Photoaffinity labeling of the N-methyltransferase domains of cyclosporin synthetase

The multifunctional polypeptide cyclosporin synthetase (CySyn) remains one of the most complex nonribosomal peptide synthetase described. In this study we used a highly specific photoaffinity labeling procedure with the natural cofactor S-adenosyl-L-methionine (AdoMet), 14C-isotopically labeled at the Sdelta methyl group to probe the concerted AdoMet-binding interaction of the N-methyltransferase (N-MTase) centers of CySyn. The binding stoichiometry for the enzyme-AdoMet complex was determined to be 1:7, which is in agreement with inferences made from analysis of the complementary DNA sequence of the simA gene encoding the CySyn polypeptide. The photolabeling of the AdoMet-binding sites displayed homotropic negative cooperativity, characterized by a curvilinear Scatchard plot with upward concavity. Although, the process of N-methyl transfer is not a critical event for peptide elongation, the destabilizing homotropic interactions between N-MTase centers that were observed may represent a mechanism whereby the enzyme preserves the proficiency of the substrate-channeling process of cyclosporin peptide assembly over a broad range of cofactor concentrations. Furthermore, we demonstrated the utility of the photolabeling procedure for tracking the enzyme during purification.     

Arylamine N-methyltransferase and thiol methyltransferase activities in cholestatic rat liver induced by common bile duct ligation

Methylation catalyzed by methyltransferases is a major metabolic pathway for an inactivation of some catecholamines, niacinamide as well as aliphatic sulfhydryl drugs and toxic hydrogen sulfides. To investigate the effects of obstructive jaundice in an animal model, common bile duct ligation (CBDL) was performed in the rat and enzyme activities of S-adenosyl-L-methionine-dependent arylamine N-methyltransferase and thiol methyltransferase were examined in liver cell fractions and serum for a period of 42 d after CBDL. Both mitochondrial and microsomal arylamine N-methyltransferase showed significant increases in their activities between the 1st through the 7th day (P < or = 0.05 to 0.001), and between the 1st through the 28th day (P < or = 0.01 to 0.001) post-ligation, although the cytosolic arylamine N-methyltransferase activity did not show a significant change compared to the activities from the sham-operated control. The mitochondrial as well as microsomal thiol methyltransferase showed significant increases in their activities between the 1st through the 28th day (P < or = 0.05 to 0.01 and P < or = 0.01 to 0.001, respectively) post-ligation, although the cytosolic thiol methyltransferase activity did not show a significant change compared to the activities from the sham-operated control. Arylamine N-methyltransferase and thiol methyltransferase in the serum from cholestatic rats also showed significant increases in their activities between the 1st through 28th day (P < or = 0.01 to 0.001), and between the 0.5th through the 42nd day (P < or = 0.05 to 0.001) post-ligation compared to the sham-operated control, respectively. Enzyme kinetic parameters (Km and Vmax) of hepatic membrane-bound arylamine N-methyltransferase and thiol methyltransferase were analyzed with the preparation from the 7th day post-ligation, using tryptamine or 4-chlorothiophenol as substrates and S-Adenosyl-L-[methyl-3H]methionine as co-substrate. The results indicate that although the Km values were about the same as the sham-operated control, the Vmax values of both enzymes increased significantly (P < or = 0.01 and 0.001, respectively). These results suggest that the biosynthesis of arylamine N-methyltransferase and thiol methyltransferase have been induced in response to obstructive jaundice.     

Construction and in vitro analysis of a new bi-modular polypeptide synthetase for synthesis of N-methylated acyl peptides

BACKGROUND: Many active peptides are synthesized by nonribosomal peptide synthetases (NRPSs), large multimodular enzymes. Each module incorporates one amino acid, and is composed of two domains: an activation domain that activates the substrate amino acid and a condensation domain for peptide-bond formation. Activation domains sometimes contain additional activities (e.g. N-methylation or epimerization). Novel peptides can be generated by swapping domains. Exchange of domains containing N-methylation activity has not been reported, however. RESULTS: The actinomycin NRPS was used to investigate domain swapping. The first two amino acids of actinomycin are threonine and valine. We replaced the valine activation domain of module 2 with an N-methyl valine (MeVal) activation domain. The recombinant NRPS (AcmTmVe) catalyzes the formation of threonyl-valine. In the presence of S-adenosyl-methionine, valine was converted to MeVal but subsequent dipeptide formation was blocked. When acyl-threonine (the natural intermediate) was present at module 1, formation of acyl-threonine-MeVal occurred. The epimerization domain of AcmTmVe was impaired. CONCLUSIONS: A simple activation domain can be replaced by one with N-methylation activity. The same condensation domain can catalyze peptide-bond formation between N-methyl and nonmethylated amino acids. Modification of the upstream amino acid (i.e. acylation of threonine), however, was required for condensation with MeVal. Steric hindrance reduces chemical reactivity of N-methyl amino acids - perfect substrate positioning may only be achieved with acylated threonine. Loss of the epimerase activity of AcmTmVe suggests N-methyltransferase and epimerase domains, not found together naturally, are incompatible.     

Two new cyclosporin folds observed in the structures of the immunosuppressant cyclosporin G and the formyl peptide receptor antagonist cyclosporin H at ultra-high resolution

Cyclosporins are cyclic undecapeptides of fungal origin the best known of which, CsA, is a lead clinical immunosuppressant; CsG is a potential clinical immunosuppressant differing from CsA in residue 2 (L-alpha-amino-butyric acid in CsA, L-norvaline in CsG); and CsH is an inverse formyl peptide receptor agonist, differing from CsA in the chiral inversion of MeVal-11 from L to D. Crystal structure determinations of CsG and CsH were undertaken to identify structural and surface features important for biological activity and the future design of new cyclosporin derivatives. Ultra-high resolution X-ray structures (0.80 to 0.87 A resolution) determined for two crystal forms of both CsH and CsG in the presence and absence of Mg2+ are described. A major outcome of this study is the observation that the local change in chirality between CsA and CsH is associated with a major structural transformation from open beta-sheet in CsA to a highly convoluted conformation in CsH. CsG also possesses a completely novel cloverleaf motif with no H-bonded secondary structure features in spite of the minimal chemical difference with CsA. Unlike CsA, the structures of both CsH and CsG are heavily solvated. This study therefore shows that the chemical differences between the three cyclosporins, CsA, CsG and CsH can invoke unpredictably major differences in their 3D structures. The 9-11 cis-peptide bond in CsA moves to 11-1 in CsG, influencing the overall molecular conformation, while the peptide bonds in the highly convoluted loop conformation of CsH are all trans.     

Wistar大鼠脑内各区的Salsolinol氮甲基转移酶活性检测

帕金森病是一种常见的神经退行性疾病,受到世界各国及研究机构的广泛重视.目前,越来越多的研究结果表明Salsoline N-甲基转移酶(SNMT)和酶活性产物(NMSal)可能是PD的关键致病因子.     

High-performance liquid chromatographic determination of histamine N-methyltransferase activity

A method for the determination of histamine N-methyltransferase (HMT) activity by high-performance liquid chromatography based on post-column derivatization with omicron-phthalaldehyde is described. The determination involves the separation of the substrate, histamine, from its product. N tau-methylhistamine, using a weak cation exchanger, followed by on-line derivatization of these imidazoleamines with omicron-phthalaldehyde and their detection and quantitation with a fluorimetric detector. This assay method is suitable for the measurement of HMT activity during enzyme purification.     

Effect of polypeptide sequence on polypeptide self-assembly

This study represents a unique example where the self-assembly of five amphiphilic polypeptides was monitored as a function of their hydrophilic-to-lipophilic balance (HLB). The HLB was tuned by preparing a series of polypeptides with aspartic acid (Asp) and phenylalanine (Phe) according to the sequence (Asp(x)Phe(y)) where x and y vary from 1 to 3. The self-assembly of the (Asp(x)Phe(y))(n) polypeptides with M(w) values ranging from 8 to 12 K was studied in solution by fluorescence quenching and nonradiative energy transfer (NRET) fluorescence experiments. Dynamic (DLS) and static (SLS) light scattering studies were used to complement the results obtained. The fluorescence quenching experiments conducted with the chromophore pyrene used as a free probe, physically bound to the polypeptides via hydrophobic interactions, demonstrated that the chromophore was less exposed to the solution for the sequences having a higher hydrophobic character. Protective quenching was also observed for those polypeptides randomly labeled with pyrene where the phenylalanine content was high, whereas pyrene was found to be fully exposed to the quencher for the polypeptides having more hydrophilic sequences. NRET used to probe interpolymeric association was not observed between a naphthalene and a pyrene labeled polypeptide for the polypeptide sequences which were richer in aspartic acid, whereas energy transfer took place with the more hydrophobic polypeptides. This observation led to the conclusion that those sequences with a higher content of aspartic acid essentially generate unimolecular polymeric micelles, whereas those with a higher content of phenylalanine generate polymeric aggregates which offer protection from the solvent to their hydrophobic cargo. The presence of these polymeric aggregates was also confirmed by DLS and SLS studies which suggest that species 100-200 nm in diameter are present in solution.     

A novel series of thromboxane A2 synthetase inhibitors with free radical scavenging and anti-peroxidative activities

A novel series of indoline derivatives with imidazole and carboxyl moieties were synthesized and evaluated for their thromboxane A2 (TXA2) synthetase inhibiting, radical scavenging and anti-peroxidative activities. Among the compounds synthesized, 3-[5-substituted-3-[2-(imidazol-1-yl)ethyl]indolin-1-yl]propionic acids showed free radical scavenging activity and inhibitory effects on lipid-peroxidation of rat brain homogenate and on arachidonate-induced TXA2-dependent aggregation of rabbit platelets. The anti-platelet and anti-peroxidative activities were related to the lipophilicity of the 5-substituent. The 5-hexyloxy derivative (13) showed about 35-fold higher inhibitory activity on TXA2 synthesis than that of ozagrel and about 100-fold higher activity on lipid peroxidation than that of alpha-tocopherol. Compound 13 showed in vivo anti-thrombotic effect in mice and ex vivo anti-peroxidative activity in rats.     

Synthesis of cyclosporin analogues

Analogues of Cyclosporin modified at positions 1 and/or 2 have been synthesised; synthesis of linear peptides was investigated by fragment condensation and stepwise synthesis and a number of cyclisation methods were evaluated.Synthesis of [(Me)(Thr¹)]-, (Hyp¹)-, (Hyp¹, Nle²)-, [(Me)Ser¹]-, (Me)Ser¹, Nle²]-, [(Me)Ser¹, Nva]- [(Me)Thr¹, Nle²]-, [(Me)Thr¹, Nva]-, [(Me)Ser¹, Thr²]- and [Dab¹]- Cyclosporins and constituent fragments are described.     

Synthetic studies of cyclosporin analogues

The syntheses of eleven analogues of Cyclosporin are described; the analogues which contain (Me)Thr, (Me)Ser, Hyp and Dab at position-1 and Abu, Nva, Nle and Thr at position-2 were prepared by stepwise assembly of the undecapeptide fragments followed by cyclisation with a variety of reagents. The highest yields were obtained using the Castro reagent, and in the best case, (Hyp¹ ,Abu²) cyclosporin, a yield of 65% was obtained.     

Lovastatin biosynthesis in Aspergillus terreus: characterization of blocked mutants, enzyme activities and a multifunctional polyketide synthase gene.

BACKGROUND: Lovastatin, an HMG-CoA reductase inhibitor produced by the fungus Aspergillus terreus, is composed of two polyketide chains. One is a nonaketide that undergoes cyclization to a hexahydronaphthalene ring system and the other is a simple diketide, 2-methylbutyrate. Fungal polyketide synthase (PKS) systems are of great interest and their genetic manipulation should lead to novel compounds. RESULTS: An A. terreus mutant (BX102) was isolated that could not synthesize the nonaketide portion of lovastatin and was missing a approximately 250 kDa polypeptide normally present under conditions of lovastatin production. Other mutants produced lovastatin intermediates without the methylbutyryl sidechain and were missing a polypeptide of approximately 220 kDa. The PKS inhibitor cerulenin reacted covalently with both polypeptides. Antiserum raised against the approximately 250 kDa polypeptide was used to isolate the corresponding gene, which complemented the BX102 mutation. The gene encodes a polypeptide of 269 kDa containing catalytic domains typical of vertebrate fatty acid and fungal PKSs, plus two additional domains not previously seen in PKSs: a centrally located methyltransferase domain and a peptide synthetase elongation domain at the carboxyl terminus. CONCLUSIONS: The results show that the nonaketide and diketide portions of lovastatin are synthesized by separate large multifunctional PKSs. Elucidation of the primary structure of the PKS that forms the lovastatin nonaketide, as well as characterization of blocked mutants, provides new details of lovastatin biosynthesis.     

Synthesis of multifunctional hydroxyethyl tetrazoles

A series of di-, tri-, and tetra-tetrazoloalkanes were synthesized from the corresponding nitrile and sodium azide. These were alkylated to give hydroxy terminated chains for possible use as high energy oligomers.     

Computational design of multifunctional materials

We propose a general scheme for the computational design of new materials using density functional theory. We then apply the scheme to two classes of materials; ferromagnetic ferroelectrics and half-metallic antiferromagnets. Our first “designer” ferromagnetic ferroelectric has subsequently been synthesized and the predicted properties verified. Our computations on half-metallic antiferromagnets have stimulated experimental study but the phenomenon remains unconfirmed.     

Knowledge-based potential for the polypeptide backbone

A knowledge-based potential for the polypeptide backbone as a function of the dihedral angles is developed and tested. The potential includes correlations due to the conformations and compositions of adjacent residues. Its purpose is to serve as a major component of a coarse-grained protein potential by including the most relevant local interactions while averaging out nonbonded ones. A probability density estimation algorithm and a multi-resolution probability combination procedure are developed to produce smooth probability distributions and dihedral angle potentials. The potential is described by a set of two-dimensional dihedral angle surfaces involving the various combinations of amino acid triplets and duplets. Several tests are carried out to evaluate the quality of the potential. Monte Carlo simulations, using only the dihedral angle potential and a coarse-grained excluded volume potential, show that the resulting dihedral angle distributions and correlations are consistent with those extracted from protein structures. Additional simulations of unfolded proteins are carried out to measure the NMR residual dipolar coupling (RDC). Significant correlations are obtained between the simulations and the corresponding experiments consistent with other simulations in the literature. Finally, the dihedral angle entropies are calculated for the 20 amino acids. In particular, the entropy difference between alanine and glycine agrees with the ones computed from molecular dynamics simulations ( approximately 0.4 kcal/mol).     

Photoinitiated cationic polymerisation of multifunctional systems

Different types of tridimensional polymer networks have been synthetised by photoinitiated cationic polymerisation of vinyl ether and epoxy-functionalised oligomers and polymers. The polymerisation kinetics was followed by real-time infrared (RTIR) spectroscopy, a technique that records directly conversion versus time profiles in a timescale as short as 1 s. The addition of a diacrylate monomer was shown to accelerate the ring-opening polymerisation of epoxidized polyisoprene, with formation of interpenetrating polymer networks having well contrasted properties. A dual polymer network has been generated by photocrosslinking of a polyisoprene functionalised with both epoxy and acrylate groups.