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1.
N-Acyl-phosphatidylethanolamines (NAPEs) are known to be precursors of bioactive N-acylethanolamines (NAEs), including the endocannabinoid arachidonoylethanolamide (anandamide) and anti-inflammatory palmitoylethanolamide. In mammals, NAPEs are produced by N-acyltransferases, which transfer an acyl chain from the sn-1 position of glycerophospholipid to the amino group of phosphatidylethanolamine (PE). Recently, the ɛ isoform of cytosolic phospholipase A2 (cPLA2ɛ) was found to be Ca2+-dependent N-acyltransferase. However, it was poorly understood which types of phospholipids serve as substrates in living cells. In the present study, we established a human embryonic kidney 293 cell line, in which doxycycline potently induces human cPLA2ɛ, and used these cells to analyze endogenous substrates and products of cPLA2ɛ with liquid chromatography-tandem mass spectrometry. When treated with doxycycline and Ca2+ ionophore, the cells produced various species of diacyl- and alkenylacyl-types of NAPEs as well as NAEs in large quantities. Moreover, the levels of diacyl- and alkenylacyl-types of PEs and diacyl-phosphatidylcholines (PCs) decreased, while those of lysophosphatidylethanolamines and lysophosphatidylcholines increased. These results suggested that cPLA2ɛ Ca2+-dependently produces NAPEs by utilizing endogenous diacyl- and alkenylacyl-types of PEs as acyl acceptors and diacyl-type PCs and diacyl-type PEs as acyl donors.  相似文献   

2.
N-Acylphosphatidylethanolamines (NAPEs) are precursors of endogenous bioactive lipids, N-acylethanolamines (NAEs). NAPEs, which occur as a minor membrane lipid, are hydrolyzed in a single enzymatic step catalyzed by a type of phospholipase D (PLD) to generate fatty acid ethanolamides. Although, the occurrence of NAPE is widespread in the plant kingdom, the physiological roles remain under appreciated due to the lack of sensitive tools to quantify the pathway metabolites. In Kilaru et al. (2012, Planta, DOI 10.1007/s00425-012-1669-z), comprehensive mass spectrometry (MS)-based methods were developed to gain a clearer understanding of the complex network of metabolites that participate in NAE metabolic pathway. This targeted lipidomics approach allowed insights to be drawn into the implications of altered NAE levels on NAPE content and composition, and the overall regulation of PLD-mediated hydrolysis in Arabidopsis. Based on these results, we point out here the important need for the identification of the precise isoform(s) of PLD in plants that is (are) involved in the regulated hydrolysis of NAPE and formation of NAE lipid mediators in vivo.  相似文献   

3.
We propose the use of Lee-Goldburg decoupling in high-resolution natural abundance 13C CP-MAS NMR spectroscopy to obtain J-resolved multiplets from membrane lipids, and the use of these in spectral assignment and to investigate changes in molecular and segmental dynamics within chemical shift-resolved lipid groups. Spectroscopic characteristics of hydrated DPPC bilayers are reported, including J1CH-couplings from the liquid crystalline and gel phases. The observed J1θCH values are scaled in the Lee-Goldburg experiment by a factor of approximately 3−1/2 and corrected J1CH values on the order of 150 Hz compare well with indirect measurements. The J-resolved multiplets show J1θCH-couplings from the chain region to be approximately 20% lower than couplings determined from the headgroup, with backbone values falling between the two. Sensitivity of Lee-Goldburg decoupling to molecular motions reveals changes in hydrocarbon chain and backbone segmental dynamics across the main phospholipid transition and reduction in headgroup mobility below the pre-transition.  相似文献   

4.
N-Formyl, N-chloroacetyl, N-glycyl, N-isobutyryl, and N-pentanoyl derivatives of chitosan have been prepared. N-Acetylchitosan was the derivative most susceptible to chitinase from Streptomyces griseus and lysozyme from chicken egg-white, but the susceptibility was not restrictive. The relative rates of hydrolysis by chitinase with respect to R in the RCONH group were CH3 > CH3CH2 > H > CH3CH2CH2 > (CH3)2CH > NH2CH2 > ClCH2. Neither enzyme hydrolysed chitosan or its N-methylene, N-benzylidene, N-benzoyl, N-nicotinyl, and N-fatty acyl (C5C18) derivatives, and lysozyme did not hydrolyse N-butyrylchitosan. N-Acetylhexanoyl-chitosans, which had d.s. ratios of ~0.7: ~0.3 and ~0.3; ~0.7, were hydrolysed at ~0.75 and ~0.04 of the rate of N-acetylchitosan (powder) by chitinase. O-Acylation of N-acylchitosans caused a decrease in the rates of hydrolysis by chitinase. N-Acetylchitosan gels were hydrolysed at 8–13 times the rate for crab-shell chitin. These results indicate that not only N- and O-substituents but also the physical form of the substrates influence the rates of hydrolysis by these enzymes.  相似文献   

5.
N-Acyl-phosphatidylethanolamines (NAPEs), a minor class of membrane glycerophospholipids, accumulate along with their bioactive metabolites, N-acylethanolamines (NAEs) during ischemia. NAPEs can be formed through N-acylation of phosphatidylethanolamine by cytosolic phospholipase A2ε (cPLA2ε, also known as PLA2G4E) or members of the phospholipase A and acyltransferase (PLAAT) family. However, the enzyme responsible for the NAPE production in brain ischemia has not yet been clarified. Here, we investigated a possible role of cPLA2ε using cPLA2ε-deficient (Pla2g4e?/?) mice. As analyzed with brain homogenates of wild-type mice, the age dependency of Ca2+-dependent NAPE-forming activity showed a bell-shape pattern being the highest at the first week of postnatal life, and the activity was completely abolished in Pla2g4e?/? mice. However, liquid chromatography-tandem mass spectrometry revealed that the NAPE levels of normal brain were similar between wild-type and Pla2g4e?/? mice. In contrast, post-mortal accumulations of NAPEs and most species of NAEs were only observed in decapitated brains of wild-type mice. These results suggested that cPLA2ε is responsible for Ca2+-dependent formation of NAPEs in the brain as well as the accumulation of NAPEs and NAEs during ischemia, while other enzyme(s) appeared to be involved in the maintenance of basal NAPE levels.  相似文献   

6.
N-Acyl phosphatidylethanolamines are negatively charged phospholipids, which are naturally occurring albeit at low abundance. In this study, we have examined how the amide-linked acyl chain affected the membrane behavior of the N-acyl-1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (N-acyl-POPE) or N-acyl-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (N-acyl-DPPE), and how the molecules interacted with cholesterol. The gel → liquid crystalline transition temperature of sonicated N-acyl phosphatidylethanolamine vesicles in water correlated positively with the number of palmitic acyl chains in the molecules. Based on diphenylhexatriene steady state anisotropy measurements, the presence of 33 mol% cholesterol in the membranes removed the phase transition from N-oleoyl-POPE bilayers, but failed to completely remove it from N-palmitoyl-DPPE and N-palmitoyl-POPE bilayers, suggesting rather weak interaction of cholesterol with the N-saturated NAPEs. The rate of cholesterol desorption from mixed monolayers containing N-palmitoyl-DPPE and cholesterol (1:1 molar ratio) was much higher compared to cholesterol/DPPE binary monolayers, suggesting a weak cholesterol interaction with N-palmitoyl-DPPE also in monolayers. In bilayer membranes, both N-palmitoyl-POPE and N-palmitoyl-DPPE failed to form sterol-rich domains, and in fact appeared to displace sterol from sterol/N-palmitoyl-sphingomyelin domains. The present data provide new information about the effects of saturated NAPEs on the lateral distribution of cholesterol in NAPE-containing membranes. These findings may be of relevance to neural cells which accumulate NAPEs during stress and cell injury.  相似文献   

7.
A new method is described for the semisynthetic preparation of mixed-acid phosphatidylethanolamine (PE) having the natural steric configuration. Any phospholipid mixture from natural sources, e.g. soya phospholipids, can be used as the starting material. In the first step, PE is reacted with tritylbromide, and the resulting N-trityl-phosphatidylethanolamine is converted to N-trityl-glycerophosphoethanolamine (N-trityl-GPE) by alkaline hydrolysis. Reaction with tritylchloride yields 1-O,N-ditrityl-GPE, which is acylated in the 2-position with, e.g. acylimidazolides. The 1-O-protecting trityl group is then selectively removed in the presence of borontrifluoride-methanol, and the second acyl moiety is introduced by acylation with fatty acid anhydrides. After N-detritylation with trifluoroacetic acid, the final product is obtained in high yield and with less than 10% of the positional isomer. The main advantages of the new method are that it requires only a few reaction steps, that some of the intermediates need not be isolated, and that no enzymatic reaction is involved. Thus, the procedure described here can be applied to the synthesis of mixed-acid PEs on a technical scale.  相似文献   

8.
N-Acylethanolamines (NAEs) are lipids involved in several physiological processes in animal and plant cells. In brain, NAEs are ligands of endocannabinoid receptors, which modulate various signaling pathways. In plant, NAEs regulate seed germination and root development, and they are involved in plant defense against pathogen attack. This signaling activity is started by an enzyme called N-acylphosphatidylethanolamine (NAPE) synthase. This catalyzes the N-acylation of phosphatidylethanolamine to form NAPE, which is most likely hydrolyzed by phospholipase D β/γ isoforms to generate NAE. This compound is further catabolized by fatty amide hydrolase. The genes encoding the enzymes involved in NAE metabolism are well characterized except for the NAPE synthase gene(s). By heterologous expression in Escherichia coli and overexpression in plants, we characterized an acyltransferase from Arabidopsis thaliana (At1g78690p) catalyzing the synthesis of lipids identified as NAPEs (two-dimensional TLC, phospholipase D hydrolysis assay, and electrospray ionization-tandem mass spectrometry analyses). The ability of free fatty acid and acyl-CoA to be used as acyl donor was compared in vitro with E. coli membranes and purified enzyme (obtained by immobilized metal ion affinity chromatography). In both cases, NAPE was synthesized only in the presence of acyl-CoA. β-Glucuronidase promoter experiments revealed a strong expression in roots and young tissues of plants. Using yellow fluorescent protein fusion, we showed that the NAPE synthase is located in the plasmalemma of plant cells.N-Acylethanolamines (NAEs)2 are bioactive lipids composed of an ethanolamine headgroup amide-linked to an acyl chain varying in length and degree of saturation. In animals, NAEs are involved in different physiological processes, such as neuroprotective action (1), embryo development (2), cell proliferation (3), apoptosis (4), nociception, anxiety, inflammation, appetite/anorexia, learning, and memory (for review, see Ref. 5). Most studies carried out with animal cells/tissues have focused on N-arachidonoylethanolamine (anandamide, NAE20:4), which is synthesized in brain neurons but also, under certain conditions, in macrophage cells (6). NAE20:4 binds CB1 cannabinoid receptors located in brain neurons (7) and also acts as ligand of vanilloid receptors for pain modulation (8). In addition, it has been shown that NAE20:4 also promotes food intake, whereas NAE18:0 and NAE18:1 exert anorexic effects by increasing satiety (911). NAE16:0 is accumulated during inflammation and has several anti-inflammatory effects (for a review, see Ref. 12).In plants, NAEs are thought to be involved in various physiological functions. For example, because NAE levels observed in various dry seeds decline rapidly after imbibition, a possible role of these compounds in the regulation of seed germination has been proposed (13). It was further observed that the addition of 25 μm NAE12:0 to growth medium of Arabidopsis thaliana leads to a decrease in the size of the main and lateral roots and in root hair formation. This reduction in growth was associated with a modification of cytoskeletal organization (14). NAE12:0 is also able to delay cut Dianthus caryophyllus (carnation) senescence by decreasing oxidative damage and enhancing antioxidant defense (15), whereas NAE14:0 inhibits the elicitor-induced medium alkalinization and activates phenylalanine ammonia lyase gene expression involved in plant defense against pathogen attack (16).Both in plant and animal cells (for a review, see Ref. 17), NAEs are formed by the hydrolysis (by PLDs) of N-acylphosphatidylethanolamine (NAPE). NAPE is an unusual derivative of phosphatidylethanolamine (PE) with a third fatty acid linked to the amine position of the ethanolamine headgroup. In animals, the formation of NAEs is catalyzed by a PLD with a high specificity toward NAPE (NAPE-PLD). In plants, PLDβ and PLDγ isoforms, but not PLDα, hydrolyzed NAPE into NAE in vitro, and this is thought to operate in response to several biotic and abiotic stresses. Both in animals and in plants, NAEs signaling is terminated by the action of fatty acid amide hydrolases, which hydrolyze NAEs to free fatty acid and ethanolamine. FAAH has been identified and characterized in mammals and plants (for a review, see Ref. 17). In Arabidopsis, FAAH has been shown to modulate NAE content. Moreover, lines overexpressing FAAH displayed enhanced seedling growth as well as increased cell size (18) and were also more susceptible to bacterial pathogens (19).Although the role of NAEs and their catabolism have been extensively investigated, little is known about their precursors, the NAPEs. NAPEs represent a minor phospholipid class but are present in all tissues of plants and animals. The principal function of NAPEs is to serve as a precursor for the production of lipid mediator NAEs, but it has also been suggested that NAPEs could serve as a membrane stabilizer to maintain cellular compartmentalization during tissue damage (20). More recently, N-palmitoyl-PE was proposed to act as an inhibitor of macrophage phagocytosis through inhibition of the activation of Rac1 and Cdc42 (21).In the animal and plant kingdoms, therefore, the signaling events mediated by NAEs appear to be involved in many physiological processes that have been extensively studied. The genes encoding the enzymes involved in the synthesis (from NAPEs) and the degradation of NAEs have been cloned and characterized. By contrast, little is known about the physiological roles of NAPEs or about the first step of this lipid signaling pathway, namely the N-acylation of PE to form NAPEs. In animals, the synthesis of NAPEs is catalyzed by an N-acyltransferase, where the O-linked acyl unit from a phospholipid donor is transferred to the ethanolamine headgroup of PE (22). Recently, a rat LRAT-like protein 1 or RLP1 was shown to display such an activity, but according to the authors, RLP-1 can function as a PE N-acyltransferase, catalytically distinguishable from the known Ca2+-dependent N-acyltransferase (23). However, a different situation is observed in plants. NAPE synthase activity was shown to directly acylate PE with free fatty acids (24, 25), but a gene encoding a NAPE synthase activity remained unidentified until now. The present work shows that the A. thaliana acyltransferase At1g78690p catalyzes the synthesis of NAPEs from PE and acyl-CoAs in vitro as well as in vivo when this enzyme is expressed in E. coli and overexpressed in plants.  相似文献   

9.
We have previously shown that simple N-acyl or N-alkyl polyamines bind to and sequester Gram-negative bacterial lipopolysaccharide, affording protection against lethality in animal models of endotoxicosis. Several iterative design-and-test cycles of SAR studies, including high-throughput screens, had converged on compounds with polyamine scaffolds which have been investigated extensively with reference to the number, position, and length of acyl or alkyl appendages. However, the polyamine backbone itself had not been explored sufficiently, and it was not known if incremental variations on the polymethylene spacing would affect LPS-binding and neutralization properties. We have now systematically explored the relationship between variously elongated spermidine [NH2–(CH2)3–NH–(CH2)4–NH2] and norspermidine [NH2–(CH2)3–NH–(CH2)3–NH2] backbones, with the N-alkyl group being held constant at C16 in order to examine if changing the spacing between the inner secondary amines may yield additional SAR information. We find that the norspermine-type compounds consistently showed higher activity compared to corresponding spermine homologues.  相似文献   

10.
The formation of N-acylphosphatidylethanolamine by N-acylation of phosphatidylethanolamine (PE) is the initial step in the biosynthetic pathway of bioactive N-acylethanolamines, including the endocannabinoid anandamide and the anti-inflammatory substance N-palmitoylethanolamine. We recently cloned a rat enzyme capable of catalyzing this reaction, and referred to the enzyme as Ca2+-independent N-acyltransferase (iNAT). Here we report cDNA cloning and characterization of human and mouse iNATs. We cloned iNAT-homologous cDNAs from human and mouse testes, and overexpressed them in COS-7 cells. The purified recombinant proteins abstracted an acyl group from both sn-1 and sn-2 positions of phosphatidylcholine, and catalyzed N-acylation of PE as well as phospholipase A1/A2-like hydrolysis. The iNAT activity was mainly detected in soluble rather than particulate fractions, and was only slightly increased by Ca2+. These results demonstrated that the human and mouse homologues function as iNAT. As for the organ distribution of iNAT, human testis and pancreas and mouse testis exhibited by far the highest expression level, suggesting its physiological importance in the specific organs. Moreover, mutagenesis studies showed crucial roles of His-154 and Cys-241 of rat iNAT in the catalysis and a possible role of the N-terminal domain in membrane association or protein–protein interaction.  相似文献   

11.
Methyl fluoride (CH3F) and dimethyl ether (DME) inhibited nitrification in washed-cell suspensions of Nitrosomonas europaea and in a variety of oxygenated soils and sediments. Headspace additions of CH3F (10% [vol/vol]) and DME (25% [vol/vol]) fully inhibited NO2- and N2O production from NH4+ in incubations of N. europaea, while lower concentrations of these gases resulted in partial inhibition. Oxidation of hydroxylamine (NH2OH) by N. europaea and oxidation of NO2- by a Nitrobacter sp. were unaffected by CH3F or DME. In nitrifying soils, CH3F and DME inhibited N2O production. In field experiments with surface flux chambers and intact cores, CH3F reduced the release of N2O from soils to the atmosphere by 20- to 30-fold. Inhibition by CH3F also resulted in decreased NO3- + NO2- levels and increased NH4+ levels in soils. CH3F did not affect patterns of dissimilatory nitrate reduction to ammonia in cell suspensions of a nitrate-respiring bacterium, nor did it affect N2O metabolism in denitrifying soils. CH3F and DME will be useful in discriminating N2O production via nitrification and denitrification when both processes occur and in decoupling these processes by blocking NO2- and NO3- production.  相似文献   

12.
In cell-free preparations of NH4+-grown cultures of the cyanobacterium Anabaena L-31 the glutamine synthetase activity is only half as much as in N2-grown cultures. Using a procedure which enables quantitative purification of the enzyme to homogeneity it has been shown that the decrease in the enzyme activity is caused by NH4+-mediated repression. Glutamine synthetase activity in both N2-grown and NH4+-grown Anabaena remains stable for more than 24 h in the presence of chloramphenicol suggesting low enzyme turnover and an enzyme half-life greater than the generation time (16–18 h) of the cyanobacterium. In N2-grown cultures, a drastic decrease in the enzyme activity by exogenous NH4+ can be discerned when fresh protein synthesis is prevented by chloramphenicol. The enzyme purified from such cultures has Km values for NH4+, glutamate Mg2+, and ATP similar to those observed for the enzyme from N2- and NH4+-grown Anabaena, but shows depression in V for all the substrates, leading to drastic decrease in specific activity. The modified enzyme also shows a sharper thermal denaturation profile. These results indicate that NH4+-mediated modification to a less active form may be a means of regulation of glutamine synthetase in N2-fixing cultures of Anabaena.  相似文献   

13.
It has been shown for the first time that deacylation is the rate-limiting step in the enteropeptidase-catalyzed hydrolysis of highly effective oligopeptide substrates containing four Asp residues in positions P2–P5. On the other hand, the rate-limiting step in the hydrolysis of low-efficiency peptide substrates containing less than four Asp or Glu residues in positions P2–P5 is acylation, as it has previously been suggested for all amide and peptide substrates of serine proteases on the basis of classical works of Bender et al. The method of introduction of an additional nucleophile or another effector that selectively affects the deacylation step was used to determine the rate-limiting step in the enteropeptidase hydrolysis of N α-benzyloxycarbonyl-L-lysine thiobenzyl ester, the highly efficient amide substrate GlyAsp4-Lys β-naphthyl amide, and the low-efficiency peptide substrate VLSAADK-GNVKAAWG (where a hyphen denotes the hydrolysis site).  相似文献   

14.
The synthesis of phosphatidylcholines (PC), phosphatidylethanolamines (PE) and phosphatidylserines (PS) containing two acyl chains of the naturally occurring sponge fatty acid (5Z,9Z)-5,9-hexacosadienoic acid as well as its hitherto unknown geometrical isomers is described. The PCs were prepared by deacylation of natural lecithins, followed by reacylation with fatty acid anhydrides. The synthesis of mixed-acid PCs is also reported: a diacyl product was converted to the lyso-PC by treatment with phospholipase A2 and subsequent acylation of the secondary hydroxyl group to give the desired mixed-acid PCs. The PEs and the PSs were prepared from the corresponding PCs by enzymatic transphosphatidylation catalyzed by phospholipase D. Structural assignments of the compounds were confirmed by spectroscopy (1H-NMR and MS). Ammonia chemical ionization mass spectrometry provided molecular ion and significant fragment peaks for PCs and PEs.  相似文献   

15.
The N-terminal segment of the Semliki Forest virus polyprotein is an intramolecular serine protease that cleaves itself off after the invariant Trp267 from a viral polyprotein and generates the mature capsid protein. After this autoproteolytic cleavage, the free carboxylic group of Trp267 interacts with the catalytic triad (His145, Asp167 and Ser219) and inactivates the enzyme. We have deleted the last 1-7 C-terminal residues of the mature capsid protease to investigate whether removal of Trp267 regenerates enzymatic activity. Although the C-terminally truncated polypeptides do not adopt a defined three-dimensional structure and show biophysical properties observed in natively unfolded proteins, they efficiently catalyse the hydrolysis of aromatic amino acid esters, with higher catalytic efficiency for tryptophan compared to tyrosine esters and kcat/KM values up to 5 × 105 s−1 M−1. The enzymatic mechanism of these deletion variants is typical of serine proteases. The pH enzyme activity profile shows a pKa1 = 6.9, and the Ser219Ala substitution destroys the enzymatic activity. In addition, the fast release of the first product of the enzymatic reaction is followed by a steady-state second phase, indicative of formation and breakdown of a covalent acyl-enzyme intermediate. The rates of acylation and deacylation are k2 = 4.4±0.6 s−1 and k3 = 1.6±0.5 s−1, respectively, for a tyrosine derivative ester substrate, and the amplitude of the burst phase indicates that 95% of the enzyme molecules are active. In summary, our data provide further evidence for the potential catalytic activity of natively unfolded proteins, and provide the basis for engineering of alphavirus capsid proteins towards hydrolytic enzymes with novel specificities.  相似文献   

16.
Two new zincophosphites [C6H14N2]0.5[Zn(H2PO3)2] 1 and [C4H12N2]0.5[(CH3)2NH2][Zn2(HPO3)3] 2 have been solvothermally synthesized in mixed solvents of N,N-dimethylformamide (DMF) and 1,4-dioxane (DOA), respectively. Single-crystal X-ray diffraction analysis reveals that compound 1 exhibits a neutral inorganic chain formed by ZnO4 and HPO2(OH) units. Interestingly, the left- and right-handed hydrogen-bonded helical chains are alternately formed via the hydrogen-bonds between two adjacent chains. Compound 2 exhibits a layer structure with 4- and 12-MRs formed by ZnO4 and HPO3 units, in which two kinds of organic amine molecules both act as countercations to compensate the overall negative electrostatic charge of the anionic network.  相似文献   

17.
A water-soluble polymer catalyst was prepared by radical polymerization of a protected hydroxamate monomer, 1-methyl-2-vinylimidazole and acrylamide, and by the subsequent NH2NH2 treatment of the polymer. The hydrolysis of p-nitrophenyl acetate by the bifunctional copolymer obeyed typical burst kinetics: rapid accumulation of acetyl hydroxamate group and its slow decomposition. The acetylation rate of the hydroxamate group was rather close to that of a polymer which does not contain the imidazole unit. However, the deacylation was markedly accelerated by the presence of the imidazole unit, and the difference in rate constants amounted to 60- to 80-fold at pH 8–9. These results indicate that the overall catalytic efficiency of the bifunctional polymer is enhanced due to the complementary action of the imidazole and hydroxamate functions.  相似文献   

18.
LigD 3′-phosphoesterase (PE) is a component of the bacterial NHEJ apparatus that performs 3′-end-healing reactions at DNA breaks. The tertiary structure, active site and substrate specificity of bacterial PE are unique vis–à-vis other end-healing enzymes. PE homologs are present in archaea, but their properties are uncharted. Here, we demonstrate the end-healing activities of two archaeal PEs—Candidatus Korarchaeum cryptofilum PE (CkoPE; 117 amino acids) and Methanosarcina barkeri PE (MbaPE; 151 amino acids)—and we report their atomic structures at 1.1 and 2.1 Å, respectively. Archaeal PEs are minimized versions of bacterial PE, consisting of an eight-stranded β barrel and a 310 helix. Their active sites are located in a crescent-shaped groove on the barrel’s outer surface, wherein two histidines and an aspartate coordinate manganese in an octahedral complex that includes two waters and a phosphate anion. The phosphate is in turn coordinated by arginine and histidine side chains. The conservation of active site architecture in bacterial and archaeal PEs, and the concordant effects of active site mutations, underscore a common catalytic mechanism, entailing transition state stabilization by manganese and the phosphate-binding arginine and histidine. Our results fortify the proposal that PEs comprise a DNA repair superfamily distributed widely among taxa.  相似文献   

19.
Cao M  Geng W  Liu L  Song C  Xie H  Guo W  Jin Y  Wang S 《Bioresource technology》2011,102(5):4251-4257
A new glutamic acid independent poly-γ-glutamic acid (γ-PGA) producing strain, which was identified as Bacillusamyloliquefaciens LL3 by analysis of 16S rDNA and gyrase subunit A gene (gyrA), was isolated from fermented food. The product had a molecular weight of 470, 801 and l-glutamate monomer content of 98.47%. The pre-optimal medium, based on single-factor tests and orthogonal design, contained 50 g/L sucrose, 2 g/L (NH4)2SO4, 0.6 g/L MgSO4, and provided well-balanced changes in processing parameters and a γ-PGA yield of 4.36 g/L in 200 L system. The γ-PGA synthetase genes pgsBCA were cloned from LL3, and successfully expressed by pTrcLpgs vector in Escherichia coli JM109, resulting the synthesis of γ-PGA without glutamate. This study demonstrates the designedly improved yield of γ-PGA in 200 L system and the first report of pgsBCA from glutamic acid independent strain, which will benefit the metabolized mechanism investigation and the wide-ranging application of γ-PGA.  相似文献   

20.
Phosphatidylethanolamine (PE) is one of the essential phospholipids in the yeast Saccharomyces cerevisiae. We have previously shown that a yeast strain, the endogenous PE synthesis of which was controllable, grew in the presence of PE containing decanoyl residues (diC10PE) when PE synthesis was repressed. In this study, we investigated the fate of diC10PE, its uptake and remodeling in yeast. Deletion of the genes encoding Lem3p/Ros3p or P-type ATPases, Dnf1p and Dnf2p, impaired the growth of the mutants in the medium containing diC10PE, suggesting the involvement of these proteins in the uptake of diC10PE. Analysis of the metabolism of deuterium-labeled diC10PE by electrospray ionization tandem mass spectrometry revealed that it was rapidly converted to deuterium-labeled PEs containing C16 or C18 acyl residues. The probable intermediate PEs that contained decanoic acid and C16 or C18 fatty acids as acyl residues were also detected. In addition, a substantial amount of decanoic acid was released into the culture medium during growth in the presence of diC10PE. These results imply that diC10PE was remodeled to PEs with longer acyl residues and used as membrane components. Defects in the remodeling of diC10PE in the deletion mutants of ALE1 and SLC1, products of which were capable of acyl-transfer to the sn− 2 position of lyso-phospholipids, suggested their involvement in the introduction of acyl residues to the sn− 2 position of lyso-phosphatidylethanolamine in the remodeling reaction of diC10PE. Our results also suggest the presence of a mechanism to maintain the physiological length of PE acyl residues in yeast.  相似文献   

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