The Candida albicans gene for mRNA 5 '-cap methyltransferase: identification of additional residues essential far catalysis

The 5'-cap structure of eukaryotic mRNA is methylated at the terminal guano sine by RNA (guanine-N-7-)-methyltransferase (cap MTase). Saccharomyces cer evisiae ABD1 (ScABD1) and human hMet (also called CMT1) genes are responsib le for this enzyme, The ABD1 homologue was cloned from the pathogenic fungu s Candida albicans and named C. albicans ABD1 (CaABD1), When expressed as a fusion with glutathione S-transferase (GST), CaAbd1p displayed cap MTase a ctivity in vitro and rescued S. cerevisiae abd1 Delta null mutants, indicat ing that CaABD1 specifies and active cap MTase. Although the human cap MTas e binds to the human capping enzyme (Hce1p), which possesses both mRNA guan ylyltransferase (mRNA GTase) and mRNA 5'-triphosphatase (mRNA TPase) activi ties, yeast two-hybrid analysis demonstrated that in yeast neither mRNA GTa se nor mRNA TPase physically interacted with the Abd1 protein, Comparison o f the amino acid sequences of known and putative cap MTases revealed a high ly conserved amino acid sequence motif, Phe/Val-Leu-Asp/Glu-Leu/Met-Xaa-Cys -Gly-Lys-Gly-Gly-Asp-Leu-Xaa-Lys, which encompasses the sequence motif char acteristic of divergent methyltransferases. Mutations in CaAbd1p of leucine at the second and the twelfth positions (so far uncharacterized) to alanin e severely diminished the enzyme activity and the functionality in vivo, wh ereas those of leucine at the fourth, cysteine at the sixth, lysine at the eighth, and glycine at the tenth positions did not. Furthermore, valine sub stitution for the twelfth, but not for the second, leucine in that motif ab olished the activity and functionality of CaAbd1p. Thus, it appears that le ucine at the second and the twelfth positions in the motif, together with a previously identified acidic residue in the third, glycine at the sixth an d glutamic acid at the eleventh positions, play important roles in the cata lysis, and that side chain length is crucial for the activity at the twelft h position in the motif.