RESUMEN
DNA methylation is important in cellular, developmental and disease processes, as well as in bacterial restriction-modification systems. Methylation of DNA at the amino groups of cytosine and adenine is a common mode of protection against restriction endonucleases afforded by the bacterial methyltransferases. The first structure of an N:6-adenine methyltransferase belonging to the beta class of bacterial methyltransferases is described here. The structure of M. RSR:I from Rhodobacter sphaeroides, which methylates the second adenine of the GAATTC sequence, was determined to 1.75 A resolution using X-ray crystallography. Like other methyltransferases, the enzyme contains the methylase fold and has well-defined substrate binding pockets. The catalytic core most closely resembles the PVU:II methyltransferase, a cytosine amino methyltransferase of the same beta group. The larger nucleotide binding pocket observed in M. RSR:I is expected because it methylates adenine. However, the most striking difference between the RSR:I methyltransferase and the other bacterial enzymes is the structure of the putative DNA target recognition domain, which is formed in part by two helices on an extended arm of the protein on the face of the enzyme opposite the active site. This observation suggests that a dramatic conformational change or oligomerization may take place during DNA binding and methylation.
Asunto(s)
Adenina/metabolismo , Rhodobacter sphaeroides/enzimología , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/química , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/clasificación , Secuencia de Aminoácidos , Secuencia de Bases , Sitios de Unión , Catálisis , Dominio Catalítico , Cristalografía por Rayos X , ADN/genética , ADN/metabolismo , Metilación de ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/clasificación , Proteínas de Unión al ADN/metabolismo , ADN-Citosina Metilasas/química , Enlace de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Nucleótidos/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , S-Adenosilmetionina/metabolismo , Alineación de Secuencia , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/metabolismo , Electricidad Estática , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The structure and function of Erwinia chrysanthemi pectate lysase C, a plant virulence factor, is reviewed to illustrate one mechanism of pathogenesis at the molecular level. Current investigative topics are discussed in this paper.
Asunto(s)
Erwinia/enzimología , Glicósido Hidrolasas/fisiología , Plantas/microbiología , Poligalacturonasa/fisiología , Polisacárido Liasas/fisiología , Virulencia , Glicósido Hidrolasas/química , Poligalacturonasa/química , Polisacárido Liasas/química , Conformación ProteicaRESUMEN
The three-dimensional structure of a complex between the pectate lyase C (PelC) R218K mutant and a plant cell wall fragment has been determined by x-ray diffraction techniques to a resolution of 2.2 A and refined to a crystallographic R factor of 18.6%. The oligosaccharide substrate, alpha-D-GalpA-([1-->4]-alpha-D-GalpA)3-(1-->4)-D-GalpA , is composed of five galacturonopyranose units (D-GalpA) linked by alpha-(1-->4) glycosidic bonds. PelC is secreted by the plant pathogen Erwinia chrysanthemi and degrades the pectate component of plant cell walls in soft rot diseases. The substrate has been trapped in crystals by using the inactive R218K mutant. Four of the five saccharide units of the substrate are well ordered and represent an atomic view of the pectate component in plant cell walls. The conformation of the pectate fragment is a mix of 21 and 31 right-handed helices. The substrate binds in a cleft, interacting primarily with positively charged groups: either lysine or arginine amino acids on PelC or the four Ca2+ ions found in the complex. The observed protein-oligosaccharide interactions provide a functional explanation for many of the invariant and conserved amino acids in the pectate lyase family of proteins. Because the R218K PelC-galacturonopentaose complex represents an intermediate in the reaction pathway, the structure also reveals important details regarding the enzymatic mechanism. Notably, the results suggest that an arginine, which is invariant in the pectate lyase superfamily, is the amino acid that initiates proton abstraction during the beta elimination cleavage of polygalacturonic acid.
Asunto(s)
Isoenzimas/química , Isoenzimas/metabolismo , Oligosacáridos/química , Oligosacáridos/metabolismo , Polisacárido Liasas/química , Polisacárido Liasas/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , Calcio/metabolismo , Conformación de Carbohidratos , Secuencia de Carbohidratos , Pared Celular , Secuencia Conservada , Cristalografía por Rayos X , Dickeya chrysanthemi/enzimología , Dickeya chrysanthemi/patogenicidad , Análisis de Fourier , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Mutagénesis Sitio-Dirigida , Plantas/microbiología , Estructura Secundaria de Proteína , Espectrometría de Masa Bombardeada por Átomos Veloces , Especificidad por SustratoRESUMEN
The crystal structure of pectate lyase E (PelE; EC 4.2.2.2) from the enterobacteria Erwinia chrysanthemi has been refined by molecular dynamics techniques to a resolution of 2.2 A and an R factor (an agreement factor between observed structure factor amplitudes) of 16.1%. The final model consists of all 355 amino acids and 157 water molecules. The root-mean-square deviation from ideality is 0.009 A for bond lengths and 1.721[deg] for bond angles. The structure of PelE bound to a lanthanum ion, which inhibits the enzymatic activity, has also been refined and compared to the metal-free protein. In addition, the structures of pectate lyase C (PelC) in the presence and absence of a lutetium ion have been refined further using an improved algorithm for identifying waters and other solvent molecules. The two putative active site regions of PelE have been compared to those in the refined structure of PelC. The analysis of the atomic details of PelE and PelC in the presence and absence of lanthanide ions provides insight into the enzymatic mechanism of pectate lyases.
RESUMEN
Several lines of evidence indicate that serine/threonine protein phosphatases may act as negative regulators of cellular growth. For example, treatment of cells with the tumor-promoter okadaic acid, an inhibitor of certain types of these phosphatases, resulted in the increased expression of several proto-oncogenes, indicating a negative role of the respective phosphatases in gene regulation. However, it was puzzling to find that okadaic acid-treated cells, even in the presence of highly expressed proto-oncogenes, did not proliferate, but were arrested at certain points of the cell cycle. To further analyze this discrepancy, we investigated the involvement of protein phosphatases in the control of other cell cycle regulatory genes, such as cdc2 which encodes an essential cell cycle regulatory kinase. We found that cdc2 gene expression was blocked by okadaic acid, but stimulated by protein phosphatase 2A. Protein phosphatase 2A is shown to be a positive regulator of cdc2 gene activity and to be required for cdc2 expression. Thus, our findings identify protein phosphatase 2A as a positive regulator of a major cell cycle regulatory gene and therefore suggest a stimulatory role of this enzyme in this aspect of cellular growth control.
Asunto(s)
Proteína Quinasa CDC2/genética , Fosfoproteínas Fosfatasas/metabolismo , Células 3T3 , Animales , Secuencia de Bases , Sitios de Unión/genética , Ciclo Celular/fisiología , División Celular/efectos de los fármacos , División Celular/genética , División Celular/fisiología , ADN/genética , ADN/metabolismo , Inhibidores Enzimáticos/farmacología , Éteres Cíclicos/farmacología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Genes Reguladores , Ratones , Datos de Secuencia Molecular , Ácido Ocadaico , Fosfoproteínas Fosfatasas/antagonistas & inhibidores , Regiones Promotoras Genéticas , Proteína Fosfatasa 2 , TransfecciónRESUMEN
The mechanism by which tumor promoters contribute to cellular transformation and tumorigenesis is not completely understood. To investigate further the molecular events involved in these processes, we used okadaic acid, a non-phorbol ester type tumor promoter that specifically inhibits certain protein phosphatases. We describe here that the continuous treatment of murine NIH 3T3 fibroblast cell cultures with okadaci acid resulted in a 50-fold amplification of two genes, mdr-1a and mdr-1b, that conferred multidrug resistance. As a consequence, the cells became cross-resistant to the cytotoxic effects of adriamycin, an antineoplastic drug used in the treatment of human tumors. Since genetic changes have been correlated with cell transformation and tumorigenesis, our results suggest that these processes may constitute an additional factor contributing to tumor promotion by okadaic acid.