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Originally published In Press as doi:10.1074/jbc.C500044200 on March 28, 2005

J. Biol. Chem., Vol. 280, Issue 21, 20185-20188, May 27, 2005
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Characterization of a New Pantothenate Kinase Isoform from Helicobacter pylori*{boxs}

Leisl A. Brand and Erick Strauss{ddagger}

From the Department of Chemistry, Stellenbosch University, Matieland 7602, South Africa

Received for publication, January 31, 2005 , and in revised form, March 9, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Pantothenate kinase (PanK) catalyzes the first step in the biosynthesis of the essential and ubiquitous cofactor coenzyme A (CoA) in all organisms. Two well characterized isoforms of the enzyme are known: a prokaryotic PanK that predominates in eubacteria and a eukaryotic isoform that has primarily been characterized from mammalian and plant sources. Curiously, the genomes of certain pathogenic bacteria, including Helicobacter pylori and Pseudomonas aeruginosa, do not contain a PanK similar to either isoform, although these organisms possess all the other biosynthetic machinery required for CoA production. In this study we cloned, overexpressed and characterized an enzyme from Bacillus subtilis and its homologue from H. pylori and show that they catalyze the ATP-dependent phosphorylation of pantothenate. These enzymes do not share sequence homology with any known PanK, and unlike the bacterial and eukaryotic PanK isoforms their activity is not regulated by either CoA or acetyl-CoA. They also do not accept the pantothenic acid antimetabolite N-pentylpantothenamide as a substrate or are inhibited by it. Taken together, these results point to the identification of a third distinct isoform of PanK that accounts for the only known activity of the enzyme in pathogens such as H. pylori and P. aeruginosa.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Coenzyme A (CoA)1 is an essential and ubiquitous cofactor in all biological systems, where it acts as the major acyl group carrier in various central metabolic reactions (1, 2). It is universally biosynthesized in five enzymatic steps from pantothenate (vitamin B5). The first of these, the ATP-dependent phosphorylation of pantothenate, is catalyzed by the enzyme pantothenate kinase (PanK). Currently two isoforms of the enzyme have been characterized: the first (Type I) is found predominantly in prokaryotic organisms and is exemplified by the Escherichia coli enzyme (EcCoaA) (3). The second (Type II) occurs mainly in eukaryotic systems, of which the murine enzyme (MmPanK1{beta}) has been the best characterized (4, 5). While these two isoforms have little primary sequence similarity, both share a common regulation mechanism based on feedback inhibition by CoA and its thioesters, although the degree of inhibition is system- and inhibitor-dependent (4–11). However, recent studies have found that this classification is not unambiguous, as the Staphylococcus aureus enzyme (SaCoaA) has a primary sequence that is closer related to Type II PanKs, and it is not regulated by feedback inhibition (12, 13).

Despite our extensive current knowledge of PanK enzymes the identification of this activity remains elusive in a subset of pathogenic bacteria that includes organisms such as Helicobacter pylori and Pseudomonas aeruginosa. This fact was highlighted in two recent studies which used a comparative genomics approach to reconstruct the universal CoA biosynthetic pathway in representative organisms of all kingdoms (14, 15). Among these, H. pylori and P. aeruginosa are examples of bacteria in which no putative PanK similar to either known isoform could be found, even though the four remaining CoA biosynthetic enzymes were clearly represented. Since PanK is an essential activity in these organisms, this suggested that at least one additional, uncharacterized isoform of PanK exists.

A recent patent application has claimed that the Bacillus subtilis genome contains two separate gene sequences which, when cloned in trans, can each suppress the effects of an Escherichia coli temperature-sensitive mutant defective in Ec-CoaA activity (16). While one of these genes encodes the predicted model prokaryotic Type I PanK (BsCoaA), the other shows no homology to any known PanK. In support of the suggested duplication of PanK activity interruption of the putative coaA gene gave a normal growing phenotype, while a double mutant defective in both gene sequences was not viable. These results suggest that simultaneous deletion of both genes is lethal to B. subtilis and that the second gene (dubbed coaX by the authors) also encodes for a protein with PanK activity.

Similarity-based genome searches using the coaX gene sequence identify homologues in a number of eubacteria (see Supplemental Table I). Included among these are various pathogenic bacteria such as H. pylori and P. aeruginosa in which the PanK-encoding gene was "missing" (15), as well as others like Bordetella pertussis (the causative agent of whooping cough) and the category A biodefense pathogen, Francisella tularensis. Interestingly, the coaX homologue in B. pertussis has been studied before and was found to be an essential gene in this organism (17, 18). However, these studies concluded that the gene product was involved in pertussis toxin production via interaction with a two component transcriptional regulator, BvgAS. Most coaX homologues are currently annotated as Bvg accessory factors or as putative transcriptional regulators.

In this paper we report the cloning, overexpression, and characterization of the coaX gene product from B. subtilis and its homologue from H. pylori and demonstrate that these proteins have PanK activity. However, our results show that in comparison to the Type I and Type II PanKs these enzymes exhibit distinctly different characteristics, suggesting that they are the first characterized examples of a third (Type III) PanK isoform.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
General—All chemicals were purchased from Aldrich, Sigma, or Fluka and were of the highest purity. ESI-MS analyses were performed at the Central Analytical Facility at Stellenbosch University on a Waters Micromass Q-TOF Ultima API mass spectrometer. SaCoaA was expressed and purified according to published methods, using BL21(DE3) (Novagen) as expression strain (13). The SaCoaA expression plasmid was a gift from Cynthia Kinsland (Cornell University). EcCoaA was prepared by published methods (19). All curve fitting analyses were performed using SigmaPlot 9.0 (Systat software).

Construction of Expression Vectors—The B. subtilis coaX gene (yacB)2 was amplified by PCR from B. subtilis 168 genomic DNA using Pfu DNA polymerase and the following primers (Inqaba Biotechnology): 5'-CAAAAGTGGTGACATATGTTGTTACTGGTTATC-3' (forward primer) and 5'-CCATATCAGTCGTTCTCGAGGCATAAGCCCGAAC-3' (reverse primer). The H. pylori coaX (HP0682) gene was amplified by PCR from H. pylori genomic DNA (a gift from Paul van Helden and Rob Warren, Stellenbosch University) using Taq DNA polymerase and the following primers: 5'-ATAAGAAGTAGGCATATGCCAGCTAGGC-3' (forward primer) and 5'-ATGCCCAAAAAACTCGAGTTGTGCATC-3' (reverse primer). Primers introduced either NdeI (forward primers) or XhoI (reverse primers) restriction sites (underlined). The resulting PCR products were digested with NdeI and XhoI and ligated to NdeI/XhoI-digested pET28a expression vector (Novagen) using T4 DNA ligase. The sequences of the resulting plasmids, named pET28a-BsCoaX and pET-28a-HpCoaX respectively, were verified by automated DNA sequencing (Inqaba Biotechnology).

Expression and Purification of Recombinant Proteins—pET28a-BsCoaX and pET28a-HpCoaX were transformed into E. coli BL21 Star(DE3) (Invitrogen). Protein expression was performed in LB media supplemented with 30 µg/ml kanamycin sulfate at 37 °C. Expression was induced by the addition of isopropyl 1-thio-{beta}-D-galactopyranoside (800 µM for BsCoaX and 100 µM for HpCoaX) at an A600 of ~0.6, and the cells were grown overnight. Harvested cells were suspended in sonication buffer (5 mM imidazole, 0.5 M NaCl, and 20 mM Tris-HCl, pH 7.9), sonicated, and centrifuged to clarify the cell-free extract. The proteins were purified using 1-ml HiTrap chelating columns (Amersham Biosciences) following the protocol provided by the suppliers and using sonication buffer as column solvent, except that BsCoaX was eluted from the column by using strip buffer (100 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl, pH 7.9) instead of imidazole. The purified protein solutions were exchanged to gel filtration buffer (5 mM MgCl2, 25 mM Tris, pH 8.0, and 5% glycerol) using HiTrap desalting columns (Amersham Biosciences) pre-equilibrated in the same buffer. Aliquots of the purified proteins were stored at –80 °C.

Determination of Kinetic Parameters—PanK activity was measured by a coupled assay as described previously (19). All reactions were performed at 25 °C in a Multiskan Spectrum multiplate spectrophotometer (Thermo Labsystems), using an extinction coefficient of 6220 M–1·cm–1 for NADH. Kinetic parameters were determined by global non-linear fitting of the initial rate data at varying ATP and pantothenate concentrations to the general equation for a steady-state bireactant model (20),

(Eq. 1)
where A is ATP, B is pantothenate, E is enzyme, KA and KB are the Michaelis constants for ATP and pantothenate, respectively, and K1A is the enzyme dissociation constant.

Each 300 µl reaction mixture contained 100 mM HEPES, pH 7.6, 20 mM KCl, 10 mM MgCl2, 2 mM PEP, 0.3 mM NADH, 5 units of lactate dehydrogenase, 2.5 units of pyruvate kinase, and 4.5 µg of PanK. ATP concentrations were varied between 0.5 and 15 mM and pantothenate concentrations between 5 and 500 µM. Reactions were initiated by the addition of pantothenate.

Inhibition Studies—Reaction mixtures for inhibition studies were identical to those described above except that the pantothenate (500 µM) and ATP (5.0 mM for BsCoaX, 10.0 mM for HpCoaX, and 1.5 mM for EcCoaA) concentrations were kept constant. Inhibitors (CoA, acetyl-CoA, or N-pentylpantothenamide (19)) were added at concentrations between 10 and 500 µM, and the reaction was initiated by the addition of pantothenate.

N-Pentylpantothenamide was also tested as a substrate for BsCoaX, HpCoaX, and SaCoaA. These reactions were performed as described above, but pantothenate was substituted with N-pentylpantothenamide and ATP concentrations remained constant at 10.0 mM for HpCoaX and BsCoaX and 1.5 mM for SaCoaA.

Testing of Alternate Phosphoryl Donors—To determine whether other phosphate-containing compounds could substitute for ATP in the pantothenate kinase reaction catalyzed by BsCoaX and HpCoaX, reaction mixtures containing 50 mM Tris, pH 7.6, 20 mM KCl, 10 mM MgCl2, 5 µg of enzyme, phosphate donor (1.5 mM concentration of either ATP, UTP, CTP, GTP, phosphoserine or phosphothreonine or 10 mM concentration of either acetylphosphate or carbamoylphosphate) and 500 µM pantothenate were incubated at 37 °C for 2 h. The reactions were placed at 95 °C for 5 min, centrifuged, and the supernatant of each sample applied to a column of Dowex 50WX8–100 resin. Columns were rinsed with deionized water and the solvent evaporated from the combined eluate. The dried samples were resuspended in 100 µl of 50% aqueous acetonitrile and analyzed by ESI-MS by direct infusion into the instrument at a rate of 20 µl/min.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Expression and Purification of Type III PanKs—The putative CoaX proteins from B. subtilis (BsCoaX) and H. pylori (Hp-CoaX) were prepared by independently cloning the respective genes into overexpression vectors coding for N-terminal His6-tagged fusion proteins. The vectors were transformed into E. coli BL21 Star(DE3) and the proteins overexpressed and purified using immobilized metal affinity chromatography.

Kinetic Characterization of Type III PanKs—The purified proteins were assayed for their ability to catalyze the ATP-dependent phosphorylation of pantothenate using a two enzyme-coupled assay that links the production of ADP to the oxidation of NADH. This allows the enzyme activity to be measured continuously by monitoring the change in A340. The assay was performed by varying the pantothenate concentration at a number of set ATP concentrations and determining the initial rates of reaction for each protein (Fig. 1). The results clearly show that both proteins have PanK activity.



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  		FIG. 1.
Initial rate plots of HpCoaX and BsCoaX. A, initial rates of HpCoaX against pantothenate concentration at an ATP concentration of 1.0 mM (•), 2.0 mM ({circ}), 3.0 mM ({blacksquare}), 5.0 mM ({square}), 10.0 mM ({blacktriangleup}), and 15.0 mM ({triangleup}). B, initial rates of BsCoaX against pantothenate concentration at an ATP concentration of 0.5 mM (•), 1.0 mM ({circ}), 2.0 mM ({blacksquare}), 5.0 mM ({square}), and 10.0 mM ({blacktriangleup}). Symbols show the average of three replicates, with error bars indicating the standard deviation. Curves show fits of the individual data sets to the Michaelis-Menten equation.

 
The initial rate data were used to determine the kinetic parameters of the enzymes by performing global non-linear fits using the most general form of the equation describing a bireactant mechanism (20). The results are summarized in Table I, which also shows the kinetic parameters of the model prokaryotic Type I PanK EcCoaA (8), the eukaryotic Type II PanK from Aspergillus nidulans (AnPanK) (11) and the recently characterized atypical Type II PanK from S. aureus, SaCoaA (12), for comparison. While the specificity constant for pantothenate varies very little among these enzymes (kcat/Km values not differing by more than a factor of 3), the CoaX enzymes exhibit a very low specificity for ATP. This is due to their surprisingly high Km values, which is nearly 10 mM in the case of HpCoaX. Such large Km values can be rationalized in the case of the BsCoaX enzyme that duplicates an activity already embodied by the more typical BsCoaA. However, since H. pylori depends exclusively on HpCoaX for its CoA biosynthetic needs, the question arises whether the low specificity constant for ATP of the enzyme determines the flux through the pathway. Further studies are currently under way to address this question.


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  		TABLE I
Kinetic parameters of the three characterized pantothenate kinase isoforms

 
Testing the Efficacy of Alternate Phosphoryl Donors—To test whether the low specificity constant exhibited by the CoaX proteins for ATP was due to their preference for other phosphoryl donors, the enzymes were incubated with pantothenate and a range of activated and non-activated phosphate-containing substrates. These included GTP, CTP, UTP, PEP, and acetyl- and carbamoylphosphate. Phosphoserine and phosphothreonine were also tested, since they are known substrates of some phosphotransferase enzymes (21, 22). The reaction mixture with PEP was subsequently analyzed by the two enzyme-coupled assay, while the rest of the reaction mixtures were analyzed by ESI-MS for the presence of 4'-phosphopantothenate. The results were compared with mixtures containing ATP as well as to control reactions with EcCoaA. Only CTP and GTP could substitute for ATP in the case of HpCoaX, albeit to a lesser extent. No other phosphoryl donor was active in the reactions catalyzed by BsCoaX (data not shown). This strongly points to ATP as the cognate phosphoryl donor in the PanK reaction catalyzed by CoaX enzymes.

Inhibition of Type III PanKs by CoA and Acetyl-CoA—With the exception of the recently characterized SaCoaA enzyme (12), all other known PanKs are inhibited by CoA or its thioesters, although the extent of inhibition is system-dependent (4–11). In all cases the inhibition serves to regulate the intracellular CoA concentration by determining the flux through the pathway. To determine whether a similar effect can be observed with the CoaX enzymes reactions containing increasing amounts of CoA or acetyl-CoA were assayed for PanK activity (Fig. 2). The experiment was also performed with EcCoaA, which has a well described inhibition profile (9), for comparison. Our results show that while EcCoaA demonstrates inhibition by CoA and to a lesser extent by acetyl-CoA as expected, neither of the CoaX enzymes are affected. This result is surprising, since the inhibition of PanK by CoA and its thioesters was until recently considered to constitute a common mechanism for the regulation of intracellular CoA levels in all organisms (12). The fact that the SaCoaA enzyme is not subject to such regulation has been rationalized in terms of the unique physiology of this organism that depends on CoA and a NADPH-dependent CoA reductase to maintain the intracellular redox balance (23, 24).



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  		FIG. 2.
Inhibition of EcCoaA, HpCoaX and BsCoaX by CoA and acetyl-CoA. Inhibition of the pantothenate kinase activity of EcCoaA (•, {circ}), BsCoaX ({blacksquare}, {square}), and HpCoaX ({blacktriangleup}, {triangleup}) in the presence of CoA (closed symbols) and acetyl-CoA (open symbols). Symbols show the average of three replicates, with error bars indicating the standard deviation.

 
Effect of Pantothenamide Antimetabolites on Type III PanKs—Recent studies on the EcCoaA and SaCoaA enzymes have shown that N-alkylpantothenamides like N-pentyl- and N-heptylpantothenamide act as antimicrobial agents through their action as CoA antimetabolites (12, 13, 19). These compounds were found to act as substrates of the CoA biosynthetic enzymes in both E. coli and S. aureus, where they are converted to inactive CoA analogues which inhibit a number of cellular targets, especially fatty acid biosynthesis (12, 19, 25). To test whether this was also the case with CoaX enzymes, N-pentylpantothenamide was substituted for pantothenate in reaction mixtures and the reactions assayed by the two enzyme-coupled system. No activity could be detected using concentrations of up to 0.5 mM (data not shown). To test whether the compound was an inhibitor of the enzyme, the activity of CoaX enzymes was assayed in the presence of increasing concentrations of N-pentylpantothenamide. No effect was observed (data not shown). It has been suggested (12) that the two enzyme-coupled system is not as sensitive as the radioactive-based assay employed in other studies as it reportedly failed to detect that N-pentylpantothenamide was a substrate for SaCoaA (13). However, in our hands we clearly detect activity when assaying SaCoaA in the presence of N-pentylpantothenamide by this method, suggesting that sensitivity is not a factor.

Gene Cluster Analysis in Support of Functional Characterization—The conservation of gene clusters across genomes can often be applied to infer gene function (26). To apply this technique to CoaX enzymes the genes coding for these proteins were aligned across a set of genomes and the respective gene clusters analyzed for the presence of other CoA and pantothenate biosynthetic genes (Fig. 3). The results show that in Mycobacterium spp., Streptomyces coelicolor, Moorella thermoacetica, and F. tularensis genes encoding CoaX proteins are found associated with two genes involved in pantothenate biosynthesis (combinations of pantothenate synthetase, aspartate 1-decarboxylase, or ketopantoate hydroxymethyltransferase), while in Bdellovibrio the cluster contains these three pantothenate biosynthetic genes as well as the putative phosphopantothenoylcysteine synthetase/decarboxylase-encoding gene. Such clustering provides strong genetic support for the direct involvement of CoaX proteins in CoA biosynthesis.



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  		FIG. 3.
Cluster analysis of the predicted coaX gene in selected organisms. Selected chromosomal contigs containing a gene predicted to encode a Type III pantothenate kinase (labeled coaX) were aligned to visualize gene clustering on the chromosome. Other genes involved in pantothenate and CoA biosynthesis are panB (ketopantoate hydroxymethyltransferase), panC (pantothenate synthetase), panD (aspartate 1-decarboxylase), and coaBC (phosphopantothenoylcysteine synthetase/decarboxylase). Other unrelated genes are also labeled using standard nomenclature for sequences with known or predicted functions or using letters A, B, and C for unknown hypothetical proteins that share high sequence similarity. The alignment was prepared from data modified from the SEED tool (TheSEED.uchicago.edu/FIG/index.cgi).

 
Multiple Sequence Alignment and Fold Prediction—An alignment of the sequences of CoaX enzymes from a diverse set of bacterial sources show that they share a set of conserved aspartate and glycine residues (see supplemental Fig. 1). These residues form part of distinct sequence motifs similar to those used in the classification of small molecule kinases, specifically those found in the ribonuclease H-like family of kinases (27). Recent studies have found that Type II PanKs are also members of this kinase family, and preliminary data suggest that CoaX proteins are indeed members of the same group.3 This indicates that Type II and Type III PanKs share the same structural fold. However, Type I PanKs are structurally distinct and belong to the family of P-loop kinases.

Conclusion—In this study we have cloned, overexpressed, and characterized two homologous proteins from B. subtilis and H. pylori and demonstrated that they have PanK activity. However, these proteins do not share sequence similarity with the other two known PanK isoforms. They also exhibit unique kinetic characteristics, showing low specificity constants for ATP and no regulation by CoA and its thioesters. These properties all distinguish them from the other PanK isoforms and strongly suggest that they are the first characterized examples of a third isoform of the PanK enzyme. Such Type III PanKs represent the enzyme in a variety of organisms in which no candidate PanK-encoding genes could be identified to date. It is still unclear what advantage, if any, these enzymes confer upon the mainly pathogenic organisms that harbor them.


   FOOTNOTES
 
* This work was supported by grants from the National Research Foundation of South Africa (GUN 2054218) and from Stellenbosch University. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{boxs} The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1 and supplemental Table I.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AY912104 [GenBank] . Back

{ddagger} To whom correspondence should be addressed: Dept. of Chemistry, Private Bag X1, Stellenbosch University, Matieland 7602, South Africa. Tel.: 27-21-808-3355; Fax: 27-21-808-3360; E-mail: estrauss{at}sun.ac.za.

1 The abbreviations used are: CoA, coenzyme A; PanK or CoaA, pantothenate kinase; EcCoaA, Escherichia coli pantothenate kinase; MmPanK1{beta}, murine pantothenate kinase; SaCoaA, Staphylococcus aureus pantothenate kinase; BsCoaA, Bacillus subtilis Type I pantothenate kinase; BsCoaX, Bacillus subtilis Type III pantothenate kinase; HpCoaX, Helicobacter pylori Type III pantothenate kinase; PEP, phosphoenolpyruvate; CoaX, Type III pantothenate kinase; AnPanK, Aspergillus nidulans pantothenate kinase; ESI-MS, electrospray ionization-mass spectrometry; contig, group of overlapping clones. Back

2 The sequence of the B. subtilis yacB gene currently deposited in the various genome databases contains a sequencing error which results in the premature termination of the protein. A corrected sequence has been submitted to GenBankTM (accession number AY912104 [GenBank] ). Back

3 K. Ginalski and N. V. Grishin, personal communication. Back


   ACKNOWLEDGMENTS
 
We extend our gratitude to Andrei Osterman for many helpful suggestions during the preparation of this manuscript, to Paul van Helden and Rob Warren (Department of Medical Biochemistry, Stellenbosch University) for a kind gift of H. pylori genomic DNA, and to Cynthia Kinsland (Department of Chemistry and Chemical Biology, Cornell University) for a kind gift of the SaCoaA expression vector. B. subtilis genomic DNA was obtained from ATCC free of charge by means of a grant from the Ellison Medical Foundation.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
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Feedback Inhibition of Pantothenate Kinase Regulates Pantothenol Uptake by the Malaria Parasite
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K. Yang, Y. Eyobo, L. A. Brand, D. Martynowski, D. Tomchick, E. Strauss, and H. Zhang
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Y.-M. Zhang, S. W. White, and C. O. Rock
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