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J. Biol. Chem., Vol. 277, Issue 39, 36137-36145, September 27, 2002

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Molecular Characterization of the 4'-Phosphopantothenoylcysteine Synthetase Domain of Bacterial Dfp Flavoproteins^*

Thomas Kupke

From the Lehrstuhl für Mikrobielle Genetik, Universität Tübingen, Auf der Morgenstelle 15, Verfügungsgebäude, 72076 Tübingen, Germany

Received for publication, June 21, 2002, and in revised form, July 22, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

In bacteria, coenzyme A is synthesized in five steps from pantothenate. The flavoprotein Dfp catalyzes the synthesis of the coenzyme A precursor 4'-phosphopantetheine in the presence of 4'-phosphopantothenate, cysteine, CTP, and Mg²⁺ (Strauss, E., Kinsland, C., Ge, Y., McLafferty, F. W., and Begley, T. P. (2001) J. Biol. Chem. 276, 13513-13516). It has been shown that the NH₂-terminal domain of Dfp has 4'-phosphopantothenoylcysteine decarboxylase activity (Kupke, T., Uebele, M., Schmid, D., Jung, G., Blaesse, M., and Steinbacher, S. (2000) J. Biol. Chem. 275, 31838-31846). Here I demonstrate that the COOH-terminal CoaB domain of Dfp catalyzes the synthesis of 4'-phosphopantothenoylcysteine. The exchange of conserved amino acid residues within the CoaB domain revealed that the synthesis of 4'-phosphopantothenoylcysteine occurs in two half-reactions. Using the mutant protein His-CoaB N210D the putative acyl-cytidylate intermediate of 4'-phosphopantothenate was detectable. The same intermediate was detectable for the wild-type CoaB enzyme if cysteine was omitted in the reaction mixture. Exchange of the conserved Lys²⁸⁹ residue, which is part of the strictly conserved ²⁸⁹KXKK²⁹² motif of the CoaB domain, resulted in complete loss of activity with neither the acyl-cytidylate intermediate nor 4'-phosphopantothenoylcysteine being detectable. Gel filtration experiments indicated that CoaB forms dimers. Residues that are important for dimerization are conserved in CoaB proteins from eubacteria, Archaea, and eukaryotes.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Coenzyme A is synthesized in five steps from pantothenate, and more than 40 years ago G. Brown showed that it is built from phosphorylated precursors (1). His initial studies were mainly continued by Y. Abiko, who characterized the various enzymatic activities of coenzyme A biosynthesis in a series of papers published in 1967 and 1968 (2-6). The conclusion of both pioneer works was that coenzyme A is synthesized in bacterial and in mammalian systems as follows (see Fig. 1). In the first step, pantothenate is phosphorylated to 4'-phosphopantothenate, which is then converted by addition of cysteine to (R)-4'-phospho-N-pantothenoylcysteine (PPC,¹ "coupling reaction"). PPC is then decarboxylated to 4'-phosphopantetheine, which is converted to coenzyme A in two further steps.

Although the pathway for coenzyme A biosynthesis has been well established since the work of Brown and Abiko, purification and detailed characterization of the enzymes involved turned out to be very difficult. Since the coenzyme A biosynthetic genes are essential, their identification and cloning was also a great challenge. Starting with cloning of the pantothenate kinase gene (coaA) from Escherichia coli in 1992 (7), it took 10 years to identify all the genes of the coenzyme A biosynthetic pathway in E. coli (8-11) and to identify the human genes by comparative genetics (12). In bacteria, the two-step conversion of 4'-phosphopantothenate to 4'-phosphopantetheine (peptide bond formation between 4'-phosphopantothenate and cysteine and then decarboxylation of the formed PPC) is catalyzed by the Dfp flavoproteins (Fig. 1 and Refs. 8 and 9), which were first described by Spitzer et al. (13, 14). Molecular characterization showed that the decarboxylase activity resides in the NH₂-terminal CoaC domain of Dfp, indicating that the COOH-terminal (CoaB) domain is involved in synthesis of PPC (8). PPC decarboxylases bind the cofactor FMN, and during the last 2 years it has been shown that decarboxylation of PPC follows the mechanism "decarboxylation by initial oxidation" of the so-called LanD enzymes (8, 15-18). In 2002 it was confirmed that the human PPC decarboxylase is also a flavoprotein (12). Earlier results classified bacterial and mammalian PPC decarboxylases as pyruvoyl-dependent enzymes (19-21), although there is no free amino group in the substrate. Begley et al. (22) first proposed a new mechanism for a pyruvoyl-dependent decarboxylation involving a N-S acyl shift to unmask the amino group, but later they confirmed that the flavoprotein Dfp catalyzes the decarboxylation of PPC as had been shown earlier by Kupke et al. (8, 9).

Until now the synthesis of PPC by Dfp has only been shown indirectly by measurement of released ¹⁴CO₂ from L-1-[¹⁴C]cysteine in the presence of 4'-phosphopantothenate, CTP, and Mg²⁺ (9). Bacterial PPC synthesis requires the cofactor CTP (1), which is converted during the reaction to CMP and inorganic pyrophosphate indicating the formation of an activated acyl-cytidylate as intermediate (Fig. 1 and Ref. 9). Human PPC synthetase uses ATP for the coupling reaction more efficiently than CTP. The synthesis of PPC was studied in this case by the release of inorganic pyrophosphate indicating that the activation mechanism of the human enzyme is comparable to that of bacterial enzymes (12). However, Abiko et al. (5) showed that the PPC synthetase from rat liver converts ATP to ADP and phosphate (and not to AMP and pyrophosphate).

View larger version (26K): [in this window] [in a new window]   Fig. 1.   The role of Dfp in coenzyme A biosynthesis. A, coenzyme A is synthesized in five steps from pantothenate. The bifunctional enzyme Dfp catalyzes the conversion of 4'-phosphopantothenic acid to 4'-phosphopantetheine. Recently it was shown that the NH2-terminal domain of Dfp has PPC decarboxylase activity and that decarboxylation is initiated by flavin-dependent oxidation of PPC. In this paper, the COOH-terminal domain of Dfp is identified as the CoaB domain, which synthesizes 4'-phosphopantothenoylcysteine (framed) from 4'-phosphopantothenic acid, CTP, and cysteine. B, the CoaB domain catalyzes the synthesis of PPC in two half-reactions starting with the formation of 4'-phosphopantothenoyl-CMP (framed, activation of the carboxyl group of 4'-phosphopantothenate). In a second step, the amide bond of PPC (see A) is formed by reaction of 4'-phosphopantothenoyl-CMP with cysteine. By using the mutant enzymes His-CoaB K289Q and His-CoaB N210D (this study) it was possible to elucidate this mechanism, which has been suggested recently by Strauss et al. (9). In summary, Dfp is an enzyme catalyzing the synthesis and the modification of a phosphopeptide-like structure.

In this study, a molecular characterization of the COOH-terminal CoaC domain of Dfp was undertaken, and its ability to catalyze the synthesis of PPC is demonstrated. In contrast to previous studies on PPC synthetases (9, 12), the formation of PPC is directly shown. Conserved sequence motifs in the CoaC domain are investigated by site-directed mutagenesis, continuing the molecular characterization of the Dfp proteins (8, 23). These studies led to detection of the proposed activated acyl-cytidylate intermediate, 4'-phosphopantothenoyl-CMP, and show that the synthesis of PPC occurs in two half-reactions. Furthermore, it is shown that CoaB forms homodimers and that residues responsible for dimerization are conserved in CoaB (Dfp) proteins from all kingdoms of life.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Plasmid Construction

General Methods-- PCR amplifications were performed with Vent-DNA polymerase (New England Biolabs). The entire sequences of the coaA and coaB coding regions of the constructed plasmids were verified. Oligonucleotides were purchased from MWG Biotech.

Cloning of the 3'-Part of the dfp Gene-- The 3'-part of the dfp gene encoding the COOH-terminal CoaB domain Ser¹⁸¹-Arg⁴⁰⁶ of Dfp was amplified by PCR and cloned into the single BamHI site of the expression vector pQE8 (Qiagen). For PCR amplification, the template pQE12 dfp (8) and the primers (i) forward, 5'-GCGGTAGCGCATAGATCTCCCGTCAACGACC-3' (the introduced BglII site is underlined) and (ii) reverse, 5'-GGTCATTACTGGATCTATCAACAGG-3' were used. The reverse primer binds downstream of the BglII site of pQE12 dfp. The amplified coaB gene was digested with BglII and cloned into pQE8 BamHI. The pQE8-derived plasmids were transformed into the expression strain E. coli M15 (pREP4) (Qiagen) by electroporation. The expression plasmid pQE8 coaB encodes an NH₂-terminal His tag fusion protein of the CoaB protein (His-CoaB: MRGSHHHHHHG-Dfp Ser¹⁸¹-Arg⁴⁰⁶).

Site-directed Mutagenesis of coaB-- All point mutations were first introduced into pQE12 dfp by using sequential PCR and appropriate mutagenesis primers as recently described (23, 24). Using the constructed mutant pQE12 dfp plasmids as templates, the mutant coaB genes were amplified and then cloned into pQE8 BamHI as described above for wt coaB.

Cloning of the coaA Gene from E. coli-- Chromosomal DNA from E. coli TB1 (New England Biolabs) was purified using the Qiagen Blood & Cell Culture DNA Mini kit. For cloning, the coaA gene (7) was amplified by PCR using the oligonucleotides (i) forward, 5'-GCTATGACCGCCGGATCCATGCTTATGAGTA-3' and (ii) reverse, 5'-GAAAGGGGAGTATTGGATCCCCTGCAAATT-3' as primers (introduced BamHI sites are underlined) and the purified chromosomal DNA as template. The amplified coaA gene was cloned into pQE8 BamHI and then transformed in E. coli M15 (pREP4) as described above. The expression plasmid pQE8 coaA encodes an NH₂-terminal His tag fusion protein of the CoaA protein (His-CoaA: MRGSHHHHHHGSML-CoaA).

Purification and Characterization of CoaB Proteins

Growth of Strains-- E. coli M15 (pREP4, pQE8) cells were grown in the presence of 100 µg/ml ampicillin and 25 µg/ml kanamycin in 0.5 liters of B-broth (10 g of casein hydrolysate 140 (Invitrogen), 5 g of yeast extract (Difco), 5 g of NaCl, 1 g of glucose, and 1 g of K₂HPO₄/liter, pH 7.3) in 2-liter shaker flasks. At A₅₇₈ = 0.4, the cells were induced with 1 mM isopropyl--D-thiogalactopyranoside and harvested 2 h after induction. The growth temperature was 37 °C.

Purification of His-CoaA and His-CoaB Proteins-- For purification of His-CoaA and His-CoaB proteins, 500 ml of isopropyl--D-thiogalactopyranoside-induced E. coli M15 (pREP4, pQE8 coaA/coaB) cells were harvested and disrupted by sonication in 10 ml of 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 4 °C) were applied to Ni-NTA spin columns (Qiagen) equilibrated with column buffer (20 mM Tris-HCl, pH 8.0, 10 mM imidazole, 300 mM NaCl). The spin columns were then washed twice with 0.65 ml of 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-NTA spin columns were centrifuged at room temperature at only 240 × g to enable effective binding of the His tag proteins. For gel filtration, a 25-µl aliquot of the Ni-NTA eluate was subjected to a Superdex 200 PC 3.2/30 column equilibrated in running buffer (20 mM Tris-HCl, pH 8.0, 200 mM NaCl) at a flow rate of 40 µl/min. The Superdex 200 PC 3.2/30 column and the standard proteins used for calibration were obtained from Amersham Biosciences. For activity assays (see below), the Ni-NTA eluates were used.

CoaB Assay-- Since 4'-phosphopantothenate is not commercially available, it was synthesized enzymatically by adding His-CoaA, pantothenate, and ATP to the His-CoaB assay mixtures. Therefore, 0.8 ml of CoaB assay mixtures contained 5 mM pantothenate, 2.5 mM MgCl₂, 5 mM ATP, 5 mM CTP, 10 mM cysteine hydrochloride, 10 mM dithiothreitol, 50 mM Tris, pH 8.0, His-CoaA (~10-15 µg), and either wt His-CoaB or mutant His-CoaB proteins in the range of 3-20 µg. After 45 min of incubation at 37 °C, the reaction mixtures were kept at 80 °C and then were successively separated by reversed phase chromatography with a µRPC C₂/C₁₈ SC 2.1/10 column on a SMART system (Amersham Biosciences). 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 µl/min. The absorbance was measured simultaneously at 214, 260, and 280 nm to enable identification of acyl-cytidylate intermediates.

SDS-PAGE-- Proteins were separated using Tricine-sodium dodecyl sulfate-polyacrylamide (10%) gel electrophoresis under reducing conditions (25). Prestained protein molecular weight standards were obtained from New England Biolabs.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Alignment of the CoaB Domains of Sequenced Dfp Proteins-- Recently the alignment of the FMN binding CoaC domains of sequenced bacterial Dfp proteins was presented, and a PPC decarboxylase signature was defined (23). Here the sequence comparison is extended to the COOH-terminal domain of Dfp proteins (Fig. 2A). There are a lot of conserved sequence motifs within the CoaB domains with the ²¹⁰NXSSGK²¹⁵ and the ²⁸⁹KXKK²⁹² motifs being the most noteworthy. In eukaryotes (and a few bacteria), the PPC decarboxylase and synthetase activities are not fused (12, 26). Human and plant PPC decarboxylases share the bacterial PPC decarboxylase signature, whereas there is only marginal similarity of the eukaryotic PPC synthetases with the bacterial CoaB domains (12, 26). The sequence comparison shown is the basis for the site-directed mutagenesis studies of the CoaB domain presented below. Interestingly, it turned out that despite the low similarity of the PPC synthetase domains, residues that are proposed to be important for dimer formation of CoaB are conserved in CoaB proteins from eukaryotes and Archaea (Fig. 2B and see below). It appears that the first Lys residue of the KXKK motif is also conserved in CoaB from eubacteria, eukaryotes, and Archaea (Fig. 2B). However, the sequence similarity is very low, and biochemical studies are required to prove that the Lys residue has the same function in all CoaB proteins.

View larger version (63K): [in this window] [in a new window]   Fig. 2.   Sequence alignment of the CoaB domains of eubacterial Dfp proteins. A, the COOH-terminal CoaB domain of the Dfp protein from E. coli was compared with eubacterial Dfp proteins (listed in alphabetical order: A. aeo., Aquifex aeolicus; B. sub., Bacillus subtilis; B. bur., Borrelia burgdorferi; B. jap., Bradyrhizobium japonicum; C. jej., Campylobacter jejuni; Hae. in., Haemophilus influenzae; Hel. p., Heliobacter pylori; Lis. m., Listeria monocytogenes; Myc. t., Mycobacterium tuberculosis; N. men., Neisseria meningitidis; Ps. ae., Pseudomonas aeruginosa; S. coe., Streptomyces coelicolor; Synech., Synechocystis sp.; T. mar., Thermotoga maritima; Z. mob., Zymomonas mobilis). Only a part of the comparison emphasizing the highly conserved motifs is shown. Conserved residues are in bold letters. Several amino acid residues were exchanged in the presented study; however, only the mutations N210D and K289Q are indicated by arrows. The exchange of the amino acid residues Thr194, Thr198, Asp203, Asn210, and Ala275 (labeled with dots) led to a significantly different elution behavior in the gel filtration experiments (Fig. 4) indicating the presence of a dimerization motif in CoaB. B, sequence comparison of the proposed dimerization motifs of CoaB (Dfp) proteins from eubacteria (E. coli: P24285), archae (Methanocaldococcus jannaschii (M. jan.): Q58323), and eukaryotes (human: XP_016228 (gi: 13638573) and yeast: P40506).

Purification of His-CoaB Proteins-- CoaB and mutant coaB genes were expressed as His tag fusion proteins, purified from the corresponding E. coli clones by immobilized metal affinity chromatography (IMAC) and additionally purified by gel filtration (Figs. 3 and 4). Gel filtration was also used to determine the apparent molecular weight and to elucidate whether native His-CoaB forms monomers or homomultimers. A comparison with standard proteins revealed an apparent molecular mass of 42 kDa for native purified His-CoaB, indicating that His-CoaB forms homodimers (Fig. 4, molecular mass of His-CoaB is 26.1 kDa). Some of the analyzed mutant His-CoaB proteins (T194V, T198V, D203N, and A275V) showed a significantly increased elution volume that corresponds exactly to the molecular weight of monomeric His-CoaB. Also the elution volume of His-CoaB N210D is increased, but it appears that the mass of this protein is slightly increased compared with monomeric His-CoaB. In general, molecular weight determination of proteins by gel filtration is not precise, but the differences of the elution volumes between wt His-CoaB and mutant His-CoaB proteins clearly show that wt enzyme forms homodimers, while several of the mutants are monomeric. The residues that are important for dimerization of E. coli CoaB are conserved in eukaryotic PPC synthetases (Fig. 2B). It appears that residues Thr¹⁹⁴-Asn²¹⁰ of E. coli CoaB form a dimerization motif.

View larger version (53K): [in this window] [in a new window]   Fig. 3.   Purification of mutant His-CoaB proteins. Purification of His-CoaB and mutant His-CoaB proteins by IMAC and gel filtration was followed by SDS-PAGE. M, molecular weight marker. His-CoaB is indicated with an arrow. Only those mutant CoaB proteins that are analyzed in more detail in the present study are shown.

View larger version (37K): [in this window] [in a new window]   Fig. 4.   Gel filtration of mutant His-CoaB proteins. IMAC-purified His-CoaB proteins were separated by gel filtration on a Superdex 200 PC 3.2/30 column, and the elution was monitored by absorbance at 278 nm. The mutant proteins were analyzed under comparable conditions together with wild-type His-CoaB in two sets of experiments (A, His-CoaB wt, His-CoaB N210D, His-CoaB S212A, His-CoaB K215Q, His-CoaB D279N, His-CoaB D279E, His-CoaB K289Q, His-CoaB K291Q, and His-CoaB K292Q; B, His-CoaB wt, His-CoaB T194V, His-CoaB T198V, His-CoaB D203N, His-CoaB R206Q, His-CoaB A275S, His-CoaB A275V, His-CoaB A276V, His-CoaB D309N, and His-CoaB F327L). The determined elution volume of wild-type His-CoaB corresponds to a molecular mass of 42 kDa. The mutations T194V, T198V, D203N, N210D, and A275V (framed) led to significant changes of the elution volume. The gel filtration column used was calibrated with standard proteins to correlate the elution volume with molecular weight information as described recently (8).

Recently we showed that Dfp is a homododecameric member of the homooligomeric flavin-containing Cys decarboxylase protein family (8, 15) and that the NH₂-terminal CoaC domain is responsible for homododecamer formation (8, 23). Mutations that prevent dimerization of CoaB do not prevent the formation of homododecameric Dfp (data not shown). Including the new results, I propose that within the Dfp homododecamers two PPC synthetase domains contact each other.

His-CoaB Synthesizes 4'-Phosphopantothenoylcysteine-- Strauss et al. (9) have shown that Dfp proteins are able to convert 4'-phosphopantothenate to 4'-phosphopantetheine when cysteine, dithiothreitol, CTP, and Mg²⁺ are present in the assay mixture, and they verified that Dfp catalyzes the decarboxylation of PPC to 4'-phosphopantetheine. Considering the known biosynthetic pathway for coenzyme A, these results show that Dfp is a bifunctional enzyme and catalyzes the synthesis of PPC and subsequently the decarboxylation of PPC to 4'-phosphopantetheine. However, Strauss et al. (9) could not directly detect PPC as the intermediate of the Dfp reaction. To analyze the biochemistry of the PPC synthetase activity of E. coli, it is necessary to separate PPC synthetase and PPC decarboxylase activities of Dfp. This can be achieved by disrupting the PPC decarboxylase activity or by separating the bifunctional Dfp protein into the two proposed domains, CoaB and CoaC. Both approaches were tried in the present study and were successful. Using the published HPLC method for the detection of PPC (8), I was able to show that the COOH-terminal CoaB domain of Dfp synthesizes PPC from 4'-phosphopantothenate and cysteine (Figs. 5-7). Biosynthesis of PPC was not only verified by comparison of the determined retention volume with that of synthetic PPC but also by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICR-MS) as described recently (8). The m/z value for PPC synthesized by CoaB was determined to be 403.09, which is in accordance with the theoretical mass of [PPC + H]⁺ = 403.0935 Da. Synthesis of PPC was also observed when Dfp C158S (26) was used instead of CoaB (Fig. 6). Recently it has been shown that the C158S mutation (Cys¹⁵⁸ is within the amino-terminal CoaC domain of Dfp) inhibits the PPC decarboxylase activity (23).

View larger version (25K): [in this window] [in a new window]   Fig. 5.   PPC synthetase activity of the CoaB domain. The synthesis of 4'-phosphopantothenoylcysteine from 4'-phosphopantothenate and cysteine was analyzed using an HPLC-based assay. 4'-Phosphopantothenate was synthesized in situ by incubation of pantothenate with ATP, Mg2+, and pantothenate kinase (His-CoaA). A, PPC is synthesized in the presence of pantothenic acid, Mg2+, ATP, CTP, cysteine, dithiothreitol, His-CoaA, and wt His-CoaB. B, when His-CoaB is omitted from the reaction mixture, no PPC is detectable. IMAC purification of the pantothenate kinase His-CoaA used was verified by SDS-PAGE as shown in the inset of B. C, no pantothenoylcysteine was detectable if pantothenic acid was incubated in the presence of CTP, cysteine, dithiothreitol, and Mg2+ with His-CoaB, indicating that only 4'-phosphopantothenate, and not pantothenate, is a substrate of CoaB. D, synthetic PPC was used as standard substance to evaluate the HPLC assays.

View larger version (35K): [in this window] [in a new window]   Fig. 6.   PPC synthetase activity of Dfp C158S and mutant CoaB proteins. Biosynthesis of PPC was analyzed using the described HPLC-based assay. PPC was detectable when wt His-CoaB, His-CoaB S212A, His-CoaB K215Q, His-CoaB K291Q, or His-CoaB K292Q was used. However, no PPC was detectable when the mutant proteins His-CoaB N210D or His-CoaB K289Q were used for the assays. Interestingly, incubation with His-CoaB N210D led to a compound (labeled with an asterisk) with high absorbances at 260 and 280 nm (not shown). This compound was later identified to be the intermediate of the CoaB reaction (compare Fig. 7). Synthesis of PPC could also be achieved with the mutant Dfp C158S protein, which is not able to decarboxylate PPC to 4'-phosphopantetheine. Synthetic PPC was used in the control experiment. The retention time of PPC was shifted to lower values compared with the data presented in Fig. 5. PPC already eluted during washing the column with H2O, 0.1% trifluoroacetic acid (gradient started at 17 min). This is due to the increased number of applications of the column used and the large amounts of compounds applied to the column during the assays.

PPC Synthetase Activity of Mutant CoaB Proteins-- In this study, the motifs ²¹⁰NXSSGK²¹⁵ and ²⁸⁹KXKK²⁹² of CoaB were investigated in more detail by site-directed mutagenesis. The mutant proteins were purified and characterized by gel filtration (see above), and the PPC synthetase activity was analyzed (Fig. 6). Although the assay used is not suitable for the determination of kinetic parameters, it can be applied to identify amino acid residues that are crucial for activity. Introduction of the point mutations N210D and K289Q led to complete loss of PPC synthetase activity. However, His-CoaB N210D was able to form 4'-phosphopantothenoyl-CMP, the proposed intermediate of PPC biosynthesis (Figs. 6 and 7 and see below). The conclusion is that PPC biosynthesis occurs in two half-reactions and that the residue Lys²⁸⁹ is important for formation of the activated acyl-cytidylate intermediate (Fig. 1B). Furthermore, it is reasonable to suggest that Lys²⁸⁹ may either be involved in binding the phosphate group of 4'-phosphopantothenate or in binding the phosphate groups of the cofactor CTP.

View larger version (27K): [in this window] [in a new window]   Fig. 7.   Synthesis of PPC occurs via an acyl-cytidylate intermediate of 4'-phosphopantothenate. The PPC synthetase activity of wt His-CoaB and His-CoaB N210D was determined using the described HPLC assay omitting either cysteine (B) or pantothenate (which is converted by the kinase His-CoaA present to 4'-phosphopantothenate, C). The absorbance was monitored at 214, 260 (not shown), and 280 nm to identify PPC and intermediates. When both pantothenate and cysteine are present (A), PPC synthesis is observed for wt His-CoaB but not for His-CoaB N210D. However, for His-CoaB N210D a compound (labeled with an asterisk) with high absorbance at 280 nm and a greater retention time than that of PPC was identified. This compound was also present when cysteine was omitted in the reaction mixture but was lacking when pantothenate was omitted (the very small detectable amounts may represent copurified intermediate bound to His-CoaB N210D). Interestingly, this compound could also be detected for wt enzyme when cysteine was omitted in the reaction mixture. Therefore, the observed compound is an intermediate of the PPC synthesis.

Biosynthesis of 4'-Phosphopantothenoylcysteine Involves a 4'-Phosphopantothenoyl-CMP Intermediate-- The activity of the mutant His-CoaB N210D protein was analyzed in more detail (Fig. 7). Only very small amounts of PPC were synthesized by His-CoaB N210D. However, an intermediate with an increased retention time compared with PPC could be identified. This intermediate was only present in very small amounts, and repeated attempts to determine the molecular mass of this compound failed. Therefore, I tried to determine whether 4'-phosphopantothenate or cysteine is converted to this intermediate. The results obtained clearly show that phosphopantothenate, but not cysteine, is converted to the intermediate (Fig. 7). As expected, this intermediate could also be detected with wt His-CoaB when cysteine was omitted in the assay mixture (Fig. 7). Further characterization of the intermediate was possible by following the elution of the compound from the used C₂/C₁₈ column at various wavelengths. This revealed high absorbances of the compound at 260 nm (not shown) and 280 nm (Fig. 7). Pantothenate does not absorb in this UV range, and the determined ratio of the absorbances, A₂₈₀/A₂₆₀ = 2.0, showed that the intermediate is derived from CTP and not ATP. Taking all these data together and including the observation of Strauss et al. (9) that CTP is converted to CMP and inorganic pyrophosphate during the Dfp reaction, I conclude that the detected compound is the proposed 4'-phosphopantothenoyl-CMP intermediate (Fig. 1B). If this intermediate is not attacked by cysteine, it will be present in an enzyme-bound form and will be released from the enzyme under acidic conditions as has been observed in the experiments shown. The conclusion is that the mutation N210D either impairs binding of cysteine or influences the nucleophilic attack on the intermediate by cysteine (Fig. 1). The experiments also indicate that formation of 4'-phosphopantothenoyl-CMP does not require a dimeric structure of CoaB since the N210D mutant is a monomer. The increased mass of purified His-CoaB N210D compared with the determined mass of monomeric His-CoaB may be explained by binding of the acyl-cytidylate.

Comparison of the Syntheses of Pantothenic Acid and 4'-Phosphopantetheine-- Pantothenate synthetase catalyzes the condensation of pantoate with -alanine in an ATP-dependent way in two half-reactions via the pantoyl-adenylate intermediate (27, 28). -Alanine is derived from aspartate by decarboxylation catalyzed by the pyruvoyl-dependent aspartate 1-decarboxylase (29, 30). It is interesting to compare this pathway with the biosynthesis of 4'-phosphopantetheine catalyzed by the Dfp protein. Both synthetases, pantothenate synthetase and CoaB, are dimers. The catalysis of peptide bond formation is similar and depends on the formation of activated acyl-cytidylate or acyl-adenylate intermediates. However, the involved decarboxylation reactions are different. Aspartate is decarboxylated before formation of the peptide bond, whereas decarboxylation of cysteine is not observed. The cysteamine residue of 4'-phosphopantetheine is introduced by FMN-dependent decarboxylation of PPC by the NH₂-terminal CoaC domain of Dfp. Recently it was shown that the structure of the NH₂-terminal domain of pantothenate synthetase is very similar to class I aminoacyl-tRNA synthetases and that pantothenate synthetase belongs to the cytidylyltransferase superfamily (31). It will be interesting to learn more about the three-dimensional structures of Dfp and CoaB by x-ray diffraction methods to elucidate how CTP is bound by the enzyme and to gain more insights into the reaction mechanism.

Conclusions-- Dfp is a bifunctional enzyme catalyzing the synthesis of 4'-phosphopantetheine in a multistep process from 4'-phosphopantothenate and cysteine. In the first step, 4'-phosphopantothenate is activated by reaction with CTP. The 4'-phosphopantothenoyl-cytidylate formed is attacked by cysteine, and PPC is synthesized. These reactions occur in the COOH-terminal CoaB domain of Dfp. The next step is the FMN-dependent oxidative decarboxylation of PPC to 4'-phosphopantothenoylaminoethenethiol, which is then reduced to 4'-phosphopantetheine (16). Oxidative decarboxylation of peptidylcysteines has been detected as an important step in the biosynthesis of the lantibiotic epidermin (17). The decarboxylation reaction is catalyzed by the NH₂-terminal CoaC domain, and presently it is not known how CoaB and CoaC domains interact to synthesize 4'-phosphopantetheine most effectively.

	ACKNOWLEDGEMENTS

I thank Regine Stemmler for excellent technical assistance, Dietmar Schmid for ESI-FTICR-MS experiments, Michael Uebele for synthesis of (R)-4'-phospho-N-pantothenoylcysteine, and Lloyd Ruddock for reading the manuscript.

	FOOTNOTES

^* This work was supported by Deutsche Forschungsgemeinschaft Grant KU869/6-1 (to T. K.).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. E-mail: Thomas. Kupke{at}t-online.de.

Published, JBC Papers in Press, July 24, 2002, DOI 10.1074/jbc.M206188200

	ABBREVIATIONS

The abbreviations used are: PPC, (R)-4'-phospho-N-pantothenoylcysteine; Ni-NTA, nickel-nitrilotriacetic acid, His-CoaB, MRGSHHHHHHG-Dfp Ser¹⁸¹-Arg⁴⁰⁶; His-CoaA, MRGSHHHHHHGSML-CoaA; ESI-FTICR-MS, electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry; IMAC, immobilized metal affinity chromatography; wt, wild-type; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; HPLC, high performance liquid chromatography.

	REFERENCES

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