins might catalyze reactions
3430 M. Blokesch et al. (Eur. J. Biochem. 271) Ó FEBS 2004
mechanistically similar to that of PurM, namely an ATP-
dependent dehydration. Indeed, HypE from E. coli dehy-
drates the carboxamido residue linked t o the C-terminal
cysteine of HypE to the protein thiocyanate [12].
Purification and properties of the HypE protein
from
E. coli
HypE protein was overproduced in the transformant of
E. coli BL21(DE3) harbouring plasmid pTE-C2 and puri-

fied following the procedure outlined under Experimental
procedures. In short, th is involved breakage of the cells by
passage through a French Press cell, preparation of the
30 000 g supernatant, anion exchange chromatography
over a DEAE column followed by ammonium sulfate
precipitation and gel filtration. Figure 1 gives the path of
purification as visualized by SDS/PAGE of the pooled
fractions after each step and staining with Serva-Blue
R-250. In a typical purification run about 14 mg apparently
homogenous HypE protein were obtained from 4 g of cells
(wet weight). Essentially the same purification protocol
could be employed to purify the following two m utant
variants of the HypE protein: (a) HypED which lack s the
C-terminal cysteine residue and (b) HypE[D83N] in which
the aspartate shown to participate in ATP binding [26] is
replaced by asparagine.
Inductively coupled atomic emission spectroscopy of the
protein showed that the purified preparation does not
contain metal in a significant amount. The UV and visible
spectrum also did not indicate the existence of a cofactor
absorbing in the wavelength range between 250 and 500 nm
(results not shown). Upon gel filtration of the protein on a
calibrated HiLoad
TM
16/60 Superdex
TM
75 pg column the
majority migrated as an apparent monomer. However,
there w as always HypE protein i n the elution position o f the
apparent homodimer. This had already been observed in the

electrophoretic separation in polyacrylamide gels under
nondenaturing conditions [12].
To assess whether this putative dimer is the product of a
chemical linkage via disulfide bridging or the result of a
monomer d imer equilibrium, samples of the purified
preparations of wild-type HypE, of HypE[D83N] and
HypED were incubated in the presence of 50 m
M
dithio-
threitol and subjected to SDS/PAGE under nondenaturing
conditions (Fig. 2A). The preincubation converted the
apparent homodimer present in the wild-type and
HypE[D83N] preparations into the monomeric form.
Because HypED, on the other hand, was devoid of the
homodimeric form these results suggest that the homodimer
is the result of disulfide bridging via the C-terminal cysteine
residues. This conclusion is supported by the results of
carbamoylation of the HypE forms by HypF protein in the
presence of carbamoylphosphate and ATP (Fig. 2 B). Incu-
bation with dithiothreitol grossly increased the capacity to
accept the label from [
14
C]carbamoylphosphate, which is in
accord with the notion that the C-terminal cysteine is
required for activity [12]. Although it is still open whether
the disulfide-bridged dimer is o f biological significance, the
results stress the necessity for the reductive activation of
HypE in order to obtain maximal acceptor activity in vitro.
HypE protein possesses intrinsic ATPase activity
As suggested by the sequence signatures, HypE protein

possesses ATPase activity delivering ADP and inorganic
Fig. 1. Purification of HypE protein from an overexpressing strain as
followed by SDS/PAGE (12.5%) of the pooled fractions of each step.
Lane 1: molec ular mass ( kDa) s tandard ( b-galactosidase, bovine serum
albumin, ovalbumin, lactate dehydrogenase, endonuclease Bsp98I,
b-lactoglobulin, lysozyme); lane 2: cell lysate of BL21(DE3)/pTE-C2
before inductio n; lane 3: cell lysate of BL21(DE3)/pTE -C2 after
induction with 1 m
M
isopropyl thio-b-
D
-galactoside; lane 4: S30
extract; lane 5: sediment of the 3 0 000 g centrifugation; lane 6: p ooled
fractions after DEAE Sepharose chromatography and ammonium
sulfate p recipitation up to 30% saturation; lane 7: HypE prote in after
gel filtration (Superdex-75) and dialysis. The gel was stained for pro-
teins with Serva Blue R-250.
Fig. 2. Migration behaviour and activity of wild -type HypE and m uta nt
variants in 10% nondenaturing SDS gels after preincubation with
dithiothreitol. (A) Serva Blue R-250 stained SDS gel. Lane 1: molecular
mass standard; lanes 2 and 3: wild -type HypE p rotein; lanes 4 an d 5:
HypE[D83N]; lanes 6 and 7: HypED. In lanes 2, 4 and 6 proteins
(12 l
M
) were preincubated with 50 m
M
dithiothreitol fo r 1h on ice.
The monomeric and dimeric formsofHypEanditsvariantsare
indicated by arrows. T he chemical basis of the migration in two fo rms
is unknown (lan es 3, 5 and 7). (B) Determination of

14
C-labelled HypE
protein and its variants by binding to nitrocellulose filters [11]. Lanes
2–7 as in (A). Results are average s of three in depe ndent experiments ±
standard deviation.
Ó FEBS 2004 Hydrogenase maturation protein HypE (Eur. J. Biochem. 271) 3431
phosphate as products (not shown). The hydrolysis rate is
linear with time and is not influenced by the presence of
carbamoylphosphate (data not presented). The D83N
exchange leads to the abolition of activity (not sho wn),
whereas the HypED variant displays about half of the
activity of the wild-type protein under the assay conditions
employed. The following kinetic constants of the ATP
cleavage reaction were determined for wild-type HypE in
five independent determinations: K
m:
25 ± 1.8 l
M
; K
cat
1.7 ± 0.16 · 10
)3
s
)1
.TheHypED mutant protein, on the
other hand, showed considerable variations in the kinetic
assays indicating stability problems. The average values
obtained in six independent determinations were K
m
:

47 ± 13.3 l
M
; K
cat
2.1 ± 2.6 · 10
)3
s
)1
.
Analysis of the transcarbamoylation reaction catalyzed
by HypF
The formation of the HypE-thiocyanate involves first the
carbamoylation of the C -terminal c ysteine of HypE b y
interaction with HypF, then the release of HypF and the
subsequent dehydration of the protein th iocarboxamide to
protein t hiocyanate [12]. T o r esolve these partial reactions it
was necessary to develop a method via which the carbamo-
ylated form of HypE could be differentiated from the
cyanated protein. Use w as made of the previous observation
that HypE-CN is labile in the presence of thiols. When
incubated in the presence of 1 m
M
dithiothreitol for 15 min
at 40 °C the yield of HypE-CN had been much lower in
comparison to samples incubated i n the absence of dithio-
threitol [12]. Alteration of the incubation conditions to
10 min at 56 °C in the presence of 100 m
M
dithiothreitol
(Ômild-denaturing Õ conditions) lead to the complete disap-

pearance of the cyanated form after SDS/PAGE (Fig. 3B,
lane 1) whereas it was still distinctly resolved upon
electrophoresis under nondenaturing conditions in the
absence of dithiothreitol and omission o f heating of the
mixture (Fig. 3A, lan e 1). On the other hand, samples from
assays that were blocked in the dehydration reaction
because of the inclusion of ADP-CH
2
-P instead of ATP in
the reaction [ 12] exhibited the presence of the HypE-
thiocarboxamide after SDS gel electrophoresis (Fig. 3B,
lane 2). It is striking that nondenaturing PAGE does not
resolve the presence of HypE-thiocarboxamide. A possible
reason could be that HypE-thiocarboxamide and HypE-
thiocyanate might possess differential stabilities under the
conditions of electrophoresis, in particular at the alkaline
pH of the gels. To follow t his assumption the HypE protein
was carbamoylated by HypF in the presence of ADP-CH
2
-
Pand[
14
C]carbamoylphosphate and the substrates were
removed by filtration. Parallel samples were incubated at
different pH values and the retention of the radioactivity
bound to HypE was a ssessed ( Fig. 3C) by Ômild-denaturing Õ
SDS/PAGE as described above. It is evident that alkaline
pH leads to the loss of the thio carboxamide moiety;
intriguingly, the apparent hydrolysis of the thiocarboxamide
requires native H ypE prote in as it is fully stable when the

samples are denatured in SDS sample buffer containing
100 m
M
dithiothreitol and s eparated in SDS g els possessing
the same pH.
The radioactive material migrating on the top of the
nondenaturing gel (Fig. 3A) coincides with a signal in
immunoblots detected bo th with anti-HypE and anti-HypF
antibodies (not shown). It therefore may denote a complex
between the HypE and HypF proteins that might constitute
an inte rmediate in the transcarbamoylation reaction. To
follow this a ssumption, transcarbamoylation/dehydration
reactions were carried out at different ratios between HypF
and HypE proteins and the products were separated by
nondenaturing PAGE (Fig. 4). (An incubation time was
chosen in which a 10-fo ld lower mount of HypF protein still
was able to convert all radioactivity on HypE i nto the
thiocyanate form, not shown). At close to stoichiometric
ratios, the major amount of radioactivity migrated in a
position i ndicated b y i mmunoblots to contain both proteins
(not shown). Decrease of HypF in the a ssay gradually
shifted the migration position of HypE, which refle cts a
Fig. 3. Differential detection of HypE-thiocarboxamide and HypE-
thiocyanate via separation by nondenaturing PAGE (A) and SDS/
PAGE (B) and instability of the HypE-thiocarboxamide (C). HypE
protein (2 l
M
) was mixed with HypF protein (0.5 l
M
),

14
C-labelled
carbamoylphosphate ([
14
C]CP; 100 l
M
) and eith er ATP (100 l
M
;lane
1) or ADP-CH
2
-P (100 l
M
;lane2)andincubatedat25°Cfor10min.
The sample for the nondenaturing PAGE was mixed prior to appli-
cation with a s ample buffer co ntaining 50 m
M
Tris/HCl (p H 6.8), 5%
glycerol and 0.025% bromophenol blue (final concentrations). The
sample for the SDS/PAGE was mixed prior to application with a
sample buffer containing 50 m
M
Tris/HCl (pH 6.8), 2% SDS, 5%
glycerol, 100 m
M
dithiothreitol, 0.025% bromophenol blue and heated
to 56 °C for 10 min. (C) Instability of HypE-thiocarboxamide in
9
dependence o n the pH. HypE (6 l
M

) and HypF (1 l
M
)proteinswere
mixedwith[
14
C]CP (100 l
M
)andADP-CH
2
-P (100 l
M
) and incuba-
ted for 15 min at 25 °C. Substrates and buffer were removed by
filtration and extensive washing (nanosep MWCO 10 kDa). Th e
protein f ractio n was further incubated for 10 min at 25 °Cin100m
M
Tris/HCl pH 7.5 (lane 1), pH 8.0 (lane 2), pH 8.5 ( lane 3), pH 8.8 (lane
4) and pH 9.2 (lane 5) followed by mixing with s ample buffer and
separation in a 10% SD S g el as ind icated f or (B).
3432 M. Blokesch et al. (Eur. J. Biochem. 271) Ó FEBS 2004
rapid equilibrium between HypF (81.9 kDa molecular
mass) and HypE (35.1 kDa molecular mass). When HypE
was in excess, HypE-CN monomer carried all the radio-
activity.
The analysis of the carbamoyltransferase reaction cata-
lyzed by HypF is dependent on the cleavage of ATP into
AMP and pyrophosphate [11]. Accordingly, ADP-CH
2
-P
serves as a substrate but not AMP-CH

2
-PP [ 12]. The easy
discrimination between HypE-thiocarboxamide and HypE-
thiocyanate a llowed the analysis whether AMP-CH
2
-PP is a
substrate in the dehydration reaction. To this end, trans-
carbamoylation/dehydration assays were carried out in the
presence of ATP, of AMP-CH
2
-PP and of different ratios
between ATP and its a nalogue and the p roducts were
analyzed (Fig. 5). It was found that the analogue is unable
to support HypF-catalyzed carbamoyltransfer to HypE as
the p rotein does not carr y any radioactivity (lane 2).
Surprisingly, it also inhibits conversion of the HypE-
thiocarboxamide into HypE-thiocyanate when offered
together with ATP: Formation of HypE-CN is gradually
decreased (Fig. 5A) and HypE-CONH
2
appears (Fig. 5B).
Mutant variants of protein HypE
It has been speculated previously that carbamoylphos-
phate, besides being the educt for synthesis of the CN
ligands, may also give rise to the formation of the
carbonyl ligand [12]. The thiocarboxamide of the HypE
protein thus could provide the carbamoyl moiety for the
reductive deamination t o deliver the carbonyl group
either at the HypE protein itself or after donating it to
the iron of the metal centre. A possibility to test this

assumption may be offered by the construction of a
mutant protein, which can accept the carbamoyl residue
but is unable to dehydrate it because of the lack of the
ATP-dependent phosphorylation activity. Position D83
was an attractive candidate as this residue was shown to
be involved in the binding of ATP in the PurM protein
[26]. Moreover, D83 is strictly conserved in all proteins
belonging to the PurM family of dehydratases [26]. A
D83N exchange was therefore introduced into HypE and
the protein was overproduced and purified. Figure 6 gives
the activity of the protein species in the transcarbamoy-
lation/dehydration reaction in comparison to that of wild-
type HypE and o f the variant lacking the C-terminal
cysteine (HypED). As expected, HypED does not act
as an acceptor in the carbamoylation reaction (lanes 3
and 4). The radioactive label in the D83N (lanes 5 and 6)
and D83N A76V variants (lanes 7 and 8) is present in the
thiocarboxamide form, irrespective of whether ATP or
ADP-CH
2
-P was present as substrate. These variants will
be analyzed in future whether they can transfer the
carboxamido moiety to the HypC · HypD complex.
Analysis of the transcarbamoylation reaction catalyzed
by the HypF protein
When the activity of the HypF protein was tested in the
absence of HypE (which acts as the natural substrate
accepting the carbamoyl group) it was shown to display
the following three activities: (a) carbamoylphosphate
Fig. 4. Autoradiograph of a nondenaturing PAG

10
in which the products
of transcarbamoylation/dehydration assays were separated. Reaction
mixtures co ntained HypE at 2 l
M
in the ratio to HypF indicated a bove
each lane (2 l
M
down to 0.2 l
M
). [
14
C]Carbamoylphosphate and ATP
were present at 100 l
M
each; i ncubation time was 30 min at 25 °C. The
identity of the material in the labelled bands was shown by immuno -
blotting (not sho wn).
Fig. 5. Inhibition of the ATP-dependent dehydration r eaction by AMP-
CH
2
-PP. HypE (2 l
M
) was incubated with HypF ( 0.2 l
M
)and
[
14
C]carbamoylphosphate (100 l
M

) in the presence o f ATP ( 100 l
M
where indicated) or/and AMP-CH
2
-PP at the concentrations indicated
on top of the gel. The samples were separated by nondenaturing
PAGE (A) or SDS/PAGE (B) and the proteins were transferred t o
nitrocellulose memb ranes t hat were a utorad iographed.
Fig. 6. Activity of wild-type and mutant HypE proteins in the trans-
carbamoylation/dehydration reaction. Four micromoles of wild-type
HypE protein (lanes 1 and 2), of HypED (lanes 3 and 4), HypE[D83N]
(lanes 5 and 6) and HypE[D83N A76V] ( lanes 7 and 8) were incubated
with 0.5 l
M
HypF, 100 l
M
[
14
C]carbamoylphosphate and 100 l
M
ATP(lanes1,3,5and7)or100l
M
ADP-CH
2
-P (lan es 2, 4, 6 and 8)
for 30 min at 25 °C. Th e reaction pro ducts were separated in non-
denaturing gels (A) and SDS gels (B), transferred to a nitrocellulose
membrane which was autoradiographed.
Ó FEBS 2004 Hydrogenase maturation protein HypE (Eur. J. Biochem. 271) 3433
phosphatase activity in the absence of ATP; (b) a car-

bamoylphosphate-dependent cleavage of ATP into AMP
and pyrophosphate and (c) a carbamoylphosphate-depend-
ent ATP-pyrophosphate exchange reaction [11]. The latter
activity, however, levelled off far before equilibrium was
reached.
Knowing that HypF transfers the carbamoyl moiety to a
free protein thiol group, it was first tested whether
nonprotein thiols can replace HypE as acceptor substrate.
To this end, determination of the carbamoylphosphate-
dependent ATP cleavage reaction in the presence or absence
of a thiol compound was tested. However, presence of
dithiothreitol in concentrations between 0.5 and 100 l
M
did
not influence the kinetics of ATP hydrolysis (data not
shown).
Analysis of HypF mutant proteins
The results described thus far support the contention that
the various activities of HypF reflect the particular
experimental condition, namely that carbamoylphosphate
phosphatase activity and carbamoylphosphate-dependent
ATP hydrolysis to AMP and pyrophosphate might
simply be side reactions followed in the absence of the
natural substrate HypE. To provide further proof fo r this
assumption, mutant variants of the HypF protein were
purified and analyzed. Two of the variants chosen,
HypF[R23Q] and HypF[R23E] have amino acid replace-
ments in the acylphosphatase motif; in particular, R23 is
part of an anion cradle of HypF which interacts with the
phosphate of carbamoylphosphate i n a crystal o f the

acylphosphatase domain complexed with the substrate
[27]. Another variant, HypF[H476A] has a replacement in
the histidine-rich motif close to the C-terminus, which is a
characteristic of O-carbamoyltransferases [11]. Previous
results had demonstrated that the replacement R23E
leads to a gene product inactive in vivo and devoid of
acylphosphatase activity in crude extracts. In contrast, the
exchange R23Q had only diminished these in vivo and
in vitro activities. The phenotype of the mutant harbour-
ing the gene for HypF[H476A] was indistinguishable from
that of the wild-type [11].
When the activity of the purified HypF variant proteins
in comparison to that of the wild-type HypF protein were
determined in the carbamoylphosphate-dependent ATP
hydrolysis reaction it was found that HypF[H476A]
possesses less than 10% of the activity of wild-type
HypF (results not shown). However, this activity
appeared to suffice for the generation of a wild-type-like
phenotype, especially when the gen e was expressed from a
plasmid [11]. From the two proteins with an exchange in
the acylphosphatase domain HypF[R23E] displays no
detectable activity whereas HypF[R23Q] has some minute
activity, ranging between 0.1 and 0.3% of wild-type
HypF (data not shown).
Discussion
The results presented above and reported in previous
communications [11,12] suggest the sequence of reactions
catalyzed by the HypF a nd HypE proteins, which are
depicted in Fig. 7. HypF catalyses the formation of a
protein-bound putative carbamoyl-adenylate with the con-

comitant liberation of pyrophosphate. The identity of the
adenylate has not been shown yet. In the absence of HypE
the a denylate is avidly h ydrolyzed into AMP and possibly
carbamate which is unstable. When HypE is present in the
reaction mixture the carbamoyl moiety is transferred to the
thiol of the C-terminal cysteine of HypE followed by its
ATP-dependent dehyd ration to the thiocyanate [12]. In the
course of the reaction HypF has to dock to the HypE
protein and this complex has been experimentally demon-
strated now (Fig. 4; and data not shown). It is intriguing
that at stoichiometric ratios of the two proteins most of the
substituted HypE is caught in the complex. This implicates
the existence of some mechanism to displace the product,
either via replacement by free HypE, some conformational
switch conferred to the HypE protein during the reaction or
the t ransfer of the HypE protein from Hyp F to the
HypC · HypD complex (our unpublished results).
An app arent ATP-pyrophosphate exchange reaction,
which i s d ependent o n carbamoylphosphate, has been
described p reviously to be catalyzed by HypF [11]. The
scheme of Fig. 7 now offers an explanation why this
exchange did not reach equilibrium. Whereas the observed
formation of r adioactive ATP from l abelled pyrophosphate
can be readily explained by the reversion of the reaction,
attainment of the equilibrium would also necessitate the
formation o f carbamoylphosphate from the postulated
enzyme-bound carbamoyladenylate at the expense of inor-
ganic phosphate. The situatio n is thus different from a
classical A TP-PP
i

exchange reaction like that catalyzed by
aminoacyl-tRNA synthetases in which the substrate (the
amino acid) drives both the forward a nd the reverse
reaction. An issue that is still open, however, i s why
carbamoylphosphate-dependent cleavage of ATP reaches a
Fig. 7. Scheme of the postulated reaction pattern of HypE (E) and
HypF (F) proteins. I indicates the carbamoylphosphate phosphatase
activity in the a bsence of ATP, II the carbamoylphosphate-dependent
cleavage of ATP into AMP and pyrophosphate, III the carbamoyl-
phosphate-depe ndent ATP-pyrophosphate exch ange re action, and IV
the ATP-dependent dehydration catalyzed by HypE.
3434 M. Blokesch et al. (Eur. J. Biochem. 271) Ó FEBS 2004
plateau at product concentrations well below equilibrium in
the absence of HypE. Inactivation of HypF during the
reaction might be one of several possible reasons.
The putative HypF-bound carbamoyl-adenylate is
extremely prone to hydrolysis, which removes it from the
reaction even in the p resence of HypE. This is also in distin ct
contrast to the p roperties of aminoacyl-adenylates, which are
shielded from water in the active site of aminoacyl-tRNA
synthetases and therefore protected from hydrolysis.
A reason for the instability o f the c arbamoyl-adenylate
might exist in th e observation that HypE can be retrieved
from cells as a complex with two other hydrogenase
maturation proteins, namely HypC and HypD (M. Blokesch
and A. Bo
¨
ck, unpublished results). This HypE in the triple
complex i s fully active and it might represent the actual state
of the protein within the cell.

Until now it was open a s to w hether HypF acts solely as a
carbamoyltransferase or whether it a lso participates in the
dehydration reaction. The property of mutant proteins w ith
amino acid exchanges in the nucleotide binding site of HypE
shows that the dehydration is catalyzed by HypE per se,as
the mutant proteins accept the carbamoyl-residue but are
unable to convert it into the thiocyanate. However, it is still
an open question whether dehydration is the result of an
intramolecular reaction or whether intermolecular HypE–
HypE interactions are in volved. The sites of carbamoyl-
binding and ATP-dependent d ehydrations definitely display
rather weak interdependence as HypE can act as acceptor
without possessing phosphorylation activity and exhibits
only marginally affected intrinsic ATPase activity in the
absence of the C-te rminal thiol. Availability of a HypE
variant that carries the c arboxamide m oiety but is unable to
convert it into the thiocyanate will facilitate the analysis
whether the carbonyl ligand also arises from carbamoyl-
phosphate.
Acknowledgements
We are greatly indebted to R. Thauer for discussion and helpful
suggestions. We t hank E. Ze helein for expert purification of HypF and
HypE, H. H artl for the ICP spectroscopy of the proteins an d
F. Lottspeich for determination of the N -terminal amino acid sequence
of HypE. This work was supported by the Deutsche Forschungsge-
meinschaft and the Fonds der C hemischen Industrie.
References
1. Vignais, P.M., Billoud, B. & Meyer, J. (2001) Classification and
phylogeny of hydrogenases. FEMS Microbiol. Rev. 25, 455–501.
2. Bo

¨
ck, A. & Sawers, G. (1996) Fermentation. In Escherichia coli
and Salmonella (Neidhardt, F.C., Curtiss,R., III, Ingraham, J.L.,
Lin, E.C.C., Low, K.B., Magasanik, B., Reznikoff, W.S., Riley,
M., Schaechter, M. & Umbarger, H.E., eds), pp. 262–282,
American Society for Microbiology, Washington, DC.
3. Frey, M., Fontecilla-Camps, J.C. & Volbeda, A. (2001) Nickel-
iron hydrogenases. In Handbook of Metalloproteins (Messer-
schmidt,A.,Huber,R.,Poulos,T.&Wieghardt,K.,eds),pp.880–
896. John Wiley & Sons, Chichester, UK.
4. Happ e, R.P., Roseboom, W., P ierik, A.J., Albracht, S. P. & Bag-
ley, K.A. (1997) Biological activation o f hydrogen. Nature 385,
126.
5. Pierik, A.J., Roseboom, W., Happe, R.P., Bagley, K.A. &
Albracht, S .P.J. (1999) C arbon monoxide and cyanide as intrinsic
ligands to iron in the active site of [NiFe]-hydrogenases. J. Biol.
Chem. 274, 3331–3337.
6. Jacobi, A., Rossmann, R. & Bo
¨
ck, A. (1992) The hyp operon gene
products are requir ed for the m aturatio n of catal ytically ac tive
hydrogenase isoenzymes in Escherichia coli. Arch. Microbiol. 158,
444–451.
7. Blokesch, M., Paschos, A., Theodoratou, E., Bauer, A., Hube, M.,
Huth,S.&Bo
¨
ck, A. (2002) Metal insertion into NiFe-hydro-
genases. Biochem. Soc. Trans. 30, 674–680.
8. Mehta, N., O lson, J.W. & Maier, R.J. (2003) Characterization of
Helicobacter pylori nickel metabolism accessory proteins needed

for m aturation of both urease and hydrogenase. J . Bacteriol. 185,
726–734.
9. Blok esch, M., Rohrmoser, M., Rode, S. & Bo
¨
ck, A. (2004) HybF,
a zinc containing protein involved in NiFe hydrogenase matura-
tion. J. Bacteriol. 186, 2603–2611.
10. Paschos, A. , Glass, R.S. & Bo
¨
ck, A. (2001) Carbamoylphosphate
requirement for synthesis of the active center of [NiFe]-hydro-
genases. FEBS Lett. 488, 9–12.
11. Paschos, A., Bauer, A., Zimmermann, A ., Zehelein, E. & Bo
¨
ck, A.
(2002) HypF, a carbamoyl phosphate-converting enzyme involved
in [NiFe] hydrogenase maturation. J. Biol. Chem. 277, 49945–
49951.
12. Reissmann, S., Hochleitner, E., Wang, H., Paschos, A., Lottspe-
ich, F., Glass, R.S. & Bo
¨
ck, A. (2003) Taming of a poison: bio-
synthesis of the NiFe-hydrogenase cyanide ligands. Science 299,
1067–1070.
13. Blokesc h, M. & Bo
¨
ck, A. (2002) Maturation of [NiFe]-hydro-
genases in Escherichia coli: the HypC cycle. J. Mol. Biol. 324, 287–
296.
14. Drapal, N. & Bo

¨
ck, A. (1998) Interaction of the hydrogenase
accessory protein HypC with HycE, the large subunit of Escher-
ichia coli hydrogenase 3 du rin g enzyme m aturation. B i oche mistr y
37, 2941–2948.
15. Magalon, A. & Bo
¨
ck, A. (2000) Analysis of the HypC-HycE
complex, a key int ermediate in the assembly of t he metal center of
the Escherichia coli hydrogenase 3. J. Biol. Chem. 275, 21114–
21220.
16. Casadaban, M.J. & Cohen, S.N. (1979) Lactose genes fused to
exogenous promoters in one step using a Mu-lac bacteriophage:
in vivo probe for transcriptional control sequences. Proc. Natl
Acad. Sci. USA 76, 4530–4533.
17. Yanisch-Perron, C., Vieira, J.&Messing,J.(1985)ImprovedM13
phage cloning vec tors a nd ho st strains: nucleotide sequences o f the
M13mp18 and pUC19 v ectors. Gene 33, 103–119.
18. Miller, J.H. ( 1992) A s hort course in bacterial ge netics. In AShort
Course in Bacterial Genetics. Cold Springer Harbor Laboratory
Press, Col Spring Harbor, New Y ork.
19. Begg, Y.A., Whyte, J.N. & Haddock, B.A. (1977) The identifica-
tion of mutants of Escherichia coli deficient in formate dehy-
drogenase and nitrate reductase activities using dye indicator
plates. FEMS Microbiol. Lett. 2, 47–50.
20. Schlensog, V. & Bo
¨
ck, A. (1990) Identification and sequence
analysis of the gene encoding the transcriptional activator of the
formate hydrogenlyase system of Escherichia c oli. Mol. Microbiol.

4, 1319–1327.
21. Ansaldi, M., Lepelletier, M. & Mejean, V. (1996) Site-specific
mutagenesis by using an accurate recombinant polymerase chain
reaction method. Anal. B iochem. 23 4, 110–111.
22. Lutz,S.,Jacobi,A.,Schlensog,V.,Bo
¨
hm,R.,Sawers,G.&Bo
¨
ck,
A. (1991) Molecular characterization of an operon (hyp) necessary
for the activity of the three hydrogenase i soenzymes in Escherichia
coli. Mol. Microbiol. 5, 123–135.
23. Ochman, H., Gerber, A.S. & Hartl, D.L. (1988) Genetic applica-
tions of an inverse polymerase chain reaction. Genetics 120,
621–623.
Ó FEBS 2004 Hydrogenase maturation protein HypE (Eur. J. Biochem. 271) 3435
24. Studier, F.W. & Moffatt, B.A. (1986) Use of bacteriophage T7
RNA polymerase to direct selective high-level expression of cloned
genes. J. Mol. Biol. 189, 113–130.
25. Laemmli, U.K. (1970) Cleavage of structural proteins during the
assembly of the he ad of b acteriophage T4. Na tu re 227, 680–685.
26. Li, C., Kappock, T.J., S tubbe, J., Weaver, T.M. & Ealick, S.E.
(1999) X-Ray crystal structure of aminoimidazole ribonucleotide
synthetase (PurM), from the Escherichia coli purine biosynthetic
pathway at 2.5 A
˚
resolution. Structure 7, 1155–1166.
27. Rosano,C.,Zuccotti,S.,Bucciantini, M., Stefani, M., Ramponi,
G. & Bolognesi, M . (2002) Crystal s tructure and anion binding i n
the prokaryotic hydrogenase maturation factor H ypF acylphos-

phatase-lik e do main. J. Mol. Biol . 321, 785–796.
3436 M. Blokesch et al. (Eur. J. Biochem. 271) Ó FEBS 2004