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Characterization of Candida albicans RNA triphosphatase and mutational analysis of its active site
1Molecular Biology Program, Sloan-Kettering Institute, 1275 York Avenue, New York, NY 10021, USA.
Abstract:
The RNA triphosphatase component (CaCet1p) of the mRNA capping apparatus of the pathogenic fungus Candida albicans differs mechanistically and structurally from the RNA triphosphatase of mammals. Hence, CaCet1p is an attractive antifungal target. Here we identify a C-terminal catalytic domain of CaCet1p from residue 257 to 520 and characterize a manganese-dependent and cobalt-dependent NTPase activity intrinsic to CaCet1p. The NTPase can be exploited to screen in vitro for inhibitors. The amino acids that comprise the active site of CaCet1p were identified by alanine-scanning mutagenesis, which was guided by the crystal structure of the homologous RNA triphosphatase from Saccharomyces cerevisiae (Cet1p). Thirteen residues required for the phosphohydrolase activity of CaCet1p (Glu287, Glu289, Asp363, Arg379, Lys396, Glu420, Arg441, Lys443, Arg445, Asp458, Glu472, Glu474 and Glu476) are located within the hydrophilic interior of an eight-strand beta barrel of Cet1p. Each of the eight strands contributes at least one essential amino acid. The essential CaCet1p residues include all of the side chains that coordinate manganese and sulfate (i.e., gamma phosphate) in the Cet1p product complex. These results suggest that the active site structure and catalytic mechanism are conserved among fungal RNA triphosphatases.
Insights
The RNA triphosphatase from Candida albicans is a potential antifungal target. Researchers identified its catalytic domain and active site, revealing conserved mechanisms with other fungal RNA triphosphatases.
Area of Science:
- Biochemistry
- Mycology
- Structural Biology
Background:
- The RNA triphosphatase component (CaCet1p) of Candida albicans is crucial for mRNA capping.
- CaCet1p's unique structure and mechanism compared to mammalian counterparts make it a promising antifungal target.
Purpose of the Study:
- To identify and characterize the catalytic domain and active site of CaCet1p.
- To explore the potential of CaCet1p's NTPase activity for inhibitor screening.
Main Methods:
- Identification of the C-terminal catalytic domain (residues 257-520).
- Characterization of manganese- and cobalt-dependent NTPase activity.
- Alanine-scanning mutagenesis guided by the crystal structure of Saccharomyces cerevisiae Cet1p.
Main Results:
- Thirteen essential residues for phosphohydrolase activity were identified within the active site's beta barrel.
- These residues are critical for coordinating manganese and sulfate (gamma phosphate).
- The active site structure and catalytic mechanism appear conserved among fungal RNA triphosphatases.
Conclusions:
- The identified catalytic domain and active site provide a basis for developing antifungal agents targeting CaCet1p.
- Conserved active site features suggest broad applicability of findings across fungal species.