Phosphopantetheine Adenylyltransferase Research Articles

Structure of a novel form of phosphopantetheine adenylyltransferase from Klebsiella pneumoniae at 2.59Å resolution.

Phosphopantetheine adenylyltransferase (EC. 2.7.7.3, PPAT) catalyzes the penultimate step of the multistep reaction in the coenzyme A (CoA) biosynthesis pathway. In this step, an adenylyl group from adenosine triphosphate (ATP) is transferred to 4'-phosphopantetheine (PNS) yielding 3'-dephospho-coenzyme A (dpCoA) and pyrophosphate (PPi). PPAT from strain C3 of Klebsiella pneumoniae (KpPPAT) was cloned, expressed and purified. It was crystallized using 0.1M HEPES buffer and PEG10000 at pH 7.5. The crystals belonged to tetragonal space group P41212 with cell dimensions of a = b = 72.82Å and c = 200.37Å. The structure was determined using the molecular replacement method and refined to values of 0.208 and 0.255 for Rcryst and Rfree factors, respectively. The structure determination showed the presence of three crystallographically independent molecules A, B and C in the asymmetric unit. The molecules A and B are observed in the form of a dimer in the asymmetric unit while molecule C belongs to the second dimer whose partner is related by crystallographic twofold symmetry. The polypeptide chain of KpPPAT folds into a β/α structure. The conformations of the side chains of several residues in the substrate binding site in KpPPAT are significantly different from those reported in other PPATs. As a result, the modes of binding of substrates, phosphopantetheine (PNS) and adenosine triphosphate (ATP) differ considerably. The binding studies using fluorescence spectroscopy indicated a KD value of 3.45 × 10-4M for ATP which is significantly lower than the corresponding values reported for PPAT from other species.

In Silico and In Vitro Identification of 1,8-Dihydroxy-4,5-dinitroanthraquinone as a New Antibacterial Agent against Staphylococcus aureus and Enterococcus faecalis.

Increasing rates of bacterial resistance to antibiotics are a growing concern worldwide. The search for potential new antibiotics has included several natural products such as anthraquinones. However, comparatively less attention has been given to anthraquinones that exhibit functional groups that are uncommon in nature. In this work, 114 anthraquinones were evaluated using in silico methods to identify inhibitors of the enzyme phosphopantetheine adenylyltransferase (PPAT) of Staphylococcus aureus, Enterococcus faecalis, and Escherichia coli. Virtual screenings based on molecular docking and the pharmacophore model, molecular dynamics simulations, and free energy calculations pointed to 1,8-dihydroxy-4,5-dinitroanthraquinone (DHDNA) as the most promising inhibitor. In addition, these analyses highlighted the contribution of the nitro group to the affinity of this anthraquinone for the nucleotide-binding site of PPAT. Furthermore, DHDNA was active in vitro towards Gram-positive bacteria with minimum inhibitory concentration (MIC) values of 31.25 µg/mL for S. aureus and 62.5 µg/mL for E. faecalis against both antibiotic-resistant isolates and reference strains but was ineffective against E. coli. Experiments on kill-time kinetics indicated that, at the tested concentrations, DHDNA produced bacteriostatic effects on both Gram-positive bacteria. Overall, our results present DHDNA as a potential PPAT inhibitor, showing antibacterial activity against antibiotic-resistant isolates of S. aureus and E. faecalis, findings that point to nitro groups as key to explaining these results.

Post-transcriptional capping generates coenzyme A-linked RNA

ABSTRACT NAD can be inserted co-transcriptionally via non-canonical initiation to form NAD-RNA. However, that mechanism is unlikely for CoA-linked RNAs due to low intracellular concentration of the required initiator nucleotide, 3’-dephospho-CoA (dpCoA). We report here that phosphopantetheine adenylyltransferase (PPAT), an enzyme of CoA biosynthetic pathway, accepts RNA transcripts as its acceptor substrate and transfers 4’-phosphopantetheine to yield CoA-RNA post-transcriptionally. Synthetic natural (RNAI) and small artificial RNAs were used to identify the features of RNA that are needed for it to serve as PPAT substrate. RNAs with 4–10 unpaired nucleotides at the 5’ terminus served as PPAT substrates, but RNAs having <4 unpaired nucleotides did not undergo capping. No capping was observed when the +1A was changed to G or when 5’ triphosphate was removed by RNA pyrophosphohydrolase (RppH), suggesting the enzyme recognizes pppA-RNA as an ATP analog. PPAT binding affinities were equivalent for transcripts with +1A, +1 G, or 5’OH (+1A), indicating that productive enzymatic recognition is driven more by local positioning effects than by overall binding affinity. Capping rates were independent of the number of unpaired nucleotides in the range of 4–10 nucleotides. Capping was strongly inhibited by ATP, reducing CoA-RNA production ~70% when equimolar ATP and substrate RNA were present. Dual bacterial expression of candidate RNAs with different 5’ structures followed by CoA-RNA CaptureSeq revealed 12-fold enrichment of the better PPAT substrate, consistent with in vivo CoA-capping of RNA transcripts by PPAT. These results suggest post-transcriptional RNA capping as a possible mechanism for the biogenesis of CoA-RNAs in bacteria.

Unravelling the role of a methionine residue in the feedback regulation process of phosphopantetheine adenylyl transferase from a highly virulent ESKAPE pathogen

Unravelling the role of a methionine residue in the feedback regulation process of phosphopantetheine adenylyl transferase from a highly virulent ESKAPE pathogen

Chemical Validation of Mycobacterium tuberculosis Phosphopantetheine Adenylyltransferase Using Fragment Linking and CRISPR Interference.

The coenzyme A (CoA) biosynthesis pathway has attracted attention as a potential target for much-needed novel antimicrobial drugs, including for the treatment of tuberculosis (TB), the lethal disease caused by Mycobacterium tuberculosis (Mtb). Seeking to identify inhibitors of Mtb phosphopantetheine adenylyltransferase (MtbPPAT), the enzyme that catalyses the penultimate step in CoA biosynthesis, we performed a fragment screen. In doing so, we discovered three series of fragments that occupy distinct regions of the MtbPPAT active site, presenting a unique opportunity for fragment linking. Here we show how, guided by X-ray crystal structures, we could link weakly-binding fragments to produce an active site binder with a KD <20 μM and on-target anti-Mtb activity, as demonstrated using CRISPR interference. This study represents a big step toward validating MtbPPAT as a potential drug target and designing a MtbPPAT-targeting anti-TB drug.

Chemical Validation of Mycobacterium tuberculosis Phosphopantetheine Adenylyltransferase Using Fragment Linking and CRISPR Interference.

The coenzyme A (CoA) biosynthesis pathway has attracted attention as a potential target for much-needed novel antimicrobial drugs, including for the treatment of tuberculosis (TB), the lethal disease caused by Mycobacterium tuberculosis (Mtb). Seeking to identify inhibitors of Mtb phosphopantetheine adenylyltransferase (MtbPPAT), the enzyme that catalyses the penultimate step in CoA biosynthesis, we performed a fragment screen. In doing so, we discovered three series of fragments that occupy distinct regions of the MtbPPAT active site, presenting a unique opportunity for fragment linking. Here we show how, guided by X-ray crystal structures, we could link weakly-binding fragments to produce an active site binder with a K D <20 μM and on-target anti-Mtb activity, as demonstrated using CRISPR interference. This study represents a big step toward validating MtbPPAT as a potential drug target and designing a MtbPPAT-targeting anti-TB drug.

Structural Characterization of Mycobacterium abscessus Phosphopantetheine Adenylyl Transferase Ligand Interactions: Implications for Fragment-Based Drug Design.

Anti-microbial resistance is a rising global healthcare concern that needs urgent attention as growing number of infections become difficult to treat with the currently available antibiotics. This is particularly true for mycobacterial infections like tuberculosis and leprosy and those with emerging opportunistic pathogens such as Mycobacterium abscessus, where multi-drug resistance leads to increased healthcare cost and mortality. M. abscessus is a highly drug-resistant non-tuberculous mycobacterium which causes life-threatening infections in people with chronic lung conditions such as cystic fibrosis. In this study, we explore M. abscessus phosphopantetheine adenylyl transferase (PPAT), an enzyme involved in the biosynthesis of Coenzyme A, as a target for the development of new antibiotics. We provide structural insights into substrate and feedback inhibitor binding modes of M. abscessus PPAT, thereby setting the basis for further chemical exploration of the enzyme. We then utilize a multi-dimensional fragment screening approach involving biophysical and structural analysis, followed by evaluation of compounds from a previous fragment-based drug discovery campaign against M. tuberculosis PPAT ortholog. This allowed the identification of an early-stage lead molecule exhibiting low micro molar affinity against M. abscessus PPAT (Kd 3.2 ± 0.8 µM) and potential new ways to design inhibitors against this enzyme. The resulting crystal structures reveal striking conformational changes and closure of solvent channel of M. abscessus PPAT hexamer providing novel strategies of inhibition. The study thus validates the ligandability of M. abscessus PPAT as an antibiotic target and identifies crucial starting points for structure-guided drug discovery against this bacterium.

Design of Novel Phosphopantetheine Adenylyltransferase Inhibitors: A Potential New Approach to Tackle Mycobacterium tuberculosis.

Tuberculosis (TB) has been a challenging disease worldwide, especially for the neglected poor populations. Presently, there are approximately 2 billion people infected with TB worldwide and 10 million people in the world fell ill with active TB, leading to 1.5 million deaths. The classic treatment is extensive and the drug- and multi-drug resistance of Mycobacterium tuberculosis has been a threat to the efficacy of the drugs currently used. Therefore, the rational design of new anti-TB candidates is urgently needed. With the aim of contributing to face this challenge, 78 compounds have been proposed based on SBDD (Structure-Based Drug Design) strategies applied to target the M. tuberculosis phosphopantetheine adenylyltransferase (MtPPAT) enzyme. Ligand-Based Drug Design (LBDD) strategies were also used for establishing Structure-Activity Relationships (SAR) and for optimizing the structures. MtPPAT is important for the biosynthesis of coenzyme A (CoA) and it has been studied recently toward the discovery of new inhibitors. After docking simulations and enthalpy calculations, the interaction of selected compounds with MtPPAT was found to be energetically favorable. The most promising compounds were then synthesized and submitted to anti-M. tuberculosis and MtPPAT inhibition assays. One of the compounds synthesized (MCP163), showed the highest activity in both of these assays.

Crystal Packing of Phosphopantetheine Adenylyltransferase from Mycobacterium tuberculosis in Two Crystal Modifications

Crystals of phosphopantetheine adenylyltransferase from Mycobacterium tuberculosis (PPATMt), which were grown using 2-methyl-2,4-pentanediol (MPD) or ammonium sulfate as the precipitant, belong to sp. grs. R32 and Р32, respectively. Crystals of the enzyme containing the ligand in the active site were obtained by the cocrystallization of the enzyme with functional substrates only in the presence of MPD (sp. gr. R32). In the presence of ammonium sulfate, the ligand was not bound in the active site, and the cocrystallization resulted only in crystals of the apo form (sp. gr. Р32). The crystal-packing patterns of the enzyme molecules and the structure of the apo form of РРАТMt in two crystal structures are compared in order to explain the binding patterns of the ligand in different crystal modifications. In the crystal modification P32, the molecules are more closely packed compared to the crystal modification R32, and intermolecular contacts restrict the access to the active site.

Structural and biochemical studies of phosphopantetheine adenylyltransferase from Acinetobacter baumannii with dephospho-coenzyme A and coenzyme A

Phosphopantetheine adenylyl transferase catalyzes a rate limiting penultimate step of the multistep reaction which produces coenzyme A (CoA) as a final product. CoA is required as an essential cofactor in a number of metabolic reactions. Therefore inhibiting the function of this enzyme will lead to cell death in bacteria. Acinetobacter baumannii is multi drug resistant pathogen and causes infections in immunocompromised patients. AbPPAT has been cloned, expressed, purified and crystallized and structures of two complexes of AbPPAT with dephospho coenzyme A (dPCoA) and coenzyme A (CoA) have been determined. Both dPCoA and CoA molecules are observed in the substrate binding site of AbPPAT. A comparison with the structures of the complexes of PPAT from other species shows that the orientations of dPCoA are identical in all the structures. On the other hand, as observed from the structures of the complexes of CoA with PPAT, the orientations of CoA are found to differ considerably. This shows that the substrates occupy identical positions in the substrate binding sites of enzymes whereas the positions of inhibitors may differ. The binding studies carried out using fluorescence method and surface plasmon resonance techniques showed that binding affinity of CoA towards AbPPAT is nearly three times higher than that of dPCoA.

Identification of Dephospho-Coenzyme A (Dephospho-CoA) Kinase in Thermococcus kodakarensis and Elucidation of the Entire CoA Biosynthesis Pathway in Archaea.

Dephospho-coenzyme A (dephospho-CoA) kinase (DPCK) catalyzes the ATP-dependent phosphorylation of dephospho-CoA, the final step in coenzyme A (CoA) biosynthesis. DPCK has been identified and characterized in bacteria and eukaryotes but not in archaea. The hyperthermophilic archaeon Thermococcus kodakarensis encodes two homologs of bacterial DPCK and the DPCK domain of eukaryotic CoA synthase, TK1334 and TK2192. We purified the recombinant TK1334 and TK2192 proteins and found that they lacked DPCK activity. Bioinformatic analyses showed that, in several archaea, the uncharacterized gene from arCOG04076 protein is fused with the gene for phosphopantetheine adenylyltransferase (PPAT), which catalyzes the reaction upstream of the DPCK reaction in CoA biosynthesis. This observation suggested that members of arCOG04076, both fused to PPAT and standalone, could be the missing archaeal DPCKs. We purified the recombinant TK1697 protein, a standalone member of arCOG04076 from T. kodakarensis, and demonstrated its GTP-dependent DPCK activity. Disruption of the TK1697 resulted in CoA auxotrophy, indicating that TK1697 encodes a DPCK that contributes to CoA biosynthesis in T. kodakarensis TK1697 homologs are widely distributed in archaea, suggesting that the arCOG04076 protein represents a novel family of DPCK that is not homologous to bacterial and eukaryotic DPCKs but is distantly related to bacterial and eukaryotic thiamine pyrophosphokinases. We also constructed and characterized gene disruption strains of TK0517 and TK2128, homologs of bifunctional phosphopantothenoylcysteine synthetase-phosphopantothenoylcysteine decarboxylase and PPAT, respectively. Both strains displayed CoA auxotrophy, indicating their contribution to CoA biosynthesis. Taken together with previous studies, the results experimentally validate the entire CoA biosynthesis pathway in T. kodakarensisIMPORTANCE CoA is utilized in a wide range of metabolic pathways, and its biosynthesis is essential for all life. Pathways for CoA biosynthesis in bacteria and eukaryotes have been established. In archaea, however, the enzyme that catalyzes the final step in CoA biosynthesis, dephospho-CoA kinase (DPCK), had not been identified. In the present study, bioinformatic analyses identified a candidate for the DPCK in archaea, which was biochemically and genetically confirmed in the hyperthermophilic archaeon Thermococcus kodakarensis Genetic analyses on genes presumed to encode bifunctional phosphopantothenoylcysteine synthetase-phosphopantothenoylcysteine decarboxylase and phosphopantetheine adenylyltransferase confirmed their involvement in CoA biosynthesis. Taken together with previous studies, the results reveal the entire pathway for CoA biosynthesis in a single archaeon and provide insight into the different mechanisms of CoA biosynthesis and their distribution in nature.

Computational investigations of gram-negative bacteria phosphopantetheine adenylyltransferase inhibitors using 3D-QSAR, molecular docking and molecular dynamic simulations

Phosphopantetheine adenylyltransferase (PPAT) has been recognized as a promising target to develop novel antimicrobial agents, which is a hexameric enzyme that catalyzes the penultimate step in coenzyme A biosynthesis. In this work, molecular modeling study was performed with a series of PPAT inhibitors using molecular docking, three-dimensional qualitative structure-activity relationship (3D-QSAR) and molecular dynamic (MD) simulations to reveal the structural determinants for their bioactivities. Molecular docking study was applied to understand the binding mode of PPAT with its inhibitors. Subsequently, 3D-QSAR model was constructed to find the features required for different substituents on the scaffolds. For the best comparative molecular field analysis (CoMFA) model, the Q2 and R2 values of which were calculated as 0.702 and 0.989, while they were calculated as 0.767 and 0.983 for the best comparative molecular similarity index analysis model. The statistical data verified the significance and accuracy of our 3D-QSAR models. Furthermore, MD simulations were carried out to evaluate the stability of the receptor–ligand contacts in physiological conditions, and the results were consistent with molecular docking studies and 3D-QSAR contour map analysis. Binding free energy was calculated with molecular mechanics generalized born surface area approach, the result of which coincided well with bioactivities and demonstrated that van der Waals accounted for the largest portion. Overall, our study provided a valuable insight for further research work on the recognition of potent PPAT inhibitors.Communicated by Ramaswamy H. Sarma

Structural and binding studies of phosphopantetheine adenylyl transferase from Acinetobacter baumannii

Phosphopantetheine adenylyltransferase (PPAT, EC. 2.7.7.3) catalyzes an essential step in the reaction that transfers an adenylyl group from adenosine tri phosphate (ATP) to 4′-phosphopantetheine (pPant) yielding 3′- dephospho-coenzyme A (dPCoA) and pyrophosphate (PP) in the coenzyme A (CoA) biosynthesis pathway. The enzyme PPAT from Acinetobacter baumannii (AbPPAT) was cloned, expressed and purified. The binding studies of AbPPAT were carried out with two compounds, tri‑sodium citrate (TSC) and l-ascorbic acid (LAA, vitamin-C) using fluorescence spectroscopic (FS) and surface Plasmon resonance (SPR) methods. Both methods provided similar values of dissociation constants for TSC and LAA which were of the order of 10−8 M and 10−5 M respectively. The computer aided docking studies indicated fewer interactions of LAA with AbPPAT as compared to those of TSC. The freshly purified samples of AbPPAT were crystallized. The crystals of AbPPAT were soaked in the solutions containing TSC and LAA. However, the crystals of the complex of AbPPAT with LAA did not diffract well and hence the structure of the complex of AbPPAT with LAA could not be determined. On the other hand, the crystals of the complex of AbPPAT with TSC diffracted well and the structure was determined at 1.76 Å resolution. It showed that TSC bound to AbPPAT at the ATP binding site and formed several intermolecular contacts including 12 hydrogen bonds. The results of binding studies for both TSC and LAA and the structure of the complex of AbPPAT with TSC clearly indicated a potential role of TSC and LAA as antibacterial agents.

Discovery and Optimization of Phosphopantetheine Adenylyltransferase Inhibitors with Gram-Negative Antibacterial Activity.

In the preceding manuscript [ Moreau et al. 2018 , 10.1021/acs.jmedchem.7b01691 ] we described a successful fragment-based lead discovery (FBLD) strategy for discovery of bacterial phosphopantetheine adenylyltransferase inhibitors (PPAT, CoaD). Following several rounds of optimization two promising lead compounds were identified: triazolopyrimidinone 3 and 4-azabenzimidazole 4. Here we disclose our efforts to further optimize these two leads for on-target potency and Gram-negative cellular activity. Enabled by a robust X-ray crystallography system, our structure-based inhibitor design approach delivered compounds with biochemical potencies 4-5 orders of magnitude greater than their respective fragment starting points. Additional optimization was guided by observations on bacterial permeability and physicochemical properties, which ultimately led to the identification of PPAT inhibitors with cellular activity against wild-type E. coli.

Fragment-Based Drug Discovery of Inhibitors of Phosphopantetheine Adenylyltransferase from Gram-Negative Bacteria.

The discovery and development of new antibiotics capable of curing infections due to multidrug-resistant and pandrug-resistant Gram-negative bacteria are a major challenge with fundamental importance to our global healthcare system. Part of our broad program at Novartis to address this urgent, unmet need includes the search for new agents that inhibit novel bacterial targets. Here we report the discovery and hit-to-lead optimization of new inhibitors of phosphopantetheine adenylyltransferase (PPAT) from Gram-negative bacteria. Utilizing a fragment-based screening approach, we discovered a number of unique scaffolds capable of interacting with the pantetheine site of E. coli PPAT and inhibiting enzymatic activity, including triazolopyrimidinone 6. Structure-based optimization resulted in the identification of two lead compounds as selective, small molecule inhibitors of bacterial PPAT: triazolopyrimidinone 53 and azabenzimidazole 54 efficiently inhibited E. coli and P. aeruginosa PPAT and displayed modest cellular potency against the efflux-deficient E. coli Δ tolC mutant strain.

Characterization and validation of Entamoeba histolytica pantothenate kinase as a novel anti-amebic drug target

The Coenzyme A (CoA), as a cofactor involved in >100 metabolic reactions, is essential to the basic biochemistry of life. Here, we investigated the CoA biosynthetic pathway of Entamoeba histolytica (E. histolytica), an enteric protozoan parasite responsible for human amebiasis. We identified four key enzymes involved in the CoA pathway: pantothenate kinase (PanK, EC 2.7.1.33), bifunctional phosphopantothenate-cysteine ligase/decarboxylase (PPCS-PPCDC), phosphopantetheine adenylyltransferase (PPAT) and dephospho-CoA kinase (DPCK). Cytosolic enzyme PanK, was selected for further biochemical, genetic, and phylogenetic characterization. Since E. histolytica PanK (EhPanK) is physiologically important and sufficiently divergent from its human orthologs, this enzyme represents an attractive target for the development of novel anti-amebic chemotherapies. Epigenetic gene silencing of PanK resulted in a significant reduction of PanK activity, intracellular CoA concentrations, and growth retardation in vitro, reinforcing the importance of this gene in E. histolytica. Furthermore, we screened the Kitasato Natural Products Library for inhibitors of recombinant EhPanK, and identified 14 such compounds. One compound demonstrated moderate inhibition of PanK activity and cell growth at a low concentration, as well as differential toxicity towards E. histolytica and human cells.

Umbrella Sampling and X-ray Crystallographic Analysis Unveil an Arg-Asp Gate Facilitating Inhibitor Binding Inside Phosphopantetheine Adenylyltransferase Allosteric Cleft.

Phosphopantetheine adenylyltransferase (PPAT) is a rate-limiting enzyme essential for biosynthesis of coenzyme A (CoA), which in turn is responsible to regulate the secretion of exotoxins via type III secretion system in Pseudomonas aeruginosa, causing severe health concerns ranging from nosocomial infections to respiratory failure. Acetyl coenzyme A (AcCoA) is a newly reported inhibitor of PPAT, believed to regulate the cellular levels of CoA and thereby the pathogenesis. Very little is known so far regarding the mechanistic details of AcCoA binding inside PPAT-binding cleft. Herein, we have used extensive umbrella sampling simulations to decipher mechanistic insight into the inhibitor accommodation inside the binding cavity. We found that R90 and D94 residues act like a gate near the binding cavity to accommodate and stabilize the incoming ligand. Mutational models concerning these residues also show considerable difference in AcCoA-binding thermodynamics. To substantiate our findings, we have solved the first crystal structure of apo-PPAT from P. aeruginosa, which also found to agree with the simulation results. Collectively, these results describe the mechanistic details of accommodation of inhibitor molecule inside PPAT-binding cavity and also offer valuable insight into regulating cellular levels of CoA/AcCoA and thus controlling the pathogenicity.

Multiplexed gene synthesis in emulsions for exploring protein functional landscapes.

Improving our ability to construct and functionally characterize DNA sequences would broadly accelerate progress in biology. Here, we introduce DropSynth, a scalable, low-cost method to build thousands of defined gene-length constructs in a pooled (multiplexed) manner. DropSynth uses a library of barcoded beads that pull down the oligonucleotides necessary for a gene's assembly, which are then processed and assembled in water-in-oil emulsions. We used DropSynth to successfully build more than 7000 synthetic genes that encode phylogenetically diverse homologs of two essential genes in Escherichia coli We tested the ability of phosphopantetheine adenylyltransferase homologs to complement a knockout E. coli strain in multiplex, revealing core functional motifs and reasons underlying homolog incompatibility. DropSynth coupled with multiplexed functional assays allows us to rationally explore sequence-function relationships at an unprecedented scale.

Efficient one-pot enzymatic synthesis of dephospho coenzyme A

Dephospho coenzyme A (depCoA) is the last intermediate for CoA biosynthesis, and it can be used as a transcription initiator to prepare CoA-linked RNA by in vitro transcription. However, commercially available depCoA is expensive. We hereby describe a simple and efficient enzymatic synthesis of depCoA in a single-step from commercially available and inexpensive oxidized pantethine (Ox-Pan) and ATP. A plasmid (pCoaDAa) was constructed to co-express and co-purify two enzymes pantothenate kinase (PanK/coaA) and phosphopantetheine adenylyltransferase (PPAT/coaD). Starting from Ox-Pan and ATP, two different synthetic routes of one-pot reaction catalyzed by PanK and PPAT, followed by a simple column purification step, afforded depCoA and its oxidized dimer (Ox-depCoA) with high yields and purity. The simplicity and low cost of our method should make depCoA easily accessible to a broad scientific community, and promote research on CoA-related areas in biology and biomedicine.

Genetic Characterization of Coenzyme A Biosynthesis Reveals Essential Distinctive Functions during Malaria Parasite Development in Blood and Mosquito.

Coenzyme A (CoA) is an essential universal cofactor for all prokaryotic and eukaryotic cells. In nearly all non-photosynthetic cells, CoA biosynthesis depends on the uptake and phosphorylation of vitamin B5 (pantothenic acid or pantothenate). Recently, putative pantothenate transporter (PAT) and pantothenate kinases (PanKs) were functionally characterized in P. yoelii. PAT and PanKs were shown to be dispensable for blood stage development, but they were essential for mosquito stages development. Yet, little is known about the cellular functions of the other enzymes of the CoA biosynthesis pathway in malaria parasite life cycle stages. All enzymes of this pathway were targeted for deletion or deletion/complementation analyses by knockout/knock-in plasmid constructs to reveal their essential roles in P. yoelii life cycle stages. The intermediate enzymes PPCS (Phosphopantothenylcysteine Synthase), PPCDC (Phosphopantothenylcysteine Decarboxylase) were shown to be dispensable for asexual and sexual blood stage development, but they were essential for oocyst development and the production of sporozoites. However, the last two enzymes of this pathway, PPAT (Phosphopantetheine Adenylyltransferase) and DPCK (Dephospho-CoA Kinase), were essential for blood stage development. These results indicate alternative first substrate requirement for the malaria parasite, other than the canonical pantothenate, for the synthesis of CoA in the blood but not inside the mosquito midgut. Collectively, our data shows that CoA de novo biosynthesis is essential for both blood and mosquito stages, and thus validates the enzymes of this pathway as potential antimalarial targets.