1,381 research outputs found

    Site of Prenylation Reaction in Synthesis of Phylloquinone (Vitamin K1) by Spinach Chloroplasts

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    In spinach chloroplasts, 1,4-dihydroxy-2-naphthoate is prenylated by phytyldiphosphate and subsequently methylated by S-adenosylmethionine to form phylloquinol. The site of the prenylation reaction is the chloroplast envelope membrane

    Design, synthesis, docking studies and monoamine oxidase inhibition of a small library of 1-acetyl- and 1-thiocarbamoyl-3,5-diphenyl-4,5-dihydro-(1h)-pyrazoles

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    New N-acetyl/N-thiocarbamoylpyrazoline derivatives were designed and synthesized in high yields to assess their inhibitory activity and selectivity against human monoamine oxidase A and B. The most important chiral compounds were separated into their single enantiomers and tested. The impact of the substituents at N1, C3 and C5 positions as well the influence of the configuration of the C5 on the biological activity were analyzed. Bulky aromatic groups at C5 were not tolerated. p-Prenyloxyaryl moiety at C3 oriented the selectivity toward the B isoform. The results were also corroborated by molecular modelling studies providing new suggestions for the synthesis of privileged structures to serve as lead compounds for the treatment of mood disorders and neurodegenerative diseases

    14-3-3 Proteins Interact with a Hybrid Prenyl-Phosphorylation Motif to Inhibit G Proteins

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    Signaling through G proteins normally involves conformational switching between GTP- and GDP-bound states. Several Rho GTPases are also regulated by RhoGDI binding and sequestering in the cytosol. Rnd proteins are atypical constitutively GTP-bound Rho proteins, whose regulation remains elusive. Here, we report a high-affinity 14-3-3-binding site at the C terminus of Rnd3 consisting of both the Cys241-farnesyl moiety and a Rho-associated coiled coil containing protein kinase (ROCK)-dependent Ser240 phosphorylation site. 14-3-3 binding to Rnd3 also involves phosphorylation of Ser218 by ROCK and/or Ser210 by protein kinase C (PKC). The crystal structure of a phosphorylated, farnesylated Rnd3 peptide with 14-3-3 reveals a hydrophobic groove in 14-3-3 proteins accommodating the farnesyl moiety. Functionally, 14-3-3 inhibits Rnd3-induced cell rounding by translocating it from the plasma membrane to the cytosol. Rnd1, Rnd2, and geranylgeranylated Rap1A interact similarly with 14-3-3. In contrast to the canonical GTP/GDP switch that regulates most Ras superfamily members, our results reveal an unprecedented mechanism for G protein inhibition by 14-3-3 proteins

    Physiological Evidence for Isopotential Tunneling in the Electron Transport Chain of Methane-Producing Archaea

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    Many, but not all, organisms use quinones to conserve energy in their electron transport chains. Fermentative bacteria and methane-producing archaea (methanogens) do not produce quinones but have devised other ways to generate ATP. Methanophenazine (MPh) is a unique membrane electron carrier found in Methanosarcina species that plays the same role as quinones in the electron transport chain. To extend the analogy between quinones and MPh, we compared the MPh pool sizes between two well-studied Methanosarcina species, Methanosarcina acetivorans C2A and Methanosarcina barkeri Fusaro, to the quinone pool size in the bacterium Escherichia coli. We found the quantity of MPh per cell increases as cultures transition from exponential growth to stationary phase, and absolute quantities of MPh were 3-fold higher in M. acetivorans than in M. barkeri. The concentration of MPh suggests the cell membrane of M. acetivorans, but not of M. barkeri, is electrically quantized as if it were a single conductive metal sheet and near optimal for rate of electron transport. Similarly, stationary (but not exponentially growing) E. coli cells also have electrically quantized membranes on the basis of quinone content. Consistent with our hypothesis, we demonstrated that the exogenous addition of phenazine increases the growth rate of M. barkeri three times that of M. acetivorans. Our work suggests electron flux through MPh is naturally higher in M. acetivorans than in M. barkeri and that hydrogen cycling is less efficient at conserving energy than scalar proton translocation using MPh

    Molecular biological and biochemical investigations on the biosynthetic enzymes of prenylated indole alkaloids from fungi

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    Prenylated indole alkaloids are widely distributed in plants, fungi and bacteria, especially in the family of Clavicipitaceae and Trichocomaceae of Ascomycota, and commonly exhibit interesting biological and pharmaceutical activities. In the biosynthetic pathway of prenylated indole alkaloids, prenylation catalyzed by prenyltransferases contributes significantly to the large structure diversity of these compounds in nature. Investigation on indole prenyltransferases would help to understand the construction of prenylated indole alkaloids in nature and also be useful for structural modification of indole derivatives and other substances to produce analogues of prenylated derivatives. Three indole prenyltransferases belonging to the dimethylallyltryptophan synthase (DMATS) superfamily were biochemically identified and characterized in vitro, including CdpC3PT from Neosartorya fischeri (N. fischeri), BrePT from Aspergillus versicolor (A. versicolor) and 5-DMATS from Aspergillus clavatus (A. clavatus). The responsible genes cdpC3PT and brePT were cloned into expression vector and heterologously expressed in Escherichia coli (E. coli). These works were carried out by Dr. Wen-Bing Yin, Suqin Yin and Qing Wang, respectively. In this thesis, CdpC3PT was confirmed to catalyze the formation of C3-prenylated products with a characteristic 6/5/5/6-fused tetracyclic ring system from tryptophan-containing cyclic dipeptides in one-step reaction. The NotF homologue BrePT showed much higher flexibility towards its aromatic substrates than NotF, and was proven to catalyze the highly regiospecific reverse prenylation at C-2 of the indole nucleus. The cloning of 5-dmats was carried out by Yan Liu. Functional proof of this gene was provided within this thesis by heterologous expression in E. coli and subsequent structure elucidation of enzyme products by mass spectrometry (MS) and nuclear magnetic resonance (NMR) analyses. 5-DMATS established high regiospecific activity to catalyze C5-prenylation on indole derivatives. Given the importance of prenylation in structure diversity and bioactivity enhancement, CdpC3PT, BrePT, 5-DMATS and other known prenyltransferases of the DMATS superfamily were applied for the chemoenzymatic synthesis of prenylated compounds. By using AnaPT, CdpC3PT and CdpNPT, eight and six stereoisomers of cis-configured prenylated pyrroloindoline diketopiperazines from cyclo-Trp-Ala and cyclo-Trp-Pro isomers were produced, respectively. The stereospecificity of AnaPT and CdpC3PT depended mainly on the configuration of tryptophanyl moiety in cyclo-Trp-Ala and cyclo-Trp-Pro isomers, while CdpNPT showed lower stereoselectivity, but higher conversion ability towards most tested substrates. 5-DMATS and FgaPT2 from Aspergillus were used for chemoenzymatic synthesis of prenylated indolocarbazoles. Reconstitution of enzyme activity of 5-DMATS and FgaPT2 in vitro revealed that they catalyzed regiospecific prenylation of indolocarbazoles at the para-position of the indole N-atom. This is the first report for prenylated indolocarbazoles. Subsequently, some indole prenyltransferases of the DMATS superfamily were found to accept also hydroxynaphthalenes and flavonoids, which were substrates for enzymes from the CloQ/NphB group and the UbiA superfamily, respectively. Nine prenylated flavonoids and twenty prenylated hydroxynaphthalenes have been isolated, and their structures were elucidated by MS and NMR analyses. It has been shown that, for an accepted hydroxynaphthalene, different enzymes produced usually the same major prenylated product, i.e. with a regular C-prenyl moiety at para- or ortho-position to a hydroxyl group. For flavonoids accepted by 7-DMATS, C-6 between two hydroxyl groups was the favorable prenylation position. The Michaelis-Menten constants (KM) and turnover numbers (kcat) of some prenyltransferases towards selected hydroxynaphthalenes are comparable to those obtained by using indole derivatives. In addition to indole prenyltransferases, other genes in the biosynthetic cluster of prenylated indole alkaloids were also investigated. A putative O-methyltransferase gene hasC and a putative cytochrome P450 gene hasH involved in the biosynthesis of hexadehydroastechrome (HAS) in Aspergillus fumigatus (A. fumigatus) were cloned into pQE60 and pESC-URA, respectively. Soluble His6-HasC was successfully overproduced in E. coli SG13009 and purified to near homogeneity by Ni-NTA. Constructs for co-expression with the reductase gene NFIA_083630 from N. fischeri in pESC-URA and for expression as His6-tagged protein in pESC-URA were also prepared for the putative cytochrome P450 gene hasH

    Structure-based protein engineering and biochemical investigations of fungal prenyltransferases for targeted production of novel prenylated indole alkaloids

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    Prenyltransferases (PTs) catalyze the transfer of one or more prenyl units with chain lengths of n x C5 (n = 1, 2 etc.) to diverse acceptors. Prenylations are essential in both the primary and secondary metabolism of all organisms. PTs are often involved in the biosynthetic pathways of secondary metabolites (SMs). Many indole alkaloids (IAs) from Ascomycota are such SMs generated by diverse enzymes and PTs of the dimethylallyl tryptophan synthase (DMATS) superfamily with L-tryptophan as a precursor. These PTs mostly use the prenyl donor dimethylallyl diphosphate (DMAPP) to catalyze metal ion-independently and regiospecific Friedel-Crafts alkylation at the indole ring. The increased lipophilicity of prenylated substances is often associated with improved bioactivity. Prenylated tryptophan-containing cyclic dipeptides (CDPs) with a 2,5 diketopiperazine ring and their derivatives are such representative structures with significant biological and pharmacological activities. Echinulin and derivatives thereof possess antiviral, antitumor and neuroprotective activities. Fumitremorgin C is a potential inhibitor of the breast cancer resistance protein. Tryprostatin A and B are known as microtubule inhibitors. The demand for new bioactive substances such as antibiotics or cancer therapeutics prompted in this thesis the targeted manipulation of PTs on the basis of protein structure data. The obtained mutants were then used for the production of new prenylated IAs. Furthermore, pharmacologically relevant substances were produced by the combination of chemical and enzymatic synthesis. In this thesis, five tryptophan PTs, FgaPT2, 5-DMATS, 5-DMATSSc, 6-DMATSSa and 7-DMATS, were investigated for their potential to prenylate the tripeptide derivative ardeemin fumiquinazoline (FQ) and its stereoisomers. Ardeemin FQ is the precursor of the reverse C3-prenylated ardeemin and 5-N-acetylardeemin. These substances block the MultiDrug Resistance (MDR) export pump in cancer cells, thereby increasing the potency of cancer therapeutics such as Vinca alkaloids in the cell. This thesis demonstrates that PTs from other biosynthetic gene clusters are also able to prenylate ardeemin FQ. The chemically synthesized stereoisomers could also be converted by these PTs, which has not been reported prior to this study. Here, the two stereo centers of these substrates impact the activity and regioselectivity of these enzymes. 18 new prenylated tripeptide derivatives were obtained. Since the tryptophan PTs catalyze only prenylations on the indole’s benzene ring of the tripeptide derivatives, five further PTs were investigated in a cooperation project with Lindsay Coby. BrePT, FtmPT1, CdpNPT, CdpC3PT and AnaPT are cyclic dipeptide PTs that prenylate their natural or best-accepted substrates at the pyrrole ring of the indole. A high conversion rate with ardeemin FQ has been determined for BrePT, FtmPT1 and CdpNPT, whereas CdpC3PT and AnaPT show less acceptance. The enantiomer was poorly converted by all enzymes. These results also show that the stereochemistry of the substrates affects their acceptance by the cyclic dipeptide PTs. 8 new pyrrole-ring prenylated tripeptide derivatives were produced. From both projects, 26 new obtained substances are available for further studies on their bioactivity. Another focus of this thesis is the structure-based protein manipulation through targeted mutation to generate PTs with novel catalytic properties and their application for the production of new prenylated IAs. For this purpose, FgaPT2, which catalyzes regular C4-prenylation of L-tryptophan, was used as a model for mutational experiments. The targeted combinational mutation of two amino acids (K174 and R244) has produced several FgaPT2 double mutants that catalyze reverse C3-prenylation of CDPs with high product yields. This work is a cooperation with Liujuan Zheng. In another study, targeted mutations were performed also on FgaPT2, which is a DMAPP-specific PT, in order to expand the prenyl donor specificity. Molecular modeling identified methionine 328 as potential key amino acid residue for the acceptance of the prenyl donor. The FgaPT2_M328G mutant shows a high turnover with GPP and low acceptance with FPP. An enzymatic geranylation or farnesylation of tryptophan as free amino acid was not described prior to this study. The decrease in DMAPP-acceptance by different FgaPT2_M328X mutants indicate that, in addition to size, the polarity of the amino acid also contributes to prenyl donor selectivity. The mutants with an amino acid smaller than methionine (FgaPT2_M328X; X = C, A, T, S, G, V, N) show high rates of GPP-utilization. The mutants FgaPT2_M328X (X = C, S, G, A) also possess FPP-activity. Due to their polar properties, high GPP- and high DMAPP-acceptance was observed by FgaPT2_M328X (X = C, T, V, N) mutants. To increase FPP-utilization, L263 and Y398 were identified as potential amino acids. The generation of the mutants FgaPT2_L263A_M328A and FgaPT2_L263A_M328A_Y398F led to a further increase in the FPP-utilization. In this study, 21 mutants were produced and 7 mutants can be used as geranyltransferases for chemoenzymatic syntheses of new IAs. In a cooperation study with Ge Liao, five additional DMATSs were selected for mutation experiments to achieve GPP-activity. The C2-PT FtmPT1 catalyzes a regular, BrePT and CdpC2PT a reverse transfer of the prenyl residue from DMAPP to the C-2 position of CDPs. The C3-PTs CdpNPT and CdpC3PT attach the prenyl residue to the C-3 position of CDPs in a reverse manner. Based on sequence alignments and molecular modeling, M364 in FtmPT1, I337 in BrePT, T351 in CdpC2PT, M349 in CdpNPT and F335 in CdpC3PT were identified to be responsible for prenyl donor selectivity and thus were replaced by glycine via site-directed mutagenesis. The generated mutants FtmPT1_M364G, BrePT_I337G, CdpNPT_M349G and CdpC3PT_F335G show clear acceptance of GPP compared to their wildtype. The CdpC2PT wildtype has already accepted GPP. The mutant CdpC2PT_T351G, however, displays a different geranylation pattern. 42 geranylated derivatives could be obtained from 15 CDPs. For cyclo-L-Trp-L-Trp the geranyl moiety of GPP could be transferred to almost all possible positions
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