4′-Phosphopantetheine Biosynthesis in Archaea*

Coenzyme A as the principal acyl carrier is required for many synthetic and degradative reactions in intermediary metabolism. It is synthesized in five steps from pantothenate, and recently the CoaA biosynthetic genes of eubacteria, plants, and human were all identified and cloned. In most bacteria, the so-called Dfp proteins catalyze the synthesis of the coenzyme A precursor 4′-phosphopantetheine. Dfp proteins are bifunctional enzymes catalyzing the synthesis of 4′-phosphopantothenoylcysteine (CoaB activity) and its decarboxylation to 4′-phosphopantetheine (CoaC activity). Here, we demonstrate the functional characterization of the CoaB and CoaC domains of an archaebacterial Dfp protein. Both domains of the Methanocaldococcus jannaschii Dfp protein were purified as His tag proteins, and their enzymatic activities were then identified and characterized by site-directed mutagenesis. Although the nucleotide binding motif II of the CoaB domain resembles that of eukaryotic enzymes, Methanocaldococcus CoaB is a CTP- and not an ATP-dependent enzyme, as shown by detection of the 4′-phosphopantothenoyl-CMP intermediate. The proposed 4′-phosphopantothenoylcysteine binding clamp of the Methanocaldococcus CoaC activity differs significantly from those of other characterized CoaC proteins. In particular, the active site cysteine residue, which otherwise is involved in the reduction of an aminoenethiol reaction intermediate, is not present. Moreover, the conserved Asn residue of the PXMNXXMW motif, which contacts the carboxyl group of 4′-phosphopantothenoylcysteine, is exchanged for His.

Coenzyme A (CoA) and 4Ј-phosphopantetheine (PP) 2 are essential cofactors for many enzymatic reactions, and acyl-CoA derivatives are key intermediates in energy metabolism. 4Ј-Phosphopantetheine is a cofactor of enzymes that play a role in the biosynthesis of fatty acids, polypeptide antibiotics, and polyketides. The thiol group of the cysteamine moiety of coenzyme A is the functional group because it activates substrates as thioesters.
Coenzyme A is synthesized from pantothenate in five steps, and all of the genes involved in the biosynthesis of the cofactor in eubacteria, plants, and human have recently been cloned and characterized. In the first step, pantothenate is phosphorylated to 4Ј-phosphopantothenate by pantothenate kinase, which is encoded by the coaA gene. Then, (R)-4Ј-phospho-N-pantothenoylcysteine (PPC) is synthesized by the addition of cysteine to 4Ј-phosphopantothenate (CoaB activity), and in the next step, PPC is decarboxylated to 4Ј-phosphopantetheine (CoaC activity). 4Ј-Phosphopantetheine is converted to coenzyme A by the enzymes 4Ј-phosphopantetheine adenylyltransferase (CoaD) and dephospho-CoA kinase (CoaE; reviewed in Ref. 1).
The key reaction in coenzyme A biosynthesis, the synthesis of the phosphopeptide-like cofactor 4Ј-phosphopantetheine from 4Ј-phosphopantothenate and cysteine, is catalyzed in Escherichia coli and in most eubacteria by the bifunctional Dfp (CoaBC) flavoprotein in a multistep process ( Fig. 1) (2). 4Ј-Phosphopantothenate is activated by reaction with CTP; the 4Ј-phosphopantothenoyl-cytidylate formed is attacked by cysteine, and PPC is synthesized (2)(3)(4). Crystal structure analysis of E. coli CoaB (5) revealed the 4Ј-phosphopantothenate and CTP binding motifs of CoaB. The nucleobase binding motif II of eubacterial CTP-binding CoaBs deviates from that of eukaryotic ATP-binding PPC synthetases. In E. coli Dfp the sequence reads 307 NPDIV, whereas the sequence is 210 VPKLL in the human enzyme (residues shown in boldface are those conserved in eubacterial/eukaryotic enzymes). In the next step, PPC is oxidatively decarboxylated to 4Ј-phosphopantothenoylaminoenethiol by the NH 2 -terminal FMNbinding CoaC domain of Dfp (6 -9). Subsequent reduction of 4Ј-phosphopantothenoylaminoenethiol to 4Ј-phosphopantetheine by the reduced cofactor FMNH 2 depends on the conserved cysteine residue of the 16-amino acid 4Ј-phosphopantothenoylcysteine binding clamp, 151 PDSGSQACGDIGPGRM (6,8,9). Binding of 4Ј-phosphopantothenoylcysteine also involves the Asn residue of the PXMNXXMW motif, which contacts the carboxyl group (8,10).
The presence of conserved CoA biosynthetic genes indicates that all Archaea convert 4Ј-phosphopantothenate into coenzyme A by using CoaB, CoaC, CoaD, and CoaE activities, although cloning and functional characterization of archaebacterial coenzyme A biosynthetic genes has not been published until now (11). In this paper, we have characterized the bifunctional Dfp protein of Methanocaldococcus jannaschii. The 5Ј-coaC part and the 3Ј-coaB part of the archaebacterial dfp gene were expressed in E. coli, and the purified His tag proteins His-CoaB and His-CoaC were characterized functionally. Although several active site residues of eubacterial and eukaryotic CoaC activities are not conserved in Methanocaldococcus Dfp (12), we were able to demonstrate its PPC decarboxylase activity. Furthermore, it is shown that His-CoaC (and thus Dfp) forms homododecamers like the E. coli Dfp protein and the related peptidyl-cysteine decarboxylases EpiD and MrsD (7,10,13). We show that M. jannaschii CoaB is a CTP-dependent enzyme, although the nucleotide binding motif II contains a Lys residue, which is characteristic for the ATP-dependent PPC synthetases (5). Cloning of coaC from M. jannaschii-The 5Ј part of the dfp gene encoding the amino-terminal CoaC domain, Met 1 -Arg 197 , of M. jannaschii Dfp was amplified by PCR and cloned into the single BamHI site of the epression vector pQE8 (Qiagen). For PCR amplification, the following primers, (i) forward, 5Ј-GGTTTAAAGATCTATGATAAGT-GAAATCATGC-3Ј, and (ii) reverse, 5Ј-CTCCGTTTAATATTAAA-GATCTATTTCCTTC-3Ј, were used. The amplified coaC gene was digested with BglII and cloned into pQE8 BamHI as described above. The expression plasmid pQE8 coaC encodes an NH 2 -terminal His tag fusion protein of the M. jannaschii CoaC protein (His-CoaC: MRGSH-HHHHHGS-Dfp-Met 1 -Arg 197 -Ser) (the additional serine residue at the COOH terminus was introduced because of the cloning procedure used). Site-directed Mutagenesis of coaB and coaC-All point mutations were introduced by using sequential PCR and appropriate mutagenesis primers as described recently (14). For the PCR mutagenesis of coaB, pQE8 coaB was used as template, and the oligonucleotides (i) forward, 5Ј-CAATTGTGAGCGGATAACAATTTCAC-3Ј, and (ii) reverse, 5Ј-CAGCTAATTAAGCTTAAGATCTAGTTTC-3Ј, were used as terminal primers (the introduced BglII site underlined). For the PCR mutagenesis of coaC, pQE8 coaC was used as template and the oligonucleotides (i) forward, 5Ј-CAATTGTGAGCGGATAACAATTTCAC-3Ј, and (ii) reverse, 5Ј-AATCCAGATGGAGATCTGAGGTCATTAC-3Ј, were used as terminal primers (introduced BglII site is underlined). The amplified coaB and coaC genes, respectively, were digested with EcoRI/BglII and cloned into pQE12 EcoRI/BglII. The pQE12-derived plasmids were transformed into the expression strain E. coli M15 (pREp4) (Qiagen) by electroporation. The entire sequences of the coding regions of the constructed pQ12 plasmids were verified. Because of the cloning procedure, the pQ12 plasmids encode the same His tag proteins as the pQE8 plasmids except for the introduced point mutation.

Purification of M. jannaschii His-CoaB and His-CoaC Proteins by Immobilized Metal Affinity Chromatography-For purification of His-
CoaB and His-CoaC proteins, 500 ml of isopropyl-␤-D-thiogalactopyranoside-induced E. coli M15 (pREP4, pQE8/12 coaB/coaC) cells were harvested and disrupted by sonication in 10 ml 20 mM Tris-HCl (pH 8.0). A cleared lysate obtained by two centrifugation steps (each for 20 min at 30,000 ϫ g at 4°C) was adjusted to 0.3 M NaCl/50 mM imidazol and then applied at room temperature to Ni-NTA resin equilibrated with column buffer (20 mM Tris-HCl, pH 8.0, 50 mM imidazole, 300 mM NaCl). His-CoaB and His-CoaC proteins, respectively, were eluted with column buffer containing 1 M instead of 50 mM imidazole. For activity assays (see below), the Ni-NTA eluates were used. For detection of copurified 4Ј-phosphopantothenoyl-CMP intermediates, His-CoaB proteins were treated with trifluoroacetic acid, denaturated proteins were centrifuged down, and the supernatant was subjected to reversed phase chromatography (RPC). 4Ј-Phosphopantothenoyl-CMP was identified by its retention time and absorbance properties.
Purification of Helper Enzymes Used in the Activity Assays-E. coli His-CoaA and Arabidopsis thaliana MBP (maltose-binding protein)-AtCoaD were purified as described recently (3,15).

Activity Assays
PPC Synthetase and PPC Decarboxylase Assay-Because 4Ј-phosphopantothenate is not commercially available, it was synthesized enzymatically by adding E. coli His-CoaA, pantothenate, and ATP to the PPC synthetase assay mixtures (3). Therefore, 1 ml of the assay mixture contained 5 mM pantothenate, 2.5 mM MgCl 2 , 5-10 mM ATP, 5 mM CTP, 5 mM L-cysteine hydrochloride, 10 mM dithiothreitol, 100 mM Tris, pH 8.0, and His-CoaA (ϳ15-25 g). After a 20 -30 min preincubation at 37°C to convert pantothenate to 4Ј-phosphopantothenate, either M. jannaschii His-CoaB or M. jannaschii His-CoaB and His-CoaC proteins together were added in the range of 5 to 25 g. After 45 min of incubation at 37 or 60°C (we did not perform the assays at the M. jannaschii growth temperature of about 80°C because of potential problems with the stability of assay components), the reaction mixtures were kept at Ϫ80°C and then were separated successively by reversed phase chromatography with a RPC C 2 /C 18 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 l/min. The absorbance was measured simultaneously at 214, 260, and 280 nm to enable identification of reaction products.
4Ј-Phosphopantetheine Adenylyltransferase Assay-Because the direct detection of PPC and PP is complicated by the low absorbance of the substances at 214 nm and the missing absorbance at 260 and 280 nm, we used a second assay in which PP was converted to dephospho-CoA by adding phosphopantetheine adenylyltransferase activity (MBP-AtCoaD) to the reaction mixture (concentrations of ATP and Mg 2ϩ were increased to 10 and 15 mM, respectively); the MBP-AtCoaD used is specific for PP and cannot ligate AMP to PPC (15). Synthesized dephospho-CoA was identified by its absorbance properties and by comparison of the retention time in RPC with chemically synthesized dephospho-CoA (Sigma) (15).

RESULTS AND DISCUSSION
Cloning and Sequence Comparison of M. jannaschii 4Ј-Phosphopantetheine Biosynthetic Genes coaB and coaC-The complete genome sequence of M. jannaschii was published as early as 1996 (17), and the dfp gene is annotated in the data banks. Several but not all residues shown to be important for PPC synthetase and decarboxylase activity and/or substrate or cofactor binding in eubacterial and eukaryotic enzymes are conserved in M. jannaschii Dfp (Fig. 2) MARCH 3, 2006 • VOLUME 281 • NUMBER 9

4-Phosphopantetheine Biosynthesis in Archaea
PPC decarboxylase functionally aligns with this active site cysteine residue. A second residue that is important for the decarboxylase activity is also not conserved in M. jannaschii Dfp. In peptidyl-cysteine decar-boxylases (LanD enzymes) and most eubacterial and eukaryotic PPC decarboxylases an Asn residue is conserved in the so-called PXM-NXXMW motif and proposed to be involved in binding of the COOH  Treatment of M. jannaschii His-CoaB N217H with trifluoroacetic acid and RPC separation of the copurified substances revealed that also in this case 4-phosphopantothenoyl-CMP is bound (Fig. 3A), indicating that the archaebacterial PPC synthetase is CTP-and not ATP-dependent. The ATP dependence of the M. jannaschii PPC synthetase was suggested recently because the nucleotide binding motif II contains a Lys residue (Lys 310 ; Fig. 2) that is conserved in eukaryotic ATP-dependent enzymes but not in eubacterial CTP-dependent ones (5).

4-Phosphopantetheine Biosynthesis in Archaea
Purified M. jannschii His-CoaC protein was green, and UV-visible spectroscopy showed absorbance maxima characteristic for flavoproteins and, additionally, a stable long-wavelength absorption with a maximum of about 706 nm (Fig. 3B), indicating the formation of a chargetransfer complex between wild-type enzyme and either the substrate PPC or the reaction product PP or with the oxidatively decarboxylated enethiolate intermediate (9). The flavin cofactor was identified by RPC as flavin mononucleotide (data not shown). Exchanging of the conserved His 87 residue to Ala led to reduced flavin binding capacity (data not shown), indicating that in this case greater structural changes were introduced. His-CoaC H87N was also purified as a green enzyme; how-

. Purification and characterization of His-CoaB and His-CoaC proteins.
A, substances copurified with His-CoaB wt (left) and His-CoaB N217H (middle), respectively, were separated by RPC. The elution from the column was followed by absorbance at 280 nm (thin line), 260 nm (thick line), and 214 nm (not shown). The higher absorbance at 280 nm compared with that at 260 nm clearly shows that 4Ј-phosphopantothenoyl-CMP (structure shown on the right) and not 4Ј-phosphopantothenoyl-AMP is bound to His-CoaB N217H and then released under denaturating acidic conditions. B, UV-visible spectra of His-CoaC wt (thick line) and His-CoaC H87N (thin line) were recorded in elution buffer ϳ20 h after purification of the proteins. C, UV-visible spectra of His-CoaC N141D (thick line), His-CoaC H139D (thin line), and His-CoaC wt (not shown; compare with panel B) were recorded in elution buffer ϳ4 h after purification of the proteins. D, Ni-NTA-purified His-CoaC wt protein was separated on a Superdex 200 PC 3.2/30 gel filtration column. To obtain molecular weight information, the elution volumes of eight standard proteins (thin lines; peaks 1-8, see "Experimental Procedures") were determined. Purified His-CoaC protein (thick line) eluted at 1.239 ml corresponding to a molecular mass of about 242 kDa. The elution of the proteins was followed by absorbance at 280 nm (and at 450 nm; not shown). MARCH 3, 2006 • VOLUME 281 • NUMBER 9 ever, the long-wavelength absorption band is not stable and is no longer visible after a few hours (Fig. 3B). The absorbance maximum of the flavin cofactor is at 448 nm for the wt enzyme and at 453 nm for the mutant enzyme H87N. From the published crystal structures of PPC decarboxylases and LanD enzymes (8,10,18,19), we know that the conserved His residue of HFCD (homo-oligomeric flavin-dependent Cys decarboxylases) enzymes is in direct neighborhood of the flavin cofactor. Therefore, it is not surprising that exchanging His 87 led to changes in the UV-visible spectrum of the bound cofactor. Because His 87 is part of the active site and proposed to be involved in substrate binding, it is also not surprising that the charge-transfer band is not as stable as in the wt enzyme.

4-Phosphopantetheine Biosynthesis in Archaea
Enzymes known to catalyze the decarboxylation of cysteine residues are trimeric (for example the PPC decarboxylase AtHAL3a (8, 18)) or dodecameric enzymes (built up from four trimers; for example the LanD enzymes EpiD and MrsD and the eubacterial Dfp proteins (10,12,13,19)) and are named HFCD proteins (10). M. jannaschii His-CoaC eluted at an apparent molecular mass of ϳ242 kDa from the gel filtration column (Fig. 3D). The calculated molecular mass of His-CoaC-FMN is 24.06 kDa. Although the observed molecular mass deviates by 46.8 kDa from the theoretical value for a homododecamer, we assume (taking the inaccuracy of molecular weight determinations by gel filtration into account) that His-CoaC builds up homododecamers. In conclusion, the M. jannaschii Dfp protein is a also a homododecameric enzyme, with the CoaC domain forming a dodecameric core structure as has recently been modeled for the E. coli enzyme (20).
Identification and Characterization of the PPC Synthetase Activity of M. jannaschii-To analyze the biochemistry of the 4Ј-phosphopantetheine synthesizing activity of M. jannaschii Dfp, it is useful to separate PPC synthetase and PPC decarboxylase activities by separating the

4-Phosphopantetheine Biosynthesis in Archaea
bifunctional Dfp protein into CoaB and CoaC. Using the published high pressure liquid chromatography method for the detection of PPC (13), we were able to show that His-CoaB synthesizes PPC from 4Ј-phosphopantothenate and cysteine in the presence of CTP (Fig. 4); 4Ј-phosphopantothenate was enzymatically synthesized in situ using E. coli CoaA. We could not observe any synthetase activity if CTP was omitted in the assay, indicating that ATP (which is always present as cofactor of the pantothenate kinase activity CoaA) is not the cofactor of the Methanocaldococcus enzyme, thus confirming the results described above. The PPC synthetase activity of His-CoaB was further confirmed in a coupled enzyme assay in which synthesized PPC was first decarboxylated to PP and then ligated with AMP to dephospho-CoA (Figs. 5 and 6). The mutant enzyme His-CoaB N217H had only residual PPC synthetase activity, and His-CoaB K310Q had very low activity (Fig. 5). From the crystal structure of E. coli His-CoaB we know that these residues are active site residues (compare also Fig. 2

) (5).
Identification of the PPC Decarboxylase Activity of M. jannaschii-Crystal structure analysis of HFCD proteins and the known site-directed mutagenesis studies showed the importance of the conserved Cys residue within the substrate recognition clamp and of the conserved Asn residue within the PXMNXXMW motif for the decarboxylase activity (6, 8 -10, 12, 19). Both residues are not present in M. jannaschii His-CoaC, and therefore we did not expect PPC decarboxylase activity.
However, when His-CoaC was added to the PPC synthetase assay and the reaction mixture separated by RPC, we observed that synthesized PPC was partially converted to a substance that was identified by its retention time as 4Ј-phosphopantetheine ( Fig. 4) (13). To verify these data in a second assay, synthesized 4Ј-phosphopantetheine was converted to dephospho-CoA by adding a 4Ј-phosphopantetheine adenylyltransferase to the assay (Figs. 5 and 6). These experiments clearly showed that His-CoaC is active in decarboxylating PPC.
Active Site Residues of the PPC Decarboxylase Domain of M. jannaschii Dfp-To characterize the PPC decarboxylase domain in more detail, the activity of several mutant enzymes was investigated. All mutants bind the flavin cofactor (however, for the H87A mutant see above) and showed charge-transfer bands indicating that no major structural changes were introduced by the amino acid exchanges. As expected, exchanging the active site residue His 87 for Asn reduced the activity of M. jannaschii His-CoaC (Fig. 6). However, the PPC decarboxylases from E. coli and A. thaliana were completely inactivated by exchanging the active site His residue for Asn (13,21). Exchanging His 139 for Asn and restoring the PXMNXXMW motif drastically reduced the decarboxylase activity rather than increasing it as had been expected. Exchanging His 139 for Asp led to a significant change in the UV-visible spectrum of the CoaC enzyme and to significant decrease in enzymatic activity (Fig. 7). The absorbance maximum of the charge-

4-Phosphopantetheine Biosynthesis in Archaea
transfer band was shifted by about 100 nm to lower wavelengths (Fig.  3C), indicating a change in the mode of substrate binding. It is possible that to some extent an Asp residue in position 139 is also present in the H139N mutant, derived by deamidation from Asn at higher temperatures (explaining the low activity of the H139N mutant?). Exchange of Asn 141 (which is not conserved in PPC decarboxylases) for Asp also led to a significant shift of the charge-transfer band (Fig. 3C) but no decrease in activity was observed (Fig. 7). Surprisingly, exchanging His 139 for Ala had no significant impact on activity. By accident, a second mutation (E168K) occurred in one of the H139A clones, and this mutant enzyme, His-CoaC H139A/E168K, had drastically reduced PPC decarboxylase activity (Fig. 6); the residue Glu 168 is within the proposed PPC recognition clamp of M. jannaschii His-CoaC. To investigate the role of the residue Glu 168 in more detail, we investigated the PPC decarboxylase activities of the mutant proteins His-CoaC E168A, His-CoaC E168D, and His-CoaC E168K (Fig. 7). We observed only residual activity for the mutant E168K, drastically reduced activity for E168A, and a significantly decreased activity for E168D. Exchange of the neighboring Glu 167 for Ala did not influence the PPC decarboxylase activity. Because the active site cysteine residue conserved in eubacterial and eukaryotic FIGURE 6. The importance of residues His 87 and His 139 for PPC decarboxylase activity. The activities of His-CoaC wt, His-CoaC H87N, His-CoaC H87A, His-CoaC H139A, His-CoaC H139A/E168K, and His-CoaC H139N were determined at 60°C by generating the CoaC substrate 4Ј-phosphopantothenoylcysteine from pantothenate using the enzymes His-CoaA (E. coli) and His-CoaB (M. jannaschii) and converting the CoaC reaction product, 4Ј-phosphopantetheine, to dephospho-CoA using the enzyme MBP-AtCoaD. In control experiments, either His-CoaC or His-CoaB was omitted. The enzymatically synthesized dephospho-CoA was detected by RPC monitoring the absorbance at 214 nm (not shown), 260 nm (not shown), and 280 nm.

4-Phosphopantetheine Biosynthesis in Archaea
PPC decarboxylases is not present in the substrate binding clamp of the M. jannaschii CoaC domain, we analyzed whether one of the four cysteine residues of CoaC is essential for activity. However, the activity of the mutant enzymes His-CoaC C54A, His-CoaC C73A, His-CoaC C94A, and His-CoaC C96A was comparable with that of the wild-type enzyme (data not shown). At the moment it is not clear whether Glu 168 of the substrate binding clamp takes over the role of the Cys residue as active site acid (in this case an increased apparent pK a value has to be assumed for Glu 168 ).
Conclusions-The biosynthetic pathway of 4Ј-phosphopantetheine from 4Ј-phosphopantothenate has now been elucidated in eubacteria, Archaea, and eukaryotes. The active site architecture of the archaebac-terial CoaC domain differs significantly from that of eubacterial and eukaryotic enzymes. The conserved Asn residue of the PXMNXXMW motif is exchanged for His, and the active site Cys residue of the substrate recognition clamp is not present. Crystal structural analysis of M. jannaschii CoaC will be useful to elucidate the reaction mechanism of archaebacterial PPC decarboxylases.