RESUMO
Recent work has demonstrated that 4-hydroxybenzoic acid is the in vivo precursor to the 1-(4-aminophenyl)-1-deoxy-D-ribitol (APDR) moiety present in the C(1) carrier coenzyme methanopterin present in the methanogenic archaea. For this transformation to occur, the hydroxyl group of the 4-hydroxybenzoic acid must be replaced with an amino group at some point in the biosynthetic pathway. Using stable isotopically labeled precursors and liquid chromatography with electrospray-ionization mass spectroscopy, the first step of this transformation in Methanocaldococcus jannaschii occurs by the reaction of 4-hydroxybenzoic acid with phosphoribosyl pyrophosphate (PRPP) to form 4-(ß-d-ribofuranosyl)hydroxybenzene 5'-phosphate (ß-RAH-P). The ß-RAH-P then condenses with l-aspartate in the presence of ATP to form 4-(ß-d-ribofuranosyl)-N-succinylaminobenzene 5'-phosphate (ß-RFSA-P). Elimination of fumarate from ß-RFSA-P produces 4-(ß-D-ribofuranosyl)aminobenzene 5'-phosphate (ß-RFA-P), the known precursor to the APDR moiety of methanopterin [White, R. H. (1996) Biochemistry 35, 3447-3456]. This work represents the first biochemical example of the conversion of a phenol to an aniline.
Assuntos
Compostos de Anilina/síntese química , Mathanococcus/enzimologia , Fenol/síntese química , Pterinas/síntese química , Ribitol/análogos & derivados , Ribitol/síntese química , Ácido 4-Aminobenzoico/síntese química , Compostos de Anilina/metabolismo , Proteínas Arqueais/síntese química , Coenzimas/síntese química , Coenzimas/metabolismo , Endorribonucleases/síntese química , Precursores Enzimáticos/síntese química , Mathanococcus/genética , Mathanococcus/crescimento & desenvolvimento , Oxo-Ácido-Liases/síntese química , Fenol/metabolismo , Pterinas/metabolismo , Ribitol/metabolismoRESUMO
Active RNase glycoprotein from three pieces: The glycoprotein enzyme ribonuclease C, which contains a complex saccharide N-glycan, was synthesized by sequential native chemical ligation. An optimized ligation and isolation protocol allowed the efficient assembly and refolding of the 124 amino acid enzyme.
Assuntos
Carboidratos/síntese química , Endorribonucleases/síntese química , Glicopeptídeos/síntese química , Glicoproteínas/síntese química , Carboidratos/química , Carboidratos/isolamento & purificação , Endorribonucleases/química , Endorribonucleases/isolamento & purificação , Enzimas/síntese química , Enzimas/química , Enzimas/isolamento & purificação , Glicopeptídeos/química , Glicopeptídeos/isolamento & purificação , Glicoproteínas/química , Glicoproteínas/isolamento & purificação , Ligadura , Dobramento de Proteína , Renaturação ProteicaRESUMO
Seven in one blow: The efficient formation of mixed disulfides on the thiol-rich fusion protein A followed by subsequent intein cleavage gave the fragment B with all seven cysteines protected against oxidation. The native chemical ligation of B with synthetic glycopeptide thioesters provides glycoproteins.
Assuntos
Endorribonucleases/síntese química , Glicopeptídeos/síntese química , Glicoproteínas/síntese química , Endorribonucleases/biossíntese , Endorribonucleases/química , Glicopeptídeos/biossíntese , Glicopeptídeos/química , Glicoproteínas/biossíntese , Glicoproteínas/química , Ligadura , Oxirredução , Engenharia de Proteínas , Dobramento de Proteína , Renaturação Proteica , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/químicaRESUMO
Plant ribosomal RNA apurinic site specific lyase (RALyase) cleaves the phosphodiester bond at the depurinated site produced by ribosome-inactivating protein, while the biological role of this enzyme is not clear. As the depurinated ribosomes retain weak translation elongation activities, it was suggested that RALyase completes the ribosome inactivation. To confirm this point, we measured the effects of the phosphodiester cleavage using a fusion of wheat RALyase produced with a cell-free protein synthesis system from wheat germ. The results indicated that RALyase diminishes the residual elongation activities of the depurinated ribosomes.
Assuntos
Endorribonucleases/metabolismo , Endorribonucleases/farmacologia , Fatores de Alongamento de Peptídeos/metabolismo , Inibidores da Síntese de Proteínas/farmacologia , Ribossomos/metabolismo , Animais , Sistema Livre de Células , Endorribonucleases/síntese química , Endorribonucleases/genética , Fator 2 de Elongação de Peptídeos/metabolismo , Peptídeos/síntese química , Poli U/metabolismo , RNA Ribossômico/metabolismo , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Suínos , TriticumAssuntos
Endorribonucleases/síntese química , Endorribonucleases/metabolismo , Ribonucleases/síntese química , Fatores de Transcrição/síntese química , Fatores de Transcrição/metabolismo , Dedos de Zinco , Dimerização , Concentração de Íons de Hidrogênio , Oligorribonucleotídeos/síntese química , Oligorribonucleotídeos/metabolismo , Ribonucleases/metabolismo , Especificidade por Substrato , Transcrição GênicaRESUMO
Fully automated solid-phase synthesis gave access to a hybrid in which 5'-phosphorylated-2'-5'-linked oligoadenylate (2-5A) is connected to the 5'-terminus of DNA which, in turn, is linked at the 3'-end to PNA [2-5A-(5')-DNA-(3')-PNA chimera]. This novel antisense molecule retains full RNase L activation potency while suffering only a slight reduction in binding affinity.
Assuntos
Endorribonucleases/síntese química , Oligonucleotídeos Antissenso/metabolismo , Ácidos Nucleicos Peptídicos , Cromatografia Líquida de Alta Pressão , Endorribonucleases/metabolismo , Ativação Enzimática , Humanos , Ligação ProteicaRESUMO
In vitro selection techniques were applied to the development of a DNA enzyme that contains three catalytically essential imidazole groups and catalyzes the cleavage of RNA substrates. Nucleic acid libraries for selection were constructed by polymerase-catalyzed incorporation of C5-imidazole-functionalized deoxyuridine in place of thymidine. Chemical synthesis was used to define a minimized catalytic domain composed of only 12 residues. The catalytic domain forms a compact hairpin structure that displays the three imidazole-containing residues. The enzyme can be made to cleave RNAs of almost any sequence by simple alteration of the two substrate-recognition domains that surround the catalytic domain. The enzyme operates with multiple turnover in the presence of micromolar concentrations of Zn2+, exhibiting saturation kinetics and a catalytic rate of >1 min-1. The imidazole-containing DNA enzyme, one of the smallest known nucleic acid enzymes, combines the substrate-recognition properties of nucleic acid enzymes and the chemical functionality of protein enzymes in a molecule that is small, yet versatile and catalytically efficient.
Assuntos
DNA/química , Desoxiuridina/análogos & derivados , Endorribonucleases/síntese química , Imidazóis/química , RNA/química , Zinco/química , Composição de Bases , Sequência de Bases , Catálise , Cátions Bivalentes , DNA/metabolismo , Endorribonucleases/química , Endorribonucleases/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Metais/química , Metais/metabolismo , RNA/metabolismo , RNA Catalítico , Especificidade por SubstratoAssuntos
Desenho de Fármacos , Endorribonucleases/biossíntese , Endorribonucleases/síntese química , Proteínas de Escherichia coli , RNA Catalítico/biossíntese , RNA Catalítico/química , RNA Catalítico/síntese química , Sequência de Bases , Primers do DNA/genética , Endorribonucleases/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Dados de Sequência Molecular , Plasmídeos/genética , RNA/genética , RNA/metabolismo , RNA Bacteriano/biossíntese , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Catalítico/genética , Ribonuclease P , Especificidade por SubstratoRESUMO
We studied the interaction of wheat germ 5S rRNA with synthetic polypeptides whose amino acid sequences were similar to that of the second zinc finger of Xenopus laevis transcriptional factor IIIA (TFIIIA). The results clearly show that in addition to weak 5S rRNA binding activity (data not shown), these two 30 amino acid long polypeptides hydrolyse some phosphodiester bonds of wheat germ 5S rRNA. The cleavage pattern of plant 5S rRNA is very specific and the cuts occur only after the pyrimidine residues. The same properties of these peptides were furthermore observed for E. coli tRNA(Phe). We found that the digestion specificity of both the zinc finger peptides is very similar to that of a pancreatic ribonuclease (RNase A).
Assuntos
Endorribonucleases/metabolismo , Fragmentos de Peptídeos/metabolismo , RNA Ribossômico 5S/metabolismo , Fatores de Transcrição/química , Dedos de Zinco , Sequência de Aminoácidos , Animais , Sequência de Bases , Endorribonucleases/síntese química , Hidrólise , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Fragmentos de Peptídeos/síntese química , Ligação Proteica , Fator de Transcrição TFIIIA , Triticum , Xenopus laevisRESUMO
The data on the synthesis of RNA-fragments with endoribonuclease activity were reviewed. In the framework of the hammerhead model refined by Haselof and Gerlach the contribution of certain nucleotides or functional groups in ribozyme catalytic activity and the role of double helices are considered in detail. The design of ribozymes with a certain specificity including the ribozymes with antiviral activity also are discussed.
Assuntos
Endorribonucleases/metabolismo , Sequência de Bases , Endorribonucleases/síntese química , Cinética , Dados de Sequência Molecular , RNA/metabolismo , RNA Catalítico/metabolismo , Especificidade por SubstratoRESUMO
The simplest variant of synthetic substrate-ribozyme complex has been proposed. The schemes of potential ribozyme "subunits" synthesis have been worked out: R1--GCUUGAAACAAA; R2--AAAAACUGAUGAAAGC. The macroscale synthesis of dinucleoside monophosphate ApU, GpC, CpU catalyzed by immobilized ribonucleases of different specificity and preparation of oligoadenylates by hydrolysis of poly-A in the presence of endonuclease Serratia marcescens, as well the synthesis of conservative sequences of potential ribozyme such as ApUpG, CpUpG, GpApU, ApApApG and others have been described.