Coenzyme A: to make it or uptake it?

The consensus has been that intracellular coenzyme A (CoA) is obtained exclusively by de novo biosynthesis via a universal, conserved five-step pathway in the cell cytosol. However, old and new evidence suggest that cells (and some microorganisms) have several strategies to obtain CoA, with 4'-phosphopantetheine (P-PantSH; the fourth intermediate in the canonical CoA biosynthetic pathway) serving as a 'nexus' metabolite.

Squalene synthase predicts poor prognosis in stage I-III colon adenocarcinoma and synergizes squalene epoxidase to promote tumor progression.

Colon adenocarcinoma (COAD) is one of the most prevalent malignancies, with poor prognosis and lack of effective treatment targets. Squalene synthase (FDFT1) is an upstream enzyme of squalene epoxidase (SQLE) in cholesterol biosynthesis. In a previous study, we revealed that SQLE promotes colon cancer cell proliferation in vitro and in vivo. Here, we investigate the prognostic value of FDFT1 in stage I-III COAD and explore the potential underlying mechanisms. Squalene synthase was significantly upregulated in stage I-III COAD and positively correlated with poor differentiation and advanced tumor stage. High expression of FDFT1 was an independent predictor of overall and relapse-free survival, and the nomograms based on FDFT1 could effectively identify patients at high risk of poor outcome. Squalene synthase accelerated colon cancer cell proliferation and promoted tumor growth. Lack of FDFT1 resulted in accumulating NAT8 and D-pantethine to lower reactive oxygen species levels and inhibit colon cancer cell proliferation. Moreover, the combined inhibition of FDFT1 and SQLE induced a greater suppressive effect on cell proliferation and tumor growth than single inhibition. Taken together, these results indicate that FDFT1 predicts poor prognosis in stage I-III COAD and has the tumor-promoting effect on COAD through regulating NAT8 and D-pantethine. Targeting both FDFT1 and SQLE is a more promising therapy than their single inhibition for stage I-III COAD.

Structures of two distinct conformations of holo-non-ribosomal peptide synthetases.

Many important natural products are produced by multidomain non-ribosomal peptide synthetases (NRPSs). During synthesis, intermediates are covalently bound to integrated carrier domains and transported to neighbouring catalytic domains in an assembly line fashion. Understanding the structural basis for catalysis with non-ribosomal peptide synthetases will facilitate bioengineering to create novel products. Here we describe the structures of two different holo-non-ribosomal peptide synthetase modules, each revealing a distinct step in the catalytic cycle. One structure depicts the carrier domain cofactor bound to the peptide bond-forming condensation domain, whereas a second structure captures the installation of the amino acid onto the cofactor within the adenylation domain. These structures demonstrate that a conformational change within the adenylation domain guides transfer of intermediates between domains. Furthermore, one structure shows that the condensation and adenylation domains simultaneously adopt their catalytic conformations, increasing the overall efficiency in a revised structural cycle. These structures and the single-particle electron microscopy analysis demonstrate a highly dynamic domain architecture and provide the foundation for understanding the structural mechanisms that could enable engineering of novel non-ribosomal peptide synthetases.

Atomic structure of the entire mammalian mitochondrial complex I.

Mitochondrial complex I (also known as NADH:ubiquinone oxidoreductase) contributes to cellular energy production by transferring electrons from NADH to ubiquinone coupled to proton translocation across the membrane. It is the largest protein assembly of the respiratory chain with a total mass of 970 kilodaltons. Here we present a nearly complete atomic structure of ovine (Ovis aries) mitochondrial complex I at 3.9 Å resolution, solved by cryo-electron microscopy with cross-linking and mass-spectrometry mapping experiments. All 14 conserved core subunits and 31 mitochondria-specific supernumerary subunits are resolved within the L-shaped molecule. The hydrophilic matrix arm comprises flavin mononucleotide and 8 iron-sulfur clusters involved in electron transfer, and the membrane arm contains 78 transmembrane helices, mostly contributed by antiporter-like subunits involved in proton translocation. Supernumerary subunits form an interlinked, stabilizing shell around the conserved core. Tightly bound lipids (including cardiolipins) further stabilize interactions between the hydrophobic subunits. Subunits with possible regulatory roles contain additional cofactors, NADPH and two phosphopantetheine molecules, which are shown to be involved in inter-subunit interactions. We observe two different conformations of the complex, which may be related to the conformationally driven coupling mechanism and to the active-deactive transition of the enzyme. Our structure provides insight into the mechanism, assembly, maturation and dysfunction of mitochondrial complex I, and allows detailed molecular analysis of disease-causing mutations.

Synthetic cycle of the initiation module of a formylating nonribosomal peptide synthetase.

Nonribosomal peptide synthetases (NRPSs) are very large proteins that produce small peptide molecules with wide-ranging biological activities, including environmentally friendly chemicals and many widely used therapeutics. NRPSs are macromolecular machines, with modular assembly-line logic, a complex catalytic cycle, moving parts and many active sites. In addition to the core domains required to link the substrates, they often include specialized tailoring domains, which introduce chemical modifications and allow the product to access a large expanse of chemical space. It is still unknown how the NRPS tailoring domains are structurally accommodated into megaenzymes or how they have adapted to function in nonribosomal peptide synthesis. Here we present a series of crystal structures of the initiation module of an antibiotic-producing NRPS, linear gramicidin synthetase. This module includes the specialized tailoring formylation domain, and states are captured that represent every major step of the assembly-line synthesis in the initiation module. The transitions between conformations are large in scale, with both the peptidyl carrier protein domain and the adenylation subdomain undergoing huge movements to transport substrate between distal active sites. The structures highlight the great versatility of NRPSs, as small domains repurpose and recycle their limited interfaces to interact with their various binding partners. Understanding tailoring domains is important if NRPSs are to be utilized in the production of novel therapeutics.

Chemical Labeling of Protein 4'-Phosphopantetheinylation.

Nature uses a diverse array of protein post-translational modifications (PTMs) to regulate protein structure, activity, localization, and function. Among them, protein 4'-phosphopantetheinylation derived from coenzyme A (CoA) is an essential PTM for the biosynthesis of fatty acids, polyketides, and nonribosomal peptides in prokaryotes and eukaryotes. To explore its functions, various chemical probes mimicking the natural structure of 4'-phosphopantetheinylation have been developed. In this minireview, we summarize these chemical probes and describe their applications in direct and metabolic labeling of proteins in bacterial and mammalian cells.

Natural Molecules and Neuroprotection: Kynurenic Acid, Pantethine and α-Lipoic Acid.

The incidence of neurodegenerative diseases has increased greatly worldwide due to the rise in life expectancy. In spite of notable development in the understanding of these disorders, there has been limited success in the development of neuroprotective agents that can slow the progression of the disease and prevent neuronal death. Some natural products and molecules are very promising neuroprotective agents because of their structural diversity and wide variety of biological activities. In addition to their neuroprotective effect, they are known for their antioxidant, anti-inflammatory and antiapoptotic effects and often serve as a starting point for drug discovery. In this review, the following natural molecules are discussed: firstly, kynurenic acid, the main neuroprotective agent formed via the kynurenine pathway of tryptophan metabolism, as it is known mainly for its role in glutamate excitotoxicity, secondly, the dietary supplement pantethine, that is many sided, well tolerated and safe, and the third molecule, α-lipoic acid is a universal antioxidant. As a conclusion, because of their beneficial properties, these molecules are potential candidates for neuroprotective therapies suitable in managing neurodegenerative diseases.

Chemical Proteomic Profiling of Protein 4'-Phosphopantetheinylation in Mammalian Cells.

Protein 4'-phosphopantetheinylation is an essential post-translational modification (PTM) in prokaryotes and eukaryotes. So far, only five protein substrates of this specific PTM have been discovered in mammalian cells. These proteins are known to perform important functions, including fatty acid biosynthesis and folate metabolism, as well as ß-alanine activation. To explore existing and new substrates of 4'-phosphopantetheinylation in mammalian proteomes, we designed and synthesized a series of new pantetheine analogue probes, enabling effective metabolic labelling of 4'-phosphopantetheinylated proteins in HepG2 cells. In combination with a quantitative chemical proteomic platform, we enriched and identified all the currently known 4'-phosphopantetheinylated proteins with high confidence, and unambiguously determined their exact sites of modification. More encouragingly, we discovered, using targeted chemical proteomics, a potential 4'-phosphopantetheinylation site in the protein of mitochondrial dehydrogenase/reductase SDR family member 2 (DHRS2).

Shifting the Hydrolysis Equilibrium of Substrate Loaded Acyl Carrier Proteins.

Acyl carrier proteins (ACP)s transport intermediates through many primary and secondary metabolic pathways. Studying the effect of substrate identity on ACP structure has been hindered by the lability of the thioester bond that attaches acyl substrates to the 4'-phosphopantetheine cofactor of ACP. Here we show that an acyl acyl-carrier protein synthetase (AasS) can be used in real time to shift the hydrolysis equilibrium toward favoring acyl-ACP during solution NMR spectroscopy. Only 0.005 molar equivalents of AasS enables 1 week of stability to palmitoyl-AcpP from Escherichia coli. 2D NMR spectra enabled with this method revealed that the tethered palmitic acid perturbs nearly every secondary structural region of AcpP. This technique will allow previously unachievable structural studies of unstable acyl-ACP species, contributing to the understanding of these complex biosynthetic pathways.

Exome sequence reveals mutations in CoA synthase as a cause of neurodegeneration with brain iron accumulation.

Neurodegeneration with brain iron accumulation (NBIA) comprises a clinically and genetically heterogeneous group of disorders with progressive extrapyramidal signs and neurological deterioration, characterized by iron accumulation in the basal ganglia. Exome sequencing revealed the presence of recessive missense mutations in COASY, encoding coenzyme A (CoA) synthase in one NBIA-affected subject. A second unrelated individual carrying mutations in COASY was identified by Sanger sequence analysis. CoA synthase is a bifunctional enzyme catalyzing the final steps of CoA biosynthesis by coupling phosphopantetheine with ATP to form dephospho-CoA and its subsequent phosphorylation to generate CoA. We demonstrate alterations in RNA and protein expression levels of CoA synthase, as well as CoA amount, in fibroblasts derived from the two clinical cases and in yeast. This is the second inborn error of coenzyme A biosynthesis to be implicated in NBIA.

Ratiometric Fluorescent Probe for Imaging of Pantetheinase in Living Cells.

Pantetheinase, which catalyzes the cleavage of pantetheine to pantothenic acid (vitamin B5) and cysteamine, is involved in the regulation of oxidative stress, pantothenate recycling and cell migration. However, further elucidating the cellular function of this enzyme is largely limited by the lack of a suitable fluorescence imaging probe. By conjugating pantothenic acid with cresyl violet, herein we develop a new fluorescence probe CV-PA for the assay of pantetheinase. The probe not only possesses long analytical wavelengths but also displays linear ratiometric (I628/582 nm) fluorescence response to pantetheinase in the range of 5-400 ng/mL with a detection limit of 4.7 ng/mL. This probe has been used to evaluate the efficiency of different inhibitors and quantitatively detect pantetheinase in serum samples, revealing that pantetheinase in fetal bovine serum and new born calf serum is much higher than that in normal human serum. Notably, with the probe the ratiometric imaging and in situ quantitative comparison of pantetheinase in different living cells (LO2 and HK-2) have been achieved for the first time. It is found that the level of pantetheinase in LO2 cells is much larger than that in HK-2 cells, as further validated by Western blot analysis. The proposed probe may be useful to better understand the specific function of pantetheinase in the pantetheinase-related pathophysiological processes.

Extracellular 4'-phosphopantetheine is a source for intracellular coenzyme A synthesis.

The metabolic cofactor coenzyme A (CoA) gained renewed attention because of its roles in neurodegeneration, protein acetylation, autophagy and signal transduction. The long-standing dogma is that eukaryotic cells obtain CoA exclusively via the uptake of extracellular precursors, especially vitamin B5, which is intracellularly converted through five conserved enzymatic reactions into CoA. This study demonstrates an alternative mechanism that allows cells and organisms to adjust intracellular CoA levels by using exogenous CoA. Here CoA was hydrolyzed extracellularly by ectonucleotide pyrophosphatases to 4'-phosphopantetheine, a biologically stable molecule able to translocate through membranes via passive diffusion. Inside the cell, 4'-phosphopantetheine was enzymatically converted back to CoA by the bifunctional enzyme CoA synthase. Phenotypes induced by intracellular CoA deprivation were reversed when exogenous CoA was provided. Our findings answer long-standing questions in fundamental cell biology and have major implications for the understanding of CoA-related diseases and therapies.

Structural basis for the recognition of peptide RJPXD33 by acyltransferases in lipid A biosynthesis.

UDP-N-acetylglucosamine acyltransferase (LpxA) and UDP-3-O-(acyl)-glucosamine acyltransferase (LpxD) constitute the essential, early acyltransferases of lipid A biosynthesis. Recently, an antimicrobial peptide inhibitor, RJPXD33, was identified with dual affinity for LpxA and LpxD. To gain a fundamental understanding of the molecular basis of inhibitor binding, we determined the crystal structure of LpxA from Escherichia coli in complex with RJPXD33 at 1.9 Å resolutions. Our results suggest that the peptide binds in a unique modality that mimics (R)-ß-hydroxyacyl pantetheine binding to LpxA and displays how the peptide binds exclusive of the native substrate, acyl-acyl carrier protein. Acyltransferase binding studies with photo-labile RJPXD33 probes and truncations of RJPXD33 validated the structure and provided fundamental insights for future design of small molecule inhibitors. Overlay of the LpxA-RJPXD33 structure with E. coli LpxD identified a complementary peptide binding pocket within LpxD and serves as a model for further biochemical characterization of RJPXD33 binding to LpxD.

The chain-flipping mechanism of ACP (acyl carrier protein)-dependent enzymes appears universal.

ACPs (acyl carrier proteins) play essential roles in the synthesis of fatty acids, polyketides and non-ribosomal polypeptides. ACP function requires the modification of the protein by attachment of 4'-phosphopantetheine to a conserved serine residue. The phosphopantetheine thiol acts to tether the starting materials and intermediates as their thioesters. ACPs are small highly soluble proteins composed of four α-helices. The helices form a bundle that acts as a hydrophobic sleeve that sequesters the acyl chains and activated thioesters from solvent. However, in the synthesis of fatty acids and complex lipids the enzymes of the pathway must access the thioester and the proximal carbon atoms in order to perform the needed chemistry. How such access is provided without exposure of the acyl chains to solvent has been a longstanding question due to the lack of acyl-ACP-enzyme complexes, a situation generally attributed to the brevity of the interactions of acyl-ACPs with their cognate enzymes. As discussed in the present review the access question has now been answered by four recent crystal structures, each of which shows that the entire acyl chain plus the 4'-phosphopantetheine prosthetic group partitions from the ACP hydrophobic sleeve into a hydrophobic pocket or groove of the enzyme protein, a process termed chain flipping.

Crystal structure of phosphopantothenate synthetase from Thermococcus kodakarensis.

Bacteria/eukaryotes share a common pathway for coenzyme A biosynthesis which involves two enzymes to convert pantoate to 4'-phosphopantothenate. These two enzymes are absent in almost all archaea. Recently, it was reported that two novel enzymes, pantoate kinase, and phosphopantothenate synthetase (PPS), are responsible for this conversion in archaea. Here, we report the crystal structure of PPS from the hyperthermophilic archaeon, Thermococcus kodakarensis and its complexes with substrates, ATP, and ATP and 4-phosphopantoate. PPS forms an asymmetric homodimer, in which two monomers composing a dimer, deviated from the exact twofold symmetry, displaying 4°-13° distortion. The structural features are consistent with the mutagenesis data and the results of biochemical experiments previously reported. Based on these structures, we discuss the catalytic mechanism by which PPS produces phosphopantoyl adenylate, which is thought to be a reaction intermediate.

Pantothenate and pantetheine antagonize the antitubercular activity of pyrazinamide.

Pyrazinamide (PZA) is a first-line tuberculosis drug that inhibits the growth of Mycobacterium tuberculosis via an as yet undefined mechanism. An M. tuberculosis laboratory strain that was auxotrophic for pantothenate was found to be insensitive to PZA and to the active form, pyrazinoic acid (POA). To determine whether this phenotype was strain or condition specific, the effect of pantothenate supplementation on PZA activity was assessed using prototrophic strains of M. tuberculosis. It was found that pantothenate and other ß-alanine-containing metabolites abolished PZA and POA susceptibility, suggesting that POA might selectively target pantothenate synthesis. However, when the pantothenate-auxotrophic strain was cultivated using a subantagonistic concentration of pantetheine in lieu of pantothenate, susceptibility to PZA and POA was restored. In addition, we found that ß-alanine could not antagonize PZA and POA activity against the pantothenate-auxotrophic strain, indicating that the antagonism is specific to pantothenate. Moreover, pantothenate-mediated antagonism was observed for structurally related compounds, including n-propyl pyrazinoate, 5-chloropyrazinamide, and nicotinamide, but not for nicotinic acid or isoniazid. Taken together, these data demonstrate that while pantothenate can interfere with the action of PZA, pantothenate synthesis is not directly targeted by PZA. Our findings suggest that targeting of pantothenate synthesis has the potential to enhance PZA efficacy and possibly to restore PZA susceptibility in isolates with panD-linked resistance.

OLD AND NEW NEUROENDOCRINE MOLECULES: SOMATOSTATIN, CYSTEAMINE, PANTETHINE AND KYNURENINE.

The aim of this review is to commemorate Hans Selye, endocrinologist, the most famous researchers of stress and to briefly summarize the major features of somatostatin (SST), cysteamine (CysA) and patethine (PAN) in neuroendocrinological aspect, which are closely related to his scientific work. In addition, some metabolites of kynurenine pathway (KP) were also mentioned in this paper, as new, possible target molecules in neuroendocrinology. R. Guillemin and A. V. Schally were the main pioneers of the discovery of SST in the 1970's. SST primarily is known as an inhibitor of growth hormone secretion and additionally reduces the gastric acid and pepsin release and also the gastroduodenal mucosal blood flow. These effects are very important in the pathophysiology of peptic ulcer bleeding, which is related to the CysA-evoked perforating duodenal ulcer experimental stress model in rats developed by Selye and Szabo. CysA is a naturally occurring duodenal ulcerogen, which depletes SST in the gastric mucosa and certain brain regions. Furthermore, in addition to depleting SST, CysA also causes adrenocortical necrosis, suggesting an interaction between the central/peripheral nervous system and the neuroendocrine system. The antioxidant PAN, formulated besides the CysA, has similar effects: it attenuates the levels of SST and prolactin in the cerebral cortex and hypothalamus through the accumulation of CysA within cells throughout the body. As new perspectives the KP may be involved in the modulation of neuroendrocrine processes: different agonists and antagonists of glutamate receptors regulate the hypothalamic-pituitary-adrenal axis and kynurenic acid augments the anxiolytic stress responses in neonatal chicks. The pro-inflammatory cytokine-induced and the toxic heavy oil contaminations-evoked alterations in the KP indirectly contribute to the development of neuroendocrine disorders. In summary, there have been highly important developments in neuroendocrinology since the early findings of Selye. Although there are as yet relatively few data about the potential role of kynurenines in neuroendocrinology, the results already achieved are extremely noteworthy and immensely promising.

Fluorometric Analysis of Carrier-Protein-Dependent Biosynthesis through a Conformationally Sensitive Solvatochromic Pantetheinamide Probe.

Carrier proteins (CPs) play a fundamental role in the biosynthesis of fatty acids, polyketides, and non-ribosomal peptides, encompassing many medicinally and pharmacologically relevant compounds. Current approaches to analyze novel carrier-protein-dependent synthetic pathways are hampered by a lack of activity-based assays for natural product biosynthesis. To fill this gap, we turned to 3-methoxychromones, highly solvatochromic fluorescent molecules whose emission intensity and wavelength are heavily dependent on their immediate molecular environment. We have developed a solvatochromic carrier-protein-targeting probe which is able to selectively fluoresce when bound to a target carrier protein. Additionally, the probe displays distinct responses upon CP binding in carrier-protein-dependent synthases. This discerning approach demonstrates the design of solvatochromic fluorophores with the ability to identify biosynthetically active CP-enzyme interactions.

Metabolic perturbation of an essential pathway: evaluation of a glycine precursor of coenzyme A.

Pantetheine and its corresponding disulfide pantethine play a key role in metabolism as building blocks of coenzyme A (CoA), an essential cofactor utilized in ~4% of primary metabolism and central to fatty acid, polyketide, and nonribosomal peptide synthases. Using a combination of recombinant engineering and chemical synthesis, we show that the disulfide of N-pantoylglycyl-2-aminoethanethiol (GlyPan), with one fewer carbon than pantetheine, can rescue a mutant E. coli strain MG1655ΔpanC lacking a functional pantothenate synthetase. Using mass spectrometry, we show that the GlyPan variant is accepted by the downstream CoA biosynthetic machinery, ultimately being incorporated into essential acyl carrier proteins. These findings point to further flexibility in CoA-dependent pathways and offer the opportunity to incorporate orthogonal analogues.

Integrated electroosmotic perfusion of tissue with online microfluidic analysis to track the metabolism of cystamine, pantethine, and coenzyme A.

We have developed an approach that integrates electroosmotic perfusion of tissue with a substrate-containing solution and online microfluidic analysis of products, in this case thiols. Using this approach we have tracked the metabolism of cystamine, pantethine and CoA in the extracellular space of organotypic hippocampal slice cultures (OHSCs). Currently, little is known about coenzyme A (CoA) biodegradation and even less is known about the regulation and kinetic characteristics for this sequential multienzyme reaction. We found that the steady state percentage yields of cysteamine from cystamine and pantethine during the transit through OHSCs were 91% ± 4% (SEM) and 0.01%-0.03%, respectively. The large difference in the yields of cysteamine can be used to explain the drugs' different toxicities and clinical effectiveness against cystinosis. The kinetic parameters of the enzyme reaction catalyzed by the ectoenzyme pantetheinase are KM,C/α = 4.4 ± 1.1 mM and Vmax,C = 29 ± 3 nM/s, where α is the percentage yield of pantethine to pantetheine through disulfide exchange. We estimate that the percentage yield of pantethine to pantetheine through disulfide exchange is approximately 0.5%. Based on the formation rate of cysteamine in the OHSCs, we obtained the overall apparent Michaelis constant and maximum reaction rate for sequential, extracellular CoA degradation in an in situ environment, which are K'M = 16 ± 4 µM, V'max = 7.1 ± 0.5 nM/s. Kinetic parameters obtained in situ, although difficult to measure, are better representations of the biochemical flux in the living organism than those from isolated enzymes in vitro.