RESUMO
The crystal structure of D-glyceraldehyde-3-phosphate dehydrogenase (PfGAPDH) from the major malaria parasite Plasmodium falciparum is solved at 2.25 A resolution. The structure of PfGAPDH is of interest due to the dependence of the malaria parasite in infected human erythrocytes on the glycolytic pathway for its energy generation. Recent evidence suggests that PfGAPDH may also be required for other critical activities such as apical complex formation. The cofactor NAD(+) is bound to all four subunits of the tetrameric enzyme displaying excellent electron densities. In addition, in all four subunits a completely unexpected large island of extra electron density in the active site is observed, approaching closely the nicotinamide ribose of the NAD(+). This density is most likely the protease inhibitor AEBSF, found in maps from two different crystals. This putative AEBSF molecule is positioned in a crucial location and hence our structure, with expected and unexpected ligands bound, can be of assistance in lead development and design of novel antimalarials.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/química , Plasmodium falciparum/enzimologia , Proteínas de Protozoários/química , Sequência de Aminoácidos , Animais , Sequência Conservada , Cristalografia por Raios X , Citoplasma/enzimologia , Gliceraldeído-3-Fosfato Desidrogenases/genética , Humanos , Ligação de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , NAD/química , NAD/metabolismo , Plasmodium falciparum/genética , Estrutura Secundária de Proteína , Subunidades Proteicas/química , Proteínas de Protozoários/genética , Proteínas Recombinantes/química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Eletricidade EstáticaRESUMO
Cloning grills are aluminum grids designed to divide an agar plate into segments, thereby multiplying the number of E. coli cultures which can be streaked out on a single plate. The grills are autoclaved and placed in square petri dishes immediately after hot agar is poured. When the agar solidifies, the grill remains embedded in the media, and each of the 12 lanes accommodates the streaking out of a single culture. As the spacing of the grill lanes is the same as that of a 96-well plate, 12 cultures can be streaked at a time using a 12-channel pipette. This allows a plate of 96 cultures to be rapidly and accurately plated for colony isolation on only eight agar plates.