The proposed intermediate of the R-4¢-phospho-N-panto-thenoylcysteine synthetase reaction, 4¢-phosphopantothe-noyl-CMP, was characterized by MALDI-TOF MS and it was shown that the interm
Trang 1Active-site residues and amino acid specificity of the bacterial
4¢-phosphopantothenoylcysteine synthetase CoaB
Thomas Kupke
Lehrstuhl fu¨r Mikrobielle Genetik, Universita¨t Tu¨bingen, Tu¨bingen, Germany
In bacteria, coenzyme A is synthesized in five steps from
D-pantothenate The Dfp flavoprotein catalyzes the
synthe-sis of the coenzyme A precursor 4¢-phosphopantetheine
from 4¢-phosphopantothenate and cysteine using the
cofac-tors CTP and flavine mononucleotide via the
phospho-peptide-like compound 4¢-phosphopantothenoylcysteine
The synthesis of 4¢-phosphopantothenoylcysteine is
cata-lyzed by the C-terminal CoaB domain of Dfp and occurs via
the acyl-cytidylate intermediate
4¢-phosphopantothenoyl-CMP in two half reactions In this new study, the molecular
characterization of the CoaB domain is continued In
addi-tion to the recently described residue Asn210, two more
active-site residues, Arg206 and Ala276, were identified
and shown to be involved in the second half reaction of
the (R)-4¢-phospho-N-pantothenoylcysteine synthetase The
proposed intermediate of the
(R)-4¢-phospho-N-panto-thenoylcysteine synthetase reaction,
4¢-phosphopantothe-noyl-CMP, was characterized by MALDI-TOF MS and it was shown that the intermediate is copurified with the mutant His-CoaB N210H/K proteins Therefore, His-CoaB N210H and His-CoaB N210K will be of interest to elucidate the crystal structure of CoaB complexed with the reaction intermediate Wild-type His-CoaB is not absolutely specific for cysteine and can couple derivatives of cysteine to 4¢-phosphopantothenate However, no phosphopeptide-like structure is formed with serine Molecular characterization
of the temperature-sensitive Escherichia coli dfp-1 mutant revealed that the residue adjacent to Ala276, Ala275 of the strictly conserved AAVAD(275–279) motif, is exchanged for Thr
Keywords: coenzyme A biosynthesis; 4¢-phosphopantethe-ine; 4¢-phosphopantothenoylcysteine synthetase; Dfp flavo-protein; cysteine metabolism
4¢-Phosphopantetheine (PP) coenzymes such as
coen-zyme A are the biochemically active forms of the vitamin
pantothenic acid In coenzyme A, 4¢-phosphopantetheine
is covalently linked to an adenylyl group, whereas it is
covalently linked to a serine hydroxyl group in acyl carrier
proteins 4¢-Phosphopantetheine is also cofactor of
enzymes that catalyze the biosynthesis of polypeptide
antibiotics [1] Lipmann discovered and characterized
coenzyme A [2] and Lynen elucidated that the thiol
group of the cysteamine moiety of coenzyme A is the
functional group by activating substrates as thioesters [3]
In Escherichia coli and most eubacteria, the synthesis of
4¢-phosphopantetheine, which is also the key reaction in
coenzyme A biosynthesis, is catalyzed from
4¢-phospho-pantothenate and cysteine by the bifunctional Dfp (CoaBC)
flavoproteins in a multistep process (Fig 1; [4–7]) In the
first step, 4¢-phosphopantothenate is activated by reaction
with CTP The 4¢-phosphopantothenoyl-cytidylate formed
is attacked by cysteine and 4¢-phosphopantothenoylcysteine (PPC) is synthesized These reactions occur at the C-terminal CoaB domain of Dfp The next step is the FMN-dependent oxidative decarboxylation of PPCto 4¢-phosphopantothenoylaminoethenethiol, which is then reduced to 4¢-phosphopantetheine; both partial reactions are catalyzed by the N-terminal CoaC domain Oxidative decarboxylation of peptidyl-cysteines had already been detected before as important step in the biosynthesis of the lantibiotics epidermin and mersacidin catalyzed by the LanD enzymes EpiD and MrsD, respectively [8–10] Flavin-dependent oxidative decarboxylation of PPCas an initial step in the conversion of PPCto PP had been proposed by Kupke et al in 2000 [6] and was later confirmed for the plant PPCdecarboxylase AtHAL3a (AtCoaC) by purifying oxidatively decarboxylated pantothenoylcysteine as a reac-tion intermediate [11] and by determining the crystal structure of AtHAL3a C175S complexed with this enethiol intermediate [12] Dfp, AtHAL3a, EpiD and MrsD belong
to a new family of flavoproteins that was named HFCD (homo-oligomeric flavin-containing Cys decarboxylases) [6,13]
In a recently published study [5], the PPC-synthetase activity of the CoaB domain of Dfp was shown, a dimerization motif within CoaB was proposed and the strictly conserved residues N210 and K289 were prelimin-ary investigated with respect to their ability to synthesize PPCand the 4¢-phosphopantothenoyl-CMP intermediate Here, the molecular characterization of the bacterial PPC
Correspondence to T Kupke, Lehrstuhl fu¨r Mikrobielle Genetik,
Universita¨t Tu¨bingen, Auf der Morgenstelle 15, Verfu¨gungsgeba¨ude,
72076 Tu¨bingen, Germany.
Fax: + 49 7071 295937, Tel.: + 49 7071 2977608,
E-mail: Thomas.Kupke@t-online.de
Abbreviations: CoaA, MRGSHHHHHHGSML-CoaA;
His-CoaB, MRGSHHHHHHG-Dfp S–R(181–406); IPTG, isopropyl
thio-b- D -galactoside; PP, 4¢-phosphopantetheine; PPC,
(R)-4¢-phospho-N-pantothenoylcysteine.
(Received 2 October 2003, accepted 11 November 2003)
Trang 2synthetase activity is continued and focused on cysteine
binding by CoaB In addition to residue N210, residues R206
and A276 are described as being important for conversion of
the 4¢-phosphopantothenoyl-CMP intermediate to the final
product PPC Moreover, the 4¢-phosphopantothenoyl-CMP
intermediate was characterized by MALDI-TOF MS and
it is shown that the 4¢-phosphopantothenoyl-CMP
inter-mediate is copurified with His-CoaB N210H/K
Dfp proteins were first purified and characterized as
flavoproteins by Spitzer and Weiss in the 1980s [14,15]
Although Spitzer et al could not determine the enzymatic functions of the Dfp proteins, they described the mutants
E coli dfp-707and E coli dfp-1 that displayed temperature-sensitive auxotrophy either forD-pantothenate or for its precursor b-alanine Molecular analysis of the conditional lethality of the dfp-707 mutant revealed a single point mutation within the N-terminal CoaC domain of Dfp [4] In this study, it is shown that in dfp-1 the residue Ala275 of the conserved AAVAD(275–279) motif of the C-terminal CoaB domain is exchanged for Thr
Fig 1 The role of Dfp (CoaBC) in 4¢-phosphopantothenoylcysteine and 4¢-phosphopantetheine biosynthesis Coenzyme A is synthesized in five steps from D -pantothenate The bifunctional enzyme Dfp catalyzes the conversion of 4¢-phosphopantothenic acid to 4¢-phosphopantetheine via the phosphopeptide-like compound 4¢-phosphopantothenoylcysteine, that is synthesized by the C-terminal CoaB domain from 4¢-phosphopantothenic acid, CTP and cysteine The synthesis of PPCoccurs in two half reactions starting with the formation of 4¢-phosphopantothenoyl-CMP (activation
of the carboxyl group of 4¢-phosphopantothenate) In a second step, the amide bond of PPC(shaded in grey) is formed by reaction of 4¢-phosphopantothenoyl-CMP with cysteine The PPC-decarboxylase activity resides in the N-terminal CoaC flavoprotein domain of Dfp In a first step, PPCis oxidatively decarboxylated to form 4¢-phosphopantothenoyl-aminoethenethiol, which is reduced in a second step to 4¢-phospho-pantetheine, the final product of the enzymatic reaction catalyzed by the enzyme Dfp.
Trang 3Materials and methods
Plasmid construction
In general, PCR amplifications were performed with
Vent-DNA-polymerase (New England Biolabs) The entire
sequences of the dfp and coaB coding regions of the
constructed plasmids were verified Used oligonucleotides
were purchased from MWG Biotech
Site-directed mutagenesis of coaB
All point mutations were first introduced into pQE12 dfp as
described recently by using sequential PCR and appropriate
mutagenesis primers [4] Using the constructed mutant
pQE12 dfp plasmids as templates, the mutant coaB genes
were amplified and then cloned into pQE8 BamHI as
described [5]
Cloning and characterization of thedfp gene
from theE coli dfp-1 mutant
The temperature-sensitive mutant E coli dfp-1 was grown
overnight in B-broth [10 g casein hydrolysate 140 (Gibco),
5 g yeast extract (Difco), 5 g NaCl, 1 g glucose, and
1 gÆL)1 K2HPO4, pH 7.3], at 30Cand chromosomal
DNA was purified using the Qiagen Blood & Cell
Cul-ture DNA Mini Kit For cloning, the mutant dfp gene
was amplified by PCR using the oligonucleotides,
CTCG-3¢ and (reverse) 5¢-CGGGTCCAAGATCTTAA
CGTCGATTTTTTTC-3¢ as primers (introduced EcoRI
and BglII sites are underlined) and the purified
chromo-somal DNA as template The amplified gene was cloned
into pQE12 EcoRI/BglII and transformed in E coli
M15 (pREP4) as described [4] Using the pQE12 dfp-1
plasmid as template, the mutant coaB-1 gene was
amplified and then cloned into pQE8 BamHI as described
[5]
Purification and characterization of Dfp and CoaB
proteins
Growth of strains E coli M15 (pREP4, pQE8/pQE12)
cells were grown in the presence of ampicillin
(100 lgÆmL)1) and kanamycin (25 lgÆmL)1) in 0.5 L of
B-broth in 2 L shaker flasks At A578¼ 0.4, the cells were
induced with 1 mMisopropyl thio-b-D-galactoside (IPTG),
and harvested 2 h after induction Growth temperature
was 37C
Purification of His-CoaA and His-CoaB proteins
For purification of His-CoaA and His-CoaB proteins,
500 mL of IPTG-induced E coli M15 (pREP4, pQE8
coaA/coaB) cells were harvested and disrupted by
sonica-tion in 10 mL 20 mM Tris/HCl (pH 8.0) 0.65–1.3 mL of
the cleared lysates obtained by two centrifugation steps
(each 20 min at 30 000 g at 4C) were applied to
Ni-nitrilotriacetic acid spin columns (Qiagen) equilibrated
with column buffer [20 mM Tris/HCl (pH 8.0), 10 mM
imidazole, 300 m NaCl] The spin columns were then
washed twice with 0.65 mL column buffer His-CoaA, His-CoaB and mutant His-CoaB proteins were eluted with 0.16 mL of column buffer containing 250 mM
instead of 10 mM imidazole The Ni-nitrilotriacetic acid spin columns were centrifuged at room temperature at only 240 g to enable effective binding of the His-tag proteins
Purification of Dfp and Dfp A275T (Dfp-1) Five hundred milliliters of IPTG-induced E coli M15 (pREP4, pQE-12 dfp) cells were harvested and disrupted
by sonication in 10 mL column buffer [20 mM Tris/HCl (pH 8.0)] Five milliliters of the cleared lysate obtained
by two centrifugation steps (each 25 min at 30 000 g at
4C) was diluted with 5 mL column buffer and loaded
on a 1 mL HiTrapQ column equilibrated with column buffer The column was then washed with 5 mL column buffer and 5 mL column buffer containing 0.1M NaCl Dfp proteins were eluted with column buffer containing 0.25M NaCl and the yellow peak fractions (approxi-mately 400 lL) were collected A 25-lL aliquot of this HiTrapQ eluate was then immediately subjected to a Superdex 200 PC3.2/30 gel filtration column equilibrated
in running buffer [20 mM Tris/HCl (pH 8.0), 200 mM
NaCl] at a flow rate of 40 lLÆmin)1 The elution was followed by absorbance at 280, 378 and 450 nm Molecular mass information was obtained as described [6], determining the elution volumes of standard proteins and the void volume of the column The Superdex 200 PC3.2/30 column and the standard proteins used for calibration were obtained from Amersham Pharmacia Biotech
CoaB assay
As 4¢-phosphopantothenate is not commercially available,
it was synthesized enzymatically in situ by adding pantothenate kinase, His-CoaA, D-pantothenate and ATP to the His-CoaB assay mixtures [5] Therefore, 0.9 mL CoaB assay mixtures contained 5 mM D -panto-thenate, 2.5 mM MgCl2, 5 mM ATP, 5 mM CTP, 5 mM
cysteine hydrochloride, 5 mM dithhiothreitol, 100 mM
Tris (pH 8.0) His-CoaA (approximately 10–15 lg) and either wild-type or mutant His-CoaB proteins in the range of 5–40 lg After 30–45 min of incubation at
37C, the reaction mixtures were kept at )80 Cand then were separated successively by reverse-phase chro-matography with a lRPCC2/C18SC2.1/10 column on a SMART system (Pharmacia) Compounds were eluted with a linear gradient of 0–50% acetonitrile/0.1% trifluoroacetic acid in 5.8 mL, with a flow rate of
200 lLÆmin)1 The absorbance was measured simulta-neously at 214, 260 and 280 nm to enable identification
of acyl-cytidylate intermediates
SDS/PAGE Purification of His-CoaB and Dfp proteins was examined using tricine-SDS/PAGE (10%) under reducing conditions [16] Prestained protein molecular mass standards were obtained from New England Biolabs
Trang 4Results and discussion
Characterization of the 4¢-phosphopantothenoyl-CMP
intermediate by MALDI-TOF MS
To confirm that the reaction intermediate of bacterial PPC
synthetases is indeed 4¢-phosphopantothenoyl-CMP,
previ-ous attempts to characterize the reaction intermediate by
mass spectrometry [5] were continued The compound was
enzymatically synthesized in larger amounts, purified by
reversed phase chromatography and then analyzed by
MALDI-TOF mass spectrometry In the mass spectrum
two prominent peaks with m/z values of 604.7 and 323.1
were present and are proposed to be
4¢-phospho-pantothenoyl-CMP (theoretical monoisotopic mass of
[M + H]+¼ 605.1 Da) and CMP (theoretical
monoiso-topic mass of [M + H]+¼ 324.1), respectively (Fig 2)
CMP was probably a by-product of 4¢-phosphopantothe-noyl-CMP, although 4¢-phosphopantothenate was not detected
Molecular characterization of His-CoaB N210 mutants The residue N210 of E coli CoaB is strictly conserved in bacterial and eukaryotic PPCsynthetases (Fig 3) To get more information on its role in PPCsynthesis, the recently carried out study [5] was continued and further N210 mutants were analyzed (N210A/E/H/K/Q) All introduced mutations drastically decreased the PPCsynthetase activity
of His-CoaB but did not inhibit the formation of the 4¢-phosphopantothenoyl-CMP intermediate (data not shown) confirming the recently obtained results for His-CoaB N210D [5] In contrast to His-His-CoaB N210D, the mutant proteins N210H and N210K had no in vitro PPC
Fig 2 Characterization of the 4¢-phospho-pantothenoyl-CMP intermediate by MALDI-TOF mass spectrometry In vitro, enzymati-cally, synthesized 4¢-phosphopantothenoyl-CMP (A; peak labeled I) and 4¢-phosphopantothenoyl-CMP (B; 5 m M ) were separated by RPC The elution from the RPCcolumn was followed by absorbance
at 280, 260 (not shown), and 214 nm (not shown) CMP did not bind to the used lRPC
C 2 /C 18 SC2.1/10 column and was found in the flow-through The 4¢-phosphopantothenoyl-CMP containing fraction was subjected to MALDI-TOF MS analysis (C).
Trang 5synthesis activity at all (data not shown) From these results
it can be proposed that residue N210 is an active-site
residue The mutant His-CoaB proteins N210A, N210E,
N210H, N210K and N210Q formed dimers as had also
been observed for the wt enzyme ([5]; Fig 4)
Residues R206, N210 and A276 are involved in the
second half reaction of the PPC synthetase
Assuming that Asn210 is not the only residue of His-CoaB
that is important for the second-half reaction (and that is
probably involved in cysteine binding), the in vitro activity
of further mutants was measured in the presence and
absence of cysteine It turned out that also for the mutant
proteins His-CoaB R206Q and His-CoaB A276V, the
4¢-phosphopantothenoyl-CMP intermediate but no or only
very little amounts of PPC are detectable in presence of
cysteine (Fig 5) Interestingly, the residues Arg206, Asn210
and Ala276 are not only conserved in bacterial Dfp/CoaB
proteins but also in eukaryotic PPCsynthetases [5,17] In
summary, it is proposed that Arg206, Asn210 and Ala276
are important for the second-half reaction and are part of
the active-site of CoaB; these residues may be directly
involved in binding of one of the substrates, the amino acid
cysteine (Fig 3) However, it cannot be excluded that
cysteine binds to another site of CoaB and that exchanges of
the residues Arg206, Asn210 or Ala276 indirectly influence
the binding of cysteine to CoaB complexed with
4¢-phosphopantothenoyl-CMP
Copurification of the 4¢-phosphopantothenoyl-CMP
intermediate
Taking into account that residues Arg206, Asn210 and
Ala276 are crucial for the second-half reaction of the
bacterial PPCsynthetase activity, it was investigated whether 4¢-phosphopantothenoyl-CMP is already bound
to mutant His-CoaB proteins purified by Ni-NTA chroma-tography from E coli cell extracts No 4¢-phosphopanto-thenoyl-CMP was copurified with wild-type His-CoaB or His-CoaB K289Q (data not shown) However copurifica-tion of low amounts of the intermediate was observed for His-CoaB R206Q, His-CoaB N210D, and His-CoaB A276 (data not shown), whereas larger amounts of the inter-mediate were copurified with His-CoaB N210H and His-CoaB N210K (Fig 4)
Ni-NTA purified protein His-CoaB N210K complexed with the 4¢-phosphopantothenoyl-CMP intermediate was incubated at 37Cwith 5 mM dithiothreitol and 5 mM
L-cysteine (in the presence of 2.5 mM MgCl2) and then analyzed by RPCto detect remaining 4¢-phosphopantothe-noyl-CMP Even after 60 min of incubation the amount of 4¢-phosphopantothenoyl-CMP only slightly decreased (data not shown) Synthesis of 4¢-phosphopantothenoyl-CMP is excluded in this experiment as 4¢-phosphopantothenate and CTP were omitted Therefore, only the occurrence of the second-half reaction is investigated and it can be concluded that either cysteine cannot bind to His-CoaB N210K-4¢-phosphopantothenoyl-CMP or that the nucleophilic attack
on 4¢-phosphopantothenoyl-CMP by cysteine is inhibited PPC synthetase binds derivatives of cysteine
To study the substrate specificity of His-CoaB, L-serine,
L-alanine, cysteamine, L-cysteine methyl ester, and D,
L-homoserine were used as potential substrates When wild-type His-CoaB was used, the 4¢-phosphopantothenoyl-CMP intermediate was converted to PPC(in presence
of cysteine) or to PP (in the presence of cysteamine) or
to 4¢-phosphopantothenoylcysteine methyl ester (in the
Fig 3 Residues that are crucial for the second-half reaction are conserved in CoaB proteins Residues of the N-terminal part of the Escherichia coli CoaB domain that are conserved in CoaB (Dfp) proteins from all kingdoms of life and that were examined in the present study are in bold letters [eubacteria, E coli: P24285; archae, Metanocaldococcus jannaschii: Q58323; eukaryotes, human: XP_016228(gi:13638573) and yeast: P40506] The mutations R206Q, N210A/D/E/H/K/Q, A275T (dfp-1), A276V, D279E/N and K289Q are indicated by arrows; mutations influencing the second-half reaction are underlined The dimerization motif is slightly longer than has been determined in the first study ([5]; T Kupke, unpublished data) One reasonable explanation of the experimental data presented in this paper is that residues R206, N210 and A276 are involved directly in binding the substrate amino acid cysteine However, this model is in contrast to published data on the crystal structure of the human PPCsynthetase [18].
Trang 6presence of cysteine methyl ester) Although the structures
of PP and 4¢-phosphopantothenoylcysteine methyl ester
reaction products were not confirmed by mass
spectrome-try, this conclusion can be made from the obtained HPLC
diagrams (data not shown) Wild-type His-CoaB was not
able to couple L-serine, L-alanine or D,L-homocysteine
to 4¢-phosphopantothenate, so that in these cases the
4¢-phosphopantothenoyl-CMP intermediate was still detect-able None of the compounds inhibited the formation of 4¢-phosphopantothenoyl-CMP or was coupled to 4¢-phos-phopantothenate by His-CoaB N210D These experiments show that the PPC-synthetase CoaB is not absolutely specific for L-cysteine, but can also bind derivatives of cysteine, as long as the -CH-SH side chain is not altered
Fig 4 Analysis of His-CoaB N210K and copurification of the 4¢-phosphopantothenoyl-CMP intermediate His-CoaB proteins (His-CoaB and His-CoaB N210K) were puri-fied from E coli cell extracts by Ni-nitrilotri-acetic acid and either characterized by gel filtration on a Superdex 200 PC3.2/30 column (A; [5]); using 20 m M Tris/HCl, pH 8.0/
200 m M NaCl as running buffer or separated
by RPC(B; approximately 150 lg of each protein were set in this case) The elution from the columns was followed by absorbance at
280 nm (thick line) and absorbance at 260 (thin line) and 214 nm (not shown) From the gel filtration column both proteins eluted at approximately 1.56 mL (showing that both proteins are dimers [5]) However, the proteins differed significantly in their UV spectra Under the acidic conditions used in the RPC experiment a compound bound to His-CoaB N210K was removed and characterized by its
UV spectrum (B, right figure) and MALDI-TOF mass spectrum (C) as 4¢-phosphopanto-thenoyl-CMP.
Trang 7The biochemical role of coenzyme A is the activation of
substrates by forming energy-rich thioester bonds
Oxo-coenzyme A (which has an ethanolamine residue instead of
a cysteamine residue) cannot fulfill this role and may inhibit
cell growth Therefore, the specificity of CoaB for the
incorporation of cysteine over serine is important However,
it is very likely that also the PPCdecarboxylase (CoaC)
activity and the 4¢-phosphopantetheine adenylyltransferase
activity contribute to the selectivity for cysteine CoaC is
related to the flavoprotein EpiD that decarboxylates
peptidyl-cysteines but not peptidyl-serines [9] and it can be
proposed that CoaC cannot decarboxylate
4¢-phospho-pantothenoylserine due to the mechanism of the
decarb-oxylation reaction [11,12]
Sequence analysis of theE coli dfp-1 mutant
In 1985, Spitzer and Weiss described the dfp gene of E coli
as a locus coding for a flavoprotein and affecting DNA
synthesis [15] Later they showed that the conditional-lethal
dfp-707 mutation requires either D-pantothenate or
b-alanine for growth at 30Cand that the dfp-1 mutation
conferred the auxotrophy but not the conditional lethality
of dfp-707 E coli dfp-1 is unable to grow on a minimal
medium at 42C, but unlike dfp-707 is not temperature
sensitive for growth on rich media Complementation
analysis suggested that the dfp-1 and dfp-707 mutations
were in the same gene [14] The nutritional requirements of the dfp-707 and dfp-1 mutants correspond to those of panD mutants, however, dfp-mutants contained wild-type levels of aspartate decarboxylase [14], which is required for synthesis
of b-alanine Therefore it looks like, that an excess of
D-pantothenate results in higher concentrations of 4¢-phos-phopantothenate and that the partial blocking of the Dfp (CoaBC) enzyme is overcome in this way
The molecular basis for the conditional lethality of the dfp-707 mutant is that one amino acid residue of the N-terminal PPC decarboxylase (CoaC) domain of Dfp is exchanged [4] For further characterization of the dfp-1 mutation, the dfp-1 gene was cloned into pQE12, sequenced and overexpressed Sequence analysis revealed that dfp-1 has a point mutation in codon 275 of the dfp gene, substituting the wild-type GCC(Ala) with ACC(Thr) (Fig 6); the G-A transition concurs with the use of hydroxylamine as the mutagenic agent [14] Therefore, the dfp-1 mutation is within the AAVAD motif of the C-terminal PPCsynthetase (CoaB) domain of Dfp (Fig 3) In contrast to wild-type His-CoaB, His-CoaB A275T is a monomeric protein, but Dfp and Dfp A275T are both dodecameric proteins (Fig 6); it was already shown that also His-CoaB A275V is a monomeric protein [5] The molecular reason for the temperature sensitivity of the
E coli dfp-1mutant has to be elucidated in more detail, but obviously increasing the temperature from 30 to 42Chas
Fig 5 Analysis of the enzymatic activity of His-CoaB R206Q and A276V The synthesis of 4¢-phosphopantothenoyl-CMP (–cysteine, upper part of the figure) and of 4¢-phosphopantothenoylcysteine (+ cysteine, lower part of the figure) by the mutant His-CoaB proteins R206Q and A276V was analyzed using the described HPLC-based assay The absorbance was monitored at 280 nm (left part of the figure), 214 nm (right part of the figure), and 260 nm (not shown) to identify PPCand the 4¢-phosphopantothenoyl-CMP intermediate (peak labeled with I) As has already been observed for the mutant N210 proteins, His-CoaB R206Q and A276V have very little PPC-synthetase activity and the 4¢-phosphopantothenoyl-CMP intermediate is also detectable in the presence of cysteine A minor portion of the detected 4¢-phosphopantothenoyl-CMP intermediate does not result from de novo synthesis but has been copurified with the used His-CoaB proteins R206 and A276, respectively.
Trang 8Fig 6 The temperature-sensitive mutant E coli dfp-1 (A) dfp-1 was cloned into pQE12 and sequence analysis revealed a single point mutation within the AAVAD motif of CoaB proteins, namely Ala275 is exchanged for Thr275 (B) Dfp and Dfp A275T (¼ Dfp-1) were enriched by anionic exchange chromatography and then purified by gel filtration chromatography from the overexpressing E coli strains grown at 37 C The elution from the Supderdex 200 PC3.2/30 gel filtration column was followed by absorbance at 280 (upper line), 378 (not shown), and 450 nm (lower line) Both wild-type and Dfp A275T proteins (peak labeled with asterisks) eluted at about 1.04 mL and bound flavin coenzyme, as shown by absorbance
at 450 nm The corresponding His-CoaB proteins were purified by IMAC from the overexpressing E coli strains grown at 37 C(same results were obtained when cells were grown at 28 C) In contrast to the Dfp proteins, the His-CoaB proteins showed different elution volumes in the gel filtration experiments indicating that His-CoaB A275T is a monomeric enzyme.
Trang 9an effect on 4¢-phosphopantetheine and coenzyme A
biosynthesis With the used assay, it is difficult to evaluate
the PPCsynthetase activity at different temperatures,
because the enzymatic activity of the present pantothenate
kinase (used for in situ synthesis of
4¢-phosphopantothe-nate) is also temperature-dependent However, increasing
the temperature from 30 to 42Csignificantly increases the
detectable amounts of PPCwhen wt His-CoaB is used,
whereas there is a slight decrease for His-CoaB A275T (data
not shown) As has been determined above, the residue
adjacent to Ala275, Ala276, is an active-site residue
probably involved in binding the substrate cysteine
There-fore, it is assumed that Ala275 is also an active-site residue
of E coli CoaB The observed PPCsynthetase activity of
His-CoaB A275T shows that dimerization of CoaB is not
essential for activity
Comparison of site-directed mutagenesis studies
with structure of human PPC synthetase
Recently, the structure of the ATP-dependent human
phosphopantothenoylcysteine synthetase was determined
at 2.3-A˚ resolution [18] This enzyme is a dimer from
identical monomers with the monomer fold having features
in common with a group of NAD-dependent enzymes on
one hand and with the ribokinase fold on the other hand
The structure of the human PPCsynthetase complexed with
substrates, the cosubstrate ATP or the intermediate
4¢-phos-phopantothenoyl-AMP was not experimentally determined
by Manoj et al [18] However, models for the
4¢-phospho-pantothenate and ATP binding sites were reported From
these models, the conserved ATP binding residues Gly43,
Ser61, Gly63, Gly66, Phe230, and Asn258 were identified in
human CoaB The identified 4¢-phosphopantothenate
bind-ing residues Asn59, Ala179, Ala180 and Asp183 from one
monomer and Arg55¢ from the adjacent monomer
corres-pond to E coli CoaB residues Asn210, Ala275, Ala276,
Asp279 and Arg206 A model of human CoaB containing
simultaneously bound 4¢-phosphopantothenoyl-AMP and
cysteine was not reported The strictly conserved lysine
residue Lys195 of human CoaB (corresponding to Lys289
of E coli CoaB) is located in a disordered loop As this
lysine residue has been identified as an active-site residue [5],
it is tempting to speculate that the loop is involved in
binding one of the (co)substrates of CoaB, indicating that
the provided models of the substrate binding sites of human
CoaB do not give a complete insight into the active-site
architecture of CoaB The data obtained from the presented
mutagenesis studies indicate that residues Arg206, Asn210
and Ala276 are important for the conversion of
4¢-phosphopantothenoyl-CMP to PPCand are not crucial
for binding 4¢-phosphopantothenate (or CTP; Fig 3) The
mutant proteins CoaB A275T, CoaB D279N, and CoaB
D279E (data not shown) were able to synthesize
4¢-phosphopantothenoylcysteine and the
4¢-phosphopanto-thenoyl-CMP intermediate was not detectable anymore
when cysteine was present in the assay This indicates that
the five residues proposed by Manoj et al to be involved in
4¢-phosphopantothenate binding are not functionally
equiv-alent, at least in the E coli protein
In order to assign a function to the conserved lysine
residue and to all the other conserved CoaB residues, it
will be extremely important to obtain not only models but also crystal structures of both the human and the bacterial CoaB proteins complexed with the (co)substrates and, most importantly, a structure of a CoaB mutant in complex with the 4¢-phosphopantothenoyl-C(A)MP inter-mediate (and cysteine) A structure of CoaB N210H/K in complex with 4¢-phosphopantothenoyl-CMP (and pos-sibly cysteine) will be indispensable to explain the experimental data presented in this paper These crystal structures will also be important to answer the question why bacterial CoaB is specific for CTP, whereas human CoaB utilizes ATP four times more efficiently than CTP [19]
Conclusions
In this study, active-site residues of E coli CoaB were identified that are involved in the second half reaction of the PPCsynthetase It was demonstrated that the 4¢-phospho-pantothenoyl-CMP intermediate is copurified with mutant His-CoaB N210 proteins This observation can enable crystal structure analysis of PPCsynthetases complexed with the reaction intermediate Copurification of the 4¢-phosphopantothenoyl-CMP intermediate with mutant CoaB proteins also shows that the in vivo function of CoaB
is the synthesis of PPC
Studying Dfp is of great general biochemical interest, as it
is this enzyme that links cysteine metabolism with the biosynthesis of coenzyme A It has also been discussed that Dfp is an interesting target for the development of new antibacterials [7,18–20] Antibacterials have already been developed against staphylococcal pantothenate kinase [21] and the bacterial phosphopantetheine adenylyltransferase activity [22]
Acknowledgements
I thank Regine Stemmler for excellent technical assistance, Bernard Weiss for providing the E coli dfp-1 mutant and Stefan Stevanovic for MALDI-TOF MS experiments This work was supported by Deutsche Forschungsgemeinschaft research grant KU869/6–1 and research fellowship KU 869/9–1 to TK.
References
1 Kleinkauf, H (2000) The role of 4¢-phosphopantetheine in the biosynthesis of fatty acids, polyketides and peptides Biofactors 11, 91–92.
2 Lipmann, F (1942–62) Development of the acetylation problem:
a personal account in Nobel lectures: Physiology or Medicine,
pp 413–438, American Elsevier, New York.
3 Lynen, F (1963–70) The Pathway from ‘Activated Acetic Acid’ to the Terpenes and Fatty Acids in Nobel Lectures: Physiology or Medicine, pp 103–138, American Elsevier, New York.
4 Kupke, T (2001) Molecular characterization of the 4¢-phospho-pantothenoylcysteine decarboxylase domain of bacterial Dfp flavoproteins J Biol Chem 276, 27597–27604.
5 Kupke, T (2002) Molecular characterization of the 4¢-phospho-pantothenoylcysteine synthetase domain of bacterial Dfp flavo-proteins J Biol Chem 277, 36137–36145.
6 Kupke, T., Uebele, M., Schmid, D., Jung, G., Blaesse, M & Steinbacher, S (2000) Molecular characterization of lantibiotic-synthesizing enzyme EpiD reveals a function for bacterial Dfp
Trang 10proteins in coenzyme A biosynthesis J Biol Chem 275, 31838–
31846.
7 Strauss, E., Kinsland, C., Ge, Y., McLafferty, F.W & Begley, T.P.
(2001) Phosphopantothenoylcysteine synthetase from Escherichia
coli: Identification and characterization of the last unidentified
coenzyme A biosynthetic enzyme in bacteria J Biol Chem 276,
13513–13516.
8 Kupke, T., Kempter, C , Gnau, V., Jung, G & Go¨tz, F (1994)
Mass spectroscopic analysis of a novel enzymatic reaction:
oxidative decarboxylation of the lantibiotic precursor peptide
EpiA catalyzed by the flavoprotein EpiD J Biol Chem 269,
5653–5659.
9 Kupke, T., Kempter, C , Jung, G & Go¨tz, F (1995) Oxidative
decarboxylation of peptides catalyzed by flavoprotein EpiD:
determination of substrate specificity using peptide libraries
and neutral loss mass spectrometry J Biol Chem 270, 11282–
11289.
10 Majer, F., Schmid, D.G., Altena, K., Bierbaum, G & Kupke, T.
(2002) The flavoprotein MrsD catalyzes the oxidative
decarbox-ylation reaction involved in formation of the peptidoglycan
biosynthesis inhibitor mersacidin J Bacteriol 184, 1234–1243.
11 Herna´ndez-Acosta, P., Schmid, D.G., Jung, G., Culia´n˜ez-Macia`,
F.A & Kupke, T (2002) Molecular characterization of the
Arabidopsis thaliana flavoprotein AtHAL3a reveals the general
reaction mechanism of 4¢-phosphopantothenoylcysteine
decarb-oxylases J Biol Chem 277, 20490–20498.
12 Steinbacher, S., Herna´ndez-Acosta, P., Bieseler, B., Blaesse, M.,
Huber, R., Culia´n˜ez-Macia`, F.A & Kupke, T (2003) Crystal
structure of the plant PPCdecarboxylase AtHAL3a complexed
with an ene-thiol reaction intermediate J Mol Biol 327, 193–202.
13 Blaesse, M., Kupke, T., Huber, R & Steinbacher, S (2000)
Crystal structure of the peptidyl-cysteine decarboxylase EpiD
complexed with a pentapeptide substrate EMBO J 19, 6299–
6310.
14 Spitzer, E.D., Jimenez-Billini, H.E & Weiss, B (1988) b-Alanine auxotrophy associated with dfp, a locus affecting DNA synthesis
in Escherichia coli J Bacteriol 170, 872–876.
15 Spitzer, E.D & Weiss, B (1985) dfp gene of Escherichia coli K-12,
a locus affecting DNA synthesis, codes for a flavoprotein.
J Bacteriol 164, 994–1003.
16 Scha¨gger, H & Jagow, G (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa Anal Biochem 166, 368–379.
17 Kupke, T., Herna´ndez-Acosta, P & Culia´n˜ez-Macia`, F.A (2003) 4¢-Phosphopantetheine and coenzyme A biosynthesis in plants.
J Biol Chem 278, 38229–38237.
18 Manoj, N., Strauss, E., Begley, T.P & Ealick, S.E (2003) Struc-ture of human phosphopantothenoylcysteine synthetase at 2.3 A˚ resolution Structure (Camb) 11, 927–936.
19 Daugherty, M., Polanuyer, B., Farrell, M., Scholle, M., Lykidis, A., De Cre´cy-Lagard, V & Osterman, A (2002) Complete reconstitution of the human coenzyme A biosynthetic pathway via comparative genomics J Biol Chem 277, 21431–21439.
20 Gerdes, S.Y., Scholle, M.D., D’Souza, M., Bernal, A., Baev, M.V., Farrell, M., Kurnasov, O.V., Daugherty, M.D., Mseeh, F., Polanuyer, B.M., Campbell, J.W., Anantha, S., Shatalin, K.Y.,
C howdhury, S.A., Fonstein, M.Y & Osterman, A.L (2002) From genetic footprinting to antimicrobial drug targets: examples in cofactor biosynthetic pathways J Bacteriol 184, 4555–4572.
21 Choudhry, A.E., Mandichak, T.L., Broskey, J.P., Egolf, R.W., Kinsland, C., Begley, T.P., Seefeld, M.A., Ku, T.W., Brown, J.R., Zalacain, M & Ratnam, K (2003) Inhibitors of pantothenate kinase: novel antibiotics for Staphylococcalinfections Antimicrob Agents Chemother 47, 2051–2055.
22 Zhao, L., Allanson, N.M., Thomson, S.P., Maclean, J.K., Barker, J.J., Primrose, W.U., Tyler, P.D & Lewendon, A (2003) Inhibitors of phosphopantetheine adenylyltransferase Eur J Med Chem 38, 345–349.