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

The results obtained with the HypE variants and also with mutant HypF forms are integrated to explain the complex reaction pattern of protein HypF.. In addition, it is shown that during

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¨t Mu¨nchen, Mu¨nchen, Germany

The hydrogenase 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 of metals or of 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 Km being 25 lM and the kcat

1.7· 10)3s)1 Removal of the C-terminal cysteine residue

of HypE which accepts the carbamoyl moiety from HypF

affected the Km (47 lM) but not significantly the kcat

(2.1· 10)3s)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 active 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 complex reaction pattern of protein HypF

Keywords: NiFe hydrogenase; maturation; CN ligand syn-thesis; hypE mutations; carbamoyl transfer

Escherichia colipossesses four hydrogenases (Hyd1–Hyd4)

which are all members of the NiFe class [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 is 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 function in the

maturation of the enzymes Most of them act 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 carboxprotein-S-carbox-amide 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 as to whether the ligandation of iron takes place at the hydrogenase large subunit apoprotein, which had accepted the iron before, or whether it occurs at some scaffold protein from which the fully substituted metal 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 migrating one from cells lacking carbamoylphosphate and a slower one when carbamoylphosphate is present but the apoprotein of the large subunit is absent 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 procedure for the purifica-tion of the HypE protein, its chemical properties and kinetic

Correspondence to A Bo¨ck, Department Biologie I, Mikrobiologie,

Ludwig-Maximilians-Universita¨t Mu¨nchen, Maria-Ward-Strasse 1a,

D-80638 Mu¨nchen, Germany Fax: + 49 89 218063857,

Tel.: + 49 89 21806116, E-mail: august.boeck@lrz.uni-muenchen.de

*Present address: 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)

characterization relative to the substrate ATP The analysis

of mutant variants of HypE demonstrates that the

carb-amoylation of the C-terminal thiol and the dehydration of

the S-carboxamide to the thiocyanate are independent

reactions and that HypE per se is able to catalyze the

dehydration reaction In addition, it is shown that during

the carbamoyltransfer reaction HypE forms a complex with

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 colistrain MC4100 [16] was used as wild-type and DH5a

[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 grown at 37C 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 growth in standing

screw cap flasks filled to the top For the maintenance of the

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 with Klenow enzyme It had a size of 5156 bp and

carried the hypE gene pHypE was then employed to

construct a variant lacking the codon for the C-terminal

cysteine It was achieved by inverse PCR [21] employing the

overlapping primers cys336del-forward (5¢-CCGCGGAT

ATGATAATAAAATTCTAAATCTCCTATAG-3¢) and

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 passage over a QIAquick Spin 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 SacII digestion and its authenticity

was confirmed by sequencing

For overproduction of the wild-type HypE protein, a

plasmid was constructed by excision of a 1.6 kb KpnI-MluI

fragment from plasmid pSA3, treatment with Klenow

enzyme to remove the protruding ends and by cloning into

the SmaI restricted vector pT7-7 In this way the GTG start

codon of the hypE gene was out of frame relative to an ATG

of the vector The plasmid was designated pTE-C2 It

represents a derivative of pT7-7 containing a

(
hypD-hypE-fhlA) 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 codon 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 amino 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, treatment 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 subsequently 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 that carried the desired mutation [23] The plasmids harbouring the wild-type and mutant hypFgenes 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 were purified by passage over

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 PRISMTM310 sequencer (PE Applied Biosystems, Weiterstadt, Germany)

Purification of HypE and HypF proteins Overproduction of HypE and of its derivatives took place in

E coli BL21(DE3) [24] transformed with the respective plasmid The following procedure was developed for purification of HypE from the wild-type and essentially the same could be adopted for the mutant variants The transformants were grown aerobically in LB-medium in 2-L Erlenmeyer flasks at 37C until the culture reached an A600

of 1 The expression was initiated by the addition of 0.5 mM isopropyl thio-b-D-galactoside followed by a further 3-h incubation period The cells were collected by centrifugation

at 3000 g

1,2 , washed in a buffer containing 50 mMTris/HCl,

1,2

pH 7.4, centrifuged again and taken up in 1 : 10 of the volume of 50 mM Tris/HCl,

acetate, 50 mMsodium chloride, 0.1 mMdithiothreitol and 0.5 lgÆmL)1each of leupeptin and pepstatin After 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 mM Tris/HCl,

50 mMsodium chloride, and 0.1 mMdithiothreitol Elution was performed with a linear gradient of sodium chloride reaching from 50 to 350 mM 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 and slowly stirred at 0C 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 Tris/HCl

acetate, 100 mM sodium chloride, 0.1 mM dithiothreitol,

dialyzed against the same buffer and subjected to gel

filtration over a HiLoadTM16/60 SuperdexTM75 pg column

(1.6· 60 cm) (Pharmacia, Freiburg, Germany) at a flow

rate of 60 mLÆh)1 Fractions 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; Pall Corp., Dreieich, Germany)

amidoblack and the membrane was subjected to a standard

immunoblotting procedure [14] Polyclonal antibodies

directed against HypE protein or HypF protein were used

in dilutions of 1 : 500 and 1 : 1000, respectively Detection

of the antibody–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 Diagnostics GmbH, Mannheim,

Ger-many) via exposure to WICORex 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 mixing the HypE and HypF

proteins at the indicated concentrations in buffer containing

50 mM Tris/HCl,

magnes-ium acetate, 0.1 mM each of ATP and 14C-labelled

car-bamoylphosphate in the presence or absence 0.1 mM

dithiothreitol The reaction was performed at 25C for

10–30 min The radioactivity transferred to HypE was

either determined by filtrating the samples through

nitro-cellulose filters and subsequent scintillation counting of the

label retained by the filters as described previously [12] or by

separating the mixture in polyacrylamide gels To detect the

14C-labelled forms of HypE, nondenaturing PAGE [14] and

a
mild-denaturing 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 mM, heated for 10 min to 56C and

subjected to SDS/PAGE afterwards All separations were

conducted in the cold room at a voltage of 110 V or less

After the separation, the proteins 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 was scanned with a Storm 840 PhosphoImager

(Amersham Biosciences, Freiburg; Germany) and data were

Determination of the ATPase activity The hydrolysis of ATP by HypE was followed in assays containing 50 mM Tris/HCl

MgCl2, 0.1 mMdithiothreitol, 50 lgÆmL)1of bovine serum albumin and HypE protein at the indicated concentration The reaction took place at 25C in a final volume of

100 lL Ten microliter samples were taken, mixed with

500 lL (5%) charcoal suspended in 50 mM KH2PO4 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 [32P]ATP[aP] After incubation, 1-lL samples were spotted onto polyethyleneimine plates (Merck, Darmstadt, Ger-many) which were developed with 0.5MKH2PO4, pH 3.4 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 Leon-Rot, Germany), New 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) or 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%

14C-labelled carbamoylphosphate was purchased from American Radiolabelled Chemicals Inc (St Louis, MO, USA) at a specific radioactivity of 7 mCiÆmmol)1 It was 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 PurM 3D structure, which suggested that the three proteins might catalyze reactions

mechanistically similar to that of PurM, namely an

ATP-dependent dehydration Indeed, HypE from E coli

dehy-drates the carboxamido residue linked to the C-terminal

cysteine of HypE to the protein thiocyanate [12]

Purification and properties of the HypE protein

fromE coli

HypE protein was overproduced in the transformant of

E coliBL21(DE3) harbouring plasmid pTE-C2 and

puri-fied following the procedure outlined under Experimental

procedures In short, this 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 mutant

variants of the HypE protein: (a) HypED which lacks 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 HiLoadTM16/60 SuperdexTM75 pg column the

majority migrated as an apparent monomer However,

there was always HypE protein in the elution position of 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 dimer equilibrium, samples of the purified preparations of wild-type HypE, of HypE[D83N] and HypED were incubated in the presence of 50 mM 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 2B) Incu-bation with dithiothreitol grossly increased the capacity to accept the label from [14C]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 of 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: molecular mass (kDa) standard (b-galactosidase, bovine serum

albumin, ovalbumin, lactate dehydrogenase, endonuclease Bsp98I,

b-lactoglobulin, lysozyme); lane 2: cell lysate of BL21(DE3)/pTE-C2

before induction; 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 30 000 g centrifugation; lane 6: pooled

fractions after DEAE Sepharose chromatography and ammonium

sulfate precipitation up to 30% saturation; lane 7: HypE protein 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 mutant 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 protein; lanes 4 and 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 for 1h on ice The monomeric and dimeric forms of HypE and its variants are indicated by arrows The chemical basis of the migration in two forms

is unknown (lanes 3, 5 and 7) (B) Determination of14C-labelled HypE protein and its variants by binding to nitrocellulose filters [11] Lanes 2–7 as in (A) Results are averages of three independent experiments ± standard deviation.

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 shown),

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: Km: 25 ± 1.8 lM; Kcat

1.7 ± 0.16· 10)3s)1 The HypED 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 Km:

47 ± 13.3 lM; Kcat2.1 ± 2.6· 10)3s)1

Analysis of the transcarbamoylation reaction catalyzed

by HypF

The formation of the HypE-thiocyanate involves first the

carbamoylation of the C-terminal cysteine of HypE by

interaction with HypF, then the release of HypF and the

subsequent dehydration of the protein thiocarboxamide to

protein thiocyanate [12] To resolve 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 was made of the previous observation

that HypE-CN is labile in the presence of thiols When

incubated in the presence of 1 mMdithiothreitol for 15 min

at 40C the yield of HypE-CN had been much lower in

comparison to samples incubated in the absence of

dithio-threitol [12] Alteration of the incubation conditions to

10 min at 56C in the presence of 100 mM dithiothreitol

(
mild-denaturing 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 of heating of the

mixture (Fig 3A, lane 1) On the other hand, samples from

assays that were blocked in the dehydration reaction

because of the inclusion of ADP-CH2-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 thiocarboxamide and

HypE-thiocyanate might possess differential stabilities under the

conditions of electrophoresis, in particular at the alkaline

pH of the gels To follow this assumption the HypE protein

was carbamoylated by HypF in the presence of ADP-CH2

-P and [14C]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 assessed (Fig 3C) by
mild-denaturing

SDS/PAGE as described above It is evident that alkaline

pH leads to the loss of the thiocarboxamide moiety;

intriguingly, the apparent hydrolysis of the thiocarboxamide

requires native HypE protein as it is fully stable when the

samples are denatured in SDS sample buffer containing

100 mMdithiothreitol and separated in SDS gels possessing

the same pH

The radioactive material migrating on the top of the

nondenaturing gel (Fig 3A) coincides with a signal in

immunoblots detected both 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 intermediate in the transcarbamoylation reaction To follow this assumption, 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-fold lower mount of HypF protein still was able to convert all radioactivity on HypE into the thiocyanate form, not shown) At close to stoichiometric ratios, the major amount of radioactivity migrated in a position indicated by immunoblots to contain both proteins (not shown) Decrease of HypF in the assay gradually shifted the migration position of HypE, which reflects a

Fig 3 Differential detection of 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 ),14C-labelled carbamoylphosphate ([14C]CP; 100 l M ) and either ATP (100 l M ; lane 1) or ADP-CH 2 -P (100 l M ; lane 2) and incubated at 25 C for 10 min The sample for the nondenaturing PAGE was mixed prior to appli-cation with a sample buffer containing 50 m M Tris/HCl (pH 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 on the pH HypE (6 l M ) and HypF (1 l M ) proteins were mixed with [ 14 C]CP (100 l M ) and ADP-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) The protein fraction was further incubated for 10 min at 25 C in 100 m 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 sample buffer and separation in a 10% SDS gel as indicated for (B).

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-CH2-P

serves as a substrate but not AMP-CH2-PP [12] The easy

discrimination between thiocarboxamide and

HypE-thiocyanate allowed the analysis whether AMP-CH2-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-CH2-PP and of different ratios

between ATP and its analogue and the products were

analyzed (Fig 5) It was found that the analogue is unable

to support HypF-catalyzed carbamoyltransfer to HypE as

the protein does not carry 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-CONH2appears (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 to 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 of 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-CH2-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 contained HypE at 2 l M in the ratio to HypF indicated above

each lane (2 l M down to 0.2 l M ) [ 14 C]Carbamoylphosphate and ATP

were present at 100 l M each; incubation time was 30 min at 25 C The

identity of the material in the labelled bands was shown by

immuno-blotting (not shown).

Fig 5 Inhibition of the ATP-dependent dehydration reaction by

AMP-CH 2 -PP HypE (2 l M ) was incubated with HypF (0.2 l M ) and

[14C]carbamoylphosphate (100 l M ) in the presence of 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 to

nitrocellulose membranes that were autoradiographed.

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 [14C]carbamoylphosphate and 100 l M

ATP (lanes 1, 3, 5 and 7) or 100 l M ADP-CH 2 -P (lanes 2, 4, 6 and 8) for 30 min at 25 C The reaction products were separated in non-denaturing gels (A) and SDS gels (B), transferred to a nitrocellulose membrane which was autoradiographed.

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 lMdid

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 for 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 in a crystal of 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 vitroactivities The phenotype of the mutant

harbour-ing the gene for HypF[H476A] was indistharbour-inguishable 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 gene 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 and 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 adenylate is avidly hydrolyzed 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 dehydration 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 transfer of the HypE protein from HypF to the HypC· HypD complex (our unpublished results)

An apparent ATP-pyrophosphate exchange reaction, which is dependent on carbamoylphosphate, has been described previously 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 radioactive ATP from labelled pyrophosphate can be readily explained by the reversion of the reaction, attainment of the equilibrium would also necessitate the formation of carbamoylphosphate from the postulated enzyme-bound carbamoyladenylate at the expense of inor-ganic phosphate The situation is thus different from a classical ATP-PPiexchange reaction like that catalyzed by aminoacyl-tRNA synthetases in which the substrate (the amino acid) drives both the forward and the reverse reaction An issue that is still open, however, is 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 absence of ATP, II the carbamoylphosphate-dependent cleavage of ATP into AMP and pyrophosphate, III the carbamoyl-phosphate-dependent ATP-pyrophosphate exchange reaction, and IV the ATP-dependent dehydration catalyzed by HypE.

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 presence of HypE This is also in distinct

contrast to the properties 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 of the carbamoyl-adenylate

might exist in the 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 is fully active and it might represent the actual state

of the protein within the cell

Until now it was open as to whether HypF acts solely as a

carbamoyltransferase or whether it also participates in the

dehydration reaction The property of mutant proteins with

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 involved The sites of

carbamoyl-binding and ATP-dependent dehydrations 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-terminal thiol Availability of a HypE

variant that carries the carboxamide moiety 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 thank E Zehelein for expert purification of HypF and

HypE, H Hartl for the ICP spectroscopy of the proteins and

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 Chemischen Industrie.

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