Structure of a novel form of phosphopantetheine adenylyltransferase from Klebsiella pneumoniae at 2.59 Å 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 0.1 M HEPES buffer and PEG10000 at pH 7.5. The crystals belonged to tetragonal space group P41212 with cell dimensions of a = b = 72.82 Å and c = 200.37 Å. 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 ß/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 × 10-4 M for ATP which is significantly lower than the corresponding values reported for PPAT from other species.

Prebiotically plausible chemoselective pantetheine synthesis in water.

Coenzyme A (CoA) is essential to all life on Earth, and its functional subunit, pantetheine, is important in many origin-of-life scenarios, but how pantetheine emerged on the early Earth remains a mystery. Earlier attempts to selectively synthesize pantetheine failed, leading to suggestions that "simpler" thiols must have preceded pantetheine at the origin of life. In this work, we report high-yielding and selective prebiotic syntheses of pantetheine in water. Chemoselective multicomponent aldol, iminolactone, and aminonitrile reactions delivered spontaneous differentiation of pantoic acid and proteinogenic amino acid syntheses, as well as the dihydroxyl, gem-dimethyl, and ß-alanine-amide moieties of pantetheine in dilute water. Our results are consistent with a role for canonical pantetheine at the outset of life on Earth.

Thiolase: A Versatile Biocatalyst Employing Coenzyme A-Thioester Chemistry for Making and Breaking C-C Bonds.

Thiolases are CoA-dependent enzymes that catalyze the thiolytic cleavage of 3-ketoacyl-CoA, as well as its reverse reaction, which is the thioester-dependent Claisen condensation reaction. Thiolases are dimers or tetramers (dimers of dimers). All thiolases have two reactive cysteines: (a) a nucleophilic cysteine, which forms a covalent intermediate, and (b) an acid/base cysteine. The best characterized thiolase is the Zoogloea ramigera thiolase, which is a bacterial biosynthetic thiolase belonging to the CT-thiolase subfamily. The thiolase active site is also characterized by two oxyanion holes, two active site waters, and four catalytic loops with characteristic amino acid sequence fingerprints. Three thiolase subfamilies can be identified, each characterized by a unique sequence fingerprint for one of their catalytic loops, which causes unique active site properties. Recent insights concerning the thiolase reaction mechanism, as obtained from recent structural studies, as well as from classical and recent enzymological studies, are addressed, and open questions are discussed.

Interaction of PKR with STCS1: an indispensable step in the biosynthesis of lunularic acid in Marchantia polymorpha.

Unlike bibenzyls derived from the vascular plants, lunularic acid (LA), a key precursor for macrocyclic bisbibenzyl synthesis in nonvascular liverworts, exhibits the absence of one hydroxy group within the A ring. It was hypothesized that both polyketide reductase (PKR) and stilbenecarboxylate synthase 1 (STCS1) were involved in the LA biosynthesis, but the underlined mechanisms have not been clarified. This study used bioinformatics analysis with molecular, biochemical and physiological approaches to characterize STCS1s and PKRs involved in the biosynthesis of LA. The results indicated that MpSTCS1s from Marchantia polymorpha catalyzed both C2→C7 aldol-type and C6→C1 Claisen-type cyclization using dihydro-p-coumaroyl-coenzyme A (CoA) and malonyl-CoA as substrates to yield a C6-C2-C6 skeleton of dihydro-resveratrol following decarboxylation and the C6-C3-C6 type of phloretin in vitro. The protein-protein interaction of PKRs with STCS1 (PPI-PS) was revealed and proved essential for LA accumulation when transiently co-expressed in Nicotiana benthamiana. Moreover, replacement of the active domain of STCS1 with an 18-amino-acid fragment from the chalcone synthase led to the PPI-PS greatly decreasing and diminishing the formation of LA. The replacement also increased the chalcone formation in STCS1s. Our results highlight a previously unrecognized PPI in planta that is indispensable for the formation of LA.

Convenient site-selective protein coupling from bacterial raw lysates to coenzyme A-modified tobacco mosaic virus (TMV) by Bacillus subtilis Sfp phosphopantetheinyl transferase.

A facile enzyme-mediated strategy enables site-specific covalent one-step coupling of genetically tagged luciferase molecules to coenzyme A-modified tobacco mosaic virus (TMV-CoA) both in solution and on solid supports. Bacillus subtilis surfactin phosphopantetheinyl transferase Sfp produced in E. coli mediated the conjugation of firefly luciferase N-terminally extended by eleven amino acids forming a 'ybbR tag' as Sfp-selective substrate, which even worked in bacterial raw lysates. The enzymes displayed on the protein coat of the TMV nanocarriers exhibited high activity. As TMV has proven a beneficial high surface-area adapter template stabilizing enzymes in different biosensing layouts in recent years, the use of TMV-CoA for fishing ybbR-tagged proteins from complex mixtures might become an advantageous concept for the versatile equipment of miniaturized devices with biologically active proteins. It comes along with new opportunities for immobilizing multiple functionalities on TMV adapter coatings, as desired, e.g., in handheld systems for point-of-care detection.

PI3K drives the de novo synthesis of coenzyme A from vitamin B5.

In response to hormones and growth factors, the class I phosphoinositide-3-kinase (PI3K) signalling network functions as a major regulator of metabolism and growth, governing cellular nutrient uptake, energy generation, reducing cofactor production and macromolecule biosynthesis1. Many of the driver mutations in cancer with the highest recurrence, including in receptor tyrosine kinases, Ras, PTEN and PI3K, pathologically activate PI3K signalling2,3. However, our understanding of the core metabolic program controlled by PI3K is almost certainly incomplete. Here, using mass-spectrometry-based metabolomics and isotope tracing, we show that PI3K signalling stimulates the de novo synthesis of one of the most pivotal metabolic cofactors: coenzyme A (CoA). CoA is the major carrier of activated acyl groups in cells4,5 and is synthesized from cysteine, ATP and the essential nutrient vitamin B5 (also known as pantothenate)6,7. We identify pantothenate kinase 2 (PANK2) and PANK4 as substrates of the PI3K effector kinase AKT8. Although PANK2 is known to catalyse the rate-determining first step of CoA synthesis, we find that the minimally characterized but highly conserved PANK49 is a rate-limiting suppressor of CoA synthesis through its metabolite phosphatase activity. Phosphorylation of PANK4 by AKT relieves this suppression. Ultimately, the PI3K-PANK4 axis regulates the abundance of acetyl-CoA and other acyl-CoAs, CoA-dependent processes such as lipid metabolism and proliferation. We propose that these regulatory mechanisms coordinate cellular CoA supplies with the demands of hormone/growth-factor-driven or oncogene-driven metabolism and growth.

A phosphopantetheinyl transferase gene restricted to Porphyromonas.

The phosphopantetheinyl transferases (PPTases) catalyze the post-translational modification of carrier proteins (CPs) from fatty acid synthases (FASs) in primary metabolism and from polyketide synthases (PKSs) and non-ribosomal polypeptide synthases (NRPSs) in secondary metabolism. Based on the conserved sequence motifs and substrate specificities, two types (AcpS-type and Sfp-type) of PPTases have been identified in prokaryotes. We present here that Porphyromonas gingivalis, the keystone pathogen in chronic periodontitis, harbors merely one PPTase, namely PptP. Complementation and gene deletion experiments clearly show that PptP can replace the function of Escherichia coli AcpS and is essential for the growth of P. gingivalis. Purified PptP transfers the 4-phosphopantetheine moiety of CoA to inactive apo-acyl carrier protein (ACP) to form holo-ACP, which functions as an active carrier of the acyl intermediates of fatty acid synthesis. Moreover, PptP exhibits broad substrate specificity, modifying all ACP substrates tested and catalyzing the transfer of coenzyme A (CoA) derivatives. The lack of sequence alignment with known PPTases together with phylogenetic analyses revealed PptP as a new class of PPTases. Identification of the new PPTase gene pptP exclusive in Porphyromonas species reveals a potential target for treating P. gingivalis infections.

Pantothenate biosynthesis is critical for chronic infection by the neurotropic parasite Toxoplasma gondii.

Coenzyme A (CoA) is an essential molecule acting in metabolism, post-translational modification, and regulation of gene expression. While all organisms synthesize CoA, many, including humans, are unable to produce its precursor, pantothenate. Intriguingly, like most plants, fungi and bacteria, parasites of the coccidian subgroup of Apicomplexa, including the human pathogen Toxoplasma gondii, possess all the enzymes required for de novo synthesis of pantothenate. Here, the importance of CoA and pantothenate biosynthesis for the acute and chronic stages of T. gondii infection is dissected through genetic, biochemical and metabolomic approaches, revealing that CoA synthesis is essential for T. gondii tachyzoites, due to the parasite's inability to salvage CoA or intermediates of the pathway. In contrast, pantothenate synthesis is only partially active in T. gondii tachyzoites, making the parasite reliant on its uptake. However, pantothenate synthesis is crucial for the establishment of chronic infection, offering a promising target for intervention against the persistent stage of T. gondii.

phytanoyl-CoA dioxygenase domain-containing protein 1 plays an important role in egg shell formation of silkworm (Bombyx mori).

Yun7Ge is a giant egg mutant found in the silkworm variety Yun7. In comparison with the giant mutant Ge, the eggs of Yun7Ge are larger. The number of laid eggs and hatching rate of Yun7Ge are reduced, which is not conducive to reproduction. In this work, the target gene controlling giant egg trait is located on the Z chromosome and was determined through genetic analysis. Transcriptome results showed that phytanoyl-CoA dioxygenase domain-containing protein 1 (PHYHD1) on the Z chromosome was silenced, and the 25 chorion genes on chromosome 2 were remarkably downregulated. Sequence analysis showed that the 73.5 kb sequence including the PHYHD1 was replaced by a ~3.0 kb sequence. After knocking out the PHYHD1 by using CRISPR/Cas9, the chorion genes were significantly downregulated. Hence, the silencing of PHYHD1 leads to the downregulation of many chorion protein genes, thus directly causing giant eggs.

Amide Bond Formation Using 4-Coumarate: CoA Ligase from Arabidopsis thaliana.

Amide bond formation is one of the most fundamental reactions in organic chemistry, and amide bonds constitute the key functional groups in natural products, peptides, and pharmaceuticals. Here we demonstrate the chemoenzymatic syntheses of 4-coumaroyl- and hexanoyl-amino acids, using 4-coumarate: CoA ligase from the model plant Arabidopsis thaliana (At4CL2). At4CL2 accepts 4-coumaric acid and hexanoic acid as the carboxylate substrates to generate acyl adenylates, which are captured by the amino group of amino acids to afford a series of N-acyl amides. This study shows the potential of 4CL for application as a biocatalyst to generate a series of biologically active amide compounds.

Structures of a non-ribosomal peptide synthetase condensation domain suggest the basis of substrate selectivity.

Non-ribosomal peptide synthetases are important enzymes for the assembly of complex peptide natural products. Within these multi-modular assembly lines, condensation domains perform the central function of chain assembly, typically by forming a peptide bond between two peptidyl carrier protein (PCP)-bound substrates. In this work, we report structural snapshots of a condensation domain in complex with an aminoacyl-PCP acceptor substrate. These structures allow the identification of a mechanism that controls access of acceptor substrates to the active site in condensation domains. The structures of this complex also allow us to demonstrate that condensation domain active sites do not contain a distinct pocket to select the side chain of the acceptor substrate during peptide assembly but that residues within the active site motif can instead serve to tune the selectivity of these central biosynthetic domains.

DpgC-Catalyzed Peroxidation of 3,5-Dihydroxyphenylacetyl-CoA (DPA-CoA): Insights into the Spin-Forbidden Transition and Charge Transfer Mechanisms*.

Despite being a very strong oxidizing agent, most organic molecules are not oxidized in the presence of O2 at room temperature because O2 is a diradical whereas most organic molecules are closed-shell. Oxidation then requires a change in the spin state of the system, which is forbidden according to non-relativistic quantum theory. To overcome this limitation, oxygenases usually rely on metal or redox cofactors to catalyze the incorporation of, at least, one oxygen atom into an organic substrate. However, some oxygenases do not require any cofactor, and the detailed mechanism followed by these enzymes remains elusive. To fill this gap, here the mechanism for the enzymatic cofactor-independent oxidation of 3,5-dihydroxyphenylacetyl-CoA (DPA-CoA) is studied by combining multireference calculations on a model system with QM/MM calculations. Our results reveal that intersystem crossing takes place without requiring the previous protonation of molecular oxygen. The characterization of the electronic states reveals that electron transfer is concomitant with the triplet-singlet transition. The enzyme plays a passive role in promoting the intersystem crossing, although spontaneous reorganization of the water wire connecting the active site with the bulk presets the substrate for subsequent chemical transformations. The results show that the stabilization of the singlet radical-pair between dioxygen and enolate is enough to promote spin-forbidden reaction without the need for neither metal cofactors nor basic residues in the active site.

Phosphopantetheine Adenylyltransferase: A promising drug target to combat antibiotic resistance.

Phosphopantetheine Adenylyltransferase (PPAT) is an enzyme that catalyzes the penultimate step in the biosynthesis of Coenzyme A (CoA), which is the active and physiologically functional form of dietary Vitamin B5. CoA serves as a cofactor for numerous metabolic reactions which makes it essential for cellular survival. This enzyme is also subject to feedback inhibition by CoA to maintain its cellular concentration. The steps of the CoA biosynthesis pathway remain conserved from prokaryotes to eukaryotes, with humans and pathogenic micro-organisms showing significant diversity on a sequence, structure and mechanistic level. This suggests that the development of selective inhibitors of microbial CoA biosynthesis should be possible using these enzymes as targets for drug development. Bacterial PPAT shows significant mechanistic difference from its human counterpart CoA synthase, which is a dual protein carrying the activity of both PPAT and next step in the pathway catalyzed by the enzyme Dephospho CoA kinase (DPCK). This review covers the detailed description of the mechanistic, structural and functional aspects of this enzyme. Also, all the attempts to design high efficiency inhibitors of this enzyme using the approach of structure based drug design have been discussed in detail. This comprehensive structural and functional discussion of PPAT will help in further exploiting it as a drug target.

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.

Structural basis for nucleotide-independent regulation of acyl-CoA thioesterase from Bacillus cereus ATCC 14579.

Acyl-CoA thioesterase is an enzyme that catalyzes the cleavage of thioester bonds and regulates the cellular concentrations of CoASH, fatty acids, and acyl-CoA. In this study, we report the crystal structure of acyl-CoA thioesterase from Bacillus cereus ATCC 14579 (BcACT1) complexed with the CoA product. BcACT1 possesses a monomeric structure of a hotdog-fold and forms a hexamer via the trimerization of three dimers. We identified the active site of BcACT1 and revealed that residues Asn23 and Asp38 are crucial for enzyme catalysis, indicating that BcACT1 belongs to the TE6 family. We also propose that BcACT1 might undergo an open-closed conformational change on the acyl-CoA binding pocket upon binding of the acyl-CoA substrate. Interestingly, the BcACT1 variants with dramatically increased activities were obtained during the site-directed mutagenesis experiments to confirm the residues involved in CoA binding. Finally, we found that BcACT1 is not nucleotide-regulated and suggest that the length and shape of the additional α2-helix are crucial in determining a regulation mode by nucleotides.

Design and synthesis of Coenzyme A analogues as Aurora kinase A inhibitors: An exploration of the roles of the pyrophosphate and pantetheine moieties.

Coenzyme A (CoA) is a highly selective inhibitor of the mitotic regulatory enzyme Aurora A kinase, with a novel mode of action. Herein we report the design and synthesis of analogues of CoA as inhibitors of Aurora A kinase. We have designed and synthesised modified CoA structures as potential inhibitors, combining dicarbonyl mimics of the pyrophosphate group with a conserved adenosine headgroup and different length pantetheine-based tail groups. An analogue with a -SH group at the end of the pantotheinate tail showed the best IC50, probably due to the formation of a covalent bond with Aurora A kinase Cys290.

Crystal structure of an acetyl-CoA acetyltransferase from PHB producing bacterium Bacillus cereus ATCC 14579.

Bacillus cereus ATCC 14579 is a known polyhydroxybutyrate (PHB)-producing microorganism that possesses genes associated with PHB synthesis such as PhaA, PhaB, and PHA synthases. PhaA (i.e., thiolase) is the first enzyme in the PHA biosynthetic pathway, which catalyze the condensation of two acetyl-CoA molecules to acetoacetyl-CoA. Our study elucidated the crystal structure of PhaA in Bacillus cereus ATCC 14579 (BcTHL) in its apo- and CoA-bound forms. BcTHL adopts a type II biosynthetic thiolase structure by forming a tetramer. The crystal structure of CoA-complexed BcTHL revealed that the substrate binding site of BcTHL is constituted by different residues compared with other known thiolases. Our study also revealed that Arg221, a residue involved in ADP binding, undergoes a positional conformational change upon the binding of the CoA molecule.

Molecular basis for N-terminal alpha-synuclein acetylation by human NatB.

NatB is one of three major N-terminal acetyltransferase (NAT) complexes (NatA-NatC), which co-translationally acetylate the N-termini of eukaryotic proteins. Its substrates account for about 21% of the human proteome, including well known proteins such as actin, tropomyosin, CDK2, and α-synuclein (αSyn). Human NatB (hNatB) mediated N-terminal acetylation of αSyn has been demonstrated to play key roles in the pathogenesis of Parkinson's disease and as a potential therapeutic target for hepatocellular carcinoma. Here we report the cryo-EM structure of hNatB bound to a CoA-αSyn conjugate, together with structure-guided analysis of mutational effects on catalysis. This analysis reveals functionally important differences with human NatA and Candida albicans NatB, resolves key hNatB protein determinants for αSyn N-terminal acetylation, and identifies important residues for substrate-specific recognition and acetylation by NatB enzymes. These studies have implications for developing small molecule NatB probes and for understanding the mode of substrate selection by NAT enzymes.

Tartryl-CoA inhibits succinyl-CoA synthetase.

Succinyl-CoA synthetase (SCS) catalyzes the only substrate-level phosphorylation step in the tricarboxylic acid cycle. Human GTP-specific SCS (GTPSCS), an αß-heterodimer, was produced in Escherichia coli. The purified protein crystallized from a solution containing tartrate, CoA and magnesium chloride, and a crystal diffracted to 1.52 Šresolution. Tartryl-CoA was discovered to be bound to GTPSCS. The CoA portion lies in the amino-terminal domain of the α-subunit and the tartryl end extends towards the catalytic histidine residue. The terminal carboxylate binds to the phosphate-binding site of GTPSCS.

Evidence of a preferred kinetic pathway in the carnitine acetyltransferase reaction.

Mammalian carnitine acetyltransferase (CrAT) is a mitochondrial enzyme that catalyzes the reversible transfer of an acetyl group from acetyl-CoA to carnitine. CrAT knockout studies have shown that this enzyme is critical to sustain metabolic flexibility, or the ability to switch between different fuel types, an underlying theme of the metabolic syndrome. These recent physiological findings imply that CrAT dysfunction, or its catalytic impairment, may lead to disease. To gain insight into the CrAT kinetic mechanism, we conducted stopped-flow experiments in various enzyme substrate/product conditions and analyzed full progress curves by global fitting. Simultaneous mixing of both substrates with CrAT produced relatively fast kinetics that follows an ordered bi bi mechanism. A great preference for ordered binding is supported by stopped-flow double mixing experiments such that premixed CrAT with acetyl-CoA or CoA demonstrated a biphasic decrease in initial rate that produces about a 100-fold attenuation in catalysis. Double mixing experiments also revealed that the CrAT initial rate is inhibited by 50% in approximately 8 s by either acetyl-CoA or CoA premixing. Analysis of available CrAT structures support a substrate conformational change between acetyl-CoA/CoA binary versus ternary complexes. Additional viscosity-based kinetic experiments yielded strong evidence that product release is the rate limiting step in the CrAT-catalyzed reaction.