Tài liệu Báo cáo khóa học: Active-site residues and amino acid specificity of the bacterial 4¢-phosphopantothenoylcysteine synthetase CoaB pptx

Active-site residues and amino acid specificity of the bacterial 4¢-phosphopantothenoylcysteine synthetase CoaB Thomas Kupke Lehrstuhl fu ¨ r Mikrobielle Genetik, Universita ¨ tTu ¨ 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 PPC to 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 PPC as an initial step in the conversion of PPC to PP had been proposed by Kupke et al. in 2000 [6] and was later confirmed for the plant PPC decarboxylase 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 PPC and the 4¢-phosphopantothenoyl-CMP intermediate. Here, the molecular characterization of the bacterial PPC Correspondence to T. Kupke, Lehrstuhl fu ¨ r Mikrobielle Genetik, Universita ¨ tTu ¨ 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: His-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) Eur. J. Biochem. 271, 163–172 (2004) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03916.x synthetase 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-707 and E. coli dfp-1 that displayed temperature- sensitive auxotrophy either for D -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 PPC occurs 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, PPC is 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. 164 T. Kupke (Eur. J. Biochem. 271) Ó FEBS 2003 Materials 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 the dfp gene from the E. 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 1gÆL )1 K 2 HPO 4 , pH 7.3], at 30 °C and 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, (forward) 5¢-GCCAGTTTT GAATTCTGGAAAGCGC CTCG-3¢ and (reverse) 5¢-CGGGTCCA AGATCTTAA 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 )in0.5Lof B-broth in 2 L shaker flasks. At A 578 ¼ 0.4, the cells were induced with 1 m M isopropyl thio-b- D -galactoside (IPTG), and harvested 2 h after induction. 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 IPTG-induced E. coli M15 (pREP4, pQE8 coaA/coaB) cells were harvested and disrupted by sonica- tion in 10 mL 20 m M 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-nitrilotriacetic acid spin columns (Qiagen) equilibrated with column buffer [20 m M Tris/HCl (pH 8.0), 10 m M imidazole, 300 m 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 m M instead of 10 m M 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 m M Tris/HCl (pH 8.0)]. Five milliliters of the cleared lysate obtained by two centrifugation steps (each 25 min at 30 000 g at 4 °C) 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 0.1 M NaCl. Dfp proteins were eluted with column buffer containing 0.25 M 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 PC 3.2/30 gel filtration column equilibrated in running buffer [20 m M Tris/HCl (pH 8.0), 200 m M NaCl] at a flow rate of 40 lLÆmin )1 .Theelutionwas 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 PC 3.2/30 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 m MD -panto- thenate, 2.5 m M MgCl 2 ,5m M ATP, 5 m M CTP, 5 m M cysteine hydrochloride, 5 m M dithhiothreitol, 100 m M 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 37 °C, the reaction mixtures were kept at )80 °C and then were separated successively by reverse-phase chro- matography with a lRPC C 2 /C 18 SC 2.1/10 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. Ó FEBS 2003 4¢-phosphopantothenoylcysteine synthetase CoaB (Eur. J. Biochem. 271) 165 Results 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 PPC synthetases (Fig. 3). To get more information on its role in PPC synthesis, 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 PPC synthetase 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-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 CMP (B; 5 m M ) were separated by RPC. The elution from the RPC column 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 SC 2.1/10 column and was found in the flow-through. The 4¢-phosphopantothenoyl- CMP containing fraction was subjected to MALDI-TOF MS analysis (C). 166 T. Kupke (Eur. J. Biochem. 271) Ó FEBS 2003 synthesis 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 PPCaredetectableinpresenceof cysteine (Fig. 5). Interestingly, the residues Arg206, Asn210 and Ala276 are not only conserved in bacterial Dfp/CoaB proteins but also in eukaryotic PPC synthetases [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 PPC synthetase 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 37 °C with 5 m M dithiothreitol and 5 m M L -cysteine(inthepresenceof2.5m M MgCl 2 )andthen analyzed by RPC to 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 PPC synthetase [18]. Ó FEBS 2003 4¢-phosphopantothenoylcysteine synthetase CoaB (Eur. J. Biochem. 271) 167 presence of cysteine methyl ester). Although the structures of PPand4¢-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 2 -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 PC 3.2/30 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 lgofeach 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. 168 T. Kupke (Eur. J. Biochem. 271) Ó FEBS 2003 The 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 PPC decarboxylase (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 the E. 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 30 °C and 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 42 °C, 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 PPC synthetase (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-1 mutant has to be elucidated in more detail, but obviously increasing the temperature from 30 to 42 °C has 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 PPC and 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. Ó FEBS 2003 4¢-phosphopantothenoylcysteine synthetase CoaB (Eur. J. Biochem. 271) 169 Fig. 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.Theelution from the Supderdex 200 PC 3.2/30 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. 170 T. Kupke (Eur. J. Biochem. 271) Ó FEBS 2003 an effect on 4¢-phosphopantetheine and coenzyme A biosynthesis. With the used assay, it is difficult to evaluate the PPC synthetase 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 42 °C significantly increases the detectable amounts of PPC when 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 PPC synthetase 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 PPC synthetase 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 PPC and 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 PPC synthetase. 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 PPC synthetases 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]. 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