Characterization of Candida albicans RNA triphosphatase and mutational analysis of its active site

The RNA triphosphatase component (CaCet1p) of the mRNA capping apparatus of the pathogenic fungus Candida albicans differs mechanistically and structu rally from the RNA triphosphatase of mammals. Hence, CaCet1p is an attracti ve antifungal target. Here we identify a C-terminal catalytic domain of CaC et1p from residue 257 to 520 and characterize a manganese-dependent and cob alt-dependent NTPase activity intrinsic to CaCet1p, The NTPase can be explo ited to screen in vitro for inhibitors. The amino acids that comprise the a ctive site of CaCet1p were identified by alanine-scanning mutagenesis, whic h was guided by the crystal structure of the homologous RNA triphosphatase from Saccharomyces cerevisiae (Cet1p). Thirteen residues required for the p hosphohydrolase activity of CaCet1p (Glu287, Glu289, Asp363, Arg379, Lys396 , Glu420, Arg441, Lys443, Arg445, Asp458, Glu472, Glu474 and Glu476) are lo cated within the hydrophilic interior of an eight-strand beta barrel of Cet 1p, Each of the eight strands contributes at least one essential amino acid . The essential CaCet1p residues include all of the side chains that coordi nate manganese and sulfate (i.e., gamma phosphate) in the Cet1p product com plex. These results suggest that the active site structure and catalytic me chanism are conserved among fungal RNA triphosphatases.