RESUMO
Although the mechanism of RNA cleavage by RNases has been studied for many years, there remain aspects that have not yet been fully clarified. We have solved the crystal structures of RNase Sa2 in the apo form and in complexes with mononucleotides. These structures provide more details about the mechanism of RNA cleavage by RNase Sa2. In addition to Glu56 and His86, which are the principal catalytic residues, an important role in the first reaction step of RNA cleavage also seems to be played by Arg67 and Arg71, which are located in the phosphate-binding site and form hydrogen bonds with the oxygens of the phosphate group of the mononucleotides. Their positive charge very likely causes polarization of the bonds between the oxygens and the phosphorus atom, leading to electron deficiency on the phosphorus atom and facilitating nucleophilic attack by O2' of the ribose on the phosphorus atom, leading to cyclophosphate formation. The negatively charged Glu56 is in position to attract the proton from O2' of the ribose. Extended molecular docking of mononucleotides, dinucleotides and trinucleotides into the active site of the enzyme allowed us to better understand the guanosine specificity of RNase Sa2 and to predict possible binding subsites for the downstream base and ribose of the second and third nucleotides.
Assuntos
Nucleotídeos/metabolismo , Ribonucleases/química , Ribonucleases/metabolismo , Apoproteínas/química , Apoproteínas/genética , Apoproteínas/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Catálise , Domínio Catalítico , Cristalografia por Raios X , Modelos Moleculares , Nucleotídeos/química , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Conformação Proteica , RNA/metabolismo , Ribonucleases/genética , Especificidade por SubstratoRESUMO
This paper describes the cloning, purification and characterization of thioredoxin (TrxA) and thioredoxin reductase (TrxR) from bacterial strain Streptomyces coelicolor . The genes of S. coelicolor encoding TrxA and TrxR were amplified by polymerase chain reaction, inserted into pET expression vector and used to overexpress these proteins in Escherichia coli . TrxA and TrxR were produced as the hexahistidine fusion proteins and were recovered from the cytoplasm as the soluble proteins. The activity of the purified recombinant proteins was demonstrated. The activity of TrxA was shown by efficient reduction of insulin and activity of NADPH-dependent TrxR was revealed by catalyses of 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) reduction reaction. The reduction reaction was fully dependent upon the presence of TrxA as intermediate electron carrier with the pH optimum 7.5 and the temperature optimum 29 degrees C. Km value of TrxR for TrxA was 0.217 +/- 0.02 microM.
Assuntos
Proteínas de Bactérias/metabolismo , Streptomyces coelicolor/enzimologia , Tiorredoxina Dissulfeto Redutase/metabolismo , Tiorredoxinas/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Ácido Ditionitrobenzoico/metabolismo , Escherichia coli/metabolismo , Insulina/metabolismo , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Streptomyces coelicolor/genética , Especificidade por Substrato , Tiorredoxina Dissulfeto Redutase/genética , Tiorredoxinas/genéticaRESUMO
Mechanistic studies of ATP-dependent proteolysis demonstrate that substrate unfolding is a prerequisite for processive peptide bond hydrolysis. We show that mitochondrial Lon also degrades folded proteins and initiates substrate cleavage non-processively. Two mitochondrial substrates with known or homology-derived three-dimensional structures were used: the mitochondrial processing peptidase alpha-subunit (MPPalpha) and the steroidogenic acute regulatory protein (StAR). Peptides generated during a time course of Lon-mediated proteolysis were identified and mapped within the primary, secondary, and tertiary structure of the substrate. Initiating cleavages occurred preferentially between hydrophobic amino acids located within highly charged environments at the surface of the folded protein. Subsequent cleavages proceeded sequentially along the primary polypeptide sequence. We propose that Lon recognizes specific surface determinants or folds, initiates proteolysis at solvent-accessible sites, and generates unfolded polypeptides that are then processively degraded.
Assuntos
Trifosfato de Adenosina/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Biologia Computacional , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Proteínas Fúngicas/metabolismo , Humanos , Hidrólise , Espectrometria de Massas , Camundongos , Mitocôndrias/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Fosfoproteínas Fosfatases/química , Fosfoproteínas/química , Protease La/química , Ligação Proteica , Dobramento de Proteína , Proteína Fosfatase 2C , Estrutura Terciária de Proteína , Proteínas/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por Substrato , Fatores de TempoRESUMO
The physico-chemical features of the NADPH-thioredoxin reductase (TRR) and two thioredoxins from Streptomyces aureofaciens (A14) are reported. The activity of pure S. aureofaciens thioredoxin reductase decreased drastically in the presence of NADPH or NADH while NADP(+), NAD(+), as well as S. aureofaciens thioredoxin-1 (TR1) activated the enzyme activity significantly. TR1 fully protected the enzyme from inactivation and also promoted its complete reactivation. S. aureofaciens thioredoxin-2 (TR2) did not protect thioredoxin reductase from NADPH inactivation. The results indicate that although the two thioredoxins from S. aureofaciens have similar biochemical properties, their essential oxidoreductase activities are not interchangeable.