Isolation and characterization of temperature-sensitive pantothenate kinase (coaA) mutants of Escherichia coli.

Escherichia coli mutants conditionally defective in the conversion of pantothenate to coenzyme A were isolated and characterized. The gene was designated coaA and localized between argEH and rpoB near min 90 of the chromosome. The coaA15(Ts) mutation caused a temperature-sensitive growth phenotype and temperature-dependent inactivation of pantothenate kinase activity assayed both in vivo and in vitro. At 30 degrees C, coaA15(Ts) extracts contained less than 20% of the wild-type pantothenate kinase activity; the kinase had near normal kinetic constants for the substrates ATP and pantothenate and was inhibited by coenzyme A to the same degree as the wild-type enzyme. These data define the coaA gene as the structural gene for pantothenate kinase.     

Biosynthesis and degradation both contribute to the regulation of coenzyme A content in Escherichia coli.

Escherichia coli mutants [coaA16(Fr); Fr indicates feedback resistance] were isolated which possessed a pantothenate kinase activity that was refractory to feedback inhibition by coenzyme A (CoA). Strains harboring this mutation had CoA levels that were significantly elevated compared with strains containing the wild-type kinase and also overproduced both intra- and extracellular 4'-phosphopantetheine. The origin of 4'-phosphopantetheine was investigated by using strain SJ135 [panD delta(aroP-aceEF)], in which synthesis of acetyl-CoA was dependent on the addition of an acetate growth supplement. Rapid degradation of CoA to 4'-phosphopantetheine was triggered by the conversion of acetyl-CoA to CoA following the removal of acetate from the media. CoA hydrolysis under these conditions appeared not to involve acyl carrier protein prosthetic group turnover since [acyl carrier protein] phosphodiesterase was inhibited equally well by acetyl-CoA or CoA. These data support the view that the total cellular CoA content is controlled by modulation of biosynthesis at the pantothenate kinase step and by degradation of CoA to 4'-phosphopantetheine.     

Analysis of the temperature-sensitive mutation of Escherichia coli pantothenate kinase reveals YbjN as a possible protein stabilizer

Pantothenate kinase (PanK), a key regulatory enzyme in the coenzyme A (CoA) biosynthetic pathway, catalyzes the rate-limiting phosphorylation of pantothenic acid to form phosphopantothenate during CoA biosynthesis. Escherichia coli ts9 strain manifests temperature-sensitive phenotype on LB media due to its mutation in the coaA gene (coaA1). Sequencing analysis revealed that coaA1 arises from a single base pair mutation that results in an amino acid change, L236F. This change, located proximate to the ATP binding site of CoaA, destabilizes both enzymatic activity and structural integrity or stability of the mutant protein in vitro. Spontaneously, revertants of ts9 were occasionally found on LB medium plates. Two groups of revertants were isolated: for those that can grow at 40 degrees C, a reversion of the original amino acid mutation L236F to L236L or other amino acid (such as L236C) occurs; for those that can grow at 37 degrees C but not 40 degrees C, a mutation at another gene or intergenic suppression is strongly indicated. Towards genetic identification of genes that might interact with coaA1, ybjN, which encodes a putative sensory transduction regulator protein, and whose over-expression is capable of ameliorating the temperature-sensitive phenotype of the structurally unstable CoaA1 or CoaA[L236F], was isolated. Over-expression of ybjN appears to suppress the temperature-sensitive phenotype of several other temperature-sensitive mutations, including coaA14 (carried by DV51 strain), coaA15 (carried by DV70 strain), and ilu-1, suggesting it not only helps CoaA1, but possibly works as a general stabilizer for some other unstable proteins.     

Role of feedback regulation of pantothenate kinase (CoaA) in control of coenzyme A levels in Escherichia coli

Pantothenate kinase (CoaA) is a key regulator of coenzyme A (CoA) biosynthesis in Escherichia coli, and its activity is controlled by feedback inhibition by CoA and its thioesters. The importance of feedback inhibition in the control of the intracellular CoA levels was tested by constructing three site-directed mutants of CoaA that were predicted to be feedback resistant based on the crystal structure of the CoaA-CoA binary complex. CoaA[R106A], CoaA[H177Q], and CoaA[F247V] were purified and shown to retain significant catalytic activity and be refractory to inhibition by CoA. CoaA[R106A] retained 50% of the catalytic activity of CoaA, whereas the CoaA[H177Q] and CoaA[F247V] mutants were less active. The importance of feedback control of CoaA to the intracellular CoA levels was assessed by expressing either CoaA or CoaA[R106A] in strain ANS3 [coaA15(Ts) panD2]. Cells expressing CoaA[R106A] had significantly higher levels of phosphorylated pantothenate-derived metabolites and CoA in vivo and excreted significantly more 4'-phosphopantetheine into the medium compared to cells expressing the wild-type protein. These data illustrate the key role of feedback regulation of pantothenate kinase in the control of intracellular CoA levels.     

Regulation of pantothenate kinase by coenzyme A and its thioesters

Pantothenate kinase catalyzes the rate-controlling step in the coenzyme A (CoA) biosynthetic pathway, and its activity is modulated by the size of the CoA pool. The effect of nonesterified CoA (CoASH) and CoA thioesters on the activity of pantothenate kinase was examined to determine which component of the CoA pool is the most effective regulator of the enzyme from Escherichia coli. CoASH was five times more potent than acetyl-CoA or other CoA thioesters as an inhibitor of pantothenate kinase activity in vitro. Inhibition by CoA thioesters was not due to their hydrolysis to CoASH. CoASH inhibition was competitive with respect to ATP, thus providing a mechanism to coordinate CoA production with the energy state of the cell. There were considerable differences in the size and composition of the CoA pool in cells grown on different carbon sources, and a carbon source shift experiment was used to test the inhibitory effect of the different CoA species in vivo. A shift from glucose to acetate as the carbon source resulted in an increase in the CoASH:acetyl-CoA ratio from 0.7 to 4.3. The alteration in the CoA pool composition was associated with the selective inhibition of pantothenate phosphorylation, consistent with CoASH being a more potent regulator of pantothenate kinase activity in vivo. These results demonstrate that CoA biosynthesis is regulated through feedback inhibition of pantothenate kinase primarily by the concentration of CoASH and secondarily by the size of the CoA thioester pool.     

Isolation of temperature-sensitive pantothenate kinase mutants of Salmonella typhimurium and mapping of the coaA gene.

Temperature-sensitive pantothenate kinase mutants of Salmonella typhimurium LT2 were selected by using the excretion of pantothenate at the nonpermissive temperature as a screening method. Thermolability of the pathothenate kinase activity in extracts of the mutants was demonstrated. The mutations were mapped at min 89 of the Salmonella chromosome, near rpoB, by transduction. As pantothenate kinase catalyzes the first step in the biosynthesis of coenzyme A from pantothenate, the new genetic locus has been designated coaA.     

A pantothenate kinase from Staphylococcus aureus refractory to feedback regulation by coenzyme A

The key regulatory step in CoA biosynthesis in bacteria and mammals is pantothenate kinase (CoaA), which governs the intracellular concentration of CoA through feedback regulation by CoA and its thioesters. CoaA from Staphylococcus aureus (SaCoaA) has a distinct primary sequence that is more similar to the mammalian pantothenate kinases than the prototypical bacterial CoaA of Escherichia coli. In contrast to all known pantothenate kinases, SaCoaA activity is not feedback-regulated by CoA or CoA thioesters. Metabolic labeling of S. aureus confirms that CoA levels are not controlled by CoaA or at steps downstream from CoaA. The pantothenic acid antimetabolite N-heptylpantothenamide (N7-Pan) possesses potent antimicrobial activity against S. aureus and has multiple cellular targets. N7-Pan is a substrate for SaCoaA and is converted to the inactive butyldethia-CoA analog by the downstream pathway enzymes. The analog is also incorporated into acyl carrier protein and D-alanyl carrier protein, the prosthetic groups of which are derived from CoA. The inactivation of acyl carrier protein and the cessation of fatty acid synthesis are the most critical causes of growth inhibition by N7-Pan because the toxicity of the drug is ameliorated by supplementing the growth medium with fatty acids. The absence of feedback regulation at the pantothenate kinase step allows the accumulation of high concentrations of intracellular CoA, consistent with the physiology of S. aureus, which lacks glutathione and relies on the CoA/CoA disulfide reductase redox system for protection from oxidative damage.     

Pantethine rescues phosphopantothenoylcysteine synthetase and phosphopantothenoylcysteine decarboxylase deficiency in Escherichia coli but not in Pseudomonas aeruginosa

Coenzyme A (CoA) plays a central and essential role in all living organisms. The pathway leading to CoA biosynthesis has been considered an attractive target for developing new antimicrobial agents with novel mechanisms of action. By using an arabinose-regulated expression system, the essentiality of coaBC, a single gene encoding a bifunctional protein catalyzing two consecutive steps in the CoA pathway converting 4'-phosphopantothenate to 4'-phosphopantetheine, was confirmed in Escherichia coli. Utilizing this regulated coaBC strain, it was further demonstrated that E. coli can effectively metabolize pantethine to bypass the requirement for coaBC. Interestingly, pantethine cannot be used by Pseudomonas aeruginosa to obviate coaBC. Through reciprocal complementation studies in combination with biochemical characterization, it was demonstrated that the differential characteristics of pantethine utilization in these two microorganisms are due to the different substrate specificities associated with endogenous pantothenate kinase, the first enzyme in the CoA biosynthetic pathway encoded by coaA in E. coli and coaX in P. aeruginosa.     

Pantothenate kinase regulation of the intracellular concentration of coenzyme A

Pantothenate kinase (PanK) is the key regulatory enzyme in the CoA biosynthetic pathway in bacteria and is thought to play a similar role in mammalian cells. We examined this hypothesis by identifying and characterizing two murine cDNAs that encoded PanK. The two cDNAs were predicted to arise from alternate splicing of the same gene to yield different mRNAs that encode two isoforms (mPanK1alpha and mPanK1beta) with distinct amino termini. The predicted protein sequence of mPanK1 was not related to bacterial PanK but exhibited significant similarity to Aspergillus nidulans PanK. mPanK1alpha was most highly expressed in heart and kidney, whereas mPanK1beta mRNA was detected primarily in liver and kidney. Pantothenate was the most abundant pathway component (42.8%) in normal cells providing clear evidence that pantothenate phosphorylation was a rate-controlling step in CoA biosynthesis. Enhanced mPanK1beta expression eliminated the intracellular pantothenate pool and triggered a 13-fold increase in intracellular CoA content. mPanK1beta activity in vitro was stimulated by CoA and strongly inhibited by acetyl-CoA illustrating that differential modulation of mPanK1beta activity by pathway end products also contributed to the management of CoA levels. These data support the concept that the expression and/or activity of PanK is a determining factor in the physiological regulation of the intracellular CoA concentration.     

Regulation of coenzyme A biosynthesis.

Coenzyme A (CoA) and acyl carrier protein are two cofactors in fatty acid metabolism, and both possess a 4'-phosphopantetheine moiety that is metabolically derived from the vitamin pantothenate. We studied the regulation of the metabolic pathway that gives rise to these two cofactors in an Escherichia coli beta-alanine auxotroph, strain SJ16. Identification and quantitation of the intracellular and extracellular beta-alanine-derived metabolites from cells grown on increasing beta-alanine concentrations were performed. The intracellular content of acyl carrier protein was relatively insensitive to beta-alanine input, whereas the CoA content increased as a function of external beta-alanine concentration, reaching a maximum at 8 microM beta-alanine. Further increase in the beta-alanine concentration led to the excretion of pantothenate into the medium. Comparing the amount of pantothenate found outside the cell to the level of intracellular metabolites demonstrates that E. coli is capable of producing 15-fold more pantoic acid than is required to maintain the intracellular CoA content. Therefore, the supply of pantoic acid is not a limiting factor in CoA biosynthesis. Wild-type cells also excreted pantothenate into the medium, showing that the beta-alanine supply is also not rate limiting in CoA biogenesis. Taken together, the results point to pantothenate kinase as the primary enzymatic step that regulates the CoA content of E. coli.     

Coenzyme A biosynthesis: reconstruction of the pathway in archaea and an evolutionary scenario based on comparative genomics

Coenzyme A (CoA) holds a central position in cellular metabolism and therefore can be assumed to be an ancient molecule. Starting from the known E. coli and human enzymes required for the biosynthesis of CoA, phylogenetic profiles and chromosomal proximity methods enabled an almost complete reconstruction of archaeal CoA biosynthesis. This includes the identification of strong candidates for archaeal pantothenate synthetase and pantothenate kinase, which are unrelated to the corresponding bacterial or eukaryotic enzymes. According to this reconstruction, the topology of CoA synthesis from common precursors is essentially conserved across the three domains of life. The CoA pathway is conserved to varying degrees in eukaryotic pathogens like Giardia lamblia or Plasmodium falciparum, indicating that these pathogens have individual uptake-mechanisms for different CoA precursors. Phylogenetic analysis and phyletic distribution of the CoA biosynthetic enzymes suggest that the enzymes required for the synthesis of phosphopantothenate were recruited independently in the bacterial and archaeal lineages by convergent evolution, and that eukaryotes inherited the genes for the synthesis of pantothenate (vitamin B5) from bacteria. Homologues to bacterial enzymes involved in pantothenate biosynthesis are present in a subset of archaeal genomes. The phylogenies of these enzymes indicate that they were acquired from bacterial thermophiles through horizontal gene transfer. Monophyly can be inferred for each of the enzymes catalyzing the four ultimate steps of CoA synthesis, the conversion of phosphopantothenate into CoA. The results support the notion that CoA was initially synthesized from a prebiotic precursor, most likely pantothenate or a related compound.     

Metabolism of 4''-phosphopantetheine in Escherichia coli.

Coenzyme A (CoA) and acyl carrier protein (ACP) contain 4'-phosphopantetheine moieties that are metabolically derived from the vitamin pantothenate. The utilization of metabolites in the biosynthetic pathway during growth was investigated by using an Escherichia coli beta-alanine auxotroph to specifically and uniformly label the pathway intermediates. Pantothenate and 4'-phosphopantetheine were the two intermediates detected in the highest concentration, both intracellularly and extracellularly. The specific cellular content of CoA and ACP was not constant during growth of strain SJ16 (panD) on 4 microM beta-[3-3H]alanine, and alterations in the utilization of 4'-phosphopantetheine and pantothenate correlated with the observed fluctuations of the intracellular pool sizes of CoA and ACP. Double-label experiments indicated that extracellular 4'-phosphopantetheine was derived from the degradation of ACP, and the extent that this intermediate was utilized by 4'-phosphopantetheine adenylyltransferase exerted control over the degradative aspect of the pathway. Control over the biosynthetic aspect of the biochemical pathway was exerted at the level of pantothenate utilization by pantothenate kinase. Reduction in the specific cellular content of CoA and ACP by 4'-phosphopantetheine excretion was irreversible since, in contrast to pantothenate, strain SJ16 was unable to assimilate exogenous 4'-phosphopantetheine into CoA or ACP.     

Human pantothenate kinase 4 is a pseudo‐pantothenate kinase

Pantothenate kinase generates 4′‐phosphopantothenate in the first and rate‐determining step of coenzyme A (CoA) biosynthesis. The human genome encodes three well‐characterized and nearly identical pantothenate kinases (PANK1‐3) plus a putative bifunctional protein (PANK4) with a predicted amino‐terminal pantothenate kinase domain fused to a carboxy‐terminal phosphatase domain. Structural and phylogenetic analyses show that all active, characterized PANKs contain the key catalytic residues Glu138 and Arg207 (HsPANK3 numbering). However, all amniote PANK4s, including human PANK4, encode Glu138Val and Arg207Trp substitutions which are predicted to inactivate kinase activity. Biochemical analysis corroborates bioinformatic predictions—human PANK4 lacks pantothenate kinase activity. Introducing Glu138Val and Arg207Trp substitutions to the human PANK3 and plant PANK4 abolished their robust pantothenate kinase activity. Introducing both catalytic residues back into human PANK4 restored kinase activity, but only to a low level. This result suggests that epistatic changes to the rest of the protein already reduced the kinase activity prior to mutation of the catalytic residues in the course of evolution. The PANK4 from frog, an anamniote living relative encoding the catalytically active residues, had only a low level of kinase activity, supporting the view that HsPANK4 had reduced kinase activity prior to the catalytic residue substitutions in amniotes. Together, our data show that human PANK4 is a pseudo‐pantothenate kinase—a catalytically deficient variant of the catalytically active PANK4 found in plants and fungi. The Glu138Val and Arg207Trp substitutions in amniotes (HsPANK3 numbering) completely deactivated the pantothenate kinase activity that had already been reduced by prior epistatic mutations.     

Increasing the acetyl-CoA pool in the presence of overexpressed phosphoenolpyruvate carboxylase or pyruvate carboxylase enhances succinate production in Escherichia coli

An in vivo strategy to apply the activation effect of acetyl-CoA on phosphoenolpyruvate carboxylase (PEPC) and pyruvate carboxylase (PYC) to increase succinate production in Escherichia coli was studied. This approach relies on the increased intracellular acetyl-CoA and CoA levels by overexpressing E. coli pantothenate kinase (PANK). The results showed that coexpression of PANK and PEPC, and PANK and PYC, did improve succinate production compared to the individual expression of PEPC and PYC, respectively. The intracellular acetyl-CoA and CoA levels were also measured, and each showed a significant increase when the PANK was overexpressed. Another effect observed was a decrease in lactate production. The least amount of lactate was produced when PANK and PEPC, and PANK and PYC, were coexpressed. This result showed increased competitiveness of the succinate pathway at the phosphoenolpyruvate and pyruvate nodes for the carbon flux, as a result reducing the carbon flux toward the lactate pathway. The study also demonstrates a feasible method for metabolic engineering to modulate enzyme activity in vivo through specific activators and inhibitors.     

A novel isoform of pantothenate synthetase in the Archaea

The linear biosynthetic pathway leading from alpha-ketoisovalerate to pantothenate (vitamin B5) and on to CoA comprises eight steps in the Bacteria and Eukaryota. Genes for up to six steps of this pathway can be identified by sequence homology in individual archaeal genomes. However, there are no archaeal homologs to known isoforms of pantothenate synthetase (PS) or pantothenate kinase. Using comparative genomics, we previously identified two conserved archaeal protein families as the best candidates for the missing steps. Here we report the characterization of the predicted PS gene from Methanosarcina mazei, which encodes a hypothetical protein (MM2281) with no obvious homologs outside its own family. When expressed in Escherichia coli, MM2281 partially complemented an auxotrophic mutant without PS activity. Purified recombinant MM2281 showed no PS activity on its own, but the enzyme enabled substantial synthesis of [14C]4'-phosphopantothenate from [14C]beta-alanine, pantoate and ATP when coupled with E. coli pantothenate kinase. ADP, but not AMP, was detected as a coproduct of the coupled reaction. MM2281 also transferred the 14C-label from [14C]beta-alanine to pantothenate in the presence of pantoate and ADP, presumably through isotope exchange. No exchange took place when pantoate was removed or ADP replaced with AMP. Our results indicate that MM2281 represents a novel type of PS that forms ADP and is strongly inhibited by its product pantothenate. These properties differ substantially from those of bacterial PS, and may explain why PS genes, in contrast to other pantothenate biosynthetic genes, were not exchanged horizontally between the Bacteria and Archaea.     

Cloning, sequence, and expression of the pantothenate permease (panF) gene of Escherichia coli.

Pantothenate permease, the product of the panF gene, catalyzes the sodium-dependent uptake of extracellular pantothenate. The panF gene was isolated from an Escherichia coli genomic DNA library and subcloned into multicopy plasmids. Increased copy number of the panF+ allele resulted in increased rates of pantothenate uptake and a significant increase in the steady-state intracellular pantothenate concentration. Despite the higher levels of pantothenate, the utilization of pantothenate for coenzyme A formation was not elevated, indicating that pantothenate kinase activity is the dominant regulator of coenzyme A biosynthesis. DNA sequencing of the panF gene revealed the presence of a single open reading frame that encoded a hydrophobic protein with a molecular weight of 51,992. Sequence analysis predicts that pantothenate permease is an integral membrane protein possessing 12 hydrophobic membrane-spanning domains connected by short hydrophilic sequences. The predicted topological profile of pantothenate permease is similar to that of other membrane carriers that catalyze cation-dependent symport.     

Acyl carrier protein is a cellular target for the antibacterial action of the pantothenamide class of pantothenate antimetabolites

Pantothenate is the precursor of the essential cofactor coenzyme A (CoA). Pantothenate kinase (CoaA) catalyzes the first and regulatory step in the CoA biosynthetic pathway. The pantothenate analogs N-pentylpantothenamide and N-heptylpantothenamide possess antibiotic activity against Escherichia coli. Both compounds are substrates for E. coli CoaA and competitively inhibit the phosphorylation of pantothenate. The phosphorylated pantothenamides are further converted to CoA analogs, which were previously predicted to act as inhibitors of CoA-dependent enzymes. Here we show that the mechanism for the toxicity of the pantothenamides is due to the inhibition of fatty acid biosynthesis through the formation and accumulation of the inactive acyl carrier protein (ACP), which was easily observed as a faster migrating protein using conformationally sensitive gel electrophoresis. E. coli treated with the pantothenamides lost the ability to incorporate [1-(14)C]acetate to its membrane lipids, indicative of the inhibition of fatty acid synthesis. Cellular CoA was maintained at the level sufficient for bacterial protein synthesis. Electrospray ionization time-of-flight mass spectrometry confirmed that the inactive ACP was the product of the transfer of the inactive phosphopantothenamide moiety of the CoA analog to apo-ACP, forming the ACP analog that lacks the sulfhydryl group for the attachment of acyl chains for fatty acid synthesis. Inactive ACP accumulated in pantothenamide-treated cells because of the active hydrolysis of regular ACP and the slow turnover of the inactive prosthetic group. Thus, the pantothenamides are pro-antibiotics that inhibit fatty acid synthesis and bacterial growth because of the covalent modification of ACP.