Conformation in solution of coenzyme A thioesters: II. Butyryl-CoA and arylacetyl-CoA compounds

The ¹H nuclear magnetic resonance (¹H-nmr) spectra of aqueous solutions of butyryl-CoA (Bu-CoA), indoleacetyl-CoA (IA-CoA), and phenylacetyl-CoA (PA-CoA) were examined at various concentrations and temperatures and compared to spectra of acetyl-CoA (Ac-CoA) and benzoyl-CoA (Bz-CoA) in order to determine to what extent, if any, each acyl-CoA compound exists in an intramolecular folded conformation. It was found previously that Ac-CoA exists predominantly in an extended conformation, whereas Bz-CoA is folded (Mieyal et al., J. Biol. Chem.249, 2633 (1974)). The present study showed: (a_ the solution behavior of Bu-CoA was essentially indistinguishable from that of Ac-CoA; thus both of these aliphatic CoA esters probably exist as extended molecules; (b) IA-CoA does form an intramolecular adenyl-indolyl complex, but the folded/unfolded ratio is only about one-half of that for Bz-CoA at physiological temperature; (c) PA-CoA apparently exists predominantly as an unfolded molecule. The diminished tendency of PA-CoA and IA-CoA to fold is probably related to the rotational mobility about the bond adjoining the respective arylmethylene group to the carbonyl moiety of the thioester link. Concentration-independent effects of the phenyl and indolyl ring currents on the ¹H-nmr signals of particular pantotheinyl methylene groups in PA-CoA and IA-CoA, respectively, are consistent with this conclusion. The outstanding conformational difference between the closely related Bz-CoA (folded) and PA-CoA (unfolded) molecules may provide insight regarding the nature of the binding sites for these molecules on the specific N-acyltransferase enzymes for which they are the best substrates (Webster et al., J. Biol. Chem.251, 3352 (1976)).     

Divergence of cofactor recognition across evolution: coenzyme A binding in a prokaryotic arylamine N-acetyltransferase

Arylamine N-acetyltransferase (NAT) enzymes are widespread in nature. They serve to acetylate xenobiotics and/or endogenous substrates using acetyl coenzyme A (CoA) as a cofactor. Conservation of the architecture of the NAT enzyme family from mammals to bacteria has been demonstrated by a series of prokaryotic NAT structures, together with the recently reported structure of human NAT1. We report here the cloning, purification, kinetic characterisation and crystallographic structure determination of NAT from Mycobacterium marinum, a close relative of the pathogenic Mycobacterium tuberculosis. We have also determined the structure of M. marinum NAT in complex with CoA, shedding the first light on cofactor recognition in prokaryotic NATs. Surprisingly, the principal CoA recognition site in M. marinum NAT is located some 30 Å from the site of CoA recognition in the recently deposited structure of human NAT2 bound to CoA. The structure explains the Ping-Pong Bi-Bi reaction mechanism of NAT enzymes and suggests mechanisms by which the acetylated enzyme intermediate may be protected. Recognition of CoA in a much wider groove in prokaryotic NATs suggests that this subfamily may accommodate larger substrates than is the case for human NATs and may assist in the identification of potential endogenous substrates. It also suggests the cofactor-binding site as a unique subsite to target in drug design directed against NAT in mycobacteria.     

The structures of Thermoplasma volcanium phosphoribosyl pyrophosphate synthetase bound to ribose-5-phosphate and ATP analogs

Phosphoribosyl pyrophosphate (PRPP) synthetase catalyzes the transfer of the pyrophosphate group from ATP to ribose-5-phosphate (R5P) yielding PRPP and AMP. PRPP is an essential metabolite that plays a central role in cellular metabolism. The enzyme from a thermophilic archaeon Thermoplasma volcanium (Tv) was expressed in Escherichia coli, crystallized, and its X-ray molecular structure was determined in a complex with its substrate R5P and with substrate analogs β,γ-methylene ATP and ADP in two monoclinic crystal forms, P2₁. The β,γ-methylene ATP- and the ADP-bound binary structures were determined from crystals grown from ammonium sulfate solutions; these crystals diffracted to 1.8 Å and 1.5 Å resolutions, respectively. Crystals of the ternary complex with ADP-Mg²⁺ and R5P were grown from a polyethylene glycol solution in the absence of sulfate ions, and they diffracted to 1.8 Å resolution; the unit cell is approximately double the size of the unit cell of the crystals grown in the presence of sulfate. The Tv PRPP synthetase adopts two conformations, open and closed, at different stages in the catalytic cycle. The binding of substrates, R5P and ATP, occurs with PRPP synthetase in the open conformation, whereas catalysis presumably takes place with PRPP synthetase in the closed conformation. The Tv PRPP synthetase forms a biological dimer in contrast to the tetrameric or hexameric quaternary structures of the Methanocaldococcus jannaschii and Bacillus subtilis PRPP synthetases, respectively.     

Comprehensive structural modeling and preparation of human 5-HT2A G-protein coupled receptor in functionally active form

The serotonin 2A receptor (5-HT_2AR) is an important member of the G-protein coupled receptor (GPCR) family involved in an array of neuromodulatory functions. Although the high-resolution structures of truncated versions of GPCRs, captured in ligand-bound conformational states, are available, the structures lack several functional regions, which have crucial roles in receptor response. Here, in order to understand the structure and dynamics of the ligand-free form of the receptor, we have performed meticulous modeling of the 5-HT_2AR with the third intracellular loop (ICL3). Our analyses revealed that the ligand-free ground state structure of 5-HT_2AR has marked distinction with ligand-bound conformations of 5-HT₂ subfamily proteins and exhibits extensive backbone flexibility across the loop regions, suggesting the importance of purifying the receptor in its native form for further studies. Hence, we have standardized a strategy that efficiently increases the expression of 5-HT_2AR by infecting Sf9 cells with a very low multiplicity of infection of baculovirus in conjunction with production boost additive and subsequently, purify the full-length receptor. Furthermore, we have optimized the selective over-expression of glycosylated and nonglycosylated forms of the receptor merely by switching the postinfection growth time, a method that has not been reported earlier.     

A specific and sensitive enzymatic-isotopic microassay of coenzyme A in pineal gland

The relationship between the concentration of CoASH and the activity of serotonin N-acetyltransferase (NAT) was studied in rat pineal glands in culture. A technique for microdetermination of CoASH was developed by utilizing acetyl CoA synthetase and partially purified rat liver NAT. Initially CoASH was acetylated with [1–³H] acetate using acetyl CoA synthetase. Subsequently, the labelled acetyl group was transferred from [1–³H] acetyl CoA to tryptamine forming [1–³H acetyl-tryptamine which was then extracted into chloroform and measured by scintillation spectrometry. A direct relationship appeared to exist between the concentrations of CoASH and [1–³H] acetyltryptamine. This method is sensitive and specific since it can detect as low as 10–15 pmoles of CoASH but not structurally related substances such as acetyl CoA, ADP, cysteamine, or D-pantothenic acid. After treating the rat pineal glands in culture with 10 μM norepinephrine for six hours, the concentration of CoASH was found to decrease significantly from 31.96 ± 0.68 to 24.44 ± 0.37 pmoles/gland, while the activity of NAT increased 68 fold. This inverse relationship indicates that CoASH does not play a direct role in NAT induction although it does protect darktime NAT activity in pineal homogenates against thermal inactivation. The sensitivity and the adaptability of this method can be utilized to measure CoASH in discrete regions of rat brain and in experimental conditions where the micromeasurement of CoASH may be required.     

Slc25a17 acts as a peroxisomal coenzyme A transporter and regulates multiorgan development in zebrafish

Slc25a17 is known as a peroxisomal solute carrier, but the in vivo role of the protein has not been demonstrated. We found that the zebrafish genome contains two slc25a17 genes that function redundantly, but additively. Notably, peroxisome function in slc25a17 knockdown embryos is severely compromised, resulting in an altered lipid composition. Along the defects found in peroxisome-associated phenotypic presentations, we highlighted that development of the swim bladder is also highly dependent on Slc25a17 function. As Slc25a17 showed substrate specificity towards coenzyme A (CoA), injecting CoA, but not NAD⁺, rescued the defective swim bladder induced by slc25a17 knockdown. These results indicated that Slc25a17 acts as a CoA transporter, involved in the maintenance of functional peroxisomes that are essential for the development of multiple organs during zebrafish embryogenesis. Given high homology in protein sequences, the role of zebrafish Slc25a17 may also be applicable to the mammalian system.     

Structural,Functional, and Inhibition Studies of a Gcn5-related N-Acetyltransferase (GNAT) Superfamily Protein PA4794: A NEW C-TERMINAL LYSINE PROTEIN ACETYLTRANSFERASE FROM PSEUDOMONAS AERUGINOSA*

The Gcn5-related N-acetyltransferase (GNAT) superfamily is a large group of evolutionarily related acetyltransferases, with multiple paralogs in organisms from all kingdoms of life. The functionally characterized GNATs have been shown to catalyze the transfer of an acetyl group from acetyl-coenzyme A (Ac-CoA) to the amine of a wide range of substrates, including small molecules and proteins. GNATs are prevalent and implicated in a myriad of aspects of eukaryotic and prokaryotic physiology, but functions of many GNATs remain unknown. In this work, we used a multi-pronged approach of x-ray crystallography and biochemical characterization to elucidate the sequence-structure-function relationship of the GNAT superfamily member PA4794 from Pseudomonas aeruginosa. We determined that PA4794 acetylates the Nϵ amine of a C-terminal lysine residue of a peptide, suggesting it is a protein acetyltransferase specific for a C-terminal lysine of a substrate protein or proteins. Furthermore, we identified a number of molecules, including cephalosporin antibiotics, which are inhibitors of PA4794 and bind in its substrate-binding site. Often, these molecules mimic the conformation of the acetylated peptide product. We have determined structures of PA4794 in the apo-form, in complexes with Ac-CoA, CoA, several antibiotics and other small molecules, and a ternary complex with the products of the reaction: CoA and acetylated peptide. Also, we analyzed PA4794 mutants to identify residues important for substrate binding and catalysis.     

Reversible covalent inhibition of a phenol sulfotransferase by coenzyme A

Phenol sulfotransferases (SULTs), which normally bind 3'-phosphoadenosine-5'-phosphosulfate as the donor substrate, are inhibited by CoA and its thioesters. Here, we report that inhibition of bovine SULT1A1 by CoA is time-dependent at neutral pH under non-reducing conditions. The rates of inactivation by CoA indicate an initial reversible SULT:CoA complex with a dissociation constant of 5.7 microM and an inactivation rate constant of 0.07 min(-1). Titrations with CoA and prolonged incubations reveal that inactivation of the dimeric enzyme is stoichiometric, consistent with the observation of complete conversion of the protein to a slightly decreased electrophoretic mobility. Both activity and normal electrophoretic migration are restored by 2-mercaptoethanol. Mutagenesis demonstrated that Cys168 is the site of CoA adduction, and a consistent model was constructed that reveals a new SULT molecular dynamic. Cysteine reaction kinetics with Ellman's reagent revealed a PAPS-induced structural change consistent with the model that accounts for binding of CoA.     

The role of acetyl coenzyme A: butyrate coenzyme A in the transferase uptake of butyrate by isolated membrane vesicles of Escherichia coli

The acetyl CoA:butyrate CoA transferase catalyzes the translocation of butyrate in membrane vesicles prepared from a strain of Escherichia coli which is depressed for the acetoacetate degradation operon. Butyrate accumulated in the membranes as butyryl CoA. The role of the transferase in uptake is supported by the following observations: (i) uptake is stimulated by acetyl CoA; (ii) the solubilized CoA transferase and uptake exhibit K_mS for butyrate, pH optima and levels inhibition by N-ethylmaleimide that are virtually identical; (iii) significant amounts of the CoA transferase are found associated with the membranes and uptake is rapidly inhibited by butyryl CoA and acetate, the products of the CoA transferase-catalyzed reaction. The fact that butyrate uptake did not exhibit saturation kinetics with increasing concentrations of acetyl CoA suggested that the transferase is not localized on the outer surface of the membrane. The level of free butyrate in the vesicles, the fact that butyrate uptake exhibited saturation kinetics with increasing concentrations of butyrate, and the observation that radioactivity was not rapidly lost from the vesicles following addition of butyryl CoA or acetate to incubation mixtures indicated that butyrate is translocated rather than trapped by the CoA transferase.     

Tuning riboswitch regulation through conformational selection

The S_MK box riboswitch, which represents one of three known classes of S-adenosylmethionine (SAM)-responsive riboswitches, regulates gene expression in bacteria at the level of translation initiation. In contrast to most riboswitches, which contain separate domains responsible for ligand recognition and gene regulation, the ligand-binding and regulatory domains of the S_MK box riboswitch are coincident. This property was exploited to allow the first atomic-level characterization of a functionally intact riboswitch in both the ligand-bound state and the ligand-free state. NMR spectroscopy revealed distinct mutually exclusive RNA conformations that are differentially populated in the presence or in the absence of the effector metabolite. Isothermal titration calorimetry and in vivo reporter assay results revealed the thermodynamic and functional consequences of this conformational equilibrium. We present a comprehensive model of the structural, thermodynamic, and functional properties of this compact RNA regulatory element.     

Acyl coenzyme A dependent retinol esterification by acyl coenzyme A:diacylglycerol acyltransferase 1

We provide biochemical evidence that enzymes involved in the synthesis of triacylglycerol, namely acyl coenzyme A:diacylglycerol acyltransferase (DGAT) and acyl coenzyme A:monoacylglycerol acyltransferase (MGAT), are capable of carrying out the acyl coenzyme A:retinol acyltransferase (ARAT) reaction. Among them, DGAT1 appears to have the highest specific activity. The apparent K_m values of recombinant DGAT1/ARAT for retinol and palmitoyl coenzyme A were determined to be 25.9 ± 2.1 μM and 13.9 ± 0.3 μM, respectively, both of which are similar to the values previously determined for ARAT in native tissues. A novel selective DGAT1 inhibitor, XP620, inhibits recombinant DGAT1/ARAT at the retinol recognition site. In the differentiated Caco-2 cell membranes, XP620 inhibits ～85% of the Caco-2/ARAT activity indicating that DGAT1/ARAT may be the major source of ARAT activity in these cells. Of the two most abundant fatty acyl retinyl esters present in the intact differentiated Caco-2 cells, XP620 selectively inhibits retinyl–oleate formation without influencing the retinyl–palmitate formation. Using this inhibitor, we estimate that ～64% of total retinyl ester formation occurs via DGAT1/ARAT. These studies suggest that DGAT1/ARAT is the major enzyme involved in retinyl ester synthesis in Caco-2 cells.     

Long Noncoding RNA Expression Profiling Reveals Upregulation of Uroplakin 1A and Uroplakin 1A Antisense RNA 1 under Hypoxic Conditions in Lung Cancer Cells

Hypoxia plays important roles in cancer progression by inducing angiogenesis, metastasis, and drug resistance. However, the effects of hypoxia on long noncoding RNA (lncRNA) expression have not been clarified. Herein, we evaluated alterations in lncRNA expression in lung cancer cells under hypoxic conditions using lncRNA microarray analyses. Among 40,173 lncRNAs, 211 and 113 lncRNAs were up- and downregulated, respectively, in both A549 and NCI-H460 cells. Uroplakin 1A (UPK1A) and UPK1A-antisense RNA 1 (AS1), which showed the highest upregulation under hypoxic conditions, were selected to investigate the effects of UPK1A-AS1 on the expression of UPK1A and the mechanisms of hypoxia-inducible expression. Following transfection of cells with small interfering RNA (siRNA) targeting hypoxia-inducible factor 1α (HIF-1α), the hypoxia-induced expression of UPK1A and UPK1A-AS1 was significantly reduced, indicating that HIF-1α played important roles in the hypoxia-induced expression of these targets. After transfection of cells with UPK1A siRNA, UPK1A and UPK1A-AS1 levels were reduced. Moreover, transfection of cells with UPK1A-AS1 siRNA downregulated both UPK1A-AS1 and UPK1A. RNase protection assays demonstrated that UPK1A and UPK1A-AS1 formed a duplex; thus, transfection with UPK1A-AS1 siRNA decreased the RNA stability of UPK1A. Overall, these results indicated that UPK1A and UPK1A-AS1 expression increased under hypoxic conditions in a HIF-1α-dependent manner and that formation of a UPK1A/UPK1A-AS1 duplex affected RNA stability, enabling each molecule to regulate the expression of the other.     

Stability of Ligand-induced Protein Conformation Influences Affinity in Maltose-binding Protein

Our understanding of what determines ligand affinity of proteins is poor, even with high-resolution structures available. Both the non-covalent ligand–protein interactions and the relative free energies of available conformations contribute to the affinity of a protein for a ligand. Distant, non-binding site residues can influence the ligand affinity by altering the free energy difference between a ligand-free and ligand-bound conformation. Our hypothesis is that when different ligands induce distinct ligand-bound conformations, it should be possible to tweak their affinities by changing the free energies of the available conformations. We tested this idea for the maltose-binding protein (MBP) from Escherichia coli. We used single-molecule Förster resonance energy transfer (smFRET) to distinguish several unique ligand-bound conformations of MBP. We engineered mutations, distant from the binding site, to affect the stabilities of different ligand-bound conformations. We show that ligand affinity can indeed be altered in a conformation-dependent manner. Our studies provide a framework for the tuning of ligand affinity, apart from modifying binding site residues.     

Purification and characterization of benzoate:coenzyme A ligase from Clarkia breweri

Benzoate:CoA ligase (BZL) was partially purified from flowers of the annual California plant Clarkia breweri. BZL catalyzes the formation of benzoyl-CoA and anthraniloyl-CoA, important intermediates for subsequent acyltransferase reactions in plant secondary metabolism. The native enzyme is active as a monomer with a molecular mass of approximately 59-64.5 kDa, and it has K(m) values of 45, 95, and 130 microM for benzoic acid, ATP, and CoA, respectively. BZL is most active in the pH range of 7.2-8.4, and its activity is strictly dependent on certain bivalent cations. BZL is an AMP-forming enzyme. Overall, its properties suggest that it is related to the family of CoA ligase enzymes that includes the plant enzyme 4-hydroxycinnamate:CoA ligase.     

Leukocyte Lysis and Cytokine Induction by the Human Sexually Transmitted Parasite Trichomonas vaginalis

Trichomonas vaginalis (Tv) is an extracellular protozoan parasite that causes the most common non-viral sexually transmitted infection: trichomoniasis. While acute symptoms in women may include vaginitis, infections are often asymptomatic, but can persist and are associated with medical complications including increased HIV susceptibility, infertility, pre-term labor, and higher incidence of cervical cancer. Heightened inflammation resulting from Tv infection could account for these complications. Effective cellular immune responses to Tv have not been characterized, and re-infection is common, suggesting a dysfunctional adaptive immune response. Using primary human leukocyte components, we have established an in vitro co-culture system to assess the interaction between Tv and the cells of the human immune system. We determined that in vitro, Tv is able to lyse T-cells and B-cells, showing a preference for B-cells. We also found that Tv lysis of lymphocytes was mediated by contact-dependent and soluble factors. Tv lysis of monocytes is far less efficient, and almost entirely contact-dependent. Interestingly, a common symbiont of Tv, Mycoplasma hominis, did not affect cytolytic activity of the parasite, but had a major impact on cytokine responses. M. hominis enabled more diverse inflammatory cytokine secretion in response to Tv and, of the cytokines tested, Tv strains cleared of M. hominis induced only IL-8 secretion from monocytes. The quality of the adaptive immune response to Tv is therefore likely influenced by Tv symbionts, commensals, and concomitant infections, and may be further complicated by direct parasite lysis of effector immune cells.