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J. Biol. Chem., Vol. 277, Issue 51, 49945-49951, December 20, 2002
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,,, and¶
From the Mikrobiologie, Department I der
Fakultät für Biologie, Ludwig-Maximilians-Universität
München, Maria-Ward-Strasse 1a, D-80638 München and
§ Ascenion GmbH, Ingolstädter Landstrasse 1,
D-85764 Oberschleißheim, Germany
Received for publication, May 10, 2002, and in revised form, August 29, 2002
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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HypF has been characterized as an auxiliary
protein whose function is required for the synthesis of active [NiFe]
hydrogenases in Escherichia coli and other bacteria. To
approach the functional analysis, in particular the involvement in
CO/CN ligand synthesis, HypF was purified from an overproducing strain
to apparent homogeneity. The purified protein behaves as a monomer on
size exclusion chromatography, and it is devoid of nickel or other
cofactors. As indicated by the existence of a sequence motif also
present in several O-carbamoyltransferases, HypF interacts
with carbamoyl phosphate as a substrate and releases inorganic
phosphate. In addition, HypF also possesses ATP cleavage activity that
gives rise to AMP and pyrophosphate as products and that is dependent
on the presence of carbamoyl phosphate. This and the fact that HypF
catalyzes a carbamoyl phosphate-dependent pyrophosphate ATP
exchange reaction suggest that the protein catalyzes activation of
carbamoyl phosphate. Extensive mutagenesis of the putative functional
motifs deduced from the derived amino acid sequence showed a full
correlation of the resulting variants between their activity in
hydrogenase maturation and the in vitro reactivity with
carbamoyl phosphate. The results are discussed in terms of the
involvement of HypF in the conversion of carbamoyl phosphate to the CN ligand.
Hydrogen metabolism in enterobacteria involves the activity of the
products of three functional classes of genes that code for structural
proteins, regulatory proteins, or for proteins involved in metal center
biosynthesis and enzyme maturation. In Escherichia coli, the
structural genes are organized in four operons, responsible for the
formation of hydrogenase 1 (hya operon), hydrogenase 2 (hyb operon), hydrogenase 3 (hyc operon), and the
putative hydrogenase 4 (hyf operon). Each operon contains
the genes for the large and small hydrogenase subunit, for redox
carriers, membrane anchor proteins plus components required for the
maturation of the hydrogenase encoded by that specific operon, like the
endopeptidase involved in proteolytic processing of the large subunit
(for review see Refs. 1 and 2). Because the hydrogenases in E. coli serve different physiological functions, the expression of
these operons is differentially regulated. Hydrogenase 3, for example,
which is a component of the formate hydrogen lyase system, is
synthesized under fermentative conditions. Its formation requires the
activity of the transcriptional activator FhlA and formate as inducer
(3).
Considerable efforts have been directed to understand the incorporation
of the [NiFe] metal center into the large hydrogenase subunit. A
scenario is emerging indicating that iron and nickel insertion proceeds
separately, whereby the incorporation of iron together with its CO and
CN ligands precedes that of nickel (1, 4, 5). The function of the HypA
and HypB proteins has been related to nickel insertion because
hypA and hypB mutations can be phenotypically
complemented by inclusion of high nickel concentrations in the medium
(6, 7) and because the HypB protein binds nickel (8). HypC, which is a
small acidic protein, forms a complex with the precursor of the large
hydrogenase subunit and, accordingly, has been postulated to play the
function of a specific chaperone, maintaining a folding state to render
the large subunit amenable for metal acquisition (9, 10). The most
detailed information for all maturation proteins is available for the
endopeptidase which cleaves the C-terminal extension from the precursor
of the large subunit once nickel has been inserted (5, 11, 12). It is
thought that the endopeptidase controls the fidelity of nickel
insertion (13) and also, by cleavage, induces the conformational shift
required to thread the finished metal center into the interior of the
large subunit (1, 5, 14).
An intriguing issue of [NiFe] center synthesis concerns the source
and the pathway of biosynthesis of the CO and CN ligands. Circumstantial evidence indicates that the HypD, HypF, and HypE proteins are involved in this process. HypD is an Fe/S protein with
unusual spectroscopy
properties,1 and HypF and
HypE display sequence signatures that implicate functions in organic
synthesis. HypF, for example, has been reported to carry a sequence
motif characteristic of acyl phosphatases (15), and HypE shares motifs
that are also found in aminoimidazole ribonucleotide synthetase and
thiamin-phosphate kinase (16, 17).
An important discovery in this connection was the finding that
maturation of [NiFe] hydrogenases requires the availability of
carbamoyl phosphate (CP).2 A
mutant of E. coli lacking CP synthetase (carAB
genes) activity was unable to synthesize active hydrogenases 1-3. The
deficiency was shown to be caused by a blockade of the maturation
process (18). Citrulline in the medium was able to complement the
lesion of the carAB mutant indicating that the carbamoyl
moiety was crucial. It was postulated that CP is the precursor of
either CO or CN or of both of them (18). In the present communication
we show that the HypF protein recognizes CP as a substrate. A detailed mutational analysis of conserved sequence traits was performed, and it
is shown that abolition of HypF function in the maturation process is
fully correlated with its ability to interact with CP as a substrate.
It is further shown that HypF cleaves ATP into AMP and pyrophosphate in
the presence of CP.
E. coli Strains, Plasmids, and Growth Conditions--
Strain
MC4100 (19) from E. coli was used as wild type, and strains
JM109 (20) and DH5
The plasmids employed in this work are listed in Table
I, together with their genotype and
source or derivation. Maintenance of plasmids in the transformants was
selected for by inclusion of the appropriate antibiotics into the
medium, ampicillin at 150 µg/ml and chloramphenicol at 30 µg/ml. If
not indicated otherwise, bacteria were grown aerobically in LB medium
(23) or anaerobically in TGYEP medium (24). Aerobic cultures were grown
in Erlenmeyer flasks under rigorous rotatory shaking, and anaerobic
growth took place in bottles filled with the medium to the top. Growth
temperature was 37 °C throughout.
Plasmid Construction--
Plasmid pUCF18 was constructed for
overexpression of the hypF gene. For this purpose, a 2.6-kb
EcoRI-BamHI fragment from plasmid pTF5 (22) was
inserted into plasmid pUC18 linearized by digestion with the same
endonucleases. pUCF18, therefore, carries a hypF gene whose
expression is under the control of the lac promoter.
Site-directed mutagenesis of the hypF gene was performed via
inverse PCR using primers that carried the desired mutation (25). Alternatively, mutations were introduced with the aid of overlapping primers according to Ansaldi et al. (26). In each instance, plasmid pAF1 (22) was used as template, and the Expand High Fidelity
PCR System from Roche Biochemicals (Penzberg, Germany) was employed.
The amplified segment was phosphorylated, religated, and transferred
into host DH5 Purification of the HypF Protein--
Cultures of strain JM109
transformed with plasmid pUCF18 were grown in 300 ml of LB medium in
2-liter Erlenmeyer flasks. After reaching an
A600 of 1.0, expression of hypF was
induced by the addition of
isopropyl-1-thio-
The cells were broken by passage through a French press cell at 118 MPa, and the homogenate was clarified first by centrifugation at
10,000 × g for 30 min (S10 supernatant) and
subsequently for 2 h at 100,000 × g (S100). The
S100 fraction was adjusted to a protein concentration of 15 mg/ml and
brought to an ammonium sulfate saturation of 35% by the addition of
solid (NH4)2SO4 followed by
stirring at 0 °C for 30 min. The precipitate formed was collected by
centrifugation (30 min at 15,000 × g), dissolved in a
minimum of buffer (10 mM Tris/Cl, pH 7.4, 1 mM
DTT), and dialyzed against the same buffer.
The dialysate was fractionated by anion exchange chromatography on a
Mono-Q HR 5/5 (Amersham Biosciences) column (1 ml) equilibrated with 10 mM Tris/Cl, pH 7.4, 1 mM DTT. Elution was with
a gradient from 0 to 1 M sodium chloride at a flow rate of
60 ml/h. Fractions (1.5 ml) were monitored for presence of HypF by
SDS-PAGE (27) and staining with Coomassie Blue. HypF-containing
fractions were combined and subjected to ammonium sulfate precipitation
at 50% saturation, and the precipitate formed was collected by centrifugation.
The sediment was dissolved in a small volume of Tris/Cl (10 mM, pH 7.4), 1 mM DTT containing ammonium
sulfate at 20% saturation and applied to a 1-ml phenyl-Superose HR 5/5
column (Amersham Biosciences) that had been equilibrated with the same
buffer. Bound proteins were eluted with a decreasing linear gradient
from 20 to 0% ammonium sulfate at a flow rate of 30 ml per min. The elution of the HypF protein was monitored by SDS-PAGE. Fractions containing HypF were combined, and the protein was concentrated by
ultrafiltration with the aid of the
MICROSEPTM-Microconcentration system (Pall Gelman
Laboratory, Dreieich, Germany).
The final purification step consisted of a gel filtration over a 300-ml
Superdex XK 26/60 (Amersham Biosciences) column. It was developed with
25 mM Tris/Cl, pH 7.4, 100 mM sodium chloride, 1 mM DTT at a flow rate of 0.5 ml per min. 1-ml fractions
were collected and assayed for HypF content by SDS-PAGE. Fractions containing HypF of apparent homogeneity were dialyzed against 25 mM Tris/Cl, pH 7.4, 1 mM DTT, 100 mM NaCl, 50% glycerol (v/v) and stored at Electrophoretic Techniques--
SDS-PAGE was performed according
to Laemmli (27) and non-denaturing PAGE as described by Drapal and
Böck (9). Immunological detection of HypF in polyacrylamide gels
after electrophoretic transfer onto nitrocellulose membranes was
achieved with anti-HypF antibodies in a 1:1000 dilution and the use of
the Lumi-Light Western blotting Substrate (Roche Diagnostics).
Antibodies directed against HypF were generated by Eurogentec (Seraing, Belgium).
Determination of Carbamoyl-phosphate Phosphatase
Activity--
CP phosphatase activity was followed via the liberation
of inorganic phosphate, determined according to Heinonen and Lahti (28). Cell extracts or purified HypF proteins were incubated in 1-ml
reaction mixtures containing 50 mM Tris/Cl, pH 7.4, 100 mM KCl, 1 mM MgCl2, 0.1 mM DTT, and the CP concentration indicated at 30 °C. At
intervals of 15 min, 200-µl samples were taken, mixed with 40 µl of
30% trichloroacetic acid, and centrifuged for 2 min at 12,000 × g. 200 µl of the supernatant were used for the colorimetric determination of phosphate. To account for the chemical instability of CP, parallel samples were incubated without HypF protein, and the values obtained were subtracted to correct for chemical hydrolysis.
Analysis of CP phosphatase activity in non-denaturing polyacrylamide
gels was carried out as described by Mizuno et al. (29).
Dependence of ATP Hydrolysis by HypF Protein on the Presence of
Carbamoyl Phosphate--
ATP cleavage by HypF was measured in 1-ml
reaction volumes containing 50 mM Tris/Cl, pH 7.4, 100 mM KCl, 1 mM MgCl2, 0.1 mM DTT, 25 nM HypF, 50 µg of bovine serum
albumin per ml, and [ [32P]PPi-ATP Exchange
Assay--
PPi-ATP exchange activity catalyzed by HypF was
determined in 1-ml assays containing 50 mM Tris/Cl, pH 7.4, 100 mM KCl, 1 mM MgCl2, 0.1 mM DTT, 0.1 mM ATP, 0.1 mM
[32P]PPi (specific radioactivity 25 µCi/µmol), carbamoyl phosphate at the indicated concentrations, and
62.5 nM HypF protein. After 0, 1, 2, 4, 8, and 16 min of
incubation at 37 °C, 100-µl samples were mixed with 25 µl of
HClO4 (30%), and the mixture was combined with 30 µl of acid-washed charcoal (12% suspension in water). After 5 min,
100 µl of 1% cold sodium pyrophosphate solution in 14%
HClO4 was added, followed by incubation for 10 min
with mixing and collection of the charcoal on Whatman GF/C glass fiber
filters. The filters were washed twice with 5 ml of ice-cold 1% sodium pyrophosphate in 14% HClO4, once with 5 ml of
ice-cold 1% sodium pyrophosphate in 1.4% HClO4,
once with 5 ml of ice-cold distilled water, and dried. Their
radioactivity was determined in a scintillation counter.
Analysis of Sequence Data--
Homology searches were conducted
with the aid of the NCBI data base of the National Institutes of Health
and the ERGO data base of Integrated Genomics Inc. (Chicago, IL). DNA
sequence analyses were performed with the GENEWORKS 2.5 program
(Intelligenetics Inc., Geel, Belgium) or the Gene Tool Lite 1.D (Bio
Tools Inc., Edmonton, Canada). Protein sequence alignments were
calculated with either GENEWORKS 2.5 (or with MEGALIGN (DNAstar Inc.,
Madison, WI). The computer-based alignments were checked and amended
"by hand" if appropriate.
Enzymes and Special Chemicals--
Enzymes for restriction and
modification of DNA were purchased from one of the following companies:
MBI Fermentas (St. Leon-Rot, Germany), New England Biolabs (Frankfurt
am Main, Germany), Stratagene (Heidelberg, Germany), Roche Molecular
Biochemicals, and Eurogentec (Köln, Germany). Oligonucleotides
were synthesized by MWG (Ebersberg, Germany) and Interactiva (Ulm,
Germany). Acetyl phosphate and other phosphorylated compounds were
obtained from Sigma. Carbamoyl phosphate was used as the di-lithium
salt and had a purity of 90-95%. It was purchased from Sigma or from
ICN Biomedical Inc. (Eschwege, Germany). [ Sequence Characteristics of the HypF Protein--
A schematic
representation of sequence motifs strongly conserved in HypF homologs
from bacteria and archaea is given in Fig. 1. The most intriguing characteristic is
the existence of two perfect apparent "zinc finger" motifs from
amino acid positions 109-184 (30). They have been implicated in the
binding of some bivalent cation, but experimental studies on such a
function are lacking thus far. On the N-terminal side of these motifs a
putative acyl phosphatase motif ranging from position 13-23 was
discovered (15), but biochemical results demonstrating such an activity and its role in hydrogenase maturation are not available yet. Finally,
in the C-terminal one-third of the protein there is a conserved
sequence segment containing three histidine residues in a
characteristic pattern. A data base search delivered best hits for
enzymes with an O-carbamoylation activity in the synthesis of antibiotics or nodulation factors (see Fig. 1, top).
Proteins possessing a similar motif, however, are also present in
organisms like Pyrococcus horikoshii (31) or
Sulfolobus tokodaii (32), which are not known to synthesize
antibiotics or nodulation factors. The existence of this motif had
prompted us to investigate whether CP is required for maturation of
hydrogenases. Indeed, a mutant of E. coli devoid of CP
synthetase activity was unable to develop active hydrogenases (18).
Purification and Properties of the HypF Protein from E. coli--
The hypF gene on plasmid pUCF18 was overexpressed
in strain JM109, and purified HypF was isolated from crude extracts by
ammonium sulfate precipitation, Mono-Q HR anion exchange
chromatography, hydrophobic interaction chromatography on a
phenyl-Superose HR 5/5 residue, and gel filtration over a Superose XK
26/60 column, as detailed under "Experimental Procedures." The path
of purification is displayed by Fig.
2A which presents a
Coomassie-stained SDS-polyacrylamide gel in which the pooled fractions
of each purification step were separated. Characteristically, from 142 mg of protein of a crude 30,000 × g supernatant 9 mg
of apparently purified HypF protein were obtained.
The UV-visible absorption spectrum of the purified protein, taken
between 200 and 500 nm, did not indicate the presence of any cofactor
absorbing in this range (data not shown). Size exclusion chromatography
on a calibrated Superdex 200 column showed that HypF eluted at a
position characteristic of a molecular mass of about 80 kDa
that corresponds with the size retrieved from the migration in SDS gels
and that delineated from the gene sequence, namely 81.9 kDa. Therefore,
HypF as purified appears to be a monomeric protein. Analysis of
purified HypF by atomic absorption spectroscopy and inductively coupled
plasma atomic emission spectroscopy revealed that the protein (dialyzed
against buffer lacking chelators) does not contain nickel, cobalt,
copper, manganese, or molybdenum. However, the protein contained 1 iron
atom per 7.8 HypF and 1 zinc atom per 2.5 HypF molecules. The
substoichiometric ratio of the two metals may indicate that they are
bound non-specifically. However, this needs further experimental analysis.
HypF Displays Carbamoyl-phosphate Phosphatase Activity--
HypF
shares a sequence signature motif with
O-carbamoyltransferases, and it was tested to determine
whether the protein purified can interact with CP. The liberation of
inorganic phosphate was taken as a measure. Table
II shows that HypF cleaves carbamoyl phosphate rather specifically; other acylphosphates or phosphoesters are only marginally hydrolyzed. The kinetics of CP cleavage by HypF
were determined by assaying the hydrolysis rate at different substrate
concentrations (Fig. 3). The liberation
of phosphate followed Michaelis-Menten-type saturation kinetics. In
several experiments Km values ranging from 260 to
330 µM were obtained.
Next it was tested whether the CP phosphatase activity exhibited by
purified HypF was also present in a freshly prepared crude extract.
Cells of a mutant of E. coli (DHP-F) carrying a deletion in
the chromosomal hypF gene were transformed either with
plasmid pAF1 carrying hypF or with the vector. Extracts were
prepared, and samples from S30 extracts were loaded on non-denaturing
polyacrylamide gels, and the gels were analyzed for CP phosphatase
activity (Fig. 2B). Enzyme activity developed specifically
in the extract of the transformant carrying hypF on a
plasmid, and it migrated in a position where the major band of the
purified HypF was detected. Therefore, CP hydrolysis in the crude
extract is catalyzed by the hypF gene product, and the
purified HypF protein exhibits the same activity.
Carbamoyl Phosphate-dependent Liberation of AMP from
ATP--
The primary structure of HypF also contains a sequence motif
(GXGXXGA) that resembles the "glycine-rich
loop" motif of a family of ATP-binding proteins
(GXGXXG(R/K)) (33). Its presence prompted the
investigation whether HypF possesses ATP cleavage activity. [
To characterize the CP-dependent ATP hydrolysis activity of
the HypF protein, initial reaction velocities were followed at different ATP concentrations keeping CP constant at 100 µM and also at varying CP concentrations in the presence
of 100 µM ATP. Fig. 4 shows
that the reaction follows Michaelis-Menten kinetics with both
substrates. The kinetic constants of HypF in the CP phosphatase and the
ATP hydrolysis reaction are summarized in Table
III.
The apparent affinity of HypF for CP in the phosphatase reaction is far
below that measured for it as substrate in the ATP hydrolysis reaction.
By taking into account the rates of the two reactions, ATP cleavage in
the presence of CP is kinetically favored.
The CP dependence of the ATP cleavage reaction indicates that CP may
form a covalent intermediate either with AMP or with pyrophosphate. If
this is the case, it should be visualized by the CP dependence of an
exchange of radioactive pyrophosphate with ATP (34, 35) catalyzed by
the HypF protein. For an assessment, radioactively labeled
pyrophosphate was incubated in the presence or absence of CP with
unlabeled ATP, and samples were taken and assayed for the generation of
radioactive ATP. The results obtained showed that such a
PPi-ATP exchange indeed takes place when CP is present in
the reaction mixture (Fig. 4B). The results also prove that
HypF cleaves ATP into AMP and pyrophosphate and not by the sequential
removal of two phosphate moieties, like in the selenophosphate
synthetase reaction (36). The entry into a plateau might either be
because of the attainment of the equilibrium in the PP-ATP exchange
reaction or be caused by CP substrate limitation. The retardation of
the reaction in the presence of 100 µM CP is unspecific
because CP at this concentration and higher inhibits HypF
activity.3
Mutational Analysis of HypF--
To gain further insight into the
function of HypF in the process of hydrogenase maturation and, in
particular, to correlate the in vitro activities of the
protein with the formation of the CO and CN ligands of the metal
center, an extensive mutagenesis of the hypF gene has been
carried out. Residues from the acylphosphatase, the zinc finger, and
the O-carbamoyltransferase motifs were replaced either
singly or in combination (see Fig. 1). The mutant gene products were
analyzed for their in vivo stability when the mutated genes
were expressed from a plasmid in strain DHP-F2 (
The consequences of the mutations carried by the different HypF
variants on their role in hydrogenase maturation was assessed by
following the proteolytic processing of the precursor of HycE, which is
the large subunit of hydrogenase 3 (Fig. 5B). The immunoblot indicates that exchange of the histidine residues in positions 475, 476, and 479 against alanine delivers gene products that are still
functional in hydrogenase maturation. All the other variants were
inactive. This was corroborated by measuring hydrogenase activity in
non-denaturing polyacrylamide gels after separation of 100 µg of
protein of crude extracts from the mutants. Upon substrate staining,
bands reflecting hydrogenase 2 and hydrogenase 1 activity were detected
in case of the mutant variants HypF-H475A, HypF-H476A, and
HypF-H479A.
Finally, it was important to correlate the proficiency of the HypF
variants in the hydrogenase maturation process with their in
vitro capacity to interact with CP. The transformants harboring the mutant alleles on a plasmid were grown anaerobically to an A600 of 1.0. The cells were harvested, and crude
extracts were prepared that were partially purified by centrifugation
at 100,000 × g and by ammonium sulfate precipitation.
200 µg of protein of the 30% sediment fraction was analyzed for CP
phosphatase activity (Table IV). Variants
carrying the H475A, H476A, or H479A exchanges displayed full activity,
whereas variant R23Q had detectable CP phosphatase activity but at a
very low level. All the other variants that produced stable HypF
protein were devoid of activity.
The synthesis of the CO and CN ligands of [NiFe] hydrogenases
and their attachment to the iron poses a number of intriguingly novel
features of bioinorganic chemistry. As some of the most toxic compounds
in biology, it is predictable that they are not synthesized in the free
state but rather bound to some adaptor, thus preventing their
interaction with susceptible and essential cellular components. It is
still unclear whether they are attached to the iron when it has been
inserted into the large hydrogenase subunit or whether they are
pre-formed, e.g. at some scaffold protein and transferred as
the complete entity into the apoprotein. Moreover, it is still
unclear how the precise stoichiometry of 2 CN and 1 CO per iron atom is achieved.
We have reported recently (18) that CP is required for the synthesis of
active [NiFe] hydrogenases. The experimental evidence is as follows:
(i) a mutant of E. coli devoid of CP synthetase activity was
unable to synthesize all three hydrogenases based on the inability to
mature and process the large subunits, and (ii) supplementation with
citrulline as a source of CP restored this capacity. It has also been
pointed out that reactions in metallo-organic chemistry have been
described that convert a carbamoyl into a carbonyl or cyanyl moiety
(18).
The results reported here convincingly show that the hydrogenase
maturation protein HypF is interacting with CP as a substrate and that
this interaction is involved in hydrogenase maturation. Purified HypF
dephosphorylates CP specifically, and this activity can be discovered
in electrophoretically separated extracts from an E. coli
strain overexpressing hypF. Because the HypF protein has
been purified under aerobic conditions, this also means that the CP
phosphatase activity is oxygen-stable. A causal connection between CP
and hydrogenase maturation by HypF is then provided by the mutational
analysis. There is a clear and quantitative parallelism between
maturation activity of HypF and the in vitro CP phosphatase
activity. Somewhat surprising is the discovery that mutations in all
three major signature motifs, the acylphosphatase, the zinc
fingers, and the O-carbamoyltransferase motifs, can lead to
the blockade of CP phosphatase activity. It indicates an integrated cooperativity between these domains in the cleavage reaction.
The delineation of the route how HypF could convert the carbamoyl
moiety into one or both of the CO/CN ligands is not possible yet. It
needs detailed analysis of the product formed in the
CP-dependent ATP cleavage reaction and, especially, also
the analysis of the activity of the HypE protein, since it has been
shown recently that HypF and HypE form a complex (37).3
We strongly emphasize in this context that the reactions assayed are
those of the sole HypF protein in the absence of HypE or putative
additional substrates. Although they present information on the
existence of substrate-binding sites and types of reactions catalyzed,
they do not preclude the possibility that the products measured are
those of side reactions when reaction partners are missing, and the
flux is blocked at the state of the same intermediate.
Bearing all this caveats in mind, speculations can be attempted on the
fate of carbamoyl phosphate when acted upon by HypF and HypE. First,
from the two reactions catalyzed by HypF, the CP phosphatase activity
is kinetically somewhat less favored in comparison to the
CP-dependent ATP cleavage. There are several explanations
for this inefficiency. First, CP hydrolysis may represent a side
reaction that is followed in the absence of other substrates, reflecting the transfer of the carbamoyl residue to a water molecule. Second, and alternatively, it could represent the carbamoyl transfer to
some acceptor group of the HypF protein that is paralleled by the
liberation of inorganic phosphate. The carbamoylated amino acid residue
could then lose its acyl group because the conversion into the adduct
is blocked, possibly due to the absence of HypE.
On the other hand, the incorporation of labeled pyrophosphate into ATP
that is catalyzed by HypF in the presence of CP supports the view that
CP is adenylated. Although exchange of the phosphoryl group of CP by an
adenyl residue cannot be excluded (energetically this would not make
sense), our favorite hypothesis is that adenylation takes place at the
hydroxyl of the tautomeric form of the carbamoyl moiety yielding the
iminoform of the carbamoyl adenylate. Removal of a hydrogen from the
imino group would directly lead to the cyano moiety. It resembles the
well known dehydration reaction with the formation of a phosphorylated
intermediate. Interestingly, HypE shares structural similarity with
PurM which catalyzes such a reaction in the purine biosynthetic pathway
(16). Further work is aimed at this possibility.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(21) served as hosts for transformations. DHP-F
(22) and DHP-F2 are derivatives of MC4100 with different deletions in
the hypF gene; DHP-F lacks the segment of the gene reaching
from amino acids 289 to 629, and DHP-F2 lacks the region from amino
acids 59 to 629. DHP-F thus forms a shortened version of HypF that is
detectable by anti-HypF antibodies in immunoblotting experiments of
crude extracts, whereas no immunologically reacting material can be
found in extracts from DHP-F2.
Plasmids used
via electroporation. Amplified fragments generated
with the use of overlapping primers (26) were purified by passage over
a QIAquick Spin column (Qiagen GmbH, Hilden, Germany) and directly used
to transform strain DH5. The authenticity of the mutant
hypF gene versions was verified by DNA sequencing using an
ABI PRISMTM 310 sequencer (PE Applied Biosystems,
Weiterstadt, Germany).
-D-galactopyranoside at 1 mM final concentration. The cells were harvested after
3 h by centrifugation, resuspended in 10 mM Tris/Cl,
pH 7.4, and sedimented again. The washed cells were suspended in 10 mM Tris/Cl, pH 7.4, 1 mM DTT (1% of the
cultivation volume), and the suspension was brought to 20 µg/ml
phenylmethylsulfonyl fluoride and DNase I each.
20 °C.
-32P]ATP (specific radioactivity
of 16.1 µCi/µmol) and carbamoyl phosphate at the indicated
concentrations. The reaction mixtures were incubated at 37 °C, and
100-µl samples were taken at the given intervals, mixed with 300 µl
of perchloric acid-washed charcoal suspension for 1 min, and
centrifuged. The radioactivity of 200-µl supernatant samples was
determined in the scintillation counter. For control, samples without
HypF protein or CP were analyzed. They were devoid of ATP cleavage activity.
-32P]ATP,
[-32P]ATP, and labeled inorganic pyrophosphate were
purchased from PerkinElmer Life Sciences.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (19K):
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Fig. 1.
Schematic representation of the HypF
sequence indicating the position of the acylphosphatase
(AP), the two zinc fingers (ZF1 and
ZF2), and the carbamoyltransferase
(O-CT) motifs. The residues replaced by targeted
mutagenesis are denoted. On the top of the figure the
sequence motifs shared between HypF and acylphosphatases and
O-carbamoyltransferases are shown. Sequences were taken from
the NCBI data base (www.ncbi.nlm.nih.gov/) for Streptomyces
(S.) lavendulae, S. spheroides, Bradyrhizobium sp. WM9, and
Mesorhizobium (M.) loti.
View larger version (27K):
[in a new window]
Fig. 2.
Purification of HypF protein from an
overexpressing strain as followed by SDS-PAGE (10%) of the pooled
fractions of each step. A: lanes 1 and 8, molecular mass standards ( -galactosidase, bovine serum albumin,
ovalbumin, lactate dehydrogenase, and restriction endonuclease
Bsp98I); lane 2, S10; lane 3, S100;
lane 4, 0-35% ammonium sulfate fraction; lane
5, MonoQ pool; lane 6, phenyl-Superose pool; lane
7, Superdex 200 pool. B shows a non-denaturing gel in
which purified HypF (lanes 5-7) and a crude extract of a
hypF strain overexpressing hypF from a plasmid
(hypF/pHypF (lanes 3 and 4) were
separated. In the control lanes 1 and 2, the
identically treated extract of the hypF strain carrying
the empty vector was applied (hypF/pACYC184). The gel of
in B was stained for CP phosphatase activity. Lanes
1 and 3 contain 60 µg of protein, and lanes
2 and 4 contain 120 µg of protein. Lanes
5-7 contain 2, 4, and 6 µg of protein,
respectively.
Liberation by HypF protein of inorganic phosphate from acylphosphates
and phosphoesters
View larger version (7K):
[in a new window]
Fig. 3.
Lineweaver-Burk plot of the substrate
concentration dependence of HypF activity in the phosphatase
reaction.
-32P]ATP was therefore incubated with HypF protein,
and the samples were applied to polyethyleneimine thin layer plates and
separated with a solvent of 0.5 M
KH2PO4 (pH 3.4). Hydrolysis of ATP with the
liberation of AMP could be observed but only when CP was included in
the reaction mixture. In its absence, no significant ATP hydrolysis took place (data not shown).
View larger version (11K):
[in a new window]
Fig. 4.
Kinetics of carbamoyl
phosphate-dependent ATP cleavage reaction.
A, Lineweaver-Burk plot of ATP cleavage at different CP
or ATP concentrations. When the CP concentration was varied, ATP was
present at 100 µM, and when ATP concentration was varied
the CP concentration was held at 100 µM.
B, [32P]PPi-ATP
exchange catalyzed by HypF at different carbamoyl phosphate
concentrations.
Kinetic constants of HypF in the CP phosphatase and the
CP-dependent ATPase reaction
hypF)
(Fig. 5A). With some
remarkable exceptions, most of the mutant alleles yielded products at
approximately the same level. The exceptions were HypF-R23K,
HypF-C162A, and HypF-H475A/H476A. Intriguingly, exchange of a
single cysteine from the C-terminal "zinc finger motif" (see Fig.
1, ZF2) destabilized the protein completely, whereas the
replacement of the distal (neighboring) one in addition yielded a
stable product (Fig. 5A, lanes 10 and
11). Surprisingly, the identical replacements at the
N-terminal zinc finger motif delivered a stable product when one of the
cysteines was exchanged but not in case of the double replacements
(Fig. 5A, lanes 8 and 9).
View larger version (18K):
[in a new window]
Fig. 5.
Analysis of mutant HypF
proteins. A, in vivo stability of the
HypF variants produced by the transformants indicated. An immunoblot is
shown from an 10% SDS gel in which lysates of 0.1 A600 units of cells were separated. The gel was
developed with antibodies directed against HypF protein.
B, processing of the precursor of the large subunit of
hydrogenase 3 from E. coli in the hypF strain
DHP-F2 transformed with plasmids carrying the wild type and the mutant
hypF alleles indicated. An immunoblot is shown of a 10% SDS
gel in which lysates of 0.1 A600 units of cells
were separated. The gel was developed with antibodies directed against
synthetic peptides of HycE. Lanes 1 and 18, wild
type hypF; lanes 2 and 17,
hypF; lanes 3-16, HypF variants;
lane 3, R23E; lane 4, R23H; lane 5,
R23K; lane 6, R23Q; lane 7, R23EV17A/V20A;
lane 8, C112A; lane 9, C109A/C112A; lane
10, C162A; lane 11, C159A/C162A; lane 12,
H475A; lane 13, H476A; lane 14, H479A; lane
15, H475A/H476A; lane 16,
H475Y/H476Y/H477Y/H479Y.
CP phosphatase activity of wild type and mutant HypF variants
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
| |
ACKNOWLEDGEMENTS |
|---|
We are greatly indebted to H. Hartl for inductively coupled plasma atomic emission spectroscopy and atomic absorption spectroscopy analysis of purified protein and to R. Klüfers for fruitful discussions.
| |
FOOTNOTES |
|---|
* This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Mikrobiologie, Department Biologie I, Universität München, Maria-Ward-Straße 1a, D-80638 München, Germany. Tel.: 49-89-2180-6120; Fax: 49-89-2180-6122; E-mail: august.boeck@lrz.uni-muenchen.de.
Published, JBC Papers in Press, October 10, 2002, DOI 10.1074/jbc.M204601200
1 N. Drapal, S. P. P. Albracht, and A. Böck, unpublished results.
3 A. Paschos, A. Bauer, and A. Böck, unpublished data.
| |
ABBREVIATIONS |
|---|
The abbreviations used are: CP, carbamoyl phosphate; DTT, dithiothreitol.
| |
REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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