RESUMEN
The DFF40-DFF45 heterodimeric complex is a primary player in apoptotic DNA fragmentation and is conserved among different species including Drosophila melanogaster. DFF40 is a novel nuclease, while DFF45 is an inhibitor that can suppress the nuclease activity of DFF40 via tight interaction. Unlike mammalian systems, apoptotic DNA fragmentation in the fly is controlled by four DFF-related proteins known as Drep1, Drep2, Drep3 and Drep4. Drep1 and Drep4 are DFF45 and DFF40 homologues, respectively. Although the exact functions of Drep2 and Drep3 are unclear, they are also involved in apoptotic DNA fragmentation via regulation of the function of Drep1 and Drep4. DFF-related proteins contain a conserved CIDE domain of â¼90 amino-acid residues that is involved in protein-protein interaction. In this study, the CIDE domains of Drep2 and Drep3 were purified in Escherichia coli, after which they formed a stable complex in vitro and were crystallized by the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to a resolution of 5.8â Å.
Asunto(s)
Proteínas de Drosophila/química , Drosophila melanogaster/química , Dominios y Motivos de Interacción de Proteínas , Animales , Cristalización , Cristalografía por Rayos XRESUMEN
ß-Transaminase (ß-TA) catalyzes the transamination reaction between ß-aminocarboxylic acids and keto acids. This enzyme is a particularly suitable candidate for use as a biocatalyst for the asymmetric synthesis of enantiochemically pure ß-amino acids for pharmaceutical purposes. The ß-TA from Mesorhizobium sp. strain LUK (ß-TAMs) belongs to a novel class in that it shows ß-transaminase activity with a broad and unique substrate specificity. In this study, ß-TAMs was overexpressed in Escherichia coli with an engineered C-terminal His tag. ß-TAMs was then purified to homogeneity and crystallized at 293â K. X-ray diffraction data were collected to a resolution of 2.5â Å from a crystal that belonged to the orthorhombic space group C222(1), with unit-cell parameters a = 90.91, b = 192.17, c = 52.75â Å.
Asunto(s)
Alphaproteobacteria/enzimología , Proteínas Bacterianas/química , Transaminasas/química , Alphaproteobacteria/genética , Secuencia de Aminoácidos , Aminoácidos/biosíntesis , Cristalización , Cristalografía por Rayos X/métodos , Difusión , Escherichia coli/enzimología , Escherichia coli/genética , Histidina/química , Transferasas Intramoleculares/química , Isopropil Tiogalactósido/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Ingeniería de Proteínas , Homología de Secuencia de Aminoácido , Estereoisomerismo , Especificidad por Sustrato , Transaminasas/aislamiento & purificación , Transaminasas/metabolismo , Transformación Bacteriana , Difracción de Rayos XRESUMEN
DNA fragmentation is the hallmark of apoptotic cells and mainly mediated by the DNA fragmentation factor DFF40(CAD)/DFF45(ICAD). DFF40 is a novel nuclease, whereas DFF45 is an inhibitor that can suppress the nuclease activity. Apoptotic DNA fragmentation in the fly is controlled by four DFF-related proteins, known as Drep1, 2, 3 and 4. However, the functions of Drep2 and Drep3 are totally unknown. Here, we found that Drep2 is a novel nuclease whose activity is inhibited by Drep3 through a tight interaction with the CIDE domain. Our results suggest that the fly has dual apoptotic DNA fragmentation systems: Drep1: Drep4 and Drep2: Drep3 complexes.